Journal of Experimental Botany, Vol. 50, No. 331, pp. 221–231, February 1999
Effects of water stress on leaf growth in tall fescue,
Italian ryegrass and their hybrid: rheological properties
of expansion zones of leaves, measured on growing
and killed tissue
Henry Thomas1, A.R. James and M.W. Humphreys
Cell Biology Department, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth,
Ceredigion SY23 3EB, UK
Received 27 May 1998; Accepted 11 September 1998
Abstract
In Expt 1, plants of tall fescue (Festuca arundinacea
Schreb.), Italian ryegrass (Lolium multiflorum Lam.)
and their F hybrid were grown in soil-based compost
1
in a controlled environment, and subjected to full or
partial irrigation for 20 d. In Expt 2, plants of the parent
species were grown in nutrient solution in the
same environment and subjected to osmotic stress
(0.76 MPa) for 2 d. In both experiments, distribution of
growth in the leaf growing zone (at the base of the
growing leaf ) was determined, and elastic and plastic
compliances were measured on methanol-killed
samples of growing zone and of mature lamina
using an extensiometer. In Expt 2 plastic compliance,
coefficient of extension, extensibility, and hydraulic
conductance were calculated from changes in leaf
extension rate occasioned by imposing linear stress.
Plastic and elastic compliances of growing zones
were 10–20 times greater than those of mature laminae. In both species, drought reduced (a) leaf extension rate, (b) the length of the growing zone, (c) the
height of maximum growth, (d) the plastic compliance
of whole bases (Expt 1), and (e) hydraulic conductance.
The elastic compliance of whole leaf bases was unaffected by drought, but when expressed per unit length
of growing zone was increased by drought. Killing with
methanol reduced the plastic compliance of leaf bases
in control plants, but not in droughted plants.
F. arundinacea differed from L. multiflorum in having
(a) a lower leaf extension rate (although drought
reduced extension by the same proportion in both
species), (b) a longer growing zone in droughted plants
in Expt 2, (c) a lower elastic and plastic compliance of
whole killed leaf bases and laminae, (d) slightly higher
plastic compliance in attached growing leaves, and (e)
lower plastic compliance per unit length of growing
zone in attached leaves. The hybrid was generally
intermediate between the parents. The results are
discussed in relation to methodology and to crop
improvement.
Key words: Extensibility, extension coefficient, hydraulic
conductance, elastic compliance, plastic compliance, leaf
growth, leaf extension rate.
Introduction
Herbage, the agriculturally-valuable part of the vegetative
grass crop, is composed mostly of leaf laminae, which are
also the principal photosynthetic organs. The rate of
increase in leaf area is, therefore, an important character
in determining plant production and economic yield.
Since leaf growth in grasses is essentially linear and a
consequence of cell expansion at the base of the leaf,
elongation rate provides a good estimate of the increase
in leaf area.
Leaf extension rate in grasses is particularly sensitive
to water stress even when turgor is maintained (Michelena
and Boyer, 1982) because the rheological characteristics
of the elongating cells at the base of the leaf are altered:
in particular, extensibility of the cell walls is reduced
(Pritchard and Tomos, 1993; Bacon et al., 1997). Water
stress also reduces the length of the growing zone and
1 To whom correspondence should be addressed. Fax: +44 1970 823242. E-mail: [email protected]
© Oxford University Press 1999
222
Thomas et al.
the distribution of cell expansion rate within the zone
(Durand et al., 1995).
Temperate grass species differ in their herbage production during drought (Garwood and Sinclair, 1979; Norris
and Thomas, 1982; Thomas, 1994). Particular attention
has been paid in recent years to the Festuca–Lolium
complex ( Humphreys et al., 1997), which includes two
inter-fertile species, the drought-resistant Festuca arundinacea Schreb. (tall fescue) and the drought-susceptible
Lolium multiflorum Lam. (Italian ryegrass). The objectives
of this study include combining desirable traits from the
two species by breeding intergeneric hybrids, and determining the mechanisms underlying complex, quantitative
traits such as drought resistance.
The first aim of this paper is to quantify the effect of
reduced water status on differences between F. arundinacea and L. multiflorum in the distribution of growth
within the basal leaf growing zone and in rheological
characters. As far as is known, there has been no published work on diversity of rheological traits between
grass species. Two growing systems were used: clonally
propagated plants were grown in soil-based compost and
subjected to restricted watering ( Expt 1), and plants
raised from seed were grown in nutrient solution and
subjected to osmotic shock ( Expt 2).
A major challenge in crop improvement is to make
practical use of basic research on fundamental physiological mechanisms, which is necessarily often conducted on
model species and systems. In attempting to use physiological measurements as criteria for selection, or to characterize genotypes or populations, sufficient useful data
have to be collected to permit statistically significant
differences to be detected. This in turn demands techniques which are rapid, repeatable, convenient, and biologically meaningful. Evaluations of leaf growing zone
rheology made by measuring stress–strain relationships
of killed material in an extensiometer fulfil the first three
of these criteria, but their relevance to growing plants is
open to doubt. Rheological characters can also be made
by measuring responses of growing leaves whilst they are
still attached to the plant, using auxanometers, which is
more ‘realistic’ but also more time-consuming. The second
aim of this paper is therefore to evaluate the application
of the two techniques to comparable plant material.
Information is also presented on characteristics of mature
tissue. Particular attention is paid to applying these
techniques to ‘non-model’ material (i.e. mature plants
rather than coleoptiles), and to presenting the rheological
quantities in a form (SI units) that permits comparisons
to be made across species and tissue types.
Materials and methods
Cultivation of soil-grown plants (Expt 1)
Plants of Lolium multiflorum cvs Tetrone (2n=4x=28) and
Tribune (2n=2x=14), of F. arundinacea ecotype Bn949
(2n=6x=42), and of the hybrid between Tetrone and Bn 949
(2n=5x=35) were grown in 1.0 l pots of John Innes No. 3 soilbased compost in a controlled environment (20 °C, 12 h day at
PPD=350 mmol m−2 s−1, VPD=0.6 kPa). When the plants had
produced at least 15 large tillers in F. arundinacea (more in L.
multiflorum), half of each population was subjected to 20 d of
controlled drought by restricted watering: sufficient water was
added to the saucer in which each pot stood to allow the pot
weight to oscillate between water deficits of 150 and 250 g. (Pot
weight at field capacity was 1.1 kg for all plants.) The aim was
to achieve a water deficit that reduced leaf extension rate by
about half and allowed newly-formed leaves to develop and
acclimate during drought over 1–2 leaf appearance intervals.
Cultivation of solution-grown plants (Expt 2)
Seedlings of L. multiflorum cv. Tribune and F. arundinacea cv.
Dovey were transplanted at the 3-leaf-stage to individual 165 ml
containers of Hoagland’s nutrient solution in a controlled
environment as above. Stress was imposed at the 6-leaf-stage
by replacing the nutrient solution with nutrient solution
containing polyethylene glycol (PEG 6000), having an osmotic
potential of 0.76 MPa (305 mOs kg−1). Great care was taken
not to disturb or cause damage to the roots, in order to avoid
uptake of PEG which might prove toxic.
Measurement of growth and water status
Leaf extension rate was calculated from daily measurements of
the length of growing leaves on two tillers of each of eight
plants of F. arundinacea and L. multiflorum. Smoothed rates
were derived by fitting (using the MLP package; Ross, 1987)
the generalized logistic curve, following Thomas and Potter
(1985):
L =1/(1+ke−b(t–m))1/k
t
where L is the length of the leaf on day t expressed as a
t
proportion of the maximum length (to represent the ‘developmental stage’ of the leaf ), m is the date of maximum extension
rate, b is proportional to maximum leaf extension rate, and k
is a symmetry parameter. Mean parameter values per species
were calculated using the MLP procedure for parallel curve
analysis.
Leaf extension rate X∞, expressed as a proportion of final leaf
t
length, was calculated as:
X∞=dL/dt=L (b/k) (1−Lk).
t
t
t
To allow effects of ontogeny to be evaluated before measurements were made, the ‘developmental stage’ of the growing leaf
was estimated as the ratio of the length of the growing leaf to
the length of the youngest fully-emerged leaf; since successive
leaves were of similar length, this value was a close approximation to L.
Water and osmotic potentials of entire tillers were measured
as described previously ( Thomas, 1987). Elastic compliance of
entire leaf laminae was measured as 1/E MPa−1, where E is an
estimate of bulk modulus of elasticity derived from simplified
pressure–volume curves as described previously (Thomas, 1987).
Estimation of length of growing zone
The length of the growing zone (L ) was measured either by
gz
pricking through the base of the tiller with a very fine needle
to mark the growing leaf (Schnyder et al., 1987) or by
measuring the gradient in cell size along the growing leaf base
(Schnyder et al., 1990).
In a preliminary experiment to determine the change in
growing-zone length with time in osmotically-stressed plants, it
Leaf rheology of fescue and ryegrass 223
was found that length declined over the first 2 d of exposure to
osmoticum, and remained stable until lamina extension slowed
and sheath extension began. Therefore, in vivo measurements
of wall rheology were made on plants treated with PEG for 2 d.
Measurements of cell wall rheology on killed tissue
In Expt 1, tillers of soil-grown plants were detached after 20 d
of restricted watering; tillers were chosen which bore at least 3
mature leaves (or their remains) and in which the youngest leaf
stage was near 0.5. In Expt 2, tillers of solution-grown plants
were exposed to osmotic shock 2 d before their predicted young
leaf stage would be 0.5–0.6, when they were detached. The
basal 45 mm of the growing leaf was dissected out by slitting,
peeling back and cutting off the enclosing sheaths of the older
leaves. In order to avoid any strain on the leaf base, which
would invalidate subsequent measurement of plastic compliance,
the exposed part of the leaf was detached before dissection, and
the sample was handled only by the enclosing sheaths or by the
(trimmed ) stem, which was left attached. The samples were
boiled and stored in 80% methanol, to destroy cell contents
and remove any effects of osmotic potential or metabolic
activity.
Subsequently, samples were rehydrated in water for 1 h, and
cell wall properties of the lower 40 mm were measured using an
extensiometer built according to van Volkenburgh et al. (1983).
Slight deformation of the instrument (L ) was measured by
inst
substituting a strip of steel for the sample, and subtracted from
measurements made on leaves. Length was increased at
1 mm min−1 to a maximum stress (s) of 100 kPa (0.1 MPa=1
bar), released at the same rate, and then reapplied in the same
way (Fig. 1a). Stress was calculated as 9.8/A kPa g−1 load,
where A is the cross-sectional area of the sample at 20 mm
above the base, estimated from the mean radius measured
under a microscope.
Compliances were calculated as in the equations below, over
the stress range from 50–100 kPa (Ds=50 kPa), when strain
was relatively linear in relation to stress (Fig. 1a).
Compliance of entire leaf base
total ‘compliance’
C =(DL −DL )/Ds
mm kPa−1
t
1
inst
elastic ‘compliance’
C =(DL −DL )/Ds
mm kPa−1
e
2
inst
plastic ‘compliance’
C =(DL −DL )/Ds
mm kPa−1
p
2
1
alternative plastic ‘compliance’
C∞ =DL /100 kPa
mm kPa−1
p
p
Mean compliance over leaf growing
zone, of length L (mm)
gz
(c , c , c , c∞ )=(C , C , C , C∞ )/L
MPa−1
t p e p
t p e p gz
Compliances are presented in two ways. Whole sample
‘compliance’ C is a measure of the characteristics of the entire
growing zone, and is particularly relevant to the interpretation
of changes in leaf extension rate. True compliance (per mm
tissue) c is calculated as C/L and hence provides a measure of
gz
the average compliance of the growing zone, but is very sensitive
to differences in L . The alternative method for measuring
gz
plastic compliance (C∞ , c∞ ) is based on the actual (plastic)
p p
increase in length (DL ) of the sample after the first stress was
p
released but, since the strain/stress curves were asymptotic
around s=0 kPa, DLp was measured at s=10 kPa on the first
and second stress event (Fig. 1a). This allows comparisons to
be made between measurements of plastic compliance of killed
tissue and of growing leaves (see next section).
Measurements of wall rheology of attached, growing leaves
The auxanometric technique used here is based on that described
by Nonami and Boyer (1990a, b) in their work on soybean
Fig. 1. Components used to derive compliance of killed samples and growth coefficients of growing leaves. (a) Increase in length L of killed leaf
bases 40 mm long, measured as stress was increased from 0 to 100 kPa (—), then relaxed (not shown) and re-stressed (A). DL , DL =change in
1
2
length as stress is increased from 50 to 100 kPa during the first and second stress events, DL =increase in length of the sample caused by the first
p
stress event (measured at s=10 kPa). Formulae for calculation of elastic and plastic compliance of entire samples (C , C , C∞ , mm kPa−1) and per
e p p
unit length of growing zone (c , c and c∞ , MPa−1) are given in the text. (b) Increase in length L (—) of growing leaf when stress s (A) was
e p
p
increased from 50 to 150 kPa. X , X are steady leaf extension rates before and after s was increased, DL is the estimated plastic deformation, and
0 1
p
t the time for 95% of DL to occur. Formulae for calculating extensibility m, extension coefficient e and hydraulic conductance l are given in the text.
p
p
224
Thomas et al.
hypocotyls, and uses similar (SI ) units to permit comparisons
to be made with other species or tissues. The auxanometers
consisted of an array of eight low-friction potentiometers fitted
with 5 mm-radius pulleys over which passed a thread connected
at one end to the tip of a growing leaf, and at the other to a
hook bearing counterweights of 4.7 or 14.1 g (see below). As
the leaf grew the potentiometer core rotated, changing the
output of the potentiometer which was measured using a
Delta-T data logger sampling every 10 s.
To measure rheological characters, plants whose growing leaf
stage was near 0.5 were attached to the extensiometer and
allowed to equilibrate for 18 h under a counterweight of 4.7 g,
equivalent to a stress s of c. 50 kPa, depending on the diameter
of the leaf base. Measurements were then made during the last
4 h of the dark period. Increase in leaf length (DL) was
measured for at least 1 h to derive the initial leaf extension rate
(X ). Then, 5 s before the logger sampled the potentiometer
0
output, a 9.4 g mass (s c. 100 kPa) was gently added to the
counterweight, and leaf length measured for a further 1–2 h
(Fig. 1b). Subsequently, an exponential decay curve was fitted
to the data (Fig. 1b), so that the change in extension rate over
time and the new stable extension rate (X ) could be estimated.
1
The rheological characters were estimated as follows (see also
definitions of variables in Fig. 1b):
Initial leaf extension rate, X , from fitted linear regression
0
of leaf length on time
L=X t
mm s−1
0
Extension rate after stress increased, X , from the fitted curve
1
L=DL +be−kt+X t
mm s−1
p
1
Increase in stress on growing leaf base of diameter r mm
Ds=9.8×9.4/(106pr2)
kPa
Time for 95% plastic deformation (0.95DL ) to occur
p
t =−[ ln(0.05)/k]
s
p
Extension coefficient
e=(X −X )/L Ds
s−1 GPa−1
1
0 gz
Increase in strain
Dc=DL /L
[ratio]
p gz
Extensibility
m=c/t Ds
s−1 GPa−1
p
Hydraulic conductance
l=me/(m−e)
s−1 GPa−1
‘drought’ in the rest of this paper, and well-watered or
unstressed treatments as ‘control’.
The growth coefficients (m, e, l ), are derived from a
number of measurements made on growing leaves, depend
on assumptions about the physiology of growth, and are
subject to cumulative errors. The various stages in computation are therefore discussed in some detail below.
Optimum leaf ‘stage’ for measurement
The aim of modelling daily leaf extension over the life of
a leaf was to identify the period when extension rate X∞
was relatively insensitive to ontogenetic effects, so that
estimates of leaf-base rheology would not be confounded
by leaf age. For both species, predicted X∞ plotted against
predicted length L (both expressed as a proportion of
final leaf length), shows that X∞ was relatively stable for
values of L=0.4–0.8 ( Fig. 2). Therefore, leaves were
chosen for measurement when they were about halfgrown (L#0.5).
Water status
Drought reduced whole-leaf water potential and osmotic
potential at the end of the dark period by 0.2–0.3 MPa
in both experiments (data not shown). The net effect in
Expt 1 was a consistent slight decrease in calculated
turgor pressure of c. 0.05 MPa in droughted plants,
whereas in Expt 2 F. arundinacea lost turgor slightly, and
L. multiflorum gained turgor slightly, but not significantly
( Table 1). These values were measured on entire leaves,
but give a reasonable approximation of effects assuming
that gradients between leaf bases and laminae were minimal at the end of the dark period. This level of turgor
(0.4–0.5 MPa) is similar to that measured in Lolium
L could not be determined on the bases of the measured
gz
leaves since they were sampled for determination of water
status; therefore mean values determined on other tillers of
similar size were used.
This technique was feasible only on the solution-grown plants
( Expt 2), which were relatively young and whose tillers were
trained vertically. Attempts on soil-grown plants were unsuccessful because the tillers grew at an angle to the vertical, and any
increase in load had an unpredictable effect on straightening
the tiller which could not be distinguished from true compliance.
Results
Differences between species, the effects of water stress,
and their interactions are generally mentioned only where
they are significant at P<0.05 as determined by ANOVA.
The two L. multiflorum populations used in Expt 1 were
very similar, and results have been combined. To avoid
confusion of water stress with physical stress (s), the
‘osmotic-stress’ treatment in Expt 2 will be referred to as
Fig. 2. Leaf extension rate X∞ versus leaf length or ‘stage’ (both
expressed as a fraction of fitted final leaf length) in F. arundinacea (—),
and L. multiflorum (A), showing ‘stage’ over which X∞ was relatively
stable. The curves were derived by fitting a generalized logistic to
measurements of leaf length versus time, using the parallel curve
algorithm in the MLP package (Ross, 1987).
Leaf rheology of fescue and ryegrass 225
Table 1. Turgor potential of whole tillers towards the end of the 12 h dark period, calculated as the difference between leaf water
potential and osmotic potential
Plants were grown in soil ( Expt 1) or nutrient solution (Expt 2). W, well-watered plants; D, plants droughted for 20 d; N, plants grown in nutrient
solution; P plants in nutrient solution plus PEG 6000 (osmotic potential −0.76 MPa).
Expt
Trait
1
TP
2
TP
Treatment
Turgor potential (MPa)
W
D
W
D
Significance of effect
F. arundinacea
Hybrid
L. multiflorum
Species
Treatment
Interaction
df
resid
0.52
0.46
0.73
0.59
0.52
0.40
—
—
0.47
0.41
0.48
0.55
ns
0.03
ns
44
0.007
ns
0.053
76
perenne swards grown under more natural conditions
under 0–4 weeks drought ( Thomas, 1991).
Growth of leaf bases
In both experiments, L. multiflorum leaves grew much
faster than F. arundinacea leaves, with the hybrid intermediate ( Table 2). Drought reduced extension rate to c.
35% of control rates in Expt 1, and c. 41% in Expt 2.
The species did not differ in sensitivity to drought.
In Expt 1, the length of growing zone L (Table 2)
gz
was similar in the three populations: c. 21 mm when wellwatered, and reduced by a third to c. 13 mm when
droughted. In Expt 2, L was estimated both from cellgz
size distribution and by marking the growing zone by
pricking-through. In watered plants L was similar
gz
(c. 25 mm) in both species, but after a 2 d drought L
gz
was reduced rather more in L. multiflorum than in
F. arundinacea.
The distribution of growth rate in the growing zone
differed between species: the point of maximum growth
rate was further from the base in F. arundinacea than in
L. multiflorum, whilst the maximum relative elemental
growth rate ( REGR) tended to be slightly slower in F.
arundinacea, as reflected in its lower leaf extension rate
( Table 2). Drought reduced both the maximum REGR
and the height at which it occurred.
In all of these traits, values determined by the pricking
technique were smaller than those made using the cellsize method. Since pricking inevitably caused some disruption to the growing leaf, and sometimes actually killed
the leaf when the needle passed through the meristematic
region, only values derived using the less-invasive cellsize technique are considered in the rest of this paper.
The possible effects of leaf ‘stage’ and final leaf length
(predicted as L , the length of the next-oldest fullyFEL
emerged leaf ) on these traits was estimated by correlation
Table 2. Leaf extension rate, length of growing zone, height and value of maximum relative elemental growth rate (REGR) in
F. arundinacea, L. multiflorum and their F hybrid when grown in soil (Expt 1) or nutrient solution (Expt 2)
1
W, well-watered plants; D, plants droughted for 20 d; N, plants grown in nutrient solution; P plants in nutrient solution plus PEG 6000 (osmotic
potential −0.76 MPa). Since drought was expected to have a proportional effect on leaf extension, statistical analysis was carried out on logtransformed data, but non-transformed means are presented. Cell-size and pricking refer to methods used to measure characteristics of growing
leaf bases: see text.
Expt
1
2
Trait
Method
Leaf extension rate
(mm d−1)
Length of zone
(mm)
Ruler
Leaf extension rate
(mm d−1)
Length of zone
(mm)
Auxanometer
Cell-size
Cell-size
Pricking
Height of max REGR Cell size
(mm)
Pricking
Max REGR
Cell size
Pricking
Treatment
Species
Significance of effects
F.arundinacea
Hybrid
L.multiflorum
Species
Treatment
Interaction
W
D
W
31.3
10.7
21.3
38.9
12.6
19.1
55.1
19.6
21.6
0.001
0.001
ns
( log-transformed data)
0.063
0.001
ns
D
W
D
N
14.0
28.7
11.6
25.9
13.3
—
—
—
13.1
36.0
15.2
26.0
0.001
0.001
ns
( log-transformed data)
0.043
0.001
0.01
P
N
P
N
P
N
P
N
P
N
P
22.7
24.6
19.0
15.3
12.1
13.3
9.8
0.047
0.041
0.030
0.019
—
—
—
—
—
—
—
—
—
—
—
17.3
24.1
14.1
12.9
9.9
10.7
7.1
0.051
0.049
0.034
0.021
0.001
0.001
0.058
0.001
0.001
ns
0.001
0.001
ns
0.05
0.05
ns
ns
0.001
ns
226
Thomas et al.
analysis. The only significant effect was in Expt 1, where
L was weakly dependent (varying by ±1 mm) on L ;
gz
FEL
therefore, in deriving rheological characters for Expt 1
(see below), L is adjusted slightly according to L . In
gz
FEL
Expt 2, the mean values in Table 2 were used.
Elastic compliance
In Expt 1, the killed, 40-mm-long, leaf bases had very similar
whole-sample elastic compliance, C , being reversibly
e
stretched by c. 22 mm kPa−1 (Fig. 3a). In Expt 2, the samples
were slightly less elastic than in Expt 1 (Fig. 3b), with L.
multiflorum being slightly more elastic than F. arundinacea,
confirming the insignificant trend in Expt 1. In neither case
did drought have any significant effect on C .
e
True compliance, c , averaged about 1.0 MPa−1: in
e
other words, length would have doubled for a stress of
1 MPa or, more realistically, have increased by 10% for
a stress of 100 kPa (=0.1 MPa=1 bar). In Expt 1, c was
e
greater in L. multiflorum than in F. arundinacea, and
actually increased by drought ( Fig. 3c). In Expt 2,
drought increased c of L. multiflorum tissue, but had no
e
effect on F. arundinacea (Fig. 3d ). These effects on c
e
reflect the influence of differences in drought-induced
reduction of L (since c =C /L ).
gz
e
e gz
Mature laminae were about 20 times stiffer than the
growing bases, having c of only about 0.06 kPa−1. Values
e
for killed lamina segments measured using an extensiometer ( Fig. 3e) or simplified pressure-volume curves
( Fig. 3f ) were very similar. In both cases, L. multiflorum
was more elastic than F. arundinacea.
Plastic compliance
Plastic (irreversible) compliance (Fig. 4) was about half
of elastic compliance. Leaf laminae were about ten times
less plastic than leaf bases (Fig. 4e). Killed leaf bases of
L. multiflorum tended to be more plastic than those of F.
arundinacea, with the hybrid intermediate. Drought
reduced C in Expt 1 (Fig. 4a) but not in Expt 2 ( Fig. 4b),
p
and increased c in L. multiflorum (Fig. 4c, d).
p
The alternative method for calculating plastic compliance (C, c) enables us to compare measurements made
on killed leaf bases and on attached, growing leaves.
Values for C and cmeasured on killed bases in Expt 1
( Fig. 5a, d) are broadly similar to C and c (Fig. 4a, c),
p
p
with L. multiflorum being most plastic, at least in the
drought treatment. Effects in Expt 2 are more complex
( Fig. 5b, e), with C and c of killed bases of F. arundinacea
being decreased by drought and of L. multiflorum being
increased. In contrast, measurements of C made on
growing leaves (Fig. 5c) indicate that F. arundinacea was
more plastic than L. multiflorum. Also, C of growing
leaves was reduced by drought. Although the two species
did not differ significantly in mean c (Fig. 5f ), L. multiflorum was less sensitive to drought than F. arundinacea.
Extensibility (m) and its components in growing leaves
The (rolled ) leaf bases of F. arundinacea were significantly
wider (greater radius) than those of L. multiflorum
( Table 3, line 1), and hence the imposed change in stress
(Ds, line 3) was rather lower. Drought was too brief (2 d)
Fig. 3. Elastic compliances of F. arundinacea (F ), Lolium multiflorum (L) and their mutual hybrid (H ), when watered (dense stippling) or droughted
( light stippling). (a, b) Elastic compliances of whole, killed, immature leaf bases C measured in an extensiometer (see Fig. 1a) in Expt 1 and Expt
e
2. (c, d) Elastic compliance per unit length of killed leaf base c (=C /L ) in Expt 1 and Expt 2. (e) Elastic compliance c of killed mature leaf
e
e gz
e
lamina measured in an extensiometer. (f ) Elastic compliance ce of living detached lamina measured in a pressure chamber. Species (s), treatment
(t) and interaction (s.t) effects significant at P<0.05, 0.01, 0.001 (*, **, ***), or no effects significant (ns).
Leaf rheology of fescue and ryegrass 227
Fig. 4. Plastic compliances of F. arundinacea (F ), Lolium multiflorum (L) and their mutual hybrid (H ), when watered (dense stippling) or droughted
( light stippling). (a, b) Plastic compliance C of whole, killed, immature leaf bases measured in an extensiometer (see Fig. 1a) in Expt 1 and Expt
p
2. (c, d) Plastic compliance per unit length of killed leaf base c (=C /L ) in Expt 1 and Expt 2. (e) Plastic compliance c of killed mature leaf
p
p gz
p
lamina measured in an extensiometer. Species (s), treatment (t) and interaction (s.t) effects significant at P<0.05, 0.01, 0.001 (*, **, ***), or no
effects significant (ns).
Fig. 5. Plastic compliances (alternative method) of F. arundinacea (F ), Lolium multiflorum (L) and their mutual hybrid (H ), when watered (dense
stippling) or droughted ( light stippling). (a, b) Plastic compliance C∞ of whole, killed, immature leaf bases measured in an extensiometer (see
p
Fig. 1a) in Expt 1 and Expt 2. (c) C∞ of growing, attached leaves measured in an auxanometer (see Fig. 1b) in Expt 2. (d, e) Plastic compliance per
p
unit length of leaf base, c∞ (=C∞ /L ) for killed tissue in Expt 1 and Expt 2. (f ) c∞ of growing leaves. Species (s), treatment (t) and interaction (s.t)
p
p gz
p
effects significant at P<0.05, 0.01, 0.001 (*, **, ***).
to affect width. The plastic increase in leaf length (DL ,
p
line 4) was almost 0.9 mm in control plants (treatment
code N ), and reduced to 0.5 mm in droughted plants
(treatment P). If it is assumed that all plasticity occurred
in the growing leaf base, then mean strain was also
reduced by drought (c, line 5). The species were very
similar for both traits. The species did differ, however, in
the time (t ) taken for this deformation to occur ( line 6):
p
it was much briefer in L. multiflorum (c. 20 min) than in
F. arundinacea (30 min). Drought reduced t by half in
p
both species.
The overall mean rate of deformation of entire leaves
228
Thomas et al.
Table 3. Derivation of growth coefficients (see Fig. 1b) of attached growing leaves of F. arundinacea and L. multiflorum cultivated in
nutrient solution before (N) or after (P) addition of PEG 6000
Where treatment effects were not significant, only means per species are shown. CV =estimate of coefficient of variation, to show the main
resid
sources of variation (due to diversity between genotypes, experimental error and random error). W=counterweight (g) imposing stress on leaf
(increased from 4.7 to 14.1 g).
Line Measurement or trait
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Diameter of leaf base=2r
Growing leaf stage
Stress (Ds=9.8W/(106 pr2))
Total plastic increase (DL )
p
Unit
mm
(ratio)
kPa
mm
Treatment
NP
NP
NP
N
P
1000×strain (c=DL /L )
N
p gz
P
Time to 95% L =t
s
N
p p
P
0.95 DL /t
mm s−1
NP
p p
0.95 DL (t .Ds)
mm s−1 GPa−1 N
p p
P
Extensibility
s−1 GPa−1
N
(m=0.95DL /(t Ds L ))
P
p p
gz
Initial extension rate (X )
mm s−1
N
0
P
Final extension rate (X )
mm s−1
N
1
P
DX=X −X
mm s−1
N
1
0
P
1000DX/L
s−1
NP
gz
Extension coefficient
s−1 GPa−1
N
(e=DX/(L Ds)
P
gz
Hydraulic conductance
s−1 GPa−1
N
(l=me/(m−e))
P
Species
Significance of effect
CV
resid
(%)
F. arundinacea
L. multiflorum
Species
Treatment
Interaction
1.19
0.55
88
879
482
33.9
21.2
1738
840
0.689
6.09
6.29
0.229
0.277
0.332
0.134
0.552
0.271
0.247
0.137
7.15
0.101
0.066
0.293
0.120
1.08
0.55
105
877
495
33.8
28.7
1254
605
0.727
6.89
8.57
0.265
0.497
0.417
0.176
0.656
0.339
0.220
0.160
9.39
0.095
0.093
0.176
0.091
0.003
ns
0.004
ns
ns
ns
ns
0.001
ns
ns
ns
ns
12
21
25
39
ns
0.003
ns
42
0.001
0.001
0.005
38
ns
0.010
ns
0.097
ns
ns
70
71
0.001
0.001
0.002
70
0.002
0.001
ns
32
0.004
0.001
ns
88
ns
0.001
ns
41
0.004
ns
ns
0.026
ns
0.031
41
40
ns
0.005
ns
(DL /t ) was 0.7 mm s−1, or 42 mm min−1 ( line 7). At this
p p
point in the calculation of extensibility, errors have accumulated, producing coefficients of variation of around
70%, so that measured species and treatment effects are
too small to be significant from one another. When this
rate is adjusted for the actual stress increase (Ds) which
varied according to leaf base width, L. multiflorum leaves
are seen ( line 8) to have deformed at a slightly greater
rate than F. arundinacea leaves. If it is assumed that all
plasticity occurred in the growing zone of the leaf (mature
tissue having plastic compliance about 5% of that in leaf
bases: Fig. 4), then mean extensibility per unit length of
growing zone, m, was higher in L. multiflorum than in F.
arundinacea, and was increased by drought, particularly
in L. multiflorum ( line 9).
Extension coefficient and hydraulic conductance of growing
leaves
Drought affected leaf extension rates of the two species
similarly, reducing X (when s #50 kPa) to c. 41%
0
of control rates ( Table 3, line 10), and X (when s
1
#150 kPa) to c. 50% ( line 11). Measurements of X had
1
a particularly high coefficient of variation, partly because
of the uncertainty of exactly quantifying a stable terminal
extension rate for leaves that were slowly, asymptotically,
assuming the new rate. The increase in extension rate
116
(DX ) caused by the increase in stress ( line 12) was reduced
by drought. Since all growth extension occurs (by definition) in the growing zone, the relative change in extension
rate (averaged over the whole zone) can be calculated as
(DX/L ); here, L. multiflorum is seen ( line 13) to be more
gz
responsive than F. arundinacea. The net effect of these
effects indicates ( line 14) that the extension coefficient e
of L. multiflorum was unaffected by drought, but that of
F. arundinacea was significantly reduced.
Hydraulic conductance l was approximately halved by
drought ( line 15). The accumulation of errors leads to a
large coefficient of variation, and masks any differences
between species.
Effects of leaf ‘stage’ and leaf length on rheological traits
and their components
Relationships were analysed by correlation analysis on
entire data sets, and on data partitioned by species and
treatment. For measurements made on killed leaf bases,
no significant correlations were detected. For measurements made on growing leaves ( Expt 2), the only significant correlation (P<0.01) was between leaf length and Ds
for control F. arundinacea plants: in other words, longer
leaves tended to have greater cross-sectional area, and
hence Ds was slightly lower.
The paucity of correlations confirms that the precau-
Leaf rheology of fescue and ryegrass 229
tions taken in selecting leaves of uniform ontogeny had
been successful.
Interrelationships between e, m, l and their components
Correlation analysis (not shown) reveals a general tendency (P<0.05 to P<0.001) for plants having high extensibility m to have higher leaf extension rates, especially
X , and high extension coefficient e. Variation in l which
0
was calculated from m and e) was very closely dependent
(r>0.9, P<0.001) on variation in e, and insensitive to
variation in m. There was no association of any rheological character or its components with water, osmotic or
turgor potential.
Discussion
Comparison of young and mature lamina tissue
The values of c for mature tissue reported here (average
e
c. 0.06 MPa−1) were consistent whether measured by
linear or volumetric stress, and were equivalent to a bulk
modulus of elasticity of 17 MPa, similar to that reported
by Thomas (1987) for Lolium perenne.
The finding that mature tissue had much lower plastic
compliance than young tissue ( Fig. 4) agrees with the
observation that extension of rye coleoptiles ceases when
the cell walls mature and lose their plasticity ( Kutschera,
1996). On the other hand, Kutschera found that coleoptile
elasticity was largely unaffected by maturation, whereas
in the present work, fully mature laminae of wellestablished plants were twenty times less elastic than
immature, growing leaf bases ( Fig. 3). Similarly, Nonami
and Boyer (1990b) showed that the elastic compliance of
soybean hypocotyls was reduced (although only to a third
of control values) after maturation.
Effects of drought on leaf growth and rheology
It is difficult to relate the actual values of rheological
characters presented here with published values, because
many authors use experiment-specific units; it is still
possible, however, to compare the magnitude and direction of the effects of drought. The elastic compliance of
whole leaf-base segments ( Fig. 3a, b) was unaffected by
drought (except in F. arundinacea in Expt 2), and this is
in general agreement with the observations of Hohl and
Schopfer (1995) on maize coleoptiles, and of Nonami
and Boyer (1990b) on soybean hypocotyls. On the other
hand, plastic compliance of whole leaf-bases was significantly reduced by drought, except in L. multiflorum in
Expt 2, which is again consistent with the cited studies.
None of these in vitro measurements fully accounts for
the effect of drought in reducing leaf extension rate, and
neither do the small changes in leaf turgor that occurred
during drought ( Table 2). The in vivo estimates of C∞ in
p
Expt 2 ( Fig. 5c) were, however, reduced to 50–55% of
control values by drought, and this is not dissimilar to
the reduction (to 41%) in leaf extension.
Nonami and Boyer (1990a, b) measured plastic deformability (extensibility, m) of control (well-watered ) soybean
hypocotyls as 0.15 s−1 MPa−1, as compared with an
average of 0.25 s−1 MPa−1 here ( Table 3), whilst their
estimate of the extension coefficient (e) was 0.05
s−1 MPa−1 compared with 0.1 s−1 MPa−1 here, and of
hydraulic conductance (l ) 0.08 s−1 MPa−1 compared
with (approximately) 0.2 here. Nonami and Boyer (1990a)
imposed a drought that reduced elongation to zero, and
caused m, e and l to fall almost to zero also. In the
present work, however, a more gentle drought that
reduced leaf extension to 41% of control values tended
to increase m, affected e inconsistently, and approximately
halved l.
A major disadvantage of using m, e and c in interpreting
the mechanisms underlying leaf growth, is that the
assumption is made that the growing zone is composed
of linearly-uniform material of length L (which is used
gz
as a divisor in the calculations). Thus, elastic and plastic
compliances measured on a tissue length basis were
apparently increased by drought, because L was reduced
gz
by drought. The measurements of the distribution of cell
growth along the growing zone ( Table 1) imply that the
zone is not uniform. Also, since growth declines asymptotically towards the top of the growing zone, it is impossible
to define L accurately: hence the use here of 95% final
gz
cell size as an upper limit to the zone. Parvez et al. (1996)
have shown that cell wall elastic compliance in the coleoptile growing zone of maize coleoptiles decreases with
age (i.e. with increasing distance from the leaf base)
presumably representing the early stages of the maturation process described above. Further work is therefore
necessary to define the gradients of rheological traits
along the growing zone. Except on very closely-defined
uniform material large enough to handle, this will be
difficult. The accumulation of errors in the derivation of
growth coefficients ( Table 3) emphasizes this point. It
would be advantageous to couple such studies with measurement of gradients of the activities of cell wall loosening
or stiffening enzymes, as described by Bacon et al. (1997).
Constitutive differences between species
The L. multiflorum population studied had a higher leaf
extension rate than the F. arundinacea population.
Volenec and Nelson (1981) have shown that in F. arundinacea extension rate is positively correlated with length of
growing zone, but this relationship did not apply to
variation between species or between individuals measured here.
F. arundinacea is characterized agronomically by its
hard, fibrous, indigestible leaves, whereas L. multiflorum
is softer and more succulent ( Thomas and Humphreys,
1991), and it is therefore not surprising to find that
230
Thomas et al.
mature laminae of F. arundinacea were both less elastic
( Fig. 3e, f ) and less plastic ( Fig. 4e) than those of
L. multiflorum.
When well-watered, the species did not differ in elasticity of whole leaf bases (Fig. 3a, b), but in Expt 1 F.
arundinacea was less plastic than L. multiflorum, with the
hybrid intermediate (Fig. 4a). Since leaf extension rate of
F. arundinacea in Expt 1 was slower than that of L.
multiflorum, it appears that growth of the fescue was
constrained by low plastic (or visco-elastic) compliance
in the basal zone.
In Expt 2, however, F. arundinacea had higher turgor
and (insignificantly) higher plasticity than L. multiflorum,
but (again insignificantly) lower extensibility and
hydraulic conductance. The lower leaf extension rate in
F. arundinacea may therefore have been due to the
overriding influence of the last two traits, or possibly to
a high yield threshold, which could not be measured in
these experiments.
Acclimatory differences between species
As far as is known, no data have been published on the
effect of drought on the distribution of growth in the
growing zone of L. multiflorum, but the results of Durand
et al. (1995) for droughted F. arundinacea show that,
when leaf extension rate fell to 40% of the control rate,
the length of the growing zone declined to 62% of the
control length, similar to that in the more slowly
droughted plants in Expt 2. In Expt 1, droughted F.
arundinacea was generally less elastic and less plastic than
L. multiflorum. Drought in Expt 2 reduced the length of
the growing zone in F. arundinacea less then in L.
multiflorum ( Table 1) but affected leaf extension (proportionally) to the same extent in both species, and hence
the maximum REGR was greater in L. multiflorum
( Table 1) as were the elastic and plastic compliances (c ,
e
c , c∞ ; Figs 3c, d; 4c, d; 5d, e).
p p
Attribute or artefact?
Any invasive physiological technique is open to the criticism that what is measured is a combination of truth and
fiction. Measurements of wall rheology made on tissue
killed by boiling methanol or by freezing are no exception.
Hence the different interpretation of extensiometer measurements of coleoptile rheology by Kutschera (1996) and
Nolte and Schopfer (1997): are growing cell walls (a)
truly plastic, i.e. is deformation truly irreversible, or (b)
are they visco-elastic, i.e. do they gradually return to their
original length? The measurements made on killed tissue
in the present work did not allow sufficient time for any
visco-elastic retraction to occur (the half-time in rye
coleoptiles is c. 70 min, according to Nolte and Schopfer,
1997), and hence the measurements referred to here as
‘plastic compliance’ may actually be visco-elastic compliance. The data of Edelmann (1995) on sunflower
hypocotyls indicate that, during growth, at least some
elastic (reversible) cell elongation is metabolically fixed,
and so the in vivo estimates of C∞ made here might well
p
represent a combination of plastic, visco-elastic and elastic
compliance.
Measurements of compliance made on killed tissue are
assumed to represent responses of the cell wall alone,
without the complication of turgor or metabolism,
although this may not always be the case (Ding and
Schopfer, 1997). Comparison of Figs 5b and c shows
that estimates of C∞ of droughted (but not control ) leaf
p
bases measured on growing and killed tissue were very
similar (c. 5 mm kPa−1). On the other hand, in control
plants, C∞ was greatly reduced by killing in methanol. (It
p
is possible that killing by freeze–thawing would have been
less likely to manufacture artefacts, but although it would
have eliminated turgor, it might not have disabled all
enzymatic activity.) C∞ of killed tissue was measured over
p
a stress of 0 to 0.1 MPa (50–100 kPa), whereas C∞ of
p
living tissue was measured by imposing a stress of c.
0.1 MPa on tissue already having an estimated turgor
pressure of 0.4–0.5 MPa. It is unlikely, however, that
differences in turgor were responsible for the discrepancy
between methods, because it occurred only in control
plants. Therefore, it would appear that killing at least
partially suppressed wall loosening effected by metabolic
activity, which would have been greater in the fastergrowing, more compliant, control leaves. Some caution
is therefore necessary in interpreting measurements made
on methanol-killed tissue.
A further error lies in estimating elastic compliance of
material that is not completely straight, such as the leaf
bases studied here, since the initial increase in length
involves straightening the tissue, and is not true strain.
Careful observation showed this to be a trivial source of
error for flaccid, rehydrated killed samples, since it
occurred with little or no measurable stress, but it invalidated estimates of c on turgid living material.
e
Conclusions
The proportional effect of drought on leaf extension rate
was very similar in the species studied here, despite the
differences in cultivation and treatment period. Therefore,
it is unlikely that the proven drought resistance of F.
arundinacea under severe drought (Humphreys and
Thomas, 1993) will be reflected in increased leaf extension
during the moderate drought. Measurements (unpublished ) of leaf extension in the field confirm this. These
experiments were conducted under short days to prevent
any flowering in L. multiflorum that might confound
rheological comparisons with F. arundinacea. In the field,
however, most swards are likely to be subjected to drought
during the flowering period, when they are particularly
sensitive, and when much of the herbage mass is composed
Leaf rheology of fescue and ryegrass 231
of culms. Under these circumstances, the most useful
agronomic trait might be the ability of moderatelydroughted plants to recover from defoliation during or
immediately after flowering. This is likely to depend as
much on the density of undecapitated tillers and on the
carbohydrate reserves available ( Volaire, 1995) as on the
inherent leaf extension rate
There was an overall tendency for elastic and plastic
compliance to be less adversely affected by drought in L.
multiflorum than in F. arundinacea, particularly in plants
that had been droughted over 1–2 leaf-appearance intervals. These would be useful traits that should be maintained in breeding Festuca–Lolium hybrids for increased
resistance to moderate drought, particularly if combined
with high turgor and low yield threshold. Compliance
seems to be a quantitative trait, since the F hybrid
1
studied here tended to be intermediate between its parents.
It would be instructive to determine the variation
(repeatability or broad-sense heritability) within these
species for leaf growth under moderate stress, and to
breed lines with extreme expression. ( There is unpublished
evidence from this laboratory that such selection is
possible in Lolium perenne.) It is likely that intergeneric hybridization between Festuca and Lolium will
induce greater diversity than exists within species. Such
plant material would be useful for ‘dissecting’ the principal traits effective in controlling leaf growth, and
hence herbage production, during moderate drought
( Humphreys et al., 1997). The in vivo extensiometry
technique is likely to be the more appropriate in this
context. In general, however, neither of the extensiometry
techniques evaluated here is likely to be appropriate as a
primary selection tool.
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