Planta (2002) 215: 327–338
DOI 10.1007/s00425-002-0747-z
O R I GI N A L A R T IC L E
Wieland Fricke
Biophysical limitation of leaf cell elongation
in source-reduced barley
Received: 23 November 2001 / Accepted: 23 January 2002 / Published online: 20 March 2002
Springer-Verlag 2002
Abstract The biophysical basis of reduced leaf elongation rate in source-reduced barley (Hordeum vulgare
L. cv Golf) was studied. Reduction in source strength was
achieved by removing the blade of leaves 1 and 2 at the
time leaf 3 had emerged 3.0–6.7 cm from the encircling
sheath. Third leaves of source-reduced plants elongated
at 10–36% lower velocities than those of control plants.
Removal of source leaves had no significant effect on
maximum relative elemental growth rates (REGRs) and
the length of the elongation zone (42–46 mm) but caused
a shift of high REGR towards the basal portion of the
elongation zone. Cell turgor was similar between treatments in the zone of maximal REGR (16–24 mm from
base), but was significantly lower in source-reduced
plants in the distal part of the elongation zone, where
REGR was also lower. Throughout the elongation zone,
osmolality and growth-associated water potential gradients were significantly smaller in source-reduced
plants; bulk concentrations of sugars (hexoses, sucrose)
were also lower. However, even in control plants, sugars
contributed little to bulk osmotic pressure (6–11%). The
most likely biophysical limitation to leaf (cell) elongation in source-reduced barley was a reduction in turgor
in the distal half of the elongation zone. It is proposed
that in the proximal half, increase in average tissue
hydraulic conductance enabled source-reduced plants to
maintain turgor and REGR at control level, while
spending less energy on solute transport.
Keywords Cell expansion Æ Hordeum (cell expansion) Æ
Leaf elongation Æ Source/sink relationship Æ Turgor Æ
Water potential
W. Fricke
Division of Biological Sciences,
University of Paisley, Paisley, PA1 2BE, UK
E-mail: [email protected]
Fax: +44-141-8483116
Abbreviations L: average hydraulic conductance of
tissue Æ m: volumetric extensibility of wall Æ P:
turgor Æ REGR: relative elemental growth rate Æ w:
water potential Æ Y: yield threshold of wall
Introduction
Leaves grow in size through the expansion of individual
cells. In grasses, increase in leaf area and photosynthetic
productivity is mainly achieved through cell elongation,
which is confined to a 20- to 50-mm-long region at the
leaf base. Cell elongation can be limited biophysically
through wall properties, water supply or osmolyte provision (Boyer et al. 1985; Cosgrove 1993).
Recent studies on barley and tall-fescue leaves have
concluded that considerable (0.1–0.3 MPa) gradients in
water potential exist between leaf xylem and (peripheral)
elongating cells. This has led to the suggestion that the
rate of tissue-water transport limits cell expansion (Fricke
et al. 1997; Fricke and Flowers 1998; Martre et al. 1999).
This finding is surprising considering the short half-times
of water exchange across plant cell membranes (Steudle
1993). In contrast, in barley exposed to high levels of
NaCl, the cell expansion rate appears to be limited by the
provision of osmolytes for osmotic adjustment (Fricke
and Peters 2002). Similarly, Thompson et al. (1997)
concluded that leaves of low-nutrient maize seedlings
subjected to water deficit elongate at reduced rates
because of failure to maintain solute reserves for turgor
generation in the elongation zone. The significance of
cell-wall-modifying proteins in the control of grass leaf
cell expansion is not known, but a recent study on fescue
(Reidy et al. 2001) suggests that xyloglucan endotransglycosylase activity is more limiting than expansins.
The aim of the present study was to further test the idea
that tissue hydraulic properties limit leaf cell elongation
in grasses. Theory predicts that growth-associated water
potential gradients between leaf xylem and peripheral
cells must approach zero when growth limitation by water
supply becomes insignificant (Boyer et al. 1985). This was
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tested by analysing the water relations of the elongating
leaf 3 of barley following removal of older leaf blades.
Blade removal reduces the source strength for the elongating leaf and is expected to limit cell expansion through
insufficient availability of energy, carbon skeletons or
sugars – but not through tissue-water transport properties. A direct biophysical limitation will only occur if
soluble sugars contribute significantly to the generation of
cell osmotic and, hence, turgor pressure.
Cell turgor was measured with the micro-pressure
probe, osmolality of extracted cell sap was determined
by picolitre osmometry and cell water potential was
calculated as the difference between the two. Cell biophysical parameters were related to the spatial profile of
relative elemental growth rates (REGRs) and subjected
to analysis by Lockhart’s (1965) growth equation. The
contribution of sugar deposition rates to rates of totalosmolyte deposition (and turgor generation) along the
growth zone was also determined.
REGR ¼ ðlnLf lnLI ÞðDtÞ1
ð1Þ
where LI and Lf denote the initial and final segment length,
respectively, and Dt stands for the duration of the experiment
(5–7 h). Data from all plants of a treatment were pooled into
consecutive 4-mm regions and average REGR was plotted against
the respective average segment midpoint, measured from the point
of leaf insertion. Pricking reduced the velocity of leaf elongation by
59% and 60% in control and source-reduced plants, respectively.
The REGR profiles were corrected for this reduction. It was
assumed that the reduction in leaf elongation velocity was spread
proportionally along the elongation zone (Schnyder et al. 1987; Hu
and Schmidhalter 2000).
Displacement velocities
The cumulative total of individual data points of REGR (control,
n=110; source-reduced, n=100) was calculated. The resulting
values were multiplied by 48, the distance along the leaf base
covered by REGR data, and divided by the number of individual
REGR data.
Analysis of bulk leaf extracts
Materials and methods
Plant material and growth conditions
Barley (Hordeum vulgare L. cv. Golf; Svalöf Weibull AB, Svalöv,
Sweden) was grown hydroponically on modified Hoagland solution
as detailed previously (Fricke et al. 1997), except that the solution
was half-strength. The photosynthetically active radiation at thirdleaf level was 250 lmol photons m–2 s–1.
To induce reduction in source strength, blades of leaves 1 and 2
were removed at the time leaf 3 had emerged from the encircling
sheath. Blades were removed just above the ligule and the cut
sealed with Vaseline. When blades of older leaves were removed at
the time leaf 3 had emerged less than 2 cm from the encircling
sheath, elongation of leaf 3 occasionally stopped for hours. In
addition, results lacked reproducibility. Therefore, older leaf blades
were removed at a later developmental stage, when leaf 3 had
emerged 3–6.7 cm from the encircling sheath. Plants were analysed
2 days later. This period was chosen as a compromise between
maximum manifestation of source reduction and analysing leaves
within the period of steady and near-maximum elongation velocities (Fricke et al. 1997).
The possibility existed that excision of older leaf blades affected
elongation of leaf 3 through factors unrelated to the provision of
photosynthate, for example through wound effects. This possibility
was tested by excising the tip 1–2 cm of leaf 3 while keeping older
leaves intact (not shown). Elongation of leaf 3 remained unaffected
by this treatment, provided the cut was sealed with Vaseline. If the
cut was left open, elongation velocities decreased, most likely as a
result of increased water loss.
Growth analysis
Leaf elongation and REGRs
Third leaves were analysed at a developmental stage in which
sheath elongation contributed insignificantly to whole-leaf elongation; the ligule was located within 2–6 mm from the leaf base.
Leaf elongation was calculated from leaf lengths measured (ruler)
twice daily.
The profile of REGR along the basal region of leaf 3 was determined in pin-pricking experiments (Fricke et al. 1997). The basal
50 mm of leaves was pricked at ca. 4-mm intervals, and the displacement of holes recorded after 5–7 h. Relative elemental growth
rate was calculated as:
Osmolality and water contents in bulk leaf extracts
Plants were placed on moistened tissue paper and the first two leaves
were removed. The basal region of leaf 3 was sectioned into either
one or three segments (for details, see figures and figure legends).
Younger leaves were removed, and segments were placed in custombuilt tubular inserts in 1.5-ml microcentrifuge tubes. The basal
opening of the inserts was covered by fine gauze. This allowed cell sap
but not tissue fragments to pass. The sample was frozen in liquid
nitrogen and thawed (two cycles) and then spun for 3 min at 11,600 g
in a microcentrifuge (Micro-Centaur; MSE, Loughborough, UK).
Samples (about 10 ll) were collected and stored under a layer of
water-saturated liquid paraffin in 0.5-ml centrifuge tubes. Small
aliquots of samples were analysed for osmolality using a picolitre
osmometer (Bangor University, UK; a short description of the
method is given further below).
Water contents
Leaves sectioned as described above were placed into pre-weighed
1.5-ml microcentrifuge tubes. Tubes were weighed again to determine fresh weights. Dry weights were obtained after drying samples
for 2 days at 55 C. Water contents per mm leaf length were calculated from fresh- and dry-weight data.
Bulk sugar concentrations
Third leaves were cut into 1- or 2-cm-long segments along the basal
leaf region and halfway along the emerged part of the blade. Sections were placed into pre-weighed 1.5-ml microcentrifuge tubes.
Tubes were weighed again to determine fresh weights. A 100-ll
sample of 25% (v/v) isopropanol, containing 50 mM Tris-acetate
(pH 7.6), was added to each tube and the leaf tissue twice frozen in
liquid nitrogen and thawed. Samples were centrifuged for 3 min at
11,600 g (Micro-Centaur) and the leaf tissue removed for determination of dry weight and calculation of initial tissue water content. The isopropanol leaf extract was used for sugar analysis.
Extracts needed to be buffered (pH 7.6) due to invertase activity of
tissues.
Hexose (glucose, fructose) and sucrose were assayed enzymatically by recording changes in absorbance at 366 nm (for
assay composition, see Fricke et al. 1994). Sucrose concentration
was determined as half the difference in hexose concentration of
samples that had been previously incubated with or without
invertase.
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Single-cell analyses
Turgor pressure
Turgor was measured in epidermal cells using the cell-pressure
probe technique (Steudle 1993). Cells were located along the
elongation zone or halfway along the emerged part of the blade.
Plants were prepared as follows to measure turgor along the
elongation zone.
The seed hull, coleoptile, and first leaf were removed, leaving
leaf 2 in its position covering the base of leaf 3. A small window
was cut into the sheath of leaf 2 under a stereomicroscope. The
window was located either at 20–24 mm (control) and 16–20 mm
(source-reduced plants), or at 28–32 mm (both treatments) above
the point of insertion of leaf 3. Plants in which leaf 3 was damaged
during this process were discarded. The window was sealed with
Vaseline and a piece of clingfilm placed on top of it. The plant was
put back into its pot and the leaf base supported with an extra piece
of foam rubber. After 4–6 h the plant was mounted on the probe
stage with roots kept in nutrient solution. The clingfilm (but not
Vaseline) was removed. Turgor measurements commenced after
10–15 min, with dim background illumination.
Using the ‘window-cut’-approach to prepare plants for turgor
and some (see below) osmolality analyses reduced elongation
velocities of leaf 3. Residual velocities (percent of elongation velocity
in plants without a window) were: turgor analysis at 16–24 mm, 37%
(control) and 29% (source-reduced); turgor analysis at 28–32 mm,
58% (control) and 54% (source-reduced); osmolality analysis at
16–24 mm, 43% (control) and 44% (source-reduced).
Osmolality
Osmolality of sap extracted from individual cells was determined
by picolitre osmometry as detailed previously (Malone et al. 1989).
In short, samples of about 30–100 pl volume were placed
underneath a droplet of water-saturated liquid paraffin on the
stage of the osmometer. The stage and samples were cooled down
(<–20 C) and then slowly heated until ice crystals in samples
disappeared. This was observed under a stereomicroscope. Melting
points of samples were converted into osmolality using standard
solutions of NaCl. To analyse cells within the elongation zone,
plants were prepared either in the same way as for turgor analyses
or older leaves were completely removed and the exposed third leaf
(lined with moist tissue) analysed within 15 min of preparation.
Both approaches gave similar osmolalities. The accuracy of
osmolality measurements, obtained through analyses of subsamples of cell extract and standards, was ± 1 mosmol kg–1.
Deposition rates
Sugars
Deposition rates were calculated using the continuity equation (Silk
and Erickson 1979) as described in Fricke and Flowers (1998).
Calculations were based on mean values of REGR, displacement
velocities and sugar concentrations of segments 0–10, 10–20, 20–30,
30–40, 40–50 and 50–60 mm from the point of leaf insertion.
Source-reduced plants were always analysed 2 days after the start
of treatment, and no consistent difference was observed between
plants analysed late morning or early afternoon. Therefore, local
rate of change with time was set to zero. Change in variable with
position was calculated for each segment as the difference in variable between adjacent distal and basal segments, divided by segment length (10 mm). For segment 0–10 mm, the difference in
variable between 0–10 and 10–20 mm was used.
Osmolality
Application of the continuity equation to osmolality gives an indication of the net rate of deposition of osmotically active solutes.
Mean osmolalities of 10-mm segments were used to calculate
deposition rates. It was assumed that 1 kg of cell sap approximated
1 l and that cell sap behaved like an ideal solution, i.e. 1 mosmol
kg–1 approximates 1 mM.
Statistics
Statistical significance of differences between data sets was evaluated by Student’s t-test. A paired t-test (Excel) was used when
various positions were analysed within the same leaves. Gauss’ law
of error propagation was used to calculate SD when overall means
were calculated from means of individual data sets.
Results
Leaf expansion in source-reduced barley
Removal of the blade of older source leaves caused a
significant reduction in elongation velocity of leaf 3.
Reductions were observed in all experiments, and ranged between 10 and 36%. Figure 1 shows results from a
representative experiment. In control plants, third leaves
elongated at near-steady and near-maximum velocities
for 4–5 days, at an average velocity of 2.02 mm h–1
(Fig. 1a). In plants that had blades of older leaves
removed within 24 h of the start of measurements, third
leaves elongated longer, but at significantly (P 0.0001)
reduced average velocities (1.48 mm h–1; Fig. 1b). Final
leaf length was significantly smaller in source-reduced
plants. This was due to shorter sheaths rather than
shorter blades (not shown). For example, in Fig. 1,
sheath length was 10.35±0.90 cm in control plants and
9.05±0.72 cm in source-reduced plants (P<0.01),
whereas blade length was 25.2±1.2 cm and
25.3±1.1 cm, respectively. Source reduction caused a
significant decrease in average blade width, as indicated
by a decrease in projected leaf-blade area. For plants
shown in Fig. 1, average blade area was 31.0±2.3 cm2
in control compared to 27.3±2.0 cm2 in source-reduced
plants (P<0.01). In addition, specific blade area was
significantly higher in source-reduced plants, in Fig. 1,
122±6 cm2 (g FW)–1 compared to 106±4 cm2 (g FW)–1
in control plants (P<0.001). This indicates that leaf 3 of
source-reduced plants was on average thinner.
Differences in elongation velocities between light
periods (8–10 h) and consecutive light/dark periods
(6–8 h/8 h) were less pronounced for source-reduced
plants (Fig. 2). This was observed in all experiments.
Relative elemental growth rate
The length of the elongation zone of leaf 3 was little
affected by source reduction and extended to 42–46 mm
from the point of leaf insertion (Fig. 3). Maximum rates
of relative elemental growth were comparable between
treatments. However, profiles of REGR differed. Control plants showed a characteristic bell-shaped profile,
whereas source-reduced plants had maximum REGR
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Fig. 2 Elongation velocity of the third leaf of barley during
consecutive light and light/dark periods. Plants were grown until
the third leaf had emerged 3.0–6.7 cm from the encircling sheath.
Then, older leaves were either left intact (control) or blades of
leaves 1 and 2 were removed (source-reduced plants). Elongation
velocities were calculated from measurement of leaf length in the
morning and late afternoon and plotted against the midpoint of the
respective time interval. Plants were grown under a 16 h/8 h light/
dark cycle, and elongation velocities were obtained for an 8- to 10-h
light period and a combined 6–8 h light/8 h dark period. In sourcereduced plants, blade removal occurred at the beginning of the first
light period (at ca. 24 h). Mean elongation velocities (±SD) from
nine (control) and six (source-reduced) plants are shown
Fig. 1a, b Increase in length with time of the third leaf of barley
(Hordeum vulgare). Plants were grown with either older leaves
intact (a, control) or with blades of leaves 1 and 2 removed (b,
source-reduced plants). Blades were removed within the first 24 h.
At 0 h, third leaves had emerged by 1.5–3 cm from the surrounding
sheath. Leaf length was measured with a ruler in the morning and
late afternoon. Results from 11 (control) and 7 (source-reduced)
plants are shown
shifted towards the base. Differences in leaf elongation
velocity between treatments were due to differences in
REGR in the distal half of the elongation zone.
Cell turgor, osmolality and water potential
Epidermal cells were analysed at two locations along the
elongation zone – where REGR of treatments was at
a maximum (16–24 mm) and comparable, and where
differences in REGR between treatments were largest
(28–32 mm from point of leaf insertion). At the zone of
highest REGR, turgor of control and source-reduced
plants was almost identical, 0.571 compared to
0.577 MPa (Fig. 4a). Cell osmolality was 0.1 MPa lower
in source-reduced plants (P<0.01), and, as a result, cell
water potential (w) was 0.1 MPa less negative.
At 28–32 mm from the point of leaf insertion, cell
turgor was significantly (P<0.05) lower in source-reduced plants. Values were 0.517±0.007 MPa for sourcereduced and 0.559±0.036 MPa for control plants
(Fig. 4b). Cell osmolality was 0.1 MPa lower and cell w
0.06 MPa less negative in source-reduced plants.
Fig. 3 Relative elemental growth rates along the basal region of
leaf 3 of barley. Plants were grown until the third leaf had emerged
3.0–6.7 cm from the encircling sheath. Then, older leaves were
either left intact (control) or blades of leaves 1 and 2 were removed
(source-reduced plants). Plants were analysed 2 days following
blade removal. Results from 11 (control) and 10 (source-reduced
plants) individual plant analyses were pooled and grouped into
4-mm-long intervals, starting at 0 mm from the point of leaf
insertion. Averages (±SD) of REGR were plotted against the
respective average distance from the point of leaf insertion
Epidermal cell turgor in the emerged blade of leaf 3
differed significantly between treatments, but in contrast
to the elongation zone, was ca. 0.14 MPa higher in
source-reduced plants (Fig. 4c). Osmolality was also
higher, by ca. 0.14 MPa. As a result, epidermal cell
water potential was identical for control and sourcereduced plants (w=–0.13 MPa). In both treatments,
331
the leaf-elongation zone. Water potential gradients
were considerable in control plants (–0.10 MPa and
–0.09 MPa), but close to zero in source-reduced plants
(–0.01 MPa and –0.03 MPa, at 16–24 and 28–32 mm
from the point of leaf insertion, respectively).
Bulk osmolality
Bulk osmolalities showed the same pattern as cell
osmolalities (Fig. 5a–d). Along the elongation zone
and adjacent non-elongation zone of leaf 3, bulk
osmolality was always higher in control than in sourcereduced plants (Fig. 5c, d). Osmolality decreased
significantly (P<0.05) towards the distal end in
source-reduced plants. Source-reduced plants, but not
control plants, showed large differences in bulk
osmolality between the emerged and elongating parts
of leaf 3 (Fig. 5e).
Sugar concentrations and bulk water content
Fig. 4a–c Turgor, osmolality and water potential in epidermal cells
of the third leaf of barley grown under control or source-reduced
conditions (see legend to Fig. 3). Cells were analysed either along
the elongation zone, a where relative elemental growth rates were at
a maximum or b where differences in relative elemental growth
rates between treatments were largest, or c halfway along the
emerged part of the blade. Third leaves had emerged 10–13 cm
from the encircling sheath. Along the elongation zone, leaves were
analysed either for cell turgor or cell osmolality, and between four
and six cells were analysed in each leaf. Cell water potential was
calculated as the difference between mean turgor and osmolality of
five to six leaf analyses, and standard deviation calculated using
Gauss’ Law of error propagation. In the emerged blade, cell turgor
and osmolality were determined in the same leaf, though different
cells, and cell water potential represents the mean (±SD) of five to
six plant analyses
epidermal cell turgor and osmolality was significantly
higher, and w less negative in the emerged blade than in
the elongation zone.
The water potential of epidermal cells in the emerged
blade was taken as the lower (most negative) approximation of xylem water potential and was used to estimate gradients in w between xylem and epidermal cell in
Fructose and, particularly, glucose concentrations
increased towards the distal half of the elongation zone
in control plants (Fig. 6a, b). Hexose concentrations
exceeded sucrose concentrations (Fig. 6c) and this was
reflected in the distribution profile of total sugar concentrations (defined here as sum of glucose, fructose and
sucrose; Fig. 6d). Most of total-sugar concentration in
source-reduced plants was attributable to glucose.
Concentrations were lower than in control plants, particularly along the distal portion of the elongation zone
(Fig. 6a–d).
In the emerged blade, sugar concentrations were also
lower in source-reduced plants (Fig. 7a, b). Unlike the
basal leaf region, sucrose constituted the main sugar. In
experiment II (Fig. 7b), leaves were 1 day advanced in
development and were analysed 10–12 h, rather than
5–7 h, into the photoperiod. This may explain higher
sugar concentrations. The concentration of sugars was
considerably lower in the emerged blade than in the
elongation zone (Fig. 6) in control, but not in sourcereduced plants.
Bulk water content increased from the basal to the
distal portion of the elongation zone and adjacent
non-elongation zone (Fig. 8a). This was observed for
both treatments. Water contents were consistently lower
in source-reduced plants.
Bulk water contents and sugar concentrations were
used to calculate total-sugar contents along the basal
leaf region. Sugar content in control plants more than
doubled towards the middle and distal portion of the
elongation zone (Fig. 8b). Beyond the elongation zone,
sugar content decreased. Source-reduced plants showed
a less obvious trend due to much lower values. On
average, control plants had a 4.3-times higher totalsugar content in the elongation zone than sourcereduced plants.
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Discussion
Profiles of REGR
Leaf 3 of source-reduced plants elongated at reduced
velocities because of reduced REGR in the distal portion
of the elongation zone. In the proximal part, REGR was
at or slightly above control level. Similar results have
been reported for grass leaves exposed to drought or
nutrient limitation (e.g. Gastal and Nelson 1994;
Durand et al. 1995; Ben-Haj Salah and Tardieu 1997;
Fricke et al. 1997) and for the root elongation zone
(Pritchard 1994; Muller et al. 1998).
Maintenance of high REGR in the proximal part of
the elongation zone is imperative to guarantee development of the growing leaf within a limited period of
time. For example, in the present study, cells of control
plants required more than 50 h to travel from 5 to
25 mm, but less than 10 h to travel from 25 to 50 mm
from the point of leaf insertion (not shown). If sourcereduced plants had maintained a high REGR in the
distal rather than proximal half, development of leaf 3
would have taken weeks rather than days.
In control plants, photosynthate was supplied from
older leaves and reached the elongation zone from the
leaf base. However, in source-reduced plants, photosynthate entered the elongation zone from the distal end.
It is unlikely that the change in direction of photosynthate supply was responsible for the spatial profile of
REGR in source-reduced plants. Allard and Nelson
(1991) observed that the pattern of 14C along the elongation zone of tall fescue leaves was the same for 14CO2
fed to older leaf blades or fed to the emerged portion of
the elongating blade.
Plant preparation for turgor analyses
Fig. 5 Cell (a, b) and bulk (c–e) osmolality at various positions
along leaf 3 of barley grown under control or source-reduced
conditions (see legend to Fig. 3). Between two and three cells were
analysed at each position (along the base) and leaf. Results are
means ± SD of five to eight leaf analyses
Deposition rates
Profiles of sugar and (net) total-osmolyte deposition rate
reflected the spatial distribution of REGR along the leaf
elongation zone. Rates of sugar (glucose + fructose +
sucrose) deposition never amounted to more than 19%
of the rate of total-osmolyte deposition in control plants
(Fig. 9a). This figure was even lower (6%) in sourcereduced plants (Fig. 9b). Differences in total-osmolyte
deposition rate between treatments were not accounted
for by differences in sugar deposition rate.
To gain access to the elongation zone of leaf 3 a small
window was cut in the sheath of the subtending leaf 2
(leaf 1 was removed). The elongation zone was analysed
4–6 h later. Averaged over all experiments, residual
elongation velocity was 46% and 42% in control and
source-reduced plants, respectively. The window-cut
approach has been used previously and percent residual
elongation velocities were either similar (Fricke et al. 1997)
or higher (Thomas et al. 1989; Pollock et al. 1990; Martre
et al. 1999). Most likely, the latter studies reduced leaf
elongation velocity less because leaf 1 was investigated,
inflicting only damage to the coleoptile, or plants were
incubated overnight following cutting of the window.
Profiles of REGR were obtained through pin-pricking. Pricking inflicted a similar reduction in leaf elongation velocity as the (window-cut) preparation of
plants for turgor analyses. Residual elongation velocities
were 40% and 41%, respectively. It is likely that both
approaches caused similar growth reductions since both
involved some injury and growth disturbance (removal
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Fig. 6a–c Bulk concentrations
of glucose (a), fructose (b) and
sucrose (c) along the basal
region of leaf 3 of barley grown
under control or source-reduced conditions (see legend to
Fig. 3). (d) The sum of glucose,
fructose and sucrose, referred to
in the text as ‘‘total-sugar’’ is also
shown. Results are means ± SD
of nine (control) and six (sourcereduced) leaf analyses. Differences in sugar concentrations
between control and source-reduced plants were always significant, except in four cases: at 0–
10, 10–20, and 50–60 mm (glucose), and at 50–60 mm (fructose) from the point of leaf
insertion
Fig. 7a, b Bulk sugar concentrations in the emerged blade of leaf 3
of barley grown under control or source-reduced conditions (see
legend to Fig. 3). Sugar concentrations were determined halfway
along the emerged part of leaf 3. a In Expt. I, plants were at their
usual developmental stage and were analysed 5–7 h into the
photoperiod. b In Expt. II, third leaves were 1 day advanced in
development and were analysed 10–12 h into the photoperiod.
Results are means ± SD of four to five leaf analyses
from nutrient solution and handling). More importantly, differences in elongation velocities between treatments were always maintained. Therefore, it appears
justified to relate cell biophysical data to REGR data.
However, how much did plant preparation affect cell
biophysical parameters?
Cell osmolality was the same, regardless of whether
plants were analysed 4–6 h after removing leaf 1 and
cutting a window in the sheath of leaf 2 or 15 min after
leaves 1 and 2 had been removed and leaf 3 lined with
moist tissue. This suggests that cell osmolality represents
values for intact, undisturbed plants.
It is possible that plant preparation decreased turgor
and, hence, resulted in substantial growth-associated w
gradients, at least in control plants. However, this need
not be the case. In a related study (Fricke and Peters
2002) turgor was the same in plants that had been prepared by the window-cut approach or analysed 15–
45 min after removal of leaves 1 and 2 and lining leaf 3
with tissue soaked in distilled water. It is therefore unlikely that cutting a window in the sheath of leaf 2 resulted in the accumulation of leaked solutes in the walls
and decrease in turgor pressure in cells of leaf 3 (e.g. red
beet, Tomos et al. 1984). Furthermore, there exist a
handful of studies in which turgor has been measured
within the elongation zone of grasses using the cellpressure probe. All these studies point to a very narrow
range of turgor values, including both, epidermal and
mesophyll cells (0.45–0.68 MPa). The studies involved
five different grasses, included a wide range of treatments, investigated leaf 1, 3 or 4, displayed large variation in absolute leaf elongation velocity and showed
large differences in residual elongation velocity following
plant preparation for turgor analyses. Yet, on average,
turgor deviated little (0.53±0.05 MPa; n=11 values;
Thiel et al. 1988; Thomas et al. 1989; Pollock et al. 1990;
Arif and Tomos 1993; Fricke et al. 1997; Thompson
et al. 1997; Martre et al. 1999; present study).
Effect of source removal on barley water relations:
the plant response
Removal of older leaves reduces the transpiring surface
drastically and it is possible that the water relations of
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Biophysical limitation of leaf (cell) elongation
During steady-state growth, the relative rate of irreversible wall expansion must equal the relative rate of
water influx (Lockhart 1965; Cosgrove 1981; Boyer
et al. 1985). The two processes are described by the
equations:
RGR ¼ mðP Y Þ
ð2Þ
RGR ¼ LðDwÞ
ð3Þ
Equation 2 relates relative growth rate (RGR) to wall
extensibility (m, MPa–1 s–1), yield threshold (Y, MPa)
and to ‘‘effective turgor’’ ([P–Y]) as the mechanical
driving force. Equation 3 relates relative growth rate to
the driving force for water uptake (here, the negative
gradient in w between xylem and expanding cell, Dw
[MPa]) and the average tissue hydraulic conductance of
the path (L, MPa–1 s–1). Both, m and L have the same
units and their relative size indicates whether cell
elongation is limited primarily by wall properties or by
tissue hydraulic conductance. This is best illustrated by
setting Eq. 2 equal to Eq. 3:
mL1 ¼ ðDwÞðP Y Þ1
Fig. 8 Bulk water content (a) and total-sugar content (b) along the
basal leaf region of leaf 3 of barley grown under control or sourcereduced conditions (see legend to Fig. 3). Total sugar content of
segments was calculated from bulk-water contents and sugar
concentrations (see Fig. 6d). ‘‘Total sugar’’ refers to the sum of
glucose, fructose and sucrose. Results are means ± SD of nine
(control) and six (source-reduced) leaf analyses
the plant are altered in general. However, this was not
the case in the present study. The water potential of the
emerged blade of leaf 3 was identical for control and
source-reduced plants (Fig. 4c). As far as the leaf is
concerned, source removal caused changes in water
potential specific to the elongation zone. It was not
tested whether root water potential of treatments was
the same, but most likely it was, since the water potential
of the root growth medium was the same.
Fig. 9 Net deposition rates of
total osmolyte and sugars along
the elongation zone of leaf 3 of
barley grown under control (a)
or source-reduced (b) conditions (see legend to Fig. 3).
Deposition rates were calculated from data on REGR
(Fig. 3), bulk osmolality
(Fig. 5c, d) and bulk sugar
concentrations (Fig. 6d) using
the continuity equation. In
some cases, sugar deposition
rates were close to zero and are
indistinguishable from the
x-axis
ð4Þ
In Table 1 the assumption was made that the yield
threshold of elongating cells was the same for control
and source-reduced plants and that differences in RGR
(or, REGR) between treatments at 28–32 mm from the
point of leaf insertion were entirely due to differences in
effective turgor. In addition, growth reductions through
the preparation technique for turgor analyses were
taken into account (see legend to Table 1). The yield
threshold for both treatments was 0.489 MPa and m
equaled 0.68 MPa–1 h–1 (Table 1). A value of Y of
around 0.5 MPa fits well within the range of cell turgor
pressures determined for the leaf elongation zone of
grasses [0.45–0.68 MPa; means ± SD, 0.53±0.05 MPa;
n=11 treatments, from 8 independent studies (cited
above)].
335
Table 1 shows that at 28–32 mm from the point of
leaf insertion, L was comparable to m and similar for
treatments. This suggests that rheological wall properties and tissue hydraulic conductance were co-limiting
cell expansion at 28–32 mm, at least in (peripheral)
epidermal cells. In contrast, at 16–24 mm from the point
of leaf insertion, L was more than 10 times larger than m
in source-reduced plants (but not in control plants);
considering that Dw was close to zero (–0.01 MPa) and
was calculated from turgor and osmolality data (error
propagation), L might have been even larger. If the
above assumptions are valid, the results suggest that in
control plants, cell elongation was always co-limited by
hydraulic and wall properties. This supports previous
studies that concluded that hydraulic properties of tissues limit cell elongation (grass leaves: Fricke et al. 1997;
Martre et al. 1999, 2001; dicot tissues: Boyer 1974; Boyer
et al. 1985; Nonami et al. 1997). Similarly, Fricke and
Flowers (1998), using anatomical and biophysical data,
concluded that on theoretical grounds (Molz and Boyer
1978) significant growth-associated w gradients in the
grass leaf zone cannot be ruled out. However, in sourcereduced plants, limitation differed for the proximal (m)
and distal portion (m, L) of the elongation zone. Values
of m and L are similar to those obtained for expanding
maize leaves (m, 0.22–1.16 MPa–1 h–1; Hsiao et al. 1998)
and soybean stems (L, 0.34 MPa–1 h–1; Boyer et al.
1985).
If source-reduced plants had maintained Lp at
16–24 mm from the point of leaf insertion at control
level, while keeping effective turgor and REGR high,
cell osmolality would have had to increase by ca.
40 mosmol kg–1. It is possible that this would have
required deposition rates of osmolytes that were energetically unsustainable at bulk level or would have
required in certain leaf tissues (mesophyll, see below)
deposition rates of sugars that were unsustainable
under source-reduced conditions. By increasing L
between leaf xylem and peripheral epidermal cells,
source-reduced plants were able to avoid these difficulties and adopt an energy-saving strategy proposed
for water-stressed plants (Hsiao and Xu 2000). Tissue
L can be increased considerably through increased
aquaporin activity at cell level, provided that the cells
in question constitute a major hydraulic barrier for
water movement between xylem and peripheral epidermal cells. The bundle sheath appears to be a prime
candidate for this mechanism of growth regulation.
Suberization of walls (O’Brien and Carr 1970; O’Brien
and Kuo 1975; Dannenhoffer et al. 1990) forces water
to move along the transcellular or symplastic path, and
high aquaporin abundance (Frangne et al. 2001) facilitates fine-tuning of cell hydraulic conductivity
through reversible phosphorylation (Kjellbom et al.
1999).
The above represents one idea to interpret the biophysical and REGR data. This idea requires only minor
(m) or no (Y) alterations in cell wall properties and
proposes that changes in effective turgor and tissue L are
Table 1 Estimation of tissue hydraulic conductance (L) and cellwall extensibility (m) in the elongation zone of leaf 3 of control and
source-reduced barley (Hordeum vulgare). The calculation rests on
the assumptions that differences in REGR at 28–32 mm from the
point of leaf insertion between control and source-reduced plants
were due to differences in effective turgor – the difference between
cell turgor (P) and wall-yield threshold (Y) – and that Y was the
same for both treatments and leaf locations. This implies also that
m was the same for both treatments at 28–32 mm. For calculation
of Y and L (Eqs. 2, 3), the REGR shown was corrected for the
reduction in leaf elongation velocity that resulted from the preparation of plants for turgor analysis. The value of Y was then used to
calculate m and L for the region 16–24 mm from the point of leaf
insertion. Rounded figures of Dw are shown
Parameter
Distance from leaf base
28–32 mm
REGR (h–1)
P (MPa)
Y (MPa)
m (MPa–1 h–1)
Dw (MPa)
L (MPa–1 h–1)
16–24 mm
Control
Sourcereduced
Control
Sourcereduced
0.0818
0.559
0.489
0.68
–0.09
0.52
0.0354
0.517
0.489
0.68
–0.03
0.58
0.0922
0.571
0.489
0.42
–0.10
0.34
0.0855
0.577
0.489
0.29
–0.01
3.67
responsible for growth reduction and maintenance,
respectively, in source-reduced plants.
The biophysical role of sugars in leaf cell elongation
Sugars played only a minor osmotic role at bulk level.
The average concentration of osmotically active sugars
along the elongation zone in control plants was 32 mM,
which is in the range of concentrations previously
reported for the grass-leaf elongation zone (Kemp 1980;
Delane et. al. 1982; Barlow 1986; Hu and Schmidhalter
1998). The concentration in source-reduced plants
was lower by 22 mM. This could account for half the
difference in osmolality between treatments. However, in
absolute terms, sugars accounted for only 6–11% and
3–4% of osmolality in control and source-reduced
plants, respectively. In comparison, Hu and Schmidhalter (1998) and Delane et al. (1982) showed for the
elongation zone of wheat and barley leaves that soluble
sugars contributed between 10 and 20% to bulk osmotic
pressure.
Between 10 and 40 mm from the leaf base, deposition
rates of sugars contributed 1–19% and 0–4% to the rate
of osmolality generation in control and source-reduced
plants, respectively (Fig. 9). It is unlikely that reduced
sugar availability affected generation rates of osmotic
(and turgor) pressure to an extent that could explain the
observed reduction in REGR – at least at bulk level.
However, at tissue and subcellular levels, sugars might
have been far more important. For example, in (fully
expanded) barley leaves, sugars are virtually absent from
the epidermis and confined to the bundle sheath and
mesophyll (Fricke et al. 1994; Koroleva et al. 2000), the
336
latter occupying ca. 57% of total leaf water volume
(Kubinova 1991). In addition, sugars are known to
function as compatible solutes by accumulating preferably in the cytosol, and this feature might have been
essential to maintain cytosolic osmolality during
expansion growth.
Morvan-Bertrand et al. (2001) showed for tall fescue
that leaf elongation velocity remained unaffected or even
increased following defoliation. The authors concluded
that leaf elongation was fuelled by fructan which was
stored in the sheath of older leaves and, particularly, in
the proximal part of the leaf elongation zone. This might
have been also the case in the present study, but probably to a lesser extent. Unlike tall fescue, barley has a
growth strategy that has been optimised for grain production rather than grazing tolerance. In addition,
defoliation removes emerged blades of all leaves,
whereas in the present study, elongating leaf 3 retained
its emerged blade.
Leaf growth in source-reduced plants:
non-biophysical observations
The present study yielded some surprising responses of
the elongating barley leaf to source removal. (i) Timing
of blade removal was crucial in an ‘‘all-or-nothing’’
manner, (ii) removal of older blades reduced elongation
of the emerging leaf (3) by as little as 10%, and (iii)
source-reduced plants showed leaf elongation velocities
during the light/dark period that were as high as during
the light-only period.
When third leaves had emerged by up to 2 cm from
the encircling sheath at the time of source removal,
elongation growth occasionally stopped entirely for
hours and in many cases only resumed at extremely low
and variable velocities. However, when leaf 3 had
emerged by 3.0–6.7 cm at the time of source removal, it
continued to elongate and at substantial velocities.
There must be a discrete threshold of a substance that is
involved in the regulation of elongation growth of the
emerging grass leaf.
At the time older leaf blades were removed, the area
of the emerged part of leaf 3 amounted to less than onetenth of the combined area of older blades. There exist
several possibilities to explain the good performance of
the source-reduced leaf 3. Emerging cereal leaves are
self-sufficient at a much earlier stage than generally
assumed and can function as an internal source. Photosynthate can be directed away from competing sinks
(root; Farrar 1985) to such a degree that it enables the
developing leaf 3 to grow at near-control velocities.
Existing leaf (Morvan-Bertrand et al. 2001) or root
tissue might be drained of fixed or stored material.
Furthermore, source-reduced plants used strategies
typical of nutrient-limited plants: decrease in leaf width
and leaf thickness (see Fig. 9), increased blade-to-sheath
ratio and extension of the period of elongation to
compensate for lower elongation velocities (Fig. 1; see
also Fricke et al. 1997).
Elongation velocities of leaf 3 were significantly
lower during the light/dark period (8 h/8 h) than during the light period (8 h) in control but not in sourcereduced plants. Diurnal differences in elongation
velocities of grass leaves have been associated with day/
night differences in evaporative demand or temperature
(Ben-Haj Salah and Tardieu 1996) and the latter might
have been the case in the present study for control
plants (night temperatures were about 4–7 C lower).
The behaviour of source-reduced plants is more difficult
to explain. Temperature as a controlling factor can be
ruled out, since source-reduced plants grew in parallel
with control plants. Source removal most likely caused
a considerable decrease in the amount of carbohydrate,
particularly fructan (Munns et al. 1982; Schnyder et al.
1988), which could be stored during the day. If so, one
would expect that source-reduced leaves would have
run out of stored carbohydrate and showed greatly
reduced elongation velocities, at least during the first
night following source removal (Christ 1978). However,
this was not the case (Fig. 2). It is striking that sourcereduced plants adjusted so quickly to the new situation.
It seems that signals that affect the partitioning of
photosynthate rather than processes providing the
photosynthate are responsible for the pattern of day/
night elongation velocities in source-reduced plants.
Munns et al. (2000) studied diurnal changes in leaf
elongation of drought-stressed barley and concluded
that plants were able to integrate growth on a 24-h
basis.
In conclusion, the present study provides a biophysical explanation for decreased leaf cell elongation rates
in source-reduced barley that does not require changes
in cell wall properties. In the distal part of the elongation zone, cells elongate at reduced elemental rates because of reduced effective turgor, whereas in the
proximal part, elemental elongation rates are maintained through increase in average tissue hydraulic
conductance between xylem and peripheral epidermal
cells. The overall response of the elongating barley leaf
suggests that older leaves affect elongation of younger
leaves through factors other than the supply of photosynthate.
Acknowledgements I thank Paisley University for financial
support, Denise Drummond for help with growing plants, and
Dr. Winfried Peters (Goethe-Universität Frankfurt, Germany) for
continued discussion about REGRs. I also thank two referees for
their helpful suggestions on earlier versions of the manuscript.
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