Journal of Experimental Botany, Vol. 47, No. 296, pp. 339-347, March 1996
Journal of
Experimental
Botany
An analysis of relative elemental growth rate, epidermal
cell size and xyloglucan endotransglycosylase activity
through the growing zone of ageing maize leaves
Stephen J. Palmer1 and William J. Davies
Division of Biological Sciences, Institute of Biological and Environmental Sciences, Lancaster University,
Bailrigg, Lancaster LA14YQ, UK
Received 12 September 1995; Accepted 27 November 1995
Abstract
The effect of development on leaf elongation rate (LER)
and the distribution of relative elemental growth rate
(REG/7), epidermal cell length, and xyloglucan endotransglycosylase (XET) activity through the growing
zone of the third leaf of maize was investigated. As
the leaf aged and leaf elongation slowed, the length
of the growing zone (initially 35 mm) and the maximal REGR (initially 0.09 mm m m " 1 h" 1 ) declined. The
decline in REGR was not uniform through the growth
profile. Leaf ageing saw a maintenance of REGR
towards the base of the leaf. Epidermal cell size was
not constant at a given position in the growing zone,
but was seen to increase as the leaf aged. There was
a peak of XET activity close to the base of the growing
zone. The peak of XET activity preceded the zone of
maximum REGR. XET activity declined as leaves aged
and their elongation rate slowed. When leaf elongation
was complete a distinct peak of XET activity remained
close to the base of the leaf.
Key words: Leaf elongation rate (LER), relative elemental
growth rate [REGR), xyloglucan endotransglycosylase
(XET).
Introduction
Maize leaves, in common with other graminaceous leaves,
have a basal growing region enclosed by older leaves
(Begg and Wright, 1962; Davidson and Milthorpe, 1966;
Boffey et al, 1980; Kemp, 1980; Volenec and Nelson,
1982; Schnyder et al, 1990). Within this region growth
is largely unidirectional, with a basal meristem producing
1
parallel files of cells (Sharman, 1942; Mac Adam et al,
1989). Cells are displaced away from the meristem by the
production and elongation of new cells. As a cell is being
displaced it undergoes expansion and differentiates. In
this way the distance of a cell from the basal meristem is
a reflection of its developmental age. This developmental
gradient makes the growing zone of maize and other
graminaceous species well suited to the study of biophysical and biochemical processes associated with growth
(Boffey et al, 1980; Schnyder and Nelson, 1987; Gandar
and Hall, 1988; Schnyder et al, 1990). Such studies
require data on the spatial distribution of growth rates
within the elongation zone. Data of this type have been
described for a number of species, e.g. leaves of tall
fescue (Festuca arundinacea Schreb.) (Schnyder and
Nelson, 1987, 1988), maize (Hsiao et al, 1985), sorghum
(Bernstein et al, 1993), and roots of maize (Sharp et al,
1988; Pritchard et al, 1993). However, only one study
(Schnyder et al, 1990) has considered the spatial distribution of growth rates at different stages of a leaf's development. The study of Schnyder et al. (1990) considered the
effect of development on the spatial distribution of growth
rate in perennial rye-grass. This study looks at the effect
of development on the spatial distribution of growth rates
in developing maize leaves. The study was undertaken as
the basis of future studies which will address the effects
of water deficits and abscisic acid on leaf elongation at a
spatial resolution. It was felt that the effect of leaf age
could, potentially, have implications for the design of
such experiments.
Over the past 4 years considerable attention has been
given to the activity of a recently discovered enzyme,
xyloglucan endotransglycosylase (XET) (Fry et al,
1992). XET has been proposed to allow cell expansion
To whom correspondence should be addressed. Fax: + 44 1524 843854. E-mail: S.Palmer©lancaster.ac.uk
© Oxford University Press 1996
340
Palmer and Davies
by cleaving xyloglucan molecules that tether adjacent
cellulose microfibrils. It is also suggested that XET
re-joins the cut ends of cleaved tethers (Smith and Fry,
1989; Fry et al., 1992). The net effect of this may be a
separation of cellulose microfibrils allowing controlled
cell expansion (Passioura and Fry, 1992). XET activity
has been shown to correlate with the spatial distribution
of elongation rate at one developmental stage in pea
stems (Fry et al., 1992) and maize primary roots
(Pritchardera/., 1993; Wu etal, 1994). To our knowledge
this is the first study of the spatial distribution of XET
activity at different stages of leaf development.
The spatial pattern of REGR, epidermal cell lengths,
and XET activity have been examined at four stages
during leaf elongation and twice after elongation was
complete. Spatial aspects of growth, epidermal cell size
and XET activity are discussed in detail.
Materials and methods
Seeds of maize (Fl Earli King, DT Brown & Company Ltd.,
Poulton-le Fylde, Lancashire, UK) were soaked overnight in
distilled water then germinated on moist tissue paper for 48 h.
Plants were grown in pots containing a commercial potting
compost (Sinclair Horticultural Products, Lincoln, UK).
Seedlings were covered in a 10 cm layer of vermiculite
(Vitagrow, Vitagrow LTD, Hull, Humberside, UK). The layer
of vermiculite promoted development of a sub-crown internode
and allowed easy excavation to access leaf bases. Plants were
watered on a daily basis and raised in a growth cabinet. The
photoperiod was set to 14 h, light intensity at plant height
c 200 ftmol m~ 2 s ~ \ daily temperature maxima c. 30 °C and
minima c. 20 °C.
Leaf length and leaf elongation rate
Eight plants were selected randomly and the length of the third
leaf measured daily with a piece of graph paper photocopied
on to acetate. Daily average leaf elongation rates (LER)
in mm h " 1 were calculated.
At six intervals through the experiment the relative elemental
growth rate (REGR), size of adaxial epidermal cells and
xyloglucan endotransglycosylase (XET) activity through the
growing zone were measured.
Spatial distribution of growth
REGR was measured by recording the displacement of marks
during short periods of time (Silk, 1984; Schnyder et al, 1987).
Eight plants were selected at random and vermiculite removed
from around the stem to allow access to the base of the third
leaf. Ten fine pins mounted in a copper block 2 mm apart were
used to mark the growing zone, which previous experiments
had shown to have a maximal length of c. 35 mm. The basal
mark was placed at the top of the crown which corresponds to
the base of the third leaf. The pins were then inserted again
with the bottom pin inserted into the hole made by the top pin
in the first insertion. This resulted in a series of 19 holes
vertically arranged from the base of the third leaf with an initial
spacing of 2 mm. Marked plants were then sampled after a
period of growth. The time before sampling was between 6 h
(fastest growing leaves) and 12 h (slowest growing leaves). Leaf
elongation between pinning and sampling never exceeded 25%
of the length of the growing zone. In order to determine the
displacement of holes in the third leaf, the coleoptile and first
two leaf pairs were removed. The distance between the holes
was then measured with an ocular graticule (Ernst Leitz Wetzlar
GmbH, D-6330 Wetzlar, Germany). Pinning reduced the
elongation rate of the leaf by c. 30%. However, a previous
study showed that this reduction is constant through the growth
zone and, therefore, does not affect the spatial distribution of
growth (Schnyder et al., 1987).
Epidermal cell length
Epidermal cell size was determined through the growing zone
of five randomly selected plants in the time interval between
pinning and sampling of marked leaves. The coleoptile and first
two leaf pairs were removed and the third leaf sectioned by
parallel razor blades spaced 2 mm apart. Sections of the third
leaf were kept in water until measurement of cell size. Epidermal
cell length was measured on files of cells adjacent to files
containing stomata. The adaxial epidermis was viewed directly
with a projection microscope at x 30 magnification. A minimum
of 10 determinations of epidermal cell length were made on
each section viewed. The mean cell length was assumed to
represent cell length at the centre of the section.
XET activity
All chemicals used were purchased from Sigma (Sigma, Poole,
Devon UK) except labelled reduced xyloglucan nonosaccharide
[ 3 H] XLLGol (Fry et al, 1993) and xyloglucan which were
kindly provided by Dr SC Fry. After sampling of pinned leaves,
8 plants were chosen at random for determination of XET
activity through the growing zone of the third leaf. The outer
two leaves and the coleoptile were removed and the third leaf
sectioned using parallel blades spaced 3 mm apart, 10 samples
through the growing zone were collected. Care was taken to
remove any younger leaves which were found within the third
leaf. This was not possible on the basal section which included
material from the crown. Samples were collected in sealed vials
held on salted ice until 8 sections had been collected when a
determination of fresh weight was made. Samples were then
stored in a freezer (—20 C C) for subsequent determination of
XET activity.
XET activity was determined according to the procedure of
Fry et al. (1992). The method utilizes the binding of long-chain
xyloglucan to cellulose (chromatography paper), the rate of
incorporation of labelled short-chain xyloglucan into long-chain
xyloglucan being measured. Briefly, samples were ground in
ice-cold buffer (Buffer A) with acid-washed sand (50 mM
succinic acid, 10 mM CaCl 2 , 1 mM dithiothreitol, pH adjusted
to 5.5 with 1 M NaOH) at a ratio of 10:1 buffer to sample
fresh weight. Care was taken to ensure tissue samples were
completely homogenized. Samples were returned to the freezer
until all samples had been ground. Samples were centrifuged at
2 000 g for 5 min and the supernatant (tissue extract) retained.
10 /A of the tissue extract was added to 50 /A of Buffer A
containing 0.3% xyloglucan (from Tropaeolum seeds), 0.5%
chlorobutanol and 70 kBq ml" 1 of [3H] XLLGol. The reaction
mixture was mixed and incubated for 1 h at 25 °C. After 1 h
the reaction was stopped by addition of 100 /j.1 of 20% formic
acid. The 160 jA of reaction mix was pipetted on to 6 x 4 cm
rectangles of Whatman 3MM chromatography paper
(Whatman International Ltd., Maidstone, Kent, U K ) and
allowed to air dry. Unreacted [3H] XLLGol was removed from
the chromatography paper by placing the rectangles of paper
in a sink of running water for 1 h. The paper was dried
overnight before being counted in a scintillation counter
Elongation dunng leaf development
(Packard 1600 TR, Packard Instrument Company, Meriden,
CT, USA) using 2ml of Ecoscint H (National Diagnostics,
Atlanta, GE, USA). XET activity was calculated from the
fraction of total label incorporated into product (labelled
xyloglucan which bound to chromatography paper). Background levels of activity on the chromatography paper were
measured by omitting the enzyme extract from the assay. This
activity (less than 1% of the total activity) was subtracted from
the measured activity. XET activities were expressed per unit
fresh weight. Activity is taken to represent the activity at the
centre of the section. The basal section which includes tissue
from the crown is shown at distance 0 mm from the base of the
third leaf.
In a follow-up experiment seedlings of maize were grown as
described previously until the third leaf had completed its
expansion. Leaf impressions were taken along the profile of the
leaf length using a 4% solution of formvar in chloroform
(Sigma, Poole, Devon, UK). Adaxial epidermal lengths were
measured on the impressions using a projection microscope at
x 30 magnification.
Results and discussion
Leaf length and LER
Leaf length follows a sigmoidal curve (Fig. 1) with final
leaf length attained c. l i d after leaf emergence.
Elongation rate initially increased from c. 1.3 mm h " 1
over the period of 2-3 d after leaf emergence to reach a
maximal rate of c. 2.8 mm h~' at around 6 d after leaf
emergence. There was a period of 4 d between 3 and 7 d
after leaf emergence when leaf elongation was maximal
and approximately constant. From 7 d after emergence,
leaf elongation rate declined to reach zero approximately
11 d after emergence. A short period of constant growth
rate was also reported for Lolium perenne (Schnyder et al.,
341
1990). However, a number of graminaceous species show
constant growth rates during an extended period of leaf
development (Robson, 1972; Wilson, 1975; Gallagher,
1979; Durand et al., 1995).
In this study leaf length was measured on a daily basis
which ignores changes in the growth rate between the
dark and light periods. In studies where the meristem
temperature was held constant LER has been observed
to be higher in the night period (Volenec and Nelson,
1982; Parrish and Wolf, 1983; Schnyder and Nelson,
1988). However, here temperature was not held constant,
being lower at night. Previous studies (data not shown)
showed that growth rates were lower in the dark period
than in the light period (see also Gallagher and Biscoe,
1979).
Spatial growth distribution
At different times during the development of the third
leaf, the spatial distribution of REGR was determined
during the light period (Fig. 2). Sampling occasions were
chosen twice during the approximately constant and
maximal growth period (days 3-7 after leaf emergence),
and twice as leaf growth started to slow. On the sampling
occasions when leaf elongation was maximal spatial variation of REGR looked symmetrical and had a maximal
value near the centre of the growth zone. For leaves in
this period of maximal growth the maximal REGR was
c. 0.09 mm mm" 1 h " 1 and the length of the growing zone
was c. 35 mm. These two values can be compared with
values of 0.07 mm mm" 1 h " 1 and 14 mm for the first leaf
of maize (DS Thompson, personal communication)
0.14
0.12 -
o.oo o 0
2
4
8
8
10
12
14
Distance from leaf base (mm)
Time after leaf emergence (days)
Fig. 1. Time series of leaf length (O) and elongation rate ( • ) of the
third leaf. Each point represents the mean ± standard error of eight
measurements.
Fig. 2. Spatial distribution of relative elemental growth rate (REGR) 4
d (O), 6 d ( • ) , 8 d ( • ) , 10 d ( • ) , and 12 d (A) after emergence of
the third leaf. Each point represents the mean ± standard error of eight
measurements.
342
Palmer and Davies
0.08 mm mm" 1 h" 1 and 90 mm for the seventh leaf of
maize, Meiri et al. (1992). This suggests that the greater
elongation rate of subsequent leaves is due largely to an
increase in the length of the growing zone.
As leaf elongation rate slowed the spatial distribution
of REGR became asymmetrical and the growth zone
progressively shorter. Thus the length of the growing
zone decreased from its maximum of c. 35 mm to
c. 30 mm 8 d after leaf emergence and was only 15 mm
10 d after leaf emergence. The position of the maximal
REGR was also displaced towards the base of the leaf
moving from 15 mm at the time of maximal elongation
rate to 7 mm 8 d after leaf emergence and 3 mm from the
leaf base 10 d after leaf emergence.
The short period with stable leaf elongation and spatial
growth distribution of REGR in maize leaves has implications for the design of experiments investigating the effect
of treatments (for example, drought stress) on leaf growth.
Treatments must be carefully timed to correspond to the
period of stable growth and be of a short duration if
results are not to be confounded by developmental effects.
In maize this would make results from, for example, a
slow soil drying treatment difficult to analyse. This can
be compared with other species such as tall fescue where
an extended period of stable growth has enabled studies
on the response of leaf growth at a spatial resolution to
a drying treatment to be performed without consideration
of developmental changes (Durand et al., 1995).
As leaf elongation slowed REGR fell. However, the
reduction in REGR was not evenly distributed over the
profile. REGR was maintained towards the leaf base. In
the region just proximal to the leaf base REGR appears
to be completely maintained 8 d after leaf emergence even
though leaf elongation rate is only 40% of the maximal
rate. This maintenance of REGR towards the base is also
seen 10 d after leaf emergence. Maintenance of REGR
towards the base of ageing maize leaves has also been
shown by Meiri et al. (1992). Maintenance of leaf growth
towards the base of the growing zone has been shown in
studies where the REGR distribution in leaves or roots
has been determined following imposition of water
stress, e.g. Sharp et al. (1988) and Durand et al. (1995).
Bernstein et al. (1993) showed the same effect when the
elongation rate of leaves of sorghum was reduced by
imposition of salt stress.
In a series of papers Sharp and co-workers (Sharp
et al., 1988; Spollen and Sharp, 1991; Wu et al., 1994)
have argued that the maintenance of REGR in the apical
region of the growing zone of maize roots, which is seen
after seedlings are transferred from high to low water
potential vermiculite, provides evidence for changes in
cell wall properties. This is based on growth being maintained even though turgor pressure falls in cells close to
the root tip. However, it is perhaps important to note
that cells at different positions in the growing zone are at
different developmental stages. Many factors may change
with development and it is possible that maintenance of
growth close to the root tip could be a result of the
developmental gradient. For example, it is possible that
the elongation rate of young cells (close to the root tip)
is limited by enzyme activity while development (increasing position from the root tip) sees a switch to growth
being limited by cell turgor pressure. Transferring seedlings to a low water potential vermiculite which reduces
turgor pressure throughout the growing zone would
reduce the REGR in the basal regions where turgor is
controlling the elongation rate of cells while it may not
affect the elongation of cells close to the root tip which
are still limited by enzyme activity. An increase in the
yield turgor pressure with age, which would potentially
have this effect, has been noted before (Randall and
Sinclair, 1989). In a similar manner the responsiveness of
cells to growth regulators may also change with development (Atkinson et al., 1989; Saab et al, 1992).
Cells in the elongation zone are being displaced away
from the leaf base by the expansion of cells closer to the
leaf base. The displacement velocity for cells in the
growing zone is, therefore, not constant, but changes with
position in the growing zone. This distribution of velocity
through the growing zone (the displacement velocity) is
shown in Fig. 3 for the third leaf of maize 4 d after leaf
emergence. The profile is a sigmoidal curve with displacement velocity increasing to a maximum at the end of the
growing zone. Thus cells close to the base of the leaf will
experience an increase in displacement velocity as they
move away from the leaf base of the growing zone. It is
possible to describe this acceleration of cell displacement
0
5
10
15
20
25
Distance from leaf base (mm)
Fig. 3. Spatial distribution of displacement velocity through the growing
zone of the third leaf 4 d after emergence. Each point represents the
mean ± standard error of eight measurements.
Elongation during leaf development
by the construction of path lines (time-position relationships; Gandar, 1983). Path lines can be constructed by
setting an initial position of an element (cell). This
position will have a velocity associated with that position.
A new position of the element may then be obtained after
a chosen time interval and the process repeated. In this
way the position of the cell over a number of time
intervals can be calculated and path lines constructed.
This process has been followed for the velocity profile
obtained from the third leaf of maize, 4 d after its
emergence. A third order regression (coefficients [bO] =
-0.0052789, [bl] = 0.0145475, [b2] = 0.0041330, [b3] =
-0.000092576, r2 value = 0.9327) was fitted to the velocity
profile. These coefficients were then used in a simple
computer program which calculated the path line for
elements at a given start position. Within the program
the displacement of the element from successive positions
was calculated by allowing 1 h of growth. Path lines for
elements with initial positions 1, 2, 3, 4, 5, 10, and 20 mm
from the leaf base are shown in Fig. 4. These path lines
clearly demonstrate that cells close to the base experience
very slow displacement. For example, a cell found 1 mm
from the base will take some 80 h to be displaced to a
position 5 mm from the base. However, cells located
5 mm from the leaf base take only 28 h to be displaced
through the remaining 30 mm of the growing zone. This
data is in close agreement both with the data of Meiri
et al. (1992) who showed that a particle located initially
6 mm from the base of the seventh leaf of maize would
take some 3 d to be displaced to the end of the growing
343
zone and MacAdam et al. (1989) who were able to show
that c. 110 h were needed for displacement of a cell
originally 5 mm from the leaf base in tall fescue to the
end of the growing zone.
Path lines enable the estimation of how long cells are
in the growing zone. Figure 4 shows that it will take over
120 h for a cell to pass through the length of the growing
zone. Path lines have only been constructed for leaves 4
d after emergence. Construction of path lines at different
developmental stages would require the use of velocity
fields from different stages. For this reason the path line
constructed for a cell 1 mm from the base of the third
leaf is likely to be slightly erroneous. To a first approximation growth of the leaf is stable for c. 4 d while the
path line constructed suggests that c. 120 h are required
for the cell to be displaced to the end of the growing
zone. It should also be noted that this analysis ignores
diurnal changes in displacement velocity which are likely
to have occurred.
Epidermal cell length distribution
The length of epidermal cells increased greatly with
distance from the leaf base at any sampling occasion
(Fig. 5). Cells from leaves sampled 4 d after leaf emergence were c. 20 /xm in length close to the leaf base and
reached 120 ^un close to the end of the growing zone
(29 mm from the base). Thus as cells pass through the
growing zone they expand. The local relative stretching
rate has been termed the relative elemental growth rate
(Erickson and Sax, 1956). However, in the maize leaf an
350 -
300
40
60
80
100
120
140
Time (hours)
Fig. 4. Path lines showing the displacement of elements spaced 1 mm
(a), 2 mm (b), 3 mm (c), 4 mm (d), 5 mm (e), 10 mm (f), and 20 mm
(g) from the leaf base at time = 0 through the growing zone of the third
leaf. The dashed line indicates the distal limit of the growing zone.
5
10
15
20
25
30
35
Disunce from leaf base (mm)
Fig. 5. Spatial distribution of adaxial epidermal cell length 4 d (O),
6 d ( • ) , 8 d (D), 10 d ( • ) , and 12 d (V) after emergence of
the third leaf. Symbols represent the mean of at least 50
measurements ± standard error.
344
Palmer and Davies
increase in cell size with distance from the leaf base is
also seen on leaves which are no longer elongating (leaves
sampled 12 d after leaf emergence). The cessation of leaf
elongation sees cells frozen in a gradient of cell size
increasing from the base.
Figure 5 shows that cell size at any one position in the
growing zone is not constant, but increases with leaf age.
Thus cells 29 mm from the growing zone are c. 120 ^m
long 4 d after leaf emergence and 280 /^m long at the
same position 10 d after leaf emergence. This means that
cells leaving the growing zone of the third leaf later in its
development will be larger than cells which completed
their expansion earlier in the development of the third
leaf. These results imply that a mature third leaf of maize
should show a profile of epidermal cell length decreasing
with distance from the leaf base (after the initial increase
through what was.the growing zone). This was checked
in a follow-up experiment where the length of adaxial
epidermal cells on files of cells adjacent to files containing
stomata were measured. Figure 6 shows that this was the
case. Cells close to the leaf tip had an average length of
125 ^m while this length increased steadily to 200 /urn at
a position just above the leaf ligule. The length of the
sheath cells (cells basal to the ligule) were still longer with
an average length of 260 /xm.
The question then becomes why do we see this effect
of leaf age on epidermal cell size? Cell size is determined
by the cumulated difference between growth rate and cell
division rate (Green, 1976; Silk, 1992). Since the REGR
has been shown to decline after emergence (Fig. 2) it
must follow that the epidermal cell division rate has
declined even more.
Distribution of XET activity through the growing zone
XET activity has been expressed on a fresh weight basis.
Expressing the results in terms of dry weight had no
significant effect on the shape of XET distribution (data
not shown). XET has been expressed on a fresh weight
basis because given adequate turgor cell elongation is
thought to be limited by the extensibility of a thin layer
of the cell wall. The effectiveness of XET at causing cell
elongation would then depend on the number of mm 2 of
this layer that each unit of XET has to work on. In the
case of a maize leaf where growth is almost uniaxial, the
important measure would be the side wall area which
should be roughly proportional to fresh weight.
XET activity is not constant through the growing zone,
but follows a typical profile. Activity is low in the crown
(0 mm from the base of the leaf), increases to a maximal
activity 3 mm from the leaf base then declines rapidly to
reach an approximately stable activity 10 mm from the
leaf base (Fig. 7). In this way there is a peak in XET
activity which occurs close to the leaf base. When comparing the spatial distribution of XET activity with the
spatial distribution of growth (Fig. 8) it is apparent that
the peak of XET activity precedes the peak in REGR.
Also XET activity was the same at the position of
maximal REGR as it was at the end of the growing zone.
This was the case at each sampling occasion (compare
350
80 -
300 -
250 -
J . 200 o>
c
CD
=
150 -
s
100 50
10
15
20
25
30
35
40
0
5
10
15
20
25
JO
Distance from leaf base (mm)
Distance from base of leaf (cm)
Fig. 6. Profile of adaxial epidermal cell length along a mature third leaf
of maize. The position of the ligule is indicated by the arrow. Symbols
represent the mean of at least 20 measurements ± standard error.
Fig. 7. Spatial distribution of XET activity 4 d ( ^ ) , 6 d ( • ) . 8 d ( ),
10 d ( • ) . 12 d C"7). and 15 d ( • ) after emergence of the third leaf.
Samples from eight plants were pooled for each measurement of
XET activity.
Elongation during leaf development
0.14
90
0 12 -
0.10 0.08 -
or 0.06 CD
0.04 -
0.02 -
0.00
5
10
15
20
25
30
35
Distance from leaf base (mm)
Fig. 8. Spatial distribution of REGR (O) and XET activity ( • ) 4 d
after the emergence of the third leaf
Fig. 2 and Fig. 7). Figure 8 shows that the peak of XET
activity occurs c. 3 mm from the leaf base while maximal
REGR occurs at c. 15 mm from the base. This relationship
between the spatial distribution of XET activity and the
spatial distribution of REGR has also been shown to
occur in roots of maize (Pritchard et ai, 1993; Wu et ai,
1994). Given the in vitro activity of XET it has been
suggested that, in maize roots, the burst of XET activity
close to the meristem may be important in allowing the
peak in REGR (Pritchard et ai, 1993). From the growth
trajectory (Fig. 4) it can be inferred that, in maize leaves,
the spatial separation represents a temporal separation of
c. 50 h. In maize roots the temporal separation between
peak XET activity and maximal REGR can be inferred
to be c. 4 h from data on the spatial distribution of XET
activity (Pritchard et ai, 1993; Wu et al., 1994) and the
growth trajectory for maize roots (Silk, 1992).
Values of XET activity are very similar to values
reported by Wu et al. (1994) in the growing zone of
maize root. Wu et al. (1994) report maximal values of
XET activity of c. 60 Bq KBq mg" 1 fr. wt. h" 1 falling to
c. 25 Bq K B q - ' m g " 1 fr. wt. h " 1 at the base of the
growing zone. The similarity in XET activity occurs
despite local REGR being c. 4 times lower in shoots than
roots (compare Fig. 2 with Fig. 2 from Wu et ai, 1994).
XET activity through the growing zone falls as the leaf
ages. 4 d after leaf emergence maximal XET activity of
78 Bq KBq ' mg ' fr. wt. h ' was found 3 mm from the
leaf base. 6 d after leaf emergence this figure was 60 Bq
K B q " ' m g ~ ' fr. wt. h " 1 reducing further to 25 Bq
KBq ~' mg ~' fr. wt. h " ' 10 d after emergence and 16
Bq KBq" 1 mg" 1 fr. wt. h" 1 15 d after emergence. It is,
however, noticeable that a distinct peak of XET activity
345
still remains close to the leaf base even where leaf elongation has ended (see profiles after 12 d and 15 d). Evidence
that the high activity of XET which occurs early in the
growing zone is important in allowing growth has been
provided by the work of Wu et al. (1994) on growing
roots of maize. In this study the maintenance of growth
seen close to the tip of roots transferred to vermiculite of
low water potential was shown to be dependent on an
abscisic acid-induced accumulation of XET activity.
However, as yet no other study that we are aware of has
gone beyond identifying a correlation between XET activity and growth rate. Such correlations can be quite
compelling, in this study XET activity has been shown to
precede maximal segmental elongation rates and the
decline in XET activity shown to precede the developmental fall in leaf growth. However, the spatial and
temporal correlations are complicated. In particular, the
residual peak in XET activity after growth cessation
weighs against a simple causal relationship. It is, however,
perhaps pertinent to note that one likely confounding
factor is that a single tissue may be limiting the extension
of the leaf (Hodick and Kutschera, 1992; Kutschera,
1992). In organs such as leaves this is often argued to be
the epidermis. Studies have shown that the epidermis and
mesophyll can have different temporal periods for cell
division and expansion (MacAdam et ai, 1989). In this
way one explanation for the persistence of the peak of
activity found at the leaf base is of XET activity not
associated with wall loosening in the growth limiting
tissue.
Conclusions
Maize leaf elongation rate has been shown to be constant
only for a short developmental period. The decline in
LER as leaves aged was related both to a decrease in the
length of the growing zone and the maximum REGR. As
leaves aged and LER fell there was a maintenance of
REGR towards the base of the growing zone. A similar
effect has been reported for maize roots growing in dry
vermiculite (Sharp et ai, 1988), sorghum leaves subjected
to salt stress (Bernstein et ai, 1993) and tall fescue leaves
subjected to drought stress (Durand et ai, 1995).
The length of epidermal cells increases from the base
of the leaf to reach a maximal value as cells cross the
distal limit of the growing zone. However, cell length is
also dependent on the age of the maize leaf. As leaves
age cells passing the distal limit of the growing zone
increase in length. This increase in the length of epidermal
cells is a result of epidermal cell division rate declining
ahead of the decline in REGR.
Maximal XET activity is shown to precede the maximal
REGR by c. 10 mm which corresponds to a temporal
separation of c. 50 h. XET activity is reduced as leaves
age and their elongation rate falls. The reduction in XET
346
Palmer and Davies
activity precedes the fall in LER. That XET activity peaks
before maximal REGR and declines ahead of the fall in
LER as leaves age may be taken as supporting evidence
for XET activity as a control point for leaf expansion.
However, the spatial and temporal correlations are complicated and the residual peak in XET activity after the
cessation of leaf elongation weighs against a simple causal
relationship.
Acknowledgements
The authors would like to thank Dr SC Fry for supplying
material for the XET analysis and critical comments on results,
Andrew Jarvis for his interest and help with analysis of results
and Dr Jeremy Pritchard and Dr Wendy Silk for their critical
reading of this paper. This research has been supported by a
grant from the EEC, contract number AIR-CT 1031.
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