Journal of Experimental Botany, Vol. 48, No. 317, pp. 2075-2085, December 1997
Journal of
Experimental
Botany
Can cell wall peroxidase activity explain the leaf growth
response of Lolium temulentum L. during drought?
Mark A. Bacon1, David S. Thompson and William J. Davies
Biological Sciences Department, Institute of Environmental and Natural Sciences, Lancaster University,
Bailrigg, Lancaster LA1 4YQ, UK
Received 14 April 1997; Accepted 18 July 1997
Abstract
In two experiments, the effect has been investigated
of a mild and a more prolonged drought on the spatial
distribution of growth, epidermal cell lengths and cell
wall peroxidase activities in the leaf elongation zone
of the grass species Lolium temulentum L. In both
experiments drought reduced the size of the elongation zone and local rates of elongation within it. Abrupt
increases in cell wall-associated peroxidase activity
occurred at or close to the position where elongation
ceased in the leaf elongation zones of well-watered
and mildly drought-stressed plants. More prolonged
drought caused a 200-300% increase in the cell wallassociated peroxidase activity in the elongation zone
only. The significant increase in the elongation zone
cell wall peroxidase activity and its spatial variation
provides evidence of a potentially causal role for cell
wall-associated peroxidase in restricting cell expansion during drought.
Key words: Cell wall peroxidase, leaf expansion, drought.
Introduction
Leaf growth is one of the main determinants of plant
productivity. For a variety of crops, dry matter production
has been shown to increase linearly with the amount of
solar radiation intercepted (Monteith, 1977), which is itself
a direct function of leaf development. Of all plant processes,
leaf growth is one of the most sensitive to drought (Hsiao,
1973) and in fertile soils, plant growth and yield appears
to be more sensitive to water availability than to any other
unfavourable factor within the environment (Boyer, 1982;
Kramer, 1974). It is therefore of considerable economic
importance to understand the processes which govern the
rate of leaf expansion during water deficit.
' To whom correspondence should be addressed. Fax: + 44 1524 843854.
© Oxford University Press 1997
Grasses are useful experimental systems as well as
forming the major crop species of the world. Grass leaf
growth is primarily unidirectional, occurring within a
basal meristem which produces parallel files of cells. These
cell files are displaced through the elongation zone by the
production and subsequent elongation of new cells from
the region at the very base of the elongation zone where
cell division occurs. As cells are displaced they expand
and differentiate, thus the distance between a cell and the
leaf base is a function of its developmental stage and age
(Schnyder et ai, 1990).
Although leaf water relations play a vital role in the
determination of the rate of leaf growth, observed reductions in leaf growth cannot always be accounted for by a
decline in leaf turgor (Termaat et ai, 1985; Thomas et ai,
1989; Van Volkenburgh and Boyer, 1985), surprisingly
even during water deficit (Gowing et al., 1990; Passioura,
1988; Puliga et al., 1995). Michelena and Boyer (1982)
demonstrated convincingly that, even though leaf elongation rates of maize growing in drying soil were significantly reduced, sufficient solutes accumulated in the
elongation zone to maintain virtually constant turgor.
Attention has therefore focused on the modification of
cell wall properties during water deficit (Pritchard and
Tomos, 1993; Neumann, 1995; Spollen et al., 1993; Wu
et al., 1994) to explain the reduction in leaf growth rate.
Several cell wall enzymes, including xyloglucan endotransglycosylase (XET), cell wall peroxidases and expansins have been implicated in the regulation of growth rate
via biochemical modification of cell wall properties (Fry,
1995; Goldberg et ai, 1987; MacAdam et ai, 1992a;
McQueen-Mason and Cosgrove, 1994; McQueen-Mason,
1995; de Silva et ai, 1994).
The formation of phenolic cross-linkages between cell
wall components, mediated by cell wall-associated peroxidase enzymes has been proposed to account for reduced
2076
Bacon et al.
plant cell expansion observed in a number of situations.
In gramineous species, ferulic acid has been found to be
ester-linked to the arabinoxylan constituent of the cell
wall (Kato and Nevins, 1985) and is able to undergo
peroxidase-mediated dimerization to produce dehydrodiferulic acid, which cross-links matrix polysaccharides.
Several dehyrodiferulates including cyclic forms are
known to be produced by cell wall-associated peroxidases
and exist in several species including Lolium (Hartly et al,
1988, 1990). Current evidence suggests that it is these
feruloylated glucuronoarabinoxylans that are the major
phenolic containing components of the gramineous cell
walls which are available for cross-linking by peroxidase
enzymes (Carpita and Gibeaut, 1993). Several studies
have established the possibility of a role for peroxidase
activity in controlling cellular expansion (CordobaPedregosa et al., 1996; Goldberg et al, 1987; Hohl et al.,
1995; Sanchez et al., 1989, 1995, 1996; Kim et al., 1989;
Valero et al., 1991; Zheng and Huystee, 1992). In the
grass species Festuca arundinacea Shreb. MacAdam et al.
(1992a, b) demonstrated that increased cell wall peroxidase activity may be responsible for controlling the decline
in cell expansion within the elongation zone and these
authors account for differences in the size of the elongation zone and consequent LER in two different genotypes,
in terms of differences in peroxidase activity.
Only one previous study (Durand et al., 1995) has
investigated drought effects on the spatial and cellular
parameters of leaf growth in a grass species and there
appear to be no reports in the literature that have
examined the role of cell wall peroxidase activity in
controlling leaf growth processes during drought. In two
experiments, the effect of a mild and a more prolonged
drought was examined on the spatial distribution of cell
wall peroxidase activity, epidermal cell length and segmental growth rates in the leaf elongation zone of Lolium
temulentum L., a useful model system in which to study
leaf growth processes in temperate C3 grasses due to its
genetic uniformity and the wealth of biochemical and
physiological studies on its leaf development (Ougham
et al., 1987; Thomas, 1983; Thomas and Stoddart, 1984;
Thomas and Potter, 1985; Thomas et al, 1989). The
possibility of a critical role for cell wall-associated peroxidase activity in controlling leaf expansion rates during
water deficit is discussed in detail. As far as is known,
this is the first time that such a high resolution investigation has demonstrated the possibility that changes in
cell wall peroxidase activity could account for the
observed reduction in leaf expansion rates during drought.
Materials and methods
Growth conditions
Seeds of the summer annual Lolium temulentum L. strain Ba
3081.83 (courtesy of I Thomas, Institute of Grassland and
Environmental Research, Aberystwyth, UK) were germinated
for 7 d in John Innes No.2 commercial potting compost (Keith
Singleton's Seaview Nurseries, Cumbria, UK). Selected uniform
seedlings were transplanted in pairs into 90 mm diameter,
250 cm3 plastic pots filled with John Innes No.2 compost and
raised in a growth cabinet with a day/night temperature of
21/15 °C, a day/night relative humidity of 30/55%, under a
photoperiod of 12 h provided by eight 400 W tungsten halide
lamps (Osram powerstar HQI-T). Plants were watered daily to
drip point and remained vegetative throughout the course of
the investigations.
Shortly before the emergence of the fourth leaf (previously
determined to be c. 21 d after sowing) half the plants in both
experiments I and II were randomly selected and water withheld
from them for the rest of the experiment (9 d).
Leaf elongation and length measurements
On emergence of the 4th leaf in experiment I and the fifth leaf
in experiment II, 15 plants from each treatment were randomly
selected and the length of the leaf measured daily half way
through the photoperiod, using graph paper photocopied on to
acetate. Daily mean leaf elongation rates (LER) were calculated
in mm h" 1 .
Spatial distnbution of growth
The spatial distribution of growth within the growing zone of
the experimental leaf was determined by the method employed
by Schnyder et al. (1987). Shortly after the beginning of the
photoperiod 10 plants from each treatment were selected and
the soil around the base of the plant removed to allow access.
10 entomology needles (Watkins and Doncaster, Hawkhurst,
Kent, UK), 0.2 mm in diameter, mounted 2 mm apart on a
piece of aluminium (20 x 30 mm) were used to mark the
elongation zone of the expanding experimental leaf. Marking
was achieved by piercing through the outer surrounding leaves
and into the elongation zone, which had previously been
determined to be c. 25-30 mm in L temulentum. Plants were
then placed back into the growth cabinet and the experimental
leaf allowed to expand a further 10 mm to allow assessment of
the spatial distribution of growth. The outer leaves were then
removed and the entire leaf dissected out. Distances between
successive holes were measured using a calibrated eye-piece
gTaticule of an objective microscope (Wild-Leitz GMBN
020-502.110, Leitz, Portugal) under the x 4 objective. The
segmenta! elongation rate (SER) in mm mm" 1 h " 1 of each
2 mm section was calculated as:
LERn
(1)
Where Ll0 is the initial distance between pin holes (2 mm) of
section /, L,j is the distance after time t, and LER^ and LERP
are the mean leaf elongation rates of unpinned and pinned
leaves during time /, respectively. The final term corrects for
the significant reduction (c. 50%) in leaf elongation caused by
pinning the leaf. The technique has, however, been shown not
to change the spatial distribution of growth rates within the
leaf base (Schnyder et al, 1987).
Epidermal cell size measurements
During the same period as spatial growth analysis five plants
were selected from each treatment and the elongation zone of
the experimental leaf dissected out, sectioned into 2 mm sections
and placed in industrial methanol (BDH Laboratory supplies,
Poole, Dorset, U K ) to solubilize the tissue pigments. After 5 d
Drought, peroxidase and leaf expansion
Extraction and assay of cell wall peroxidase activity
At the same time as samples were taken for cell size
determination 10 plants were randomly chosen from each
treatment and the experimental leaf dissected out and sectioned
in the same manner as for cell size determination. Care
was taken to remove younger leaves before sectioning.
Corresponding sections from each of the 10 leaves were bulked
to form one sample on which analysis could take place. Samples
were collected in sealed, PCR tubes held on ice. After collection
the fresh weights of the bulked samples were determined before
being stored in the freezer at — 20 °C for subsequent determination of cell wall peroxidase activity.
Initially the samples were homogenized with quartz sand
(Sigma, Poole, Dorset, UK) in ice-cold buffer (50 mM sodium
succinate, 10 mM calcium chloride, 1 mM dithiothreitol, all
purchased from Sigma, Poole, UK) at a ratio of 10:1 buffer to
sample fresh weight. After samples had been ground they were
centrifuged at 2000 g for 5 min. The pellet was washed twice
again in the same volume of 50mM sodium succinate (Sigma,
Poole, Dorset, UK) to remove cytoplasmic peroxidase activity,
before being resuspended in an equal volume of the final
extraction buffer containing 50 mM sodium succinate and 1 M
sodium chloride (Sigma, Poole, Dorset, UK) to disrupt the
association of activity with the cell wall. Each of the three low
ionic concentration buffer washing stages were determined to
reduce activity derived from tissue by 90% at all points in the
elongation zone.
Activity was determined by assaying a 100^1 sample of the
supernatant (equivalent to lOmg fresh weight) using the
Guaiacol test detailed by Chance and Maehly (1955). This
100^1 sample was added to 1 ml of 20 mM sodium phosphate
buffer, which contained 276 /A of guaiacol (Sigma, Poole,
Dorset, UK) per 50 m] of buffer. The reaction was started by
adding 200 ^1 of 0.03% hydrogen peroxide in distilled water
(w/w) (Sigma, Poole, Dorset, UK) The concentration of
hydrogen peroxidase (the H donor) and guaiacol (the H
acceptor) used, gave a linear change in absorbency over 20 +
min with both extracted peroxidase and horseradish peroxidase
solutions. The reaction was mixed and incubated at 25 CC in
1.5 ml spectrophotometric cuvettes (BDH, supplied by Merk
Ltd., Lutterworth, Leicestershire, U K ) . The absorbency of the
solutions at 470 nm was then measured after 20 min using a
Cecil series 2 spectrophotometer (Cecil Instruments, Cambridge,
UK). A horseradish peroxidase solution (Sigma, Poole, Dorset,
UK) of similar activity (0.004 units) to extracted activity was
also run in the assay system to allow conversion of rates of
extracted activity (AA470 min" 1 ) into standard units of activity
of horseradish peroxidase, so-called horseradish peroxidase
equivalence units (HRPEU). One unit of activity is able to
form 1.0 mg purpurogallin from pryogallol in 20 s at pH 6.0 at
20 °C in the pyrogallol test (Chance and Maehly, 1955). The
effect of a series of pH values between 3.5 and 8.5 on extracted
activity were assessed. The greatest change in absorbance
occurred between pH 4.5 and 7.5. Therefore, all buffer extraction
and assay solutions were corrected to pH 5.5 a presumed pH
of the expanding plant cell wall under well-watered conditions.
Results
Leaf elongation rate
In experiment I leaf elongation was followed until the
leaf had completed its expansion. In well-watered plants
leaf elongation reached maximal rates of around
1.2-1.3 mm h~' 2-5 d after emergence (Fig. 1). In wellwatered plants this maximal rate of expansion began to
decline after day 5 until the leaf had completed its
expansion 9 d after emergence. A significant decrease in
LER (P<0.05) caused by the drought treatment was first
detected 3 d after withholding water when rates within
control plants were still maximal. Analysis of the effects
of drought on the spatial distribution of growth, peroxidase activity and abaxial epidermal cell length took place
at this point (indicated on Fig. 1C). Expansion of leaves
in draughted plants continued at lower rates, ceasing 9 d
after emergence, as in control leaves. The significantly
lower rates of elongation after day 3 however, resulted in
a c. 20% reduction in the final length of the fourth leaf
as seen in Fig. 1C which shows the sigmoidal nature of
cumulative leaf length increase from emergence to growth
cessation.
In experiment II, LER and leaf length of the fifth leaf
EXPERIMENT I
E
E.
1.4 - A
12 -
aCO 1.0 0.8 3 06 -
tr
UJ
04 -
eaf
in methanol, when the tissue was nearly clear, sections were
transferred to 8 5 + % solution of lactic acid (Sigma, Poole,
Dorset, UK) in water to finish clearing and for preservation.
Each section was then later examined under the objective
microscope at x40 and x 4 magnification and the lengths of
10 abaxial epidermal cell lengths were determined for each
section.
2077
02 -
_ j
•
EXPERIMENT II
! •
R \r
1 \
\ ^
V"•A
\
0.0 -
Days of sofl drying
Fig. I. Leaf elongation rates (LER) in mm h" 1 (A, B) and cumulative
leaf lengths in mm (C, D) of experimental leaves from well-watered ( • )
and unwatered ( • ) plants in experiments I (A, C) and II (B, D) during
the soil drying period. Each point represents the mean ± standard error
of 15 plants. The periods of growth analysis are marked in C and D
by black bars.
2078
Bacon et al.
were recorded until shortly after the period of spatial
growth analysis. One day after emergence and 5 d since
last watering, the LER of the fifth leaf in unwatered
plants was significantly reduced (/><0.01) from c.
1.2mmh"' to 0.8mmh"' (Fig. IB). The LER of
unwatered plants continued to decline while control rates
remained near steady. Nine days after withholding water,
when control rates remained at c. 1.2 mm h" 1 , the leaf
growth rate of the fifth leaf of drought-exposed plants
had fallen to c. 0.4 mm h" 1 , one-third of the rate of
control plant LER. Spatial analysis of growth, cell wall
peroxidase activity and epidermal cell lengths within the
expanding meristem took place 7-8 d after withholding
water and 3-4 d after leaf emergence (indicated on
Fig. ID).
024
E
E
0 24 -ice
HI
v
Spatial distribution of growth and epidermal cell lengths
Figure 2A and B illustrates the spatial distribution of
growth within the elongation zone of leaves analysed in
experiments I and II. Third order regression lines (95%
confidence) were fitted to the data with r2 values of 0.85
and 0.91 for the well-watered and drought treatment,
respectively, in experiment I (Fig. 2A) and 0.86 and 0.83
for the well-watered and drought treatment, respectively,
in experiment II. Figure 2A and B demonstrates that the
observed reduction in LER in unwatered plants is due to
both a reduction in growth rates and length of the
elongation zone. In experiment I the maximal growth
rate within well-watered plants was c. 0.10 mm mm" 1
h" 1 compared to c. 0.08 mm mm" 1 IT 1 in unwatered
leaves, a c. 20% reduction. The length of the elongation
zone was reduced marginally from c. 23 mm to c. 20 mm
(c. 15%). The spatial distribution of growth rates through
the elongation zone in both treatments was approximately
symmetrical. Growth rates close to the leaf base of
unwatered plants were maintained at values close to those
recorded in the leaf bases of well-watered plants.
Figure 2B shows that in experiment II, the length of
the elongation zone was again reduced marginally from
c. 23 mm to c. 20 mm. Growth rates throughout the leaf
elongation zone of unwatered plants were reduced significantly by the more prolonged drought. The maximal
growth rate within the elongation zone of unwatered
leaves was c. 0.05 mm mm" 1 h" 1 is compared to c.
0.16 mm mm ~' h ~' in well-watered controls.
The insets of Figure 2A and B show the spatial distribution of abaxial epidermal cell lengths for leaves of both
treatments. In experiment I, cell lengths increase from a
mean length of c. 30 p.m up to a final cell length of c.
700 fxtn in well-watered plants and c. 550 f^m in leaves of
unwatered plants. In experiment II thefinalcell length of
cells from thefifthleaf of well-watered plants is c. 700 ^m
the same as in the fourth leaf of well-watered plants. The
mature epidermal cell length is reduced even further by
0.22 -]
B
0.20 0.18 0 16 0.14 0.12 0.10 0.08 0.06 0.04 0 02 0 00 5
10
15
20
25
30
Distance from leaf base (mm)
Fig. 2. Spatial distribution of segmental elongation rates (SER)
in mm mm" 1 h" 1 for the experimental leaf in experiment I (A) and II
(B) from well-watered ( • ) and unwatered plants ( • ) . Each point
represents the mean ± standard error of at least six measurements.
Third order regression lines were fitted to SER values (r2 values between
0.83-0.98, 95% confidence). Inset. The spatial distribution of mean
abaxial epidermal cell lengths in experimental leaves from well-watered
(O) and unwatered ( • ) plants. Each point is the mean of 50 cell length
determinations.
the longer drought exposure in experiment II with final
mean cell lengths of c. 420 ^.m.
Spatial distribution of cell wall-associated peroxidase
activity
The spatial variation in cell wall-associated peroxidase
activity and SER along the elongation zones of leaves in
both experiments I and II is shown in Figs 3 and 4. Cell
wall peroxidase activities through the fourth leaf of
watered and unwatered plants in experiment I (Fig. 3)
are comparable to those determined for the fifth leaf of
watered plants in experiment II (between 1 and 3 x 10~4
horseradish peroxidase equivalent units [HRPEU] of
activity mg" 1 FW). Peroxidase activities through the
elongation zone of the fourth leaf of plants which had
been left unwatered for 3 d were marginally higher than
in well-watered controls for most points in the elongation
Drought, peroxidase and leaf expansion
2079
•r
o
X
Q.
E
E
e
E
13
ID
CO
u.
8
UJ
X
0.0
10
15
20
25
30
0
35
Distance from leaf base (mm)
Fig. 3. Spatial distributions of cell wall peroxidase activities ( • ) and
segmental elongation rates (represented by the same third order
regression used to describe data in Fig. 2A) through the leaf elongation
zone of plants which had been well-watered (A) or left unwatered (B)
for 3 d in experiment I. Peroxidase activity was determined from a
bulked sample containing tissue collected from 10 individual plants.
Activities are expressed as equivalent units of horseradish peroxidase
activity (HRPEU).
zone. The spatial distribution of activities showed a
characteristic pattern with distinctive peaks in activity c.
23 mm from the leaf base in leaves from well-watered
plants and 2 mm closer to the leaf base in leaves from
unwatered plants. In well-watered leaves in experiment II,
this peak is even more pronounced (c. 6.5 x 10~4
HRPEU mg" 1 FW) and again appears 21 mm from the
leaf base. A second less pronounced peak c. 9 mm from
the leaf base in well-watered and unwatered leaves studied
in experiment 1 and the well-watered leaves in
experiment II is also apparent.
In experiment II, the leaf bases of unwatered plants
displayed a far higher level of assayed cell wall-associated
peroxidase activity, with activities 200-300% that of those
recorded in the well-watered controls. Activity peaked c.
25 mm from the leaf base and then returned close to
control levels of activity outside the zone of expansion.
Figures 3 and 4 also display the spatial distribution of
5
10
15
20
25
30
Distance from leaf base (mm)
Fig. 4. Spatial distribution of cell wall peroxidase activities ( • ) and
segmental elongation rates (described by the third order regression
fitted to data in Fig. 2B) through the leaf elongation zone of plants
which had been well-watered (A) or left unwatered for 8 d (B) in
experiment II. Peroxidase activity was determined from a bulked sample
containing tissue collected from 10 individual plants. Activities arc
expressed as equivalent units of horseradish peroxidase activity
(HRPEU).
SER in those leaves, described by the regression curves
fitted in Fig. 2. Comparing SER estimates of the length
of the elongation zone with the profile of cell wall
associated-peroxidase activity shows that peaks of activity
in the leaf bases of both watered and unwatered plants
in experiment I and well-watered plants of experiment II
occur at or close to the point of growth termination
within the elongation zone.
Discussion
Leaf expansion
The kinetics of leaf expansion observed are similar to
those reported by Thomas and Stoddart (1984) for L
temulentum L. Similar patterns of expansion, with a short
period of maximal and constant growth rate have also
been reported for Lolium perenne L. (Schnyder et ai,
1990). Longer periods of constant growth rate have also
2080 Bacon et al.
been reported in other more slow growing temperate
grass species such as Festuca arundinacea L. (Robson,
1972; Durand et al., 1995).
If the analysis of drought effects on leaf growth processes is not to be confounded by developmental changes
then treatments must be carefully timed to have a significant effect during this period of maximal and near
constant growth (Schnyder et al., 1990; Palmer and
Davies, 1996). In both experiments I and II, significant
differences in LER between watered and unwatered plants
appeared when control leaves were undergoing maximal
expansion and experimental leaves from both wellwatered and unwatered plants emerged at the same time.
Spatial distribution of growth and epidermal cell lengths
The distinctive pattern of the spatial distribution of SER
through the meristem approximately described by third
order regressions has been reported in several studies
identifying changes in the spatial distribution of growth
in response to treatments such as soil compaction, salt
stress, age, temperature, photoperiod, low water potential,
and drought (Beemster et al., 1996; Ben-Haj-Salah and
Tardieu, 1995; Bernstein et al., 1993; Pritchard et al.,
1993; Durand et al., 1995; Palmer and Davies, 1996;
Schnyder and Nelson; 1988; Schnyder et al., 1990). The
study of leaf development in L. perenne by Schnyder et al.
(1990) recorded spatial growth distributions and rates
comparable to those observed in the well-watered leaves
of L. temulentum in experiments I and II. Only one other
study has investigated drought effects on the spatial
distribution and cellular regulation of growth in the grass
leaf (Durand et al., 1995). As in the present study, the
authors reported a reduction in the length of the elongation zone and rates of segmental growth, with maintenance of growth rates occurring at the very base of F.
arundinacea leaves for the first few days of drought
exposure. With increasing drought exposure segmental
growth rates were increasingly reduced, as observed in
this investigation. It would appear from results presented
in that study and those presented here, that drought
effects are more pronounced on cells in the distal half of
the leaf base.
Sharp et al. (1988), Spollen and Sharp (1991) and Wu
et al. (1994) have provided evidence for the maintenance
of growth in the apical region of the maize primary root
at low water potential by biochemical modification of the
cell wall, permitting continued expansion even though
turgors of those cells close to the root tip are seen to fall.
Wu et al. (1994) present evidence for its mediation by
elevated XET activity in this region. XET is able to cleave
and re-join xyloglucan constituents of the cell wall and
therefore participate in the control of cellular expansion
(Fry et al., 1992: Passioura and Fry, 1992). Preliminary
evidence has recently been presented to suggest that the
maintenance of growth rates at the leaf base during the
first few days of water deficit observed in this investigation, may indeed be mediated by elevation of XET
activity (Thompson et al., 1997).
Attention is focused on epidermal cells as this is often
considered to be the limiting tissue layer to growth
(Kutschera et al., 1987). The distance from the leaf base
where cell size first becomes constant, is the point where
elongation ceases (Fig. 2, inset). Comparison of this
information with that obtained from pinning of leaves
suggests that the pinning method underestimates the
position at which cessation of growth actually occurs by
3-5 mm. For example, in experiment II, cell length data
indicates that the leaf elongation zone in well-watered
plants extends up to c. 28 mm from the leaf base whereas
pinning data suggests that this distance is 23 mm. Durand
et al. (1995) stated that cell length data in well-watered
leaves of F. arundinacea show that the length of the
elongation zone was c. 40 mm. However, the authors
present SER measurements derived from pinning which
indicate that the length of the elongation zone is c. 30 mm.
It would therefore appear that some discrepancy is found
when comparing spatial growth distributions obtained by
cell length or pinning measurements during the imposition
of a non-steady-state treatment such as soil drying.
An assessment of the spatial distribution of growth
must be both elemental and instantaneous (Erickson and
Silk, 1980). Segments of the elongation zone should
elongate little between pinning and observation of their
displacement. However, it is also necessary to allow
pinned segments to expand for sufficient time to be able
to detect any displacement. Allowing equivalent increases
in elongation allows the comparison of the effect of a
drought treatment on the spatial growth distribution
relative to a control, even if it is not possible to localize
specific growth rates within the elongation zone. Other
workers (MacAdam et al., 1992a, b; Schnyder et al.,
1990) have used records of epidermal cell lengths, together
with the methodology developed by Erickson (1976) and
widely used by Silk and co-workers (Silk, 1992: Morris
and Silk, 1992) to estimate segmental elongation rates
within the elongation zone. These mathematical methods
however, assume growth to be in steady-state and cell
division not to be occurring within the elongation zone.
During the imposition of drought, it is unlikely that
growth will be in steady state for any significant time
period. A cell at the base of a leaf which is experiencing
water deficit may take up to 3 d (three photoperiods) to
pass through the elongation zone (see below), three times
longer than a comparable cell in a well-watered plant.
During these 3 d of increasing water deficit it is unlikely
that cellular expansion will be occurring at a steady rate
for any considerable period of time. Such indirect determination of SER for elongation zones experiencing an
increasing water deficit is therefore unreliable. Estimation
Drought, peroxidase and leaf expansion
of SER in non-steadily expanding elongation zones is
possible (Gandar and Chalabi, 1989), but requires data
on local time rates of change in cell number density which
are only available if multiple sampling events have
taken place.
Interpretation of the effect of non-steady-state treatments on spatial growth profiles obtained from either
pinning or cell size measurements should therefore be
done with caution, particularly when comparing with
other growth-related variables such as cell wall-associated
peroxidase activity.
Peroxidase activity
Spatial differences in dry weight distribution through the
elongation zone was not determined, but previous work
on maize in this laboratory (Palmer and Davies, unpublished results) and in Festuca arundinacea L. (MacAdam
et al., 1992a) demonstrate that dry weight per millimetre
of leaf length rapidly decreases with increasing distance
from the leaf base in a cell size-dependent manner and
cannot therefore explain the distinct patterns of activity
reported here. It would, however, explain the presence of
basal peaks in activity, attributable to the high density of
cells in the crown tissue and the increased efficiency of
extraction from these fragile, newly synthesized cells at
the very base of the meristem. On freeze-drying whole
elongation zones lost c. 80% of their mass irrespective of
the treatment (data not shown). The spatial variation in
soluble protein was determined (not shown), but plots of
specific activity using this variable are inappropriate as
the interest is in the activity associated with the cell-wall,
specifically the side walls, not peroxidase activity within
the cytoplasm. It is also possible to express activities on
a cell length basis (not shown), but again this is inappropriate. In uniaxially expanding organs it is the walls of
cells which are in parallel with this axis which are of
primary interest. In the case of experiment II, plotting
activity on a cell length basis would further exaggerate
differences in the amount of activity in the distal half of
the elongation zone due to significantly smaller cell lengths
in the drought treatment. Although no change in the
spatial pattern of activity would occur on expression in
terms of dry weight, expression of activities on a fresh
weight basis is considered more appropriate when it is
considered that the interest is in the effect of activity on
the side walls of cells. This side wall area should to be
roughly proportional to fresh weight (Palmer and
Davies, 1996).
Several authors have shown that changes in peroxidase
activity which accompany changes in growth, are usually
associated with the apoplast rather than with the cytoplasm of assayed tissue (Cordoba-Pedregosa et al., 1996;
Goldberg etal., 1987; Sanchez et al., 1989, 1996; Schopfer,
1994; Valero et al., 1991). Changes in cytoplasmic activity
2081
are often associated with induction of plant antioxidant
systems. Eight days after withholding water, Zhang and
Kirkham (1996) did not detect any significant increase in
cytoplasmic associated peroxidase activity in sunflower
plants. Ascorbate peroxidase activities associated with
mesophyll and bundle sheath cells of maize did not
increase in response to 18 d without water, even though
soil water content (v/v) fell close to zero (Brown et al.,
1995). In this and similar investigations in this laboratory
(Puliga et al., 1995), soil water content did not fall below
60%. This would be considered as a moderate soil drying
treatment under which reduced rates of growth are maintained. Observations on the different compartments of
activity and their roles in the plants' physiology and the
fact that a massive increase in cell wall associated activity
in leaf bases of plants left unwatered for several days,
returns to control levels outside of the elongation zone
(2.39 x 10" 3 and 2.83 x 10" 3 HRPEU at 37 mm from the
leaf base, in well-watered and unwatered leaves, respectively; data not shown), suggests that the response of
activity to drought reported here may have a specific role
in the control of cellular expansion by mediating changes
in cell wall properties.
Growth and peroxidase activity
If it is to be believed that elevated levels of peroxidase
activity are responsible for the reduced rates of cell wall
expansion then it may be possible to explain the leaf
growth response to drought reported in these experiments.
In experiment I, the marginally higher peak in activity
in the elongation zone of unwatered plants appears 2 mm
closer to the leaf base. This may account for the apparent
reduction in the length of the elongation zone and the
consequent reduction in LER. A similar peak in activity
close to the end of the elongation zone in well-watered
plants in experiment II strengthens the suggestion of the
possible involvement of this activity in controlling cellular
expansion in some way. In experiment II plants which
had been left unwatered for several days, displayed
enhanced activity 7 mm closer to the leaf base than the
peak of activity in the well-watered control and rose
dramatically as SER declined. Figure 4B shows that cell
wall-associated peroxidase activity in the base of leaves
which developed during several days of water deficit are
significantly higher, distal to the basal 13 mm of the
elongation zone. Activities are a remarkable 200-300%
higher than control activities in this region of the elongation zone. However, activities in both well-watered and
unwatered plants return to comparable values once cells
had stopped expanding. The far higher levels of activity
within the cell walls of the elongation zone are associated
with drastically reduced rates of elongation.
MacAdam et al. (1992a) reported a steady increase in
cell wall-associated activity, which peaked at approxi-
2082 Bacon et al.
(Gandar and Hall, 1988). Figure 5 displays SER and
mately 15 and 22 mm from the ligule in two well-watered
peroxidase activities for experiments I and II on a temgenotypes of Festuca arundinacea L. This steady increase
poral basis. Analysed over time, peroxidase activity
in activity was accompanied by decreasing rates of SER.
observed in both well-watered and unwatered leaves of
Cell wall activity in that investigation was assumed to be
experiment I, began to increase c. 5 h before total cessathe difference between total extractable activity and soltion of elongation and peaked less than 2 h before elongauble activity. The authors did not observe the smaller
tion ceased in the elongation zones of leaves from wellpeak of activity closer to the leaf base observed in this
watered and unwatered plants. In experiment II the
investigation in well-watered plants. In both treatments
increase in activity in the elongation zone of leaves from
of experiment 1 and the well-watered treatment in
well-watered plants precedes cessation of growth by c.
experiment II (three separate observations), the smaller
3 h and again peaked less than 2 h before cessation of
peak in activity occurs at the point where growth begins
cellular expansion. However, in the leaf bases of plants
to decline. In the unwatered plants of experiment II, this
which had been left unwatered for a total of 8 d, the first
peak is absent or masked by the amount of activity at
significant rise in peroxidase activity was much earlier, c.
the point at which this smaller peak is observed. Activity
10 h before total growth cessation and peaked as cessation
was determined for 2 mm sections of tissue in this investiof expansion occurred. Schopfer (1996) has demonstrated
gation. MacAdam et al. (1992a, b) sampled 5 mm secthat external application of horseradish peroxidase solutions. These smaller peaks in activity span no more than
tions in the concentration range of 10-10000 /^mol I" 1 to
6 mm which suggests that their study may not have
maize coleoptile segments, began to significantly inhibit
detected such changes in activity at such a resolution.
elongation growth (in vivo) and decrease cell wall extensibCan any physiological significance be attached to these
ility (in vitro), less than 2 h after application. MacAdam
minor peaks? They occur at the point were growth
et al. (1992a) reported rises in activity 10 h before growth
deceleration begins and may, therefore, have physiological
cessation and proposed that in well-watered plants, this
significance. The peak although of no great magnitude
steady but significant increase in activity was primarily
may be sufficient to commence the onset of growth
responsible for the decreasing rates of cellular expansion.
deceleration in the well-watered plant by catalysing the
This may also be the case for well-watered plants studied
formation of phenolic cross-links between the cellulose
in this investigation.
microfibrils in the cell walls.
Mader et al. (1986) has suggested that some cell wallassociated activity is in fact an artefact of the homoj
tion which takes place to extract activity. Ionic p<
ases within the cytoplasm may associate with the a
upon homogenization. MacAdam et al. (1992a, b) d
strated that this did not appear to be occurring ii
leaf tissue by comparison of apoplastic activities ot
by homogenization in a high ionic concentration
or vacuum infiltration and centrifugation. In this i
E:
gation it was not possible to assess whether the two
in activity in the well-watered and mildly drought-si
leaf bases are related in terms of their involvem
controlling expansion. The peaks may relate to th<
or different isoforms of peroxidase which may ha\
similar or different physiological functions. The p:
ent peak in activity observed repeatedly in well-w
plants and its correlation with growth would sugge
this activity may have physiological significance.
In experiment II the significantly higher levels of
able activity in unwatered plants, its dramatic ir
70-60-50-40
-70-60-50-40-30-20-10 0 -70 -«0 -50 -A0 -30 -20 -10 0
through the growing zone and its association with s<
reduced growth rates through the elongation zone suggest
Fig. 5. Cell wall peroxidase activities ( • ) shown in Figs 3 and 4 and
that cell wall-associated peroxidase activity may be
segmental elongation rates (O) shown in Fig. 2A and B for the
responsible for the leaf growth response to water deficit.
elongation zone of experimental leaves in experiments I (A, B) and II
(C, D) expressed as a function of time until cessation of cell elongation,
It is important to consider these growth events on a
determined by position-time analysis (Gander and Hall, 1988). Third
temporal basis. This is achieved by associating the time
order regression lines (r ! >0.9, 95% confidence) are fitted to SER values.
until cessation of expansion to a particular position in
Figures 3A and C relate to well-watered leaves and Fig. 3B and D
relate to leaves which have been left without water for 3 d (B) or 8 d (D).
the elongation zone, so-called position-time analysis
Drought, peroxidase and leaf expansion
Alternatively, it has been proposed that peroxidase
activity in the cell walls of well-watered plants may not
function in controlling the rate of cell elongation, but in
fixing turgor-driven viscoelatically extended wall structures (Hohl et at., 1995; Schopfer, 1996). Removal of
peroxidase activity in the leaf bases of well-watered plants
using ascorbate as an inhibitor of activity results in
reduced rates of irreversible extension (unpublished
results) as also reported by Schopfer (1996) in isolated
maize coleoptiles. Rapid increases in peroxidase activity
reported here, which appear at or close to the end of the
zone of elongation may therefore support this hypothesis.
While the rate of cellular expansion is turgor-dependent
it may also be dependent on other biochemical modifications to the cell wall. Cell wall 'expansins' have been
suggested as the biochemical mediator of cell expansion
rates (McQueen-Mason, 1995).
During a sustained period of increasing water deficit,
the central interest in this study, the considerable upregulation of activity (200-300%) within the elongation zone
of the leaf blade provides evidence that peroxidases may
play a central role in controlling the expansion rate of
cells in a plant under drought conditions. Under such
environmental conditions, cells being displaced through
the elongation zone experiencing far higher levels of
activity and are also subject to increased exposure to this
activity as a result of the slower rates of displacement.
The cumulative effect of increased exposure to higher
rates of peroxidase-mediated phenolic cross-wall linking
is a highly attractive hypothesis to explain the growth
response of grass leaves to drought.
Conclusions
This investigation has provided evidence for the involvement of enhanced, elongation zone cell wall-associated
peroxidase activity in limiting cell expansion during water
deficit. It is necessary to investigate this correlation further
in order to discover a causal relationship. This will involve
the in vivo manipulation of cell wall peroxidase activity
biochemically and genetically. The water relations of these
cells are also being investigated in order to gain a clearer
understanding of the growth processes during water deficit. Regulation of cell wall-associated activity must also
be addressed. Recent reports have suggested that the
drought stress-induced plant growth regulator abscisic
acid (ABA) can cause changes in peroxidase activity
which are correlated with changes in growth rates (Basra
et al., 1992; Lee and Lin, 1996). ABA is known to
accumulate in the grass leaf meristem during water deficit
(Dodd and Davies, 1996) and affect the redox state of
ascorbate, a well understood regulator of peroxidase
activity (Takahama, 1994). It is therefore tempting to
speculate on the possibility of a role for ABA in control-
2083
ling leaf expansion during water deficit via modulation
of cell wall-associated peroxidase activity.
Acknowledgements
The authors would like to thank the genetic resources
department of the Institute of Grassland and Environmental
Research (IGER), Aberystwyth, Wales, for providing seed;
C. Dobson, D. Pennington and G. Tinney for practical
assistance and Dr Stephen Palmer for useful discussion of this
work. MAB is grateful to the Natural Environment Research
Council (NERC) for the award of a studentship. This research
has also been supported by a grant from the EU (contract no.
AIR-CT93 1031).
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