J Agro Crop Sci (2015) ISSN 0931-2250

DROUGHT STRESS

Photosynthesis is Reduced, and Seeds Fail to Set and Fill at

Similar Soil Water Contents in Grass Pea (Lathyrus sativus L.)
Subjected to Terminal Drought
H. Kong1,2, J. A. Palta3,4, K. H. M. Siddique2, K. Stefanova2, Y.-C. Xiong1 & N. C. Turner2,5
1 State Key Laboratory of Grassland Agroecosystems, Institute of Arid Agroecology, School of Life Sciences, Lanzhou University, Lanzhou, China
2 The UWA Institute of Agriculture, The University of Western Australia, Crawley, WA, Australia
3 CSIRO Plant Industry, Wembley, WA, Australia
4 School of Plant Biology, The University of Western Australia, Crawley, WA, Australia
5 Centre for Plant Genetics and Breeding, The University of Western Australia, Crawley, WA, Australia

Keywords Abstract
13
C labelling; flower abortion; pod abortion;
stomatal conductance; water stress; b-N- Grass pea (Lathyrus sativus L.) is an indeterminate grain legume considered
oxalyl-L-a, b-diaminopropionic acid (b-ODAP) adapted to dry environments, but the mechanisms of its adaptation are not well
understood. Grass pea plants were exposed to terminal drought from podding,
Correspondence and the development of water deficit was measured together with its effects on
N. C. Turner
leaf photosynthesis, stomatal conductance, carbon remobilisation to the seeds,
The UWA Institute of Agriculture and Centre
flower production and abortion, pod production and abortion, seed set, seed
for Plant Genetics and Breeding
M080, The University of Western Australia growth and the neurotoxin b-N-oxalyl-L-a, b-diaminopropionic acid (b-ODAP)
35 Stirling Highway concentration. Predawn leaf water potential (Ψleaf), stomatal conductance (gs),
Crawley, WA 6009, Australia rate of leaf photosynthesis (Pn), flower production, pod production, filled pod
Tel.: +61 8 6488 4723 number, seed number, seed size and yield decreased, while flower abortion, pod
Fax: +61 8 6488 1140 abortion and seed abortion increased, and the concentration of b-ODAP was
Email: neil.turner@uwa.edu.au
unchanged under terminal drought conditions. gs and Pn began to decrease at a
and
higher plant-available soil water content (PAWC) (67.2
2.3 % and
Y.-C. Xiong
State Key Laboratory of Grassland 62.9
2.3 %) than Ψleaf (43.7
1.1 %). Flowers and pods ceased being pro-
Agroecosystems duced only when the PAWC decreased below 40.1
4.6 % and 35.3
3.0 %,
Institute of Arid Agroecology, respectively, but seed set and seed growth ceased when PAWC decreased below
School of Life Sciences 55.5
1.6 % and 58.0
3.7 %, respectively. The mobilization of 13C labelled
Lanzhou University, Lanzhou 730000 assimilates from the stems was greater under terminal drought than under well-
Gansu Province, China
watered conditions, but the transfer to the seed was small. We conclude that seed
Tel: +86-931-8914500
set and seed growth decreased as the soil dried due to a reduction in current
Fax: +86-931-8914500
Email: xiongyc@lzu.edu.cn photosynthesis as a result of stomatal closure.

Accepted September 4, 2014

doi:10.1111/jac.12102

Indian subcontinent, Ethiopia and to a lesser extent to Eur-

Introduction
Grass pea (Lathyrus sativus L.), a multipurpose grain
legume with an indeterminate growth habit, is well adapted
to adverse environments such as drought and flooding
(Siddique et al. 1996, 1999, Campbell 1997, Thomson et al.
1997, Leport et al. 1998, Sarker et al. 2001, Solaiman et al.
2007). However, grass pea is now mainly confined to the
ope, Australia, Asia and North Africa (Campbell et al.
1994, Campbell 1997, Hanbury et al. 1999) because of the
presence of the neuro-excitatory non-protein amino acid,
b-N-oxalyl-L-a, b-diaminopropionic acid (b-ODAP) in the
seeds. b-ODAP is considered the causative agent of lathy-
rism, an irreversible paralysis of the lower limbs (Kusama-
Eguchi et al. 2010, Van Moorhem et al. 2010, Nunn et al.

© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252 241

Kong et al.
2011). Considerable effort has been focussed in selecting
low-toxin genotypes, but as the species is cross pollinated,
low b-ODAP is difficult to maintain in the field if high-b-
ODAP genotypes are grown nearby. High levels of b-ODAP
biosynthesis in different tissues of the grass pea plant have
been shown to be positively associated with more severe
water stress (Lambein et al. 1990, Yan et al. 2006, Haque
et al. 2011, Kumar et al. 2011). The effects of water stress
on the levels of b-ODAP in the seeds were higher in the
low-toxin cultivar than in the high-toxin cultivar when
plants were grown in pots and under glasshouse conditions
(Hussain et al. 1997).
The impact of a water deficit on floral organs, which are
directly linked to seed production, is critical in the under-
standing of the adaptation of grass pea to dry environ-
ments. The sensitivity of growth, flowers and pods to water
deficits has been investigated in several grain legumes.
Water deficits during reproductive growth reduced flower
production by reducing node numbers, increased pod and
seed abortion, resulting in decreased yield in chickpea
(Leport et al. 2006, Fang et al. 2010). Water deficits during
reproductive growth of grass pea reduced dry matter, seed
yield, harvest index and water use efficiency, without affect-
ing b-ODAP concentration and seed size (Herwig et al.
2001, Polignano et al. 2009, Gusmao et al. 2012).
In a number of species, the remobilisation of pre-anthe-
sis stored reserves and post-anthesis carbon assimilates can
be important contributors of resources for seed filling,
particularly when seed filling occurs under water deficits
(Atkins and Smith 2007, Zaman-Allah et al. 2011). For
instance, studies using 13C showed that <20 % of the seed
carbon in chickpea came from pre-podding stored assimi-
lates when plants were water-stressed, proportions signifi-
cantly less than those calculated from changes in dry
weight (Davies et al. 1999, 2000, Leport et al. 1999, Fur-
bank et al. 2004). In water-stressed chickpea, the water
stress suppressed the net CO2 exchange by both leaves and
pods during seed filling, with high rates of CO2 recycling
inside the pods helping to compensate for the low rates of
photosynthesis in the leaves and providing an important
source of carbon for seed growth, additional to the previ-
ously-reported carbon redistribution from pod walls,
leaves and stems (Davies et al. 1999, 2000, Leport et al.
1999). The contribution of pre-anthesis and post-anthesis
carbon assimilates varies depending on species, genotypes
and environments (Fang et al. 2010, Kobata et al. 2012,
Aranjuelo et al. 2013).
Few studies have been focused on how carbon assimilates
influence seed and b-ODAP concentration in grass pea
under terminal drought. In this study, grass pea (Lathyrus
sativus L.) cv. Ceora, a cultivar with low levels (<0.1 %) of
the toxin b-ODAP (Siddique et al. 2006) was used to deter-
mine the contribution of pre-podding stored assimilates
242
and post-podding carbon assimilation to seed set, seed
growth, seed yield and b-ODAP concentration when a
water deficit was induced from podding. The aims of this
study were to (i) determine the effect of terminal drought
on flower and pod production, seed set, seed abortion, seed
growth and seed yield of grass pea; (ii) determine the con-
tribution of pre-podding stored carbon assimilates and
post-podding carbon assimilation to seed filling and seed
yield under terminal drought conditions; and (iii) deter-
mine the effect of terminal drought on the b-ODAP
concentration and amount in the seed.
Materials and Methods
Plant material and growth conditions
The experiment was conducted in a glasshouse from May
to October 2013 at The University of Western Australia,
Perth, Australia (31°570 S, 115°470 E). The average maxi-
mum and minimum temperatures over this period were
28 °C and 10 °C, respectively, with a mean relative humid-
ity of 57 %. Plants were grown in PVC columns, 0.15 m in
diameter that were filled to a depth of 0.4 m with 10 kg of
a 1 : 1 mixture of sieved, air-dried river sand and a clay
loam soil (clay = 50 %, silt = 22 %, sand = 27 %, pH 7.8
in 0.01 M CaCl2) collected from a farm site at Bindi Bindi,
Western Australia, dried and sieved through a 4-mm sieve
to remove stones and gravel. The bottom of each pot had
four 12-mm-diameter drainage holes that were covered
with a piece of filter paper before filling to prevent soil loss.
Prior to filling the pots, superphosphate (2.0 g), potassium
sulphate (2.0 g) and 0.8 g of a trace element mix (S, Fe,
Zn, Ca, Mn, B, Mg and Mo) were well mixed with each
10 kg of soil. Before sowing, 2.5 g of a commercial peat-
based Group F Rhizobium was also incorporated into the
top 30–40 mm of soil in each pot.
On 1st May, five seeds of L. sativus L. cv. Ceora were
sown 30–40 mm deep in each pot containing soil that had
previously been watered to excess and then allowed to
drain for 24 h before weighing to determine the field
capacity (FC) of the soil. The soil mixture had a water-
holding capacity of 2.3 l available water per pot. The plants
were thinned to three seedlings per pot at 10 days after
sowing (DAS) and reduced to one plant per pot 20 DAS.
The soil surface of each pot was covered with 150 g of plas-
tic beads (15 mm thick) to minimize soil evaporation.
Watering treatments
All pots were watered to bring the soil content to 100 % FC
by weighing every 2–3 days until two watering treatments
were imposed on 114 DAS when all of plants were podding:
(i) half of the pots (43 pots) were well watered every day to
© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252

maintain soil water content close to 100 % FC (well
watered, WW); (ii) water was withheld from the other half
of the pots (43 pots) to impose a slow dry down of the soil
water content by rewatering the pots so that a maximum of
100 ml of soil water was lost each day until water loss was
below 100 ml (water stress, WS) (Zaman-Allah et al. 2011).
The plants were harvested on four occasions: 114 DAS
(the day water was withheld, H1); 129 DAS (H2); 135 DAS
(H3) and 155 DAS (H4, physiological maturity, that is,
pods fully ripe). At each harvest, four plants per treatment
were harvested. In addition, 16 additional plants (8 WW
and 8 WS) were harvested at physiological maturity: eight
(4 WW and 4 WS) were used to measure the natural abun-
dance of 13CO2, while 8 plants (4 WW and 4 WS) were
used to measure b-ODAP concentration. Ten plants (5
WW and 5 WS) were used for destructive measurements of
predawn leaf water potential (Ψleaf), leaf net photosynthesis
(Pn) and stomatal conductance (gs). Twenty-eight addi-
tional plants (14 WW and 14 WS) were labelled three times
with 13CO2 during vegetative growth. The times of labelling
were (i) 30 DAS; (ii) 49 DAS; and (iii) 58 DAS. The labelled
plants were harvested on two occasions: 114 DAS, immedi-
ately before water was withheld (H1) and 155 DAS at phys-
iological maturity (H4). Pots were kept free of weeds and
moved every 2–3 days to minimise positional effects within
the glasshouse.
13
C labelling
During the vegetative phase, 28 plants were exposed to
13
CO2-enriched air at three times to label the photosyn-
thetic carbon and the pre-podding accumulation of carbon
in the leaves, stems and roots (Palta et al. 1994, Palta and
Gregory 1997). A 200 lm thick Mylar film (Dow Co., Mel-
bourne, Australia) chamber with aluminium frame was
placed over the plants and sealed to a groundsheet of poly-
ethylene film beneath the pots. Air inside the chamber was
stirred with a 30-cm-diameter fan. Canopy labelling
of 13CO2 was carried out between 10:00 and 13:00 h [Aus-
tralian Western Standard Time (AWST)] on clear sunny
days when the photosynthetic photon flux density (PPFD)
at the top of the canopy was above 1100 lmol m2 s1.
Before 13CO2 labelling, a column of soda lime was intro-
duced to the air circulation system to remove the excess of
CO2 inside the chamber. Once the air CO2 concentration
had declined to about 200 ll l1, the soda lime was discon-
nected and 13CO2 (99.9 % atom) gas was injected into the
chamber until the CO2 concentration reached 500–600 ll
l1, and the plants were allowed to deplete the CO2 in the
chamber to about 220 ll l1. This procedure was repeated
3–4 times during each labelling. The CO2 concentration in
the chamber was monitored with a LI-6251 CO2 infrared
gas analyzer (LI-COR Inc., Lincoln, NE, USA).
© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252
Water Stress, Photosynthesis and Seed Set in Grass Pea
Plant-available water content (PAWC)
The pots in the WW treatment were weighed between
09.00 h and 11.00 h (AWST) every 2–3 days throughout
the growth period to determine the water use (transpira-
tion), while the pots in the WS treatment were weighed
daily between 09.00 h and 11.00 h AWST as the soil dried
until no further water loss was recorded. This value was
taken as the lower limit of plant-available water in the soil.
The plant-available water content (PAWC) is then the
water in the soil between 100 % FC and this lower limit
and was calculated at the end of the experiment once the
lower limit had been determined. The PAWC values are
presented as a percentage between 100 % (100 % FC) and
the lower limit of 0 %.
Flower and pod tagging
The start of flowering and podding was recorded for each
plant to determine the mean time of flowering and pod-
ding. All new flowers and pods were tagged every 2–3 days
with the date of flowering and podding noted on the tags
so that flower number, flower abortion, pod number, pod
abortion, filled pod number and seed number could be
determined at physiological maturity.
Leaf water potential (Ψleaf)
The predawnΨleaf (05.00–06.00 h AWST) of one fully-
expanded leaf from the plant in four of the five pots per
treatment was measured every two days from the time the
two water treatments were imposed using a pressure cham-
ber (Model 1000, PMS Instrument Co., Albany, OR, USA).
Precautions, such as slow pressurisation, were followed as
recommended by Turner (1988).
Net rate of leaf photosynthesis (Pn) and stomatal
conductance (gs)
Pn and gs of plants in the WW and WS treatments were
measured every day between 13.00 h and 15.00 h AWST
during the period in which water was withheld. One fully-
expanded leaf from each pot with good light exposure was
chosen and measured using a LI-6400 portable photosyn-
thesis system (LI-COR Inc., Lincoln, NE, USA) at a photo-
synthetically active radiation of 1200 lmol m2 s1 and a
CO2 concentration of 380 ll l1.
Biomass, yield and yield components
At each harvest, plants were sampled by cutting the shoots
from the roots at the crown and the shoots were divided
into leaves, stems, pods and seeds. Immediately after the
shoots were harvested, the soil from each pot was carefully
243
Kong et al.
emptied on to a plastic sheet and the roots were recovered
from the soil by repeated sieving on a 1.4-mm sieve to pro-
duce a clean sample as described by Liao et al. (2006) and
Palta et al. (2007). All plant parts were dried at 70 °C for
72 h before weighing. At final harvest, the seeds were
grouped by date of pod set and were oven-dried at 60 °C
for 72 h and then weighed. Seed dry weights with different
podding dates were summed for yield and calculation of
harvest index and water use efficiency (WUEgr)
13
C abundance analysis
The plants labelled three times with 13CO2 during the pre-
podding phase were harvested at podding before the two
water treatments were imposed at 114 DAS and at final
harvest (155 DAS). Unlabelled plants were also harvested
for natural 13C abundance. As described above, plants were
cut at the soil level and the shoots divided into leaves,
stems, pods and seeds, and the roots from each pot were
recovered from the soil. All plant parts were dried at 70 °C
for 72 h, weighed ground to a fine powder using a ball mill
for analysis and measurement of 13C. Analysis for C con-
centration and the isotopic 13C enrichment was determined
using a VG-Micro mass Sira-10 (V-G Isogas Ltd, Middle-
wich, England) connected to a Europa Roboprep C-N ana-
lyser (Europa Scientific Ltd, Crewe, England). The amount
of C in the seed (Cs) that was derived from the vegetative
parts prior to podding and that which was derived from
post-podding assimilation and fixation was calculated. This
calculation was made using the 13C concentration in the
seed at maturity (13Csm), and the content in the vegetative
parts (vp) of unlabelled C (12C) and 13C (Cvp, 13Cvp) at
podding according to the following:
Cs = 13
Csm (Cvp/13Cvp)
It was assumed that there was no discrimination in the
remobilisation of 13C and 12C when C was translocated
from the vegetative tissues to the seed. Consequently, it was
assumed that unlabelled C was remobilised in a consistent
ratio with its stable isotope from podding onwards. In this
way, the total contribution of pre-podding remobilised C
to the seed was estimated. Post-podding assimilation and
remobilisation of C were calculated by subtracting the pre-
podding remobilised C from the total seed C.
Determination of b-ODAP concentration in seeds
The b-ODAP concentration was determined following the
method described by Wang et al. (2000) and Yan et al.
(2005). Eighty air-dry seeds, randomly chosen, were
ground to a powder and then 0.5 g was extracted with
4.5 ml of ethanol/water (3/1, v/v), shaken briefly, extracted
for 24 h, centrifuged for 0.25 h at 150 000 g and then
244
0.5 ml of supernatant was then dried at 50 °C for 24 h.
The residue was dissolved in 10 ll of a 0.5 M NaHCO3
solution, 100 ll of the reagent (100 mg of 1-fluoro-2, 4-
dinitrobenzene in 10 ml of acetonitrile) was added and the
solution thoroughly mixed. The derivatisation was com-
pleted in 0.75 h at 60 °C. After cooling to room tempera-
ture, the reaction mixture was added to 0.8 ml of 0.01 M
KH2PO4 solution, vortexed for several seconds and an ali-
quot of 20 ll of the product was taken for HPLC analysis.
An Agilent 1100 series HPLC liquid chromatography sys-
tem (Agilent Technologies, Santa Clara, CA, USA)
equipped with a C18 column of 3.9 9 150 mm was used.
The mobile phase solutions were filtered through a
0.45 lm membrane filter and degassed before use. The col-
umn was maintained at 40 °C and operated at a flow rate
of 1 ml min1 and then the absorbance of the sample was
recorded at 360 nm. Purified b-ODAP was used as external
standard for quantitative analysis.
Statistical analysis
The treatment structure of the experiment consisted of two
watering regimes – well watered (WW) and water stressed
(WS). There were four harvests, where each harvest and
each treatment level was replicated four times. A rando-
mised complete block design was used, accounting for the
treatment structure and using the four harvests as blocking
structure. All experimental data were analysed using linear
mixed model (LMM) techniques. The types of LMM used
in the analyses reflect the nature of the response variables
and the experimental procedure, in particular the treat-
ment structure and the experimental design/layout. In gen-
eral, the fixed terms in the model included treatment main
effects and their interactions and the random terms
accounted for the blocking structure.
The group of response variables related to counts,
namely flower number, pod number, filled pod number and
seed number were analysed using generalized linear mixed
models (GLMM). The count data were assumed to be Pois-
son distributed and the logarithmic (log) link function was
used. The response variable seed weight was normally dis-
tributed and a LMM showed a good fit. The second group
of analyses was related to measurements repeated in time
on the same experimental units (pots). These measure-
ments are correlated and the LMM techniques allow proper
modelling of the correlation/covariance structure. There
were 12 measurements, usually every 3 days between 23
August and 30 September. Additionally, PAWC was mea-
sured daily in parallel, so two separate types of analyses
were performed for all responses with explanatory vari-
ables, Time and PAWC, respectively.
The statistical model with time explanatory variable was
similar to the previously described LMM, except that in the
© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252

random part of the model the interaction term podtime was
added. The covariance structure of the latter was modelled
to account for the repeated measures correlations.
The statistical model with PAWC explanatory variable
used a ‘broken stick’ regression to assess the threshold
PAWC for different responses, namely Pn, gs, Ψleaf, cumula-
tive flower number, cumulative pod number, cumulative filled
pod number, cumulative seed number and cumulative seed
weight.
All of the analyses were performed using GenStat 15th
Edition (2013, VSN International, Rothamsted, UK), AS-
Reml-R (Butler et al. 2009), R 3.01 (R Core Development
Team, Austria, 2013), GenStat procedure R2 LINES
(A.W.A. Murray & J.T. Wood) and R package SIZER (Toms
and Lesperance 2003).
Results
Crop phenology
Flowering commenced in the first plants 59 DAS with a
mean time to the first flower of 65
3.7 DAS. Podding
first commenced at 73 DAS and the time from when a
flower first opened to when the pod was visible was
5
0.6 days. The first flower always aborted. The WS
treatment was imposed at 50 % podding (114 DAS) and
reduced the length of the flowering and podding period by
12 and 15 days, respectively. The plants in the WS treat-
ment reached physiological maturity 10 days earlier than
those in the WW treatment.
Plant-available water content (PAWC), stomatal
conductance (gs), photosynthesis (Pn) and predawn leaf
water potential (Ψleaf)
PAWC of the soil in the WW treatment was maintained
close to 100 % FC by watering the pots every 2–3 days
(Fig. 1). The PAWC of the soil in the WS treatment
decreased steadily for the first 13 days after water was with-
held (DAW) when water loss was restricted to a maximum
of 100 ml per day and then decreased very slowly from 13
DAW to near zero at 33 DAW (Fig. 1).
The gs and Pn of plants in the WW treatment were main-
tained high, near 200 mmol m2 s1 and 16 lmol m2
s1, respectively (Fig. 2a,b). The values of gs and Pn of
plants in the WS treatment decreased significantly after
seven DAW (Fig. 2a,b) when PAWC fell below
67.2
2.3 % and 62.9
2.3 %, respectively (Fig. 3a). gs
and Pn of the plants in the WS treatment decreased steadily
as the PAWC decreased to reach zero when the PAWC was
10.0
0.7 % (Fig. 3a).The predawn Ψleaf in the WW
treatment was maintained at 0.5 to 0.6 MPa (Fig. 2c).
Ψleaf in the WS treatment was similar to that in the WW
© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252
Water Stress, Photosynthesis and Seed Set in Grass Pea
Fig. 1 Change in plant-available water content (PAWC) in the soil
growing grass pea in the well-watered (WW) treatment and the water-
stressed (WW) treatment with time after water was withheld. Values
are means
one S.E. of the mean (n = 5) (all smaller than the
symbols).
treatment for the first 7 DAW, but then decreased after the
PAWC fell below 43.7
1.1 % (Fig. 3b) at a rate of
0.22 MPa day1 (P < 0.05) and reached 3.3 MPa when
PAWC was about 10 % (Fig. 3b).
Flower and pod production, flower and pod abortion, and
seed set
When the WS treatment was imposed, plants had devel-
oped 124
10 flowers. Flower production in the plants in
the WW treatment increased steadily and reached
200
11 flowers at about 30 DAW (Fig. 4a). Flower pro-
duction in the plants in the WS treatment increased faster
after water was withheld than in the WW treatment
(Fig. 4a), and the cumulative number of flowers increased
to a maximum of 196
14 flowers at 15 DAW so that at
18 DAW there was no significant difference (P > 0.05) in
the cumulative flower number between the WW and the
WS treatments (Fig. 4a). In the WS treatment, the cumula-
tive flower number increased linearly with PAWC until
PAWC decreased below 40.1
4.6 %.
The WS treatment had a significant effect on the total
number of pods and seeds produced (Fig. 4a–d). Fifteen
DAW when the PAWC was below 35.3
3.0 %, no more
pods were produced in the WS treatment resulting in a
total of 122
8 pods plant1 being produced by physio-
logical maturity, compared to 174
8 pods plant1 pro-
duced in plants under the WW treatment (Fig. 4a,b).
However, the number of filled pods did not increase from
9 DAW when the PAWC fell below 52.4
3.2 % in the
WS treatment (Fig. 4a,b), so that by physiological maturity
the plants in this treatment produced only 75.4
7.3 filled
245
Kong et al.
(a)
(b)
(c)
pods plant1 compared to 128.5
4.5 filled pods plant1
in the WW treatment (Table 1, Fig. 4a,b). When the
PAWC in the WS treatment fell below 55.5
1.6 %, at 9
DAW, no more seeds were set (Fig 4c,d), and the final
number of seeds was 124.0
14.3 seeds plant1in the WS
treatment compared to 269.5
10.9 seeds plant1 in the
WW treatment (Table 1, Fig. 4c,d). In the WW treatment,
the seed weight continued to increase for 24 DAW in the
WS treatment to reach a final weight of 31.8
1.3 g
plant1 at physiological maturity, whereas when PAWC
decreased to below 58.0
3.7 % in the WS treatment, the
seed weight stopped increasing to give a final seed weight
of 10.8
1.4 g plant1 (Table 1, Fig. 4e,f).
Flower abortion (the number of flowers that failed to
produce a pod, measured at physiological maturity)
246
Fig. 2 The change with time after water was
withheld in stomatal conductance (gs, a), pho-
tosynthesis (Pn, b) and predawn leaf water
potential (Ψleaf, c) of grass pea in the water-
stressed (WS) treatment and well-watered
(WW) treatment. Values are means
one S.E.
of the mean (n = 5) when larger than the
symbol.
increased from 16 % (31 of the 200 tagged flowers) in
the WW plants to 38 % (74 of the 196 tagged flowers)
in the WS plants (Table 2). There was no significant dif-
ference in the number of pods that did not produce a
seed (Table 2), but because of the different number of
pods produced in the WW and WS treatments (Table 1,
Fig. 4a), the percentage of pods that aborted by maturity
was 38 % in the WS treatment compared to 28 % in
the WW treatment.
Dry matter production, yield, yield components and
b-ODAP concentration
The WS treatment had a significant effect on the stem, leaf
and pod dry matter accumulation compared with the WW
© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252

(a)
(b)
Fig. 3 Relationships between (a) stomatal conductance (gs, % well-
watered treatment) and photosynthesis (Pn, % well-watered treatment)
and plant-available water content (PAWC), and (b) predawn leaf water
potential (Ψleaf, % well-watered treatment) and plant-available water
content (PAWC). Lines in (a) and (b) were fitted with a ‘broken stick’
regression.
treatment (Fig. 5). In the WW treatment, the total dry
matter increased at each harvest, but in the WS treatment,
there was no significant (P > 0.05) increase in total dry
weight (DW) in the reproductive phase (H2–H4) as a result
of a decrease in leaf DW, stem DW and root DW, but an
increase in pod DW (Fig. 5).
At physiological maturity, the WS treatment reduced
the aboveground DW by 39 % (P < 0.01), the root DW
by 22 % (P < 0.05) and seed yield by 66 % (P < 0.001)
compared with the WW plants (Table 1). The WS treat-
ment reduced the number of filled pods plant1 by
41 % (P < 0.001), reduced the seed number plant1 by
54 % (P < 0.001), reduced the seed number pod1 from
2.1 to 1.6 (P < 0.05) and seed size from 120 to 90 mg
seed1 (P < 0.05) (Table 1). Harvest index (HI)
decreased significantly (P < 0.01) from 0.46 in the WW
treatment to 0.26 in the WS treatment (Table 1). The
water use efficiency in the production of yield (WUEgr)
was also reduced from 1.68 g l1 in plants in the WW
treatment to 0.96 g l1 in the WS treatment. There was
no difference in b-ODAP concentration in the seeds in
the two treatments (Table 1), but as seed yield was sig-
nificantly lower in the WS treatment, the total amount
of b-ODAP accumulated in the seeds was significantly
lower at 1.42 g plant1 in the WS treatment compared
with 4.10 g plant1 in the WW treatment.
© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252
Water Stress, Photosynthesis and Seed Set in Grass Pea
13
C redistribution
At podding (114 DAS), the amount of 13C in the leaves,
stems, pods and roots was 34.9
3.7 mg, 36.4
2.6 mg,
0.7
0.1 mg and 12.5
0.8 mg, respectively, corre-
sponding with a percentage allocation of 13C of 40.6

3.4 %, 43.7
3.3 %, 0.9
0.1 % and 14.8
0.8 %,
respectively (Fig. 6). At physiological maturity (155 DAS),
the 13C stored in leaves was a little higher than at podding,
but there was no significant difference (P > 0.05) between
the WW plants and WS plants, whereas the 13C stored in
stems decreased significantly between podding and matu-
rity by more than 6.0 %, but there was no significant
difference between WW and WS plants (P > 0.05) in the
13
C in the stems at maturity. The contribution of pre-pod-
ding C from vegetative tissues to seed C was <5 % in both
the WS and the WW treatments with no significant differ-
ence between the WW and WS treatments in the amount
or proportion of pre-podding C in the seeds (Fig. 6). The
majority of the 13C redistributed from vegetative tissues to
the seed was to the first-formed seeds that developed before
the water stress became severe (Fig. 6).
Discussion
Terminal drought, imposed during podding had a major
impact on the accumulation of shoot and root DW,
seed yield, HI, water use and WUEgr in grass pea. The
reduction in seed yield arose from a reduction in flower
production, pod production, seed set and seed growth,
and an increase in flower, pod and seed abortion. Inter-
estingly, the PAWC at which the production of flowers
and pods stopped and the PAWC at which seed set
ceased were different. Seed set ceased and seed filling
slowed at higher values of PAWC than flower and pod
production, but the flowers and pods that were pro-
duced at these low values of PAWC aborted and did
not produce any seeds. The PAWC at which seed set
and seed growth were affected coincided with the
PAWC at which gs and Pn began to decrease, suggesting
that seed set and seed filling, but not flower production
and initial pod development, were limited by the avail-
ability of current assimilates.
In the present study, the soil water content was con-
trolled for the first 15 DAW so that it decreased steadily
and similarly in all pots, irrespective of the size and leaf
area of the plants, until the PAWC was below 25 %. This
slow and steady decrease in soil water content enabled the
threshold PAWC at which the stomata began to close and
Pn to decrease to be identified and shown to be about
65 %, at which PAWC the predawn Ψleaf was still the same
as in the WW plants. The threshold PAWC at which the
predawn Ψleaf began to decrease was 44 %. The results
247
Kong et al.
(a) (b)
(c) (d)
(e) (f)
suggest that gs and Pn did not decrease as a result of leaf
dehydration and loss of turgor, but decreased as a result of
the production of phytohormones, such as abscisic acid
(ABA), by the roots (Davies and Zhang 1991, Davies et al.
2005, Turner and Hartung 2012). ABA in the xylem and
leaves was not measured in this study, but the results are
similar to those observed in wheat when subjected to a
water deficit in which ABA in the leaves was measured
(Xiong et al. 2006, Du et al. 2013). However, flower pro-
duction and initial pod development continued until the
PAWC was below 40 %, that is below the PAWC at which
the Ψleaf began to decrease. Although the flower and small
pod water potential were not measured in this study, the
results are consistent with the results in Brassica rapa in
which the flower bud had lower temperatures, indicative of
248
Fig. 4 Changes in (a. b) the cumulative num-
ber of flowers, pods and filled pods plant1,
(c, d) the cumulative number of seeds plant1,
and (e, f) cumulative seed weight plant1 of
grass pea with time after water was withheld
in the water-stressed (WS) treatment and well-
watered (WW) treatment (a, c, e), and with
plant-available water content in the WS treat-
ment (b, d, f). Values are means
one S.E. of
the mean (n = 12) when larger than the
symbol.
stomatal opening, to lower soil water contents than the leaf
temperature (Guo et al. 2013).
Grass pea is known to be well adapted to dry environ-
ments where water deficits occur (Siddique et al. 1996,
1999, Campbell 1997, Thomson et al. 1997, Gusmao et al.
2012). However, the water deficit that the plants developed
in the terminal drought reduced seed yield by 66 % com-
pared to the plants in the WW treatment. This was due to a
significant reduction in number of filled pods plant1
(41 %), seeds plant1 (54 %) and seeds pod1 (22 %)
compared with WW plants, corresponding with less flower
and pod production and more flower and pod abortion.
The water deficit decreased HI and WUEgr by 43 % com-
pared to the plants in the WW treatment. These results are
similar to the results on chickpea and lentil (Leport et al.
© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252
 Water Stress, Photosynthesis and Seed Set in Grass Pea

Table 1 Aboveground dry weight (DW), root DW, seed yield, yield
components, harvest index, water use, water use efficiency of grain
(WUEgr) and b-ODAP concentration of the seeds of grass pea in the
well-watered (WW) treatment and water-stressed (WS) treatment at
physiological maturity

P-
Components WW WS value

Aboveground DW (g 97.7
2.5 60.0
3.0 **

plant1)
Root DW (g plant1) 6.0
0.4 4.7
0.2 *
Seed yield (g plant1) 31.8
1.3 10.8
1.4 ***
Filled pod number 128.5
4.5 75.4
7.3 ***
(plant1)
Seed number (plant1) 269.5
10.9 124.0
14.3 ***
Seed number (pod1) 2.10
0.06 1.64
0.06 *
Seed size (g seed1) 0.12
0.03 0.09
0.02 *
Harvest index 0.46
0.02 0.26
0.02 **
Water use (l pot1) 18.2
1.3 11.3
1.9 ** Fig. 5 Root dry weight (DW, g plant1), stem DW (g plant1), leaf DW
WUEgr (g l1) 1.68
0.17 0.96
0.09 ** (g plant1) and pod DW (g plant1) of grass pea at four harvests on 114
b-ODAP (%) 0.129
0.004 0.132
0.008 ns DAS (H1), 129 DAS (H2), 135 DAS (H3) and physiological maturity (155
DAS, H4) in the well-watered (WW) treatment and water-stressed (WS)
Values are means
one S.E. of the mean. (n = 12). The significance (P
treatment. Bars are
one S.E. of the mean (n = 4 for H1 and n = 12
value) is also given. Significant at ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05;
for H2–H4)
ns, not significant at P > 0.05.

Table 2 The number of aborted flowers and aborted pods of grass pea
at physiological maturity in the well-watered (WW) treatment and the
water-stressed (WS) treatment. Values are means
one S.E. of the
mean (n = 12)
Flowers/pods WW WS
Aborted flowers (plant1) 31
3.5 74
5.1
Aborted pods (plant-1) 46
4.9 46
3.7
Significant differences are also shown: ***P ≤ 0.001, and ns, not
significant.
1998, 1999, 2006, Behboudian et al. 2001, Shrestha et al.
2006, Fang et al. 2010, 2011, Gusmao et al. 2012). The seed
size of ‘Ceora’ in response to water deficit can be useful in
the adaptation of grass pea to the Mediterranean-type envi-
ronment of southern Australia (Gusmao et al. 2012). Con-
trary to results with chickpea (Fang et al. 2010, 2011) and a
previous study with grass pea (Gusmao et al. 2012), termi-
nal drought decreased seed size by 25 % compared to WW
plants, primarily in seeds that were filling late in the drying
cycle.
The present study strongly suggests that seed set and seed
filling were reduced in the plants in the WS treatment as a
consequence of a shortage of assimilates induced by the
decrease in the rate of leaf photosynthesis (Palta et al. 2008,
2012). The number of filled pods, number of seeds plant1,
seed weight and pod weight all stopped increasing when
the PAWC was between 53 % and 58 %, while Pn began to
decrease when the PAWC was 62
2.3 %. Seed set and
seed filling in indeterminate grain legumes are heavily
dependent on current photosynthesis from leaves and pos-
sibly pod walls (Furbank et al. 2004), as only small quanti-
ties of pre-anthesis carbon are transferred to the seeds
P-value
where it contributes mainly to the protein fraction of the
*** seed (Singh and Pandey 1980, Palta et al. 2008, 2012).The
ns 13
C study showed that <5 % of the carbon in the seed was
retranslocated from vegetative tissues and that the amount
of carbon redistributed from vegetative tissues to the seed
was similar in the plants in both the WS and the WS treat-
ments. Most of the pre-podding assimilated 13C was allo-
cated to the leaves and stems, but after podding this carbon
was largely retranslocated to the newly-formed leaves.
Grass pea is an indeterminate legume, and most indetermi-
nate legumes have high growth rates and continue leaf pro-
duction after flowering (Singh and Pandey 1980) that
depend on pre-flowering stored assimilates (Palta et al.
2008, 2012). Some pre-podding assimilated carbon was
retranslocated from the stems and roots to the seed during
seed filling, but the amount was small and could not com-
pensate for the shortage of current assimilates that resulted
from the reduction in Pn with terminal drought after pod-
ding. Other studies with legumes have shown that <20 %
of seed carbon in chickpea came from pre-podding stored
assimilates when plants were water-stressed (Davies et al.
2000). Other studies, using 14C and 13C labelling, found
that current assimilation by the leaves and pods was the
primary source of carbon for seed growth (Singh and
Pandey 1980, Ma et al. 2001, Furbank et al. 2004).

© 2014 Blackwell Verlag GmbH, 201 (2015) 241–252 249

Kong et al.
(a)
(b)
Fig. 6 Change in the allocation of 13C in leaves, stems, pods, roots and
seeds (Seeds 1 were produced before water was withheld, Seeds 2
were produced 0–5 days after water was withheld (DAW), Seeds 3
were produced 6–11 DAW and Seeds 4 were produced more than 11
DAW) of grass pea in the well-watered (WW) treatment at the podding
stage (P) just before the watering treatments were imposed, and in the
WW treatment and water-stressed (WS) treatment at physiological
maturity (final harvest, FH). Values are means
one S.E. of the mean
(n = 4) when larger than the bar graph.
The b-ODAP concentration in seeds was not signifi-
cantly different between WW and WS plants. Gusmao et al.
(2012) had a similar result in which a water deficit had little
effect on b-ODAP concentration of ‘Ceora’ seeds, but this
contrasts with previous studies showing that water stress
increased b-ODAP concentration in the seed (Herwig et al.
2001, Yang et al. 2004, Haque et al. 2011, Kumar et al.
2011). Moreover, these experiments used high-b-ODAP
lines, whereas Ceora is a very low-b-ODAP line. Neverthe-
less, as seed yield decreased, the amount of b-ODAP syn-
thesised and accumulated in the seed by WS plants was
only 35 % of that synthesised by WW plants, which is simi-
lar to the 13–50 % reduction in synthesis in previous stud-
ies (Herwig et al. 2001, Yang et al. 2004, Haque et al. 2011,
Kumar et al. 2011, Gusmao et al. 2012). Moreover, some
studies showed that b-ODAP concentration is most
strongly influenced by the genotype by environment inter-
action (Hanbury et al. 1999) indicating that further studies
on the effect of water deficit on b-ODAP concentration in
a range of genotypes and water-stress scenarios are
warranted.
250
Conclusions
The slow steady decrease in soil water content has shown
that gs, Pn, seed set and seed growth all decreased when the
PAWC was about 60 %, that is when only 40 % of the
plant-available water in the soil had been transpired, and at
PAWC about 10 % higher than when the Ψleaf began to
decrease. This suggests that seed set and seed growth were
limited by current assimilate supply as a result of stomatal
closure induced by root-sourced phytohormones, such as
abscisic acid. Nevertheless, we cannot rule out the possibil-
ity that the phytohormones directly affected the fertilisa-
tion and early growth of the seeds, rather than the supply
of assimilates. The role of phytohormones and assimilates
in seed set and seed growth is worthy of further investiga-
tion. What is clear from the study is that stored assimilates
in the leaves, stems and roots, although remobilised during
seed filling, did not contribute any more carbon to seed fill-
ing than in the well-watered plants, even when the terminal
drought reduced current assimilation.
Acknowledgements
The Chinese Scholarships Council, the Overseas Masters
Program of the Ministry of Education of China
(Ms2011LZDX059), the National Meteorological Industrial
Program (GYHY201106029 -2), the Chinese Ministry of
Education, Lanzhou University, CSIRO Plant Industry, the
UWA Institute of Agriculture and Centre for Plant Genetics
and Breeding at The University of Western Australia are
thanked for support for this project. The School of Plant
Biology at The University of Western Australia is thanked
for the loan of equipment.
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