DEPOSITED BY THE FACULTY OF
GRADUATE STUDIES AND RESEARCH
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UNACC.DTE1933
ACC. NO.
THE RELATION OF STOMATAL OPENING
TO TEMPERATURE AND OTHER FACTORS
A thesis submitted for the
degree of Master of Science
to McGill University, Montreal
by
J. H. Whyte, B.Sc. (Agric. Edin.)
TABLE OF CONTENTS
INTRODUCTION
1.
HISTORICAL
Temperature and Stomatal Movement.
Leaf Turgor and Stomatal Movement.
2.
6.
EXPERIMENTAL WORK
Methods-.
Experiments devised to obtain the Relationship
between Temperature and Stomatal Movement in
the Dark.
'/•
14.
Discussion of the Theories on the Effect of
Leaf Turgor on Stomatal Movement.
19.
Experiments on the Direct Effects of Leaf
Turgor on Stomatal Posture.
22.
The Indirect Effect of Leaf Turgor on
Stomatal Movement in the Dark.
26.
The Effect of Temperature on the Uptake
of Y/ater by Leaves.
27.
The Correlation between Leaf Turgor and the
Opening of Stomata in the Dark.
27.
The Effect of the Interference with the
Gas Exchange on Stomatal Movement.
30.
Experiments on the Effects of the Lack of
Oxygen on Stomatal Movement in the Dark.
33.
DISCUSSION
Review and Discussion of Temperature and its
Effect on Stomatal Movement in the Dark.
35.
Night Opening of Stomata.
38.
Respiration and Some Factors Affecting it.
40.
General Summary and Conclusions.
43.
References.
45.
Introduction.
This work began as a further study of the effect
of temperature on stomatal movement as commenced by Scarth
and Brown (unpub.).
As temperature effects cannot be studied
alone, the other environmental factors were taken into account,
especially turgidity of leaf and factors governing night
opening of stomata and, as it turned out, the most important
result of the investigation concerns the latter.
This work was. undertaken at the Department of
Botany of McGill University.
My thanks are due to Professor
Scarth, by whom the problem was suggested( and under whose
supervision the work was brought to a successful conclusion,
and to Professor Lloyd, who kindly placed at my disposal
the facilities of the Department.
2.
Temperature and Stomatal Movement.
A Historical Review.
Muller (1878,), whose experiments are the first
on record, found that, within limits, heat acts in a manner
similar to that of light in causing stomata to open.
Schwendener (1882) came to the conculsion that, with leaves
in the dark either in an atmosphere saturated with moisture
or submerged in water, a rise of temperature from 15° - 17°c.
to 27°- 32°c. gave no opening of the stomata.
The findings
of Leitgeb (1886) were in agreement with this but he also
observed that, with a rise in temperature in the light, an
increased opening of the stomata occurred.
Working with
various plants, Kohl (1886) failed to observe any stomatal
opening with a similar increase of temperature of the surroundings when warmed by the radiation from a hot plate.
Using Trianaea bogotensis. a plant with which Kohl had also
experimented, Eberdt (1889) found that the opening of the
stomata was quickly caused by drawing a current of moist air
at 30°c. over the plant, but was followed by complete
closure in two or three minutes. Darwin (1898), whose
hygroscopic method indicates stomatal movement indirectly
and requires dry air for its use, decided that in some oases
the closure in the dark is more rapid when the air is coolf
while in other cases, a change of temperature seemed to make
very little difference. The factors at work in these experiments are, however, very complex.
In addition to this type of investigation in which
3.
the leaves are usually kept in darkness and their temperature
governed mainly by conduction to and from the surrounding
medium, the same authors studied the effect of radiant heat
which might be transformed into other forms of energy as well
as to raise the temperature of the leaf.
Kohl found that by
cutting out a large proportion of the infra-red (heat) rays
from sunlight by an alum plate or solution, leaves of Trianaea
floating on the water required about three or four times
longer an exposure than when exposed to direct sunlight.
Eberdt, in addition to confirming this, cut out the visible
rays of the sun and left only the infra-red, by passing them
through a solution of I in CSp. He observed that whereas shut
stomata opened in the invisible rays, open stomata also shut.
Jj'rom this type of experiment, Darwin found that stomata of
Campanula pyramidates remained open for two or three hours.
The actual temperatures of the leaves in these experiments
are not known.
Recent work also seems to be just as contradictory
as that carried out in the past. Kuijper (1915) found that
high temperatures in a dark chamber prevent closure in various
plants, while blogteren (1917) observed that a high temperature
in a moist, well-lighted greenhouse induced closure in fficus
elastica.
In experimenting with alfalfa, Loftfield (1921)
concluded that temperature markedly affected stomatal movement
between 0° and 40°c. and that in general, for every rise of
10°c. the rate of stomatal movement is doubled; that is, the
accelerating effect of temperature obeys van!t Hofffs Law.
Zalenski (1921) showed that when the temperature rises to
4.
35°- 500c. the starch in the guard cells is transformed to
maltose and that the turgor of these cells rises, and in
consequence, the stomata open widely, a fact which is also
observed in wilted plants. Not only do the stomata open more
widely at such temperatures, but they lose the capacity to
close.
He postulates as an explanation of these facts that at
higher temperatures the plasma passes into a dormant state, the
synthetic processes thereby being inhibited whereas those of
hydrolysis still remain active.
Working with leaves of a large
number of plants, Kisselew (1928) found that, on exposing them
to a temperature of 40°c for 18 - 20 hours, he obtained an
increase in the hydrolysis of starch in both the mesophyll
and the guard cells, hydrolysis being more marked in the latter.
btalfelt (1928) found, when working with detached leaves of
bean in moist air with temperatures between 20°c. and 34°c,
that no movement of the stomata occurred in darkness and the
rate of opening in the light was in no way affected,
while
studying the effects of light, Scarth (1932) observed that
with excised leaves of Zebrina. an increase of temperature
brought about an increase in the pH of the vacuole of the
guard cells and that temperatures of 38°- 40°c. not only
accentuated stomatal opening in the light but tended to cause
it, or prevented closure in the darkness, as the case might
be, while low temperatures (0°- 8°c.) prevented opening in
the light, bcarth and Brown (unpub.), in a more detailed
study of the effect of temperature on stomatal movement in
excised leaves offlebrina,decided that temperature markedly
5.
affects the turgor and posture of the guard cells by influencing
the starch-sugar equilibrium, and at high temperatures, by
modifying their permeability.
At low temperatures (2°c.)
condensation of starch predominates and stomata may close even
in powerful light. As the temperature increased, both the
rate of hydrolysis and of condensation increase, the latter,
o N
however, more slowly, so that at high temperatures (40 c.)
it is overcome by hydrolysis, even in darkness. Nevertheless,
rise of turgor in the guard cells at these temperatures is
largely prevented by their increased permeability.
6.
Leaf Turgor and stomatal Movement.
A Historical Review.
Scarth (1932) sums up the position of our knowledge
in relation to leaf turgor and stomatal movement.
"The
relation of stomatal opening to the amount of water in the
leaf is a very complex one. Whether turgidity be above or
below normal, the result varies with the duration of the
abnormal state.
It also varies considerably with the species
of plant and the age and position of the leaves, while, of
course, it is modified by other external factors which
regulate opening."
Tort Mohl (1856) and Leitgeb (1886)
attributed greater activity to the epidermal cells than to
the guard cells in stomatal movement.
Von Mohl (1856) stated
that if the epidermis of most plants with open stomata be
mounted in water, the stomata close immediately.
This state-
ment is not true of all plants with mobile stomata (Copeland,
1902).
Schaefer (1888), in explaining this phenomenon,
ppints out that when this closure occurs, it is due to the
structure of the epidermal cells, which permits them to take
up water more rapidly than the guard cells and that this
preliminary closure is prevented if the sections be thick
enough to include part of the mesophyll, thus preventing the
water from coming into close contact with the inner wall
of the epidermis.
It has also been found that on cutting
tangential sections of the under epidermis offlebrinathe
stomata close in the vicinity of the wound.
Arends (1925)
7.
writes that starch is rapidly formed in the guard cells as
the stomata close.
Scarth (1932) also observed this closure
but failed to get this rapid formation of starch and ascribes
the loss of turgor in the guard cells to increased permeability
and escape of sugars.
Leitgeb believed that, if stomata
closed at night, it must be due to the increased turgescence
of the cells surrounding the guard cells forcing them to close.
he also observed that on brightly illuminated plants which
had transpired freely and where, therefore, the pressure against
the guard cells would not be at its greatest, the stomata were
closed and also that there was a disappearance of solid matter
in the guard cells before the pore opened,
schaefer showed
that for some stomata at least any movement
occurring was
independent of the surrounding epidermal cells.
From data obtained by the use of porometers,
Darwin (1898), Darwin and Pertz (1911), and Laidlaw and Knight
(1916) postulated an initial opening of the stomata on
wilting.
Lloyd 11908) was unable to observe this initial
opening.
He found no effect on stomatal movement arising from
an increase in atmospheric humidity and stated that wilting
has to be pronounced before it results in closure, a finding
which later workers have confirmed.
He also observed that
stomata continued to open when the water content of the leaves
was decreasing and concluded, as did Knight (1917), that the
stomata were inefiective in maintaining a constant water
supply in the leaf and were thus of little or no value in
controlling the water loss.
Loftfield (1921) criticizes
these conclusions on the grounds that there is in the turgid
8.
leaf a certain amount of the water present which may be
regarded as a working margin and which may be last without
interfering with the opening of the stomata or the various
other functions of the leaf. After the water loss falls to
or below this margin, the stomata begins to close, due to a
fall in the turgor of the guard cells.
Of this margin, he
states that the maximum leaf turgor increases after rains
or irrigation and thereafter decreases until the next watering
and that the percentage water-loss at which the working margin
disappears also fluctuates between applications of water.
He observed that if a leaf be wetted either by dew, rain or
artifically, the stomata tend to open and when the water dries
the stomata tend to close partially or completely.
Of the
cause of day closure and night opening, he writes:- "Changes of
leaf turgor offer the best explanation regarding the mechanism
of mid-day closure and night opening.
It has always been found
that mid-day closure occurs when the leaf water has been
reduced to a point which is the safe minimum for a given water
content.
The stomata do not reopen until the percent of water
rises once more above this point and the leaf has a margin with
which to safely operate
Night opening occurs only in
those leaves in which turgor is recovered faster than starch
is stored in the guard cells." Working with peach trees,
Henrickson (1926) found that the decrease in moisture content
of the leaves appeared quite a while before the maximum
opening of the stomata was reached,
bayre (1926) records
that when he forced water into the leaf stalk under a pressure
9.
of three atmospheres, the stomata, previously opened, closed
to 10$ of their maximum, returning to their previous condition
on the release of the pressure.
Conversely, on wilting,
stomata were found to open from 10% to 18.4% after fifteen
minutes or so. This was followed by a closure movement which
is probably due to the effect of secondary changes within
the guard cells, acting like darkness in reducing their osmotic
pressure.
In a paper on the stomatal reaction to water
balance, Stalfelt (1929) distinguished between three types
of systems controlling stomatal movement:The passive system.
The photo-active system.
The hydro-active system.
The first system occurs when the water content is supraoptimal.
The idea being that, should the stomata be open
and the water content reach this supra-optimal stage, they
tend to close, opening again as the water content falls.
The reaction mechanism is the pressure of the turgescent
cells of the adjacent epidermis and mesophyll.
This system
he calls the passive system, as the guard cells play no
active part in the change.
In the photo-active system the
primary cause of the reaction is light, the stomata opening
in light and closing in darkness. The light affects the
osmotically active substances in the guard cells, causing
a change in the osmotic value. This system is most sensitive
when the water content is optimal: should it increase above
this, the passive system begins to act and at a certain
10.
content a delicate balance between these two systems results.
If the water content falls below the optimal state, that is,
sub-optimal, the stomatal movement is still dominated by
changes in the osmotically active cell substances.
The lower
the water content falls, the greater the tendency to close.
This system is called the hydro-active, as the guard cells
play an important part. The hydro-active system comes into
play not immediately but a few minutes afterwards.
Here
again, at a certain water content there is a delicate balance
between the hydro-active and photo-active systems.
11.
Methods.
The material used for experiment was mainly
potted plants, cuttings and excised leaves of Zebrina pendula.
In certain instances a variety of other plants was used.
As a method of temperature control, a temperature
gradient tank which gave a range of more or less constant
temperatures was constructed.
During the time of an experiment
it was found that the temperature of each compartment did not
vary by more than one or two degrees centigrade.
The experiments were carried out both in light
and in darkness but mainly in the dark.
As a source of light,
a series of 100 watt lamps was placed directly above the
containers at a distance of six inches above the water level
of the temperature gradient tank.
Large tin cans, 6-| inches in diameter and 11 inches
deep, lined with filter paper which was kept moist, were used
as containers for the plants in the temperature gradient tank.
When excised leaves were used, they were placed
in corked bottles which were either filled with water or
contained some damp cotton wool.
The latter have been desig-
nated "moist chambers"•
In observing the stomata, the direct method
I Lloyd, 1913) was used whenever possible and where it was
necessary, Lloyd's 11908) Alcohol stripping method was used.
When it was desirable to test for starch in the
guard cells, tangential sections of the epidermis were
12.
taken, and after decolourizing in alcohol, were stained in
EI.
Observations on the pH . of the guard cells were
carried out as detailed by Scarth (1929 and 1932) and Scarth
and Brown (unpub.).
Thin sections of the epidermis were
taken and placed in the stain(methyl red) for a standard time,
circa two minutes.
In forming an opinion as to the colour
of the indicator in the guard cells, care was taken to disregard stbmata whose auxiliary cells were injured in cutting
the section, because it was observed that the guard cell
vacuome of such stomata tended to be more acid than that of
a stomata whose auxiliary cells were normal.
The cause of
this probably lay in the production in the maltreated cells
of "acid of injury" and its diffusion into the guard cells.
The principal distinction looked for was whether the indicator
accumulated in the guard cells in the red or pale yellow form.
The latter is invisible but its presence can be revealed by
treating with dilute acetic acid, which quickly changes the
colour to the more brilliant red hue.
In all cases the
method was to compare the section under consideration with
sections from leaves in light and sections from leaves in
the dark.
In experiments carried out in oil, the leaves
were placed directly into Sujol in glass containers with loose
lids.
The apparatus used in the experiments with oxygen
free air was a U-tube as shown in figure 1.
One side of the
13.
tube dipped into alkaline pyrogallic acid and the corked side
into water, thus making a complete seal. The mixture of
pyrogallol and potassium hydroxide used was that recommended
by Cumming and Kay (1919), viz:Pyrogallic acid - 7 grams; water - 25cc.
Potassium hydroxide - 50 .grams; water - llOcc.
The solutions were allowed to cool before mixing. When it
was desired to prevent any further absorption of the oxygen
from the air in the U-tube, a thick layer of liquid paraffin
(fiujol) was introduced over the alkali pyrogallol in the tube
arm by means of a bent pipette.
Fig. 1
14.
Experiments DeviSed to Obtain the Relationship
between Temperature and Stomatal Opening in
the Dark.
Potted plants of Zebrina pendula which had been
kept in the dark for a 12 - 24 hour period were placed at
different temperatures.
The posture of the stomata was
determined by the direct method.
In any one leaf, the variation in the rate and
amount of opening was found to be considerable, but in general,
the greatest opening occurred between 20°- 24°c. and was about
25% of the maximum.
In only one experiment at temperatures above 27°c.
was opening recorded, death usually occurring, if not before
24 hours, always after.
At temperatures below 20°c, slight
opening occurred and in one case, opening to 25% of the
maximum was observed at 12°c.Maximum opening was generally
reacted after a period of 20 - 24 hours, after which, at the
higher temperatures especially, closing of the stomata occurred.
It was noticed that, in the lower leaves of a
plant, opening occurred more rapidly than in the upper ones
and in all experiments carried out with excised leaves or
leaves attached to the plant, only the two uppermost were used.
It was also found that in healthy leaves the first stomata
to open were those nearest the edge of the leaf.
At the very
edge of the leaf there occur stomata which are very much
smaller than the stomata in other parts of the leaf.
Frequently
those at the edge of the leaf are open while the others remain
15.
closed.
In a previous study by Scarth and Brown (unpub.),
there was found to be a difference in behaviour between leaves
in water and leaves in moist air.
«ith leaves immersed in
water they observed the maximum opening to be at 30 c.
At
45°c, rapid but slight opening occurred, whereas at temperatures
between 8 c. and 30 c , the stomata showed a tendency to open
gradually.
At lower temperatures than this (2 c ) , no opening
occurred even after 96 hours.
"With Reaves in the moist
chamber, stomatal movement proved to be very erratic and was,
on the whole, slight throughout the whole temperature range,
with a tendency to opening."
The containers used in the foregoing experiments
were glass dishes with loosely fitting lids and it was thought
that the difference in the results was caused partly by a
loss in turgor in the case of the leaves in the moist chambers,
due to the fact that, the glass dishes emerged partly above
the water and there was therefore a difference in temperature
above and below, thus causing the condensation of moisture
on the lid and evaporation from the leaves at high temperatures
of the water bath and a condensation in the leaves at low
temperatures.
They also pointed out that one cannot "assume
that the difference between the results obtained with leaves
in moist air and in water respectively is due entirely to
turgor effects, since immersion must also interfere with gas
exchange".
These experiments were repeated, using rubber
16.
stoppered bottles in which the leaves were immersed in water
or put on moist cotton wool (moist chamber) and placed beneath
the water surface in the temperature tank.
The results obtained when the leaves were immersed
in water at different temperatures agreed with those of Scarth
and Brown, but the results in regard to the moisture chamber
agreed more nearly with the water immersed leaves than with
those of the original moist chamber.
Table 1
Average results compiled from experiments on the
opening of stomata in moist chamber (corked bottles) at
different temperatures (24 hours).
Temperature
Percent of maximum opening
35°- 34°c.
12-|
30°c.
36
27°- 26°c.
30
22°c.
13i
17°c.
10
12°c.
10
7°e.
no opening
Although the results were more in harmony with leaves immersed
in water than with the leaves in the orignal moist chambers,
the percentage opening occurring was not so great as found in
the former.
Table 2
Experiments with leaves immersed in water (24 hours)•
(Scarth and Brown).
17.
Temperature
Percent of maximum opening
45° c.
dead
30°c.
43
20°c.
12*
8°c.
6
2°c.
no opening
Table 3
Experiments with potted plants (24 hours).
Temperature
Percent of maximum opening
27°c.
12f
24° - 20 °c.
25
19°- 12°o.
12i
10°c.
no opening
From the examination of tables 1, 2 and 3, it can be seen
that the general tendency is the same, except that the
maximum opening varies as does the optimum temperature,
especially with reference to the experiments with plants.
In searching for an explanation for the occurrence of such
a phenomenon, it was thought that perhaps leaf turgor was
the main factor which controlled stomatal movement in the
dark, because it was found that leaves on plants and in
moist chambers were flaccid at high temperatures and that
plants placed in tin cans, such as described in "Methods",
had open stomata, whereas the stomata of plants which had
been in a cupboard for the same length of time remained closed
The assumption made was that plants in an enclosed atmosphere,
a tin can in this case, become more turgid.
18.
The
,pH of the guard cells was also studied and
it was found that when opening occurred in the dark their
reaction tended to be much more alkaline than leaves with
closed stomata and comparable to guard cells of plants with
open stomata in the light.
Our observations with regard to closure, or,in
some cases, the failure to open to any great extent, of stomata
at high temperatures in the dark are in agreement with Scarth
and Brown in that it is not due to the lack of formation of
osmotically active substances but rather to an increased
permeability of the guard cells, which thus allows to escape
the sugar formed by the hydrolysis of starch.
19.
Discussion of Theories on the Effect of
Leaf Turgor on Stomatal Movement.
If leaf turgor be the controlling factor of
stomatal movement, it will be instructive to discuss the points
of view of previous workers before examining our experimental
results.
We have already seen that there are two main
systems recognized, viz:- the active or indirect, and the
passive or direct systems. As to the operation of the passive
system, experiments are contradictory and opinions divergent
but the more general opinion is perhaps such as suggested by
Loftfield's results. He found that, in general, leaf turgor
favoured opening whether in light or in darkness; also wrhen
the leaves of a plant are ?;et artificially, or by rain or
dew, the stomata tend to open and, if partially open already,
to open more widely, and when the water dried, the stomata
closed wholly or partially.
The slight extent of the change
in leaf water content, the rate of the response and the
absence of correlated starch changes in some cases, point
to the passive rather than to the active type of mechanism
but the indication is by no means certain.
On the other hand,
Stalfelt (1929), who has studied the effects of water balance
in great detail, has arrived at the conclusion that the passive
system acts njuite in the opposite way to that which is
indicated above and in addition, that it is frequently the
dominant factor in stomatal movement.
Thus, according to
20.
his hypothesis, high leaf turgor through direct mechanical
4
action may completely inhibit photo-active opening of stomata
under natural conditions and the failure of stomata to open
on cold, damp days is, he believes, due to this cause.
Possibly these contradictory results may be
explained in part by differences in stomatal mechanism in
the plants studied and in part by difference between temporary
and lasting effects. Copeland (1902) suggests that large,
thin-wailed guard cells lose or absorb water more rapidly
than the other epidermal cells, while small, thick-walled
guard cells do so more slowly. The latter seems to be the
case in Zebrina (Scarth 1927). Its guard cells seem both
to obtain and lose water mainly by way of the subsidiary
cells so that a temporary closing movement follows rapid
absorption of water by the leaf and a temporary opening by
a rapid loss.
This transient phase of the effect of variation
in water balance is of slight biological significance
compared v/ith the more enduring efiects. Still putting aside
those which are indirect, let us consider theoretically what
enduring direct results are to be expected.
In so far as the guard cells alone are concerned,
increased turgidity alone leads, of course, to increased
opening of the pore. On the other hand, the relation of the
guard cells to the subsidiary cells in many plants at least
is such that an equal increase of turgor pressure in both
might tend to compression of the former. This follows from
21.
the fact that the guard cells usually bulge into the subsidiary cells and that with increased tension on the wall
dividing them and no change in the difference of pressure
between the two sides, the curvature of the wall must be
reduced.
This would tend to flatten the cells in the vertical
plane and so reduce the width of the whole stomata.
what
would be the net result in pore width of these two opposing
pressures in and on the guard cells can hardly be predicted
without a very precipe knowledge of the mechanism- of the
particular type of stomata.
There are other types of stomata in which, as
Haberlandt (1887) and Copeland (1902) have shown, the action
of the guard cells is purely of the bellows type.
The cells
expand and contract only in the vertical plane and the
position of their dorsal walls is little affected by changes
in pressure of the adjoining cells.
In such cases, the
passive mechanism, being a function of the guard cells alone,
must lead to opening when leaf turgor is high and to closing
when it is low.
The general conclusion from these theoretical
considerations is that the direct effect of leaf turgor on
stomata depends upon their structure and may be directly
opposite in different types.
It is necessary to put it to
the test of experiment for the species with which we are
working.
22.
Experiments on Direct Effects
of Leaf Turgor on Stomatal Posture.
The principal difficulty in determining the direct
and indirect effects of leaf turgor is that, if we allow ample
time to lapse after a change in water relation for equilibrium of suction tension to be established between the
guard cells and the subsidiary cells, indirect effects may
have begun to appear, especially in a change from or to the
wilted state.
On these early indirect effects we have little
check because slight changes in the osmotic value of the
guard cells would be difficult to detect by plasmotic methods
and may easily cause stomatal movement. We know, however,
as already explained, in what direction at least the indirect
effects, if any, ought to act and so when we cannot eliminate
theiptwe can make allowances for them.
One series of experiments was carried out in
which the osmotic value of the guard cells was maintained
as low as possible.
Cuttings of Zebrina were set in water
and kept in the dark overnight. Leaves or half leaves were
than excised and either allowed to wilt or were immersed in
water and still kept in the dark. After a suitable internal,
the average width of the stomatal apparatus was determined
on the leaves so treated and compared with that of the control
leaves or half leaves still attached to the currings. After
two hours1 immersion in water, the width of the stomatal
apparatus was about bfo less than in the
23,
controls and after forty-five minutes' wilting, 10% or so
greater than in the controls.
In neither case was there
likely to be any considerable osmotic change in the guard
cells.
If any did occur, it would act oppositely to the
observed effect which must therefore be a direct one. No
opening of the stomatal aperture attended the widening of
the whole stomata on wilting leaves of Zebrina but it was
sometimes observed on Vicia fabia.
In another series of experiments, the osmotic
value of the guard cells was maintained as high as possible.
Turgid leaves with wide open stomata and no starch in the
guard cells were placed lower side up under a strong light
which was filtered through an alum solution to reduce the
heating effect.
Some of the leaves or halves of longitudinally
divided leaves were just covered by water and other similar
leaves exposed to the air.
The latter wilted rapidly.
In
thirty minutes, when the epidermal walls of the wilting leaves
had become markedly wrinkled, the average measurement of a
large number of stomata in each set was as follows:Average results of experiments devised to discover
the direct effect of leaf turgor on stomatal posture in light.
Slit
Stoma
Leaves in water. Turgid.
19
57
Leaves in air.
Wilted.
21
61
After a further period, the stomata of the wilted leaves
proceeded to close and it is probable that the expansion of
the stomata during wilting would have been somewhat greater
if the osmotic value of the guard cells had not already begun
24.
to fall before maximum opeMng was reached.
In neither of the above series of tests did the
stomatal aperture vary as much absolutely as the whole stoma.
This relation was still more clearly brought out in a comparison of the width of whole stomata having the same aperture
on turgid and wilted leaves respectively.
With a mere slit
showing in both cases, the stomatal apparatus was 10% or even
20% wider in severely wilted leaves. This applies to
measurements made at the widest part of the guard cells,'
which, as indicated in our theoretical discussion, loop
deeply into the subsidiary cell as wilting creates tensions
in the latter.
Taking normal leaves as the standard, the effect
of high leaf turgor is small as compared with the direct
effect of wilting.
This was brought out in the aforementioned
series of tests with leaves in the dark.
In light it is
unusual to find any evidence of passive resistance to opening
due to high leaf turgor.
Under favourable conditions of
light and temperature, opening is as good in leaves immersed
in water for varying lengths of time as in less turgid leaves
either attached to the plant or detached.
This applies not
only to Zebrina but to Vicia and all other plants tested.
The absence of an optimum water content for opening - which,
we are led to expect on the basis of the experiments in the
dark - may be due to the swamping of the passive mechanism
by the photo-active, especially if the latter on its part
is actually promoted by leaf turgor up to the limit.
It was
25.
only at low temperatures and in rather weak light that we
found opening proceeding distinctly more rapidly in non-turgid
leaves or even in slightly wilted ones than in those which
were saturated.
To sum up the passive system of stomatal
movement, it acts according to our results after the manner
that Stalfelt describes but in a lesser degree. Except
possibly at low temperatures or in light of limited intensity,
the passive system does not, in our experience, oppose photoactive opening and except, perhaps, in some plants under
conditions of extreme wilting, it does not prevent hydroactive
closure.
Ordinarily, therefore, it seems that the passive
system is of little biological significance.
26.
The Indirect Effect of Leaf Turgor on Stomatal
Movement in the Dark.
The only feasible explanation so far offered to
account for night opening of stomata is that of leaf turgor see section on night opening of stomata.
since the guard
cells of stomata which have opened in the dark are more or
less devoid of starch, the opening must be of the active
type and the effect of leaf turgor must be an indirect one.
The experimental results of ocarth and Brown (unpub.) and our
own with leaves in atmospheres of different humidity, with
regard to the response of the stomatal apparatus to temperature, seem to ppint to leaf turgor having this indirect effect.
A S well as the experiments already reported, the following one
seems to uphold the leaf turgor theory.
The stomata of a
leaf kept in the air overnight averaged 35/A- diameter with no
opening; on a similar leaf immersed in water they averaged
40/awith a slit of 3.0yUL.If such be the case, it will be
necessary to put the theory to the test.
It was thought
that perhaps at different temperatures there would be a
difference in the uptake of water which would explain the
mechanism of temperature effects.
27.
Experiment to Determine the Effect of Temperature
on the Uptake of Water by leaves.
Leaves which had been kept in the dark for a
12 to 24 hour period were weighed and placed in the dark in
water at different temperatures. At different time intervals
the leaves were dried between two sheets of blotting paper
and weighed.
No constant relation was found between tempera-
ture and uptake of water; for example, at 34 - 35 c , there
was an average increase of weight of Z% after if hours and
one of Q% after 48 hours but the increase varied from 1 to
ie/o; at 26°- 27°c. there was an average increase of weight of
3.7$ after 24 hours, the range being from 1 to 8$, whereas
after 3t hours the average increase was 4.6$, varying from
3 to 6$. There is a tendency, especially at temperatures
above 20°c, for the infiltration of the air spaces of the
leaf, which thus gives greater difference in weight, but even
disregarding such cases the variation at any one temperature
was just as great as between the different temperatures.
Thus it would appear that there is no correlation between
temperature and uptake of water by the leaf.
An experiment designed to find the correlation
between leaf turgor and opening of stomata in the dark.
Another series of experiments was undertaken
at room temperature in an effort to discover the leaf water
28.
content which brings about stomatal movement in the dark.
Leaves were detached from cuttings which had been kept in
the dark overnight.
They were then weighed and placed between
sheets of wet blotting paper and examined again after 24 hours,
with the following results.
Duration of experiment - 24 hours.
The stomata were closed at the beginning of the experiment.
Condition of Stomata
No. of Leaves
fo Gain in Weight
25% of maximum opening
2
20 and 16
25$ of maximum opening
at edge of leaf only
2
21 and 9.5
Slight slits all
over the leaf
3
21, 18 and 16,5
Slight slite at edge
of leaf only
2
Closed
5
17.9 and 5
23, 17, 14, 8.4
and 6.5
On the whole, except in the case of closure in
the light due to wilting, there is no evidence to show that
leaf turgor has any marked effect on stomatal movement and
another explanation must be found to account for the difference
in the results obtained by Scarth and Brown (unpub.) when
using the moist chamber as compared with our own results and
also those obtained with plants and excised leaves. By
immersing leaves in water it is obvious that one must interfere with the gas exchange and experiments were carried out
29.
to discover the possibility of this having some effect
on stomatal movement.
30.
Experiments on the Effect of the Interference
with Gas Exchange on Stomatal Movement.
It was seen that the stomata of leaves immersed
in water opened more widely than those of leaves in moist
chambers but that this was further complicated by infiltration.
In order to discover the answer to the above question
without the complication of infiltration of water, a series
of experiments was carried out in which the leaves were
immersed in oil (Nujol).
As had been done previously, before
investigating the temperature effects, leaves were experimented on at room temperature.
If leaves with closed stomata
were placed in oil, the stomata were found to open widely
and furthermore, this opening took place not only in
Zebrina but in all other plants experimented with, although
the opening was not always of such great magnitude.
List of plants in which opening occurred in the
stomata when placed in oil;
Tradescanlia sp.
Zebrina pendula
Cobea Scandens
Chrysanthemum sp.
Euschia sp.
Vicia fabia
The pH of the guard cells at the time of opening
showed an alkaline reaction to methyl red; also their
31.
starch content was much less than in closed stomata in air.
In experiments to determine the effeet of
temperature on stomatal movement, $nere was clear indication
that a rise in temperature accelerated opening in the dark.
In one series run for eight hours, the following results
were obtained:
Opening Shown by Stomata
Temperature
36°c.
687a of maximum opening
30 °c;
60% of maximum opening
27 c.
42% of maximum opening
20°c.
22% of maximum opening
ll°c.
12% of maximum opening
In another of twenty-four hours' duration, these results
were obtained:
Temperature
Opening Shown by Stomata
0
40 c.
35°c.
65% of maximum opening
80% of maximum opening
0
„„
30 c.
65% of maximum opening
27 c.
70% of maximum opening
23&c;
65% of maximum opening
In considering the cause of this opening it
was thought that it could be due to either 6f two causes:(1) The accumulation of C 0 2
(2) The lack of oxygen.
Prom experiments carried out with different concentrations
of C 0 2 it was found that the opening was not so rapid or
of such magnitude.
The time required was usually over twenty-
four hours - Scarth (1932) Ferguson and Whyte (unpub. research)
32.
Also the pH of the guard cells does not favour this point
of view. Experiments were then undertaken to find the
effect of lack of oxygen on stomatal movement.
33.
Experiments on the Effect of the Lack of Oxygen
on stomatal Movement in the Dark.
The experiments were more of a qualitative than
of a quantitative nature and further work will be necessary
to clear up some of the finer details but the results obtained
were such that there can be no doubt as to their significance.
If all the oxygen be removed from the air in the
container, death of the leaf soon occurs. After trial and
error it was found that opening occurs most rapidly when
almost all the oxygen is removed.
The chief drawback of the
method employed is that a considerable period passes between
placing the leaves over the oxygen absorbing mixture and when
it is deemed that sufficient oxygen has been removed,
further
experiments will be carried out by placing leaves in atmospheres of known oxygen concentration which will permit of a
better comparison between the different experiments being made.
As it was, a measured rise of the alkali pyrogallol was
allowed to take place and then a further absorption of oxygen
prevented by running a thick layer of oil over it.
In experiments conducted at room temperature and
allowed to tun for nineteen hours, the stomata of wilted and
turgid leaves opened widely.
Others, in which the effect
of temperature was considered, shewed clearly the accelerating
effect of an increase in temperature.
Duration of experiment - 3 hours
34.
Temperature
Opening Shown by Stomata
37°c.
45% of maximum opening
31°c.
60% of maximum opening
25°c.
30Jo of maximum opening
13 c.
15% of maximum opening
The behaviour of the starch in the guard cells as well as
the
pH, on opening was found to agree with our previous
experiments with stomatal opening in the dark.
35.
Review and Discussion of Temperature and its
Effects on Stomatal Movement in the Dark.
In all our experiments in relation to temperature
and stomatal movement there is a clear indication that* with
an increase in temperature, there occurs up to a point, an
increase in the rate and amount of opening; above this point
rapid but slight opening takes places, followed by closure.
At lower temperatures than the optimum, opening is progressively
slower until, at very low temperatures, no movement of the
guard cells occurs. As between excised leaves and leaves
still attached to the plant, there is a difference in the
amount of opening occurring as well as in the optimum temperature.
With leaves surrounded with different media, the rate
and amount of opening do not agree, the relative rates of
opening being of the follovfing order;Leaves in oil, in almost completely deoxygenated air, in
water, in moist chamber, and leaves in air while still attached
to the plant.
The starch in the guard cells of open stomata
has to a great extent disappeared and also, with increase in
temperature, the rate of hydrolysis of the starch also increases.
At high temperatures (40°c.+), where the stomata open rapidly
but soon close again, the starch in the guard cells continues
to disappear even after the closure of the stomata has
occurred.
The explanation given for the lack of movement of
the stomatal apparatus even with an increase of osmotically
36.
active substances in the guard cells agrees with that of
Scarth and Brown (unpub.) and is that there must be a change
in the permeability of the guard cells which permits the
sugar formed by the hydrolysis of the starch to escape, thus
preventing any increase in turgor and ultimately, movement.
The p.H of the vacuoles of the guard cells of open stomata
were more alkaline than those of closed stomata.
Shy should there be a variation in the rate of
opening of stomata when leaves are in different environments?
This appears to be due to the degree of interference with the gas exchange of the leaves. By placing
leaves in oil, communication with the atmosphere is almost
entirely cut off.
This applies also to leaves in an oxygen
'deficient atmosphere and it is only a matter of experiment
until one should be able, by regulating the amount of the
oxygen present, to obtain the same rate of opening as with
leaves in oil. With leaves in water, the isolation is not
quite so complete but more so than in the case with leaves
in the moist chamber.
The puzzling feature is why should
there be a lower optimum temperature in the case of leaves
still attached to the plant.
In the light of the evidence
available, it would appear that the only feasible
explanation is that, due to the fact that the containers
projected a few inches above the surface of the water and
that in all probability the lids were not truly airtight,
above and below room temperature the differences in the
inside and outside temperatures of the tins would cause air
37.
currents to circulate through them and in this manner
preventing the occurrence of an oxygen deficiency.
This
explanation is probably the one that holds for the differences
obtained between the moist chambers as originally used by
Scarth and Brown and the ones used in this present project.
Therefore, although at first sight it would appear that
temperature directly affects the rate of stomatal movement
in the dark, in view of the evidence presented, it is
obvious that the effect is an indirect one, acting through
its effect on respiration, the rate of which is increased
by a rise in temperature, thus causing an oxygen deficiency
conducive to opening to be reached sooner than at a lower
temperature.
Thus the effect of temperature on stomatal
movement is threefold.
(l) At high temperatures by causing an increase in
permeability of the guard cells and thus preventing opeming.
(2) Increasing the rate of starch hydrolysis.
(3) By increasing the rate o£ respiration and thus
bringing about a deficiency of oxygen which in all probability
produces a suitable
p H for the hydrolysis of starch.
38.
Night Opening of Stomata
While the statement that stomata open in the light
and close in darkness is generally made, it has been known
for a considerable time that this is too sweeping a generality,
as the stomata of some plants show night movements while
those of others never close (Leitgeb 1888, Darwin 1398,
Lloyd 1908, stahl 1919, aurgenstein 1920, Loftfield 1921,
-iaximov 1928).
Eor sake of clarity, four types of stomatal
movement may be denoted:(a) Those which never close.
(b) Those which normally open in the light and close in
the dark.
(c) Those which normally open in the light and close in
the dark but may show night opening.
( d) Those which remain closed by day and open at night.
It must be noted that such a classification is not a clear
cut one, aj there are intermediate types which show a
gradation of behaviour between the four main classes.
Leitgeb found that with prolonged darkness there
is a tendency for stomata to open but that it is very erratic.
It was his belief that the opening is due rather to the
continued effect a moist atmosphere than to darkness - a
belief which is not consistent with his views in regard to
closure.
Darwin criticized this and stated that in all
probability the opening was owing to the epidermal cells
being less resistant and losing their turgor before the
39.
guard cells do.
Lloyd agreed with Darwin's criticism but,
while he suggested that it is possible that the osmotic
adjustments which occur during darkness between the cells of
the stomata and the epidermis on the one handfand the cells
of the epidermis arid the-chlorenchyma on the other, may
account for the movement, he put forward the view that the
opening is probably of an enzymatic nature and this is in alj.
likelihood much nearer the truth than any of the other views.
Loftfield, as already quoted, explains night opening as due
to the increase, or rather recovery, of leaf turgor.
In our experiments in the dark with turgid and
non-turgid leaves placed in oil or. in an oxygen deficient
atmosphere, it was found that the stomata opened widely and
thus it would appear that leaf turgor is not the controlling
factor of stomatal movement in the dark but that they open
because of a lack oi oxygen.
If we consider the case of the thin-f.leave".d
mesophytes which generally open in light and close in
darkness but, as Loftfield reports, under certain conditions
may show night opening after day closure, it is suggested
that this longer period with the stomata closed permits
of a condition of oxygen deficiency occurring in the leaf
which causes the stomata tp open, and not due, as Loftfield
states, to increase in turgor.
It would be fitting, here, if we considered the
work carried out by various workers on the effect of
environmental factors on respiration.
40.
Respiration and Some factors Affecting It.
Many e±perimenters have considered the effects
of the various environmental factors on respiration.
The
material used has been very varied and this variety perhaps
helps to explain the unsatisfactory position of the literature
on this topic. Usually, seeds are used for experimental
study, but f^ngi, algae, seedlings and discs of potato, carrot
and, in some cases, leaves and fruits have been used.
Temperature
Those who have studied this problem seem to be
more or less in one accord that, with increasing temperature,
the rate of respiration increases very much in accordance with
Van't Hoff's rule, the temperature coefficient being 2 - 3 .
Kostychev (1927), Lundegurdh (1931), Millar (1931).
Moisture Content
Starting with "dry" material, e.g. seeds, it
has been found that the rate of respiration increases with
an increase in the water content.
As far as the higher plants
are concerned, such data is of little value as such a state
as "air dried" would mean the death of the plant.
Smith
(1916), using a vacuum dessecator reduced the water content
of leaves of snowdrop and stem tips of nasturtium and young
asparagus and found that there was an increase of respiration
until 30/o of the water had been withdrawn.
The rate then
remained constant until the total withdrawal of water reached
50%, after which, until complete dryness was reached, the
rate fell in direct proportion to the water lost.
41.
Oxygen Concentration
Saussure (1833) reported that no effect was
observed on the rate of respiration when the oxygen content
of the air was reduced to half the normal concentration.
Wilson (J885) subsequently proved that the plants respire
normally in an artificial mixture of 0.2 parts atmosphere
air and 0.8 parts hydrogen, the rate being the same as in
normal air.
^ven with an oxygen content of 1%, no checking
of plant respiration could be noted I see ilostychev, 1927).
On the other hand, stich (1891) states that the absorption
of oxygen becomes insufficient for most plants when the
supply in the air falls below 5 - 8%.
Xidd, west and sriggs
(1921) found that the carbon dioxide output from plant
material, starting trom low pressures of oxygen in the
atmosphere, increases with rising partial pressures, at
first rapidly, then more slowly, until a maximum is reached,
ana then decreases,
uhevillard, Hamon, Mayer and r'lanlefol
I 1930) have shown that the respiratory quotient in air is
approximately unity, though it may deviate somewhat from
this value in mixtures with low oxygen content.
According
to Steward, bright and Berry (1952), with changes in the
oxygen supply there appeared corresponding changes in the
respiration, as measured by carbon dioxide produced.
These
later, workers used discs of potato, a highly aerobic type of
plant material.
Of the factors affecting the rate of respiration,
it will be seen that temperature markedly affects the rate,
42.
which is in agreement with our views on the mechanism of
temperature in its relationship to the opening of stomata
in the dark.
With regard to moisture content, it is hard
just to know what will happen in the plant.
Workers starting
with "air dry" material believe that there is an increase
in respiration with increased water content, until, in some
cases, the latter is 600^ greater than in the beginning,
whereas Smith (19J.6) shows that with a 30% decrease of
water content there is an increase in the rate of respiration.
As far as material 4sed in the experiments is concerned, that
of Smith is nearer to our own but before any statement can
be made further wori would need to be carried out with
material comparable to our own.
In the case of the effect of oxygen content of
the air on respiration, the view we tend to uphold is that
of £idd, West and Briggs and of Stewart, etc., and is that
with low oxygen pressures the rate of respiration is slow.
This problem of the effects of external factors
on respiration in relation to stomatal movement will need
to be studied more thoroughly before any true understanding
of the mechanism of night opening can be reached.
43.
Summary of Experimental Results
Temperature
(l) The rate of stomatal movement in the dark is increased
with a rise in temperature.
(2) The hydrolysis of starch to sugar is increased with a
rise in temperature.
(3) At high temperatures the stomata do not open because
of an increased permeability of the guard cells.
(4) The pH of the guard cell vacuoles of stomata that
have opened in the dark is higher than in that of closed
stomata and is comparable to that of those which have
opened in the light.
Turgor
(l) The passive system of stomatal movement acts 4fter that
of StSlfelt, but in a lesser degree, and ordinarily seems
to be of little biological significance.
(2) There is no correlation between leaf turgor and stomatal
movement in the dark.
Gas Exchange
(i) The stomata of non-turgid and turgid leaves placed in
an oxygen deficient atmosphere opened widely.
(2)
opening from a similar cause was also obtained by
placing leaves in Nujol, water, etc.
Conclusions
It is suggested that night opening of stomata
is due to a lack of oxygen and not, as Loftfield writes, to
44.
leaf turgor being regained.
The effect of temperature is an indirect one
and acts by increasing the rate of respiration, thus bringing
about more speedily than at lower temperatures an oxygen
deficiency.
In all probability, this produces a suitable
p,H. for the initiation of the hydrolysis of starch to
sugar, which, once it is started, is also accelerated by
an increase in temperature.
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