PHYSIOLOGIA PLANTARUM 119: 211–220.
2003
Printed in Denmark – all rights reserved
Low irradiance modifies the effect of water stress on survival and
growth-related parameters during the early developmental stages of
buckwheat (Fagopyrum esculentum)
C. Delpérée, J. M. Kinet and S. Lutts*
Unite´ de Biologie Ve´ge´tale, Institut des Sciences de la Vie, Universite´ catholique de Louvain, 5 (Bte 13) Place Croix du Sud, B-1348
Louvain-la-Neuve, Belgium
*Corresponding author, e-mail: lutts@bota.ucl.ac.be
Received 14 October 2002; revised 24 March 2003
In order to characterize physiological modifications encoun-
tered by buckwheat plants exposed to both drought and low-
light stresses, seedlings (cv. La Harpe) were exposed under
controlled environmental conditions, to a progressive decline
in soil volumetric water content under two light regimes: low
irradiance (80 mmol m
2 s
1) or moderate irradiance (160
mmol m
2 s
1). Phenological evolution of the whole plant
until the macroscopic appearance of the reproductive structure
and physiological properties of leaves in relation to their
position on the main axis were quantified. Water stress
reduced net assimilation rate (NAR) before specific leaf area
(SLA) and induced a decrease in stomatal conductance (gl)
and carbon isotope discrimination (D). Water consumption by
Introduction
Drought stress is a major constraint to crop production
and yield stability in rainfed regions. Transient water
stresses also occur in temperate climates and may have
a strong impact on plant development. An efficient use
of limited water resources and a sustained growth when
water supply is limited are therefore desirable traits to
improve crop performance under drought conditions.
Plant response to environmental stresses varies during
ontogenesis and development. Stress can alter leaf struc-
ture considerably and the dynamic of stress interacts
with the dynamic of development of structure and func-
tions in growing tissues, resulting in very different
responses to stress in leaves of different developmental
Abbreviations – A, photosynthetic rate indicated by the assimilation of CO2; chl, chlorophyll; Ci/Ca, ratio of internal leaf CO2 concentration
to ambient CO2 concentration; D, carbon, isotope discrimination; yv, soil volumetric water content; d13C: carbon isotope composition; FWt,
fresh weight at full turgor; gl, leaf stomatal conductance; LA, leaf area; NAR, net assimilation rate; RWC, relative water content; SLA, specific
leaf area; Cs, osmotic potential.
Physiol. Plant. 119, 2003
Copyright # Physiologia Plantarum 2003
ISSN 0031-9317
stressed plants was similar under both light treatments.
Water-stressed plants under moderate irradiance exhibited
higher growth, NAR, osmotic adjustment, and lower SLA
than plants maintained under low irradiance. However, the
former died after 27 days of treatment while the latter still
remained alive until the experiment was discontinued
(40 days). We concluded that the physiological strategy
adopted by the water-stressed plants maintained under mod-
erate irradiance did not afford a long-term advantage in terms
of survival. The effects of a combination of low-light and
water stress on chlorophyll concentration and carbon isotope
discrimination (D) are discussed in relation to growth param-
eters.
situations (Roggatz et al. 1999). During drought stress
growing leaves may develop source functions at smaller
leaf size, often before specific physiological adaptations
to drought occur (Schurr et al. 2000).
Adaptive morphophysiological traits of cultivated
plants to water deficit result from the ecological con-
straint the crop faced with, as well as from associated
agronomic practices. Among cereal species, common
buckwheat (Fagopyrum esculentum Moench.) exhibits
several peculiar characteristics: it is a dicotyledonous
species showing a high level of allogamy due to hetero-
stily, a very rapid floral initiation, and a non-determined
flowering habit leading to a simultaneous production of
211
leaves and inflorescences. Flower production and seed
maturation are not synchronized and a given inflorescence
may thus contain flowers and seeds at different stages
of maturity (Marshall and Pomeranz 1982, Björkman
1995, Taylor and Obendorf 2001). Slawinska and
Obendorf (2001) recently demonstrated that water
stress has an important impact on seed set and that
the effect of water deficit continues after rewatering
and is subsequently expressed as a reduction of fertility
in newly formed flowers. Although field studies suggest
that buckwheat requires high amounts of water before
flowering (Marshall and Pomeranz 1982), no physio-
logical data on water stress impact during the vegetative
stage are available. Water stress episodes in field con-
ditions are frequently associated with high radiation.
Because of its very rapid growth, buckwheat is often
used as a catch crop in spring or autumn. It may
therefore be exposed simultaneously to transient low
water availability and low radiation. Unfortunately,
data concerning the impact of combined water stress
and low irradiance on plants are crucially lacking.
Since the response of the whole plant at the vegetative
stage results from the co-ordinated integration of individ-
ual leaf responses, a thorough study aiming to get a
better understanding of multiple stresses impact on
plants should consider different organs at different devel-
opmental stages. The aims of this study are therefore:
(1) to determine if low radiation has an impact on the
response of buckwheat exposed to progressive water
stress at the young seedling stage in terms of survival
and growth-related parameters; and (2) to develop a
comprehensive approach of such an impact by a sequen-
tial analysis of what occurs in individual leaves at differ-
ent positions on the plant during their development
under these unfavourable conditions.
Materials and methods
Plant material, growth conditions and modalities of stress
imposition
Seeds from the diploid cultivar La Harpe were obtained
from AgriObtention (France). They were allowed to ger-
minate on a compost substrate previously treated with a
fungicide (Rovral, 2.5 g l
1) in 20 containers (3.2 l; 40
seeds per container) placed in a growth chamber at 21 C
during the day and 18 C during the night, under a rela-
tive humidity of 78±5% and a day night/cycle of 16/8 h.
Light was supplied by Philips lamps (Philips Lighting
S.A., Brussels, Belgium) (HPIT 400 Watts). Half of the
containers were maintained under a PAR of 160±20
mmol m
2 s
1 (moderate radiation) and the other half
under a PAR of 80±20 mmol m
2 s
1 (low radiation).
Light radiation for both treatments was carefully
checked and re-adjusted every 2 days.
Seedlings (480) developing one pair of green cotyle-
dons were then carefully transplanted in individual pots
(1.0 l) filled with a mixture of silt (29.7%), clay (7.8%)
and sand (62.5%). The pH value of the substrate was 6.7
212
(pH KCl). Substrate contains (in mg per 100 g DW): 17 P,
126 K, 33 Mg, 322 Ca, 1215 total N and 845 total C. At
the beginning of the treatment, substrate was watered to
the field capacity (water content of 28%) and plants were
randomly distributed in two groups. For both light levels,
a first group of 120 plants (referred to as ‘control’) was
watered daily while a second group of 120 plants (referred
to as ‘water stressed’) was deprived of additional watering
until the end of the experiment. Soil water content (yv) for
each treatment was estimated daily in 10 randomly chosen
pots using the method of responding changes in the
apparent dielectric constant and a calibrated ThetaProbe
soil moisture sensor type ML1 (Delta-T Devices Ltd,
UK). The soil water potential (Cws) was determined
according to the model of van Genuchten (1980) on the
basis of calibration obtained for the considered substrate
with samples of known gravimetric water content.
Phenological evolution, growth parameter and osmotic
potential
Plants were considered at the following vegetative stages:
unfolded cotyledons, 2, 4 and 6 unfolded leaves. Because
of the death of water-stressed plants (see Results), sub-
sequent stages were not considered. The time required to
reach a given developmental stage was recorded for each
plant. The date of emergence of the first inflorescence
was also determined, as well as the number of leaves
present at the time of the inflorescence appearance and
the date of first anthesis.
At each developmental stage, leaves belonging to five
randomly chosen individual plants per treatment were
collected. Cotyledons and pairs of true leaves (leaves
no. 1 and 2; 3 and 4; 5 and 6, numbered acropeticaly) were
always considered separately. Leaf area was determined
by an Image Analysis System (Delta-T Devices LTD,
Cambridge, UK) coupled to WinDias software. Leaves
were then immediately weighed to determine FW and
incubated for 4 h in Petri dishes in darkness at 4 C on
two layers of filter paper (Whatman No. 1) moistened
with sterile deionized water. Turgid fresh weight (FWt)
was estimated and leaves were then maintained in an
oven at 70 C for 48 h prior to DW estimation. Relative
water content (RWC) was calculated by the formula:
RWCðin percentageÞ ¼ ½ðFW
DWÞ=ðFWt
DWÞ
 100
Specific leaf area (SLA) was defined as:
SLA ¼ LA=DW
where LA is the leaf area.
Data from consecutive sampling were used to compute the
Net Assimilation Rate (NAR) by the following equation:
NAR ¼ ð1=LA2Þ ððDW2
DW1Þ=ðt2
t1 ÞÞ
where 1 and 2 refer to harvest at time t1 and t2, respect-
ively.
Physiol. Plant. 119, 2003
 For osmotic potential (Cs) determination, leaves belong-
ing to each pair were separately collected at 14 : 00 h
(corresponding to the mid-photoperiod) on 10 plants, cut in
small segments, placed in Eppendorf tubes perforated with
four small holes and immediately frozen in liquid nitrogen.
After being encased individually in a second intact Eppendorf
tube, they were allowed to thaw for 30 min and centrifuged
at 15 000 g for 15 min at 4 C. Osmolarity of the extracted
sap was assessed with a vapour pressure osmometer (Wescor
5500, Wescor Inc., Utah, USA) and converted from mos-
mol kg
1 to MPa using the Van’t Hoff equation.
In order to quantify osmotic adjustment, and consid-
ering the putative loss of water in stressed tissues, osmotic
potential was readjusted according to:
ccorr ¼ cs ðWCs =WCc Þ
where WCs and WCc are the water content of stressed
and control leaves, respectively.
Biochemical analysis
Leaves from 10 plants per treatment were harvested,
frozen in liquid nitrogen and kept at
80 C until ana-
lysis. For chlorophyll (chl) determination, 100 mg of FW
were ground in a prechilled mortar in the presence of
8 ml acetone 80%. After complete extraction, the mixture
was filtered and the volume was adjusted to 10 ml with
cold acetone. The absorbance of the extract was read at
645 and 663 nm using a Beckman DU640 spectrophoto-
meter and concentration for chl a and chl b were calcu-
lated using the formula of McKinney (1941).
For free soluble sugar extraction, 1 g of frozen samples
was ground in liquid nitrogen to a fine powder and then
extracted with 4 ml of 70% ethanol. Extracts were centri-
fuged for 15 min at 6600 g. Total soluble sugars in the super-
natant were estimated according to Yemm and Willis (1954).
For the identification and quantification of individual
sugars, a fixed volume of the ethanol fraction was evap-
orated under vacuum to dryness, resuspended in 150 ml
of HPLC-grade water (Acros-Organics BVBA SPRL Geel,
Belgium) and filtered through a 0.2-mm filter (Acrodisc
LC 13 mm – PVDF Merck-Eurolab SA, Leuven, Belgium).
Sugars were fractionated using a Bio-Rad 2700 HPLC
system and an Aminex HPX-87C resin-based column
(7.8  300 mm; Bio-Rad 125-0095, Bio-Rad SA, Nazar-
eth EKE, Belgium) in degassed and prewarmed (70 C)
HPLC water at a flow rate of 0.6 ml min
1. Individual
sugars were detected using a refractive index monitor
(Model 1755) and quantified by the Valuechrom soft-
ware (Bio-Rad) integrating data from standard curves
obtained using commercially available sugars (Sigma
Chemical, Sigma-Aldrich, Bornem, Belgium).
Proline was extracted from 200 mg FW in 3 ml sulfo-
salicylic acid 3% (v/v) centrifuged for 15 min at 8100 g
and quantified using the ninhydrin method as described
by Bates et al. (1973).
For a given sample, each measure was performed in
triplicate. The concentrations of organic compounds were
expressed on a fresh matter basis, except chlorophyll, which
Physiol. Plant. 119, 2003
was expressed on a leaf area basis. However, in order to
consider accumulation independently of tissue water losses,
concentrations were readjusted to the same water content
as the controls, as stated above for osmotic potential.
Stomatal conductance and carbon isotope discrimination
Leaf stomatal conductance (gl) was measured on the
abaxial surface of leaves from 10 randomly chosen plants
per treatment, using an automatic porometer (MKIII;
Delta-T Devices LTD, Cambridge, UK). Measurements
were performed at each developmental stage and on each
pair of leaves, considering the mean value for the two
leaves belonging to the same pair.
For carbon isotope discrimination, leaves were col-
lected on 5 randomly chosen plants per treatment for
each stage, and immediately oven-dried for 48 h at
70 C. Approximately 1 g of tissue for each sample was
then ground to a fine powder. Carbon isotope compo-
sition (d13C) was determined in duplicate for each sample
with an isotope mass spectrometer (Isotope Services Inc,
Los Alamos, NM, USA) and calculated with respect to
PeeDeeBelemnite reference as d13C (%) ¼ [(Rsample/
Rreference)
1  1000] where R is 13C/12C ratio. Carbon
isotope discrimination (D) was calculated according to
the formula of Hubick et al. (1986): D ¼ [(da
dp)/
(1 1 dp)]*1000 where dp is the d13C of the leaf sample
and da is the d13C of the atmospheric CO2 (
8%).
Statistical analysis
Each experiment was repeated twice (in autumn 1999
and in spring 2000) under controlled growth chamber
conditions and lead to statistically similar results. Data
presented hereafter are from one single experiment. An
analysis of variance was performed on all data set using the
SAS software (SAS Institutes, Tervuren, Belgium) using
variance analysis. Since the number of analysed pairs of
leaves depends on the considered developmental stage, two
separate three-way ANOVA were performed using radiation,
water availability and the developmental stage for a given
pair of leaves on the one hand, and the level of leaf at a
given developmental stage on the other hand. General
mean values were compared using the Scheffé method.
Results
Soil water content and plant phenology
In control treatment, water content of the soil (Fig. 1)
remained between 20 and 25% (Cws ¼
0.01 to
0.12
MPa) throughout the experiment while yv progressively
decreased to near 10% after 10 days (Cws ¼
1.24 MPa)
and plunged under 5% (Cws ¼
2.73 MPa) after
15 days in the water stress treatment. No difference in
this respect was observed between the low and moderate
irradiance treatment. Under control conditions, all
plants reached the 6-leaf stage but this growth stage was
delayed by 2 days under low irradiance compared to
213
 30 Fig. 1. Mean water content of
substrate used for buckwheat
culture under low irradiance (80
25 mmol m
2 s
1; LI) and moderate
Water content of substrate (%)

irradiance (160 mmol m
2 s
1; MI).

Plants were maintained under
20 control (C) or water stress (WS)
conditions. Water stress was
imposed by withholding watering.
15 Plants maintained for 27 days in
C-MI moderate irradiance died (arrow)
WS-MI and the experiment therefore
C-LI continued under low irradiance
10
WS-LI only. Measurements are the mean
of 10 randomly chosen pots and
vertical bars are standard errors.
5

0
0 5 10 15 20 25 30 35
Time from the beginning of the stress (day)

moderate irradiance. Water stress strongly decreased the Growth parameters and water status
rhythm of leaf appearance as shown in Fig. 2. As a
Low irradiance slightly decreased DW of cotyledons at
consequence, water-stressed plants reached the 4-leaf
the cotyledonary stage but had no impact on this param-
stage 15 (moderate irradiance) and 13 days (low irradiance)
eter at the 2-leaf stage (Fig. 3). Water stress had no
after control plants. Water-stressed plants under moder-
significant impact on DW at these stages, except for the
ate irradiance exhibited 100% mortality after 27 days. In
first pair of leaves under low irradiance. At the 4-leaf
both experiments, they suddenly lost turgor within 48 h
stage, water stress reduced DW of all leaves, including
and all leaves appeared fully necrosed; these plants were
cotyledons; a significant interaction light  water stress
consequently considered as dead while water-stressed
was observed at this stage, since the lowest DW for true
plants maintained under low irradiance still remained
leaves was observed in stressed plants maintained under
alive but appeared unable to reach the 6-leaf stage.
low irradiance. At the 6-leaf stage, well-watered plants
When the first inflorescence appeared, some leaves
maintained under low irradiance exhibited higher DW
were unfolded while others were still folded and clustered
than plants experiencing moderate irradiance.
just below the inflorescence. Irradiance had no impact on
Relative water content (RWC; Table 1) of cotyledons
the total (unfolded 1 folded) number of leaves present at
was not affected by light or water stress treatment at the
the time of macroscopic appearance of inflorescence, and
cotyledonary stage. It decreased in response to water
water stress only slightly reduced their number (from 5.4
stress in both cotyledons and the first pair of leaves at
in control to 5.0 in water-stressed plants). When anthesis
the 2-leaf stage and to a similar extent, under low and
of the first flower occurred, the mean number of
moderate irradiance. At the 4-leaf stage, RWC of all leaf
unfolded leaves was 6.1 and 3.3 in control and water
levels decreased in response to water stress and to a
deficit conditions, respectively.

8
Number of unfolded leaves

7 C-MI
WS-MI
6 C-LI
WS-LI
5

3 Fig. 2. Number of unfolded leaves

as a function of time in buckwheat
2 exposed to a combination of low
(80 mmol m
2 s
1; LI) or moderate
1 irradiance (160 mmol m
2 s
1; MI)
and well-watered (C) or drought
0 stress (WS) conditions. Each point
0 5 10 15 20 25 30 35 is the mean of 5 replicates per
treatment and vertical bars are
Time from sowing (day) standard errors.

214 Physiol. Plant. 119, 2003

Fig. 3. Evolution of the DW of cotyledons, first, second and third pair of leaves in buckwheat maintained to a combination of low (80 mmol
m
2 s
1; LI) or moderate irradiance (160 mmol m
2 s
1; MI) and well watered (C) or water stress (WS) conditions. Weights were considered at
the cotyledonary, 2-, 4- and 6-leaf stages (*, all plants were dead for this treatment; ND, not determined because the plant never reached this
stage). Each value is the mean of 10 replicates per treatment and vertical bars are standard errors.

higher extent in cotyledons than in true leaves. Irradi-
ance had no clear impact on RWC. In contrast, the
ability for osmotic adjustment in water stress conditions
(Fig. 4) was clearly higher under a moderate compared to
a low irradiance, as indicated by the lower Ccorr values
recorded in organs at the 2- and 4-leaf stage. In the
specific case of water stressed plants at the 4-leaf stage,
Ccorr values increased from the older to the younger
leaves under both irradiance treatment; such a gradient
was not observed under well-watered conditions at the
4-leaf stage, but became obvious for control plants, espe-
cially under moderate irradiance at the 6-leaf stage. At
this latter stage, control plants maintained under low
irradiance exhibited higher Ccorr than control plants
maintained under moderate irradiance.
In control plants, leaf area of all organs (except coty-
ledons) was higher under low than under moderate
irradiance (detailed data not shown). Water stress
reduced the total leaf area of the plants (Fig. 5) (except
at the cotyledonary stage) and irradiance had no impact
on LA under water stress conditions. In well-watered
plants, SLA of plants maintained under low and mod-
erate irradiance only differed at the 2- and 4-leaf stages,
thus indicating that an increase in the total LA at the
6-leaf stage for plants maintained under low irradiance
induced a concomitant and proportional increase in
DW. In water-stressed plants, SLA increased at the
2-leaf stage compared to the cotyledonary stage, but then
dropped at the 4-leaf stage; it still remained significantly
higher under low compared to moderate irradiance (Fig. 5).

Table 1. Relative water content (RWC; in percentage) of cotyledons, first, second and third pair of leaves in buckwheat maintained under a
combination of low (80 mmol m
2 s
1; LI) or moderate irradiance (160 mmol m
2 s
1; MI) and well-watered (C) or water stress (WS) conditions.
RWC was considered at the cotyledonary, 2-, 4- and 6-leaf stages (*, all plants were dead for this treatment; ND, not determined (no plants
reached the 6-leaf stage)). Each value is the mean of 10 replicates per treatment (±SE).

Cotyledons Pair n 1 Pair n 2 Pair n 3

Stages LI MI LI MI LI MI LI MI
Cotyledonary C 93.0±1.7 91.7±0.8
WS 89.2±1.4 92.0±0.9
2 leaves C 91.3±1.5 92.1±0.6 92.0±1.3 89.6±1.1
WS 87.4±1.2 86.9±1.6 87.3±0.7 86.5±0.7
4 leaves C 89.1±1.0 92.8±0.6 90.2±0.6 92.7±0.5 87.9±0.8 91.9±0.8
WS 56.1±4.5 41.5±10.8 74.2±2.5 75.9±3.3 76.2±3.0 69.9±4.6
6 leaves C 91.1±1.2 85.5±3.0 91.7±0.4 88.9±1.4 95.7±6.1 87.6±0.9 85.9±0.7 87.4±1.1
WS ND * ND * ND * ND *

Physiol. Plant. 119, 2003 215

Fig. 4. Osmotic potential (adjusted to water content of controls, Ccorr, in MPa) of cotyledons, first, second and third pair of leaves in
buckwheat maintained to a combination of low (80 mmol m
2 s
1; LI) or moderate irradiance (160 mmol m
2 s
1; MI) and well watered (C) or
water stress (WS) conditions. Osmotic potentials were considered at the cotyledonary, 2-, 4- and 6-leaf stages (*, all plants were dead for this
treatment; ND, not determined because the plants never reached this stage). Each value is the mean of 10 replicates per treatment and vertical
bars are standard errors.

The evolution of the NAR considered at the whole Chlorophyll, sugars and proline content
plant level between specific stages of development is
Water stress unexpectedly increased the total chl content
indicated in Table 2. Under well-watered conditions,
expressed on an area basis; this was particularly obvious
low irradiance decreased NAR comparatively to moder-
at the 4-leaf stage (Fig. 6). Total chlorophyll concentration
ate irradiance between the cotyledonary and 2-leaf
of well-watered plants was higher only under low com-
stages, as well as between the 2- and 4-leaf stages but
pared to moderate irradiance at the 6-leaf stage; the dif-
not between the 4- and 6-leaf stages. Water stress drastic-
ference between the two light treatments increased with
ally reduced NAR values and, from a relative point of
leaf age (Fig. 6). Such an increase was also observed if the
view, the impact of drought was higher between the
chl concentration was expressed on a FW basis (data not
2- and 4-leaf stages than between the cotyledonary and
shown). Both chl a and chl b contributed to this increase:
2-leaf stages. In water-stressed plants, NAR was still
for a given pair of leaves, the ratio chl a/chl b remained
higher in moderate compared to low irradiance treat-
constant whatever the water and light regimes.
ment.
Water stress and irradiance had no impact on proline
In well-watered conditions, low irradiance reduced sto-
or soluble sugar concentrations of leaf organs at cotyle-
matal conductance (Table 3) in all leaves and at all the
donary and 2-leaf stages. The concentration in these
developmental stages considered in the present study. At
compounds are therefore reported in Table 4 for leaf
the 4-leaf stage, stomatal conductance decreased with the
pair no. 2 collected at the 4-leaf stage only. In well-
age of the leaf in control plants but this, however, was no
watered conditions, proline concentration was far higher
longer the case in water stress conditions: a very low sto-
in low compared to moderate irradiance. Water stress
matal conductance was recorded in response to water stress
induced a significant increase in proline concentration
and at the 2- and 4-leaves stages, no significant difference
under both light conditions; from a relative point of
for this parameter was recorded between organs or light
view, however, such an increase was higher under mod-
treatment. Carbon isotope discrimination (D; Table 3) was
erate than under low irradiance.
always higher under low than moderate irradiance. Water
In well-watered conditions, hexose concentrations
stress clearly reduced the D values compared to the corres-
were higher under low compared to moderate irradiance
ponding control and, for a given level and developmental
while an opposite trend was observed for sucrose. Water
stage, the lowest D value was always recorded for stressed
stress induced an obvious increase in soluble sugar
plants maintained under moderate irradiance.
concentrations, which was always higher in moderate

216 Physiol. Plant. 119, 2003

 70 some cultivars (Marshall and Pomeranz 1982, Baugmärtner
C-MI et al. 1998). Although most studies devoted to the
60
WS-MI impact of drought on this species focused on the repro-
50 C-LI ductive phase (Marshall and Pomeranz 1982, Slawinska
LA (cm2)

40 WS-LI and Obendorf 2001), transient water shortage episodes

30 may also occur in spring or autumn under low light
conditions and have a strong impact during the early
20
stages of the plant cycle, mainly due to the very poor
10 root system of the seedlings. Plants acclimatize and
0 adapt to light limitation by changes in morphology,
Cotyledonary 2 Leaves 4 Leaves 6 Leaves anatomy and physiology. Those changes also affect
Stage Stage Stage Stage plant response to water stress. Thus, an important
250 point is to determine how response to water stress is
affected by changes in light intensity.
SLA (m2 kg–1 DW)

200

150
100
50
0
Cotyledonary 2 Leaves 4 Leaves 6 Leaves
Stage Stage Stage Stage
Stages
Fig. 5. Total leaf area (LA; in cm2) and specific leaf area (SLA; in
m2 kg
1 DW) in buckwheat maintained under a combination of low
(80 mmol m
2 s
1; LI) or moderate irradiance (160 mmol m
2 s
1;
MI) and well-watered (C) or water stress (WS) conditions. The
evolution of these parameters are reported during the cotyledonary,
2-, 4- and 6-leaf stages. Each value is the mean of 10 replicates per
treatment and vertical bars are standard errors.
compared to low irradiance, except for the case of Glu in
the first pair of leaves. In response to drought, total
soluble sugars considered at the whole plant level by
the anthrone reagent method increased by 459% and
284% under moderate and low irradiance, respectively.
Discussion
Common buckwheat is characterized by a very short
vegetative phase in its development, since flowering
may be observed as early as 35 days after sowing in
Table 2. Net assimilation rate (NAR; in g m
2 day
1) in buckwheat
maintained under a combination of low (80 mmol m
2 s
1; LI) or
moderate irradiance (160 mmol m
2 s
1; MI) and well watered (C) or
water stress (WS) conditions. Values were considered separately for
time intervals separating the following developmental stages:
cotyledonary, 2-, 4-, and 6-leaf stages (*, all plants were dead for
this treatment; ND, not determined because no plant reached the
6-leaf stage). Each value is the mean of 10 replicates per treatment
(±SE).
MI LI
Time Interval C WS C WS
cotyl. – 2 leaves 0.40±0.05 0.32±0.02 0.29±0.01 0.23±0.01
2–4 leaves 0.76±0.05 0.29±0.08 0.52±0.05 0.14±0.04
4–6 leaves 0.33±0.07 * 0.37±0.011 ND
Low irradiance modifies the response of buckwheat to
water stress
The present work shows that low irradiance on the one
hand and water stress on the other hand may induce
changes in similar directions for some parameters but
in opposite directions for others. Both stresses induced
a decrease in NAR (Table 2) and gl (Table 3), as well as
an increase in proline content (Table 4), while an antag-
onistic effect was recorded for leaf area (Fig. 5), osmotic
adjustment (Fig. 4), carbon isotope discrimination
(Table 3) and sucrose content (Table 4). Some param-
eters, such as water consumption and RWC were
affected by water stress but were not significantly
affected by low irradiance. The overall consequence is
that plants exposed to both stresses surprizingly had a
higher survival rate than those exposed to water stress
under moderate irradiance.
Leaves exposed to low light intensity usually exhibit a
higher leaf area, a decrease in leaf thickness, in cell
abundance and stomata density and an increase in cell
size and in the proportion of stacked membranes in the
chloroplast (Nilsen and Orcutt 1996). These features are
thought to maximize photon penetration into the leaf
and potential absorption of photons in an environment
of low photon flux density. As expected, the SLA of
buckwheat was higher under low compared to moderate
irradiance. This strategy, however, does not compensate
for the lower potential of photosynthesis, since NAR
values were lower under low irradiance than under mod-
erate irradiance, except between the 4- and 6-leaf stages
(Table 2).
It is frequently reported that low-light-adapted leaves
have less total chlorophyll per leaf area and a smaller chl
a/chl b ratio, which reflects the changes in the relative
abundance of light-harvesting complexes (Nilsen and
Orcutt 1996). We did not observe such a trend in our
experiments: low-irradiance had no impact on chl con-
tent at the cotyledonary, 2- and 4-leaf stages and
increased both chl a and chl b at the 6-leaf stages (Fig. 6).
It is also noteworthy that in well-watered plants, gl was
always higher under moderate than under low irradi-
ance. The impact of light on the regulation of stomatal
aperture is a well-known phenomenon (Mott et al. 1999):

Physiol. Plant. 119, 2003 217

Table 3. Stomatal conductance (gl; in mmol m
2 s
1) and carbon isotope discrimination (D; in %) of cotyledons, first, second and third pair of
leaves in buckwheat maintained under a combination of low (80 mmol m
2 s
1; LI) or moderate irradiance (160 mmol m
2 s
1; MI) and well
watered (C) or water stress (WS) conditions. RWC was considered at the cotyledonary, 2-, 4- and 6-leaf stages (*, all plants were dead for this
treatment; ND, not determined). Each value is the mean of 10 replicates per treatment (±SE) for stomatal conductance and 3 replicates per
treatment for carbon isotope discrimination.

Cotyledons Pair n 1 Pair n 2 Pair n 3

LI MI LI MI LI MI LI MI

g1
cotyledonary C 110.8±11.3 152.9±18.6
WS 74.5±6.4 120.0±23.9
2 leaves C 214.0±11.7 306.7±12.5 247.8±18.8 298.2±9.4
WS 26.8±5.6 19.7±2.9 34.1±5.3 41.4±7.3
4 leaves C 57.3±7.9 201.1±17.6 99.8±10.6 279.7±21.3 178.3±13.8 364.3±23.2
WS 4.1±0.5 6.5±2.8 9.8±1.8 5.9±1.7 21.3±1.8 17.6±3.3
6 leaves C 173.3±45.5 167.1±40.0 73.3±5.3 208.2±14.9 65.2±7.6 143.7±12.8 94.2±14.2 144.6±18.5
WS ND * ND * ND * ND *
D
cotyledonary C ND ND
WS ND ND
2 leaves C 30.12±0.03 29.15±0.01 29.70±0.01 29.56±0.02
WS 28.73±0.05 27.24±0.05 31.19±0.12 29.10±0.10
4 leaves C 31.14±0.04 29.54±0.02 32.80±0.10 30.21±0.06 33.05±0.02 29.63±0.01
WS 28.17±0.04 27.27±0.06 29.68±0.11 26.67±0.08 27.30±0.09 24.37±0.02
6 leaves C 30.86±0.07 29.12±0.02 31.66±0.04 30.85±0.07 30.32±0.09 28.66±0.11 30.27±0.11 28.28±0.05
WS ND * ND * ND * ND *

one may expect that a partial stomatal closure would
decrease Ci/Ca and thus D values. The present work
demonstrates that this was not the case in buckwheat
since D values for leaves of plants maintained under low
irradiance were always higher than those recorded for
plants maintained under a moderate irradiance, whatever
the water regime (Table 3). Our results also show that
this trend was observed for almost all leaves and that
ontogeny did not modify the impact of light on D values.
The influence of environmental parameters such as water
availability, temperature and external CO2 concentration
(Körner et al. 1991, Tognetti et al. 2000, Merah et al.
2001) on carbon isotope discrimination was previously
analysed, but the influence of light has been neglected.
Since in our buckwheat well-watered plants, gl values are
negatively correlated to D, it may be hypothesized that
low light drastically reduced the intrinsic photosynthetic
capacity of the leaves and that such an effect had a
higher impact on D values than stomatal properties. It
is well known that Rubisco is light activated but that the
amount of light required to activate this enzyme is very
small compared to light available, even in a low-light
environment. A higher D value in low irradiance-exposed
plants might be a consequence of leaf structure modifica-
tion since specific leaf DW is negatively correlated with D
(Araus et al. 1997), thus implying that the correlation
between SLA and D is positive, as shown in our study.
It has to be noticed that the physiological strategy
adopted by the water-stressed plants maintained under
moderate irradiance did not afford a long-term advan-
tage, since all of them died before reaching the 6-leaf
stage. Thus, osmotic adjustment, which is more efficient

Fig. 6. Total chlorophyll concentration (in mg cm
2) in cotyledons, first, second and third pair of leaves in buckwheat maintained under a
combination of low (80 mmol m
2 s
1; LI) or moderate irradiance (160 mmol m
2 s
1; MI) and well-watered (C) or water stress (WS) conditions.
Analyses were performed at the 4- and 6-leaf stages (*, all plants were dead for this treatment; ND, not determined because the plants never
reached this stage). Values are calculated from the means of chlorophyll content estimated on a FW basis and the means of leaf area estimated
for each pair of leaves.

218 Physiol. Plant. 119, 2003

Table 4. Sucrose, glucose, fructose concentrations (in mmol g
1
FW; adjusted to water content of controls) and proline
concentrations (in nmol g
1 FW; adjusted to water content of
controls) in the second pair of leaf of buckwheat maintained under a
combination of low (80 mmol m
2 s
1; LI) or moderate irradiance
(160 mmol m
2 s
1; MI) and well watered (C) or water stress (WS)
conditions. Analyses were performed at the 4-leaf stage. Values are
the mean of 3 replicates ±SE.
MI LI
Compound C WS C WS
Sucrose 2.00±0.08 8.84±0.02 1.53±0.09 4.25±0.06
Glucose 0.29±0.05 3.84±0.31 0.50±0.04 2.26±0.07
Fructose 0.36±0.04 5.24±0.15 0.53±0.19 2.64±0.08
Proline 116.9±14.8 1119.4±12.3 536.2±14.8 844.8±27.9
under moderate than under low irradiance may sustain
protection for temporary stress but not for permanent
water shortage. The fact that proline content was higher
in control plants under low irradiance compared to mod-
erate irradiance conditions suggests that the former were
stressed independently of any drought treatment and
that total soluble sugars, which were present at higher
concentrations under moderate irradiance than under
low irradiance, are at least partly responsible for osmotic
adjustment. This reinforces the hypothesis that proline
accumulation is a general symptom of stress rather than
a strategy of resistance (Lutts et al. 1999). We may even
hypothesize that, although NAR values were higher
under moderate than under low irradiance, osmotic
adjustment has a metabolic cost, which may compromise
plant survival under permanent water stress. Although
water-stressed plants maintained under low irradiance
stopped their development at the 4-leaf stage, they still
remained alive until the end of the treatment and thus
appear to develop a more efficient physiological strategy.
Winkel et al. (2001) suggested that a decrease in growth,
or accelerated leaf senescence in response to drought
reduce crop water requirement and hence can extend
the duration of soil water availability. This hypothesis
does not fit with our experimental data since a similar
reduction in soil water content was noticed in water stress
treatment for both low andmoderate irradiance. Nilsen and
Orcutt (1996) reported that leaves of low-light adapted
plants have an extended longevity, which may constitute
an advantage during water shortage episode and for long
periods of stress. Deo and Biswal (2001) recently demon-
strated that the loss of D1 protein in water-stressed
cotyledons of cluster bean is arrested in seedlings experi-
encing low light intensities in relation to a higher level of
b-carotene and a lower rate of lipid peroxidation.
Buckwheat is sensitive to water stress at the seedling stage
Although stress imposition was progressive in our study,
buckwheat appears as a very drought-sensitive species.
Indeed, in comparison to other species, its water poten-
tial is quite high and it was not possible to precisely
quantify this parameter with the classical pressure cham-
ber method (Delpérée, unpublished results). The high level
Physiol. Plant. 119, 2003
of drought-sensitivity at the seedling stage may also be
related to the high values observed for carbon isotope
discrimination and to the poor ability of the species to
accumulate putative soluble osmoprotectants. In our work,
however, the amount of water available for the plants in
stressed treatments was fixed. Plants are indeed not able
to absorb water from the soil below a minimal Cws,
which depends on the soil property, and results could
therefore be different if the experiment is conducted in
pots of another size or in natural field conditions.
Theoretically, D values are positively correlated to Ci/Ca
ratios in C3 species and inversely related to transpir-
ation efficiency (Farquahr and Richards 1984). Since tran-
spiration efficiency is among the main determinants of
water use efficiency, one may assume that a high D value
is an indicator of a poor ability to efficiently regulate the
leaf water status. To the best of our knowledge, carbon
isotope discrimination was never previously quantified in
buckwheat. D values decreased in water stress conditions,
as expected on the basis of the well documented stress-
induced increase in water use efficiency. The values
recorded in Table 3, however, are always higher than
those reported in the literature for other species (Körner
et al. 1991, Tognetti et al. 2000, Merah et al. 2001).
Whatever the considered phenological stage, the plants
seemed able to regulate stomatal aperture in water stress
conditions, as suggested by the very low value recorded for
gl (Table 3). Thus, the stress-induced decrease in D may be
associated with a decrease in stomatal conductance,
leading to an increased ratio of A/gl but the very high D
value in buckwheat recorded even in control conditions
should be related to a low photosynthetic capacity leading
to a high Ci/Ca ratio.
Buckwheat seeds were shown to accumulate an
unusual galactosyl cyclitol (fagopyritol B1: O-a-D-
galactopyranosyl- (1!2)-D-chiro-inositol) associated with
the acquisition of dessication tolerance (Horbowicz et al.
1998). The presence of fagopyritol was never reported in
the vegetative organs of whole plants and we failed to
detect any unidentified peak with our refractive index
monitor after HPLC separation, even in cotyledons, thus
suggesting that this unusual sugar is absent, or present
only in undetectable amounts in our tissues.
Influence of leaf age
Acceleration of leaf senescence in plants subjected to vary-
ing degrees of water deficits has been described for a
number of species (Olsson 1995, Lutts et al. 1996, Deo
and Biswal 2001). Therefore, the sensitivity of leaf tissues
to drought is likely to increase with leaf age. This is not
the case in buckwheat since the youngest leaves often
appeared more sensitive than the oldest leaves on the
basis of their relative water content or abilities for osmotic
adjustment. Schurr et al. (2000) demonstrated that in
Ricinus communis submitted to water stress, growing
leaves developed source functions at a smaller size before
physiological adaptation to drought occurred. According
to Weber et al. (1996), sink to source transition implies a
219
developmentally regulated stimulation of sucrose-
phosphate-synthase leading to an obvious increase
in sucrose to hexose ratios. It was not the case in water-
stressed buckwheat and such a ratio even decreased, at
least under MI conditions. This suggests that sink-to-
source transition was not accelerated in our material or
that other factors, such as a stimulation of sucrose trans-
location or stress-induced stimulation of sucrose synthase
and/or invertases may concomitantly occur. In water-
stressed potato plants, Büssis et al. (1998) showed that
young leaves growing after the imposition of water stress
were smaller than control leaves but that their water use
efficiency remained unchanged. Our results with buck-
wheat present a different trend since at the 4-leaf stage
under both irradiances, D values were clearly reduced even
in youngest leaves (Table 3). These differences may be
related to species properties, but also to the modalities
of stress imposition. Büssis et al. (1998) exposed plants to
polyethyleneglycol in hydroponic cultures and thus to a
sudden osmotic shock while we used a progressive expos-
ure on solid substrate similar to constraints occurring in
natural conditions. Considering the progressive decrease
in yv (Fig. 1), it is not possible to assess precisely when the
plants actually begin to be stressed, although some physio-
logical modifications may already be observed at the
2-leaf stage. This also implies that in our system, organs of
different ages were exposed to different stress intensities.
Using a similar experimental strategy, David et al. (1998)
showed that the way Ci and A/gl changed with drought in
lupin was strongly dependent on leaf age: A/gl increase
only in the youngest leaves and soluble sugars decrease as
leaves aged, as shown in the present study.
Acknowledgements – This research was supported by the Fonds
National de la Recherche Scientifique (FNRS – Grant no.
2.4562.99). The authors are very grateful to Dr J. F. Ledent for
stimulating discussion, to Dr H. Batoko for his careful reading and
comments on the manuscript, to Dr B. McIntree for her valuable
help in the carbon isotope discrimination estimation and to Mrs
D. Masquelier and B. Vanpee for their valuable technical assistance.
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