A title in the European Plant Biology Series
Consulting Editor
Professor M.B. Wilkins
University of Nottingham
Physiology of
Stomata
Hans Meidner
Reader, Department of Horticulture, University of Readihg
and T. A. Mansfield
Lecturer. Department of Biological Sciences. University of Lancaster
TATA McGRAW-HILL PUBLISHING COMPANY LTO.
Bombay - New Delhi
198
PHYSIOLOGY OF STOMATA
I
L'l-E G :551-1 Hi?'1K. ?,
Copyright @ 1968 McGraw-Hili Book Company (U.K.) Limited
An Rights Reserved.
No part of this publication may be reproduced, stored in a retrieval system,
or transmitted, in any form or by any means, electronic, mechanical,
photocopying, recording or othetwise, without the prior permission
of McGraw-HilI Book Company (U.K.) limited.
T M H Edition
Reprinted in India by arrangement with McGraw-Hili Bool< Company (U.K.)
Limited, MAIDENHEAD; BERKSHIRE; ENGLAND.
This edition can be exported {rom India only by the publishers,
Tata McGraw~Hill Publishing Company Ltd.
Published by Tata McCraw-HIli Pu'blishlOg Company Limited and
Printed by Mohan Makhijanlat Rekha Prlnler•• Pvl. Ltd. New Delhl-15.
Preface
The stomatal apparatus and the mechanism of its operation are
usually dealt with in connection with transpiration and photosynthesis, because both these processes are to a considerable extent
controlled by the stomata. However, stomatal responses to various
stimuli are so complex that the physiology of the stomata might well
be treated as a subject in its own right, and we have tried, therefore,
to bring together in this book the important known facts about the
.fJIC\...<'.i1nnTt=SiIT (fl' ~'tI.JJ.Ttat(fI' iiW9errte.L1't:)'.
Stomatal morphology, the technology of stomatal investigations,
and the physical aspects of diffusion through stomata are so generally
agreed upon that we have been able to present these, in chapters 1 to
3, in textbook fashion, although we have taken care to direct the
reader to the original research papers upon which we have based our
statements. On the other hand, knowledge is less complete about the
responses of stomata to stimuli, their rhythms and oscillations, and
the biochemical changes that constitute the mechanisms behind the
movements; consequently, in dealing with these subjects in chapters
4 to 7, we discuss the available experimental results, the hypotheses
vii
PREFACE
that have been formulated on this evidence, and the conflicting
evidence that questions the validity of the various hypotheses. On
these topics we have deliberately presented the evidence as a survey
of the experimental results with as many attempts at synthesis t~wards
a generally valid hypothesis as appeared permissible.
The theoretical study of any subject matter gains much from
experimental work and, therefore, we have included an appendIX with
some suggestions for practical work on stomata. Experiments on
stomata have a place in ceurses on general plant physiology, and
teachers as well as students may find here some new and useful
suggestions.
The renewed interest in stomatal physiology during recent years has
been partly directed towards finding ways and means of imposing on
stomata, especially those of crop plants, artificial controls that would
reduce transpirational water loss without seriously reducing photosynthetic yield. For the pursuance of such an aim a thorough understanding of stomatal physiology is essential, and we hope that this
bringing together of the important information on stomata will help
students and research workers. If this book contributes to a better
understanding of this complex subject matter by stimulating further
research into the physiology of stomata we shall have achieved our
purpose.
viii
AcklUJwledgement.
We should like to acknowledge our indebtedness to Professor
O. V. S. Heath, F.R.S., University of Reading, who taught us much of
what we know about stomata. In particular, we have benefited from
lectures given by Professor Heath. and from papers by Dr H. L.
Penman, F.R.S., on the theory of diffusion through stomata dealt
with in chapter 3. However, we are entirely responsible for the
presentation of the subject matter and any errors that may be found.
One of us (H.M.) remembers with gratitude Emeritus Professor
A. W. Bayer, University of Natal, for his teaching of Botany.
To the undermentioned we are indebted for permission to use
diagrams and tables from their published work: Dr G. G. J. Bange
for Fig. 3.3; Blackwell Scientific Publications, Ltd for Fig. 7.2; the
Carnegie Institution of Washington for Figs 4.12 and 4.15, and the
quotation on p. 87; DrM. Fujinofordata in Table 6.1; DrP. Gaastra
for Fig. 4.6; Professor O. V. S. Heath for Figs 4.4, 4.5, 4.11, and
4.13; Dr P. J. C. Kuiper for Fig. 4.7; Dr K. Nishida for data in
Table 5.2; North Holland Publishing Company for Fig. 3.3;
Professor M. Shaw and the National Research Council of Canada
for Fig. 4.3; Professor M. G. Stalfelt for Figs 4.16 and 5.6; Dr J. P.
Ting for Figs 5.11 and 5.12; Professor H. J. Virgin for Figs 4.2, 4.9,
and 4.10; Professor W. T. Williams for Fig. 7.3, and Dr A. J. Willis
for Figs .4.18 and 7.2.
HANS MEIDNER
T. A.
MANSFIELD
ix
Contents
Chapter 1 MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
1.1 The occurrence of stomata in the plant
kingdom
1.2 The differentiation of stomata
1.3 Dimensions and structure of guard cells
1.4 The mechanism of guard cell movement
1.5 The contents of guard cells
1.6 Properties and functions of subsidiary cells
1.7 Modifications of the stomatal apparatus with
habitat
Chapter 2 THE MEASUREMENT OF STOMATAL APERTURE
2.1 The choice of plant material
2.2 Microscopic measuring techniques
2.3 Macroscopic measuring techniques
2.4 Viscous flow parometers
2.5 Diffusive flow porometers
1
6
10
14
18
22
23
26
27
29
31
32
42
xi
CONTENTS
Chapter 3 THE THEORY OF DIFFUSION THROUGH STOMATA
Chapter 4
49
49
3.1 The rate of diffusion of a gas
3.2 Details of the diffusion path between leaves
and the atmosphere
3.3 Stomatal control of transpiration
3.4 Stomatal control of photosynthesis
3.5 The experimental control of stomatal aperture
55
59
63
66
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
69
4.1 Light and carbon dioxide
70
4.2 Transmission of an opening or closing
stimulus
85
4.3 Temperature
87
4.4 Water supply
92
4.5 Relative humidity ofthe atmosphere
100
4.6 Interactions between environmental factors
101
Chapter S THE ROLE OF RHYTIIMSIN STOMATAL BEHAVIOUR
Chapter 6
102
5.1 Endogenous rhythms
5.2 Night opening of stomata
5.3 Rhythms and the normal diurnal movements
of stomata
5.4 After-effects of environmental factors on
rhythms
5.S Stomatal behaviour in succulent plants
5.6 Short-period fluctuations
103
109
STOMATAL OPENING AND CLOSING REACTIONS
119
6.1
Sources of energy for stomatal movements
6.2 Dynamics of stomatal movements
113
113
114
11&
119
128
6.3
Distinction between the maintenance and the
131
production of opening
6.4 Effects of temperature on opening and closing
movements
131
6.5 Ionic effects
132
Chapter 7
xii
HYPOTHESES OLD AND NEW
7.1 The glycollate hypothesis
7.2 Dark carboxylation reactions
135
135
138
CONTENTS
7.3
7.4
7.5
7.6
Appendix
The role of starch? sugar interconversion
Permeability changes
Blue ligbt elfects
What next?
141
145
146
147
SOME SUGGESTIONS FOR PRACTICAL WORK
150
A.I Observations and measurements on epidermal
strips
A.2 Measuring stomatal aperture in intact leaves
A.3 Stomatal responses to stimuli
A.4 Experiments on gaseous diffusion
150
J <;4
155
157
Bibliography
160
Index
170
1. Morphology and physiology
of stomatal cells
The water relations and the metabolism of land plants depend to a
large extent on the diffusion of water vapour and of gases through the
stomatal pores which occur on their aerial parts. These pores are
situated between two s]lecialized el'idermal cells, the guard cells,
which by changes in their dinlonsions and shapes bring about openin;;
and closing movements of the stomata. These movements distinguish
stomata from other pores found in plant organs, as for example,
pneumathodes, hydathodes, lenticels, and the breathing pores found
in the thalli of liverworts.
1.1
The occurrence of stomata in the
plant kingdom
True stomatal pores, which can change from the wide open to an
apparently completely closed condition, are a feature of land plants
above the evolutionary level of Anthoceros. Thus, stomata have been
PHYSIOWGY OF STOMATA
Table 1.1 Stomatal frequencies, dimensions and pore areas as percentages of
total leaf areas. AI! dimensions in p. {,u.m in 51 symbols)
Frequencies
permm2
Dimensions
of slomatal
apparatus
Length of
'"
...------'-----
~
pore
'"
Bore area
as average
percentage
of lolaI
leaf area;
pore width
takl!D as
6",
upper lower upper lower upper lower
Osmunda
67
56x38
30
0·5
regalis
Phyllitis
59
77 x42
38
0·55
85
46x28
21
0'45
IlO
49x28
17
0'45
sc%pendr;um
Pteridium
aquilinum
Ahies
nordmanniana
Cedrus
84
85
38 x 31
38 x 31
24
24
1·0
deodara
Pinus
120
120
28 x28
18 x28
20
20
1-2
39
14
39
16
49x45
42x26
49x45
42x26
12
20
12
20
0·25
0·15
175
50
70
175
45
85
42x38
52 x 31
42x21
42x38
56x26
38x21
24
20
17
24
19
17
2'0
0·5
0·65
50
40
56 x 31
53 x Us
28
28
0·63
98
108
38 x 19
43 x24
12
16
0·7
sylvestris
Picca pungens
Larix decidua
Alliumcepa
Avena sativa
Hordeum
vulgare
Triticum
vulgare
Zeamays
Aesculus hippo-
210
28x 14
13
0-7
170
32x24
13
0·6
370
31 x23
10
0·9
340
370
28x18
25 x 18
10
10
0-8
0·9
castanum
Carpinus
betulus
Eucalyptus
globulus
Quercus robuT
Tilia europea
2
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
Table 1.1
Continued
Pore area
as average
percentage
of total
Frequencies
permmz
Dimensions
of stomatal
apparatus
leaf area;
,.,.
Length of
pore width
pore
taken as
,.,.
6,.,.
,..------.-"--,--~ ~
upper lower upper
lIelianthus
annuus
Medicago
sativa
Nicotiana
tahacum
Pelargonium
zonale
Ricinus
communis
Viciafaba
Xanthium
pennsylvanicum
Sedum
spec/ahilis
Tradescantia
lower
upper
lower
120
115
35 x 25
32x29
IS
17
1·1
169
188
26x 17
25 x 17
9
13
0·8
50
190
31 x 25
31 x 25
14
14
0·8
29
179
48 x40
44x38
24
23
1·2
182
270
31 x 21
38 x24
12
24
2'1
65
173
75
177
46x25
39 x25
46x25
39 x 25
28
17
28
17
1'0
1'5
28
35
32x 32
33 x 31
21
20
0'32
7
23
67 x 38
70x42
49
52
0'35
virginiana
Note. Unless specifically stated, all the data in Tables 1.1 to 1.4 were
collected by the authors. The data should be regarded as the basis for some
of the statements made in the text; they would not, necessarily, be representative of other plants, although the same tendencies would, no doubt,
be illustrated with data collected in another survey.
MeaSUIements were made on plants growing in one locality or speciaIJy
grO\Vll for the purpose in the glasshouse. Each figureIepresents the average
of eighty·one measurements made on three leaves selected for uniformity
of appearance and circumstances of growth. From each of these leaves,
three epidermal strips were taken, and on each strip three areas of 0·25 cm2
were selected for making three counts or measurements in each.
found in fossil Psilophytales (Rhynia) and are present in both fossil
and living members of the Spermatophyta, both Angiospermae and
Gymnospermae. In the Cycadales and the fossil gymnosperms, the
differentiation of stomata from protoderm cells has been used as a
2
3
PHYSIOLOGY OF STOMATA
diagnostic character for the classification of fossil plants. (21) In the
present-day flora, stomata are found in the sporophytic capsules of
Anthoceros and most mosses, which are partly independent of their
gametophytcs for carbohydrate food, and they are present in the
epidermes of practically all aerial parts of our land flora.
Although stomata are commonly associated with leaves only, they
may occur in the epidermis of herbaceous stems and petioles, in the
parts of the angiosperm flower, and in many fmits (banana, bean,
cucumber, pea). Stomata can also be found in the awns of most
graminaceous inflorescences and in modified leaves such as tendrils
(Pisum sativtlm). In some of these structures the stomata may be nonfunctioning under ordinary conditioru; of illumination, carbon dioxide
concentration, water supply, and humidity.
The number of stomata in the lower epidermis of leaves often
exceeds the number in the upper epidermis, where they may be
altogether absent. Leaves with stomata in both epidermes are called
amphistomatous, those with stomata in the lower epidermis only are
called hypostomatous.
The number of stomata per unit area varies not only betwer'l
species but also within anyone species owing to the influence of
environmental factors during growth. This is due not so much to an
inftuence on the frequency with which stomata are differentiated as
to an influence on cell size in general, including, of course, the guard
cells. Thus, there are more cells per unit area and more stomata per
unit area in sun leaves than in shade leaves, and more also in leaves
of plants growing in dry soil and low humidity compared with those
growing in moist soil and high humidity. Also, on a single plant,
t~"ie W. a \en&"i\C"j {'U"i \'t'b.'Y~ 'Uf ril~\\'C1 irfl,tlti'\'lu \'U ha"Ve ~ma~\et
stomata and more per unit area than leaves of lower insertion on the
stem (see Table 1.2 (a)). Even within a single leaf the number of
stomata per unit area varies, being influenced, for instance, by the
distance from the sheath (see Table 1.2 (b)) and by the distance from
major veins.
Inspection of the data in Table 1.1 will show that there is a tendency
for stomata to be smaller where they are more numerous. Thus, the
ratio of total pore area/leaf area varies surprisingly little among comparable plants. It may be mentioned here that the choice of 61'- for the
calculation of the toral pore areaS shown in Table 1.1 was made
because such a degree of opening is commonly met with in all types
of plants. In many species the maximum stomatal opening occurring
4
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
Table 1,2 Changes in stomatal frequency. (a) with height of insertion on
plant. (b) with distance from leaf sheath, (c) with stage of leaf development.
All counts expressed as number per mm 2
Height of insertion
_---------
_--_.
(a) Polygonotum
multifiorum
(after Salisbury,
1927)(115)
TUfa europea
Triticum vulgare
(b) Zea ma),s
em
Upper
epidermis
Lower
epidermis
_---_---_-
-~--
21,5·
27,0
30'0
50'0
56,5
53-6
59'0
76,0
73·0
91,0
370
420
500
1,500
Third leaf below flag
leaf
First leaf below flag
leaf
Blade near sheath
Middle region of
blade
Near tip of blade
39
30
50
40
77
98
82
108
108
118
Leafsize
em
(c) Impatiens holstii
0'2 x 0,8
300 ofwhich 150
0·7 x
2,0 x
2,5 x
4,0 x
530
330
300
250
incomplete
NieD/iana
1,3
2'5
3'5
6,0
4,0 x 9'0
60
220 of which 120
9,0 x 17,0
110
i7'0 x 30·0
50
390 of which 130
incomplete
190
0'5xi'5
153
2,0 x 4-0
68
incomplete
tabacum
Viciafaba
170 of which 140
incomplete
76
under natural conditions may be in the region of lOlL, which would
increase the percentages quoted in Table 1.1 by about two-thirds.
In order to take into account cell size in general, Saiisbury(115)
5
PHYSIOLOGY OF STOMATA
introduced the concept of Sloma tal Index, which relates the number
of stomata per unit area to the number of epidermal cells per unit
area; this index tends to remain fairly constant for anyone plant.
Stomatal Index ~
100
number of stomata per unit area
.
x
number of stomata + number of epIdermal
per unit area
cells per unit area
Submerged leaves rarely bear stomata, and when there is dimorphism the aerial parts alone have stomata; in floating leaves the
upper epidermis bears stomata while the lower usually has nonesuch leaves are termed epislomatous.
1.2 The differentiation of stomata
Stomata are differentiated from protoderm cells early in the development of plant organs, but differentiation continues for some time as
the organ grows. The expansion of leaf blades results mainly from
the division of marginal and intercalary meristematic cells leading to
the differentiation of the epidermis (including the stomata) and other
leaf tissues. Cell enlargement follows cell division and, therefore,
counts of stomata per unit area carried out at different stages in leaf
development will differ, tending in the earlier stages to be higher than
in later ones (see Table L2 (c)). Individual stomata, once differentiated, grow in size and may change in shape as the leaf blade
expands.
Several processes of differentiation of stomata have been observed:
Stomata of elliptical shape. The most commonly occurring stomata
are elliptical in shape and differentiate from a protodermal cell by
division into two guard cells which soon assume their typical shapelike a bean in surface view. By separating slightly in the centre, the
guard cells form the stomatal pore between them. There is no radical
change in shape of the guard cells as they grow in size except that the
early rounded shape changes into a more elongated, elliptical one.
Adjacent epidermal cells mayor may not be distinctive in appearance,
but they usually function as subsidiary cells (see p. 22).
Graminaceous stomata. In most members of the Graminae and
Cyperaceae, differentiation of a stoma begins with the division of two
protoderm cells on either side of a stoma mother cell. The two
daughter cells reSUlting from these divisions, which lie adjacent to the
stoma mother cells, are the two future subsidiary cells; they are clearly
6
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
stoma mother cell
stoma mother cell
enlargemen.:t::.o.:..f__~
stoma
mother
cell and
beginning
of division
~ ~;oma mother cell begins
two guard cells have
been
they
remain
joined but
)\"~
two guard
cells formed
the
matrix
becomes
change. ~(\ shap.e
conspicuous
-",Ud'U cell
cell
epidermal cell
(a)
(b)
Fig. 1.1 Four stages in the differentiation of (a) elliptically shaped and (b)
graminaceous stomata
7
PHYSIOLOGY OF STOMATA
distinguishable in shape from other epidermal cells. The stoma
mother cell divides next to form the guard cells, between which the
stomatal pore appears. At this stage the ~raminaceous stoma r'rmstoma mother cell
stoma mother
ce:ow
epidermal cell
A
first division of
stoma mother
cell
diViSiO~Of
stoma
mother cell
second division resulting
in two subsidiary cells
on either side of central
cell
1
guard cell
epidermal cell
probably modified
into subsidiary cell
division of central
ce111nto two guard ceffs
(ar
pore
guard cell
(b)
Fig.1.2 Two types of differentiation found in the gymnosperms: (a) h3pl0.
cheilie, (b) syndetocheilic
bles the elliptical one in shape, but a further stage in its development
results in an elongation of the guard cells which finally assUlne the
characteristic dumb-bell shape (see p. 17).
I
8
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
The stomata of the gymnosperms. In the gymnosperms two processes of differentiation have been identified by Florin. (21) In the one
case a stoma mother cell divides into two guard cells, enclosing the
pore; subsidiary cells, if present, result from the modification of
(8)
(b)
subsidiary cell
,,-~-----
\ 1111 \ I I)
(e)
(c)
(d)
Fig. 1.3 Stomatal arrangement in: (a) Graminae (maize), (b) Liliaceae
(onion). (c) Gymnospermae (pine), (d) plants with elliptical stomata (Vicia
(aba). (e) most succulent plants (Sedum spectabilis)
adjacent epidermal cells-the process is similar to that of the elliptically shaped stomata and has been termed 'haplocheilic'. In the
alternative sequence of events the stoma mother cell divides twice,
producing, first, two specialized subsidiary cells on either side of a
central cell which divides later to give rise to the two guard cells; this
process has been termed 'syndetocheilic'.
Arrangement of stomata in the epidermis. Practically all graminaceous stomata occur in fairly regular patterns. There are rows of
9
PHYSIOLOGY OF STOMATA
epidermal cells running parallel to the length of the leaf which are
alternately with and without stomata; in addition, there is also an
alternation of stomata in the lateral direction. Stomata usually occur
in longitudinal rows also in the gymnosperms and in the Liliaceae
where the patterns resemble those found in the Graminae. In the
majority of species with elliptical stomata these occur scattered in the
epidermis in what appears to be a random arrangement. Blinning,(15)
analysing processes of .differentiation in general, distinguished
between true meristematic protoderm cells and so-called 'meristemoids'. The latter are cells that have regained their power of cell
division after the most active growing centre in their neighbourhood
has ceased to suppress meristematic activity in its field of inhibition.
Blinning also found that in the immediate vicinity of an active meristem, cell division is stimulated, giving]"ise to numerous smaller cells
surrounding the meristemoid.
Stoma mother cells are regarded as such meristemoids in the
epidermis that have regained their power of cell division when the
period of most active embryonality in the growing leaf has been
completed. The stoma mother cell divides to produce the two guard
cells, and stimulates cell division in some of its neighbours so that the
latter produce small cells surrounding the guard cells. A good example
of these developments can be seen in epidermal strips of rhubarb
petioles. The origin of subsidiary cells, often smaller than ordinary
epidermal cells, may also be explained on this basis. The field of
inhibition belonging to each stoma mother cell would be the cause of
the spacing of stomata in the epidermis and, in so far as the distances
between stomata are a function of these zones of inhibition, the
distribution of stomata is therefore never truly random.
1.3 Dimensions and structure of guard
cells
In the longitudinal direction, the stomatal apparatus is situated like
a bridge across the substomatal cavity in the mesophyll tissue, with
the bridge-heads rather firmly anchored against the adjacent epidermal
cells. Only small changes in the length of the stomatal apparatus are,
therefore, possible. In Fig. 1.4 (a) the position of the stomatal
apparatus is shown diagrammatically with the necessary technical
terms. In order to obtain a picture of stomata that relates their
structure to their mode of operation a three-dimensional view is
10
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
desirable. The transverse sections of guard cells along their longer
axis, and ofa stoma, shown in Fig. 1.4 (b) and (c), will complete the
picture.
antechamber
thin
dorsal
wall
I
wall
strengthening I
ridges
I
:
.
strengthening
" - - ___ ~ ridges
WIDTH
(a)
guard
cell
mesophyll cells
mesophyll cells
(b)
(c)
Fig. 1.4 (a) Section through elliptical stoma shown as a perspectjve diagram,
(b) transverse section'through an elliptical stoma and the sub-stomatal cavity,
(c) longitudinal section through one guard cell and the sub-stomatal cavity
of an elliptical stoma
Examples of the dimensions of different types of stomata are listed
in Table 1.3, together with changes in dimensions on opening. From
the figures quoted it appears that the length of the stomatal apparatus
does not change during the opening and closing movements. This is
11
PHYSIOLOGY OF STOMATA
partly on account of the firm anchoring of the ends of the guard celis
mentioned above. However, changes in the length of the stomatal pore
itself do occur, butthey are difficult to measure and we have, therefore,
not quoted any figures for such changes. During the opening and
closing movements the pointed ends of the potes must become so
narrow that their dimensions approach those of the mean free path
of the diffusing gas molecules (in the region of 0'06/"), when the
normally quoted diffusion coefficients (see p. 51) are no longer valid.
Even if opposite walls at the ends do not actually touch, effective
diffusion will probably be prevented at the ends before the pore is
closed at its centre.
Table 1.3 Changes in the dimensions of stomata during the opening and
closing movements. All measurements in J.L
Overall
dimensions
~-.....______,
open
closed
Allium cepa
38 x 38 38 x 33
40x30 40x23
Vidafaba
45 x 35 45 x28
Ranullculus
bulbo,a,
The following data after:
Haberlandt, 1904(32)
Mnium
51 x 42 51 x 42
Guard cell
dimensions
~~-----,
open
closed
38 x 14 38 x 17
40x9 40% 11
45 x 13 45 x 14
51x1751x20
Pore
dimensions
r--"'-----.
open
closed
19 x 10 19 x 1
25x 12 25x 1
19 x 9 19 x 1
?x8
?x2
?xll
?xO
cuspidatum
Schwendener, 1881(127)
? x 43
? x 36
Tradescantia
?x16
?x17
discolor
The overall width of the stomata increases as they open, but the
width of individual guard cells changes only in some stomata;
frequently, when it does, the maximum width is attained when the
pore is about half open, decreasing towards its original width with
further opening. Reliable measurements of the changes in depth of
stomata during the opening and closing process are not available but
these must presumably occur when the guard-cell volume changes
without appreciable changes in the length and width.
The spicules shown in Fig. 1.4 (b) are the sectional views of the two
strengthening ridges, described in detail below. They occur almost
12
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
universally on the guard cells of elliptical stomata, but are not equally
prominent in all such stomata; they may be composed of cutin alone
or of cellulose wall material covered with cutin. Their configuration
varies greatly. In some cases the strengthening ridges of a pair of
guard cells interlock down their depth, not unlike the cogs of gearwheels, or they may overlap when the stomata are closed. Often, the
ridges enclose an antechamber outside the central pore or throat
between the ventral walls of the guard cells. Such antechambers are
spaces somewhat protected from air movements, enclosing an
atmosphere of relatively high water-vapour density (see p. 55), and
when the stomata are open they are continuous with the sub-stomatal
cavity.
The layer of cutin covering the outer wall of the guard cell forms
part of the spicules or ridges and extends as a thin hydrophobic
covering on to the walls bordering the sub-stomatal cavity. This
internal cuticle offers a resistance to water-vapour loss directly from
the guard-cell walls into the airspace system of the leaf. The presence
of the internal cuticle has been questioned, but the hydrophobic
nature of the walls facing the sub-stomatal cavity and the results of
detailed studies should remove doubts. (109)
Guard-cell walls have a distinctive pattern of arrangement of their
micellae(l17); in the walls of bean-shaped guard cells the micellae
radiate out from the pore and, since the extensibility of a cell wall is
greatest in the direction at right angles to the orientation of the
micellae, these guard cells extend in a manner that causes a change
from the slightly curved shape to a more pronounced concave shape
when the stomata open. This deformation into a concave shape is
helped, also, by the firm anchoring of the narrow ends of the guard
cells and by the presence of the two strengthening ridges on the
ventral walls which tend to prevent their stretching lengthwise and,
thus, promote their bending concavely so as to form a pore between
them.
In the thin walls common to guard cells and subsidiary cells there
occur few plasmodesmata, less frequent than between subsidiary cells
and ordinary epidermal cells, and they seem to be altogether absent
from the graminaceous stomata. (14) The occurrence of plasmodesmata between guard cells and epidermal cells has been a matter
of dispute, but the observations of Sievers(132) leave little doubt as to
their existence in some cases. Ectodesmata are to be found in the
anticlinal walls of epidermal cells and in the outer walls of gu ard
13
PHYSIOLOGY OF STOMATA
radial arrangement of
micellae permitting extenSion
at right angles
(b)
Fig. 1.5 Diagram indicating the orientation of the micellae in the guard cell
walls of: (a) an elliptical stoma, and (b) a graminaceous stoma
cells; they are especially prominent at the rims of the stomatal
pore(221 (see p. 124).
1.4 The mechanism of guard cell movement
Ordinary cells, including epidermal cells, tend to shrink or extend
more or less uniformly in all directions as a result of changes in their
14
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
turgor, but when the turgor relations between epidermal and guard
cells change, the guard cells do not shrink or extend uniformly
because of the unequal thickening and elasticity of their walls.
Consequently, the guard cells undergo changes in their shape, and
this occurs the more readily because they bridge the sub-stomatal
cavity so that three of their six major walls are free and not cemented
on to any other cell wall; this applies to the outer wall and those
facing the sub-stomatal cavity as well as the ventral walls between
which the stomatal pore appears. These walls are, therefore, able to
stretch or bend under the influence of pressures and tensions. Of the
other three major walls, those at the narrow ends of the guard cells
are rather rigidly anchored, but the dorsal walls adjacent to the
subsidiary cells are as a rule delicate structures capable of being
deformed under pressure.
When the stomatal pore is closed, the guard cells are in the relaxed
state, perhaps slightly pressed together by the surrounding epidermal
cells whose turgor is then either equal to or in excess of that of the
guard cells. When the stomatal pore opens, guard-cell turgor exceeds
that of the epidermal cells either owing to an increase in their own
turgor or a decrease in that of the epidermal cells or both (see Table
1.4). It may be emphasized here that stomatal movements should be
regarded as the resultant of changes in the turgor relations between
guard cells and surrounding epidermal cells and not as a consequence
of changes in turgor pressure of the guard cells alone.
The elliptically shaped stomata. In some stomata, for instance
those of the moss Mnium, the strip of thin ventral wall between the
upper anb ine 'lower strenginenmg floges \nilges sllg'nflY outwarbs
when the guard cells are in the relaxed state so that the two oppositely
situated bulging walls meet to close the pore. When the turgor
pressure of the guard cells increases and exceeds that of the epidermal
cells, the former assume a more circular shape in cross section as
distinct from the elliptical one in the relaxed state. As a result, the
outward bulging walls become less curved and the pore opens (see
Fig. 1.6 (a». In this type of stoma, this is the major mechanism for
opening and closing the pore. According to Schwendener(127) a
similar mechanism operates in the stomata of species of Hellehorus
and Tradescantin. In most plants, however, the mechanism of stomatal
movement is more complex. Increased turgor pressure in the guard
cells causes the thin dorsal walls to extend and to bulge into neighbouring epidermal or subsidiary cells, whereas the structure of the
15
PHYSIOLOGY OF STOMATA
guard cell
,
(aJ Mnium cuspid.lum
dorsal walls of guard ;
cells bulge into subsidiary
cells and
:
alignment
.
of guard
cells in - - epidermis may
change
(bJ most commonly found
elliptical stoma
thin end parts of
guard cells swell
and pull stiff dorsal
walls apart at end
thick rigid ventral
walls
I
~ canal connecting thin end
parts of s;ngle guard cell
fairly /_!_!lIJL---.'
thick
dorsal wall
(c) graminaceous stoma
Fig. 1.6
16
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
ventral walls prevents them from extending to the same degree. When
opposite walls of an elastic body extend unequally, curvature results,
thus the dorsal walls bend inwards, that is, in the only direction in
which their two strengthening ridges can bend, a direction that is also
dictated by the orientation of the micellae in the walls as mentioned
above (see p. 14). Thus, the ventral walls of the two guard cells become
concave, Le., they move in opposite directions and open a pore
between them (see Fig. 1.6. (b».
At the upper and lower edges of the dorsal walls of guard cells there
are especially thin regions, originally called 'Hautgelenke' by
Schwendener, and thought to facilitate a movement of the guard cells
as if they were 'hinged'. It is probably more correct to ascribe to these
regions the function of making it possible for the dorsal walls to be
bent into the subsidiary cells when they stretch under turgor pressure.
Nevertheless, some guard cells appear to change their alignment in
the epidermis during the opening and closing movements, as indicated
in Fig. 1.6 (b).
It should be noted that during the movement of the guard cells, the
dimensions of the antechamber between the ridges may remain
unaltered or, if the ridges do move when the guard cells alter their
shape, such movements need not necessarily be of the same magnitude
or even in the same direction as those of the ventral walls. If stomata
are observed microscopically (see p. 29) it is, therefore, of great
importance to verify whether the pore between the ridges or the
central throat between the ventral walls is in focus, and if stomatal
measurements are carried ont by micro-relief :nethods (see p. 30) it
should be remembered that it is usually the ante-chamber that is
replicated, not the throat of the stoma.
The graminaceous stomata. In the guard cells of graminaceous
stomata the wall structure is altogether different. Each guard cell has
two thin-walled end-parts which are connected by a narrow tube with
strongly thickened walls and a very narrow lumen. The thick walls of
the central region of the guard cells are the dorsal and ventral walls
Fig. 1.6 Diagrammatic representation of changes in the shape of guard cells
during the opening movement. (a) Helleborus type in which guard cells
change so that in transverse section they appear not elliptical but circular.
(b) the ordinary elliptical tYP8 in which the extensible dorsal wall bulges into
the subsidiary cell, thereby pulling the less-extensible ventral wall into a
concave shape and away from its opposite wall, (c) the graminaceous type in
which the movement of the wal!s at the ends of the pore causes the parallel
walls delimiting the pore to move apart
17
PHYSIOLOGY OF STOMATA
respectively, and they are practically unable to bend, so that, when
the stoma opens, a pore with almost parallel sides is formed between
these inflexible walls (see Fig. 1.6 (c)). In the graminaceous guard
cells there is only one centrally placed strengthening ridge in the
ventral wall, which confers great rigidity. Figure 1.6 (c) shows the
configuration of the guard cell walls where the thickened dorsal and
ventral sections go over into the thinner sections of wall surrounding
the enlarged end-parts. The thin wall separating the two guard cells
where they meet has, on occasions, been found to be incomplete, so
that the protoplasts of the two guard cells are confluent, and the
vacuoles of the two cells may combine to form one single larger
vacuole. (89, 14)
The opening of the pore in the graminaceous type of stoma is also
the result of increased turgor pressure in the guard cells relative to
that in the epidermal cells. The enlarged end-parts, connected by the
thin canal in the (hick-walled central region, expand and swell into
more spherical bodies, pressing against each other where they meet
and pulling the V-shaped walls out into a less acute angle (see Fig.
1.6 (c)). By this change in the shape of the end-parts the rigid ventral
walls are pushed apart at each end, and the pore opens. In several
species of grasses the enlarged end-parts of the guard cells are filled
with cytoplasm, embedded in which are plastids but no chloroplasts,
nor could a vacuole be found. In fact, the end-parts are of about the
same diameter as a single chloroplast from the mesophyll of the
blade.(14)
1.5 The contents of guard cells
Guard cells not only differ from other epidermal cells by their morphology and anatomical features, but also by their cytoplasmic
inclusions, vacuolar properties and, indeed, their metabolism,
especially as affected by light. However, our knowledge of the content
of guard cells is very incomplete, partly on account of the difficulty
of obtaining reliable information about single cells embedded in the
epidermal tissue. It is probably reasonable to assume that guard cells
contain most of the metabolites found in mesophyll cells, and we shall
restrict ourselves to mentioning those features that distinguish guard
cells from surrounding epidermal cells and whose presence or absence
has been experimentally verified.
Chloroplasts do occur in the epidermal cells of some leaves, but the
presence of chloroplasts in practically all guard cells is a significant
18
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
feature. Guard cell chloroplasts are usually smaller and less numerous
than those in the mesophyll cells, but occasionally they can be surprisingly numerous and densely packed, as in the guard cells of the
fern Phyllitis scolopendrium. The chloroplasts in the guard celIs often
appear less rounded and somewhat dissected, suggesting a comparatively large surface-to-volume ratio, and they appear to be less densely
packed with grana. Numbers of chloroplasts in guard celIs have been
used as an indicator of ploidy in plants. In sugar cane, for instance,
75 per cent of tetraploid plants have been found to have significantly
greater numbers of chloroplasts in their guard cells than triploid and
diploid plants. (120)
Guard cell chloroplasts are exceptionally rich in starch, and in
epidermal strips stained with iodine the guard cells ,tand out because
of the characteristic reaction with starch; they may also be made
conspicuous by treatment with one per cent silver nitrate solution.
Exceptions to these observations exist; the guard cells of onion do
not contain disyrete chloroplasts, but chlorophyll is present in a
finely dispersed form, and they are devoid of starch, as is the rest of
the onion leaf. In many guard cells, especially those of evergreen
leaves, large oil drops can be observed. Electron microscope studies
of the guard cells of rye leaves revealed exceptionally large numbers
of mitochondria. ('9)
The presence of phosphorylase in guard cells distinguishes them
from other epidermal cells and suggests that sugar-starch conversion
occurs in most guard cells (see p. 141); this could explain the observable changes in the starch content of guard cells. During the day, the
amount of starch tends to diminish, and at night it accumulates, which
is in marked contrast to the rest of the lear in which starch content
diminishes in the dark and builds up under illumination. Apparently
associated with changes in starch content of the cytoplasm there occur
changes in the pH value of the vacuolar sap (see Table 1.4); all the
available evidence shows a rise in pH from about 5·0 to 6·5 as the
stomata open.
In addition to phosphorylase, adenosinetriphosphatase and
peroxidase occur in guard cells, the amount of ATP-ase probably
being greater in closed than in open stomata (see p. 122). Associated
with these changes there occur changes in calcium and potassium ion
concentration in the guard cells; these ions accumulate against the
concentration gradient in open stomata when the ATP content is
high(24) (see also the discussion on p. 132).
19
PHYSIOLOGY OF STOMATA
The nucleus of guard cells is generally more prominent than that
of other epidermal cells and can be observed by placing epidermal
strips in neutral red dye, when it remains colourless. In the graminaceous guard cells the nucleus is situated in the enlarged end-parts near
the mouths of the connecting canal, and its two portions are held
together by a slender strand traversing this canal; on occasion this
fine connection has been observed to break. Functional changes in
the cytoplasm and in the nucleus of guard cells of Vicia faba have
been described by Ilieller and Resch. (55) When the stomata are closed,
as for instance after a period of darkness or owing to treatment with
solutions of KCNS, the nuclei of guard cells are elongated with the
chromatic material distributed in the normal manner, and the cytoplasm of the cell is of low viscosity. When the stomata are open, after
illumination or owing to treatment with KNO, solution, the nuclei
are more rounded in appearance with the chromatic material homogeneously distributed; the cytoplasm in this condition is of a higher
degree of viscosity.
Anthocyanin, found in the vacuoles of many epidermal cells, is, as
a rule, absent from guard cells, but the Occurrence of anthoxanthine
pigments has been reported (11,61) (see also the discussion in chapter 7).
Similarly, proteinaceous spindle crystals occurring in nuclei, and
calcium oxalate crystals in the vacuoles of epidermal cells of some
species, are hardly ever found in the guard cells. In some virus-infected
leaves the content of epidermal cells is found to coagulate, a phenomenon not observed in guard cells, suggesting that the permeabilities of the membranes of epidermal and guard cells differ. Also,
it was found that when epidermal strips of Bellis perennis were
placed in a mixture of neutral red and methylene green dyes, the
guard cells stained red whereas the other epidermal cells stained
blue. (157,158)
Exceptional permeabilities of guard cell membranes have been the
subject of several hypotheses of the mechanism of stomatal action
(see p. 145). Changes in permeability on illumination have been
postulated, for instance, as possibly explaining the opening response
to blue light, while chemically induced increases in permeability have
been observed to be followed by stomatal closure. The permeability
of epidermal cell membranes appears generally greater than that of
the guard cells, which can be verified by the use of neutral red dye and
a plasmolysing solution. However, consideration of the ratio cellsurface/cell-volume for epidermal and guard cells makes a valid com20
MORPHOLOGY
AND PHYSIOLOGY OFST6MATAL CllIJ:S"
parison between the rates of plasmolysis and de-plasmolysis very
difficult.
The osmotic relations between epidermal and guard-cell saps have
been investigated by plasmolytic methods and all results agree that
the osmotic pressure of the guard cell sap of open stomata is higher
than that of the sap of neighbouring epidermal cells and that, when
stomata are closed, the surplus in osmotic potential of the guard cells
Table 1.4 Osmotic pressures in bar and pH values of the vacuolar sap of
guard cells and epidermal cells
Veronica beccabunga
Allium cepa
Rumex patientia
Cyclamen
Iresine
Sugar beet
Vicia/aba
floating
Guard cells
Epidermal cells
~
~
open
closed
open
closed
Author
25
18
19
21·7
40·0
32
10·3
20
15·3
13
13·1
22
22
8·7
12
5·5
14
1l·2
11·5
12
6·5
12
5·5
13
11·2
11·5
12
6·5
Mouravieff(97)
15·0
6·0
5·8
4·2
(96)
"
Sayre(122)
Wiggans(160)
"
Stalfelt(l47)
disc~
Viciajaba
intact leaves
Ranuflculus bulboslis
pH values:
Rumex acetosa
14·0
15·0
9,0
H
6·3
6·7
4·5
5·7
5·0
5·6
Meidner,
unpublished
19·0 Glink.,
unpublished
7·7
Pekarek(106)
Pallas{lOS)
Scarth(123)
over that of the epidermal cells is reduced. However, in some cases
it is the sap of the guard cells and, in others, that of the epidermal cells
that undergoes changes in osmotic potential, and in other cases
again, both change. In Table 1.4 some entries show the osmotic
potential of the epidermal cells to be fairly constant while that of the
guard ceUs changed with illumination; in others the osmotic potential
of lhe epidermal cells underwent changes as well. The osmotic
potentials shown for Vida faba are those of external solutions in
which 50 per cent of the cells were to some degree plasmolysed; in the
intact leaves, osmotic potentials of open guard cells ranged from 13 to
21.
PHYSIOLOGY OF STOMATA
28 bar with the 50 per cent value at 15 bar. When open, the stomatal
pores of Vicia laba measured at least 91-', but many had a pore
diameter of 151-', and it was the osmotic potential of these very
wide-open stomata that reached a value of 28 bar.
Generally, guard cells have been found to be more resistant to
adverse conditions such as low temperatures and noxious vapours
than other epidermal cells. During senescence, epidermal cells begin
to deteriorate long before the guard cells. Guard cells withstand
limited periods of drought conditions much better than epidermal
cells, and even if drought conditi~ns have killed the leaf, the guard
cells can be found turgid and with green chloroplasts, though the
stomata are usually no longer able to open and close, probably on
account of the rigidity of the dead epidermal ceils that surround
them.
Cytoplasmic streaming can be observed in guard cells When
the stomata are in the process of opening; it often comes to a halt
when a certain degree of opening has been reached. Streaming in the
guard cells appears to be associated with changing stomatal apertures
rather than with the maintenance of fully open or closed stomata. (105)
The form 01 plasmolysis of the guard cells of closed stomata of Vida
laba is usually convex and that of guard cells of open stomata concave;
the protoplast remains attached to the ventral wall in both cases,
indicating either a greater permeability of the dorsal wall or an effect
of the numerous ectodesmata holding the protoplast to the ventral
walL
1.6
Properties and functions of subsidiary
cells
The epidermal cells adjacent to the guard cells may be distinguished
by their size or shape, as for instance in the graminaceous stoma or
those of species of Sedum, where they are obviously subsidiary cells
and belong to the stomatal apparatus. Often, however, neighbouring
cells are not distinguishable from other epidermal cells by microscopic
observation although they may well function as subsidiary cells.
One feature that distinguishes subsidiary cells from other epidermal
cells is that they are suspended, wholly or partly, above the substomatal cavity; compared with ordinary epidermal cells, they have,
therefore, an additional free wall which does not abut on another.
This circumstance enables the subsidiary cell to give under pressure
22
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
from other cells to a greater degree than other epidermal cells that are
hemmed in on all but their outer surface by neighbouring cells.
Subsidiary cells can thus act like elastic buffers between guard cells
and epidermal cells. Often, the walls of subsidiary cells are noticeably
thin and tend to bulge slightly into neighbouring cells or into the substomatal cavity; in some species they may also bulge upwards (see
p. 25 and Fig. 1.7 (b» and over the ante-chamber. In succulent leaves,
like those of different species of Sedum, the subsidiary cells surround
the guard cells completely and here, as elsewhere, their function
appears to be to allow for greater ease of movement for the guard cells
by their mechanical buffering property. This is the more necessary in
thick succulent leaves that undergo a considerable degree of shrinkage
and expansion with changes in water content, so that the stomatal
apparatus would be exposed to pressures and tensions which might
counteract stomatal movements, e.g., those in response to the stimuli
of light and carbon dioxide concentration (see p. 70). Guard cells
completely surrounded by subsidiary cells would be protected from
some of these pressures and tensions.
An indication that subsidiary cells (and epidermal cells) may be
actively involved in the stomatal mechanism has been obtained with
leaves of Galium rnollugo and Tradescantia virginiana. The starch
content of the subsidiary cells of these species undergoes changes
which are in the opposite direction to those occurring in the guard
cells, i.e., when the stomata open and guard cell starch decreases, the
starch content of the subsidiary cells increases.
A curious distiuctiml between subsidiary cells and ordinary
epidermal cells was observed by MO\lra~ieff(9') when pla,molysing
epidermal strips of Aponagetan distachys L. in 0·7 M glucose solution:
epidermal cells plasmolysed mostly in the convex manner and
subsidiary cells concavely (guard cells did not plasmolyse in this
solution).
1.7
Modifications of the stomatal
apparatus with habitat
Plants growing in different habitats display several anatomical and
morphological adaptations which fit them to their particular environment. In this respect stomata are no exception and modifications are
numerous. As the exchange of gases and water vapour between the
plant and the atmosphere proceeds mainly through the stomata it is
23
PHYSIOLOGY OF STOMATA
not surprising that extreme conditions of water supply to plants are
found to cause the most numerous modifications of the stomatal
apparatus. The position of the stomata relative to the leaf epidermis
is most commonly affected by a special environment. In mesophytic
stomata in very shallow
depression of epidermis
(a) Canna mdica
~
(atter Schwendener)
averarching subsidiary
cells forming extensive
antechamber
(b) Allium cepa
chimney of cutin
surrounds and extends
antechamber
(e) Euphorbia tirucalli
1OQ0P~DD(
pore formed by
spicules only
(d) Lemna minor
(e) Pinus sylvestris
FJg.1.7
Modified stomata. (a) Canna indica, slightly be/ow the leaf surface,
(b) Allium cepa, sunken, Cc) Euphorbia tirucal/i, protected by chimney formed
from cuticle. (d) Lemnaminor,sunken with subsidiary and epidermal cells above
guard cells, (e) Pinus sylvestris, reduced in structure, opening and closing
achieved by spicules alone. (After Haberlandt.(32»
24
MORPHOLOGY AND PHYSIOLOGY OF STOMATAL CELLS
plants the stomata are more or less level with the epidermal cells and
the ante-chamber is not too conspicuous. In dry habitats the stomata
are often situated below the level of the epidermal cells, at the bottom
of shallow depressions (see Fig. 1.7 (a». In several cases these depressions are arched over on the outside by bulges from the subsidiary
cells (see p. 23 and Fig. 1.7 (b». In some leaves a chimney formed
from cutin is placed above the stomatal pore, as is shown in Fig. 1.7 (c).
A further development can be seen, for instance, in Nerium oleander,
where the depression in the leaf surface has become a fairly extensive
cavity which accommodates several stomata around its periphery;
the opening of the cavity is partially obscured by numerous hairs
growing from epidermal cells.
All these modifications provide an airspace immediately outside the
stomatal pore, which is protected from air movements and which
must, therefore, contain air with a comparatively high water vapour
content; this will help to increase the external layer of water vapour
close to the leaf surface, with the result that trans pi ration a I water
vapour loss will be slowed down (see p. 54).
Modifications of the opposite kind can also be found, e.g., plants
growing in moist habitats display a modified stomatal apparatus in
which the opening and closing of the pore is brought about only by
the position of the strengthening ridges relative to each other, as
shown in Fig. 1.7 (d).
25
2. The measurement of
stomatal aperture
The measurement of stomatal aperture has been attemped ever since
it was realized that the gas exchange between plants and the atmosphere is often regulated by the degree of opening of the stomata.
Basically, there are two alternative methods: the dimensions of
individual guard cells and stomatal pores can be measured, or the
resistances offered collectively by many stomata to gaseous diffusion
or viscous flow, or to the infiltration of suitable liquids, can be
assessed. (Diffusion occurs on account of a difference in partial
pressure of a gas, viscous flow on account of a difference in total
pressure.) However, it must be realized that changes in visible
stomatal dimensions do not necessarily occur when there are changes
in stomatal resistances. Therefore, in choosing an appropriate
tecbnique it is necessary to keep in mind the limitations of the different
methods, and also the particular properties of the stomatal apparatus
that are to be measured.
26
THE MEASUREMENT OF STOMATAL APERTURE
2.1 The choice of plant material
Since some techniques require the use of specially prepared plant
material, it will be useful, first, to consider limitations that can be set
by the nature of the material itself.
Attached and detached leaves. In the intact leaf, stomatal movements occur ultimately because of changes in the turgor relations
between epidermal and guard cells, and they should not be studied in
material in which these turgor relations bave been altered. Tbis is
especially relevant to kinetic studies that aim at measuring rates of
6
30
60
90
120
150
180
210
240
minutes
Fig. 2.1
The course of stomatal opening from dark in leaves of Xanthium
pennsylvanicum. Measurements with a Wheatstone bridge parameter,
illumination 15,000 lux, 25°C(27)
movements. There is some evidence that leaves remaining attached to
a well-watered plant respond to many stimuli in the same way as
detached leaves supplied with water through their petioles,I'6) but,
even here, evidence has been obtained which suggests that some types
of rhythmic changes in stomatal aperture occur only in leaves that
remain attacbed to a rooted planUS)
Leaf discs watered via their cut edges. The turgor pressure of leaf
tissue supplied with water via the cut edges appears to be higher than
that of intact leaves; this is probably due to the greater ease with
which water lost by transpiration can be replenished. Figure 2.1 shows
a comparison between intact leaves and leaf discs supplied with water
via the cut edges; both the rate of movement and the final steady-state
opening were greatly affected.
27
PHYSIOLOGY OF STOMATA
Floating leaf discs. Turgor conditions are yet more drastically
affected when leaf discs are floated on water. because transpiration
cannot occur from the epidermis in contact with water and because
the water required to replace that lost from the epidermis facing the
atmosphere has to traverse only the leaf thickness. In addition, carbon
dioxide is comparatively unavailable to the stomata in contact with
water and, therefore, the carbon dioxide concentration in the intercellular spaces of floating leaf discs wiII be reduced, with important
consequences for the stomatal mechanism (see pp. 70-84). Results
in Table 2.1 demonstrate both the turgidity effect and the carbon
dioxide effect.
Table 2.1 Stomatal responses to illumination and concentration of carbon
dioxide in tobacco leaf discs floating on water or on 0·12 M mannitol solution.
Illumination 15,000 lux, measurements carried out on lower epidermis by
silicone rubber impreSSions and scafing(27). Encircled figures represent the
percentage score of open stomata.
Concentration of
carbon dioxide
Zero
e
.1ower
epidermis
down
.••..•
:
52
: .,' :
.~
~
lower
epidermis
down
,.; .. ,
:
1
~
lower
epidermis
up
e:
·S6
:
:
:
:
~
CO 2:eff'ect
;
absence of e")
80 •.•••••••.•.••••• ·SI
:
COreifect
1
····®.co'.etrect·e
SiBnificant dHference at P < 0-02 = 10·4<0,05= s-t
<0-10= 6·3
28
••••
:
effects
:
:
tur;jdjly
~
effect
:
COreffect
:
:.......
that epidermis
in contact with
conditions
t~rgidity III- This ~hows tJlat
!
Il. . .®'
:
®
:
:
: ®. ·.c02-eft'ect·
lower
epidermis
up
This shows
................. 7,'V, +- water is virtua1ly
C02~effect
: ",
in COrfree
:':
turgidity :
effFtCO : ~
:
2-euect
::
WATER
0'12M
MANNITOL
SOLUTION
absence of
~,~
even m floatmg
leaf discs the
epidermis facing
-the atmosphere
is less turgid
than the ~ne in
contact WItn
water
THE MEASUREMENT OF STOMATAL APERTURE
Epidermal strips and isolated stomata. In epidermal strips the turgor
pressure must be very high compared with that of intact leaves.
However, it remains practically constant-a condition otherwise very
difficult to' achieve. The guard cells of epidermal strips floating on
water, as distinct from those of leaf discs, can readily be treated
with different concentrations of carbon dioxide since they are
exposed to the atmosphere as well as to the water; they can also be
treated with solutions of different molarities or containing different
chemicals, and they are, therefore, very suitable material for certain
studies of the stomatal mechanism.l241 The same is true of isolated
stomata. Naturally, turgor differences between epidermal and guard
cells have been eliminated once the stomata have been isolated, but
only with this material can the specific responses of the guard cells to
various stimuli be studied.
2.2 Microscopic measuring techniques
The most obvious and natural method of attempting to measure
stomatal aperture is by microscopic observation of intact leaves;
however, this is not easy. Ifit is desired to follow the course of opening
or closing of particular stomata, the tissue must be fixed to the microscope stage and illuminated in this position. Dependil)g on leaf
thickness and surface features, it is possible to obtain a clear view
with illumination either from below or from above. (421 An alternative
procedure has been developed by Stalfelt( 141 1 who illuminated and
treated leaves as desired before cutting leaf squares (5 x 10 mm) at
intervals, transferring each quickly on to a microsco?e slide, and
immersing it in liquid ?araffin. Observation was carried out without
coversli?, through an oil-immersion objective. To ?revent the lear
square from sticking to the objective during focusing, it is necessary
to hold the tissue down with weights or light springs. It is also recommended that the observations are carried out in a weakly illuminated
room.
Mouravieff(931 obtains 'isolated' stomata from paradermal sections
of leaves. Epidermal cells adjacent to such isolated stomata are
damaged by the razor cut and have, therefore, a turgor ?ressure of
zero. Stomata situated at the edge of the section are most suitable for
observation. Such isolated stomata can be treated as desired and can
be observed with great clarity under the microscope. However, the
?reparation requires considerable skill and the method is really one
for experienced research workers.
29
PHYSIOLOGY OF STOMATA
Direct microscopic observation of stomata has some shortcomings;
firstly, it is a time-consuming process, and secondly, with the considerable variation in pore width that exists in most leaves, a very
large number of stomata -has to be measured to give reliable mean
values. There is a third, most important consideration: it has been
shown that the stomatal pore proper, i.e., the throat, is formed
between the ventral walls of the guard celIs (see pp. 10--14) and is
often arched over by the strengthening ridges. From above, only by
very careful focusing is it possible to distinguish between the pore
bounded by these ridges (antechamber) and that bounded by the
guard-ceIl walls (throat). Moreover, these two pores do not always
move in unison, and the antechamber may remain unaltered while
the throat changes in dimensions. From the point of view of gaseous
diffusion, the smalier of the two pores will be the controlling one and,
usually, this is the pare between the guard cell waIls; however, at very
wide stomatal openings the antechamber may become the controlling one.
Lloyd's strips. Direct microscopic measuremenfs can also be carried
out on Lloyd's strips(69) taken from the leaf under investigation and
immediately immersed in absolute alcohol for fiKation and preservation. Observation is easy and can be carried out when convenient.
However, in addition to the difficulties common to all microscopic
methods mentioned above, Lloyd's strips suffer from the additional
disadvantage that fixation in alcohol may cause distortion of the
stomatal apparatus. Nevertheless, useful investigations and field
surveys have been carried out employing this method.
Micro-relief methOds. Micro-relief impressions of leaf epidermes
were introduced in J90J, (16) w.bm lranspaIenl negatiye cDllodion
impressions were prepared and examined microscopically. Nowadays,
silicone rubber formulae(l16) are used so that the leaf is not harmed
when the impressions are made. From the silicone-rubber impressions
a positive replica of the epidermis is made with nail varnish and used
for microscopic examination. The same difficulties attend this method
as the other microscopic ones, but with the added disadvantage that
the pore replicated in the nail varnish film is usually the antechamber
because the silicone rubber does not enter readily into the throat of
most stomatal pores. Table 2.2 shows that the method can, therefore,
be insensitive as compared with porometer readings, especially in
certain stages of stomatal opening and closing. Zelitch (173) has
developed a technique of sto~atal assay which does not involve the
30
THE MEASUREMENT OF STOMATAL APERTURE
accurate measurement of stomatal pores but depends on scoring open
and closed stomata in several high-power fields of the microscope.
Table 2.2 Different degrees of steady slate stomatal opening as measured
with a resistance poromete-J' and siHcone rubber impressions taken inside the
perometer cup, Xanthium pennsylvanicum
~
J
(Conductance) 1/2
Width of pore in JL
_______________
2·00
2·36
2·68
3·34
4·48
5·75
2.3
Less than 2
Less than 2
2-4
3·1
4·0
4·9
Macroscopic measuring techniques
The degree of opening of the stomata can be assessed by the ease with
which specially selected liquids infiltrate into the leaf tissue or by the
rate with which water vapour diffuses through the stomata from
inside the leaf.
Cobalt chloride paper method. This method is due to Stahl(138) and
dates back to 1894. Specially prepared (see p. 154) cobalt chloride
papers are dried and kept in a desiccator, or a tin containing some
silica gel. When a stomatal determination is to be made, the papers
are inserted in a simple perspex holding clip and placed on the leaf
under investigation. The cobalt chloride paper is blue when dry, and
becomes paler as it absorbs moisture, turning pink eventually. To
make the measurement of this colour change more precise, it is usual
to place a dark-blue colour standard on one side of the test paper and
a light-blue one on the other side, and the time is measured for the
cobalt chloride paper to change from one colour standard to the
other. Figure 2.2 shows the holding clamp and the papers.
Provided the intercellular spaces of the leaf are saturated with water
vapour, the rate of diffusion of water vapour from the leaf to the
initially dry cobalt chloride paper will indeed be a function of the
diffusive resistance of the stomatal pores and that of the cuticle. There
is one disadvantage to the method: by placing the papers on the leaf,
light will be excluded, and if the measurement takes a long time
stomatal closure may set in. When stomata are fully open, the colour
31
PHYSIOLOGY OF STOMATA
change occurs within a minute or two, but if the stomata are only
slightly open (or if there are only rew stomata per mm 2 in the epidermis
under investigation) the colour change may be completed only after
five or ten minutes.
CoCI 2 paper
~~~pe~rs~pe~x~p~lat~e~:~L:",~
3 x25 x 15 mm
(a)
light blue
standard
dark blue
standard
(b)
Fig. 2.2 (a) Perspex clip for cobalt chloride papers. (b) A properly prepared
cobalt chloride paper
Infiltration methods. Infiltration methods date from 19055143)
Alcohol, benzol and xylol are some of the liquids that have been used
as infiltrating agents. Drops of uniform size arc placed on the epidermis under investigation and the time is measured for the drop to
disappear by infiltration into the leaf. Xylol can be used on the same
spot of a leaf for several measurements, without apparently harming
the leaf, although presumably it may affect gas exchange. An improved infiltration method was introduced by Alvim and Havis.I') A
graded series of solutions of different viscosities and surface tensions
is used. The solutions are made from liquid paraffin and kerosene,
mixed in proportions 1:9, 2: 8, etc., with increasing viscosity over the
series. By trial and error, the solution is found that just infiltrates
while the next one in the series remains on the epidermis; thus, ten
different stages in stomatal opening can be defined.
2.4 Viscous flow porometers
Viscous flow porometers work best with amphistomatolls leaves, and
especially wei! with those that are also heterobaric, i.e., leaves that
have stomata opposite each other in the two epidermes, between
32
THE MEASUREMENT OF STOMATAL APERTURE
which run more or less vertical canal-like air spaces through the
mesophyll tisslie so that there is practically no lateral connection
between air spaces. Xanthium pennsylvanicum is an example having
such a leaf structure. However, viscous flow parameters can be used
with hypostomatous leaves, bur in these the air must flow into the
lear in the area outside the parameter cup, and traverse the mesophyll
in a plane parallel to the epidermes between the washers of the
parameter clamp and into the cup through the stomata. Resistance
to viscous flow therefore includes that offered by the airspace system
in the mesophyll tissue.
Mesophyll resistance varies not only with the water content of
leaves but also with the degree of stomatal opening because it is this
that determines the extent of the mesophyll tissue through which air
will be drawn. In hypostomatous leaves the mesophyll resistance
constitutes a real source of error for viscous flow porameter readings.
Nevertheless, parameter results obtained with hypostomatous leaves
give a reasonably valid picture of changes in stomatal resistance.
Basically, all viscous flow porometers measure the rate at which air
can be drawn through a leaf when a pressure difference is applied
across the leaf. Results from viscous flow parameters can be expressed
in units of resistance offered collectively by the stomata enclosed in
the parameter cup, and by the mesophyll tissue involved. From
Table 1.1 it can be seen that the number of stomata in 0·2 cm2 of a
leaf may be anything between 700 and 7,000, so that parameter
results are representative of very much greater numbers of stomata
than can be surveyed and measured microscopically.
The first porometer of Darwin and Pertz. The first viscous flow
parameter was constructed by Darwin and Pertz in 1911.(20) It
consisted of a small glass cup, similar in shape to a thistle funnel,
which could be glued on to a leaf surface containing stomata by means
of a mixture of Vaseline and beeswax. A column of water in a tube
was connected to this cup by a glass T-piece provided with a three-way
stopcock (see Fig. 2.3). When the stopcock connected the cup with
the water column, the level of the water in the tube descended as it
drew a known volume of air through the leaf. The rate of descent was
mainly a function of the stomatal resistance to viscous flow. By
timing the travel of the meniscus over a measured distance in the tube
a direct measure of the stomatal resistance could be obtained.
The leaf attachment clamp, The permanent attachment of the
Darwin and Pertz instrument with beeswax was an unsatisfactory
33
PHYSIOLOGY OF STOMATA
feature, and modern porometers employ detachable or air-swept
attachment clamps that seal the leaf hermetically to the porometer
cup. In 1948, Heath and Williams!'4) drew attention to the fact that,
in a permanently attached cup, the carbon dioxide content is quickly
exhausted by the photosynthetic activity of the leaf, so that within a
leaf
cup
descending
meniscus
three-way
stopcock
o
30
Fig. 2.3
o
bulb used to pull up
column·of water
30 em
Diagram of a Darwin and Pertz parameter
short time the stomata inside the porometer cup are exposed to a very
low concentration of carbon dioxide. As will be seen in chapter 4, low
concentrations of carbon dioxide cause wide stomatal opening and.
hence, results obtained with permanently attached cups are unrepresentative of the state of stomata in the leaf oetside the cup.
While it is desirable that porometer clamps remain attached to a
selected spot of the leaf under investigation, and be absolutely airtight
34
THE MEASUREMENT OF STOMATAL APERTURE
wing nuts for - ___
prassing perspex plate
upper perspex plate
and loaf-onto leaf
washer
leaf
:
leaf washer
movable portion of leaf
clamp pressed into positon
by spring when clamp closed
washerlor
closing cup
I
r_
- - a i r inlet to or frem parameter
_,n",.",,, pieces 3 x30 x25 mm
leaf w •• h,,,• ..../
1'-"""- B7 male and female
ground glass joint
t air inlet from porameter
Fig. 2.4 (a) A detachable porameter
parameter clip(82)
CUp.(137)
(b) A simple detachable
35
PHYSIOLOGY OF STOMATA
for the duration of the measurement, they must also be open to the
atmosphere between readings so that carbon dioxide can diffuse
freely to the leaf. Two designs of porameter clamps that fulfil these
requirements are shown in Fig. 2.4; clamp (a) is modelled after that
designed by Spanner and Heath, (13"/) and clamp (b) is a simple version
more suitable for field porameters.(82)
leaf disc
upper:~~b,er:=1~;;;§[:~::~~;;~~
inletinlet to outer
compartment
upper chamber
outlet
outlet from outer
compartment
water
Gaca washers, O-ring
movable platform
cBirying lower chamber
leaf disc
washers
screw cup used to move
lower chamber upwards till
leaf disc hl;!ld airtight
I
inlet
between washers
outlet
to lower leaf chamber
Fig. 2.5 A modified version of Raschke's porameter cup for leaf discs.<27, 112)
Diameter of external compartment 45 mm, diameter of upper and lower leaf
chambers 10 mm, diameter of screw cup 60 mm, height of assembly 60 mm
Instead of a detachable porameter cup a ventilated one can be
used, Le., a cup through which an air stream passes when readings
are not being taken, so that the air within the cup is continuously
renewed. This principle is used in a porameter cl2mp designed by
Raschke,OI2) who chose to work with leaf discs instead of with intact leaves. A modified design of this porometer clamp is shown in
Fig. 2.5.
The seal between the clamp and the leaf can be achieved by gelatine
washers, but since these melt in moderately high temperatures and
need frequent renewal because they dry out, washers made of soft,
pliable plastic materials are to be preferred (see p. 156). With most
leaves, circular washers of about 5 mm internal diameter and about
36
THE MEASUREMENT OF STOMATAL APERTURE
sphygmomanometer
<a)
double-bulb bellows used to
pump up to initial pressure
tapped for 10 BA thread --...,
(Ao B'b 50] }upper
! 1il~ VL~;;'~"
, ~O~;1~~~p~a:rt___ gUide
,.,
Lr
pin
-,OSr-'-8" ]Iower
plastic _ _--<:""7.-;-A-'
washer
B7female----ll
part
~~IJ
l
l"
10BAthread
ground glass
joint
B7 male-----fl
ground glass
joint
compression
spring 1~" long
(b)
Fig.2.6
i" bolt, 21" long
(a) Diagram of an Alvim field porometer,(2) (b) diagram of a simple
hand porometer(B2)
37
PHYSIOLOGY OF STOMATA
1-2 mm width work very well; for monocotyledonous leaves, rectangular washers with slits of 2-mm width and 10-mm length have
been found suitable; in both types of washers about 0·2 cm2 of leaf
is enclosed.
A/rim's parameter. (iJ The measurement of the rate of air flow
through a leaf by timing with a stop-watch is still used in Alvim's
porometer, which employs a sphygmomanometer (blood pressure
gauge), and in Meidner's hand porometer(B21-both are useful and
convenient field instruments (Fig. 2.6). In Alvim's porometer the time
cup
S,
C
B,
A
B,
from gasometer·
type aspirator
selector taps
0-
0-
-0
-0
manometer I
manometer II
Fig. 2..7
filled with
Ii~uid paraffin
Diagram of a Gregory and Pearse resistance porometer(30,1 37 J
,
is measured for the pressure to decrease over a certain range on the
dial; in the other instrument, the time is measured for the rubber teat
to inflate. However, for greater precision and meaSllrements in
absolute units, the instruments shown in Figs 2. 7 and 2.9 must be used.
The Gregory and Pearse resistance parameter. (30) In the resistance
porometer, an air suction from a constant pressure aspirator ~ A,
communicates via one of three fixed capillary resistances of known
magnitude, Dh D2 or D3, with the parameter cup C. The passage of
air through tbe leaf depends mainly on the degree of stomatal opening,
bence the resistance to air flow through the leaf varies with the degree
of stomatal opening. The leaf resistance is compared with the fixed
capillary resistance by manometers, which indicate the pressure
38
THE MEASUREMEi-rr OF STOMATAL APERTURE
difference in the system as a whole (PJ - PI) and across the leaf alone
(P2 -1'1) (Fig. 2.85· Manometer I is attached between the aspirator
and the fixed capillary resistance, and manometer II between the
capillary resistance and the leaf. Figure 2.8 shows diagrammatically
the pressure relations that exist in the resistance parameter so that the
resistance offered by the leaf can be calculated and expressed in the
same units as that offered by the fixed capillary resistance.
When the stomata are closed, the two manometers register the same
reading because no air flow occurs. As the stomata open, manometer II
Ii
P, -p,
selected fixed
capillary,
standard
resistance
R'=
,
Fig. 2.8
leaf resistance
RL unknown
R (P -P)
'
2
, ,
P-P
,
Diagrammatic representation of the pressure relations in a resistance
porometer(137)
gradually falls. The instrument is most sensitive when the leaf resistance and that of the capillary are approximately equal. When the
leaf resistance becomes much smaller than the capillary resistance,
the instrument is at the end of its useful range. To extend the range,
two or three different capillary resistances can be incorporated in the
instrument and shunted into the system by glass stopcocks as the
occasion demands.
The instrument described above is an improved type, (137) and a
recording version of the resistance porometer has been constructed
by Heath and Mansfield.(44)
The Wheatstone bridge porometer, This instrument was constructed
by Heath and Russell in 1951 ;(51) it is a further refinement in porometry, incorporating principles analogous to those of an electrical
39
PHYSIOLOGY OF STOMATA
Wheatstone bridg~, and is more accurate than the resistance porometer, responding quickly to any change in the stomatal resistance to
viscous flow becatlse it is a null point instrument. A diagram of the
Edwards' needle
valve, type LBI
Brodie's
fluid
manometer
B
fixed resistance
F
A_~~~~======~
parameter air
H
inlet from
aspirator
Fig. 2.9
Diagram of a Wheatstone bridge porometer(60)
Wheatstone-bridge porometer is shown in Fig. 2.9. The pressure of
an air stream frorn an aspirator, A, is balanced in the arms of a
Wheatstone bridge to obtain a null reading of the single manometer,
B, of this instrument. The manometer is filled with Brodie's solution,
of specific gravity J'03, and topped up with liquid paraffin of specific
40
THE MEASUREMENT OF STOMATAL APERTURE
gravity 0·83; because of the difference in the densities of the two
manometer liquids, such a manometer becomes five times as sensitive
as the normal type, and is called a differential manometer. The tap, C,
placed across the manometer permits adjustments to be made to the
assembly without the danger of upsetting the manometer liquids.
The differential manometer is placed across the arms of the
Wheatstone bridge. One of these arms consists of anyone of three
known capillary resistances, D I • D 2 , D 3, in series with the unknown
resistance offered by the leaf, E. The other arm of the bridge consists
of a known fixed capillary resistance, F, in series with the calibrated
adjustable resistance of a fine needle valve, G. Resistance Fis of about
the same magnitude as resistance D 1.
In operation, the airstream divides and passes into the one arm of
the bridge at point H, through one of the selected standard resistances
D 1 , D z or D 3 , and then through the leaf into the atmosphere. In the
other arm of the - bridge the airstream passes through the fixed
resistance F and then through the adjustable needle valve G into the
atmosphere. In order to obtain the null reading of the manometer the
needle valve must be adjusted until the airstreams in the two arms of
the bridge are equal.
Units of porometer measurements. In their resistance parameter,
Gregory and Pearse('O) used an arbitrary unit of resistance, and
subsequent investigators using this unit reported many of their
results in 'Gregory and Pearse' units. Such measurements should
perhaps be expressed in absolute units, and we recall that Heath (36)
calibrated capillary tubes having a known resistance in Gregory and
Pearse units, in absolute units of the e.g.s. system by measuring the
flow of air through these tubes under known pressure differences. The
data so obtained were used in Poiseulle's equation for rates of viscous
flow:
.
rrr 4p
Rate of VlSCOUS flow ~ - 81)/
where r and I are the radius and the length of the tube respectively,
p is the pressure difference in dyne cm-2 (Nm-Z) under which the flow
occurs, and 1) is the coefficient of viscosity, which has the dimensions
dyne cm- 2 s (Nm- 2 s), and varies with the gas flowing. Dimensionany,
therefore, Poiseulle's equation reads:
Rate of viscous flow
em' dyne cm-2
~ em 3 s-1
em dyne em-2 s
~ -~~
41
PHYSIOLOGY OF STOMATA
Applying the same argument, as on p. 56 in connection with the
calculation of resistance to diffusion, the resistance to viscous flow is
expressed by:
·
fI ow resistance
.
8'11
= ----:;
VlSCOUS
7rr
Using the viscosity coefficient for air in this equation, Heath
obtained a value for the effective dimensions l/r 4 ofa tube of resistance
of one Gregory and Pearse unit as 3·77 x 10' cm-', and this term can
be used as a conversion factor for Gregory and Pearse units to
absolute units. However, since both water vapour and carbon dioxide
move through the stomata by diffusion, a more appropriate mode of
expressing viscous flow porometer results would be as diffusive
conductance. Both theoretical considerations and experimental
results (see p. 46) show that diffusive conductance has a square root
relation to viscous flow resistance so that viscous flow porometer
results are best quoted as
.
I .
)1/2 or (viscous conductance)I/2
( VISCOUS
resistance
Viscous flow porometer results re!lect most accurately changes in
stomatal conductance, but they are not immediately relevant when
the aim of an investigation is to estimate rates of transpiration or
photosynthesis (discussed in chapter 3). For such estimates, the
resistance of the diffusion path through the stomata must be known
or, as will be shown in chapter 3, the 'effective length' of that path.
It is, therefore, most desirable for a viscous flow parometer to be
calibrated in 'effective length' by using it in conjunction with the
instrument described on pp. 46-47.
2.5 Diffusive flow porometers
For investigations concerned with stomatal control of transpiration
or assimilation, neither stomatal pore dimensions nor stomatal
resistance to viscous flow are as valuable as a knowledge of the
diffusive conductance of stomata. This can be assessed by porometers that measure the rate of diffusion of a gas or water vapour
through or out of the leaf when the stomata are at different degrees
of opening.
The Gregory and Armstrong hydrogen diffusion porometer. (29) In
this porometer, the leaf cup, A, in Fig. 2.10, is connected to a
42
THE MEASUREMENT OF STOMATAL APERTURE
three-way tap, B, which can be opened either to a hydrogen cylinder,
C, or to a hydrogen generating electrolysis apparatus, D. To prepare for a reading of the porometer, the whole system has to be
A
gas escape vent E
B
three way tap
D~~========~
manometer tap
H,
G
L
IIII
c
cylinder
battery
D
micro-ammeter
sliding resistance
manometer
,~
electrolysis apparatus
Fig. 2.10
Diagrammatic representation of the apparatus for a hydrogen
diffusion poromster(29)
filled with hydrogen and thoroughly purged of all air-this is done
by using the hydrogen cylinder supply. During this preparatory
stage a gas escape vent, E, attached to the porometer cup is kept
open while the manometer tap, F, remains closed. When the system
43
PHYSIOLOGY OF STOMATA
is filled with hydrogen, the gas escape vent is closed so that
hydrogen can escape only by diffusion through the leaf; this outward
diffusion of hydrogen proceeds faster than the inward diffusion of air
(nitrogen and oxygen) so that a reduced pressure develops in the
system, indicated by the readings of the manometer, G (see pp. 158).
By adjustment of the sliding resistance the rate of evolution of
hydrogen by electrolysis can be so regulated that when the system is
first closed it just balances the rate of outward diffusion of hydrogen
through the stomata. The microammeter, records the flow of current
from battery, L, required to achieve this balance, and represents
the reading of the instrument; the greater the stomatal opening
the greater the rate of hydrogen diffusion out of the system; hence
the greater the current required to generate hydrogen at a rate equal
to its diffusion through the stomata. Long exposure to hydrogen was
found to have an effect on the stomatal mechanism, and the instrument h" not been used extensively.
The Dufour effect diffusion porometer. Another hydrogen diffusion
porometer, which dispenses with the need to expose the leaf to
hydrogen for prolonged periods, and with the need to enclose the
leaf in an airtight chamber, was designed by Spanner. " J6) The
operation of this instrument is based on the Dufour effect, which
consists of a considerable temperature wave when two gases interdiffuse. In this case, the two gases are hydrogen, and nitrogen (and
oxygen) of the air. A finejet of hydrogen is directed on to a selected
spot of a leaf for a split second. As the hydrogen diffuses through the
leaf and emerges on the other side, it sets up the temperature wave
mentioned. An exceedingly fine thermocouple (51" diameter wires),
placed below the spot on to which the hydrogen jet is directeD, picks
up the. temperature difference, which is recorded on a very sensitive
mirror galvanometer (0'1 second period, 30 mm deflection per I" Y,
30 Q coil resistance and 2,000 Q critical damping). The mirror
galvanometer registers a sudden flick of the spot within one fifth of
a second, and the magnitude of this initial flick is a measure of the
speed of diffusion of the hydrogen through the stomata of the leaf.
The instrument can be used only with amphistomatous leaves and,
probably owing to the specialized galvanometer and the great skill
required to make the sensitive thermocouple, it has not been used
extensively.
The nitrous oxide diffusion porometer. A diffusion porameter, in
which nitrous oxide is used as the indicator gas, has been designed hy
44
THE MEASUREMENT OF STOMATAL APERTURE
Slatyer and Jarvis.(lJ4) It can be used only with amphistomatous
leaves, which are placed in an airtight chamber in which the leaf
itself forms a dividing membrane. An airstream containing a known
amount of nitrous oxide passes across one side of the leaf, and an
airstream initially free from nitrous oxide passes across the other
side. The nitrous oxide diffuses on account of its own partial pressure
difference through the leaf at rates which depend mainly on the
stomatal diffusive conductance. The airstreams entering and leaving
the leaf chamber are led through infra-rcd gas analysers so that the
rate of diffusion of nitrous oxide can be calculated from the concentrations recorded by the analyser. Simultaneously with the measurement of stomatal diffusive conductance, rates of carbon assimilation
and water vapour loss from the leaf can be measured by means of
additional gas analysers.
The apparatus is a valuable, but complex, research porometer; the
relation of viscous flow to diffusive flow found by the differential
transpiration porometer below was verified with this instrument.
The differential transpiration porometer. This porometer uses the
rate of outward diffusion of water vapour through the stomata and
does not rely on the use of extraneous gases for its operation. The
instrument was first used by Meidner and Spanner ;(88) it measures
the difference in leaf temperature produced in two adjacent spots of a
leaf by the differentillol cooling effect of different transpiration rates.
This is achieved by blowing two airstreams of d;ffe(ent water vapour
deficits on the leaf. The water vapour deficits are controlled and kept
constant by bubbling through saturared solutions of KCl (85 per
cent R.H.) and NaCI (75 per cent R.H.). As the airstreams impinge
on one side of the leaf, they cause different transpiration rates, owing
to their different vapour-pressure deficits, which produce a temperature difference that is picked up by a thermopile on the other side of
the leaf. The leaf holder is shown in Fig. 2.11. It is essential that the
two airstreams have the same temperature and the same rate of flow.
For this reason, the saturated salt solutions are kept together in one
water bath for temperature control, and on each line there is a
calibrated manometer and needle valve for regulation of the rate of
flow (about 40 cm J min-I). In order to take a reading, the airstreams
are commutated so that the previously warmer spot on the leaf
becomes the cooler and vice versa; by this commutation the galvanometer connected to the thermopile reverses its deflection and doubles
it, making the instrument more sensitive.
45
PHYSIOLOGY OF STOMATA
The leaf need only be held lightly in the holder, and both amphistomatous and hypostomatous leaves can he used, but since temperature
control is necessary the instrument is not suitable for use outdoors.
Leaves of unusual shapes can be used, like those of onion,(B4) and the
instrument also measures transpirational vapour loss when stomatal
pores are apparently closed (cuticular transpiration), or at least
closed to such a degree that viscous flow porometers register practically infinite resistance. In combination with a viscous flow porometer the differential transpiration porometer is suitable for
investigations of stomata! control of transpiration. (88) Measurements
leads to galvanometer
./-dc)uble hinge ansuring
para".1 position of upper
and lower perspex pieces
_ _-upp,,, perspox piece
leaf
~iiiliilii5i~:::: inlet 1,75% R.H.
'"
. I
inlet 2,85% R.H.
L~i'rsp",,-e,,'-soft plastic pad to
1 and 2
ensure isolation
of air spaces 1 a"d 2
Fig. 2.11
Leaf holder with paralle! moving ieaf supports fer the differential
transpiration porometer(a8)
made with this porometer reflect the rate of outward diffusion of water
vapour through the stomata under defined and not unnatural conditions of atmospheric moisture. The instrument has been used to
calibrate the Wheatstone bridge porometer in terms of diffusive
conductance. The relation of viscous flow conductance to diffusive
conductance has been found to he different for the graminaceous type
and elliptical-shaped stoma. Although the relationship does not hold
at very small (see p. 12) and at very large openings, it is a reasonable
approximation to state that, for graminaceous 5tomata, diffusive
conductance has a cube-root relation to viscous flow resistance, and
for elliptical stomata a square-root relationship.
The sensor element diffusion porometer. Another porometer in
which the outward diffusion of water vapour is the basis of measure46
THE MEASUREMENT OF STOMATAL APERTURE
ment has been designed by van Bavel, Nakayama and Ehrler;(15o: an
improved construction has been produced by Stiles (unpublished).
In this instrument, the leaf must be attached to an airtight cup in
l-" perspex
bulldog clip
silicone rubber
washers --=:!tr
!L._syru,gebarrel
1-+1-- SY'rlnlJ6 slem
electronic
components
sliding lube
carrying thermistor
for placing against
leaf or in centre
of parameter cup
drying air inlet
c;;,....::::::~~=:c.,bleta instrument box
Fig.2.12
Diagram of a leaf attachment clamp for the sensor element diffusion
porameter
which is situated a sensorelemcnt that changes its electrical resistance
with moisture content. The element is fixed at a definite distance from
the leaf (preferably I cm), and water vapour diffuses out of the leaf,
47
PHYSIOLOGY OF STOMATA
across the I cm distance, on to the initially dry element at rates
depending chiefly on the degree of stomatal opening. The conditions
for diffusion are initially constant and defined, though somewhat
unnatural. The leaf clamp, shown diagrammatically in Fig. 2.12 has
been made from a 20 em disposable syringe. The sensor element can
be positioned at different distances from the mouth of the clamp and
calibration is therefore easy. The range of the instrument has also
been extended by the possibility of putting the sensor element at
different distances from the leaf.
Before a reading is taken a small battery-operated pump blows
an air stream over silica gel and into the cup so that the sensor
becomes dry and offers an infinite resistance to current flow; the
conductance of the sensor is then at its lowest, and is indicated by a
microammeter which measures the current flowing through the
sensor from a regulated source in the instrument case. As the sensor
becomes moist, its conductivity increases, and a gradually increasing
current flows through the sensor. When the microammeter registers
a certain current (e.g. 20I'A), a stop-watch is started, and the time
measured for the current to increase to, say, 60 /LA. The rate of
increase in conductivity of the sensor is directly proportional to the
rate of outward diffusion of water vapour from the leaf. A temperature
correction has to be made, and for this purpose a thermistor is built
into the leaf clamp so that the temperature of the leaf can be measured
after every porometer reading by operating a switch.
This instrument is ideally suited for measuring the diffusive
conductance of leaves in the field and in the laboratory; its operation
as a porometer assumes lilat water vapour diffusion out of a leaf into
dry air is regulated by the degree of opening of the stomata (neglecting
cuticular transpiration), and that the air space system of the leaf is
saturated with water vapour. The instrument is usually calibrated in
'effective length' (see p. 56) and results obtained with it can, therefore,
be used in estimating rates of transpiration and photosynthesis (as
discussed in chapter 3). Thus, there is a need to calibrate a viscous
flowporometer in 'effective length' by matching it with this instrument.
48
3. The theory of diffusion
through stomata
The rate of water vapour loss from a lear in still air is not very different
from the rate of evaporation from an open water surface of the same
size as the leaf, although the combined pore area of the open stomata
amounts to only 0·5 to 2·0 per cent of the total leaf area when both
epidermes are taken into account (Table 1.1). This 'efficiency' of
stomatal pores as pathways for gaseous diffusion depends essentially
on their size and on their distribution in the epidermis. Since the loss
of vapour from a leaf, as well as the entry of carbon dioxide into a
leaf, occurs by diffusion, it will be necessary to examine the nature of
gaseous diffusion through small pores.
3.1
The rate of diffusion of a gas
The mechanism of a diffusive flow, of water vapour forinstance, can be
visualized by assuming two adjacent open air spaces of equal volume,
of which the one on the right contains a larger number of vapour
49
PHYSIOLOGY OF STOMATA
molecules than the one on the left. All molecules are in constant
random thermal motion and, since there are more vapour molecules
on the right than on the left, in their random movements more will
cross the dividing boundary between the two air spaces from right to
left than vice versa; this will continue unlll the numbers of vapour
molecules in the two air spaces are substantially equal. At this stage,
the numbers passing from one space to the other will be the same in
both directions-a dynamic equilibrium will have been established.
Fick's Law of diffusion and the diffusion coefficient. A generaJly
valid equation for the rate of diffusion of a gas is expressed by Fick's
Law, which states that the rate of diffusion of a gas is directly proportional to the cross-sectional area of the path and to Ihe difference in
density of the gas along the path, and it is inversely proportional to the
length of the path.
In symbols we can write:
m
DaJp
(I)
t=-I-
where m!t is the mass of gas, measured in grammes, diffusing in one
second; a is the cross-sectional area of the path in cm2; Jp is the
difference in density in g cm-J and I is the length of the path in em.
D is the coefficient of diffusion; by definition it is the mass of gas, m,
diffusing in unit time, t, across unit cross~sectional area, G, of a path of
unit length, I, under unit d(fJerence in density, dp. The dimensions of
Dare cm2 S-I, as can be seen by solving equation (1) for D:
D= ml ;
taJp
gem
s cm2 gcm
cm2 5- 1
J
(2)
It is often convenient to use the difference in partial pressure of the
diffusing gas instead of the difference in density, in which case the
rate of diffusion will be expressed in terms of volume at unit pressure.
If the partial pressures are measured in bar, the rate of diffusion will
be stated as a volume at 1 bar diffusing per second; if the partial
pressures are in mbar it will be as a volume at I mbar. The value and
the dimensions of D remain the same.
The diffusion coefficient is practically constant over the range of
densities of water vapour and carbon dioxide with which we are
concerned; but, since diffusion occurs as a consequence of the
random thermal movements of molecules, it is dependent on the
so
THE THEORY OF DIFFUSION THROUGH STOMATA
temperature (which affects the velocity of these movements) and on
the atmospheric pressure (which affects their mean free path). The
effects are not very large; the QIO for gaseous diffusion is about 1·06
at ordinary temperatures, and the rate varies inversely as the barometric pressure. In addition, the velocity of thermal movements
depends on the molecular weight of the diffusing gas; the lighter the
gas the faster its movement and, hence, its diffusion. Generally, Ihe
relative rates oj diffusion oj two gases are inversely proportional to the
square rools oj their densities or molecular weights (Graham's Law).
Thus, diffusion coefficients are not the same for all gases; those for
water vapour, carbon dioxide, oxygen, and hydrogen diffusing in air
at 20"C and 1013 mbar are as in Table 3.1:
Table 3.1
Diffusion coefficients of various gases
Diffusion
Molecular
weight
coefficient
H,O
18
CO,
0,
44
0·249
0·159
0·201
0'760
Gas
H,
32
2
(em' 5-')
Corrections for temperature and pressure are made according to
the equation:
D ~ D20
(.I.)m
~013
293
p'
where m ~ 2 for CO, and 1·75 for the other gases.
On page 12 we mentioned that at very narrow stomatal openings
the dimensions of the pore, especially at the tapered ends, approach
those of the mean free path of the diffusing molecules (about 0'06fL);
under these conditions the diffusion coefficients, and indeed the laws
of diffusive flow themselves, change. The effects of very narrow
apertures are said to become noticeable when the pore diameter
becomes less than one micron, but except for short periods during
the opening and closing movements, pore widths greatly exceed one
micron, so that ordinarily Fick's Law may be applied in estimating
diffusive flow through stomatal tubes. However, another circumstance complicates matters, because it is not the density difference
SI
PHYSIOLOGY OF STOMATA
between the apertures at the ends of the stomatal tube that has to be
taken intI' account when estimating diffusive flow, but the difference
in densities between regions some distance away from the open ends
of the stomatal tube. Therefore, diffusion through pores of the
dimensions of stomata involves not only the passage through the
stomatal tube but, also, entry into the aperture at one end and
diffusion away from the aperture at the other end. In estimating
diffusion rates through stomata, allowance has to be made for these
effects at the ends (see p. 56).
Diffusion through small apertures. Diffusion away from the narrow
end of a tube can be compared with the movement of water vapour
away from a small open water surface when evaporation occurs. As
the data in Table 3.2 show, the rate of evaporation from such open
water surfaces in still air, calculated per unit area, is greater the
smaller the diameter of the surface.
Table 3.2
Rates of evaporation from open water surfaces in fairly still air.
(After Sayre, 1926)<122)
Diameter
of dish
em
Relative
area
2·64
1-60
0·80
0·40
0·37
0·09
0·02
H)()
Absolute
evaporation
g h- 1
per unit area
2·66
1·51
0·76
0·39
2·66
4·08
8-42
19·52
Evaporation
Diffusion away from an open water surface thus appears to be more
nearly proportional to the diameter, and hence the circumference of
the surface, and not as with diffusion through a tube, to the cross·
sectional area. The data in Table 3.2 confirm Stefan's Law of 1881,
which states that diffusion through small apertures is proportional to
the diameter, d, of the aperture and to the difference in density, Ap,
measured between the underside of the pore and a point some distance
away from it. We may write:
Rate of diffusion ~ 2DApd; cm2 S-l g cm- 3 em ~ g S-l
(3)
The physical explanation for Stofan's Law lies in the fact that in
still air the pattern of diffusive flow away from an open surface
remains geometrically similar when the surface is magnified in size.
52
THE THEORY OF DIFFUSION THROUGH STOMATA
Unless the overall differences in vapour density are also magnified,
the density gradients above the magnified area will be reduced by the
scale factor of magnification.!
_- -_
~
vapour density at
half value contour
surface, density
p/2 at one diameter
distance
sunace=p
....-
_--
"., / '".,-/
vapour density..at
half value eQnlou,
surface, density
p/2 at one: diameter
surface=p
distance
Fig. 3.1 Diagrammatic representation of diffusive flow liries above two open
water surfaces in perfectly still air. Surface WI! has twice the diameter of WI;
scale factor f = 2. Dotted lines represent contour surfaces of equal vapour
density, the half~value contour surfaces are indicated by fully drawn lines.
Bold arrows represent the diffusive flow lines fanning out from the surtace
In Pig. 3.1, W' and W' represent two surfaces drawn to scale, W'
having twice the diameter of W'. The diffusion patterns are indicated
by bold arrows fanning out at right angles to the contour surfaces of
equal densities. We may imagine those contour surfaces at which
the vapour density will be half its original value to be at a distance of
one diameter above each surface (represented by the fully drawn-out
53
PHYSIOLOGY OF STOMATA
semicircles in Fig. 3.1). While the area of W' has increased as the
sqnare off, the length of the diffusion path from W' to the half-value
contour surface has increased by /; the result, based on Fick's Law,
eq. (1), is Stefan's Law: the rate of diffusion is proportional to
/2// ~ f, or in other words the rate is proportional to the diameter of
the evaporating surface, and not its area.
Another feature of the diffusion pattern shown in Fig. 3.1 may be
noted; the fanning out flow lines of steepest gradient indicate that
the fastest diffusion occurs at the circumference of the aperture, which
sutfaces of equal
V.P.
external resista:lce
of adhering air layer
)
~
edge diffusion
etige dif1usion
mutual interference of neighbouring
diffusive flow lines
Fig. 3.2
Diffusive flow Jines in perfectly still air above a perforated septum
such as an epidermis with stomatal pores. The magnitude of the external
resistance of a leaf depends on the degree of confluence of the flow lines
from individual pores and on the wind speed
has the important consequence that the water at the edges will be
cooler than the water at the centre, and the saturation water vapour
pressure immediately above the water will, therefore, be lower at the
edges than in the centre. Thus, conditions exist for the occurrence of
convection currents and for diffusive flow of vapour so that truly
'still' conditions can never be realized (see p. 60).
Nevertheless, conditions of 'still' air permit individual stomata to
be comparatively efficient paths of gaseous diffusion, provided their
spacing is such that their flow lines do not markedly interfere with
each other (Fig. 3.2 and p. 61). The greater the distance between
stomata the smaller will be the degree of interference between neighbouring flow lines; as stomata open, interference will tend to increase
54
THE THEORY OF DIFFUSION THROUGH STOMATA
and the 'diameter law' will tend progressively to give place to the
'area law', making the stomata, per unit pore area, less efficient as
diffusion pathways. However, as can be seen from Fig. 3.2, even
when the stomata are not fully open, the contour surfaces of equal
vapour density above individual stomatal pores will fuse at some
. distance to form corresponding contour surfaces above the leaf as
a whole. These constitute a region of relatively high vapour density
outside the leaf through which molecules diffusing away from the
leaf will have to pass.
Effects of wind. It follows from the foregoing that air movements
which reduce the degree of accumulation of vapour outside the leaf
effectively will have a considerable influence on transpiration rates.
The air immediately above the leaf surface is known as the boundary
layer, and even strong winds cannot totally remove it. In a wind of
5 km h- 1 for instance, the boundary layer extends to about one
millimeter above the leaf surface, and vapours and gases move
through it by diffusion, whereas in the layer of turbulent flow beyond
the boundary layer the rate of movement is largely a function of the
wind speed. The effects of wind on the rate of transpiration are,
therefore, due to the alteration of the length of the non-turbulent
diffusion path external to the leaf. In addition, wind has an effect on
the temperature of the leaf, which is of major importance for transpiration. Allowance for the effect of wind on the length of the external
diffusion path must be made in any attempt to estimate transpiration
rates (see p. 60).
3.2 Details of the diffusion path between
leaves and the atmosphere
Wherever a difference in density exists for a particular gas, diffusion
of this gas will naturally occur, unless flow is totally prevented by an
impermeable barrier. For water vapour moving out of a leaf, the path
can be analysed into the following sections in series:
(a) the air space system within the leaf:
(b) the entrance into the stomatal tube:
(c) the passage through the stomatal tube;
(d) the exit from the stomatal tube;
(e) the external diffusion system attributable to the leaf as a whole.
The diffusion of carbon dioxide into the leaf follows the same
sequence in the reverse order, but once inside the leaf air-space
S5
PHYSIOLOGY OF STOMATA
~ystem,
carbon dioxide will dissolve in the water film in the
mesophyll cell walls, and diffusion to the chloroplasts will be in the
liquid phase. Diffusion in the liquid phase is slower than in the gas
phase by a factor of about 10,000, so that for carbon dioxide intake
these diffusion paths in the liquid phase must be taken into account
(see p. 64). However, it is possible that in the dissolved state, carbon
dioxide moves not only by diffusion but also by a form of cytoplasmic
transport based on streaming movements, which would partly offset
the slowness of diffusion in the liquid phase.
The resistance of diffusion paths, the 'End Correction' and
'Effecti.. Le"gths'. Both Fick's and Stefan's Laws imply that the rate
of diffusion is a function of the geometry of the diffusion paths.
However, we have mentioned (see p. 52) that an estimate of diffusion
through narrow tubes, such as the stomata, cannot easily be based
solely on the dimensions of the stomatal tube since the densities of the
diffusing gas are not usually known at its exact ends. Owing to the
fact that they are known only at some distance from the ends, diffusion
through a narrow tube must be calculated as if it were through a tube
of the same cross-sectional area but having a length, the 'effective
length', greater than the actual length by the so-called 'end corrections'. These depend on the diameter of the apertures at the ends only.
Further, in assessing the resistance of a diffusion path, its 'equivalent length' must be evaluated in order to take account of variations
in the cross section of the path. Thus, all resistances are expressed as
equivalent length of open air path of unit cross-sectional area.
In order to arrive at a definition of 'resistance to diffusive flow', We
may consider diffusion as analogous to the flow of water in a restricting
pipe between two pools of water situated at different heights, or to the
flow of electricity in a resistance wire connecting two terminals
between which there is a difference in electrical potential. We may
consider tbe density difference between the ends of a diffusion path
as a driving force, and the diffusive flow along the path as resulting
from this. The relation between driving force, flow, and resistance,
analogous to Ohm's Law, is:
Ra teo f fI oW=
density difference
.
resistance
and, bence,
.
density difference
reSIStance =
f fI
rate 0 ow
S6
THE THEORY OF DIFFUSION THROUGH STOMATA
We can now develop an expression for the resistance offered by the
different sections of the diffusion path through the stomata. Using
eq. (I) for the resistance inside the stomatal tube we have:
.
Jp
I
ReSlstance,ub, = DaJp/1 = Da;
em
cm2 s 1 cm2
s cm- 3
(4)
Then, using eq. (3) for the end effects, i.e., the resistance to diffusion
away from open pores:
.
Jp
)
ReSlstance
po" = 2DJ pd = 2Dd
(5
with the same dimensions as the Resistance,u""
On the electrical analogy, resistances arranged in series may be
added, and hence we obtain a value for the resistance to diffusion
through and away from a stomatal tube by adding eqs (4) and (5):
I
I
Da + 2Dd'
4iubstitution of ",d 2/4 for a results in
Bringing back the symbol a for the area, we obtain:
Resistance,u"'+Po" =
~a (I + ~d)
(6)
The term, 1+ 7rd/8, represents the 'effective length' of the path in
em, I being the actual length, and ",d/S the end correction. In subsequent calculations corrections will have to be applied for the effects
at both ends of the stomatal tube, so that the total resistance to
diffusion up to, through, and away from stomatal tubes becomes:
(I
..!._ + 7rd)
Da
4
(7)
If I'ff is used as symbol for effective length, its relatien to the diffusive
resistance is:
DI'ff"
uSlve resIstance = -I'ff ,
Da
57
PHYSIOLOGY OF STOMATA
It may be noted that the diffusion coefficient, D, remains in the
expression defining the diffusive resistance, and therefore the diffusive resistance of a path will vary with the nature of the diffusing
gas; it will, for instance, be smaller for the diffusion of water vapour
than for that of carbon dioxide. Diffusive resistances are often
expressed as resistance per cm z when the dimensions are s em-I;
this usage has advantages, especially in studies in which resistances
of crops and vegetation cover are compared. By making the calculations in eqs (6) and (7) for unit area, resistances per cm2 with
the dimensions s cm- 1 are obtained.
The quantitative estimation of the diffusive resistance of a leaf. The
resistances of the five sections of the diffusion path listed above (see
p. 55) can now be quantitatively expressed by the use of eqs (4), (5),
and (7). For convenience the following symbols will be used:
L ~ length of the diffusion path in the air space system of the leaf,
/ ~ length or depth of the stomatal tube,
A ~ area of the whole leaf surface,
a ~ cross-sectional area of one stoma,
D ~ diameter of the whole leaf area,
d ~ diameter of the stomatal tube,
n ~ number of stomata in the leaf surface.
Resistance of the path in the air space system (cf. eq. (4»
L
DA
Resistance of n stomatal paths, each comprising entry at one end,
passage through the tube and exit from the open end (cf. eq. (7)
_1 (/+ 1Td)
4
nDa
Resistance external to the leaf in perfectly still air (cf. eq. (5»
2DD
Resistances in sefies may be summed, so that we have:
1
R~-
DA
58
[ L+-+-/+A
A ( 1Td)].
2D na
4
THE THEORY OF DIFFUSION THROUGH STOMATA
As the area of the leaf has to be calculated from its diameter, it is
convenient once more to substitute 1TD'/4 for A in the term for the
external diffusion path, thus obtaining:
R=
I
DA
[
L
+
+
common term effective
transforming length of
effective
internal air
lengths into
space system;
resistances in estimated as
terms of
average
distance
equivalent
between
air paths
mesophyli
cell walls
and entrance
effective
length of
external
diffusion
path in
perfectly still
air
A
Ita
proportion
of total leaf
area to
stomatal
pore area
(see p. 2)
( 1+ 1T:)]
(S)
effective
length of the
diffusion
path of onc
stoma
to stomata
In this conventional way of writing the equation, two end correc·
tions of the value 1Td18 each, i.e., 1TdI4, have been applied for the
calculation of the effective length of the stomatal diffusion path,
since the gas has to enter the stomatal tube as well as escape from it;
on the other hand, the effective length of the external diffusion path
away from the leaf remains 1T DIS, i.e., it is in the form of a single 'end
correction' (cf. eq. (6». The term 1T DIS assumes perfectly still condi·
tions, and, as we have seen, these are unattainable, hence the effective
length of the diffusion path external to the leaf will be overestimated
by using this expression. Experimental results have shown that a more
realistic estimate of the effective length of the external path in
nominally still air would be obtained by using the term 1T DO.6/S. In
conditions of moving air, the effective length of the external path
(boundary layer and turbulent region) depends, of course. on the
wind speed; the following expression permits an estimate of the
effective length of the external path in wind:
O'S9DD-46
VO· 56
where D is the diameter of the leaf in em, as before, and v is the wind
velocity in cm S-I.
3.3 Stomatal control of transpiration
Since the rate of diffusion is proportional to the difference in vapour
density between the leaf air space system and the atmosphere, and
59
PHYSIOLOGY OF STOMATA
inversely proportional to the resistance of the diffusion path, we can
write:
Rate of transpiration
LlpDA .
(mass of water vapour ~
Tr D A ( Trd)
per second)
L + - + - 1+ (9)
8
na
4
LlpDA
~
1;;;.
If we consider a hypostomatous leaf of 10 em diameter, 0·05 em
thick, with 35,000 stomata per em>, an average pore width of 0·0006
em, and a depth of pore of 0·003 em, the resistance of the diffusion
path in the air space system of the leaf, (I/DA)L, is about 0·0012
s cm- 3, that of the external diffusion path, (I/DA)TrD/8, amounts to
about 0·2 s cm-3 (or if we use the term 11 DO·"/8 ~ 0·08 s cm-3), and
that of the stomata
_I .naA (I + Trd)]
[DA
4
to about 0·017 s cm- 3. It is therefore clear that in still air the stomatal
resistance is very small compared with the external resistance, and
stomatal control of the rate of vapour loss must be limited to smaller
openings when the stomatal resistance becomes comparable in
magnitude to the external resistance. At a pore width of 0·0002 cm,
for instance, the resistance of the stomatal diffusion path would rise
to 0·14 s cm-3, and as such it would contribute markedly to the
control of the rate of vapour loss. The limited stomatal control of
transpiration in still air has been experimentally verified as shown by
the results represented in curve (a) of Fig. 3.3; results obtained in
moving air are represented by curve (b). The effect of air movements
in reducing the external resistance has two aspects: it permits a
considerable increase in the maximum transpiration rate, and it
allows for a high degree of stomatal control of transpiration, because
under conditions of moving air it is the stomatal resistance to diffusion
that is the highest in the series of resistances in the diffusion path.
It is worth noting that in the two water vapour diffusion poro"
meters described in chapter 2, the external resistance to diffusion has
been reduced and, in one, practically eliminated; in the differential
transpiration porometer it is kept at an extremely low value by the
continuous airstreams impinging directly on the leaf, and in the sensor"
60
THE THEORY OF DIFFUSION THROUGH STOMATA
element diffusion porometer, although measurements are carried out
in stilI air, the hygroscopic element, fitted at 1 cm distance from the
leaf surface, also keeps the external resistance at a low value. Results
300
250
/
~
I
E
• I
."
~
•
I
••, • • 1
.'
:. I
~
I
• .. I •
,"
I
.,
...
150
., I
'c.
I
'"c:
o
I
• Of
~
~E
0
• I
200
x
'"oc:
o
' .. I
,."
6'"
-(b)
I
•
/
100
/
I
I
ell
•
I,
-E
0
".
•I
•
'r •
..
to / .
.'
•• 0
••••
or..
~!- __ .·-fi--
•
__ -
-(a)
50 :.,,1 •• •••~-- ••••
• 1 • __..-<'.o
.•t:/o,
•
., j / '
o =-'empirical values in still air
• =-~empirical values in wind
If.
o,~--~--~--~~~
5
10
15
20
Stomatal aperture in Jl
Fig. 3.3 The relation between pore diameter and rate of transpiration in still
air and in moving air (Zebrina pendula). E=evaporation in still air. (After
Bange, 1953(7»
obtained with both these instruments confirm the high degree of
stomatal control of transpiration in the absence of a large external
resistance to diffusion.
'Mutual interference' and 'incipient drying'. At this stage we must
mention the contradictory views found in the literature concerning the
61
PHYSIOLOGY OF STOMATA
stomatal control of transpiration. One cause of the confusion is the
fact that Brown and Escombe(lJ), the pioneer workers in this subject,
made no allowance for the external resistance, particularly important
in still air. Their calculated rates of transpiration were, therefore,
consistently greater than the measured ones. In order to explain these
discrepancies, Brown and Escombe postulated increases in the stomatal resistance to diffusion owing to 'mutual interference' of the
flow lines when stomatal pores were wide open. Some investigators
inferred from this that stomatal control was limited to the state when
stomata were less than one-third open, because, at larger openings,
mutual interference was supposed to decrease their efficiency. Others
postulated a non-stomatal resistance to diffusion inside the leaf, such
as 'incipient drying' during high ratesoftranspiration at wide stomatal
openings. Evidence for the occurrence of incipient drying, i.e., a
condition when the air space system of the leaf is appreciably below
its saturation vapour pressure, has been indirect only. Thus, under
severe experimental conditions, changes in rates of vapour loss from
leaves at constant stomatal opening have been measured, (31) and the
possibility that this occurs under extreme and rapidly changing
climatic conditions cannot be ruled ont; usually, severe decreases in
leaf water potential are follo"ed by stomatal elosure.
Various mechanisms that could be responsible for the occurrence
of incipient drying have been considered, but none of them appears
feasible on theoretical grounds. The possibility of a withdrawal of the
water menisci into the interior of the mesophyll colI walls has been
investigated, and calculations indicate that to effect withdrawal of the
menisci, the leaf water potential would have to decrease to values so
low that they would kill the leaf. Similarly, calculations show that the
accumulation of solutes at the evaporation sites is not likely to
become so high as to lower the vapour pressure of the solution
sufficiently to cause incipient drying. ([33) The naturally occurring
increases in the concentration of the cell sap would not cause mOre
than a slight reduction in the vapour pressure of the sap; for instance,
if the sap in a leaf at 20°C is equivalent to a molar solution with an
osmotic pressure of about 22 bar, its vapour pressure would be
22·90 mbar as compared with that of pure water of 23·31 mbar
(Raoult's Law). If, owing to transpirational water loss, the sap were
to become so much more concentrated that it would be equivalent to
a three molar solution with an osmotic potential of about 66 bar, its
vapour pressure would be reduced by only 1·39 mbar. The effect
62
,
THE THEORY OF DIFFUSION THROUGH STOMATA
on the rate of evaporation into the leaf air space system would be too
small to bring about incipient drying.
Thus, a mechanism resulting in a water vapour pressure of the air
space system significantly below that at saturation is not known,
although some evidence has been obtained to show that a steep
gradient of water potential can develop across cell walls so that the
water potential at the surface of the walls is far from zero-in maize
leaves under mild drought conditions it has been estimated at about
-80 bar.(130)
3.4 Stomatal control of photosynthesis
The diffusion of carbon dioxide from the atmosphere into the air
space system of the leaf, and then in the dissolved state through the
cell membranes and aqueous medium to the ,hloroplasts, proceeds in
light because of the gradient in density for carbon dioxide. In still air,
the density immediately outside the stomata may be about 4 x 10-4
mg cm- J (corresponding to a volume content of CO 2 0[0·02 per cent)
and in moving air about 6 x 10-4 mg cm-3 (0'03 po< cent), whereas
at the chloroplasts it may be considered to be near zero. The resistances
to diffusion of carbon dioxide in the gas phase are of the same nature
as those for water vapour but somewhat greater because ofthe smaller
diffusion coefficient of carbon dioxide; however, there is an additional
resistance to diffusion of carbon dioxide in the dissolved state, which
we have referred to on p. 56 as liquid phase re,istance. When this
liquid phase resistance is much the greatest in the series of resistances,
the rate of diffusion of carbon dioxide into the leaf is largely controlled by it. Gaastra (25) estimated the external resistance to be about
2 s em-I when the wind speed was about 230 m h- I ; the stomatal
resistance was estimated to be about 3 scm-I, but the liquid phase
resistance was 2-10 scm-I.
When carbon dioxide is limiting the rate of photosynthesis, as often
occurs under natural conditions in sunlight, a cMnge in stomatal
resistance, such as may be brought about hy a decrease in leaf water
potential, will begin markedly to affect the rate of diffusion of carbon
dioxide once the stomata! resistance becomes about equal to the sum
of the external resistance and the liquid phase resistance. In this case,
a doubling of the stomatal resistance will cause a reduction in the rate
of photosynthesis by one-third. (This estimate, as well as those cited
on p. 64, was made by Gaastra(25), whose detailed analysis of this
problem should be consulted.)
63
PHYSIOLOGY OF STOMATA
As an example, we may calculate the rate of inward diffusion of
carbon dioxide, using eq. (9). For the leaf specified on p. 60 the terms
in the equation will be:
.
.lip ~ 6 X 10-4 mg cm-' (C02 density in normal air);
D co , at 20°C ~ 0·159 cm2 S-I;
A~80cm2;
L~0'025cm;
",Do·.
-g- = 1·6 cm for nominally still air;
A
- ~ 100'
na
'
6 X 10- 4 X 0·159 x gO
0'025 + 1·6 + 100(0·0035)
rate of CO 2 intake by
leaf of gO cm2 per second
~
0·00384 mg CO 2 per second.
Rates of photosynthetic intake of carbon dioxide are customarily
expressed in mg dm- 2 h- I , and if we make allowance for this we
obtain a rate of 17 mg CO 2 dm- 2 !r l • If we wish to compare this
calculated rate with one that has been experimentally measured, we
shall have to reduce further the term for the resistance external to the
leaf, as most experiments are carried out in moving air. At a wind
speed of 5 km h- I the length of the external diffusion path will reduce
from 1·6 to about 0'2, and recalculation results in a rate of 60 mg
CO 2 dm- 2 h- I ; this compares with a calculated stomatal diffusion
capacity of Zebrina pendula leaves under similar conditions to tbose
assumed in our example, and quoted by Gaastra (25) as 46 mg CO 2
dm- 2 b- I for stomatal pores of 5,. diameter.
So far, we bave not made any allowance for the resistance to
diffusion in the liquid pbase. Information on the magnitude of this
resistance is available only from a few experimental measurements.
Gaastra estimated the effective length of this resistance to be one-tofour times that of the stomatal path; we chose, therefore, a value of 1·2
for the effective length of the resistance in the liquid phase. Using tiiis
64
THE THEORY OF DIFFUSION THROUGH STOMATA
in the equation, we obtain a rate of carbon dioxide intake of 19 mg
dm- 2 h-' which compares well with typical rates measured experimentally; for instance, Gaastra quotes a rate of20 mg dm- 2 h-' for a
sugar beet leaf at a light intensity between 10 and 20 J m-2 s-'.
It may be noted that in our example the combined pore area of the
61-' wide stomata occupied only about one per cent of the leaf area; in
this condition the stomatal resistance to the diffusion of CO 2 amounted
to 0·028 s cm- 3 and that of the liquid phase resistance 0·1 s cm-';
hence partial stomatal control of the rate of carbon dioxide intake
could be expected.
At low light intensities « 40 J m- 2 s-') and at an external density
of carbon dioxide of 6 x 10- 4 mg cm-3 (0·03 per cent) the rate of
diffusion is much greater than the rate of the chemical processes
in photosynthesis, so that over a considerable range of stomatal
resistances the rate of photosynthesis remains controlled by the
chemical processes and practically unaffected by stomatal aperture.
At higher light intensities (> 50 J m-2 s-') with the stomata
wide open and an external density of about 6 x 10-4 mg cm-', the
. rate of diffusion of carbon dioxide limits the rate of photosynthesis.
Variations in light intensity will cause only slight changes in stomatal
resistance, hence the liquid phase resistance, assumed to be large and
unaffected by changes in light intensity, controls the rate of diffusion.
Besides the possible intervention of the liquid phase resistance in
the control of the rate of diffusion of carbon dioxide, other circumstances make stomatal control of photosynthesis rather complex. For
instance, at high light intensities, when the density of carbon dioxide
external to the leaf is raised from 8 x 10- 4 mg cm-' (0·04 per cent) to
2 x 10-' mg cm- J (0·1 per cent), stomatal resistances increase by
about one-third, because partial stomatal closure is brought about
by the high concentration of carbon dioxide (see chapter 4). Nevertheless, the rate of photosynthesis remains unaffected because the
greater difference in densities of carbon dioxide allows for an unchanged rate of diffusion. At low light intensities, when stomatal
resistances increase, owing to an increase in the external density of
carbon dioxide from 2 x 10- 4 mg cm-3 (0·01 per cent) to 4 x 10-4 mg
cm- 3 (0·02 per cent), the rate of photosynthesis will increase because
the greater difference in densities of carbon dioxide more than
compensates for the stomatal closure that occurs.
Summarizing, one may say that stomatal control of photosynthesis
is most marked when carbon dioxide is limiting and the liquid phase
6S
PHYSIOLOGY OF STOMATA
resistance is comparatively low. When light intensity is limiting,
stomatal control of photosynthesis is relatively small.
3.5 The experimental control of stomatal
aperture
In agricultural and horticultural practice a reduction in the rate of
transpirational water loss would often be very desirable provided this
could be achieved without adversely affecting the general metabolism
of the plants and, especially, the rate of photosynthesis. Various
chemicals that cause partial or complete stomatal closure, and
surface films covering, the leaves have been used as ~antitranspirants'.
Foreseeable consequences of reducing transpiration rates are increased leaf temperatures and, possibly, reduced rates of ion uptake
and translocation. (26) The cooling effect of transpiration varies, of
course, with insolation and wind speed, but it is commonly between
2' and 4'C in temperate climates and may reach SoC in hotter areas.
In the complete absence of this cooling effect, leaf temperatures may
come dangerously near the death point of protoplasm, especially in
hot areas where antitranspirants would be most useful. However,
the use of antitranspirants aims at restricting transpiration and not
at preventing it entirely; thus, only part of the cooling effect would be
lost by their use. Therefore, if leaf water content can be conserved
during critical periods by applying antitranspirants and thereby
avoiding wilting and even higher leaf temperatures, the plants may
recover when more favourable conditions return.
There remains the fact that, at certain light intensities, higher leaf
temperatures will affect respiration more than photosynthesis, so that
the rate of increase in dry weight may be slightly retarded-a small
disadvantage if the use of antitranspirants can prevent the desiccation
of the crop.
The effects of a reduotion in transpiration on ion uptake and transiocation(26) are as real as the effects on leaf temperature, but their
temporary restriction could hardly affect the metabolism of the
plants very seriously. Again, it is a choice between the possibility of
losing a crop during a critical period or saving it at the price of a
retardation in growth.
Enrichment of the air with carbon dioxide. From the examples given
in the previous section (see p. 60), and in view of the differential effect
of partial stomatal closure on the rates of photosynthesis ana trans66
THE THEORY OF DIFFUSION THROUGH STOMATA
piration, the enrichme1.t of the air by an additional carbon dioxide
supply would seem to offer the simplest approach. Thus, Moss
et al. (91) reported a reduction in transpiration of 23 per cent following
an increase in the carbon dioxide concentration of the air from 0·031
to 0'575 per cent; at the same time, in spite of the stomatal closure
that must have occurred, photosynthesis increased by 30 per cent,
presumably because of the improved partial-pressure gradient for
carbon dioxide into the leaf and because photosynthesis was severely
carbon dioxide limited at 0·031 per cent. The enrichment of the air
with carbon dioxide is a practical and economic procedure in glasshouses but would be much more difficult and expensive for field crops.
Epidermalfilms. Other methods of reducing transpiration have been
tried, such as covering the leaves with films that are pervious to
carbon dioxide but impervious to water vapour, or films that increase
the reflectivity of the plant surface, thus keeping the temperature of
the plant down, with a consequent lessening of the difference in
vapour density between the plant and the atmosphere. (26) These
methods do not operate via an effect on stomatal aperture and do not
concern us here. There are, however, chemicals that affect the
metabolism of the plant in such a way that partial stomatal closure
occurs, and the use of such chemicals as antitranspirants must be
considered.
Chemical antitranspirants. The fungicide phenylmercuric acetate
(PMA) at 5 x 10-5 M concentration and O!-hydroxysulphonate at
3 x 10-2 M have been used to reduce the rate of transpiration by
causing partial closure of the stomata.
PMA sprayed on leaves reduced transpiration of tobacco by
40 per cent([74) and that of cotton by 60 per cent ;(!3J) these reductions
in transpiration were accompanied by decreased rates of carbon
dioxide intake and it is most likely that stomatal closure following
PMA treatment was the result of changes in \he concentration of
carbon dioxide in the guard cells, since closure produced by PMA
can be prevented or reversed by treatment of epidermal strips with
carbon dioxide-free air.(76) Although PMA is a poison that can
seriously inhibit the process of photophosphorylation, when it is
sprayed in solution on to the leaf surface it may not enter much
beyond the guard cells. Solutes from sprays are readily taken up by
guard cells (see p. 13) and they are often translocated to the rest of
the leaf, but PMA has a low mobility within the plant and its effect
is probably restricted mainly to the guard cells. Thus, without acting
67
PHYSIOLOGY OF STOMATA
as a general poison it can cause stomatal closure and, hence, reduce
transpiration.
IX-Hydroxysulphonates inhibit the oxidation of glycollate, an early
product of photosynthesis, and a product of degradation of more
complex photosynthates in the absence of carbon dioxide. One
hypothesis of the stomatal mechanism (see pp. 135 to 138) postulates
that glycollate metabolism is essential for stomatal opening and that
its inhibition results in stomatal closure. However, the action of
IX-hydroxysulphonates in causing stomatal closure at leaf temperatures of about 25°C and at light intensities between 5,000 and 15,000
lux is almost certainly due to their adverse effect on photosynthesis,
which results in an increased concentration of carbon dioxide in the
leaf. At leaf temperatures around 35°C, IX-hydroxysulphonates have
been reported to improve the rate of carbon dioxide intake by
illuminated tobacco leaves within minutes of their applicationsignificantly, stomatal closure was delayed under these conditions
and certainly did not occur during the period when the improved
rates of photosynthesis were measured.
The mode of action of an ideal chemical antitranspirant would be
to cause partial stomatal closure and thereby a reduction in the
transpiration rate. Whether this would be achieved without equally
affecting the rate of photosynthesis would depend on the prevailing
light intensity (see p. 65) and concentration of carbon dioxide outside
the leaf, as well as on the comparative magnitude of the diffusion
resistance for carbon dioxide in the liquid phase. There is more than
a suspicion that some of the chemical antitranspirants used so far
operate in the reverse order: they appear to decrease the rate of
photosynthesis and thereby cause partial stomatal closure. This is rar
from the desired mechanism, but even so, it may often be preferable
to save a crop from severe drought by using an antitranspirant even
if it does reduce the rate of photosynthesis. The effects of sprays of
PMA and of o<-hydroxysulphonates wear off after about two weeks. If
they can reduce transpiratiollal water loss during a critical time their
use will be well worth while.
68
4. Stomatal responses to
environmental factors
Stomatal movements, in common with many other activities in plants,
are greatly affected by the environment. However, aperture is not
always determined by the external factors prevailing at the time of
observation, for the plant retains a considerable measure of control
through its endogenous rhythms, and this important subject will be
covered separately in chapter 5.
The remarkable reactivity of stomata has interested plant physiologists for well over a century and the number of investigations reported
in the literature is vast. There have been many disagreements about
responses to various stimuJi, and sometimes quite contrary accounts
are to be found. This is probably a direct outcome of the reactivity of
stomata, which have responded to a factor the worker failed to
control, and probably did not even realize was important. Stomatal
sensitivity to atmospheric carbon dioxide concentration has been
fully recognized only for about twenty years, and in earlier work the
effect of the researcher breathing on to his plants must have been a
69
PHYSIOLOGY OF STOMATA
major experimental hazard! In the following account of the responses
of stomata we have found it necessary to ignore some of the more
doubtful work, but we have been unable to dispose of the confusion
in certain fields.
4.1
Light and carbon dioxide
Light is probably more import~nt in determining the course of stomatal behaviour than any other component of the environment. In
:[
C>
c:
'c
"c.0
'0 4
"~
'0
"
C>
2
o
30
60
90
120
150
time (minutes)
Fig. 4.1
Opening in Xanthium pennsylvanicum is under way after a few
minutes' illumination. Illumination of 20,000 lux from zero time, the preced!ng
dark period being eight hours in length. (After Mansfield and" Heath(77»
most plants, stomata tend to open during the'llay and close at night,
although they may not be open all day and closed all night. Certainly,
they do not always open in light and close in the dark. Stomata
illuminated during the ~:ght hours are usually reluctant to open, and
darkness during the day may not produce complete closure because
of the overriding effect of endogenous rhythms.
Rhythms apart, the usual effect of light is to produce an opening
movement. The maximum speed of response varies considerably from
species to species. In soroe cases the complete opening movement may
take several hours, whereas in others the movement is under way in a
70
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
few minutes and may be nearly complete in half an hour, as, for
example, in Xanthium pennsylvanicum (Fig. 4.1).
The minimal intensity for opening is difficult to determine because
the stomata of so many species can open in complete darkness,
particularly when the dark period is prolonged over the time when
the plant is normally illuminated in the morning. It is extremely
difficult to distinguish between such rhythmic opening and the
reaction to low-intensity light. The most reliable work is probably
that on cereals, whose stomata are unusual in that they do not
normally show night opening (70) (see p. 109), yet the stomata of wheat
opened distinctly in light of 900 lux, i.e., I or 2 per cent of full sunlight.(53) Virgin(153) found a small increase in the transpiration of
wheat seedlings with a light intensity as low as 10 lux (about I-footcandle) and it is probable that this was due to stomatal opening
(Fig. 4.9).* The material in these experiments had been grown under
constant temperature in continuous darkness, and consequently an
acquired rhythm was unlikely to have been involved. The plants
were, of course, etiolated after being grown in the dark, and Virgin
observed that chlorophyll formation took place during the course of
the experiment. The observations were, therefore, made in somewhat
unusual circumstances, but nonetheless the apparent response of the
stomata to irradiation of such low intensity is of considerable interest.
Opening increases with light intensity up to a saturating value that
is often of the same order of magnitude as moderate sunlight. Figure
4.2 shows the time course of stomatal opening in wheat, as shown by
changes in transpiration rate in a wide range of light intensities. The
plants in these experiments had been in darkness for only thirty
minutes before the light was switched on and the measurements made.
This probably accounts for the unusually rapid response to light, since
it is unlikely that the stomata were fully closed after such a short time
in the dark. Transpiration would be increased independently of
stomatal aperture if leaf temperature increased under high light
• Irradiation intensities are expressed in different units by different
authors, and we have not thought it desirable to convert to a uniform unit
since this is a hazardous procedure, particularly where photometric (e.g.
lux, foot~candles) and energy (e.g. J m~2 S-1) units are concerned. The conversion from one to the other is not by a simple factor, but depends on the
spectral distribution of the light source used. As a rough guide there are
usually 3 to 4·5 m J m-2 5- 1 per lux, over the visible range of the spectrum.
A valuable conversion table can be found on page 24 of the paper by
Gaastra. (25)
71
PHYSIOLOGY OF STOMATA
intensity, which would mean that the curves contain a component
not due to changes in the stomata. However, Virgin did attempt to
reduce heating of the leaf to a minimum by absorbing infra-red
~
'"
C>
c
.
'E
60
~
E
"0
-
50
:§
40
"
.t::
~
0
~
E
"
"
.">
..
16000
0
~
.t::
"!!
32000
c
8000
4000
2000
g
30
0
5
10
15
20
25
minutes
Fig. 4.2 Transpiration from wheat leaves measured with the 'Corona hygro~
meter'. Before illumination the leaves had been in darkness for 30 minutes.
The transpiration value in darkness was 32 per cent. (After Virgin(152)
radiation from the lamp with a water screen, and so the artefacts in
the measurements were probably not too great.
The role o/photosynthesis. The first theory of the mechanism behind
stomatal movements, put forward by Von Mohl in 1856,(90) was that,
in light, the guard cell chloroplasts produced soluble assimilates that
were osmotically active in producing the increase in turgor necessary
for opening. Interest in the role of photosynthesis has continued to
the present day, but whether or not the products of guard cell chloroplasts are an important feature of the mechanism remains unknown.
There is no longer any doubt that the guard cells can take in CO 2
when illuminated. Microautoradiographs of onion leaf epidermis
after exposure to "C0 2 in light are shown in Fig. 4.3, in which can
be seen pronounced activity in the region of the guard cells. Shaw and
MacLachlan, (129) who carried out this work, calculated that the rate
of photosynthesis per guard cell was in the region of 0·0210·025 x 10- 12 moles CO2 per hour. From camera lucida drawings of
stomata in cross section and surface view, guard cell volume was
found to be 6·5 x 10-9 em', and assuming a vacuolar volume 20 per
72
,
.'Q'
"
",
"
" ':, 'I
'
... ,-,::. "', '1
"
"
,
•
.,0
\~
(a)
,
"I'
"
....
' • . • '. I
M
{
i'
~
(b)
, ~ ".', (~)i
• . ,. _'_~2' ;~~: . ,: -,-'j,,' .••-_.~ .~:
~
.1
(d) :
"
-.~.
,',
II"
~\
(e)1 ; "i,)
-
-,
'
,'.
Fig. 4.3 (a) Autoradiograph of onion epidermis after three hours in radioactive carbon dioxice (14C0 2) in light. (b) autoradiograph of onion epidermis
after three hours in 14C02 in darkness, (c-f) autoradiographs of sil1g1e stomata
of onion, barley, Sedum and rradescantia, respectively, after three hours in
14C0 2 in light. (After Shaw and Maclachlan. Reproduced by permission of the
National Research Council of Canada from the Canadian Journal of Botany,
32,784--94,1954)
STOMATAL RESPONSES TO ENVIRONMENTAL FACfORS
cent of this, and that all carbon was converted to hexose, they deduced
that the rate of photosynthesis per guard cell corresponded to an
increase in osmotic potential of up toO'34 bar in three hours. Sayre(122)
found in a different species (Rumex patientia) an increase of about
six bar in osmotic pressure in open, compared with closed, stomata
(see Table 1.4). Supposing the correctness of Shaw and MacLachlan's
assumptions, the measured rate of fixation would not account for
such an increase. Even if vacuolar volume were slightly overestimated
and the products of photosynthesis were not hexoses but two-carbon
compounds (e.g., glycollic acid CHzOH. COOH), the increase in
osmotic pressure measured by Sayre would not be attained. Furthermore, stomata can, under favourable circumstances, open to more or
less full aperture in less than an hour (Figs 4.1 and 4.2) whereas Shaw
and MacLachlan's calculations were based on the fixation of CO 2
over three hours.
Thus, it seems that the guard cell chloroplasts do not carry out
sufficient photosynthesis to produce the fairly rapid osmotic change
that would be necessary to bring about the observed increase in
guard cell turgor in stomatal opening. Of course, the possibility that
photosynthetic products playa part other than as osmotically active
solutes cannot be ruled out by the above considerations.
The role of carbon dioxide. In 1932, Scarth suggested the possibility
that light-dark responses of stomata might be entirely due to photosynthetic removal and respiratory production of COz inside the leaf.
This was an inspired guess rather than a deduction from experimental
evidence, but a few years later the necessary evidence was forthcoming. Freudenberger(Z3) tested the effect on stomata of different
COz concentrations over a range within physiological levels (that is,
around 0·03 per cent). The results showed clearly that increases in concentration caused stomatal closure, and decreases caused opening.
This finding applied to illuminated as well as to darkened leaves,
and to etiolated leaves (in which chlorophyll could not be detected by
fluorescence microscopy) as well as to normal leaves. Similar work
and more detailed studies were carried out by Heath and his colleagues(37,39,48,53) who finally established the close relationship
between CO 2 concentration and stomatal aperture. Figure 4.4 is a
three-dimensional graph drawn on isometric paper, showing theresults
of an experiment carried out on wheat in which the relationship
between stomatal aperture, rate of air flow, and carbon-dioxide
concentration was observed in a light intensity of 10,000. lux.
73
PHYSIOLOGY OF STOMATA
The air flow was directed across both surfaces 'of the leaf. Note that
increased CO 2 concentration caused stomatal closure (shown as an
increased resistance) as also did increased rate of air flow. The latter
probably led to closure because of the increased supply of CO 2, and
perhaps because of mild water stress imposed by a greater rate of
Fig. 4.4
Opening of wheat stomata as affected by rate of air flow and CO 2
concentration. Light intensity 10,000 lux. Ordinate scale is 'Jog e resistance'
measured with a parameter (a higher value indicates stomatal closure). (After
Heath and Milthorpe(48)
transpiration. Heath and Russell carried out a more complex experiment in which, in an attempt to regulate the CO2 concentration in the
sub-stomatal cavity, they forced air through the leaf as well as across
the surface. The relationship they found between stomatal aperture,
CO 2 concentration, and light intensity is shown in Fig. 4.5. The
important point to notice here is that aperture was determined by
both light intensity and CO 2 concentration: the higher the light
intensity the higher the concentration required to produce a given
degree of stomatal closure. This would be explained by a greater CO 2
consumption in photosynthesis at higher light intensity, so that a
greater external concentration was required to maintain a given
concentration inside the cells. Many other workers have since confirmed the close relationship between CO 2 concentration and stomatal
aperture. The work of Gaastra (25) showed particularly well how
transpiration could be affected by changes in CO2 concentration
(Fig. 4.6). A most important finding, which is evident from the data
74
STOMATAL RESPONSES TO ENVIRONMENTAL FACfORS
0
~
I
Z
~
~
.~
~
~
-!!
~
""'0
0q.,
"''''a<>c.
rpa
e-..,.......
~'" ~
~
-q..,,>
0. ""
0:11...;:.
C''''1:::J
O?)!,.
Q":
-Do:
0
Fig. 4.5 Opening of wheat stomata as affected by light intensity and CO 2
concentration. As in Fig. 4.4, a higher value on the ordinate indicates stomatal
closure. (After Heath and Russell(53»
of Figs 4.5 and 4.6, is that C02~free air prevented complete closure in
the dark. Many other workers have confirmed that stomata respond
to changes in CO 2 concentration in the dark as well as in the light.
However, it has been found that stomata that are closed completely
0,042%
0·127%
0·031%
o 60
120
180
240 300
minutes
360
420
480
Fig.4.6 Transpiration in turnip as affected by changes in CO 2 concentration.
light intensity 1·6'5 J m-2 $-1, (After Gaastra(25»
75
PHYSIOLOGY OF STOMATA
in the dark do not open if air surrounding the leaf is devoid of
CO2 , (39.125) This shows that a reduction of the concentration in the
sub-stomatal cavity rather than on the outer surfaces of the guard
cells is necessary in order to produce opening. Probably, the difference
in thickness and permeability of inner and outer walls of the guard
cells plays a part here.
The stomata of most plants appear to be insensitive to a reduction
in CO 2 concentration below about 0·01 per cent, as will be evident
from a consideration of Figs 4.4 to 4.6. In the leaves of most species,
the CO 2 concentration in the intercellular atmosphere does not fall
below about 0·01 per cent when the surrounding air contains the
normal 0·03 per cent. This is the case even in quite high light intensity.
Consequently, the lack of further stomatal opening between 0·01 per
cent and zero CO 2 is not entirely surprising, since it is below the range
encountered in nature. Zea mays, however, is unusual in that it can
utilize CO 2 from the intercellular atmosphere even when the concentration is near zero, and it is of interest that the stomata of this
species are sensitive to changes in CO2 concentration right down to
zero.(80)
Although much research has nOw been done on the responses of
stomata to changing CO2 concentration in the physiological range
little is known about the effect of concentrations above this range.
Pallas(104) carried out measurements of trans)Jiration in CO 2 concentrations up to 0·4 per cent. Transpiration rate declined rapidly as the
concentration increased from zero to 0·08 per cent, but in two of the
species tested it increased slightly between 0·2 and 0·4 per cent.
Further investigation of this phenomenon is desirable. As well as
being of physiological interest it is of practical importance in view of
the increasing use of CO 2 enrichment in horticulture and agriculture
(see chapter 3).
With the possible exception of this work by Pallas, the evidence
relating to the COz-control mechanism is against the old idea of
osmotically active assimilates being important since, according to
that theory, one would expect higher CO2 concentrations to be
favourable for opening, as is increased light intensity. On the other
hand, the suggestion of Scarth that stomatal movements in nature
might be brought about solely by changes in CO2 concentration
produced by photosynthesis and respiration is not contradicted.
There is, however, other evidence relevant to this question, which we
shall consider in the next section.
76
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
Light effects independent of carbon dioxide concentration. The study
ofthe relative efficiency of different wavelengths in producing opening
is one line of approach that has been considered relevant to the
question of whether or not photosynthesis participates in the stomatal
light response. Here, the early investigators ran into difficulty, for it
is not easy to obtain light of different wavelengths of sufficient purity
for experimental purposes, and measurement of the'energy of irradiation is impossible without elaborate instrumentation. In view of the
inadequate facilities they had available it is not at all surprising that
contradictory resu'ts were obtained by different workers. Darwin,(19)
for example, found red light efficient in producing opening, and blue
hardly effective at all; Paetz(102) found red light about twice as
efficient as blue, but then Sierp(I3I) and Harms(34) found blue more
efficient than red.
If we turn to more recent work, where the techniques were more
refined, there is general agreement that, on a quantum basis, blue
light is considerably more effective than red. Mouravieff(97) found
that the energy required for producing 'full opening' in Veronica
beccabunga was seventeen times as great at 660 run (red) as at 464 nm
(blue).' Kuiper(65) published an action spectrum for stomatal
opening in epidermal strips of Senecio odoris showing that an intensity
of 1·14 nano Einstein cm-2 S-I produced apertures of about 8·51'
in the blue region, and only 4'51' in the red (Fig. 4.7). Mansfield and
Meidner(7B) compared opening in Xanlhium pennsylvanicum in
different wavebands obtained with broad band interference filters,
and obtained results very similar to those of Kuiper.
If stomatal opening depended only upon a reduction of CO 2
concentration brought about by photosynthesis, then red and blue
light, which are the main parts of the spectrum utilized in photosynthesis, should not differ greatly in the efficiency with which they
produce opening. The observations of considerably greater opening
in blue light appeared to suggest a response not dependent upon CO 2
concentration. A reaction to light independent of CO, concentration
* When intensity is quoted in energy units, allowance has to be made for
the fact that the energy content per quantum (which is the relevant unit for
photochemical reactions) is greater at shorter wavelengths. The energy of
irradiation in blue light needs to be about 1·4 times that in red for a comparable quantum supply. The unit used by Kuiper (mma Einstein cm- 2 S-I)
requires no such correction. For blue light of 439 nm,l nE == 2.7 Jm- 2 s- 1•
and for red light of 679 nrn, 1 nE" 1.8 J rn- 2 S-I.
77
PHYSIOLOGY OF STOMATA
had been postulated by Heath and Russell (53) to explain opening due
to increased light intensity which occurred while the intercellular
space CO2 concentration was maintained at a low level (Fig. 4.5).
Maru;field and Meidner(7S) carried out an experiment to determine
whether the greater opening in blue light was indeed due to a CO,.
independent reaction. Before describing the experiment it might be
helpful if we first explain what is meant by 'carbon dioxide compensation point'.
When a leaf is placed in a completely closed system and illuminated,
it cannot remove all the CO2 from the atmosphere surrounding it.
-;:
8
~
m
'5
6
""c.
'"
"j§
4
'"
2
E
~
0
400
500
600
700
wavelength (nm)
Fig.4.7 Effect of wavelength of illumination on stomatal opening in Senecio
odorjs. Intensity of irradiation 1·14 nano Einstein cm 2 S-1. (After Kuiper(65»
For example, if a leaf is placed in a transparent chamber containing
normal air (0'03 per cent CO,) and illuminated with light of, say,
10,000 lux, the CO, concentration will q_uite q_uickly be reduced to
somewhere below 0·01 per cent where it will remain constant (this
applies to the majority of species, but there are a few, for example
maize and sugar cane, which are exceptional in that they can reduce
the CO2 concentration in an enclosed space to nearly zero). The CO2compensation point is also reached if the closed system is initially
exhausted of CO" in which case there is, of course, first a net production of CO2 ,
The CO 2-compensation point is a concentration that is kept
steady by a lear under constant conditions, and net CO 2 exchange
occurs neither between the exterior of the lear and the intercellular
space system, nor between the latter and the leaf tissues as a whole
(there will be a small net CO2 uptake in the cells containing chloro78
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
plasts resulting from the fixation of CO 2 produced by respiration
in nongreen cells). Since there is no net movement of CO 2 into
or out of the leaf there can be no concentration gradient between the
external atmosphtTe and the intercellular spaces. Consequently, it
can be assumed that a measurement of the concentration external to
the leaf, at equilibrium in a closed system, represents the concentratili>n
in the intercellular atmosphere.
In the experiments in question, the closed system consisted of an
infra-red gas analyser for measuring the CO 2 concentration, joined
to an air-tight' .perspex lear chamber with a built-in parameter for
measuring stomatal aperture. In the leaf chamber were placed
detached leaves of Xanlhiurn pennsyivanicurn. The system was
initially flushed with air free of COb and the leaf was illuminated
with red and blue light.
The results of one experiment are shown in Table 4.1.
Table 4.1 Carbon dioxide compensation points and stomatal opening in
blue light of 4,55 J m- 2 S-1 and red of 3'40 J m- 2 5-'. Xanthium pennsylvanicum
Stomatal opening based
CO 2 concentration
(per cent)
blue
0·014
0·011
0·011
0·013
0·015
0·012
0·012
0·014
Means: 0·013
on square root of
conductance to viscous
flow
red
blue
red
0·014
0·011
0·011
0·011
0'014
0·013
0·015
0·012
0'013
20·9
16·6
26·2
21·4
30·1
16·6
IS'O
25·2
21·9
2'54
2'09
2'95
2'09
2'54
2·95
N5
2·19
N7
Eight different leaves were used in this experiment, and each line
across the table represents the data obtained from a single leaf. The
energies of irradiation were adjusted to supply approximately equal
numbers of quanta, since for photochemical reactions light operates
in quanta. There was some leaf to leaf variation but it was quite clear
79
PHYSIOLOGY OF STOMATA
that although the CO2-compensation point differed very little between
red and blue light, stomatal opening was very much greater in blue.
In another experiment, leaves were irradiated under a range of
intensities in red and blue, and stomatal openings and C02"compensation points were measured. In Fig. 4.8 is shown the relationship'
blue
20
red
•
o
6
4
J m- 2
8
S-l
Fig.4.8 Effect of intensity of irradiation on stomatal opening in red and blue
light in Xanthium pennsylvanicum. The experiments were in a closed system
with the CO 2 concentration established by the leaf itself. Ordinate scale is
square-root conductance measured wLth a parometer (increasing values
indicate stomatal opening). (After Mansfield and Meidner(78»
between light intensity and the degree of opening. These experiments
left little doubt about the great superiority of blue light for producing
stomatal opening. From the regression lines in Fig. 4.8 it can be seen
that the energy of red light required to produce an opening of 10 units
was about seven times that in blue, and this was in spite of the fact
that with such an energy difference red light led to a much lower CO 2compensation point.
Raschke!ll]) obtained some similar results using Zea mays, which
he kept in CO 2-free air to ensure that any effects due to CO2 Were
excluded. The energy of red light (679 nm) required to produce a
given opening was seven times that of blue (439 nm).
These experiments, together with the action spectra obtained by
other workers, provided strong evidence of a C02"independent effect
of blue light on stomata. The conclusions would be erroneous only
if CO 2 uptake in the guard cells themselves were much greater in blue
light than in red. Even if this were so, it could be safely said that the
stomata are to a large extent independent of the CO2 concentration
80
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
in the intercellular spaces; their movements are, therefore, not
entirely controlled by the photosynthetic activity within the mesophyll
tissue.
It seems quite reasonable to conclude from this evidence that both
blue light and the removal of carbon dioxide are capable of opening
stomata, and their effects are to some extent independent. A striking
feature of the results of Mansfield and Meidner was the degree of
opening produced by blue light at surprisingly low intensities. An
intensity of 1·29 J m-2 S-1 produced a readily measurable opening,
although the leaves were not able, at this low intensity, to hold a
steady CO 2 concentration below 0·03 per cent (in other words, the
intensity was below the light compensation point). This suggests that,
in nature, when the light intensity falls below the level at which
photosynthesis just balances respiration, stomatal opening will, in
part, be maintained by the shorter wavelengths in sunlight. In cloudy
weather with light intensity changing frequently, large Iluctuations
would be to some extent avoided.
The role of chlorophyll. Experiments on the effects of different CO2
concentrations on aperture in light lent little support to the idea that
soluble products of photosynthesis play an important part in increasing guard-cell turgor. Photosynthesis does, hOwever, indirectly
regulate stomatal aperture by affecting CO 2 concentration in the
guard cells. The removal of CO 2 in the dark can lead to stomatal
opening, thus simulating part of the light effect. This evidence
indicates that light absorption by chlorophyll is not an essential part
of the opening mechanism.
On the other hand, there is evidence that in the absence of chlorophyll the stomata do not open. Virgin(153) could detect no lightinduced transpiration of etiolated wheat leaves until the chlorophyll
content had reached a certain level. Wheat seedlings that had been
grown in complete darkness were illuminated with light over a wide
range of intensities from 0·8 to 64,000 lux. Mter varying periods of
illumination (one, two, three, four, five, and six hours) the light
intensity was increased to 64,000 lux to determine the opening
response of the stomata. At the two lowest intensities (0'8 and 2 lux)
little chlorophyll formation took place during six hours and the
increase in transpiration induced by exposure to 64,000 lux was small.
The highest transpiration values were obtained when the intensity of
the prolonged exposure was 10,000 lux, at which intensity chlorophyll
formation was rapid. When the prolonged exposure was to 50,000 or
81
PHYSIOLOGY OF STOMATA
64,000 lux, chlorophyll was destroyed more rapidly !han it was
formed, and the total content therefore remained low. From Fig. 4.9
it will be seen that transpiration was closely related to this pattern of
'E~-= ~g[
.
;0-
10,000 lux
'
L=~I==J~_~h-~~r=~!~~~!~==
~gr
L
1000 lux
+
L
hours
Fig. 4.9
Transpiration from etiolated wheat leaves as influenced by light of
different intensities. In each case the continuous line indicates the course of
transpiration when the plants were irradiated for seven hours under the
intensity shown. At intensities below 64,000 lux a proportion of the material
was removed at hourly intervals and placed under 64,000 lux. The transpiration
induced by this treatment is shown by the line which commences under each
small arrow. (After V(rgin(153»
chlorophyll formation; the continuous line shows the transpiration
over the six hours of constant illumination, and each short line shows
the increase in transpiration resulting from the exposure to 64,000 lux,
The remarkable steady increase in transpiration in very low light
82
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
intensity, presumably due to stomatal opening, was commented upon
earlier (p. 71). Virgin thought that the ability of the stomata to respond
to light was closely related to the chlorophyll a content of the tissue;
in the absence of chlorophyll a a transpiration increase was not
1-0
?;
'in
C
•
"
"
"
" ,,
0"-
"C
'0;
I
barley
A
",
1:10
A: normal
,,
C: albina
I I
I I
I
8: xantha
A
'"c
,
0·5
E
,,
\
'"
B
1:1
"
.;"
C
1 :1 ,
...........
~
\
,
\
0
'0 60
.~
0
400
500
A
600
wavelength ml'
"C
'f
"
.""
.c
>
50
'"
~
40
light
minutes
Fig. 4.10 Transpiration of three varieties of barley having different pigment
contents. The inset shows the absorption spectra of 75 cm 3 ether extracts:
(a) 0'550 9 of 'normal' leaves: (b) 0·540 9 of 'Xantha'leaves; (c) 1'100 9 of
'Albina' leaves. The dilution prior to spectrophotometer readings is shown on
the figure. (After Virgin(154»
detected. In subsequent work, Virgin(t,.) studied the effect oflight on
transpiration in three types of barley, (I) a normal green variety,
(2) 'xantha', a pale green variety and (3) 'albina', an almost white
variety. The absorption spectra of extracts of pigments from these
varieties are shown in Fig. 4.10, together with curves showing the
response of transpiration to light. Transpiration appeared to be
7
83
PHYSIOLOGY OF STOMATA
closely related to chlorophyll content, and in the albino no increase
was detected.
Shaw(·2') also experimented with an albino variety of barley. The
plants were not absolutely free of chlorophyll but the amount present
was minute compared with that in a normal barley. Shaw found only
1 to 2 p.g per gramme fresh weight in the albino compared with 770 p.g
in a green barley. This small amount of chlorophyll enabled the albino
to photosynthesise, but at a very low rate. The guard cells were quite
normal in appearance, having small plastids containing starch but
with no detectable chlorophyll. No photosynthesis in the guard cells
was detected by microautoradiography following exposure to
labelled CO2 , The starch in the guard cell plastids was probably
derived from the food reserves of the seed, for when these were
exhausted it disappeared. Shaw experimented extensively with this
plant and was unable to obtain stomatal opening either in light, or in
CO,-free air.
The findings of Virgin and Shaw raise two important questions:
1. Is the presence of chlorophyll necessary for stomatal opening in
response to CO2 removal? 2. Do the chloroplast pigments absorb the
energy for the CO,-independent opening reaction? Neither of these
questions can be answered satisfactorily on the evidence available.
First, it has been reported by several workers that the stomata of
normal leaves will not open in CO2-free air in darkness, if the stomata
are completely closed (see p. 76). If the stomata are only slightly open
in the dark, CO,-free air blown through the leaf will produce opening.
This suggests that a reduction in CO 2 concentration at the internal
surfaces of the guard cells is necessary to initiate the opening reaction.
Consequently, exposing the leaves of albino plants with tightly closed
stomata to CO 2-free air is not a satisfactory test of their CO2 sensitivity. Shaw exposed intact seedlings or detached leaves to CO,-free
air, but he did not report having used detached epidermis. The
exposure of detached epidermis from an albino plant to CO 2-free air
would appear to be a critical experiment; this would ensure that the
treatment affected the more permeable inner surface of the guard cells.
The second question may be considered in relation to the evidence
from action spectra, which suggests that if chloroplast pigments are
involved in the CO,-independent mechanism, then blue light is either
absorbed or utilized more efficiently than red. If the guard cell
chloroplasts take up CO2 more rapidly in light of shorter wavelengths,
the proposed CO2-independent mechanism (p. 77) might be a mis84
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
conception-the CO2 cOllcentr~tion determined within the guard cells
by their own chloroplasts could be the important factor. There is no
direct evidence against this, but indirect evidence does not support it.
In the experiments of Mansfield and Meidner (see p. 79) the opening
in blue light was enormously greater than in red when leaves were
at the COz-compensation point in a closed system, that is, when there
was no net uptake of CO 2 by the leaf as a whole. The CO2 concentra"
tion at the compensation point would be determined mainly by the
mesophyIl, and would presumably be lower than that which could
be established by guard ceIJs whose chloroplasts have a lower chloro"
phyll content. It is difficult to believe that under these conditions
there would be much photosynthetic CO2 uptake in the guard cells
at all. In the same series of experiments, blue light of an intensity
below the light compensation point produced a readily measurable
stomatal opening. The guard cells would have to fix CO 2 at a rate
greater than mesophyll cells to achieve a eoncentration below normal,
if the opening were due to reduced CO2 concentration. It seems
unlikely that they could do this, for the guard cell chloroplasts are
usually smaller and less pigmented than those of mesophyll cells. (129)
Thus, the evidence there is does not support the view that the
guard cells take in more CO2 in blue light than red, thereby pro"
ducing a greater opening through the COz-sensitive mechanism.
However, direct evidence is always better, and investigation of the
CO 2 fixation by guard cells under different wavelengths is necessary.
4.2 Transmission of an opening or closing
stimulus
Several people have reported finding that stomatal movements are
not confined to the area of leaf where an external stimulus is applied.
Scarth(l24) found stomatal opening on the white parts of variegated
leaves, particularly in the area nearest the green tissue. This might
have been due to a lowering of CO2 concentration in the intercellular
space system brought about by the activity of the green mesophyll. On
the other hand, Heath and Russell ('2) found evidence of transmission
along wheat leaves that was not so easily explained. They illuminated
a control area with white light of 2700 lux, and varied the
illumination in another area over three intensities, namely 900, 2700
and 8000 lux. The intercellular space system of both these areas
was swept with CO 2"free air throughout the experiment. There
8S
PHYSIOLOGY OF STOMATA
was a distance of 1· 7 cm between the two areas and they kept this at
8000 lux. In spite of this 'light barrie!, a change in light intensity
in one area brought about a change in stomatal aperture in the
other area (Fig. 4.11). Heath and Russell thought, in view of the
precautions they took, that transmission was unlikely to have been
through the intercellular space system. They suggested that transportation of the stimulus might have been by either an electrical or a
chemical means. Later, Kuiper(6J) worked with Pelargonium and
found that stomata in different areas could interfere with one another.
rO
J,
.E
2'·5
~ ~:
PO'01
PO'05
o-. ____________________
11
PO·02
PO'05
o
2·0'-;;~,___-.....,.-__,;_:~__;;~0
8000
2700 900
light intensity (lux)
Fig. 4.11 Light effect transmitted over a distance of 1·7 em in wheat. Continuous line: experimental area~ light intensity varied. Broken line: control
area at 2700 lux throughout. (After Heath and Russell(52»
The mechanism of transmission of the stimulus is not known.
Williams(164) obtained some evidence that heat-shock stimuli affecting
stomata could be transmitted along the phloem, and the same might,
perhaps, be true of the stimuli due to light and darkness. Zelitch(17J)
found that inhibitors of stomatal opening were present in the epidermis of leaves kept in the dark, but did not identify the substances
involved. The observations of Heath and Russell depicted in Fig. 4.11
could be explained in terms of an inhibitor produced in greater
quantity the lower the light intensity, and translocated from the
experimental to the control area.
The observations of transmission raise the possibility that the
operation of the guard cells is affected by substances produced in other
lear cells. On the other hand, there is considerable evidence that the
guard cells can function independently of the rest of the leaf-several
workers have studied stomatal movements on detached epidermis,
and Mouravieff(95) used stomata that were isolated, for his technique
killed the neighbouring epidermal cells (see p. 29).
86
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
4.3 Temperature
There are differing reports on the effects of temperature on stomatal
aperture. Some workers, for example, found that temperature had no
effect, which was surprising considering the great sensitivity of
stomata to other environmental factors. There are other reports
showing that opening may be either favoured or suppressed by a
temperature increase. There is, fortunately, a quite straightforward
explanation of these apparently conflicting findings. We have seen
that stomatal movements are greatly influenced by changes of CO2
concentration, and this was taken into account when interpreting the
responses to light. Similarly, the interaction of temperature with CO 2
concentration is important, and it provides an explanation of the
various temperature effects that have been reported.
A particularly good example of the way temperature affects
stomatal aperture through CO2 concentration is to be found in the
so-called 'midday closure'. A frequently observed pattern of behaviour
in plants growing in hot climates is wide stomatal opening in the
morning, partial or even complete closure sometime after midday,
followed by reopening in the afternoon. Loftfield(70) wrote:
'The curve for stomatal movement in the upper epidermis of
Alfalfa follows that of sunlight, except for the sharp dip commencing
at noon. The curve for sunlight shows that the day was cloudless
and totally free from haze. At 9.00 a.m. both were at 60 per cent of
maximum; at 10.00 a.m. they reached 90 per cent, at 11.00 a.m.
they were at 99 per cent; and at 12.00 noon both reached maximum.
Sunlight continued at maximum for the next hour and a half, and
was still 98 per cent at 2.00 p.m. In the meantime the ~tomata
started to close and reached total closure at 2.00 p.m. At 3.00 p.m.
they had begun to open again, and at 4.00 p.m. were 80 per cent
open, the maximum for the afternoon. This closure at 2.00 p.m.
was very puzzling. At the time it was believed that some mistake
had been made, that inadvertently a functionless leaf had been
stripped at 2.00 p.m.'
However, Loftfield's subsequent observations showed that there
was no mistake, for he found the phenomenon in many other plants
too. The observations for alfalfa quoted above are reproduced in
Fig. 4.12. Loftfield himself thought that water strain was the cause,
as did some later workers, but Nutman(101) suggested that a depression
87
PHYSIOLOGY OF STOMATA
of assimilation by high light intensity was the cause. The true nature
of the effect was not elucidated until Heath and Orchard('.) studied
the influence of temperature on the CO2-compensation point (see
p. 78). They worked with amon, coffee and Peiargonium, and in all
three, the CO 2 concentration rose with temperature over the range
10' to 35'C. However, in onion and coffee the rise was particularly
steep between 30' and 35'C (Fig. 4.13). These two species were both
known to exhibit midday closure, and Heath and Orchard put forward
100
V
90
:1
80
E 70 fIf/
'x
E 60
s···.
1\
\
'.
1/I'\.
".
'.
E
E
"E
'x
'"
E
'b
'" 50
Cl 40
c
'b
'"
A
'c
~ 30
20
10
9
10
11 noon 1
2
3
4
5
flg. 4.12 A.. Movemems of upper stomata ot aUana growing out of doors.
Broken line, B. indicates intensity of sunlight. (After Loftfield(70»
the suggestion that the cause was a temperature effect on the intercellular space CO 2 concentration. It was also shown that if the central
leaf cavity of onion was opened at the tip, and flushed with CO 2-free
air, the stomatal closure, which normally occurred in response to a
temperature increase from 25' to 35'C, was prevented. (46.84) Figure
4.14 demonstrates how stomatal opening increased progressively
with temperature if the onion leaf was swept with air of normal CO2
content (0'03 per cent); when sweeping ceased at the higher temperature, closure soon set in. Onion provides a particularly good example
of midday closure; it is presumahly because of the large amount of
non-chlorophyllous tissue present that the CO 2-compensation point
88
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
is so sensitive to temperature, and the temperature stimulation of
respiratory activity in this tissue can be looked upon as the cause of
stomatal closure. COrcompensation point is a steady-state value
c:
0
.~
~
c:
"
c:
..,"0
c5
u
atmospheric C O , -
5·5
5·0
4·5
4·0
_--onion
d>
.2 3·5
b---------coffee
O-'-'-'peJargonium
3·0
10
15
30
20
25
leaf temperature ('C)
35
Fig. 4.13 Effect of temperature on carbon dioxide compensation point.
(After Heath and Orchard(50)
5·0
~-<><>
!
d' ,._ sweeping
4·0
?
.- '. ceases
i:: j/ \--E
c..>
1.0
...-Ji:~~\. temperature
/O--~o
11·00 13·00 15·00
17·00
time
Fig.4.14 Stomatal conductance (measured as em galvanometer deflection
with a differential transpiration parameter) and lesf temperature, in an onion
leaf. The leaf cavity was swept with normal air until the time indicated. (After
Meidner and Heath<El4)
representing the balance between photosynthesis and respiration, and
the difference in Q10 of the two processes leads to a different balance
at different temperatures.
When the accumulation of CO2 in the leaf cavity of onion was
89
PHYSIOLOGY OF STOMATA
prevented, higher temperatures increased both the rate of opening
and the final aperture. In many plants the complicating CO 2 effects
of 'midday closure' do not occur, and under normal circumstances
opening is favoured by higher temperatures. In alfalfa for instance,
Loftfield(70) found that the time taken for opening to full aperture
depended on temperature. His measurements were made out of doors,
and although all were in sunlight, factors other than temperature
must have varied. Nevertheless, the relationship between temperature
and opening (Fig. 4.15) is not unlike that obtained in more recent
hours
8
\
7
6
5
4
\
\
\.
3
'\
2
\..
"I
r-.....
-
.
'C 0
10
20
30
Fig.4.15 Effect of temperature on the time taken for full stomataf opening in
Alfalfa. (After Loftfield(7O»
work under controlled conditions. Stalfelt(l·5) carri,d out a careful
study of the effect of temperature on opening in the broad bean,
Vicia jaba, and found a closely similar relationship in light of 20,000
lux, and in CO 2-free air in darkness (Fig. 4.16). Meidner and
Heath(") found a QIO of2·2 for rate of opening in onion in a low light
intensity (3000 lux) and suggested that a chemical 'dark' reaction
was involved, and this view was reinforced by StaIfelt's observations. In Xanthiurn pennsyivanicurn, wide opening could be induced in darkness by a temperature increase from 27' to 36'C
without any treatment to remove CO2 from the leaf (Fig. 4.17).
Observations such as these have led to a reappraisal of the role of light
in stomatal opening. High temperature and removal of CO 2 can
90
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
together lead to wide opening. These factors are normally experienced
by leaves in sunlight, and it is understandable that the main role
should originally have been attributed to light. However, it is now
180
u
/\
A
0 140
co;
1il
'"
'"""0
"0
....
Co
o
200
0
100
60
20
-20 5
15
25
35
45
temperature °C
temperature cC
Fig. 4.16 Effect of temperature on stomatal opening in Vicia (aba. (A) in light
of 20,000 lux; (8) in CO 2 -free air in darkness. Dots = aperture of stomata
plus width of guard cells. Circles = aperture of stomata only. Ordinates show
opening as a percentage of that at 20o e. (After SdUfelt(145»
5
"'"
4
00
3
'""
"0
"e;,"
'0
"
2
--~
o
2
3
4
time (hr)
Fig. 4.17 Continuous tine: stomatal Q(lenin(! in darkness in response to a
temperature increase from 27° to 36°C. Broken line: control leaves kept at
2JOC throughout. Each curve is the mean of observations on six leaves of
Xanthium pennsylvanicum. (After Mansfield(75)
clear that light energy as such is not necessary for the opening reaction
to occur, but light may playa specific role in the CO 2-independent
opening reaction (see previous section), and its effect in determining
the phase of endogenous rhythms is of major importance (chapter 5).
91
PHYSIOLOGY OF STOMATA
4.4 Water supply
,
Stomatal movements are greatly affected by water supply to the plant.
The way in which aperture is related to hydration is by no means
simple, in fact it is known that several different mechanisms are
involved. Stalfelt(142) has distinguished passive and active movements,
the distinction being that the former are detlrmined by forces outside
the guard cells, and the latter by the participation of the guard cells
themselves. Evidence of the general usefulness of these terms is
provided by the number of other workers who have since adopted
them. It is, however, unfortunate that the term 'active' is also used in
another context, namely in describing a process that is dependent
upon metabolic energy. Present evidence does not enable us to assert
that all stomatal movements for which the guard cells themselves are
responsible are dependent on metabolic energy, indeed there is good
reason for believing that this is not so-for example, stomatal closure
in darkness is not impeded by the absence of oxygen (see chapter 6).
In view of the wide acceptance of Stalfelt's terminology we shall
continue to make use of his distinction between active and passive
movements for the discussion in this section, and the words will be
printed in italics. At a later stage in the book it will be necessary to use
the term 'active' in the other sense, to refer to a process dependent on
metabolic energy.
Heath(35) found that puncturing a subsidiary celi with a microneedle caused a sudden further opening of the pore on that side of the
stoma. This was a passive movement. Opening of the stoma was
normally restricted by the pressure exerted by the turgid subsidiary
ceIl. (See experiment, p. 153.)
Any change in the pressure exerted by the epidermis can cause a
passive movement of the stomata. For example, rapid water loss from
the mesophyll reduces the turgor of the epidermal celIs before the
guard cells are affected so that when a turgid leaf is excised from the
plant, the stomata open widely for a few minutes before the guard
cells also lose turgor and closure sets in (see experiment, p. 157). (There
is no direct connection between guard cells and mesophyll cells, see
chapter 1.) If the leaf is already under water stress at the time of
excision, then the temporary opening is not observed (Fig. 4.18).
This type of passive opening is usually explained by assuming that
the guard cells are at the end of a chain of cells and, therefore, are the
last to be affected: after excision of the leaf, evaporation from the
92
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS.
mesophyll cells into the intercellular spaces continues, but the water
supply from the xylem has ceased and so the water content of the cells
decreases; the mesophyll cells then withdraw water from the epidermal cells, and then from the subsidiary cells, and the release of
pressure allows the guard cells to bulge outwards, opening the pores.
Closure occurs when the water content of the subsidiary cells
decreases sufficiently to cause water to move out of the guard cells.
0·81-----===~;;]
/0-°-<::>
...............4;"1:' ..
v
t
"uc:
O'H--
4··..6···
p"
,4'"
/
/
I'
-<>-"-1
/
I
I
~
tl
.~
leaf excision \
O~
/
I
I
I'
\
\
~
\
\
I
I
I
\
\
o
"
I
?
\/
20
,
40
60
time (minutes)
Fig.4.18
Changes in stomatal aperture brought about by wilting (after leaf
excision) in leaves of Vicia faba. The leaves had different water deficits prior
to excision: 0 = 0·2 per cent; • = 10·7 per cent; 6. = 20 per cent. (After
Willis, Yemm, and Balasubramaniam(168»
At the moment of excision of a transpiring leaf, there is a sudden
release of tension in the xylem sap, and the water remaining within
the xylem will be more freely available to the leaf. For a short time
therefore, the leaf should actually gain in turgor, and the epidermal
cells should present an increased resistance to the guard cells, causing
stomatal closure. Meidner (81) looked for this effect and found that
during the first few seconds after excision, a very small passive
closing movement of the stomata did indeed occur (Fig. 4.19).
Only after some delay did passive opening of the type shown in Fig.
4.18 take place.
93
PHYSIOLOGY OF STOMATA
Stomatal opening is, therefore, closely dependent upon the hydrostatic pressure within the epidermis. When the epidermis is fully
turgid, the guard cells encounter resistance, which prevents them from
opening to the fullest extent. According to StillfeJtl 1421 there is then a
suboptimal water deficit. Stalfelt also found an optimal deficit, when
opening in light met little or no resistance from the epidermis, and a
supraoptimaJ deficit which Jed to partial or complete stomatal closure.
2'5
Isecond"'\.
a:
I
!closing ~
fmovem~nt
________ ~ __ L_
~~oLl..---·-100---·-J
dS~~. sec.
1-5
e ay first
closing
movement
0·0
seconds
opening
movement
60
120
180
seconds
Fig.4.19
Changes in stomatal aperture following leaf excision in Phaseo/us
vulgaris. (After Meidner(81»
This was termed hydroactive closure, for it appeared that the guard
cells played an active part in producing it. Stalfelt found that with a
supraoptimal deficit the stomata became less sensitive to light; that
is, they opened less widely in a given light intensity. Unpublished
work by Z. Glinka confirms the existence of an optimal water deficit,
although it is not a single point but a range of deficit values, i.e., there
is quite a broad plateau.
Subsequent work has shown that there may be more than one
factor involved in hydroactive closure. First, it has been found that
the carbon dioxide compensation point (see p. 78) increases in leaves
with a high water deficit. Heath and Meidner{471 treated detached
leaves of wheat to produce different water deficits by immersing the
leaf sheaths in 0'2 M and 0·4 M mannitol. The carbon dioxide compensation point was normally about 80 ppm in a light intensity of
9000 lux at 2ye but increased to 94 ppm and II 6 ppm due to
0·2 M and 0·4 M mannitol respectively. 0·4 M mannitol caused complete stomatal closure, and an examination of Fig. 4.5 reveals
94
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
that such an effect would not be expected with an increase in CO 2
concentration from 80 to 116 ppm, particularly in a light intensity as
high as 9000 lux. However, it must be remembered that these
experiments were carried out under conditions for measuring the
CO 2-compensation point, when the concentration is the same both
inside and outside the leaf. With a leaf in normal air, the effect of
water stress on the intercellular space CO 2 concentration must be
greater. Nevertheless, in the experiments under discussion. the major
effect of mannitol was unlikely to have been due to increased CO2
concentration and so another explanation of hydroactive closure must
be sought. A possible one was provided by some experiments of Heath
and Mansfield.(44) It was found that the stomatal sensilivity to CO 2
increased enormously in leaves with a high water deficit. Plants in
pots were allowed to go without water, and the stomata showed the
increased CO 2 sensitivity some hours before there was visible wilting
of the leaves. As the water deficit increased, the stomata became more
closed, and the CO 2 sensitivity increased progressively. Eventually,
they were so sensitive that forcing normal air through the leaffor three
minutes caused a 50 per cent reduction in the opening as measured
by the porometer.
A mechanism for increasing sensitivity to CO 2 when water deficits
develop is clearly of great advantage to the plant, for it must assist in
the cutting down of transpirational water loss. So long as water is in
sufficient supply, wide stomatal opening permits a high rate of
photosynthesis. With decreased availability of water, the plant
reduces the stomatal aperture considerably, cutting down transpiration, and although photosynthesis is also reduced the proportional drop is probably less than that for transpiration (see chapter 3).
An increased sensitivity to CO 2 is a most suitable means for reducing
stomatal aperture, for under the most drying conditions, that is in
wind, the ambient CO 2 concentration of 0·03 per cent will be maintained near the leaf surface, and so the concentration surrounding the
guard cells will be greater.
After-effects of wilting. Stomatal closure occurs during the early
stages of water shortage, sometimes several hours before actual
wilting occurs. By the time the plant shows visible symptoms of·
distress the stomata are usually completely closed, and they remain
closed if the water shortage continues. If the water supply is suddenly
renewed, the plant may quickly regain a normal external appearance,
so long as the wilting has not been excessive. However, physiological
9S
PHYSIOWGY OF STOMATA
activities do not always recover so readily, and the stomata in particular are known to exhibit prolonged after-effects. Iljin(59) reports
that in Centaurea orientalis only 8 per cent of the stomata retained the
ability to open although the leaves had regained full turgor. Iljin
speaks of the other stomata as being 'rendered inactive' or 'killed',
but other workers have observed a gradual recovery over a period of
days, the stomata eventually regaining the capacity to open to an
aperture as wide as that achieved before the period of water strain.
In this case they certainly cannot have been killed, and the term
'inactive' may be misleading. The failure to open over a period should
Fig. 4.20 Diagrammatic representation of stomatal behaviour during and
after wilting (continuous line). Note the ptonounced after-effect (opening
below that of the control plant, the behaviour of which is shown by the
broken line).
probably be looked upon as an essential part of a protective mechanism against drought; the stomata therefore, far from being inactive,
are displaying a flexibility that enables the plant to adjust to different
environmental conditions. Figure 4.20 shOWS, diagrammatically,
stomatal behaviour in a normal mesophyte before, during, and after
water deficit (the drawing is based mainly on the observations of
lfeath and Mansfield(44) on the common dandelion, Taraxacum
officinale).
The stomatal contribution to the protective mechanism against
serious water shortage thus appears to be twofold:
1. When water deficits develop, the sensitivity to CO2 increases
greatly, which has the effect of causing stomatal closure. In
wind, the reduction of aperture will be more rapid than in still
air, and so the tendency for increased water loss under these
conditions will be prevented.
96
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
2. When the water supply is renewed, the stomata do not im-
mediately open to their full aperture. This guards against the
rapid consumption of the uew supply.
From the point of view of the mesophyte which may experience
arid conditions over short periods ouly, this method of protectiou is
particularly advantageous. It is certainly preferable to possessing
xerophytic features, which normally reduce surface to volume ratio
and, hence, reduce the efficiency of the leaves as photosynthetic
organs.
Glover(2') made some interesting observations of the effects of
drought on stomatal behaviour in maize and sorghum crops in Kenya.
In maize, the stomata closed under severe drought of three or four
days' duration but recovered in two days when the water supply was
renewed. If the period of drought was longer, such as a week or more,
the after-effects on the stomata were permanent and they opened to
less than half their normal aperture, yet the leaves had completely
recovered their turgidity and, in external appearance, were quite
normal. New leaves developing after the period of drought were not
affected, and eventually there existed on the same plant two sets of
leaves, those which had been present at the time of the drought and
whose stomata opened only partially, and those of later development
with normal stomatal behaviour. From the economic point of view,
drought at an early stage of development would appear to be better,
for then a smallu proportion of the plant's leaves are permanently
affected. Glover drew attention to the practice of Mrican farmers,
who plant maize before the rainy season so that the young plants are
exposed to drought. With the onset of the rains they recover and
9roduce a reasonable cro9, whereas those 9lanted later may suffer
drought near maturity when all the leaves are affected, and the result
is more serious.
Sorghum was found to be much less sensitive to drought than maize,
and even after fourteen days of severe drought there was little aftereffect on the behaviour of the stomata. The reason for the difference
in drought sensitivity between the two species has not been
investigated.
The nature of the after-effect of drought on maize was not examined
by Glover, and it was not known whether the guard cells themselves
were affected, or whether the photosynthetic capacity of the mesophyll
was reduced, and the stomatal closure therefore brought about by an
increase in the intercellular space CO 2 concentration.
97
PHYSIOLOGY OF STOMATA
Meidner(80) examined the after-effect of water'Strain on the carbon
dioxide compensation point in maize leaves. Detached leaves were
placed with their bases in 0·25 M mannitol, or were allowed to dry out
in air, and the CO 2-compensation points were determined before,
during, and after these treatments (Table 4.2). Although there was a
significant difference in the value for the compensation point before
and after water stress, the magnitude of the increase was relatively
small. It would not be expected to lead to much reduction in stomatal
aperture, judging from the measurements of the relation between
concentration and aperture in Table 4.2.
Table 4.2
{after MeidnerCao)
A. CO 2 compensation points in maize leaves supplied with water,
and under water stress
Light intensity 60()() lux
CO, concentration (ppm)
Treatment
Water (before water stress)
4·8
22.1
0·25 M mannitol
Leaf base drf
Water (after water stress)
Least significant difference for P 0·001
23'S
8·8
~
R·8
2· 3
B. Stomatal conductance of maize leal'es at different C02
concentrations
Light intensity 60()() lux
CO 2 concentration
Stomatal conductance
30 ppm
3·48
zero
3'72
(arbitrary units)
Least significant difference for P O'()()l
~
60 ppm
3'21
0'21.
The large after-effects found by Glover may have involved other
factors in addition to small increases in CO2 -compensation point like
those observed in these experiments with detached leaves. However,
the water deficits suffered by plants in the field were undoubtedly much
greater, and so after-effects on the processes which determine CO,compensation points may also have been greater. If this were the
98
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
cause of the failure of the stomata to open fully after re-watering,
then the stomata should not be looked upon as the cause of reduced
photosynthesis.
Effects of water deficits on photosynthesis. Since stomatal movements are so sensitive to changes in CO 2 concentration, any change
brought about by high water deficits on photosynthetic rate in the
mesophyll would be expected to exert an effect on aperture. A large
amount of work has been carried out to determine the relationship
between rate of photosynthesis and hydration of the photosynthesizing
tissue. The findings of various workers have differed, but rail into two
main categories:
1. There is an optimal water deficit at which rate of photosynthesis
is higher than if the tissue is water saturated. Water deficits
greater than the optimum lead to a reduction in photosynthetic
rate (e.g., Brilliant).(l2)
2. Photosynthesis is greatest at water saturation and the rate
declines as water content falls (e.g., Slavik).(13l)
Brilliant found, in Hedera helix, that apparent assimilation rate
increased with water deficits of up to 25 per cent, and in young leaves
of Impatiens parviflora, with deficits up to 40 per cent. Higher deficits
led to a reduction in rate. Similar results were found in leaves from
which the epidermis had been removed, and, therefore, changes in
stomatal aperture could not have been responsible for the observed
effects. Heath and Meidner(47) found that the first effect of high water
deficits in wheat was to decrease the CO,-compensation point. This
was compatible with the results of Brilliant. The preliminary stomatal
opening with reduced turgor in wheat was accompanied by the
decrease in CO 2 concentration, but Heath and Meidner thought that
the latter was unlikely to have been the cause of the opening, for the
concentrations involved were within the range of 0--100 ppm, to
which wheat stomata do not respond (see Fig. 4.5). (There was no
question of the stomatal opening being the cause of the decrease in
CO,-compensation point, because when the latter is established there
is no concentration gradient across the stomata and, therefore,
stomatal diffusive resistance is of no consequence so long as it is
below infinity.)
In the alternatives (I) and (2) above, there is agreement that large
water deficits lead to a reduction in assimilation rate, and this is likely 8
~
PHYSIOLOGY OF STOMATA
to contribute towards stomatal closure. However, for the reasons
discussed above (p. 98) it seems unlikely that changes in assimilation
rate are the most important factor in the stomatal responses, indeed
they probably playa relatively minor role in the overall movements
accompanying water shortage. Quite small water deficits can often
lead to complete stomatal closure, in spite of the fact that photosynthesis is not completely suppressed until a plant has lost a major
portion of its water content. (1171
4.5
Relative humidity of the atmosphere
Although stomatal behaviour is very sensitive to the turgor relations
of the plant, it is comparatively unaffected by changes in the relative
humidity of the ambient air. Wilson(169) found that the effect of
relative humidity on stomatal aperture was very small indeed at
temperatures of lS'C and below. Even at 30'C the stomata remained
fully open in a high light intensity as the relative humidity changed in
the range 50-100 per cent. Below 50 per cent there was, however, a
reduction in aperture. Wilson's experiments were carried out in the
open, and he collected an enormous amount of data showing how
stomatal behaviourinLiguslrum and Camellia was influenced by light,
temperature, and relative huntidity of the atmosphere. He did not take
account of wind speed, and the observations were all made with
permanently attached porometer cups before the disadvantages of
these were discovered. (54) In view of this, his beautiful three-dimensional graphs must present an imperfect evaluation; nevertheless the
conclusion that the effect of humidity is ofless importance than other
factors is probably valid.
For a plant growing out of doors, it is the actual rate of transpiration
that is important, and this is influenced by wind speed as well as by
huntidity. In still air the external resistance to transpiration is high at
all huntidities (see chapter 3), but in wind the diffusion gradient
depends much more on huntidity. Stomatal closure may occur in
wind if transpiration rate increases sufficiently to cause a water deficit
in the leaf. In wind, the external diffusion resistance to CO2 intake
will be reduced, and the increased sensitivity to this CO2 , which
occurs under water deficit, will tend to close the stomata (see p. 96).
The closure observed by Wilson at lower relative humidities could,
therefore, be the result of an interaction between CO2 and the drying
power of the atmosphere.
100
STOMATAL RESPONSES TO ENVIRONMENTAL FACTORS
4.6
Interactions between environmental
factors
The usual experimental approach in stomatal physiology has been to
study environmental factors singly, without regard to interactions
with other factors. This is the simplest approach and, for preliminary
'Nork, it is the correct one, but if we are to understand fully how
stomatal behaviour is regulated by the plant's environment, more
sophisticated studies will be required to determine how factors
interact. Few such studies have yet been attempted, probably because
they require much more technical skill. The factorial experiment of
Heath and Russell (Fig. 4.5) is a line example of the rewarding results
that can be obtained.
Because of the lack of suitable information we have been obliged
to discuss factors individually, and the limitations of the available
evidence are sometimes very apparent. Nevertheless, we have seen,
for example, that the influence of temperature on the stomatal
mechanism often consists of an interaction with other factors, such
as the concentration of carbon dioxide in the leaf air-space system
(p. 87). Several other examples of interactions between different
factors will be found in the preceding pages but special emphasis must
be placed on the interactions between water supply and other factors
because, ultimately, changes in the turgor relations between epidermal
and guard cells determine the direction and rate of stomatal
movements.
Interactions between water supply and environmental factors like
light intensity must occur almost all the time in natural conditions.
The daily opening r"'pome to light in the morning and closure
towards evening must be considerably influenced by the inevitable
changes in leaf water content, and in these instances the passive
components (St!llfel!'s terminology, see p. 92) will reinforce, and thus
speed up, movements in response to light and darkness. Less common,
at least in temperate climates, will be interactions between passive and
active components where the two oppose each other. Such a situation
can Occur on hot, dry days when a fall in leaf water content enforces
partial stomatal closure in spite of a good light supply sufficient for a
rate of photosynthesis that would keep the concentration of CO 2 in
the leaf at a low level. Many more examples of important interactions
involving both internal and external factors are likely to be revealed
by future research.
101
5. The role of rhythms in
stomatal behaviour
It is only comparatively recently that rhythmic changes in the rate of
physiological processes have been thoroughly investigated. As a
result of work during the past twenty years or so it is known that
rhythms occur in many processes in plants and animals. Rhythms in
stomatal movements are among those that have been studied, and
they have been shown to be of great importance in determining the
course of stomatal behaviour.
The study of rhythms is a subject that has its own terminology, and
some of the more simple terms will need to be defined before proceeding further. The characteristics of a rhythm are defined by
reference to the amplitude and the period (Fig. 5.1). As the rhythm
progresses there is said to be a change in the phase, and the peaks and
troughs constitute precisely opposite phases. By suitable treatment a
phase-shift can be achieved (Fig. 5.2).
Stomata have been found to exhibit rhythms of considerable
amplitude with a period ofthe order of twenty-four hours. In addition,
102
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
there are fiuctuations in aperture of low amplitude, where the period
may be only a matter of minutes. Many biological processes exhibit
rhythms having a period of around twenty-four hours which persist
in constant conditions, and the term 'endogenous rhythm' is often
used in describing this behaviour. The short-term fluctuations do not
time_.,
Fig. 5.1 Diagrammatic representation of a rhythm to indicate the meaning of
the terms 'amplitude' and 'period'
time____..,
Fig.5.2 Two rhythms showing the same amplitude and period but which are
out of phase
rail into this same category but it seems appropriate to mention them
in this context, and they will be covered in a short section at the end
of the chapter.
5.1
Endogenous rhythms
Observations of the rhythmic behaviour of stomata fall into two main
categories. First, there are rhythmic changes in stomatal aperture
under constant conditions both in light and in darkness, and secondly,
there are rhythmic changes in the response to a stimulus, particularly
the rate of opening in light or closing in darkness.
103
PHYSIOLOGY OF STOMATA
•
Rhythmic changes in aperture. Sayre(12l) reported that plants of
Rumex palientia kept in continuous darkness exhibited a period of
stomatal opening on the first morning to 10-15 per cent of the full
aperture in light, and this was repeated on the second day; after that
the rhythm seemed to have died out. Maskell (79) observed rhythmic
stomatal movements in light in Prunus laurocerasus, which accompanied rhythmic fluctuations in the rate of photosynthesis. If the leaf
surface was so cut that CO 2-intake could occur independently of the
7
g> 6
'2 5
~
o 4
'0
~t,
,
12p.m.
,
12 noon
time of day
12p.m.
Fig. 5.3 Stomatal rhythm in wheat in continuous light of 20,000 lux at 25°C.
Each point represents four observations on different leaves. (After Meidner
and Mansfield(B!5»
stomata, the rhythm in photosynthesis did not occur. Maskell therefore reached the important conclusion that the stomata were the cause
of the rhythm in photosynthesis, rather than vice versa.
Apart fmm the fact that obfoervatim'$ have been made on a variety
of species, and more refined techniques have been employed, there has
been little further contribution to our knowledge of stomatal rhythms
in light since tbe work of Maskell. Indeed, from the evidence available
the possibility that the rhythms are not truly endogenous cannot be
ruled out. To establish that the source of a rhythm is endogenous, the
observations should be carried out under conditions where environmental factors such as temperature, light intensity, and CO 2 concentration are maintained constant. Unfortunately, much of the evidence
has been collected under semi-controlled conditions, which is unsatisfactory since stomata are so sensitive to small changes in the
environment. It is panicularly important to impose stringent conditions for experiments of this type.
104
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
Quite simple experiments can establish whether a rhythm is
endogenous. For example, if the period deviates from twenty-four
hours, thi, indicates that the rhythm is not caused by an environmental factor flnctuating according to time of day. Stomatal rhythms
have often proved to have a period close to twenty-four hours (Fig.
5.3), but there are cases of deviation from this (e.g., Fig. 5.4). An even
Ei12~6~1~2~6~1~2~6~1~2~6~1~2~6~12
p.m. a.m. p.m. a.m. p.m. a.m.
time of day
Fig.5.4 Stomatal rhythm in soybean in oontinuous light of 20,000 lux at 27 c C.
Each point is the mean of four observations on different plants. (After Heath
and Mansfield(44»
/.
2
p.m.
E
ECl
.c
x·ro c
E~
/
/
/:
noon
10
/;
0
o_
m",
E.~
8
'';:; c
~
6
a.m.
6
10
8
p.m.
12
2
a.m.
time of termination
of light of 1,500 lux
Fig. 5.5 Light-induced phase-shift in the rhythm in darkness in Xanthium
pennsYlvanicum. The time of night opening depended on the time of termination of illumination (1,500 lux). The broken line indicates the relationship that
would have existed if the extent of phase-shift had equalled the increase in
duration of the preceding light period; the significant deviation from this
suggests that the rhythm did progress slowly in light. (After Mansfield and
Heath(?1)
105
PHYSIOLOGY OF STOMATA
better indication that the rhythm is not imposed by the environment
is obtained if a phase-shift can be produced by a suitable treatment.
In many processes the phase is determined, for rhythms both in light
and in darkness, by the time light or darkness respectively begin. An
excellent example of this is to be found in the rhythm of CO2 output
in succulent plants.(161, 162) The time of occurrence of rhythmic
stomatal opening in the dark has been shown to depend on the
time of termination of light (Fig. 5.5), but so far as we know the
production of phase-shift in stomatal rhythms in light has not been
investigated. This is an important omission that needs to be made
good in future research. On balance, however, the evidence Seems to
favour the view that the source of rhythms in stomatal aperture is not
the environment, but that they are endogenous in nature.
Rhythmic changes in response to stimuli. Stalfelt, (146) in a most
painstaking piece of research, obtained some invaluable records
demonstrating how the width of the guard cells, as well as the width
of the stomatal pore, exhibited a rhythm in continuous darkness.
Guard cell width proved to be the more sensitive indicator of the
rhythm (Fig. 5.6). Stalfelt's work demonstrates very well how the
stomata become 'prepared' for opening during the course of the night,
and even though they may not actually open in darkness, by the
morning they have gained considerably in turgor so that they ~an
open rapidly when illuminated.
35
34
33
32
31
30
29
28
0
/I
,
0
I
I
<: 5
~ 4
t:
" 3
0.
I
0
,
0
\
0
0
\
1\
\
I
0
0
'0
I
\
"
\
<0
'm 2
~
\
\
~
'iii
\
°,
/
I
,
0'
,0
8
°'0
\
\
0,
0-0
1
0
\
4
,
~
::;
a;
<>
11
~
"
'0'"
-"
-0
.~
27
16 20
time of day
Fig. 5.S Rhythm in stomatal aperture, and total width of the guard cells on a
plant of Vicia faba kept in continuous darkness. (After St{llfelt(146))
106
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
The progress ofthe rhythm in darkness has a considerable influence
on the opening response to light. m) After three hours in the dark the
stomata were not in a state of 'preparedness' and opened only slowly,
even though the light intensity was high (20,000 lux); the curve for
opening contrasted sharply with that following a nine-hour night,
when opening was achieved rapidly (Fig. 5.7). At this stage the
stomata were sometimes found to be slightly open, although this was
not always so. After longer times in the dark, e.g., seventeen and
twenty-one hours, the stomata responded more slowly to light,
indicating the continued course of the rhythm.
C>
8
'"""
-"
6
___ 3hr
0
4
"
2
- - 9hr
-17hr
0---<> 21 hr
0.
0
~
C>
'0
o
30 60 90 120150180
time (min)
Fig. 5.7 Rate of stomatal opening in Xanthium pennsy/vanicum as affected by
the preceding length of night. Light intensity 20,000 lux, (After Mansfield and
Heath(77»
There has been no similar thorough investigation of the effect of the
progress of the rhythm on the rate of closure in darkness. However,
several authors have noted, in passing, that stomatal closure is more
rapid in the afternoon than in the morning, and it therefore seems
likely that rate of closure, like opening, is influenced by the phase of
the rhythm. It is not known, as far as we are aware, whether COzsensitivity changes rhythmically.
There have been reports that wide stomatal opening can be induced
in darkness simply by a suitable temperature increase. The degree of
opening achieved has been shown to be in1luenced by the phase of the
rhythm, (75) and it appears that a temperature increase is effective only
if applied at a time when the stomata are in the state of 'preparedness'
for opening. The difference in response to temperature after long and
short periods in the dark was enormous (Fig. 5.8).
Phase-skiji. The phase of rhythms in biological processes can often
be changed by a suitable stimulus from the environment. In plants,
107
PHYSIOLOGY OF STOMATA
the phase of rhythms in light is frequently determined by the time
illumination begins and, similarly, the phase of rhythms in darkness
depends on the time at which illumination terminates.
Studies of the stomatal rhythm in darkness have shown that its
phase is regulated, to a large extent, by the time of termination of
light, for prolonging the light period produced a phase-shift in the
rhythm.(17) It was of considerable interest that the phase-shift could
be brought about by very low-intensity light, 0·1 J m-2 S-1 being
sufficient, (72) since a response to such a low intensity makes it unlikely
that photosynthesis is the process involved. Studies with light of
~
~
5
'"~
,!;
'"c:
'c
"c.0
4
~
Z
~
3 c.
E
'0 2 J!l
"~
'0
"'"
+
0
•
2
0
3
time (hr)
Fig. 5.S
Stomatal opening in Xanthium pennsylvanicum in response to a
temperature increase from 2]0 to 36°C after four hours (open circles) and
16 hours in the dark (closed circles). (After Mansfield(75»
different wavelengths showed that tb.e red region of the spectrum w~s
the most effective, with a peak action in the region of 700 nm.
Although some non-photosynthetic effects of light have been well
established in other processes in plants, they are usually either due to
blue light (in phototropism, for example) or red light of 660 nm, when
reversal of the effect can be achieved by 730 nm. In the latter case the
photoreversible pigment 'phytochrome' is involved. The stomatal
response, however, falls between the wavelengths absorbed by the
two forms of phytochrome, and furthermore, reversal by 730 nm has
not been achieved. (72) The physiological observations give an indication of a pigment absorbing at 700 nm, but we have no information
whatever of its nature.
Synchronization throughout the plant. The rhythm affecting
stomatal behaviour seems, under normal circumstances, to be at the
!O8
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
same phase in different parts of a plant. In many of the experiments
reported by Mansfield and Heath, (77) simultaneous measurements
were made on two leaves of plants of Xanthium pennsyivanicum, and
although the leaves were of different ages the rhythm seemed to
operate similarly in both.
Comparisons of the rate of opening after lo"g and short nights
(i.e., at two phases of the rhythm) on attached and detached leaves of
Xanthium showed no significant differences in the behaviour of
detached leaves.(86) This showed that the source of the rhythm
existed independently in each leaf, rather than being dependent on a
central control mechanism in the plant. Further experiments showed
that desynchronization of the rhythm in two leaves attached to a
single plant could easily be achieved; by a suitable irradiation of one
of them a phase-shift was induced in its rhythm while the rhythm in
the other leaf continued as before.
The above information suggests that a plant growing in natural
conditions is likely to show a synchronization of stomatal rhythms in
different leaves, However, uneven illumination such as might be
produced by heavy shading of some leaves by others could perhaps
lead to some desynchronization.
5.2
Night opening of stomata
The endogenous rhythm that has been observed to operate under
experimental conditions of continuous darkness must under natural
conditions contribute towards opening in the morning. Indeed, it has
been observed that stomatal opening can begin before dawn on plants
growing out of doors. The phenomenon has usually been termed
'night opening' in the literature, but in most cases (with the exception
of succulents-see below) it would probably be better called 'predawn opening' since the stomata remain closed for most of the night
and open only in the hour or so preceding dawn. In the majority of
plants the opening in the absence of light is quite small, being only
just detectable by a sensitive parameter method (chapter 2). Figure 5.9
shows the course of stomatal behaviour on a cabbage plant (var.
Large York) that had been grown in a light regime of eight hours of
light (8.00 a.m.-4.00 p.m.) and sixteen hours of darkness. The
observations were made in constant temperature (25"C) in a growth
cabinet, where the dark period extended from 5.30 p.m. to 9.30 a.m.
It will be seen that a very small opening movement of the stomata
109
PHYSIOLOGY OF STOMATA
lx:gan about three hours before the accustomed time of illumination.
This pattern of hehaviour is probably similar to that found in most
plants. In the majority of mesophytes it seems unlikely that the stomata open to more than a small aperture in darkness under normal
couditions. There have been reports of much wider opening in
darkness, but in some cases at least, the plants were covered with
'black boxes' durillg the day, alld the temperature rise may, therefore,
have been quite considerable. A large temperature increase can lead
to wide opening in the dark in a species normally showing only a
small opening (see below). However, there are some plants where
night opening apparently takes p'lace to wide apertures. Loftfield (70)
7
Cl
6
..
co
'EQ> 5
C.
0
0
Q>
4
~
3
"
2
Cl
'0
....
1
6
9
12
a.m.
noon
Fig. 5.9 The course of stomatal behaviour in Brassica oleracea. Ught
1
3
6
p.m.
9
12
p.m.
3
intensity 20,000 lux: dark period from 5.30 p.m. to 9.30 a.m.; temperature
25°C. Note the small opening towards the end of the night. Our data. previously unpublished
made extensive observations on the course of stomatal movements in
potato. Stomatal closure occurred for a short period only, in the
lower light intensity towards the end of the day. During the early
part of the night, opening occurred again to full aperture and the
stomata then remained wide open until the following evening.
The results of Loftfield's extensive investigations on potato and
many other species led him to distinguish three basic patterns of
stomatal behaviour. Potato belonged to his group of plants that
normally had stomata wide open at night. The cereals constituted a
group whose stomata never opened except in light. In the third group
the stomata normally opened during the day and closed at night, but
110
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
under certain circumstances night ope'ning could OCCUf, for example
when there had been closure due to water stress during the day. As
far as we know, night opening has not been detected in cereals by
subsequent workers, but there has been little evidence as to the
validity or otherwise of Loftfield's other two categories. One of the
plants listed in the 'potato group' is cabbage (Brassica oleraeea), and
it will be apparent from Fig. 5.9 that our own findings for this species
do not agree with those of Loftfield. It would be useful if a survey
using modern techniques could be undertaken to enable Loftfield's
categories to be reassessed.
Effects of temperature on night opening. Darwin(19) reported that
stomatal opening could be induced in darkness by a high temperature
treatment. This finding has been confirmed in a recent study of night
opening in Xanthium pennsyivanicum, in which a temperature increase
at the appropriate phase of the rhythm produced wide stomatal
opening in darkness (Fig. 5.8). The occurrence of such a reaction
raises an interesting question concerning the CO 2 responses of
stomata in darkness. Increased temperature will stimulate the
respiratory production of CO2 and one would, therefore, expect
stomatal closure rather than opening. The fact that opening occurs,
suggests that the sensitivity to CO 2 becomes reduced at higher
temperatures. Whether this is so has not been determined, although
it has been shown that the stomata do retain a CO2 response at the
higher temperature (Fig. 5.10).
In Xanthium pennsylvanicum the opening response to high temperature took place only if the temperature increase occurred during the
period of night opening. (75) The reaction that produced a small night
opening in ordinary temperatures appeared to be greatly enhanced by
a sudden temperature rise. However, if the temperature increase was
given throughout the night the effect was quite different. At 15"C,
opening began after about nine hours in the dark, whereas at 35°C it
did not take place until twelve hours had elapsed. Moreover, the
degree of opening achieved was no greater at 35° than at 15"C.
Jt is not unusual for processes in plants to show quite different
responses to environmental factors at opposite phases of the endogenous rhythm. This is probably what was happening in this case. A
temperature increase at the opening phase of the rhythm in darkness
greatly enhanced the opening, but theTe was no such effect when the
treatment was given during the opposite (clqsed) phase. The temperature effect on endogenous rhythms is sometimes closely analogous to
111
PHYSIOLOGY OF STOMATA
the effect of light(163) and the same seems to be true for the stomatal
rhythm in darkness. At the time of night opening, light causes a
further opening movement, but low intensity light given throughout
6
'c'"
""'
'"
1?
c:
5
"0.0
4 1?
'0
'"II!
'C
'"
C>
3
.""
"
N
"
0.
E
2 $
1
i
0
3
2
1
4
time (hr)
Fig.5.10 Opening of Xanthium pennsylvanicum stomata in air (0'03% CO 2 )
and CO 2 -free air, in continuous darkness. Means of observations on four
leaves which were younger than those in the experiment recorded in Fig. 5.S,
hence the smaJ/er opening achieved in air. Arrow indicates the time of temperature increase from 27° to 36°C. {After Mansfield(75)
the night can actually prevent opening. This was shown by an experiment in which X. pennsylvanicum was kept for twenty hours either
in darkness or in low-intensity light (10 lux) in temperatures of 15°,
Table 5.1 Observations on Xanthium pennsy!vanicum. Number of leaves out
at eight show'fng n'lght open'lng during 20 hours of darkness or at low Intens'lty
light
29°
Darkness
7
8
10 lux
8
5
(After Mansfield and Heath(71")
8
o
6
()
22', 29' and 36'C (Table 5.1). At higher temperatures light was
effective in preventing night opening, this presumably being a phaseshift effect on the rhythm. An interesting observation was the temperature dependency of this low light effect.
112
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
5.3
Rhythms and the normal diurnal
movements of stomata
The gas exchange required by the aerial parts of a plant takes place
mainly through the stomata, and the requirements, especially those
for photosynthesis, vary according to the prevailing external condi·
tioll<. The ol'ening reaction to light is of obvious value since it
provides for opening just when CO2 intake is required for photo·
synthesis. What then is the significance of the rhythmic opening that
can occur in the absence oflight? This question is not readily answered
on present evidence. The opening due to CO 2 removal apparently
ensures that the stomata are at their widest aperture when CO 2 fixation
is most rapid. However, opening in light depends not only upon CO2
removal; there is the blue light effect to be considered as well (chapter
4), which would also ensure the occurrence of opening under condi·
tions favourable for photosynthesis. The value of possessing these
distinct opening mechanisms is not entirely clear, although it is known
that the CO 2·effect plays a special role in the protective mechanism
against water stress (chapter 4). Perhaps the value of the 'endogenous'
contribution to opening during the day is to overrule, to some extent,
the wild fluctuations in aperture that might otherwise occur in the
rapidly fluctuating light intensities in nature. It could ensure that the
stomata remain partially open when the light intensity falls, with the
beneficial effect of no undue restriction to photosynthesis when
sunlight suddenly returns.
5.4 II.fter·effects of environmental
factors on rhythms
The study of endogenous rhythms has shown that stomata are affected
by environmental factors not only at the time they are experienced,
but that, in addition, there can be after·effects. This is principally due
to phase·shift, which has been shown to be brought about by light
and which can probably result from other factors too, particularly
temperature. For the physiologist working on stomata it is clearly
important to standardize the pretreatment of plant material in any
one experiment to ensure that after·effects do not obscure the treat·
ment effects. In other branches of plant physiology too, the behaviour
of stomata should not be ignored. The practice in photoperiodic
experiments of giving long·day treatments with a period of low·
intensity light to extend the day will cause a phase.shift in the stomatal
113
PHYSIOLOGY OF STOMATA
rhythm so that opening is reduced on the succeeding day. This
difficulty can be overcome by using a short night-interruption instead
of a long-day treatment, which does not appear to influence the
subsequent behaviour of stomata.(n)
5.5
Stomatal behaviour in succulent plants
Plants that have adaptations to enable them to live in arid conditions,
such as the members of the Cactaceae and Crasslilaceae, have
low stomatal frequencies and other anatomical modifications, all
of which reduce transpiration. There is considerable evidence,
however, that reduction in stomatal numbers and changes in anatomy
are not the only adaptations, for stomatal behaviour can apparently
differ from that found in mesophytes. It has been reported for many
succulent species that the stomata open at night and close during the
day. James(60) records that such behaviour is found for cacti growing
in American deserts, although Opuntia growing in Britain showed the
normal behaviour of opening during the day and closing at night.
The most extensive investigation of stomatal behaviour in succulents was carried out by Nishida,llOO) who found night opening in
members of the Cactaceae, Crassu/aceae, and Liliaceae. Some
Crassulaceae growing under normal conditions, e.g., Bryophyllum
calycinum, Kalanchoe blossfeldiana, and Cotyledon peacockii showed
stomatal opening at night and closure during the day, where".s others,
e.g., Sedum verticillatum, showed the usual daytime opening.
Nishida found that night opening began rather slowly after sunset,
and throughout the night there was a gradual progression to a wider
aperture. After sunrise the stomata initially opened further, but after
two or three hours they closed and remained so for the rest of the day.
Many Crassulaceae are known to be capable of a large-scale
fixation of CO, at night, leading to organic-acid accumulation in the
cell vacuoles, and Nishida obtained evidence of some correlation
between organic acid content and the opening of the stomata.
S. verticil/atum, whose stomata did not open.at night, showed little
diurnal fluctuation in acidity, whereas Bryophyllum daigremontianum
showed a large accumulation of acids at night and accompanying
stomatal opening (Table 5.2).
Nishida obtained further evidence that stomatal opening and dark
acidification were correlated from experiments in which different light
intensities were given during the day: higher light intensities over the
114
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
range 3,000 to 13,000 lux during the photoperiod led to increased
acidification at night, and there was a corresponding increase in both
the duration and the magnitude of stomatal opening.
Table 5.2
Acidity {cm3 of 0·05 N NaOH required for titration with 1 cm 3 of
expressed
sap;
phenolphthalein
indicator)
and
stomatal
opening·
in
Bryophyllum daigremontianum at different times of day
Stomatal opening
(arbitrary
Acidity
porometer units)
12 noon
2-62
Zero
3 p.m.
6 p.m.
9p.m.
11 p.m.
4a.m.
1·66
2·03
3·05
3·16
8'76
1J.42
8·64
4'72
1-40
Zero
Zero
6
8
8 a.m.
10 a.m.
12 noon
3 p.m.
11
20
11
Zero
Zero
.. Read as accurately as possible from Nishida's(100) graph of stomatal
behaviour.
Nishida thought from this apparent correlation that dark fixation
was the direct cause of stomatal opening at night. It is difficult to
accept this without further evidence, for there remains considerable
doubt about which is cause and which effect. Stomata of mesophytes
are known to open in response to CO 2 removal by photosynthesis, and
it seems not unreasonable to suppose that CO 2 removal by dark
fixation could have a similar effect in succulents. Some recent work
of Ting ef al. (148) has shown that under certain temperature regimes
(e.g., 26°C during the day, 21 at night) the stomata of Kalanchoe
blossfeldiana were much more widely open during the day than at
night (Fig. 5.11). There was some opening during the night but this
did not approach the level achieved during the day, and the pattern
of stomatal behaviour resembled that of a mesophyte except that
night opening began earlier. Ting ef al. studied the behaviour under
various temperature regimes and concluded that under some, e.g.,
day 30"C, night 18°C, the stomata were more open at night than
during the day. However, this was due to closure induced by the
0
115
PHYSIOLOGY OF STOMATA
higher temperature during the day rather than to an opening at night,
as will be seen from Fig. 5.12. Closure during the day could thus be
due to a temperature effect on CO,-compensation point (see p. 88),
i.e., it might be comparable with the midday closure found in many
mesophytes. The experiments of Ting et al. do not support the view
that stomatal behaviour in succulents is vastly different from that in
mesophytes. Like Nishida, they found that the first response to light
100
90
80
70
1
"
rr:
.!!!-
60
50
40
30
20
10
08
10 12 14 16 18 20 22 24 2
4
6
8
10 12
time (hrs)
Fig. 5."
Diutna) changes in leaf resistance to water vapour Joss (indicating
changes in stomatal aperture) in the succulent Kalanchoe blossfeldiana. Note
that a higher value indicates stomatal closure. Ught and darkness shown
beneath abscissa. Temperature regime: day 26°C, night 21 CC. (After ling,
Thompson, and Dugger(148»
was an opening movement, and the first response to darkness was
closure. In mesophytes the stomata frequently open a little towards
the end of the night and partially close in the afternoon even if full
light intensity is maintained. Both effects are probably due to the
endogenous rhythm. We have elsewhere suggested that stomatal
behaviour in succulents is a simple development from this common
pattern of behaviour (see Fig. 5.13) and this view is supported by
Ting et al. who say that their results do not indicate an opening
mechanism different from the normal.
Levittl66) has tried to explain stomatal behaviour at night in
succulents in the following manner. Initially, CO2 fixation in the dark
leads to stomatal closure by decreasing guard cell pH (as was also
116
THE ROLE OF RHYTHMS IN STOMATAL BEHAVIOUR
suggested for non-succulents, see chapter 7). Eventually, however,
acids accumulate in the vacuoles to such an extent that an increase
in osmotic pressure is achieved sufficient to produce opening. The
latter is similar to a suggestion put forward by Nishida (100) who
80
~
r------------------,
60
E
"
~
ri
Relight
40
20
o
15
21
26
32
day temp CCl
Fig. 5.12 Effect of day temperature on stomatal behaviour in Kalanchoe
blossfeldiana. Night temperature was 15"C when day temperatures were
15°C, 21 cC, or 26"C; and la"e when the day temperature was 30"C. At
higher temperatures the stomata close in light, and observations of stomatal
behaviour in a temperature regime of 30° day, 18° night. would indicate an
appeuent opening at night. Stomatal behaviour was shown by observations
of lea1 resistance to water vapour transfer, and consequently a higher value
indicates closure. (After Ting, Thompson and Dugger(14S»
Fig. 5.13 Diagrammatic representation of the daily course of stomatal
behaviour in a succulent plant (continuous line) and a normal mesophyte
(broken line). (After Meidner and Mansfield(85)
produced, in support of it, evidence that the guard cells really did
fix CO, in darkness.
Evidence against this view is provided both by the work of Nishida
himself and the recent study by Ting et af. (1481 This is the observation
that the first effect of illumination in the morning is to cause further
117
PHYSIOLOGY OF STOMATA
stomatal opening. If organic acids in the vacuoles were the cause of
the opening, the stomata should close in response to light, which is
known to cause deacidification in succulents.
5.6
Short-period fluctuations
Short-period fluctuations have been found using a variety of methods
of observation, and there can be little doubt as to their reality. In 1929
Stalfelt recorded them by direct microscopic observation, and found
that the period was around twenty minutes. Later, Gregory and
Pearse()l) found them superimposed on a normal endogenous
rhythm, and from the published records the period also seems to be
about twenty minutes. More recently, Howe(58) examined oscillations
in photosynthetic activity, the cycle length of which was about thirty
minutes, and concluded that they coincided with stomatal oscillations.
Whether one was the cause of the other, or whether both were due to
a common cause, was not clear.
Stalfelt's(143) explanation of this phenomenon is that after a certain
period of wide opening a water deficit develops which causes a closing
movement. Then transpiration is reduced, the water deficit is
corrected, and stomatal opening occurs again. The sequenc~ goes on
ad infinitum.
Raschke(111) discovered some oscillations of a rather different
character. He thought the stomata were operating as an oscillating
regulator effecting a constant CO 2 supply to the leaves. Unlike those
found by many other workers, Raschke's oscillations had a period of
five Dr six minutes and were of variable amplitude. A change in the
environment, for example of light intensity, set up oscillations of
decreasing amplitude.
Very recently, Barrs and Klepper(8) have made the important
observation that certain kinds of oscillations can be stopped by cutting
off the root system and leaving the cut end of the stem in water. It is
probably the removal of the resistance to water uptake in the roots
that is responsible, and Stalfel!,s conception of the mechanism is
supported by this finding. A good test ofStalfelt's interpretation would
be to measure the period of the oscillations on plants in various
atmospheric vapour pressure deficits. When transpiration rate is
high, one would expect each cycle of the oscillations to be completed
more quickly.
liB
6. Stomatal opening and
closing reactions
Specific investigations of the processes of opening and closing are
fewer in number than studies in which stomatal apertures have been
observed at equilibrium under given conditions. Some recent investigations have shown that the mechanisms involved in opening and
closing are of particular interest. Evidence has come to light that has
led some authors to suggest that opening and closing do not depend
upon a simple reversal of the same reaction, and it has also been
suggested that there is a process involved in the production of opening
that is not necessary for its maintenance. In this chapter we shall
examine some physiological and biochemical· evidence regarding
the nature of the opening and closing mechanisms.
6.1
Sources of energy for stomatal
movements
Stomatal movement appears to be a mechanical effect depending upon
a change in the turgor difference between the guard cells and the
119
PHYSIOLOGY OF STOMATA
surrounding epidermal cells. When pressures within the epidermal
cells are relaxed, as occurs in the early stages of wilting, the stomata
open more widely until the guard cells themselves lose turgor, when
closure sets in. The existence of the initial opening provides a good
indication that mechanical pressure is exerted by the guard cells as
the stomata open, and that resistance is offered by the epidermal cells;
a lowering of this resistance leads to further opening. Heath's(JS)
studies of the effects of puncturing guard and subsidiary cells strongly
support the view that opening occurs against epidermal resistance.
Looking at the condition of the guard cells from the mechanical viewpoint, therefore, closure appears to be the 'relaxed' state; and if we
have understood Ihe mechanism aright, a supply of free energy will
be required for opening to occur.
The mechanical work represented by opening against epidermal
resistance must be provided for and the necessary energy could be the
outcome of the metabolic activities of the guard cells. Guard cells, like
other photosynthetic cells in plants, have two possible sources of ready
energy: oxidative phosphorylation, and photosynthetic phosphorylation. Oxygen is an essential requirement for the former but not for
the latter and, consequently, studies of stomatal behaviour in anaerobic conditions are of value in assessing the contribution of oxidative
phosphorylation.
Heath and Orchard(49) carried out an experiment to determine the
oxygen requirement for stomatal closure. They exposed leaves of
wheat to three environmental factors known to produce closure,
namely high COrconcentration, darkness, and low humidity, each
being given under aerobic and anaerobic conditions. Lack of oxygen
did not prevent closure under any of the three treatments, and it
significantly increased the closing tendency during the initial stages.
It thus appeared that oxidative phosphorylation was not required for
stomatal closure, and when darkness was the closing treatment, photophosphorylation could not have been involved as an alternative source
of energy. Stomatal closure, therefore, apparently takes place without
the expenditure of energy produced metabolically in the guard cells.
In the above series of experiments, Heath and Orchard included
control leaves to which no treatments were applied. In these leaves,
anaerobic conditions had relatively little effect on the aperture
maintained in light. In a more recent investigation, Walker and
Zelitch(156) found that lack of oxygen did not induce closure in
stomata that were already open in light. Anaerobic conditions did,
120
STOMATAL OPENING AND CLOSING REACTIONS
however, inhibit opening in the light, which suggests that this, unlike
stomatal closure, is an energy-requiring process.
Studies of photosynthetic phosphorylation have been intensively
carried out during the past decade and, inevitably, it has been suggested as the energy source for stomatal movements. Experiments to
determine whether or not this is so have produced uncertain results,
but one thing is certain, photophosphorylation does not play an
essential part in the reaction to CO,. This is shown by the fact that
stomatal response to CO, occurs not only in light, but in darkness as
well. However, the whole of the stomatal light response cannot be
explained in terms of opening due to CO, removal. The COr
independent reaction (chapter 4) could represent the utilization of the
additional ATP available in light, but there is little evidence in favour
of this possibility. Kuiper l65 ) found that stomata closed when epidermal strips were treated with 3-(3,4-dichlorophenyl)-I, I-dimethylurea (DCMV), which is known to inhibit the process known as noncyclic photophosphorylation in isolated chloroplasts. 14) A related
compound, 3-(4-chlorophenyl)-I,I-dimethylurea (CMU) has been
found to cause stomatal closure in intact leaves, but this was readily
reversed if the intercellular spaces were flushed with CO,-free air.(1)
This suggested that stomatal closure was not due specifically to an
inhibition of photophosphorylation, but very probably to the
accumulating CO 2 of respiration following inhibition of photosynthesis by CMU.
DCMU and CMU block non-cyclic but not cyclic photophosphcrylation in isolated chloroplasts. There is some doubt of the
existence of cyclic photophosphorylation in intact cells, although
Arnon et al. 15 ) cite evidence that it can occur. If it occurs in guard cells
then this might perhaps have been the source of energy during opening
in CMU-treated leaves. It seems unlikely, however, that cyclic
photophosphorylation is behind the CO2-independent opening
reaction, for it is favoured by far-red rather than red light, whereas
the opposite is true for stomatal opening.
There has not been general agreement with Heath and Orchard'sI49)
finding that lack of oxygen actually accelerated stomatal closure.
Walker and Zelitch(156) observed the rate of closure in air, helium,
and nitrogen, and they found only slight differences in rate, which
were in the direction of slower closure in helium and nitrogen. Their
results suggest, however, that at most there is only a small oxygenrequiring component in closure. Recently, on the other hand,
121
PHYSIOLOGY OF STOMATA
Fujino(24) has come firmly to the conclusion that both opening and
closing are energy-consuming processes. He reports a detailed study
of the role of ATP and ATPase in stomatal movements. ATPase
(adenosine triphosphatase) catalyses the reaction
A TP + H 20
-+
ADP + inorganic phosphate.
The energy released by this hydrolysis might be used for active
transport in or out of the guard celis. It has been suggested that it
participates in transport processes in animal tissues, in fact the view
has been expressed that there may be a general relationship between
ATPase action ar,d transport against a concentration gradient.(18)
Fujino attempted to measure ATPase activity in epidermal strips
in light and darkness, using a histochemical technique. Strips were
first fixed in acetone and then incubated in a medium containing
0·05 M ATP (sodium salt) and lead nitrate. After three hours the
strips were washed and then immersed in ammonium sulphide
solution. In the initial incubation, inorganic phosphate produced
from ATP hydrolysis is precipitated as lead phosphate, and the
subsequent immersion in ammonium sulphide leads to formation of
black lead sulphide. This technique revealed weak activity in guard
cells in light, but strong activity in the dark, and Fujino concluded
that loss of solutes by the guard ceils is 'by use of energy which is
released from ATP by ATPase'.
It is interesting to speculate how ATP utilization in the dark could
be linked to the CO 2 effect in causing closure. There is an ATPdependent pyruvate carboxylase that may operate in conjunction
with other enzymes in the production ofphosphoenolypyruvate. One
overall mechanism suggested by Wood and U ttcr(170) is
pyruvate + ATP + GTP
~
phosphoenolpyruvate + ADP + GDP + inorganic phosphate
(GTP, GDP ~ guanosine tri- and diphosphates)
Phosphoenolpyruvate could enter into COz-fixation reactions to
produce organic acids, which might lead to closure either by the
lowering of pH, or by some other influence. The possible participation
of carboxylation mechanisms in the CO 2 response will be discussed in
chapter 7.
The apparently conflicting observations, that ATP is most rapidly
122
STOMATAL OPENING AND CLOSING REACTIONS
hydrolysed by guard cells in the dark, and that oxygen is not
required for closure, are not irreconcilable. Consider, for example,
the situation if the following is true: (a) opening is an energy-requiring
process involving uptake of substances into the guard cells against a
concentration gradient; (b) operation of the uptake mechanism
requires relatively high pH values in the guard cells; (c) in darkness
(Le., in high [C0 2 Dan ATP-requiring CO 2-fixation process takes place
forming organic acids that reduce guard-cell pH, which inhibits
operation of the uptake mechanism.
The above would be the normal state of affairs in aerobic conditions. However, lack of oxygen would also inhibit the uptake mechanism, and consequently this, too, would cause stomatal closure. This,
of course, is highly speculative but it does accommodate many of the
experimental observations. If ATI hydrolysis is required directly for
the transport of substances out of the guard cells, then this is difficult
to reconcile with the observed effect of anaerobic conditions, whereas
a less direct involvement, as suggested above, can be accommodated.
In view oftlie mechanics of the situation, which we considered at the
beginning of this section, it would be surprising if closnre were
absolutely dependent on consumption of metabolic energy by the
guard cells, since, once the guard cells are unable to maintain their
turgor, energy for closure is immediately available in the pressure
exerted by the epidermis.
Although ATPase activity was apparently much lower in epidermal
strips that had been in light than in those that had been in darkness,
nevertheless a supply of A TP in light caused opening to a much wider
aperture, particularly if a supply of potassium ions was present in the
medium. (24) It was concluded from this that active transport of
potassium into the guard cells led to stomatal opening. This conclusion would be supported by the fact that oxygen is required for
opening, and the finding by several workers that potassium ions in
the medium enhance opening in epidermal strips. This evidence is
discussed below (p. 132).
One or two curious features about Fujino's results are not easily
explained. Why did added ATP lead to stomatal opening in the dark?
He claimed that release of energy from ATP by ATPase was required
for ioss of solutes by the guard cells. If he is correct in saying that
both opening and closing are active processes, it is by no means clear
why added ATP does not stimulate closure in conditions in which
closure normally occurs. Further, why was ATPase action not detected
123
PHYSIOWGY OF STOMATA
in the light when added ATP so clearly led to stomatal opening? One
wonders if the method of assaying ATPase led to erroneous conclusions. The method would be valid only if the inorganic phosphate
released in the hydrolysis of ATP remained in an unbound form in the
guard cells. It could, perhaps, be immediately utilized in the conversion of starch to glucose-I-phosphate; the enzyme for this conversion
has been detected in guard cells (chapters 1 and 7) and might possibly
operate in acetone-fixed tissue along with the ATP hydrolysis. The
pH of guard cells in the light is favourable for starch hydrolysis
through the action of phosphorylase. Thus, the deduction that
ATPase activity is greater in the dark than in the light might be
erroneous. The situation would be less confusing if it were!
From a theoretical point of view, the idea that active transport
might contribute to the stomatal mechanism seems quite feasible.
Accumulation of solutes against a concentration gradient has been
well demonstrated in other plant cells. For example, Bieleski(9) has
shown for phloem tissue that accumulation of phosphate can occur
against a concentration ratio of 5,000 times, and accumulation of
sucrose against a ratio of 60 times.
It can easily be observed that guard cells do accumulate solutes at
a great rate from the surrounding medium. An epidermal strip from
broad bean placed in neutral red solution very quickly shows an
intense coloration in the guard-cell vacuoles, whereas there is a much
slower uptake into epidermal cells. (105) There appears to be an
accumulation against a concentration gradient although whether
this is dependent on metabolic energy is not known (the possibility
that the stain might be removed from solution once it is in the cells
cannot be excluded). There is, however, other good evidence that
guard cells are particularly active in taking up solutes. Pallas(103)
showed that sugars could be transported along the epidermis into the
guard cells, where they accumulated as starch. The fungicide phenylmercuric acetate, which has a low mobility within the plant, accumulates most markedly in guard cells (see chapter 3). Sargent and
Blackman{1l9) found that weedkillers sprayed in solution on to leaves
were rapidly taken into guard cells, and from there they were
apparently transloc&ted to the rest of the leaf. Ectodesmata III the
outer walls of guard cells (see chapter I) are the obvious pathway for
uptake from solutions on the leaf surface.
The difference in osmotic potential between open and closed
stomata may be as high as 10 bar but it is unlikely that this is
124
STOMATAL OPENING AND CLOSING REACTIONS
achieved entirely by transfer of solutes from outside the guard cells.
There are considerable carbohydrate reserves in guard cells and, on
balance, the evidence suggests that starch _,. sugar conversion plays
at least some part in the increase of turgor (chapter 7).
Opening in anaerobic conditions. Although, as we have seen, there
is strong evidence that closure can occur in the absence of oxygen,
there is other evidence that, under some circumstances, prolonged
lack of oxygen can lead to opening in darkness. This was first observed
by Scarth et a/.(126) who thought it might explain opening at night
(which is now considered to be an expression of an endogenous
rhythm-see chapter 6). Freudenberger(2J) found that opening
occurred in COrfree air in darkness, but was not maintained
in the absence of oxygen. However, a prolonged exposure to an
oxygen-free atmosphere in darkness led to a reopening. Heath and
Orchard(49) reported one experiment in which wide opening occurred
in darkness in anaerobic conditions, although their general finding
was that lack of oxygen accelerated closure. Their 'exceptional'
result, however, confirmed that the phenomenon could occur in the
manner described by Scarth et al. and Freudenberger. Recently,
Raschke(114) has shown that even a short exposure to anaerobic
conditions can lead to further opening. In Vicia labo, stomata which
had reached a steady aperture in COrfree air in blue light opened
more widely within fifteen minutes when the air was replaced by
nitrogen. Curiously, this effect could not be obtained more than once
on the same portion of leaf.
The effects of anaerobic conditions on stomatal behaviour are so
variable that they contribute little to our understanding of the
mechanism. Since one has reason to believe that opening is energyrequiring, the achievement of wider apertures in the absence of
oxygen is surprising. One possibility is that damage to the epidermal cells occurs, their loss of turgor permitting stomatal opening.
This would be comparable with the 'passive' opening that occurs
before wilting.
Experiments with azide and other inhibitors. Attempts to determine
whether opening and closing reactions depend on the respiratory
release of energy have been made with the use of metabolic inhibitors,
particularly sodium azide (NaN J). Azide is a much-used inhibitor, but
its effects in vivo are not always easily definable. It has been shown
to be an inhibitor of terminal oxidases, i.e., enzymes catalysing the
final transfer of hydrogen from the substrate to oxygen. However,
125
PHYSIOLOGY OF STOMATA
in vivo, at higher pH values it has been found actually to stimulate
oxygen uptake, and it may act as an uncoupier of oxidative
phosphorylation. (57)
The first investigation was by Mouravieff,(94) who found that
stomatal opening in light was inhibited by azide. Later, Stalfelt(144)
presented evidence that azide prevented stomatal closure, and concluded that there is a 'non-osmotic component' in the water loss by
guard cells, meaning, presumably, that it depends upon the expenditure of respiratory energy.
This conclusion has been criticized on the ground that the high
concentration of azide (10-2 M) might have killed the subsidiary cells,
reducing their mechanical effect and so preventing closure. (42) This
is always likely to be a hazard in inhibition studies, for guard cells are
known to have a greater capacity for survival than other epidermal
cells (see chapter I). This criticism was supported by the experimental
finding that 0·01 per cent azide (less than one-sixth of the concentration used by Stalfelt) caused death of the subsidiary cells within
four hours. (94)
An attempt to resolve the difficulties raised by these earlier studies
was made by Walker and Zelitch.(156) They found that azide concentrations of 10- 4 M and above prevented opening in the light, but they
also found that a concentration of 5 x 10- 4 M prevented closure when
stomata, opened in the light, were placed in darkness. Studies of azide
effects on stomata already open and kept in continuous light proved
to be of additional interest. At a concentration of 10-4 M it caused
closure, but at both higher and lower concentrations than this the
effect was reduced, and was virtually absent at 5 x ]0-5 and 10-3 M.
They suggested that this surprising response could be explained if
there are different reactions leading to opening and closing (i.e., there
is not a simple reversal of one reaction) and if any given stomatal
aperture is determined by the balance between these reactions.
10-4 M azide was thought to inhibit the opening reaction without
affecting the closing mechanism, the net effect being closure. At higher
concentrations, both opening and closing mechanisms were com~
pletely inhibited, and the stomata remained open.
This is an ingenious explanation of the experimental observations,
but one must be cautious about accepting any implication that
closure is a mechanism requiring expenditure of energy by the guard
cells. The possibility of damage to the epidermal cells (see above)
could equally be applicable here. Perhaps the closing mechanism
i26
STOMATAL OPENING AND CLOSING REACTIONS
inhibited by higher azide concentrations is simply the ability of the
-epidermal cells to maintain their own turgor.
There are many difficulties in the application of metabolic inhibitors
in vivo, for there is often uncertainty about how many enzyme systems
are affected directly, and always about how many side-effects occur
in other processes following a blockage in one vital reaction. Any
inhibitor that disturbs the CO, balance in the leaf by stimulating or
inhibiting photosynthesis or respiration is likely to cause a change in
stomat"l aperture. Sodium azide inhibits both respiration and photosynthesis, so the effect on stomatal behaviour might well include the
result of some disturbance in CO, balance. Many substances have
been reported to cause stomatal closure, and sometimes too much
has been deduced about the stomatal mechanism from their effects.
For example, Zelitch,(173) following up the work discussed above,
argued that if a given stomatal aperture represents a balance between
opening and closing processes, then with inhibitors one should find
the same end point of stomatal aperture in light, starting either with
closed or fully open stomata. The fact that several inhibitors did just
this was considered by Zelitch to support the idea of separate processes. This deduction goes beyond what is reasonably permitted by
the nature of the evidence. One of the inhibitors was phenylmercuric
acetate, which interferes with photophosphorylation and, presumably.
with photosynthesis. It probably causes an increase in intracellular
CO, concentration and, hence,leads to stomatal closure, which might
happen without any more direct effect on the special mechanism in
the guard cells. That this is probably the case with phenylmercuric
acetate has been shown by experiments in which its effect was reversed
by CO,-free air. (76)
This deficiency in one piece of evidence cited in support does not
invalidate Walker and Zelitch's idea of separate opening and closing
processes. However, on present evidence we are unable to accept that
both require expenditure of metabolic energy by the guard cells. Of
course, if opening is active and closing is not, there is not a simple
reversal of the same mechanism, which fits in just as well with their
suggestion.
Studies with 2,4-dinitrophenol, the well-known uncoupler of
oxidative phosphorylation, are consistent with the view that opening
depends on metabolic energy. Mouravieff(9Z) found that it inhibited
the water uptake by guard cells, and Ventura(151) found that it
decreased stomatal aperture in light.
127
PHYSIOLOGY OF STOMATA
6.2
Dynamics of stomatal movements-
The closing movement. The idea that closing is an energy-requiring
process first came, in 1954, from Williams' delving into the hitherto
obscure field that he termed 'stomatal dynamics'.11.51 One of the main
pieces of evidence cited was some unpublished work of Cope, who had
studied the reversal of stomatal movement by the sudden darkening
of opening stomata or the sudden illumination of closing stomata. It
was found that, if stomata that were fully open in the light were
14
1l
12
"
U
10
8"
8
::I
"C
'>
•
6
4
2
o
-5
0
5
10
15
20 25
30 35
40
45
time (minutes)
Fig. 6.1
Effect of temperature on overshoot of the closing reaction in
'Charter" wheat. Ught intensity 45,000 lux
darkened long enough to allow the closing reaction to develop at full
rate, reillumination did not quickly arrest the movement.
We have made some observations looking for overshoot of the
closing reaction, and have confirmed that it exists. If it occurs because
closure is 'active', then overshoot might be expected to be greater at
a higher temperature. Our experiments have not shown this, but that
if anything, the converse is true (Fig. 6.1).
Gregory and Pearse!3l) gave some detailed consideration to the
shape of the curves for opening and closing: 'During closure, after the
preliminary lag period, the stomatal aperture at first decreases rapidly
128
STOMATAL OPENING AND CLOSING REACTIONS
with time, the rate of change progressively becomes slower, the whole
resembling a logarithmic decrement curve. The stomatal opening, on
the other hand, shows a curve of variation in aperture with time of
the sigmoid form.'
Some results from our own recent studies l78) have indicated that
closure is more complicated than originally thought. With the aid of
a continuously recording parameter it was found, for Xanthium
pennsy!vanicwn, that there was a noticeable discontinuity between an
initial rapid phase and a subsequent slower movement (Fig. 6.2). The
recognition of the CO 2-dependent and -independent effects of light
in producing opening (see p. 77) offered a possible explanation of
120
Fig. 6.2 Continuous record of closing in darkness in Xanthium pennsy!vani·
cum, showing initial rapid and subsequent slower stages. Arrow indicates time
of darkening. Illumination prior to darkening was 15,000 lux. (After Mansfield
and Meidner{7B»)
closure curves of this type. Since opening in light consists in part of
effects not dependent on CO, removal, closure in darkness will not
depend merely upon the accumulation of respiratory CO,. When the
leaf intercellular spaces were flushed with CO 2-free air during closure _
in darkness, the initial rapid movement still occurred, but the second
slower stage was altogether prevented (Fig. 6.3). Thus, it seemed that
the first stage was CO 2 -independent, and the second stage dependent
upon the steady accumulation of respiratory CO 2 in the leaf. Of
Course, the CO 2-effect must begin to operate during the first stage,
and the CO 2-independent effect is unlikely to end abruptly; hence a
sharp transition is not to be expected. In individual records like
Fig. 6.2 it was, however, remarkably distinct. (Fig. 6.3 shows the
means of six leaves for each treatment, and the transition, the time of
which differed a little from leaf to leaf, is somewhat obscured).
This work suggests that closure in darkness occurs due to two
129
PHYSIOLOGY OF STOMATA
distinct opening mechanisms being cut off: one ends abruptly when
the light is extinguished, the other ends more gradually as CO 2
accumulates in the leaf. The independence of the two mechanisms
leading to opening is thus reflected in the closing movement.
80
70
60
""c
50
"C
40
'"::>
lJ
C
0
"
'>
30
20
0·03% CO,
10
0
-15
0
10 20 30 40 50 60
time (min)
Fig. 6.3 Stomatal closure upon darkening (time indicated by arrow) with a
stream of air (0-03 per cent CO 2 ) or CO 2 -free air passing through the Ipaf.
Illumination prior to darkening was 15,000 lux. (After M;:msfield and
Meidner(76)
The opening movement. Like Gregory and Pearse, (31) other workers
have found that opening curves often have a sigmoid form. However,
the shape depends a great deal on the endogenous component, as was
shown in Fig. 5.7. An important suggestion concerning the nature of
the opening mechanism was made by Stalfelt as long ago as 1927.([39)
He distinguished between Spannungsphase, during which preparatory
processes occurred in the guard cells, but which did not produce any
opening of the pore, and Motorphase, during which the actual
opening occurred. Later workers(34,67, 13[) were able to distinguish
between these phases on the basis of their different light requirements.
A relatively low energy supply can apparently set in motion the
Spannungsphase but the Motorphase requires a considerably higher
light intensity.
It is unlikely that the two phases correspond to CO 2-dependent and
-independent light effects because both these are capable of producing
actual opening.
130
STOMATAL OPENING AND CLOSING REACTIONS
6.3
Distinction between the maintenance and
the production of opening
A suggestion that there is a distinction between maintenance and
production of opening came from the work of Walker and Zelitch. (156)
They found. in tobacco, an oxygen requirement for the opening
movement in response to light, but that lack of oxygen in light did not
induce closure in stomata that were already open. The total energy
requirements for the production of opening might be greater than for
its maintenance, and since photophosphorylation can go on in the
absence of oxygen this might provide enough energy to keep the
stomata open, whereas additional energy from oxidative phosphorylation might be required to produce opening, starting from the closed
condition. This is rather speculative but, unfortunately, there is little
other useful evidence to which we can turn. It is interesting that
stomata recovering from a closing treatment often overshoot quite
considerably on the reopening, before settling down again at a steady
aperture. (74) A reaction participating in the recovery process, but not
persisting to maintain opening, could perhaps explain this.
6.4 Effects of temperature on opening and
closing movements
A process depending upon metabolism, such as has been suggested is
the case with stomatal opening, should be favoured by higher temperatures. This appears to be so in the onion, for which a QIO of 2'2
for rate of opening in light of 3,000 lux has been found.(S<) Many
investigators agree that steady-state aperture is increased by temperature, but there have been few other attempts to determine the effect
on rate of movement. However, for tobacco, increased temperature
has been reported to stimulate not only the extent but also the rate
of opening, whereas the rate of closure was 'largely temperature
independent'. (173) This agrees with the findings of several observers
Who have noted that rate of closure is either not affected or is actually
slowed by higher temperatures. A good example of a reduced rate at
higher temperature was found in Xanthium pennsy/vanicum (Fig. 6.4).
A temperature coefficient below unity is most likely to occur when
the response being observed is the outcome of two opposing processes
that have different temperature coefficients. The idea of 'opposing
processes' in stomatal movements, which has received support from
10
I3l
PHYSIOLOGY OF STOMATA
several writers, is further supported by the observed temperature
effects on closure.
time (hr)
Fig. 6.4
Effect of temperature on rate of stomatal closure in darkness in
Xanthium pennsy/vanicum (beginning at time indicated by arrow). light
intensity prior to darkening was 15,000 lux. (After Mansfield(75»
6.5 Ion ic effects
Studies with detached epidermis have revealed that the nature of the
ions present in the medium can exert a considerable influence on
stomatal behaviour. I1jin('9) found that the presence of alkali metals
(lithium, sodium, potassium, rubidium, and caesium) stimulated
stomatal opening, whereas some of the metals of group II (of the
periodic table), magnesium, calcium, and strontium, could nullify
their activity. He wrote: 'By the use of salts, changing their concentration and the relation among the elements, one can control stomatal
movement.'
Fujino(24) has presented some results which, in many respects, agree
with I1jin's findings.,Stomata of Commelina communis did not open
when epidermal strips were placed in distilled water, but he found that
opening was enhanced if 10-2 M ATP (sodium salt) was present in the
medium, and addition of 1(IS M potassium chloride nearly trebled
the aperture achieved in ATP alone. This and other results led him
to the belief that stomatal movement is largely dependent on potassium migration in and out of the guard cclls. Microchemical tests
showed that the concentration of potassium increased as the degree
132
STOMATAL OPENING AND CLOSING REACTIONS
of opening increased. Also, like many other workers (p. 138) he found
an increased pH in the guard cells of open stomata.
Stomata on epidermal strips of CommeUna immersed in a medium
of 18·7 cm' of 1/15 M phosphate buffer plus 1-3 cm' of 1 M KCl opened
to an aperture of 9/-, in four hours, but addition of 10-4 M CaCl,
depressed opening to 3/-" and 10-' M CaCl, prevented it altogether
(Table 6.1).
Table 6.1 Effect of CeCI 2 on stomatal opening (shown in microns) on
epidermal strips of Commelina communis in light of 10,000 lux
Time (hrs)
Control (buffer + KCl)
1O-4 M CaCl,
10-' M CaCl,
0
0
0
0
1
3·0
1-1
0
2
6'2
2'2
0
3
8·4
2·2
0
4
9'0
3·1
0
(After Fujino('4»
This appears to be similar to the antagonistic effect of calcium found
by Iljin. However, unlike Iljin, Fujino found little indication of such
an effect with magnesium.
Fujino considered the possibility that calcium might have an effect
on guard cell membranes, inhibiting the penetration of potassium. In
the presence of calcium, potassium uptake did not occur and its
excretion by the guard cells was accelerated. Fujino took the view
that there are separate mechanisms for potassium uptake and loss by
the guard cells, and that the calcium effect is to accelerate the latter.
Fujino concludes from his extensive investigation that stomatal
m"''Iement h b,,,,'Ught ",oo'Ut b.., "'''
t''''''''P'',t ",f 'P"t",,,,'Um '"
and out of the guard cells. Certainly his results with epidermal strips
indicated an enbancement of opening with potassium in the medium,
and it appeared to be transported against a gradient into the guard
cells when opening occurred. It is questionable, however, whether it
accounted for the whole of the osmotic pressure increase associated
with opening, and whether, in the intact plant, potassium transport
between guard and subsidiary cells takes place to any extent. Nevertheless, the existence of a contra-gradient transport mechanism is of
great inter.est, and in the intact leaf other solutes could probably be
taken in as well as the potassium ion.
The presence or absence of stomata is known to be of significance
in the absorption of a variety of materials, from solutions applied to
"'' 'h' ",
133
PHYSIOLOGY OF STOMATA
the leaf surface. Foliar absorption of weedkillers and other substances
is of considerable practical importance, and much attention has been
paid recently to the method of uptake. It has been shown for widely
differing substances, from 2,4-dichIorophenoxyacetic acid to iron in
solution, that entry is first into the guard cells, from where translocation may take place to the rest of the leaf.(118) Thus, organic compounds as well as metallic ions can be absorbed into the guard cells,
and transferred from guard to suhsidiary cells. If active transport
contributes towards stomatal movements substances other than
potassium might well be involved.
134
7. Hypotheses old and new
New hypotheses of the stomatal mechanism have appeared at fairly
regular intervals over the years, and although evidence in favour of
them has often been scanty, so has the evidence against, the result
being that most of the hypotheses are still extant. Of the four that will
be discussed below, one is quite new, and the other three have received
support from different research workers in the last few years.
An acceptable hypothesis should explain how stomata respond to
stimuli from the environment, and the sequence of events in the guard
cells by which the stimuli are translated into the turgor changes
leading to opening and closing movements. The first two we shall
consider attempt to explain the stomatal response to changes in
carbon dioxide concentration, and the way this is linked to the
turgor change.
7.1
The glycollate hypothesis
This is the most recent of the four, having been put forward by Zelitch
in 1963.(173) It is an interesting hypothesis and has brought a little
135
PHYSIOLOGY OF STOMATA
wholesome controversy to the subject, which for a number of years
had appeared to be in an impasse.
In some of his earlier work, Zelitch had found that a-hydroxysulphonic acids, which are compounds having the general formula
R. CHOH. S03H, were inhibitors of the oxidase of glycollic acid. The
",-hydroxysulphonic acids are analogues of glycollic acid, which has
the formula H.CHOH.COOH. Later, a chance observation was
made that leaves to which the inhibitors had been supplied wilted less
rapidly than control leaves. Subsequent investigation showed that
treatment with ",-hydroxysulphonates prevented opening of the
stomata in the light, and induced closure when they were already
open, which suggested that glycollate metabolism might be of
importance in the stomatal opening process. It was known that the
relative abundance of labelled glycollate after photosynthesis of
I'C02 was greater at low than at high CO 2-concentrations, and
Zelitch suggested that if glycollate played a role in the reactions
leading to stomatal opening, then here was a key to a possible explanation of the CO2 responses of stomata.
Some tentative evidence was obtained that a supply of glycollate
could partly reverse closure produced by high COz-concentration,
which is cited in further support of the hypothesis.
Since stomata have to open against the resistance of the surrounding
epidermal cells, it seems reasonable to suppose that energy is required
for the opening movement, and for the maintenance of opening (see
chapter 6). Zelitch suggested a way in which energy available in the
form of ATP might be dependent on the supply of glycollate. He drew
attention to the following scheme for cyclic photophosphorylation
involving (out SUGl,"",,,;ive reaction<, which had been <ugge<red by
Butt and Peel.(17)
.
light
(I) NADP + ADP + Pi + H,O --> NADPH, + ATP + 102'
(ii) NADPH 2 + CHO.COOH __,.
NADP + CH 20H.COOH (glyoxylate reduction)
(iii) CH,OH.COOH + 0, -+
CHO.COOH + H,O, (glycollate oxidation)
(iv) H 20 2 __,. H 20
+ 102
light
Balance:ADP+P,-->- ATP_
• NADP = nicotinamide·adenine dinucleotide phosphate. Pi .... inor-
ganic phosphate.
136
HYPOTHESES OLD AND NEW
Reaction (i) represents non-cyclic photophosphorylation, (4) and it
was suggested that at low CO,_concentrations, less NADPH 2 would
be oxidized in photosynthetic CO 2-reduction and it would tend to
accumulate, slowing reaction (i) and, hence, the formation of A TP.
The presence of glycollate and glyoxylate would permit the operation
of reactions (ii) and (iii), which represent a glycollate-glyoxylate cycle
achieving the oxidation of NADPH 2 • ATP formed as the overall
product of (i) to (iv) would be able to participate in an energyrequiring mechanism for increasing and maintaining guard cell
turgor. The availability of ATP for this purpose might depend on a
low CO,_concentration, which increases the amount of glycollate
formed in light in green cells.
At first sight this hypothesis has many attractions, but closer
examination reveals some shortcomings, the most important of which
is its failure to explain the fact that stomata respond to CO 2-concentration changes in darkness (probably by the same mechanism as in
light). Removal of CO 2 has been shown to lead to wide opening in the
dark, (63,73,147) and in this case the energy supply clearly cannot
depend on photophosphorylation. Also, it has been shown that the
formation of glycollate is strictly light dependent.(159) Zelitch-has
suggested that perhaps the mechanism in the dark is different from
that in the light but there is no evidence that this is the case, indeed it
seems unlikely that a normal mesophyte would possess a special
mechanism for opening its stomata in the dark in response to CO2
removal, since this is something it never encounters in nature.
The main piece of evidence favouring the hypothesis is that the
",-hydroxysulphonates, which inhibit the operation of glycollate
oxidase (reaction (iii) above), cause stomatal dosure. H.owever, these
substances appear to have an inhibitory effect on photosynthetic
CO2 uptake when applied in the concentration (0'01 M) that caused
stomatal closure.(6, 87, 149, 176) Stomatal closure is probably brought
about by the increase in the intercellular-space CO2-concentration
that occurs due to the inhibition of photosynthesis, and this view is
supported by the observation that flushing the intercellular spaces
with COrfree air re-opened stomata that had closed under the
influence of an ",-hydroxysulphonate (Fig. 7.1).
If, as has been suggested,(176) it is correct that glycollate is formed
from CO 2 as an early product of photosynthesis, then the glycollate
hypothesis is not favoured by the fact that stomata open most widely
in air entirely devoid of CO 2 , Under CO,-free conditions there can,
137
PHYSIOLOGY OF STOMATA
of course, be no photosynthesis except for some refixation of the C02
of respiration.
Taking into account all the information available at present, it
appears that the glycollate hypothesis does not provide a feasible
explanation of the CO2 responses of stomata. However, it does not
necessarily follow that glycollate plays no part whatever in the
slomatal mechanism, for this compound appears to be an important
metabolite in green cells.
CO 2 -free air
6
I
Cl
""- 4
~
.
~
-"
0
0
i!!
"'"
'0
2
,
",'
,
,
+
,._.
......
'
... ,
...,
", ,
\
,,
,
2
3
4
time (h)
Fig. 7.1 Stomata on a detached leaf of Xanthium pennsvlvanicum. in the
course of opening in light of 5,000 lUX, begin to close when 1a- 2 M a:-hydroxy~
sulphonate is fed thrOugh the petioie (time of application indicated by arrow).
Flushing the leaf with CO 2 -free air completely reverses the effect of the
Inhibitor. (After Heath, Mansfield and Meidner(45)}
7.2 Dark carboxylation reactions
Before considering the possible role of dark carboxylation reactions
it will be useful to mention briefly some evidence that guard cell pH
varies with stomatal aperture.
In his consideration of the starch"" sugar hypothesis of stomatal
movement, Sayre(121.122) made observations of the pH changes in
guard-cell vacuoles because, 'the H-ion concentration of the medium
is important in the hydrolytic action of enzymes'. He used bromophenol blue as an indicator and found that the guard cells were less
acid when open than when closed. Because of the difficulty of accurate
colour comparison under the microscope he did not attempt to
measure pH precisely but, instead, studied the effect on the stomata
of immersing pieces of epidermis in various buffer solutions. He tried
138
HYPOTHESES OLD AND NEW
to find a non-toxic solution, using for the most part expressed leaf sap
adjusted to various pH values by the addition of small quantities of
dilute hydrochloric acid or sodium hydroxide. The stomata remained
closed over the ranges 3·6-4·0 and 4'6-5'0, but opened after two hours
in the solutions of pH 4·2 and 4·4.
In some· carefully controlled experiments on Zebrina pendula,
Scarth(124) observed pH changes with indicators such as methyl red,
neutral red, and bromocresol purple. He found that the natural pH
in the guard cells ranged from about 5·0 in the dark to between 6·0
and 7·4 in the light. Several other studies were subsequently carried out
with buffer solutions, but Mouravieff(93) demonstrated that in these,
as well as those of Sayre, there may have been artefacts due to toxicity,
and consequently we shall not call upon the evidence from these
experiments. The careful work of Scarth, together with the preliminary observations of Sayre does, however, appear to show that
pH changes occur in guard cells, and a correlation with the degree of
opening seems likely, the acidity increasing during the dark.
Sayre suggested that the increase in acidity in darkness in guard
cells might be due to a process of acidification similar to that in cacti,
but the discovery that dark COrfixation in succulents leads to the
production of organic acids was not made until many years later
when, in 1948, Bonner and Bonner(lO) found that organic-acid
formation in Bryophy/lum increased greatly with COrconcentration
over the range 0-0·1 per cent. Since this is the range over which
stomata are sensitive, this could be the mechanism by which small
changes in COrconcentration might bring about significant changes
in pH, which, in turn, might regulate stomatal aliening. A "hange in
CO r concen1ration of itself causes a l'H change in a solution, but,
over the range of stomatal sensitivity the magnitude of the change is
only a matter of 0, I to 0, 3 units on the l'H scale, and is insignificant.
It is now thought that the following process is responsible for the
dark fixation of C02 which occurs in green plant8;(I55)
phosphoenolpyruvate + CO2 + H 20 "" oxaloacetate + Pi
Malic and other organic acids are formed subsequently from oxaloacetate, and in succulents like Bryophyllum they accumulate in the
cell vacuoles on a massive scale, which produces a large reduction
in pH.
Walker(155) calculated that 18 molecules of CO2 could be incorporated into 12 molecules of malate, with an energy requirement of
139
PHYSIOLOGY OF STOMATA
42 A TP equivalents. If CO 2 affects stomata by regulating the rate of
phosphoenolpyruvate carboxylation and the subsequent formation
of malate, then, in darkness, the ATP required for acid accumulation
would presumably be derived from oxidative phosphorylation, and
consequently a dependence of stomatal closure on oxygen might be
expected. Contrary to this, there is evidence that stomatal closure can
proceed in the absence of oxygen (see chapter 6), and one is tempted
to reach the conclusion that the above carboxylation mechanism,
with the further conversion of oxaloacetate to malate, is not involved
in stomatal closure. This kind of deduction from physiological
evidence is Dot infallible and there is at least one circumstance in
which it might be wrong. Suppose that the turgor increase on stomatal
opening requires oxygen, while closure is simply a passive loss of
turgor. Suppose, moreover, that the opening reaction is somehow
stimulated by an increase in pH, as might occur with cessation of
organic acid formation by dark fixation. Then, under normal circumstances stomatal closure would be induced if increased CO2-concentration stimulated acid formation but, in the absence of oxygen,
water uptake by the cells would cease in any case and closure would
result from their passive loss of turgor whether or not acid formation
took place.
Carbon dioxide control over stomatal movements can be demonstrated in light as well as in darkness, but this does not preclude
phosphoenolpyruvate carboxylation from being involved. In succulents, acidification can take place in light if the supply of CO2 is
sufficient. Ranson and Thomas lllO) said that, in light, acidification
ceases because CO 2 is preferentially used in photosynthesis, and it is
only the failure of the supply at carboxylation centres that prevents
acid formation. Many workers have drawn attention to the fact that
guard-cell chloroplasts are usually smaller than those of the mesophyll and contain less chlorophyll (see p. 19). It is likely, therefore,
that photosynthesis in guard cells is a less successful competitor for
CO 2 than in normal photosynthetic tissue, and dark carboxylation
could perhaps remain active in the light even at relatively low CO 2
concentrations. Thus, one could explain the stomatal response to
small changes in CO 2-concentration in the light (see Figs 4.4 to 4.6).
The above is practically all one can say in support of this hypothesis,
for there is precious little experimental evidence to call upon. Shaw
and Maclachlan ll29 ) showed, by means of autoradiography, that
guard cells do take up CO 2 in darkness, but this may not be of especial
140
HYPOTHESES OLD AND NEW
significance since the ability to fix CO 2 in the dark in small quantities
appears to be a property of chloropbyll-containing cells generally.
The reality of the CO 2 response of stomata is unquestionable, and
if CO 2 exerts a metabolic effect on the guard cells it is most likely to
do so by entering into combination with an acceptor, which must have
a high affinity for it since the stomatal response is at such low CO 2
levels. There are several carboxylation mechanisms known in plants,
but the one involving phosphoenolpyruvate is the most likely candidate in view of its high affinity for CO 2 ,
The 'carboxylation' hypothesis is incomplete since it postulates
only upon the biochemistry of the CO 2 effect, making no attempt to
explain the mechanism of the turgor change. This difficulty has been
overcome by linking it to the starch"" sugar hypothesis, which we
shall discuss next.
7.3 The role of starch "" sugar i nterconversion
The starch"" sugar hypothesis, which was in vogue for about forty
years after its conception by Lloyd in 1908,(69) eventually fell out of
favour not because it was proved wrong, but because unambiguous
evidence in favour of it could not be obtained. It has received vigorous
renewed support recently from a number of workers. Briefly, the
hypothesis states that the turgor change for stomatal movement is
brought about by the osmotic effect of conversion of starch to sugar,
and vice versa, in the guard cells.
Guard cells are quite exceptional among leaf cells in the ability of
their chloroplasts to accumulate starch, and to hold on to it for long
periods in the dark when starvation should surely compel them to
utilize it (see p. 19). Lloyd could detect no change in the starch
content of Iris guard cells even after thirty days in the dark. Mesophyll cells, on the other hand, lose starch easily, and in many species
it disappears overnight. Pallas(I03) found that guard cells readily
accumulated starch when pieces of epidermis were floated on sugar
solutions in the dark. Tn experiments on whole plants, he noted that
starch build-up took place in the guard cells in the first few days in
the dark and 'frequently the plant body succumbed to starvation
while the guard cells were still gorged with starch'. Pallas also
showed that externally applied sugars were translocated along the
epidermis to the guard cells in the dark, and there converted into
starch. From this it seems very likely that the accumulation of
starch in guard cells is at the expense of other leaf cells.
141
PHYSIOLOGY OF STOMATA
Yin and Tung(172) found that guard ceUs previously freed of starch
were able to accumulate it when epidermal pieces were placed in a
medium containing glucose-I-phosphate, from which they deduced
that the enzyme phosphorylase was present in the guard ceUs. Phosphorylase catalyses the process known as phosphorolysis, in which
terminal glucose groups of the polysaccharide chain are successively
removed and attached to inorganic phosphate groups, forming
glucose- I -phosphate. Phosphorylase is highly specific for glucose 1-4
bonds, and the amylose component of starch, which contains linkages
only of the <X- I -4 type, may be completely converted to glucose- 1phosphate. The amylopectin component, in which <x-1-6Iinkages also
occur, is not completely degraded.
The occurrence of phosphorolysis alone in guard cells would not
account for any osmotic change because, in the formation of each
soluble glucose-I-phosphate unit, inorganic phosphate is consumed.
Further hydrolysis of glucose-I-phosphate would have to occur in
order to produce an increase in the number of soluble molecules.
Phosphorolysis has a pH-dependent equilibrium point, which fits
nicely with the observations of guard cell pH changes (p. 138). Hanes
and Maskell(33) found that with a pH change from 7·0 to 5'0, the
ratio of total inorganic phosphate to glucose-I-phosphate increased
from 3: 1. to 11: I, i.e., the equilibrium shifted in favour of starch
synthesis. Scarth(124) found guard-cell pH to be 5·0 in the dark, and
perhaps as high as 7·4 in the light, a range that would clearly affect
phosphorylase a<:tivity.
Some work of Heath(40) has sometimes been looked upon as having
disproved the starch"" sugar hypothesis. He showed that onion
Btomata exhibited a normal Jjght-dark .re'pome although they contained uo starch, but Heath himself recommended caution in using
this evidence as counting against the hypothesis. A water-soluble
fructosan might be involved in onion, the hydrolysis of this to fructose
fulfilling the same role as starcb hydrolysis in other plauts. However,
it must not be forgotten that the turgor change is controlled by the
environment. The CO 2 mechanism operating through pH changes
could control starch hydrolysis, but there is no indication that
hydrolysis of fructosans is similarly pH dependent, and onion
stomata show the normal COz-sensitivity.
Much importance has been attached to the fact that guard cell
starch content falls during the day when stomata open, and increases
at night when they close. The way in which starch content falls as
142
HYPOTHESES OLD AND NEW
opening occurs during the day is quite impressive (see, for example,
Fig. 7.2). Unfortunately, one cannot safely deduce anything in terms
of cause or effect from this evidence, for the two occurrences could be
quite independent of each other. More suitable evidence would be a
correlation between starch content and aperture when light and
darkness are given at other than the normal time of day, and when
stomatal movements are brought about by other environmental
factors. Heath(38) carried out some experiments in which he reversed
the normal light-dark sequence for Peiargonium, and measured
stomatal starch content in epidermal strips. He found no obvious
0
~
~
5
0~"\
I
4
"
0.
"
-;;;
'"
3
E
2
I
I
j'
\;,--0
.
\
t:
;;
'bA 0 \
5
!!!
4
"E"
0-00
"'"'"
'0
c
2c
/
CD
~
a;
0
"~
.c
0
3
a.m.
5
7
9
11
3
p.m.
5
7
0
£l
G.M.T.
Fig.7.2 Changes in stomatal aperture (open circles) and guard cell starch
content (closed circles) during the course of the day in Hydlocotyle vulgaris.
(After Willis and Jefferies(167»
correlation between starch content and aperture, there being no
significant difference in the amount of starch in stomata that were
open in the light and those that were closed in darkness. Heath could
only find an apparent correlation when the plants were receiving their
normal daily light-dark cycle, and it appeared that starch content may
have varied according to an endogenous rhythm which functioned
without change of phase even when darkness was given during the
day, and light at night.
On the other hand, Williams and Barratt (166) claimed to have found
some correlation between aperture and starch content from experiments involving a variety of treatments (see Fig. 7.3). Although light
per se appeared to have little effect, removal of CO 2 did, and consequently there might be an effect of light as a result of photosynthetic
143
PHYSIOLOGY OF STOMATA
removal of CO,. Mouraviejf(97} found that blue light caused hydrolysis
of stomatal starch while red did not, and as Williams and Barratt used
tungsten filament light, which is relatively rich in red and deficient in
blue, their observations might not be inconsistent with those of
Mouravieft'. Williams and Barratt deduced from their collected results
that although stomatal aperture could change without a change in
starch, a change in starch invariably resulted in a change in aperture.
If true, this could account for discrepancies between the observations
of different workers. The work of Pallas, mentioned above, shows that
o
o
o
o
o
starch content
Fig. 7.3 Correlation (or lack of it) between starch content and aperture.
Ordinate scale is iaglo resistance as measured with a porometer-inareasing
values indicate stomatal closure. (After Williams and 8a'ratt(166~)
the guard cells can acquire sugars from other leaf cells, clearly
demonstrating that a guard cell should not be regarded as a closed
system. To expect a perfect correlation between starch content and
aperture would be justified only if all the osmotically active material
came from starch, and vice versa. Since guard cells both produce
sugars by photosynthesis and consume them by respiration, this
cannot in any Case be true, but it is the acquirin~ of solutes from an
external source that would be likely to lead to the biggest departure
from the expected correlation.
Although most attention has been paid to the light effect on
stomatal starch, some workers have considered the effect of water
relations. Yemm and Willisll71) found in Chrysanthemum that the
stomatal closure occurring upon wilting was accompanied by an
144
HYPOTHESES OLD AND NEW
increase in guard cell starch content. A change from sugar to starch
leading to stomatal closure thus might playa role in the protective
mechanism against water stress (.0. 96).
It is difficult to believe, in view of all the evidence, that starch "'"
sugar changes do not make some contribution towards the opening
of stomata by increasing the osmotic pressure of the guard cells, but
this is probably only one contributory mechanism operating along
with others.
The tenacity with which guard cells retain starch in the dark must,
in some way, be connected with their specialized function. Perhaps
the energy re'luirements for stomatal opening in the light are sufficient
to merit a large store of carbohydrate within the guard cells.
7.4
Permeabilitv c/tanges
Another old hypothesis, which has also received renewed attention
recently, suggested that there might be permeability changes in
guard cell membranes in light and darkness. \62. 6~) Kuiper(64) found
that alkenylsuccinic acids greatly increased the permeability of bean
roots to water, and Zelitch(174) investigated the effect of these substances on stomatal behaviour in the light and found they prevented
opening. He suggested that they increased the permeability of the
membranes oflhe guard cells, thereby causing loss of turgor. Although
several other substances known to increase membrane permeability
cause stomata to close, and probably do so by affecting the retention
of materials by the guard ce11s,(175) this by no means indicates that
permeability changes contribute towards the normal oper.'ltiDn Df
stomata.
If changes in CO 2 concentration and/or light intensity eKert an
effect on membrane permeability, this might take part in the stomatal
mechanism, but how it would operate to advantage is not immediately
clear. An increase in permeability in the light would be advantageous
if the guard cells actively took up dissolved substances (followed by
water) from the surrounding cells, in order to increase turgor. However, closure would require a loss of these substances, and a decrease
in permeability in the dark would not be favourable to the closing
process. Moreover, an increase in permeability in the light would
make the retention of solutes more difficult, and so more energy
Would have to be expended by the guard cells.
A more obviously useful system would be a unidirectional increase
145
PHYSIOLOGY OF STOMATA
in permeability, in effect a valve. It is not inconceivable that such a
system operates, but there is no evidence whatever to support the idea.
7.5
Blue light effects
The discovery of the C0z-independent effects of blue light in producing stomatal opening (pp. 77-81) post-dates the main hypotheses,
and it will be interesting to see how those we have discussed manage
to accommodate this new information. From what is known at the
moment we can make only a few comments.
When Mouravieff(97) found that blue light was so much more
efficient than red in producing opening, he examined the possibility
that the explanation lay in the effect of light on starch hydrolysis. He
had some difficulty in trying to determine the fate of starch, because
a large amount persisted even after four or five hours in the Jight, and
when the starch content is above a certain level it is virtually impossible
to determine, visually, an increase or decrease in the amount.
Mouravieff found that if he kept leaves in the dark for several days
the guard cells lost some of their starch, and he was then able to
determine more readily the changes in content brought about by the
expeIimental treatments. In blue light he found a reduction in the
amount or even a total disappearance of starch from guard cell
chloroplasts, while at the same time the mesophyll chloroplasts grew
rich in starch. In red light, on the other hand, the guard cells and
mesophyll cells behaved in a similar manner, the chloroplasts forming
abundant starch. He thought that both the hydrolysis of starch
already present and the failure of the newly-fixed carbon to appear as
starch would contribute to the greater opening in blue light. It has
since been shown that the much wider opening in blue light still
occurred in leaves illuminated in a system initially free of CO,,(781
indicating little, if any, role for substances formed from newly-fixed
carbon. In onion, the guard cells of which do not form starch, blue
light was more effective than red for producing opening by a factor
of about three. (831 Although the difference between red and blue light
was smaller in onion than in Xantltium pennsy!vanicum, whose
stomata do contain starch, the fact that onion shows enhanced
opening in blue light might be taken as indicating that the effect is not
brought about via starch hydrolysis. However, the possibility must
remain that soluble polysacch.arides could replace the role of starch.
Raschke(1131 thought the blue light effect could be explained in
146
HYPOTHESES OLD AND NEW
terms of permeability changes. 'He found that stomata which had
been open in blue light closed more slowly during the first three to
five minutes of darkness than those which had been in red light. He
concluded that blue light reduced the permeability of guard cell
membranes more than red light. and that this effect persisted during
the first few minutes in darkness. In the absence of other evidence
this must be looked upon as purely speculative. One can think of
various other explanations-for example there might be sufficient
activated pigment for an effect to persist for a minute or two in
darkness.
The blue light effects raise the whole question of the pigment
systems operative in guard cells. The action spectra for opening, and
micro spectrophotometer measurements of light absorption in guard
cells!9') suggest that a yellow pigment is fairly abundant (see p. 84).
This might be a carotenoid, but there is no further evidence to confirm this. The relation between guard-cell pigments and stomatal
behaviour is clearly quite complex. We know that chlorophylls are
important in the photosynthetic removal of CO 2 , and along with the
yellow pigment they contribute to opening in the light. Then there is
the separate pigment absorbing mainly in the red region, which is
concerned in regulating the phase of endogenous rhythms. Mouravieff
has recently pioneered the use of microspectroscopic analysis of the
guard cell, and it is probable that more work along these lines will
prove fruitful in the future.
7.6 What next 7
The fact that different hypotheses are currently in competition
demonstrates that none of them is entirely satisfactory. In the future
we may expect to see existing hypotheses drastically modified, or
completely replaced by new oneS.
None of the hypotheses manages to explain the versatility of the
stomatal apparatus. It should be evident from the account of the
responses in chapter 4 that stomata can respond similarly to very
different stimuli and that, frequently, several of these operate simultaneously. The situation is extremely complicated and it may well be
that several aspects of different hypotheses will combine finally to
form an acceptable working hypothesis.
Stalfelt's work has shown the importance of considering the precise
turgor relations between epidermal and guard cells, rather than only
147
PHYSIOLOGY OF STOMATA
the events in the guard cells themselves. Several possibilities can be
distinguished as the immediate causes of stomatal movements:
I. Changes in the turgor of the guard cells owing to changes in the
osmotic potential of their sap (there mayor may not be an
'active' contribution, Le., expenditure of metabolic energy by
the guard cells).
2. Changes in the turgor of the epidermal cells owing to changes in
the osmotic potential of the mesophyll sap; these would include
changes following photosynthesis or other metabolic activities,
for instance those that might occur during the night when
stored starch is mobilized.
3. Changes in the osmotic values of the epidermal cells occ.urring
independently of those in the mesophyll or guard cells. In some
cases it appears that these changes may be of considerabie
importance. (1<7)
4. Changes in the turgor of the epidermal cells owing to an outside
influence, such as transpirational water loss or sudden water
inflow at a time when leaf water content is very low.
Up to now, hypotheses have referred to the stomatal mechanism,
but when just a few of the complications are considered such a simple
approach seems unlikely to lead to a final solution.
Significant further advances in the field are likely to be dependent
on the development of new techniques of investigation. The biggest
problem in stomatal physiology is the situation of the guard cells;
placed among epidermal cells of quite different character, they have
hardly been studied individually. Mouravieff has done some work
with 'isolated stomata', which were on pieces of epidermis in which
the other cells had been killed by the sectioning. This is a tedious
technique that would be unsuitable for obtaining guard cells in
sufficient quantity to enable their biochemistry to be studied.
Many chemicals have been found to close stomata, but with one
exception they are all metabolic inhibitors or substances suspected of
being toxic in some way. The exception is carbon dioxide, the role of
which appears to be of great significance. If the action of CO2 is to
regulate the rate of a carboxylation process it could be expected that
the first product of the carboxylation would also be effective in
closing stomata. No metabolite has been reported to close stomata
nearly as effectively as CO2 , although perhaps not many have been
148
HYPOTHESES OLD AND NEW
tried. Difficulties in this approach can he anticipated; for example,
other cells mis-'lt consume the metabolite if it is fed to whole leaves,
and consequently epidermal strips or isolated stomata ought to he
used. Then there is the problem of permeability-C0 2 readily gains
access to cells, but the same is not true of many intermediary
metabolites.
The question of active transport has been brought to the fore in
recent papers, and we can expect to hear more of this in the next few
years. There is now positive evidence (pp. 134, 141) of the translocation
of substances into and out of the guard cells, showing that they are not
as isolated as was once thought. The translocation of a solute might
occur against a concentration gradient with the expenditure of
respiratory energy, as we saw in chapter 6.
Fer the future, only one thing is really clear: research into the
stomatal mechanism is going to become increasingly difficult. All the
reSources of modern plant physiology are likely to be called upon,
and many specially-devised techniques besides.
149
Appendix: Some suggestions
for practical work
A.1
Observations and measurements on
epidermal strips
Selection of material. Some leaves yield epidermal strips more
readily than others and therefore we have concentrated on the most
suitable and easily obtainable plants we know. However. the use of
leaves from the local flora should be encouraged. and a survey may
lead to the discovery of material just as suitable as that mentioned
below. For all work fully turgid-leaves are the most satisfactory.
Succulent leave •. These leaves, among which we include those of
onion plants, strip the most easily and need be cut slightly on one side
only before being broken so that the cut opens up and the unharmed
epidermis on the other side peels off. Pieces of leaf about 2 em long
and 0·3 em wide can be handled comfortably and provide pieces of
epidermis about 0·3 )( 0·3 em for microscopic work. Before placing
150
SOME SUGGESTIONS FOR PRACTICAL WORK
the cover slip in position, a slight tapping with a dissecting needle
will expel adhering air bubbles from the tissue.
Non-succulent leaves from which the epidermis strips easily. The
lower epidermis of leaves of Vida Jaba, Pisum salivum, Tradescantia
virginiana and Commelina coelestis strips off easily once an oblique cut
has been made into the leaf blade. The loose wedge-shaped flap of
tissue can be grasped with a pointed, sharp-edged pair of tweezers in'
order to pull off a strip of epidermis about 0·1 to 0·2 em wide and
0·3 em long.
More difficult leaves. Leaves of grasses and needles of conifers can
be bent over the index finger and held in position with thumb and
middle finger of one hand. With a sharp blad~, an oblique cut is made
into the epidermis. By levelling the blade, a small piece of epidermis
can be shaved off; even a piece 0·1 x 0'1 em is sufficient. but usually
slightly larger pieces can be obtained.
Leaves that can be squashed to yield strips of epidermis. Pieces of
leaves of Impatiens holsrii, or Vitia/aba, about 0·3 x 0·3 em in area,
or smaller, are placed between two microscope slides and pressure is
exerted with thumb and forefinger so that the sap issues from the cut
edges of the tissue; while the pressure is applied, the slides are moved
sideways, gliding across each other for a small distance. Either both
or one of the epidermes will be seen to adhere to the slides, which
must be separated gently while a few drops of water are run between
them. If the epidermis has become folded on the slide, it can be
flattened easily by floating on a drop of water and manipulating with
dissecting needles. The lower epidermis is usually fairly clean, and
the upper epidermis appears green on account of adhering palisade
tissue~
Frequencies, patterns, and differentiation of stomata. We reCommend the use of an epidermal strip of a green onion leaf, counting the
number of stomata in several high-power fields of the microscope,
calibrated in mm2, and drawing the pattern in which stomata appear
in this epidermis. We also recommend drawing the patterns in epidermal strips of Vida/aba, a grass blade, and a pine needle.
Epidermal strips taken from very small leaves (0'2 to 0·3 em long)
from near the growing point of Impatiens holstii, prepared by squashing as outlined above, and from slightly larger leaves, make it possible
to distinguish about three stages in the development of stomata.
Similar observations can be made in epidermal strips from leaves of
Vicia /abo about 1·5 em long and 0'5 em wide; the strips are also
151
PHYSIOLOGY OF STOMATA
prepared by squashing, and all stages of differentiation will be seen.
Counts of stomata per unit area of such small leaves should be
compared with those made on mature leaves.
The two stages in the differentiation or the graminaceous stoma can
be observed in barley and wheat, where the stomata at the tip of the
coleoptile will be found to be of the elliptical type, and those on the
blades of the graminaceous type. Intermediate stages are most likely
to be found on the base of the coleoptile.
Guard cell contents. An epidermal strip from a Sedum leaf shows
large oil drops in the guard cells and numerous chloroplasts. With a
piece of filter paper, draw a drop of iodine stain under the cover glass
and observe the characteristic colour of the starch iodine stain. When
applying stains, especially vital stains (see below), or any other
solution to epidermal strips, the temperature of the reagents and
that of the room should not be less than 20'C.
Observations on leaves of Pisum sativum or Vicia faba, some of
which have been in the light and others kept in the dark for a day or
two, will show that while the mesophyll cells ofleaves kept in the dark
are almost entirely devoid of starch, guard cells in epidermal strips
taken from the same leaves are rich in starch. It is, however,
possible that a difference in guard cell starch content between the
leaves exposed to light and those kept in the dark can be detected by
the intensity of the stain.
If it is desirable not to interfere with the state of stomatal opening
when investigating guard cell starch, Heath's reagent should be used
instead of the usual iodine stain: 2'5 g iodine are dissolved in 100 em 3
phenol (liquefied with a minimum of water) containing an excess of
potassium iodide.
Osmotic pressure of epidermal and guard cell sap. An epidermal
strip from a turgid piece of Fuchsia sepal, O' 3 em wide and 2 em long,
can be obtained in the same way as from a succulent leaf. Many of the
epidermal cells contain anthocyanin and, although there are only a
few stomata per mm', they stand out owing to their conspicuous
chloroplasts and the absence of anthocyanin. Both upper and lower
epidermes are suitable, but, depending on the variety used, the one or
the other may prove better. Stomata adjacent to coloured epidermal
cells should be selected for observation. Place portions of epidermis
for 20 to 30 minutes in watch·glasses containing water, 0'1, 0·2, ... ,
0·5 M solutions of mannitol; re-examine the portions of epidermal
strip and count the number of plasmolysed and non-plasmolysed
152
SOME SUGGESTIONS FOR PRACTICAL WORK
epidermal cells in five high power fields of the microscope for each
molarity. Plot the percentage of plasmolysed cells against the
molarities of the solutions and, from the graph obtained, determine
the molarity of the solution in which 50 per cent of cells would be
expected to be plasmolysed. Solutions of inorganic saits must not bo
used as their ions are more likely to move into the epidermal and
guard cells and, thereby, give elevated results. Continuing with more
concentrated solutions, an attempt can be made to find the concentration of solution that will just cause plasmolysis of the guard cells;
because of the absence of anthocyanin, it will not be possible to
decide clearly which cells are plasmolysed, but at a more advanced
stage of plasmolysis the chloroplasts crowd together, and this is
easily observed.
Vital staining of epidermal strips. Similar measurements can be
made with epidermal strips of Vicia faba, Commelina coelestis, or
Ranunculus bulbosus that have been stained with neutral red dye
solution (0'1 g 1-1). Plasmolysis can be observed very readily in both
epidermal and guard cells once their vacuoles have been stained.
The technique of vital staining should be practised also with strips
of epidermis from green leaves of onion. The dye is pulled under the
cover glass with the aid of some blotting paper. Many of the epidermal
cells are usually damaged by the strippi~g process and will therefore
not take up any stain. Moreover, because the damaged epidermal
cells have lost their turgor and offer no resistance to guard cell
movement, many stomata will be seen to be open. The majority of
the guard cells will gradually stain a deeper red than the outside
Solulion, suggesling an acave accumulation of the dye inside the
vacuolar sap of the guard cells. Note that the nuclei remain unstained.
Some stomata will have one guard cell open, while the other remains
closed-as a rule, the closed guard cell will be found to be adjacent
to epidermal cells that were not damaged and that have stained pinkillustrating the importance of epidermal turgor relations for guardcell movements.
Subsidiary cells and modified stomata. Different types of subsidiary
cells can be seen in epidermal strips of grass or cereal leaves, a succulent leaf such as Sedum, a pine needle and Vida faba.
Modifications of the stomatal apparatus must be looked for in
transverse sections of the leaf blade. Any of the types mentioned on
pp. 23-25 are worth studying, but any collection of ecologically
different types of leaves would show interesting modifications.
1'3
PHYSIOLOGY OF STOMATA
A.2
Measuring stomatal aperture in intact
leaves
Preparation of cobalt chloride papers. Test papers impregnated with
cobalt chloride, together with the colour standards, require careful
preparation if consistent results are to be obtained, Henderson{")
has given the following reliable instructions,
150 g dry cobalt chloride is dissolved in I litre of distilled water,
black strips
to be cut in half
I .,'
smear
~f:~ CoCI
~gIU~
~
dark
light
c:>5
t
'/ue}~ '~:c
//';;
/
mmwide
7'
'ri.
~:r
~
~pearanc:;;f
II
t
•
5 mm distance
a single test
paper strip
/
I
II II II II II 1'1 II
; ~li312~
sequence of blue standard,
test paper, light blue standard
Fig. A.1
papers
154
Diagram to aid the preparation of three-colour cobalt chloride test
SOME SUGGESTIONS FOR PRACfICAL WORK
Stock solutions of 1 g methylene blue, and of 0·1 g eosin both in
I litre of distilled water are required for the preparation of the two
colour standards.
Whatman No. I filter paper is used for the three kinds of coloured
strips. The sheet of filter paper is first wetted evenly, like a photographic print, by edge-on immersion in water for one minute,
squeegeed between blotting papers, and thereafter immersed for
one minute in the cobalt chloride solution, or in the dark colour
standard, which is one-eighth the strength of the methylene hlue
stock solution, or in the light blue colour standard, which is a 1 in 32
dilution of the stock solution. The papers are squeegeed before
being hung up to dry. The light blue colour standard, when dry, is
given a further one-minute immersion in the eosin solution, squee-
geed, and hung up once more to dry.
Strips, 0·5 em wide, of each of the coloured sheets are cut off with
a guillotine, a straight line of glue is placed on a fairly stiff cardboard,
and the coloured strips are then glued down at one end in a repeating
pattern: light standard-cobalt chloride-dark standard, as shown
in Fig. A.!, When the colour strips have been assembled on the
cardboard, strips, 0·3 em wide, of black photographic wrapping
paper are glued across the coloured strips at about 0·5 em intervals
(Fig. A.l). When ihe glue has dried, the black paper strips are cut
lengthwise, giving a strip of three-colour test papers which can now
be separated into individual three-colour papers with scissors. The
test papers must be kept in a dry and cool place.
Stomatal frequency and diffusive resistance. Using potted plants
with greatly different numbers of stomata in the upper and lower
epidermes, such as Pelargonium or tobacco, make cobalt-chloride
paper measurements on both epidermes at zero, 20, 40, 60, and 90
minutes after switching on the light.
A.3 Stomatal responses to stimuli
Light. The course of stomatal opeuing on illumination can be
followed by using non-succulent leaves from which the epidermis
strips easily, for instance, Vida faba. Three methods of estimating
stomatal aperture are recommended, and three conditions of plant
material may be used, namely potted plants, detached leaves kept
with their petioles in beakers of water, and leaf discs floating with the
upper epidermis on water. Keep the material in adark place for several
155
PHYSIOWGY OF STOMATA
hours and take Lloyd's strips, silicone rubber impressions, or carry
out an infiltration test with Alvim's series, or xylol, in subdued light.
Repeat the measurements after twenty, forty, sixty, and ninety
minutes of illumination, using the lower epidermis. The silicone
rubber compounds used by dentists are usually quite suitable for
making leaf impressions as they are non-toxic.
Carbon dioxide. The effect of carbon dioxide on the stomatal
mechanism can be demonstrated using any of tbe methods described
on pp. 29-48. Keep the material in normal air in a room in which
there are no people. When the stomata are fairly wide open, record the
measurement and stay next to the plant, breathing directly on its
leaves, and take a second series of readings after about five minutes.
Conversely, use two detached leaves, kept with their petioles in
beakers of water, and take readings in subdued light. Place a dish of
a concentrated solution of potassium hydroxide next to one of the
beakers, cover both with a bell jar, and place another bell jar over the
other leaf with its rim on three inverted beakers so that air can freely
circulate. Illuminate both bell jars equally and measure the degree of
stomatal opening achieved after one hour.
The use of porcmeters. There are no bard and fast rules about
whicb type of porometer is most suitable for stomatal investigations
because both the anatomy of the leaf and the special topic of the
investigation may demand the use of a particular bnd of instrument.
Generally, viscous flow porometers are more convenient to use than
the majority of diffusiou porometers, but it must never be overlooked
that viscous flow porometers, especially when used with hypos tomatous leaves, measure not only stomatal conductance but also
mesopn1l\ tissue conductance. On the other hand, when interpreting
results obtained with tlle most practical of the diffusion porometers,
the seusor type of instrument, it is assumed that the water-vapour
pressure in the leaf air space system remains constant and practically
at saturation; this instrument may, therefore, not be suitable when
investigating the influence of waler stress on stomatal aperture.
Viscous flow porometers require a reliable method of airtight
attachment to the leaf by means of washers. In the past, these were
usually made of gelatine, water, and glycerol. but since they are liable
to melt at moderately high leaf temperatures, washers of the kind
described below are to be preferred.
Preparation of porometer washers. An annulus of commercially
available silicone rubber, about 0·1 em high and 0·2 em wide, is
156
SOME SUGGESTIONS FOR PRACTICAL WORK
placed concentrically about the hole in the porometer clamp by means
of a pointed knife. A fiat, shiny surface is obtained by placing a
lightly greased microscope slide on the annulus after it has dried for
ten to fifteen minutes. When the slide is removed after about six hours,
the washer sticks fast to the porometer clamp and, if need be, it can
be trimmed in its width with a razor blade; it is not possible to trim
its height.
Leaf water content. With a simple resistance porometer and the
attachment clamp shown in Fig. 2.4 held in a retort stand, measure
the course of stomatal opening in an amphistomatous leaf such as
Xanthiumpennsylvanicum, Helianthus annuus, Vidafaba, or Phaseolus
vulgaris, the large first leaves of the last being especially suitable. Do
not omit to detach the porometer from the clamp at the ground-glass
joint between readings. When the stomala are fairly wide open and
the leaf transpires strongly, after about sixty to ninety minutes,
refrain from detaching the porometer from the clamp and, while a
reading is being taken, sever the petiole of the leaf with a sharp, wet
blade. Observe the changes that follow in the manometer reading
(see Fig. 2.7).
By taking frequent readings with a simple parameter, as shown on
p. 37, this phenomenon of stomatal opening on rapid wilting can also
be observed, but the changes in the manometer readings of a resistance
porometer are much more impressive (the Wheatstone bridge
porometer is even better for showing these changes).
A.4
Experiments on gaseous diffusion
Differential rates of diffusion of different gases. The principle underlying the hydrogen-diffusion porometer can be demonstrated by the
following experiment, which also demonstrates that diffusive flow
can set up a considerable pressure in a system, and that rates of diffusion of different gases vary (see p. 51).
Mount a porous pot on the end of a U-tube manometer of about
50-em length, half-filled with indicator liquid. Invert an empty
beaker over the porous pot, and support with a retort stand. Lead
some hydrogen from a supply into the inverted beaker, and observe
the changes in the manometer; remove the inverted beaker and
observe again.
Diffusioll away from all open wat.r surface. Prepare three dishes
with cross-sectional areas roughly in the ratio 4: 2: 1 and place them
IS7
PHYSIOLOGY OF STOMATA
on reasonably sensitive open balances so that the dishes are exposed
to the same conditions of humidity and temperature. Fill each dish
to the brim with distilled water and record vapour losses over a
convenient period. Calculate the rate of evaporation per unit area.
H,J
Fig. A.2
Diagram of apparatus suitable for demonstrating the principle of the
hydrogen diffusion parameter
Make determinations in 'still air', i.e., in a room without undue
draughts, and in moving air, i.e., with a slow fan blowing evenly
across all three dishes, but be careful not to place one dish in front
of another.
Diffusion through perforated septa. Prepare three dishes over which
a metal septum can be placed neatly. In the centre of one septum make
158
SOME SUGGESTIONS FOR PRACTICAL WORK
a hole of8 mm diameter, in the second, four holes of 4 mm diameter,
each situated with their centres on a l2-mm diameter circle, and in
the third septum make sixteen holes of 2 mm diameter each, the
centre of eight of these holes to be situated on the l2-mm circle, and
the centres of the remaining eight on a circle of 6-mm diameter_
Determine the losses in weight over a convenient period in 'still' air
and in moving air. Calculate the vapour losses per unit area and as a
function of the total pore diameters.
The leaf as a diffusive flow resistance. Place a leaf into the convenient porometer clamp shown in Fig. A.2. Purge the apparatus of
air by gently passing hydrogen from a supply via tap B and out at
tap A. Close both taps to the outside air and observe the changes in
the manometer. Carry out the measurements at the beginning of
illumination, and after forty and ninety minutes; avoid exposing the
leaf to hydrogen for too long a period, and detach the clamp at the
ground-glass joint between readings so that the leaf is exposed to
ordinar~! air.
The effect of antitranspirants. Use potted plants, and spray some
of the leaves with solutions of PMA (5 x 10-5 M) and lX-hydroxysui phonates (2 x 10-2 M), and compare the course of stomatal
opening in treated and untreated leaves by any of the methods
mentioned in chapter 2. Ensure that the leaves used receive the same
illumination.
Look for reductions in the rate of water consumption in whole
plants treated with antitranspirants. At the same time, determine
whether dry weight increase is adversely affected.
159
Bibliography
I. Allaway, W. G. and Mansfield, T. A. (1967) Stomatal responses to
changes in carbon dioxide concentration in leaves treated with
3-(4-chlorophenyl)-I,I-dimethyl urea. New Phyla!', 66, 57-63.
2. Alvim, P. de T. (1965) A new type of porometer for measuring stomatal
opening and its use in irrigation studies. Unesco Arid Zone Res., 25,
325-9.
3. Alvim, P. de T. and Havis, J. R. (1954) An improved infiltration
series for studying stomatal opening as illustrated with coffee. Pl.
4.
5.
6.
7_
160
Physio!., Lancasler, 29, 97-8.
Arnon, D. I. (1959) Conversion of light into chemical energy in
photosynthesis. Nature. London, 184, 10.
Arnon, D. I., Tsujimoto, H. Y., and McSwain, B. V. (1967) Ferredoxin
and photosynthetic photophosphorylation. Nalure, Landon, 214,
562-6.
Asada, K. and Kasai, Z. (1962) Inhibition of the photosynthetic
carbon dioxide fixation of green plants by a-hydroxysulvhonates and
its effects on the assimilation products. Plant and Cell Physiol., 3,
125-36.
Bange, G. G. J. (1953) On the quantitative explanation of stomatal
transpiration. Acta Bot. Neerlandia, 2, 255-96.
BIBLIOGRAPHY
8. Barrs, H. D. and Klepper, Betty (1968) Cyclic variations in plant
properties under constant environmental conditions. Physiol. Plant.,
Zl,711-30.
9. Bieleski, R. L. (1966) Accumulation of phosphate, sulfate and
sucrose by excised phloem tissue. Plant Physio/.,Lancasler,41, 447-54.
10. Bonner, W. and Bonner, J. (1948) The role of carbon dioxide in acid
formation by succulent plants. Amer. J. Bot., 35, 113-17.
11. Brat, L. and Weber, F. (1951) Anthoxanthin in den Schliesszel!en der
KronenbHitter von Victoria amazonica und Nymphea zanzibarensis,
Phylon, 3, 22-7.
12. Brilliant, B. (1924) Le teneur en eau dans les feuilles et I'energie
assimilatrice. C.R. des Sc. Acad. Sci., Paris, 178, 2122-5.
13. Brown, H. and Escombe, F. (1900) Static diffusion of gases and liquids
in relation to the assimilation of carbon and translocation in plants.
Phil. Trans. R. Soc. B., 193, 223.
14. Brown, W. V. and Johnson, Sr. C. (1962) The fine structure of the
grass guard cell. Amer. J. Bot., 49,110-15.
15. Blinning, E. (1956) General processes of differentiation in the growth
of leaves. In The Growth 0/ Leaves (ed. Milthorpe, F. L.), pp. 18-29.
Butterworth Sc. Publ., London.
16. Buscaloni, L. and Pollacci, G. (1901) L'applicazione delle pellicole di
collodio aIlo studio di alcuni processi fisiologici delle piante ad in
particolar modo pella traspirazione. Atti 1st. bot. ecc.,Pavia, 2.
17. Butt, V. S. and Peel, M. (1963) The participation of glycollate oxidation in glucose uptake by illuminated Chlorella suspensions. Biochem.
18.
19.
20.
21.
J.,83,31 P.
Christensen, H. V. (1962) Biological Transport. W. A. Benjamin
Inc., New York.
Darwin, F. (1898) Observations on stomata. Phil. Trans. R. Soc., B.,
190,531--621.
Darwin, F. and Pertz, D. F. M. (1911) On a new method of estimating
the aperture of stomata. Proc. Roy. Soc., B., 84, 136-54.
Florin, R. (1951) Evolution in CordaiteS and Conifers. Acta florti.
Bergiafla, 15, 285-388.
22. Franke, W. (1967) Ektodesmen und die Peristomatiire Transpiration.
Planta, 73, 138-54.
23. Freudenberger, H. (1940) Die Reaktion der Schliesszellen auf
Kohlensiiure und Sauerstoff Entzug. Protop/asma, 35, 15-54.
24. Fujino, M. (1967) Role of adenosinetriphosphate and adenosinetriphosphatase in stomatal movement. Sc. Bull. Fae. Educ., Nagasaki
Univ., 18, 1-47.
25. Gaastra, P. (1959) Photosynthesis of crop plants as influenced by
light, carbon dioxide, temperature and stomatal diffusion resistance.
Meded. r.d. Landbouwhogeschool., Wageningen, 59, 1--68.
26. Gale, J. and Hagan, R. M. (1966) Plant anti-transpirants. Ann. Rev.
PI. Physiol., 17, 269-82.
27. Glinka, Z. and Meidner, H. (1968) The measurement of stomatal
responses to stimuli in leaves and leaf discs. J. expo Bot., 19, 152-66.
161
PHYSIOLOGY OF STOMATA
28. Glover, J. (1959) The apparent behaviour of maize and sorghum
stomata during and after drought. J. Agric. Sci., 53, 412-16.
29. Gregory, F. G. and Armstrong, J. I. (1936) The diffusion porometer.
Proc. Roy. Sot., B., 121, 27-42.
30. Gregory, F. G. and Pearse, H. L. (1934) The resistance porometer and
its application to the study of stomatal movement. Proc. Roy. Soc,:
B., 114,477-93.
31. Gregory, F. G. and Pearse, H. L. (1937) The effect on the behaviour
of the stomata. of alternating periods of light and darkness of short
duration. Ann. Bot., NS I, 3-10.
32. Haberlandt, O. (1904) Physiologische PfJanzenanatomie. Wilhelm
Engelmann, Leipzig.
33. Hanes, C. J. and Maskell, E. J. (1942) The influence of hydrogen·ion
concentration upon the equilibrium state in phosphorylase systems,
Biochem. J., 36, 76.
34. Harms, H. (1936) Beziehungen zwischen Stomataweite, Lichtstarke
und Lichtfarbe. Planta, 25, 155-93.
35. Heath, O. V. S. (1938) An experimental investigation of the mechanism
of stomatal movement, with some preliminary observations on the
response of the guard cells to shock. New Phyto!., 37, 385-95.
36. Heath, O. V. S. (1939) Experimental studies of the relation between
carbon assimilation and stomatal movement. Ann. Bot., NS 3,
469-95.
37. Heath, O. V. S. (1948) Control of stomatal movement by a reduction
, in the normal carbon dioxide content of the air. Nature, London,
161,179-81.
38. Heath, O. V. S. (1949) The role of starch in the light response of
stomata I. New Phytol., 48,186-211.
39. Heath, O. V. s, (1950) The role of carbon dioxide in the light response
of stomata I. J. expo Bot., I, 29-{)2.
40. Heath, O. V. S. (1952) The role of starch in the light response of
stomata II. NeW Phytol., 51, 30-47.
41. Heath, O. V. S. (1959) Light and carbon dioxide in stomatal movements. In Handbuch der PfJanzenphysiologie, 17/1, 415-{)4. Springer
Verlag, Berlin.
42. Heath, O. V. S. (1959) The water relations of stomatal cells and the
mechanism of stomatal movement. In Plant Physiology (a treatise,
ed. Steward, F. C.), Vol. II. pp. 193-250. Academic Press, New York.
43. Heath, O. V. S. (1968) Physiological Aspects of Photosynthesis.
Wm. Heinemann, London.
44. Heath, O. V. S. and Mansfield, T. A. (1962). A recording poromoter
with detachable cups operating on four separate leaves. Proc. Roy.
Soc., B., 156, 1-13.
45. Heath, O. V. S., Mansfield, T. A., and Meidner, H. (1965) Lightinduced stomatal opening and the postulated role of glycollic acid.
Nature, London, 207, 960-2.
46. Heath, O. V. S. and Meidner, H. (1957) Effects of carbon dioxide and
temperature on stomata of Allium cepa L. Nature, London, 180, 181-2.
162
BIBLIOGRAPHY
47. Heath, O. V. S. and Meidner, H. (1961) The influence of water
strain on the minimum interceUular space carbon dioxide concen~
tration and stomatal movements in wheat leaves. J. expo Bot" 12,
226-42.
48. Heath, O. V. S. and Milthorpe, F. L. (1950) Tile role of carbon dioxide
in the light response of stomata II.J. expo Bot., 1, 227--43.
49. Heath, O. V. S. and Orchard, B. (1956) Effects of anaerobic conditions
upon stomatal movements-a test of Williams' hypothesis of the
stomatal mechanism. J. expo Bot., 7, 313-25.
50. Heath, O. V. S. and Orchard, B. (1957) Temperature effects an the
minimum intercellular space carbon dioxide concentration. Nature,
London, 180, 180-1.
51. Heath, O. V. S. and Russell, J. (1951). The Wheatstone bridge
parameter. J. expo Bot., 2, 111-16.
52. Heath, O. V. S. and Russell, J. (l954a) An investigation of the light
responses of wheat stomata with the attempted elimination of control
by the mesophyll. Part I. EfIects of light independent of carbon
dioxide. J. expo Bot. 5, I-IS.
53. Heath, O. V. S. and Russell, J. (1954b) Part II. Interactions with carbon
dioxide. J. expo Bot., 5, 269-92.
54. Heath, O. V. S. and Williams, W. T. (1948) Studies in stomatal action.
Nature, London, 161, 178-9.
55. Heller, F. O. and Resch, A. (1967) Funktionell bedingter Strukturwechsel der Zellkerne in den Schliesszellen von Vicia/aba. Planta, 75,
243-52.
56. Henderson, F. Y. (1936) The preparation of three-colour strips for
transpiration measurements. Ann. Bot.) L., 321-4.
57. Hewitt, E. J. and Nicholas, D. J. D. (1963) Cations and Anions:
inhibitions and interactions in metabolism and enzyme activity.
In Metabolic Inhibitors, eds. Hochster, R. M. and QuasteI, J. H.,
VeI.Il., j)j). 311-436. Academic Press, New York.
58. Howe, G. F. (1964) Time course of photosynthetic rhythms in
Phaseo1us vulgaris L. as re)ated to changes in degree oi stomata)
opening. Ohio J. Sc., 64, 378-84.
59. Iljin, W. S. (1957) Drought resistance in plants and physiological
processes, Ann. Rev. PI. Physiol., 8, 257-74.
60. James, W. O. (1958) Succulent plants. Endeavour, 17, 90-3.
61. Kenda, G., Thaler, I., and Weber, F. (1951) Kern Kristalloide in
Stomatazellen. ProlOplasma, XL, 624-32.
62. Kissilew, N. (1925) Veriinderung der Durchliissigkeit des Protoplasmas der Schliesszellen im Zusammenhange mit stomatarer
Bewegung. Beih. Bot. ZBI., 41.287-308.
63. Kuiper, P. J. C. (1961) The effect of factors on the transpiration of
leaves with special reference to the stomatal light response, Meded.
Landbouwhogeschool.. Wageningen, 61,1-49.
64. Kuiper, P. J. C. (1964a) Water transport across root cell membranes:
effect of alkenylsuccinic acid. Science, 143, 690-1.
163
PHYSIOLOGY OF STOMATA
65. Kuiper. P. J. C. (1964b) Dependence upon wavelength of stomatal
movement in epidermal tissue of Senecio odoris. Plan' Physiol.,
Lancaster, 39, 952-5.
66. Levitt, J. (1967) The mechanism of stomatal action. Planla, 74,
101-18.
67. Liebig, M. (1942) Untersuchungen tiber die Abhiingigkeit der Spaltenweite der Stomata von Intensitat und Qualitat der Strahlung. PIan/a •.
33,206-57.
68. Linsbauer, K. (1916) Beitrage zur Kenntnis der Spaltoffnungsbewegung. Flora, N.F., 9, 100-43.
69. Lloyd, F. E. (1908) The physiology of stomata. Publ. Carlleg;e Insln.,
Wash., 82,1-142.
70. Loftfield, J. V. G. (1921) The behaviour of stomata. Publ. Carnegie
Insln., Wash., 314, 1-104.
71. Mansfield, T. A. (1964) A stomatal light reaction sensitive to wavelengths in the region of 700 mIL. Nature, London, 201, 470--2.
72. Mansfield, T. A. (1965a) The low intensity light reaction of stomataeffects of red light on rhythmic stomatal behaviour in Xanthium
pennsyivanicum. Proc. Roy. Soc., B., 162, 567-74.
73. Mansfield, T. A. (1965b) Glycollic acid metabolism and the movements
of stomata. Nalure, London, 205, 617-18.
74. Mansfield, T. A. (l965c) Responses of stomata to short duration
increases in carbon u:oxide concentration. Physioi. Plant., 18, 79-84.
75. Mansfield, T. A. (I965d) Stomatal opening in high temperature in
darkness. J. expo Bol., 16, 721-31.
76. Mansfield, T. A. (1967) Stomatal behaviour following treatment with
auxin-like substances and PMA. New Phytol., 66, 325-30.
77. Mansfield, T. A. and Heath, O. V. S. (1963) Photoperiodic effects on
rhythmic phenomena in stomata of Xanthium pennsylvanicllm.
J. expo BOI., 14, 334-52.
77a.Mansfield, T. A. and Heath, O. V. S. (1964) Studies in Stomatal
Behaviour X. J. expo Bal., IS, 114-24.
78. Mansfield, T. A. and Meidner, H. (1966) Stomatal opening in light of
different wavelengths: effects of blue light independent of carbondioxide concentration. I. expo Bot., 17, 510-21.
79. Maskell, E. J. (1928) Experimental researches in vegetable assimilation. XVllI. Proc. Roy. Soc., B., 102, 488-533.
80. Meidner, H. (1962) The minimum intercellular space carbon dioxide
concentration of maize leaves and its influence on stomatal movements. J. expo BOI., 13, 284-93.
81. Meidner, H. (1965) Stomatal control of transpirational water loss.
In 'The state of water in living organisms'. Symp. Soc. Exptl. BioI.,
XIX, 185-204.
82. Meidner, H. (1965b) A simple parameter for measuring the resistance
to air flow offered by stomata. School Sc. Rev., 47, 149-51.
83. Meidner, H. (1967) The effect of blue (459 nm) and red (656 nm) light
on the stomata of Allium cepa L. and Xanthium pennsy[J'anicum.
J. expo Bol., 18, 146-51.
164
BIBLIOGRAPHY
84. Meidner, H. and Heath, O. V. S. (1959) Stomatal responses to temperature and carbon dioxide concentrati.on in Allium cepa L. and their
relevance to midday closure. J. expo Bot., 10, 206-19.
85. Meidner, H. and Mansfield, T. A. (1965) Stomatal responses to
i1lumination. BioI. Rev" 40, 483-509.
86. Meidner, H. and Mansfield, T. A. (1965) Further observations on
stomatal responses to night length. J. expo Bot., 16, 145-50.
87. Meidner, H. and Mansfleld, T. A. (1966) Rates of photosynthesis and
respiration in relation to stomatal movements in leaves treated with
",.hydraxysulphonates. J. expo Bot., 17, 502-9.
88. Meidner, H. and Spanner, D. C. (1959) The differential transpiration
parameter. J. expo Bot., 10, 190-205.
89. Miroslavov, E. A. (1966) Electron microscopic studies of the stomata
of rye leaves. Bot. Zh., 51, 446-9.
90. Mohl, von H. (1856) Welche Ursachen bewirken die Erweiterung und
Verengung der Spaltiiffnungen. Bot. Z., 14,697-704.
91. Moss, D. N., Musgrave, R. B., and Lemon, E. R. (1961) Photo·
synthesis under field conditions. III. Crop Sc., 1, 83-7.
92. Mouravieff, I. (1953) Influence de diverses substances sur la teneur en
amidon et hydration des cellules stomatiques de l'Aponogeton
distachys. C. R. Acad. Sci., Paris, 236, 1434.
93. Mouravieff,1. (1954) Action des solutions tampons et de leur constituants sur les cellules stomatique d'Aponogeton distachys L. Ann.
Univ. de Lyon, C8, 87-115.
94. MouravieIf, I. (1956a) Action du CO2 el de la lumiere sur I'appareil
stomatique sipare du mesophylle. II. Botaniste (Paris), n, 195212.
95. MouravieIf,1. (I 956b) Action du gaz carbonique el de la lurni"re sur
l'appareil stomatique isole. C. R. Acad. Sci., Paris, 242, 926-7.
96. MouravieIf, 1. (1957) Action du CO 2 et de la lumiere sur l'appareil
stomatique separe du mesophylle. Experiences sur Allium ursinum L.
Botaniste, 41, 1-11.
~1. Mouravieff, 1. (195%) Action de la lumiere sur la cellule vegetale.
Bull. Soc. Bar. Fr., 105, 467-75.
98. Mouravieff, I. (1959) Observations sur la plasmolyse des cellules
annexes de I'appareil slomatique. Bull. Soc. Bot. Fr., 106, 301-5.
99. Mouravieff, I. (1966) Sur les proprietees opliques des cellules
slomatiques. 1. Bull. Soc. Bot. Fr., 113. 220-3.
100. Nishida, K. (1963) Studies on stomalal movement of Crassulacean
plants in relation to the acid metabolism. Plant Physiol., Lancaster,
16,281-98.
101. Nutman, F. J. (1937) Studies of the physiology of Coffee arabica. II.
Ann. Bot., N.s. 1, 681-94.
.
102. Paetz, K. W. (1930) Untersuchungen fiber die Zusammenhange
zwischen stomatarer Offnungsweite und bekannten Intensitaten
bestimmter Spektralbereiche. Planta, 10, 61 1-65.
103. Pallas J. E. (1964) Guard cell starch retenlion and accumulation in
the dark. Bot. Gaz., 125, 102-7.
12'
165
PHYSIOLOGY OF STOMATA
104. Pallas, J. E. (1965) Transpiration and stomatal opening with changes
in carbon dioxide content of the air. Science, 147, 171-3.
105. Pallas, J. E. (1966) Mechanism of guard cell action. Quarl. Rev. Bioi.,
41,365-83.
106. Pekarek, J. (1936) Ober die Azididatsverhiiltnisse in den Epidermen
und Schliesszellen bei Rumex acelosa im Licht und Dunkeln. Planla,
11,419-46.
107. Penman, H. L. (1942) Theory of porometers used in the study of
stomatal movements in leaves. Proc. Roy. Soc., B, 130, 416-33.
108. Penman, H. L. and Schofield, R. K. (1951) Some physical aspects of
assimilation and transpiration. Symp. Soc. exptl Bioi., V, 115-29.
109. Priestley, J. H. (1943) The cuticle in angiosperms. Bot. Rev., 9, 593-616.
110. Ranson, S. L. and Thomas, M. (1960) Crassulacean acid metabolism.
Ann. Rev. Plant Physiol., 11, 81-1I0.
Ill. Raschke, K. (1965) Die Stomata als Glieder eines schwingungsfiihigen CO 2-Regelsyslems. Z. Naturiorsch., lOb, 1261-70.
112. Raschke, K. (1965b) Eignung und Konstruktion registrierender
Porometer flir das Studium der Schliesszellen Physiologie. Planta, 67,
225-41.
113. Raschke, K. (1967) Der Einfluss von Rot und Blau Licht auf die
(}fnnungs und Schliess Geschwindigkeit der Stomata von Zea mays.
Naturwiss., 54, 72-3.
1I4. Raschke, K. (1967) Erweiterung der Spaltiiffnungen von Viciaiaba
durch Stickstoff. Naturwiss., 54, 73.
115. Salisbury, E. J. (1928) On the causes and ecological significance of
stomatal frequency with special reference to the woodland flora.
Phil. Trans. R. Soc., B., 216. 1-65.
116. Sampson, J. (1961) A method of replicating dry or moist surfaces for
examination by light microscopy. Nature, London, 191, 932.
1I7. Santarius, K. A. (1967) Das Verhalten von CO 2-Assimilation, NADP
und PGS Reduktion und ATP Synthese intakter Blattzellen in
Abhiingigkeit vom Wassergehalt. Planta, 73, 228-42.
118. Sargent, J. A. (1965) The pe.nelratjon of growth regulators jnto leaves.
Ann. Rev. Plant Physiol., 16, 1-12.
119. Sargent, J. A. and Blackman, G. E. (1962) Studies on foliar penetration. I. J. expo Bot., 13, 348-68.
120. Savitzky, H. (1966) Effectiveness of selection for tetraploid plants in
Co generation on the basis of the number of chloroplasts in stomata.
J. Amer. Soc. Sugar Beet Techn., 13, 655-61.
121. Sayre, J. D. (1923) Physiology of stomata of Rumexpatientia. Science,
57,205-6.
122. Sayre, J. D. (1926) Physiology of stomata of Rumex patienlia. OkioJ.
Sc., 26, 233-66.
123. Scarth, G. W. (1929) The influence of hydrogen ion concentration on
the turgor and movement of plant cells with special reference to
stomatal behaviour. Int. Congr. Plant Sci., Ithaca, Proc., 1151-62.
124. Scarth, G. W. (1932) Mechanism of the action of light and other
factors on stomatal movement. Plant Physioi.,lAncaster, 7, 481-504.
166
BIDLlOGRAPHY
125. Scarth, G. W. and Shaw, M. (1951) Stomatal movement and photosynthesis in Pelargonium. I. Plant Physio{., Lancaster, 26, 201-2S.
126. Scarth, G. W., Whyte, J., and Brown, A. (1933) On the cause of night
opening of stomata. Trans, Roy, Soc, Canada, Sect. V, 27, 115-17,
127. Schwendener, S, (1881) Bau und Mechanik der Spaltiiffnungen, Ber,
Akad. Wiss" Berlin, 833--67,
128. Shaw, M, (1958) The physiology of stomata, II, Can.J. Bot" 36, 575-79.
129, Shaw, M, and Maclachlan, G. A. (1954) The physiology of stomata. 1.
Can, J, Bot" 32, 784--94.
130, Shimshi, D, (1963) Effect of chemical closure of stomata on transpiration in varied soil and atmospheric environments. Plant Physio{.,
Lancaster, 38, 709-12.
131. Sierp, H, (1934) Untersuchungen ilber die Offnungsbewegung der
Stomata in verschiedenen Spektralbezirken, Flora, 128, 269.
132, Sievers, A. (1959) Untersuchungen Uber die Darstellbarkeit der
Ektodesmen und ihre Beeinflussung durch physikalische Faktoren.
Flora, 147, 263-316.
133, 'Slatyer, R, 0, (1967) Plant-Water Relationships, Academic Press,
New York,
134. Slatyer, R. O. and Jarvis, P. G, ([966) Gaseous diffusion porometer
for continuous measurement of diffusive resistance of leaves. Science,
151, 574--6,
J35, Slavik, B, (1965) The influence of decreasing hydration level on
photosynthetic rate in the thalli of the hepatic ConocephalJum conicllm.
In Waterstress in Plants, W, Junk, The Hague., pp, 195-201.
136. Spanner, D, C, (1953) On a new method for measuring the stomatal
aperture of leaves. J, exp, Bot" 4, 283-95,
137. Spanner, D. C, and Heath, 0, V, s, (l951) Experimental studies oftb
relation between carbon assimilation and stomatal movement. Ann.
Bot" N.S. XV, 319-431.
138. Stahl, E. (1894) Einige Versllche fiber Transpiration und Assimilation.
Bol, Ztg" 52,117.
139. Stalfelt, M, G, (1927) Die photischen Reaktionen im Spalt6ffnungsmechanismus, Fiora, IZl, J.36-7Z,
140. StAlfelt, M. G. (1929) Pulsierende Blattgewebe, Planta, 7, 720-34,
141. StAlfelt, M. G, (1932) Die stomatare Regulation in der pflanzlichen
Transpiration. Planta, 17, 22-32.
142. StAlrelt, M, G, ([955) The stomata as a hydrophotic regulator of the
water deficit of the plant. physiol, Plant" 8, 572-93,
143, Stalfelt, M, G. (1956) Die stomatare Transpiration und die Physiologie der Spalt6ffnungen. In Handbuch der Pjlanzenphysi%gie, III,
351-426, Springer Verlag, Berlin.
144, StAlfelt, M, G, (1957) The water output of the guard cells of the
stomata, Physiol. Plant" 10, 752-73.
145, StAlfelt, M, G, (1962) The effect of temperature on opening of the
stomatal cells, Physiol, Plant" 15, 772-9,
146. StAlfelt, M. G, (1963) Diurnal dark reactions in the stomatal movements, Physiol, Plant" 16, 756·-66.
167
PHYSIOLOGY OF STOMATA
147. StAlfeit, M. O. (1967) The components of the CO,-induced stomatal
movements. Physiol. Plant., 20, 634-42.
148. Ting, P., Thompson, M. L. D., and Dugger, W. M. (1967) Leaf
resistance to water vapour transfer in succulent plants: effect of
thermoperiod. Am. J. Bot., 54, 245-51.
149. Tolbert, N. E. and Hess, J. L. (1966) The effect of ~.hydroxymethane.
sulphonates on ''C0 2 photosynthesis by algae. J. BioI. Chem., 241,
5712-15.
150. Van Bavel, C. H. M., Nakayama, F. S., and Ehrler, W. L. (1965)
Measuring transpiration resjstance of leaves. Plant Physiol.. Lancaster,
40,535-40.
151. Ventura, M. M. (1954) Action of enzymaL inhibitors on transpiration
and the behaviour of stomata. II. Rev. Bras. Bioi., 14, 153-61.
152. Virgin, H. I. (1956a) Light induced stomatal movements in wheat
leaves recorded as transpiration. Experiments with the corona
hygrometer. Physiol. Plant., 9, 28(}-303.
153. Virgin, H. 1. (1956b) Light induced stomatal transpiration of etiolated
wheat leaves as related to chlorophyll a content. Physiol. Plant., 9,
482-93.
154. Virgin, H. I. (1957) Stomatal transpiration of some variegated plants
and of chlorophyll·deficient mutants of harley. Physiol. Plont., 10,
17(}-86.
ISS. Walker, D. A. (1966) Carboxylation in plants. Endeavour, 25, 21-{J.
156. Walker, D. A. and Zelitch, I. (1963) Some effects of metabolic inhibi·
tors and anaerobic conditions on stomatal movement. Plant Physioi.,
Lancaster, 38, 39(}-6.
157. Weber, F. (1933) Zur Permeabilitiit der Schliesszellen. Protopiasma,
XIX,452-4.
158. Weber, F. (1951) Viruskorper fehlen den Stomazellen. Protoplasma,
XL,636-9.
159. Whittingham, C. P., Coombs, J., and Marker, A. F. H. (1966) The
role of glycollate in photosynthetic carbon fixation. Biochemistry of
Chloroplasts, 2, 155-173.
160. Wiggans, R. O. (1921) Variations in the osmotic concentrations of
the guard cells during opening and closing of the stomata. Amer. J.
Bot., 8, 30-41.
161. Wilkins, M. B. (1959) An endogenous rhythm in the rate of carbon
dioxide output of Bryophyllum. I. J. expo Bot., 10, 377-90.
162. Wilkins, M. B. (1960) An endogenous rhythm in the rate of carbon
dioxide output of Bryophyllum. II. J. expo Bot., 11, 269-88.
163. Wilkins, M. B. (1962) An endogenous rhythm in the rate of carhon
dioxide output of Bryophyllum. III. Proc. Roy. Soc., R, 156, 22(}41.
164. Williams, W. T. (1948) Stomatal movement induced by heat shock
stimuli and the transmission of such stimuli across the leaves of
Pelargonium zonale. Ann. Bot., N.S. 12, 35-51.
165. Williams, W. T. (1954) A new theory of the mechanism of stomatal
movement. J. expo Bot., 5, 343-52.
168
BIBLIOGRAPHY
166. Williams, W. T. and Barratt, F. A. (19S4)The effect ofextemalfactors
on stomatal starch. Physiol. Plant., 7, 298-311.
167. Willis, A. J. and Jefferies, R. L. (1963) Investigations on the watt!
relations of sand~dune plants. under natural conditions. In The Water
Relations ofPlants, pp. 168-89. Blackwell Sc. Pub!., Oxford.
168. Willis, A. J., Yemm, E. W., and Balasubramaniam, S. ([963)
Transpiration phenomena in detached leaves. Nature, London, 199,
265--J;6.
[69. Wilson, C. C. (1948) The effect of some environmental factors on the
movements of guard cells. Plant Physiol., Lancastert 23, 5-37.
170. Wood, H. G. and Utter, M. F. (I965) The role of CO 2-fixation in
metabolism. Essays in Biochemistry. Vo1. I, pp. 1-27. Academic
Press, New York.
171. Yemm, E. W. and Willis, A. J. (1954) Stomatal movements and
changes of carbohydrate in leaves of Chrysanthemum maximum. New
PhytoL, '53, ,1,-%.
172. Yin, H. C. and Tung, Y. T. (l948) Ph'''phorylalle ;n guard cells.
Science, 108, 87-8.
! 73. Zelitch, I. (1963) The control and mechanism of stomatal movement.
In 'Stomata and water relations in plallts', Bull. 664) Conn. Exp. Sta.,
18-36.
174. Zelitch, I. (1964) Reduction of transpiration in leaves through stomatal
closure induced by alkenyl succinic acids. Science, 143, 692-3.
175. Zelitch, I. (l965) Environmental and biochemical control of stomatal
movements in leaves. BioI. Rev. 40,463-82.
176. Zelitch, I. (1966) Increased rate of net photosynthetic carbon dioxide
uptake caused by the inhibition of glycollate oxidase. Plant Physioi.,
Lancaster,41,1623-31.
177. Ziegenspeck, H. (1955) Das Vorkommen von Fila in radialer Anordnung in den Schliesszellen. Protoplasma, 44, 385.
169
Index
Where a page number next to an author's name is followed by a
bibliography reference number in brackets (thus: Asada, K. 137''') this
means that the author's name may not appear in the text on that page
although his work is discussed there.
Acidification, 114-118
Action spectrum, for stomatal
opening, 77-81, 84, 147
'Active' movements, 92-95, 101
Active transport, 122-124, 133-134,
14&-149
Adenosinetriphosphate, 121-124,
132
Adenosinetriphosphatase, 19, 122..,
124
After-etfects of wilting, 95
Airspace system resistance, 33, 55,
S8
Albino barley, 83-84
Alcohol for infiltration, 32
Alfalfa, 87, 90
Alkenylsuccinic acids, 145
170
Allaway, W. G., 121 (1), 160
Alphahydroxysulphollates, 68, 136138
Alvim, P. de T., 32, 37, 38, 160
Arnphistornatous leaves, 4, 32, 46
Arru;llit.ude of [h.~thrn, 102
Anaerobic conditions, 120-121, 123,
125, 131, 140
Antechamber, 13, 17, 25, 30
Anthoceros, 1,4
Anthocyanin, 20
Anthoxanthin., 20
Antitranspirants, 159
alphahydroxysulphonates, 68
carbon dioxide, 66
chemical, 67
films on leaves, 66
INDEX
Aponogeton distachys, 23
Armstrong, J. I., 42, 162
Arnon, D. I., 121(4" 137(4), 160
Arrangement of stomata, 9, 151
Asada,!(. 137(6), 160
Assay ofstomatal widths, 30
Attached leaves, 2i
Attachment clamps for leaves, 33,
34-35
Autoradiographs of guard cells, 72,
84, 140
Awns (stomata in), 4
Azide 125-127
Caesium ions, 132
Calcium ions, 19, 132, 133
Calcium oxalate crystals, 20
Camellia, 100
Canal in graminaceous guard cells,
18
Capillary resistance, 38, 41
Carbon dioxide, 72-86, 95-96,
100-101, 111-112, 115-117,
120--12),12,
as antitranspirant, 66
as stomatal stimulus) 73, 156
compensation point, 78-81, 85,
88-89,94-95,98-99,116
concentration in guard cells, 87,
Bange, G. G. J., 61, 160
127
Barley, a1bino, 83
Barratt, F. A., 143, 169
Barrs, H. D., 27(8), 118, 161
enrichment, 66, 76
interaction with water stress,
Bavel, C. H. M., van, 47, 168
Bellis perennis, 20
--effect in floating leaf discs, 28
-free air, 28, 74-76, 84, 127,
Benzol for infiltration, 32
Bieleski, R. L., 124, 161
Blackman, G. E., 124, 166
Blue light (see Light)
Bonner, J., 139, 161
Bonner, W., 139, 161
Boundary layer, 55
Brassica o/eracea, 109-111
Brat, L., 20(11),161
Brilliant, B., 99, 161
Broad bean, 90, 93, 106, 124-125
'Brodie's solution, 40
Bromocresol purple, 139
Bromophenol blue, 138
Brown, A., 125 (26 ), 167
Brown, H., 62, 161
Brown, W. Y., 13(14), 18(14" 161
Bryophyllurn caiyeinurn, 114,139
Byrophyl/um daigremontianum, 114115
BUnning, E., 10, 161
Buscaloni, L., 30(16), 161
Butt, Y. S., 136, 161
95-96
129-130
-independent
77-81, 84,
146-147
light
121,
reaction,
129-130,
Carboxylation reactions, 114-118.
122, 138-141, 148
Cavity, substomatal, 10, 13
Centaurea orientatis, 96
Cereals, night opening in, 110
3-(4-Chlorophenyl)-I, l-dimenthylurea (CMU), 12
Chlorophyll, role of in stomatal
movements, 81
Chloroplasts in guard cells, 18,
81-85
Christensen, H. Y., 122(18), 161
Chromatic material in guard cells,
20
Chrysanthemum, 144-145
Clamps for porometers, 33
Closing, hydroactive, 94-95
passive, 92-93,101, 125
midday, 87-89
Closing movements, 128
Closing, reactions, 119-134
Cabbage, 109-111
Cactaceae, 114, 139
Cobalt chloride paper, 31, 154
Coz (see Carbon dioxide)
171
INDEX
Coefficient of diffusion, 12, 50, 58
Coffea arabica, 88-89
Commelinacommllnis, 132-133
Conductance, viscous and diffusive,
42,46
Contour surfaces of equal vapour
density, 53-55
Convection, 54
Coombs, ].,137"59',168
Corona hygrometer, 72
Cotyledon peacockii, 114
Diffusion, diffusive
area law, 50, 55
coefficient, 12, 58, 59
conductance, 42, 46, 48
• diameter law, 52-55
experiments in, 157
Fick's law, 50, 54
in liquid phase, 56
in moving air, 54, 55, 59, 60, 61.
100
in still air, 59, j 00
Crassulaceae, 114
mechanism
Crystals in guard cells, 20
Cups for leaf attachment, 33, 34-35
Cuticle, internal, 13
path,56
rate of, 49
Cuticular transpiration, 46
temperature coefficient of, 51
Cutin, 13
Cycadales, 3
Cyclic photophosphorylation, 121,
136-137
Cyperaceae, 6
Cytoplasmic
inclusions, 18
streaming as transport mechan·
ism, 56
streaming in guard cells, 22
viscosity, 20
Dark carboxylation (fixation), 114118,122,138-141,148
Dark opening and temperature, III
Darwin, F., 33, 161
Density of carbon dioxide in air, 63
Desynchronization of rhythms, 109
Detachable cups, 35
Detached epidermis, 86
Detached leaves, 27
Diameter law of diffusion, 52-55
2,4-Dichlrophenoxyacetic acid, 134
3-(3,4-Dichlorophenyl)-I, I-dimethylurea (DCMU), 121
Differential manometer, 41
Differential transpiration poro~
meter 45, 60
Differentiation, of guard cells, 6, lSI
haplocheilic, 8
syndetocheilic, 8
172
of~
49
resistance, 46, 56, 58
Dimensions of stomata, 2, 3
Dimorphism, 6
2,4-Dinitrophenol, 127
Discs (leaf), 27
Dorsal wall of guard cell, 17
Drought resistance, 22
Drying, incipient, 61
Dufour effect, 44
Dugger, W. M., 115-117\148), 168
Dynamics of stomatal movements,
128-130
Ectodesmata, 13, 22, 124
Effective length, 56
of extemal path, 54-59
of internal path, 59
of stomatal path, 59
Ehrler, W. L., 47,168
Elliptical stomata, 6, 15
End correction, 56
End effects, 52
Endogenous rhythms, 69, 70, 103118, 125, 147
Energy for stomatal movements,
119-127, 136, 148
Epidermal
films, 61
strips, 29, 115
turgor, 27, 92-94, 120, 123, 148
E?\stomatous ~aves. ()
Equivalent length, 56, 59
INDEX
Escombe, F., 62, 161
Etiolated plants, 73
Exli!rnal diffusion path, 59
Evaporation, rate of, 49
External resistance, 54-59
Far-red light, 121
Fick's law, 50, 54
Fieid porameter, 38
Fixed resistance, capiIIary, 38, 41
Floating lear discs, 28
Florin, R., 4, 9, 161
Flowers (stomata in) 4
Flow lines of diffusion, 54
mutuB? interference of, 54, 61
Fossil gymnosperms, 3
Franke, W., 14(22), 161
Frequencies of stomata, 2, 3, 5, 6,
151,155
Freudenberger, H., 73, 125, 161
Fructosans, 142
Fruits (stomata in), 4
Fujino, M., 19(24), 29(24), 122-124,
132-133,161
Gaastra, P., 63, 64, 161
Gale, J., 61Mi7(26l, 161
Glinka, Z., 21, 27(27), 28(27), 36(27\
94,161
Glover, J., 97, 162
Glucose-I-phosphate, 142
Glycollate, 68, 73
hypothesis, 135-138
Glyoxylate, 137
Graham's law, 51
Graminae, 10
Graminaceous stomata, 6, 7, 17
Gregory, F. G., 38,42,118,128-130,
162
Gregory and Pearse resistance porometer, 38
Gregory and Pearse units, 42
Guanosine di- and triphosphate, 122
Guard cell, 6-22, 30, 152
ATPase, 19, 122-124
calcium ions, 19, 132, 133
Guard cell-continued
canal, 19,20
chloroplasts, 18, 81-85, 140141, 146
chromatic material, 20
content, 18-22, 152
cytoplasmic streaming, 22
differentiation, 6
dimensions, 10, 12
dorsal walls, 17
mitochondria, 19
nucleus, 20
oil drops, 19
osmotic potential, 21, 73, 117,
124,133,145,148,152
permeability, 20, 145-146
peroxidase, 19
pH, 19, 116-118, 122, 138-140,
142
phosphorylase, 19, 124, 142
pigments, 81-85
plasmolysis, 22
potassium ions, 19, 123, 132-134
sap, 21,116-118,152
senescence, 22
spicules, 12
starch, 18, 141-145
strengthening ridges, 12, 30
tUIgor, 17, 120, 148
vaCUOle, 20, 116-118
ventral wall, 13, 17
viscosity of cytoplasm, 20
wall thickening, 15-18
width, 12, 106
Gymnospermae, 3, 8, 9
Haberlandt, G., 12,24,162
Habitat, 23
Hagan, R. M., 66, 67(26), 161
Hanes, C. J., 142, 162
Haplocheilic differentiation, 8
Harms, H., 77, 130(34), 162
Hautgelenke, 16, 17
Havis, J. R., 32, 160
Heath, O. V. S., 29(42), 34, 36, 39, 41,
46(84),70,71 (53),73,75,78,85,86,
88-89,92,94-96,99, 100(54), 101,
173
INDEX
Heath, O. V. S.-continued
105, 107, 109, 112, 120, 121, 125,
131 (84), 138, 142, 143, 162, 163
Heath's reagent, 152
H""t shock stimulus, 86
Iodine stain for starch, 152
Ionic effects, 132-134
Iris, 141
Iron, 134
Isolated stomata, 29, 86
Hedera helix, 99
Height of insertion of leaves and
stomatal number, 5
Helium, stomatal behaviour in, 121
He((oborus, 15
Heller, F. 0., 20, 163
Henderson, F. Y., 154, 163
Hess, J. L., 137\1<'1), 168
Heterobaric leaves~ 32
Hewitt, E. J., 126(''', 163
Howe, G. F., 118, 163
Humidity, 100, 120
Hydathodes, 1
Hydroactive closure, 94-95
Hydrocotyle vulgariS, 143
Hydrogen diffusion porometen,
42-44
Hygrometer, cor<>na, 72
Hypostomatous leaves, 4, 33, 46
Hypothesis
dark fixation, 138-141
glycollate, 135--138
permeability, 145-146
starch ",sugar, 138, 141-145
Jam.., W.O., 114, 163
Jarvis, P. G., 45, 167
Jefferies, R. L.,143, 169
Johnson, Sr. C., 13(14), 18(14), 161
Kalancho. bloss/eldiana, 114, 116117
Kasai, Z., 137(6), 160
KCNS,20
Kenda, G., 20 (6 1), 163
Kenya, maize and sorghum crops,
97
Kerosene for infiltration, 32
Kinetics of stomatal movements,
27,128-132
Kissilew, N., 145(62),163
Klepper, Betty, 27(8), 118, 161
Kuiper, P. J. C., 77, 86, 121, 137(63),
145
Lear
Iljin, W. S., 96,132,133,163
impatieru partJij!ora... 99
Impressions, silicone rubber, 30
Incipient drying ofleaves, 61
Index, stomatal, 6
Infiltration, 26, 32
Infra-red gas analyser in porometery,45
Insertion hejght of leaves and
stomatal number, 5
Intact leaves, 27
clamps, 33
cups,34
discs,27
excision, 92-94
senescence, 22
temperature and transpiration, 66
water content, 151
Leaves
amphistomatous, 4, 32, 46
attached, 27
detached, 27
epistomatolls, 6
Interactions between environmental
heterobaric, 32
factors, 101
Interference of !low lines, 54, 61
hypostomatous, 4, 33, 46
incipient drying of, 61
inta-ct, 2'1
succulent, 23, 150
variegated, 85
Internal airspace system, resistance
of, 33, 55, 58, 59
Internal cuticle, 13
174
INDEX
Lemon, E. R., 67(911, 165
Lenticels, 1
Levitt, J., 116-117, 164
VobiS, M., 130(67), 164
Light
as stimulus, 70-86, 91, 101, 109110, 112, 116, 120, 128-131,
135-149, 155
b\ue, 20, 77-7S, S5, 108, II" 125,
146-147
effects independent of CO" 7781,84,121,129-130,146-147
far-red, 121
Jow~intensity reaction, 108, 112
red, 77-81, 85,108,121,146
Light inlemily
effect on stomatal aperture, 72, 81,
82,86
interaction with CO" 74-75
minimum for opening, 11
Mansfield, T. A.-contin...d
107-109,112,114(72),117,121(1),
127(76), 129-130, 131 (74), 132,
137(73), 146(78), 164
Marker, A. F. H., 137(mJ, 168
Maskell, E. J., 142, 162
Mean free path, 12, 51
Mechanical work in opening, 120
Meidner, H., 21. 27_28(271, 36(27),
38 45 46"') 62"0 76('°) 77-in
85:
93-94, 98,' 104, '109(86):
89,
117, 129-130, 131 1" " 137("',
13S, 146(83), 164, 165
Meristemoid, 10
Mesophyll resistance, 33, 55, 58, 59
~,.,;>\\~tk ;>!ants, 14
MetaboBc inhibitors, 125-127, 14S
Micellae in guard cell walls, 13, 17
Micro-relief, 17, 30
Microscopic observation, 29
units of measurement. 7]
Liguslrurn, 100
Midday closure, 87-89
Milthorpe, F. L., 73(48), 74; 163
Liliaceae, 10, 14
Lin,bauer, K., 145(68), 164
Liquid paraffin for infiltralion, 32
Liquid phase
MirosJavov, E. A., 18(89),165
diffusion in, 56
resistance, 63-66
Lithium ions, 132
Lloyd, F. E., 30, 141, 164
Lloyd's strips,
Loftfield, J. V. G., 71(70), 87-90,
110-111,164
Long-day Ireatment, \13
Low-intensity light reaction, 108,
112
,0
Mitochondria, 19
Mnium, 15
Mohl, von H., 72, 165
Moss, D. N., 67, 165
Moss plants, 4
Mother cell of guard cells, 6
Motorphase, 130
Mouravieff, I., 21, 23, 29, 77, 86,
126-127, 139, 144, 146-148
Moving air, effect on diffusion, 5455, 59, 60-61, 100
Movements of stomata, 128-130
Musgrave, R. B., 67(90, 165
Mutua} interference of flow lines, 54,
61
Maclachlan, G. A., 72-73, 85(129),
140-141, 167
McSwain, B. V., 121(5), 160
Magnesium, {32
Maize, 76, 80, 97-98
Malic acid, 139
Mannitol, 94
Manometer, differential, 40-41
Mansfield, T. A., 27('6), 39, 67(76),
70,77-81,85-91,95-96,104-105,
Nan varnish, use in micro-relief, 30
Nakayama, F. S., 47, 168
Needle valve, in porometer, 40-41
Nerium oleandert 25
Neutral red, 124, 139, 153
Nicholas, D. J. D., 126(57J, 163
Night interruptions, 114
Night length effect, 107, 113-114
175
INDEX
Night opening, 105, 109-112, 114117
and temperature, 11 t
Nicotinamide-adenine dinucleotide
phosphate (NADP), 136
Nishida, K, 114-115, 117, 165
Nitrogen, stomatal behaviour in,
121,125
Nitrous oxide diffusion porometer,
44
Non-cyclic photophosphorylation,
121, 136-137
Nucleus of guard cell, 20
Nutman, F. J., 87,165
Ohm's law, 56
Oil drops in guard cells, 19
Onion, 19, 72, 88-89, 142, 146, 150,
151,153
Opening movements, 130
in dark, 111
Opuntia, 114
Orchard, B., 88-89, 120-121, 125,
163
Organic acids, 114-118, 122, 138141
Oscillatory movements, 118
Osmotic pressure of guard cell sap,
21,73,117,152
Oxidative phosphorylation, 120,
127, 13l
()xygen,9Z, 12a-fZf, f31, i4a
Paetz, K. W., 77, 165
Pallas, J. E., 21, 76, 124(105), 141,
144,165,166
Paraffin, liquid for infiltration. 32
Partial pressure, 26, 50
'Passive' movements, 92-93, 101,
125
Pearse, H. L., 38, 118, 128-130, 162
Peel, M., 06, 161
Pekarek, J., 21, 166
Pelargonium, 86, 88-89, 143
Penman, H. L., ix, 166
Period of rhythms, 102-106
176
Permanently attached clamps, 33
Permeability hypothesis, 145-147
Permeabiliiy of membranes, 20
Pertz, D. F. M., 33, 161
Petioles (stomata in), 4
Peroxidase, 19
pH of guard cell sap, 21,138-142
Phase of rhythm, 102-109, III
Phase shift, 102-109, 112-114
Phenyimercuricacetate, 67, 124, 127
Phosphate buffer, 133
Phosphoenolpyruvate, 122, 139-141
Phosphorylase, 19, 124, 142
Photoperiodic experiments, 113-114
Photophosphorylation, 120-121,
127,131,136-137
Photosynthesis
by guard cell chloroplasts, 72-73,
81-85,121
effect of water delict, 99, 100
rate, control by stomata, 64
role in stomatal movements,
72-76,81-85,118,121
Phyllitis sc%pendrium, 19
Phytochrome, 108
Plasmodesmata, 13
Plasmolysis in guard cells, 22
Ploidy and chloroplast number in
guard cells, 19
Pneumathodes, 1
Poisseuille's equation, 41
Pollacci, G., 30(16),161
Porometer
Alvim's 37
clamps detachable, 35-36
cups,permanentlyattached,33-34
swept and ventilated, 35-36
Darwin and Pertz', 34
differential transpiration-, 45, 46,
60
diffusion, 42-48
Dufour, 44
field,37
hydrogen, 43,44
methods of use, 156
Raschke's, 36
resistance, 38
sensor element, 47. 61
INDEX
Porometer~continued
units, 41
van Bavel, 47
viscous flow, 33
washers, 36, 156
Wheatstone bridge, 40
Potassium ions, 19, 123, 132-134
Potato, I 1(}-11 I
Pressure
osmotic,21
partial, 26, 50
turgor, 27
within epidermis, 92-94, 120,
123,148
Priestley, J. H., 13(109), 166
Protective mechanism against water
shortage, 96-99
Protoderm, 6
Prunus laurocerasus, ] 04
Psilophytales, 3
Puncturing of guard cells, 120
Pyruvate carboxylase, 122
Rhubarb petiole (stomata in), 10
Rhynia,3
Rhythm
amplitude of, 102
endogenous, 69, 70,103-118,125,
147
period of, 102-106
phase of, 102-109, III
short period, 103, 118
stomatal, 69-70, 102-118
synchronization of, 108-109
Rhythmic changes in aperture, 27,
102-118
Rhythmic changes in response to
stimuli, 106
Ridges, strengthening, 12, 30
Rubber, silicone impressions, 30
washers, 36, 156
Rubidium ions, 132
Russell, J., 39, 71 (53', 73(53', 74-75,
78, 85-86, 10 I, 163
Q 10 of diffusion, 51
QIO of stomatal opening, 89-91,131
Salisbury, E. J., 5, 166
Sampson, J., 30(116), 166
Santarius, K. A., 100(117), 166
Sargeant, J. A., 124, 166
Savitzky, H., 19(120" 166
Sayre,J. 0.,21, 52, 73,104,138-139,
166
Scarth, G. W., 21, 73, 76(125), 85,
125,139,142,166,167
Schofield, R. K., ix, 166
Schwendener, S., 15,17, 167
Sedum,22
Senecio odoris, 77
Senescence ofleaves and guard cells,
22
Sensor element diffusion porometer,
46,60,61
Shaw, M., 72, 76(125" 84, 140, 167
Shimshi, D., 63(130), 167
Short period fluctuations, 103, 118
Sierp, H., 77, 130(131), 167
Sievers, A., 13, 167
Silicone rubber impressions, 30
Slayter, R. 0., 45, 62, 67, 167
Slavik, B., 99, 167
Ranson, S, L., 140, 166
Raoult's law, 62
Raschke, K.,36,80,118,125,146,166
Rate of opening, 70, 73
Red light (see Light)
Relative humidity, 100, 120
Resch, A., 20, 163
Resistance
airspace system, 33, 55, 58, 59
capillary, 38, 41
diffusive, 56, 58
e>.'ternal, 54-59
fixed, 38, 41
leaf as a whole, 58
liquid phase, 63-66
mesophyll, 33, 55, 58, 59
of diffusion path, 56
of stomatal tube, 58
per cm2, 58
porometer, 38
stomatal, 59
viscous, 33, 42
117
INDEX
Sodium ions, 132
Sodium azide, 125-127
Sorghum, 97
Soybean, lOS
Stomata, stomatal-conllnued
pores, 6
position, alignment in epidermis,
17
Spanner, D. C., 36, 39 J 44, 45, 46(88),
resistance, S9
167
Spannungsphase, 130
Spermatophyta, 3
Sphygmomanometer, 38
Spicules, 12
Spindle crystals, 20
Stahl, B., 31, 167
Staining of epidermal strips, 153
StMfelt, M. G., 21, 29, 90-92, 94,
106, 118, 126, 130, 137(147), 167,
168
Starch, 18, 124-125, 138, 141-146
hypothesis 138,141-145
Stefan's law, 52
Sterns (stomata in), 4
Stiles, W., 47
rhythm, 69-70, 102-108
Still air, diffusion in, 59, 100
Stoma mother cells, 6, 10
Stomata, stomatal
antechamber, 13, 25, 30
assay, 30
cavity, 10, 13
closing movements, 128
closing reactions, 119-134
control of photosynthesis, 63
control of transpiration, 60
differentiation of, 6, 151
diffusion path, 59
dimensions, 2, 3
dynamics, 128-130
elliptical, 6, IS
frequencies, 2, 3, 5, 6, lSI, ISS
graminaceous, 6, 7 14. 16, 17
guard cells, 6
index, 6
isolated, 29
mother cells, 6
opening in anaerobic conditions,
125
opening movements, 130
opening reactions, 119-134
pattern, 9, 10, 151
pore area, 2, 3, 6
J
178
spicules, 12
subsidiary cells, 6, 25
throat, 13, 30
tube as diffusion path, 55, 58
resistance, 58
Strengthening ridges, 12, 30
Strips, epidermal, 29, lSI
Strontium ions, 132
Suboptimal water dtiicit, 94
Subsidiary cells, 6, 22, 25, 92-93,
126,153
Substomatal cavity, 10, 13
Succulent leaves, 23, 150
Succulent plants, stomata! behaviour of, 114-118
Supraoptimal water deficit, 94
Surface films as antitranspirants, 66
Swept porometer cups, 34
Synchronization of rhythms, 108-
109
Syndetocheilic differentiation, 8
Taraxacum o./licinale, 96
Temperature, 87-91, 101, 115-117,
131-132
coefficient of diffusion, 51
coefficient of stomatal opening,
89-91,131
effect on opening in darkness,
90-91,108,110-112,115-117
interaction with CO2• 87-91,
101,1J1-112
of 1eaf and transpiration, 66
Tendril, stomata in, 4
Thaler, I., 20(61), 166
Thermocouple in porameter, 44, 46
Thermopile in parameter, 45
Thomas, M., 140, 166
Thompson, M. L. D., 115_117(148),
168
Throat of stomata, 13, 30
INDEX
Ting, 1. P., 115-117, 168
Tobacco, 131
Tolbert, N. E., 137(149), 168
Tradescantia, 15
Transmission of stimuli, 85-86
Transpiration
cuticular, 46
effects of [CO,), 66
parometer differential, 45, 46, 60
rate, control by stomata, 60
Tsujimoto, H. Y., 121(5), 160
Tung, Y. T., 142, 169
Turbulent flow, 55
Turgidity effects, 28
Turgor pressure in guard ceUs, 15,
17,27
Water deficit, effect on photosynthesis, 99
protection against, 96--99
suboptimal, 94
supraoptimaI,94
Water stress, interaction with COl
95-96
Weber, F., 20(157), 168
Wcedkillers, entry through stomata,
124, 136
Wheat, 71-72, 82, 99, 104, 120, 128
Wheatstone bridge porometer, 39
Whittingham, C. P., 137(159), 168
Whyte, J., 125(126), 168
Wiggans, R. G., 21,168
Williams, W. 1., 34, 86, 1001")' 128,
143, 168
Wllki:TI's, M.B., 1{}6{1()1) (161), 112n(3),
Units in porometery, 41
Utter, M. F., 122, 169
Vacuole of guard cell, 20
van Bavel, C. H. M., 47, 168
Vapour density, 53-56
Vapour pressure deficit, 118
Variegated leaves, 85
Ventilated cup, 36
Ventral wall of guard cells, 13, 17
Ventura, M. M., 127(151), 168
Veronica heccabunga, 77
Vicia/aba, 90, 93, 106, 125-125
Virgin, H. I., 71-72, 81-84, 168
Virus -infecfions, 20
Viscosity of cytoplasm, 20
Viscous conductance 42, 46
Viscous flow porometry, 26, 33
168
Willis, A. J., 93,143-145,169
Wilson, C. c., 100, 169
Wilting, after-effects of, 95-99
and stomatal closure, 92-93, 101,
125
Wind effects on diffusion, 55, 59, 100
Wood, H. G., 122, 169
Xanthiumpennsy[vanicum, 33,70-71,
77,79-81,90, 105,107-109,111112,129,131,138,146
Xylol for infiltration, 32
Yemm, E. W., 93, 144, 169
Yin, II. c., 142, 169
Viscous flow resistance, 46
Vital staining, 153
Walker, D. A., 120--121, 126-127,
131, 139, 168
Washers for porometers, 36, 156
Zea mays, 76, 80, 97-98
Zebrina pendula, 139
Zelitch, 1., 30, 86, 120--121, 126-127,
131, 135-137, 145, 169
Ziegenspeck, H., 13(177), 169
. • 0,
I 9 '3
.
PHYSIOLOGY OF STOMATA
..
By H. MEIDNER, University of Reading, and
T. A. MANSFIELD, University ofLanc_ter
This book Is written for those who require Information
about the structure and functioning of stomata at a
reasonably advanced level. This topic has been neglected
until recently when It has been given much attention by
students and researchers in plant physiology in general,
and water-relations of plants and crops In particular. The
authors employ a truly physiological approach In combining
physical, biochemical, and anatomical aspects.
TATA McGRAW-HILL PUBLISHING COMPANY LTD.
BOMBAY
•
NEW DELHI