CSIRO PUBLISHING

Crop & Pasture Science, 2016, 67, 553–562

[Link]

Comparative responses to water deficit stress and subsequent

recovery in the cultivated beet Beta vulgaris and its wild
relative B. macrocarpa

Inès Slama A,C, Asma Jdey A, Aida Rouached A,B, Ons Talbi A, Ahmed Debez A, Tahar Ghnaya A,
Mohamed Anis Limami B, and Chedly Abdelly A
A
Laboratoire des Plantes Extrêmophiles, BP 901, Centre de Biotechnologie, Technopole de Borj Cédria,
Hammam-Lif 2050, Tunisia.
B
University of Angers, UMR 1345 Research Institute of Horticulture and Seeds (INRA, Agrocampus-Ouest,
University of Angers), SFR 4207 Quasav, 2 Bd Lavoisier, 49045 Angers Cedex, France.
C
Corresponding author. Email: slama_ines@[Link]

Abstract. The effects of water deficit stress and recovery on growth, photosynthesis, physiological and biochemical
parameters were investigated in the cultivated Beta vulgaris and in two Tunisian provenances (Soliman and Enfidha) of its
wild relative B. macrocarpa. Seedlings were cultivated for 4 weeks under optimal or limiting water supply (respectively,
100% and 25% of field capacity, FC). After 2 weeks of treatment, a lot of stressed plants were rehydrated to 100% FC. In
the Control, B. vulgaris was more productive than B. macrocarpa, whereas Enfidha provenance showed the highest
biomass production (1.6- and 3-fold compared with B. vulgaris and Soliman, respectively), under water deficit stress.
A partial re-establishment of growth occurred in both species upon recovery at 100% FC. The sensitivity of B. vulgaris
and Soliman provenance to drought was associated with the disturbance of leaf water status and the sharp decrease in net
CO2 assimilation (–66% and –82% as compared with the Control, respectively). On the contrary, the better behaviour of
Enfidha provenance was related to its better photosynthetic capacity and leaf relative water content, along with a higher
accumulation of amino acids (proline, glycine, and glutamine) implied in the osmotic adjustment. Leaf hexose concentration
increased significantly under drought stress in both species whereas leaf sucrose concentration declined only in drought-
stressed B. vulgaris and Soliman provenance. Leaf glutamate dehydrogenase activity increased under water deficit in both
species despite to a higher extent in B. vulgaris. As glutamate dehydrogenase is implied in catabolism of glutamate to
oxoglutarate, it might contribute to provide stressed plants with carbon skeletons.
Enfidha provenance of the spontaneous species B. macrocarpa could be used in the marginal arid ecosystems in order
to limit the deficit in fodder and to improve the pastoral value of these regions. In addition, this species could serve as a
source of genes for genetic improvement to water deficit stress.

Additional keywords: amino acid, nitrogen metabolism, photosynthesis, soluble sugars, water statue.

Received 11 May 2015, accepted 16 December 2015, published online 5 April 2016

Introduction of drought-tolerant plants with fodder potentialities would

Drought is among the major constraints affecting yield stability enable to create new production zones in order to limit the
and fodder production in arid and semiarid regions, where deficit in fodder and to improve the pastoral value of these
climate is generally characterised by extreme seasonal marginal arid ecosystems. Selection of plants resistant to water
variations in temperatures in addition to low rainfall. This, deficit is of paramount importance and the identification
together with scarcity and low quality of water resources, of physiological proprieties used by some chenopodiaceae
hinders a quick recovery of the soil plant covering (Save et al. species to cope with drought is therefore of great interest
1999; Hessini et al. 2008). For these reasons, the identification (Sambatti and Caylor 2007).

Abbreviations: ATP, adenosine triphosphate; CBBC, Centre of Biotechnology of Borj Cedria; DAS, days after sowing; DW, dry
weight; EC, electrical conductivity; EL, electrolyte leakage; FC, field capacity; FW, fresh weight; GABA, g-aminobutyric acid; GDH,
glutamate dehydrogenase; LWC, leaf water content; NAD+, nicotinamide adénine dinucléotide; NADH, nicotinamide adenine
dinucleotide hydrogen; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; P5CS, pyrroline-5-carboxylate synthase;
PAR, photosynthetically active radiation; ROS, reactive oxygen species; VPD, vapour pressure deficit.;

Journal compilation  CSIRO 2016 [Link]/journals/cp

554 Crop & Pasture Science I. Slama et al.

Environmental stress can disrupt cellular structures and In Tunisia, it is found on salty soils, among halophytes tufts, and
impair key physiological functions (Larcher 2003). Drought at the edge of sebkhas. As a halophytic or semi-halophytic
generates an osmotic stress that can lead to turgor loss. species, this herbaceous annual species can potentially be used
Membranes may become disorganised, protein may undergo to maintain the plant cover in salt-affected regions (Abdelly
loss of activity or be denatured, and often, excessive levels 1997). B. macrocarpa shows high biomass production in the
of reactive oxygen species are produced leading to oxidative presence of 200 mM NaCl and is able to use ammoniacal N,
damage (Krasensky and Jonak 2012) as a consequence. The forms relatively available in the saline biotopes. Hessini et al.
inhibition of photosynthesis, metabolic dysfunction and damage (2009) studied responses of B. macrocarpa to salinity and
of cellular structures contribute to growth disturbances. Drought demonstrated that unlike other Chenopodiaceae, this species
stress decreases photosynthetic rate and disrupts carbohydrate accumulates NaCl at low rates not exceeding 5.4% of dry
metabolism in leaves too (Pelleschi et al. 1997; Kim et al. 2000). matter. Its protein content (18–34%) makes it possible to
Both may lead to a reduced amount of assimilate available for classify it like plants of interesting fodder quality (9–19%).
export to sink organ. The physiology of B. macrocarpa under limiting water
In plants, the main end products of photosynthesis are starch supply has not been yet studied. Thus, the present work aims
and sucrose. The increase in leaf hexose concentration under to investigate the behaviour of two Tunisian provenances of
water deficit stress has also been observed in soybean (Huber B. macrocarpa to water deficit stress and to evaluate their
et al. 1984), pigeonpea (Keller and Ludlow 1993). Hexose recovery capacity after a period of water deficit. In addition,
accumulation in plant leaves under drought stress may B. macrocarpa provenances were compared with their relative
contribute to turgor maintenance and it has been thought to be cultivated species B. vulgaris.
part of plant strategies to adapt to drought stress (Turner 1996).
However, a high hexose concentration may also play a feedback Material and methods
role in depressing photosynthesis (Goldschmidt and Huber Experimental design and measured parameters
1992).
Drought-tolerant plants can use several mechanisms to adapt Experiment was carried out in pots in a greenhouse at the
Centre of Biotechnology of Borj Cedria under semi-controlled
to water stress. These include the reduction of water loss by
conditions (14-h photoperiod, mean temperature and relative
increased water uptake via the development of a large and deep
root system (Parsons and Howe 1984). Metabolic adjustments humidity were, respectively, 29
38C, 52
4% day and
in response to unfavourable conditions are dynamic and 15
38C, 90
3% night). Every pot was filled with 2 kg of
soil from a parcel located at Centre of Biotechnology of Borj
multiple and not only depend on the type and strength of the
stress, but also on the cultivar and the plant species. At the Cedria. It is a clay-sandy soil containing 0.95, 0.25, 0.65 and
cellular level, plants tolerate drought stress to some degree by 0.05 mEqn 100 g1 dry soil K+, Na+, Ca2+, Cl, respectively, and
0.24 and 0.45 g kg1 of dry soil of P2O5 and total N, respectively.
accumulating osmolytes (Bohnert et al. 1995). Most of these
osmolytes are nitrogenous compounds and hence N metabolism The pH and the electrical conductivity of the aqueous extract
is of central importance under stressful conditions. Further, the (1/10) were 6.6 and 0.05 mmhos cm1, respectively. The wilting
point of our soil is 2.6%. The field capacity (FC) of soil, measured
increase in amino acid pool has been shown to occur in a variety
according to the technique of Bouyoucos (1983), was 11.25%.
of plant species as a response to water stress (Dubey 1994),
accounting for osmotic adjustment. For instance, it has been Culture was conducted in the greenhouse in the Centre of
Biotechnology of Borj Cedria. Seeds were sown at a rate of six
suggested that glycine betaine protects photosystem II,
seeds per pot. Emerged seedlings of uniform size (one plant per
stabilises membranes, and mitigates oxidative damage in
addition to its role as an osmolyte. In addition to glycine the pot) were subjected to a 40-day-long pre-treatment phase during
role of proline in osmotolerance was documented (Szabados which soil was maintained at 100% FC. Plants (40 days after
sowing (DAS)) were then divided into two batches: Control
and Savouré 2010). Proline biosynthesis is activated and its
catabolism repressed during dehydration, whereas rehydration (100% FC) and stressed (25% FC) plants. After 2 weeks of
triggers the opposite regulation (Xue et al. 2009). treatment (54 DAS), a lot of dehydrated plants was rewatered
at 100% FC. After 1 month of treatment (70 DAS), a final
Plant tolerance to water deficit conditions depends on their
harvest was carried out and plants were separated into shoots
ability to activate several mechanisms including osmotic
adjustment. Increased concentrations of free amino acids together and roots.
with organic acids and quaternary ammonium compounds serve Growth, water relation measurements and gas exchange
as compatible cytoplasmic solutes to maintain the osmotic
balance under water stress (Dubey 1994). Plant fresh weight (FW) was determined upon harvesting. Dry
Water deficit stress affects N metabolism. In this way, weight (DW) was obtained after oven drying the samples at 608C
glutamate dehydrogenase (GDH) can also contribute to until a constant weight was reached. Turgid weight (TW) was
ammonium assimilation. Despite its abundance in plant tissues, obtained after soaking leaves in distilled water in test tubes for
under stressed conditions, the precise function of GDH remains 12 h at room temperature (~208C), under low light conditions.
unclear. GDH is mainly involved in the oxidative deamination Relative water content was determined using the following
process to provide carbon skeletons to the citric acid cycle equation: RWC (%) = ((FW – DW)/(TW – DW).
(Robinson et al. 1991; Taiz and Zeiger 1998).
Leaf water content was determined as:
The annual B. macrocarpa (Chenopodiaceae) is a widespread
species in the west Mediterranean basin (Pottier-Alapetite 1979). LWC ðmL g1 DWÞ ¼ ðFW  DWÞ=DW:
Effects of water deficit stress in Beta macrocarpa Crop & Pasture Science 555

Net CO2 assimilation was recorded on fully expanded polyvinylpolypyrrolidone. The homogenate was centrifuged
mature leaves with a portable photosynthesis system (LCA4). (15 000g, 48C, 15 min) and the supernatant was used as the
Measurement conditions were as follows: 25
38C leaf crude extract.
temperature, 1300 mmol m2 s1 (photosynthetically active The GDH activity was assayed by following the oxidation of
radiation), 360 mmol mol–1 ambient CO2 concentration and NADH and the reduction of nicotinamide adénine dinucléotide
1.2 kPa vapour pressure deficit. Measurements were carried out at 340 nm for 30 min at 308C, respectively, according to
in the morning between 10 : 00 and 12 : 00 a.m. Data were Glévarec et al. (2004). P5CS activity was measured using
automatically collected every minute after the photosynthesis 20 mL of the crude extract in a reaction mixture containing
rate had stabilised. Tris-HCl buffer (100 mM, pH 7.2) supplemented with MgCl2
(20 mM), glutamate (75 mM), and ATP (5 mM). The reaction was
Electrolyte leakage initiated by the addition of nicotinamide adenine dinucleotide
Electrolyte leakage (EL) was determined according to Dionisio- phosphate hydrogen (NADPH) (0.4 mM) substrate of the P5CS.
Sese and Tobita (1998) and calculated according to the following The oxidation of NADPH was followed spectrophotometrically
equation: at 340 nm for 30 min at 308C.
EL ¼ ðEC1=EC2Þ  100:
Soluble proteins
Amino acid extraction and high-performance liquid Soluble protein concentrations were determined using
chromatography (HPLC) analyses Coomassie brilliant blue (Bradford 1976) with bovine serum
Amino acids were extracted from leaves in 96% (v/v) ethanol at albumin as a protein standard.
48C for 1 h. After centrifugation (9500g, 48C, 15 min), the ethanol
fraction was removed and the same process was repeated with Statistical analyses
deionised water. The ethanol and water fractions were combined. Results are expressed as mean of four replicates. Data were
After evaporation of the extract under vacuum, organic residues analysed with the MSUSTAT software version 4.12 (Lund
were dissolved in deionised water and extracted with the same 1989). A comparison among the means to determine significant
volume of chloroform. After centrifugation (14 000g, 48C, 15 min), differences (P < 0.05) was performed using the l.s.d. test.
an aqueous phase containing amino acids was vacuum-dried. The
amino acids were then redissolved in deionised water. Samples Results
were passed through a nylon syringe filter (0.45 mm) and then Plant growth
analysed by HPLC (Waters Corporation, Milford, Massachusetts,
Under Control conditions, shoot and root DW were significantly
USA). The amino acids were determined by the AccQTag
higher in the cultivated species B. vulgaris when compared
method, which uses AccQFluor reagent (Waters Corp. Milford,
with the two provenances of the spontaneous species
MA, USA) to derivatise the amino acids. The reagent is a highly
B. macrocarpa (Soliman and Enfidha) (Fig. 1a, b). Water
reactive compound (6-aminoquinolyl-N-hydroxy-succinimidyl
deficit stress reduced shoot DW (Fig. 1a), especially in
carbamate) that forms stable derivatives with primary and
Soliman provenance (75%), followed by B. vulgaris (62%)
secondary amino acids. Derivatives were separated by reverse-
and Enfidha provenance (37%). Under water deficit stress,
phase (C18 column) HPLC and quantified by fluorescence
Soliman provenance produced significantly lesser biomass
detection (the excitation and emission wavelengths were,
when compared with B. vulgaris and Enfidha provenance.
respectively, 250 and 395 nm). Each sample was analysed over
Shoot DW recovery was partial in all studied species. Yet,
1 h. Amino Acid Hydrolysate Standard and an AccQ-Tag Amino
B. vulgaris showed higher root biomass than Soliman and
Acid Analysis Column (3.9  150 mm, 4 mm) were used for
Enfidha. Water deficit stress decreased significantly root DW
amino acid determination. The run time is 60 min and the
in B. vulgaris (50%), whereas no effect was observed in the
temperature was 378C.
spontaneous species B. macrocarpa.
Soluble sugars extraction and HPLC analyses
Leaf water content
Sugars were extracted by the same method as amino acid, and
they were analysed by HPLC on a Carbopac PA1 (Dionex Corp., In Control plants, leaf RWC was similar for B. vulgaris and
Sunnyvale, CA, USA) using pulsed amperometric detection B. macrocarpa (~70%) (Fig. 2a). Water deficit stress reduced
EC 2000 (Thermo Scientific, Vernon Hills, IL, USA), Sugars leaf RWC in both species by 33%, 27% and 8.5%, respectively,
were chromatographed on a CarboPac PA100 4 250-mm column in B. vulgaris and the provenance Soliman and Enfidha.
(Dionex Corp.) preceded by a guard column (CarboPac PA100, In water deficit-stressed plants, leaf RWC was significantly
4 50 mm). Software Borwin (JMBS Developments, Les Ulis, higher in the provenance Enfidha than B. vulgaris and Soliman
France) was used to collect and analyse data. Carbohydrate provenance. Rehydration restored partially this parameter in
standards (glucose, fructose, sucrose, raffinose, stachyose and B. vulgaris and in the provenance Enfidha. Leaf water content
melizitose) was used for soluble sugar determination. decreased by water deficit stress conditions in B. vulgaris and
in the two provenances of B. macrocarpa by ~30% (Fig. 2b).
Pyrroline-5-carboxylate synthase (P5CS) GDH activity
Glutamate dehydrogenase and P5CS were extracted from Net CO2 assimilation
frozen seedlings in 2 mL of phosphate buffer (Na2HPO4/ Net CO2 assimilation (Fig. 3) changes were similar to those of
KH2PO4; 10 mM, pH 7.5) supplemented with 1% (w/v) shoot DW variation. The highest value of net CO2 assimilation
556 Crop & Pasture Science I. Slama et al.

Control Stressed Rehydrated Control Stressed Rehydrated

5 100
f
(a) (a)
90
e d d

Leaf relative water content %

4 80 d
c c
de c
Shoot DW g plant–1

d 70 b
d
b
3 c 60
a
50
b
2 b 40

30
a
1 20

10

0 0
3.0 16
c (b) (b)
14 b b

Leaf water content (mL g–1 DW)

b b
b
2.5 b
12
Root DW g plant–1

10 a a
2.0
b b a
8

1.5 a a 6
a
a a a
4
0.5
2

0 0
Soliman Enfidha Soliman Enfidha
Beta vulgaris Beta macrocarpa Beta vulgaris Beta macrocarpa

Fig. 1. (a) Shoot and (b) root dry weight of Beta vulgaris and two Fig. 2. Leaf water content of Beta vulgaris and two provenances of Beta
provenances of Beta macrocarpa. Plants were harvested after 1 month of macrocarpa. Plants were harvested after 1 month of treatment. Control
treatment. Control plants were irrigated at 100% field capacity (FC) (open plants were irrigated at 100% field capacity (FC) (open columns).
columns). Drought-stressed plants were irrigated at 25% FC (closed Drought-stressed plants were irrigated at 25% FC (closed columns). Plants
columns). Plants were rewatered to 100% FC after 15 days of treatment were rewatered to 100% FC after 15 days of treatment (grey columns).
(grey columns). Mean of four replicates. Bars with different letters are Mean of four replicates. Bars with different letters are significantly
significantly different according to l.s.d. test at P < 0.05. different according to l.s.d. test at P < 0.05.

was observed in Control plants of B. vulgaris (15.73 mmol CO2 plants of Soliman provenance showed EL value close to that
m–2 s–1). When subjected to water deficit stress, this parameter of the stressed plants.
decreased significantly in both species. This trend was however
more marked in the provenance Soliman (–82%). In B. vulgaris Soluble sugar content
and the provenance Enfidha, this reduction reached 66% and
Leaf hexose (fructose and glucose) concentration was
38%, respectively. Under water deficit stress conditions, Enfidha
significantly higher in drought-stressed plants of both species
exhibited the highest value of net CO2 assimilation compared
compared with their respective Control. The increase magnitude
with B. vulgaris and to the provenance Soliman. Rehydration
was both species- and provenance-dependent. Leaf sucrose
of plants partially restored net CO2 assimilation values in
level declined upon exposure to water stress in B. vulgaris and
B. vulgaris and in both provenances (Soliman and Enfidha) of
in the provenance Soliman of B. macrocarpa (–95% and –91%
B. macrocarpa.
in water deficit-stressed plants as compared with well-watered
ones) (Fig. 5c). These values increased after rehydration to
Electolyte leakage reach 45% and 52% of the Control. In the provenance
Enfidha, water deficit stress had no effect on leaf sucrose content.
The EL values ranged between 30% and 50% (Fig. 4). No
significant differences in EL were found among treatments
in B. vulgaris and in the provenance Enfidha. However, in Soluble proteins and total amino acid
Soliman provenance, this parameter increased significantly by The total protein content was significantly higher in the
40% under water deficit stress conditions. When rehydrated, stressed plants of B. vulgaris and B. macrocarpa (Fig. 6a).
Effects of water deficit stress in Beta macrocarpa Crop & Pasture Science 557

Control Stressed Rehydrated Control Stressed Rehydrated

20 180
(a)
f f
f ef
16 140 d

Fructose, µg mg–1 MF
ef c c
µmol CO2 m–2 s–1

e
12 100 b b
d d
cd c a
8 80

4 40
a

0
0 300
Soliman Enfidha (b)
Beta vulgaris Beta macrocarpa e e
Fig. 3. Net CO2 assimilation of Beta vulgaris and two provenances of Beta

Glucose, µg mg–1 MF
macrocarpa. Plants were harvested after 1 month of treatment. Control plants de d c
were irrigated at 100% field capacity (FC) (open columns). Drought-stressed 200
plants were irrigated at 25% FC (closed columns). Plants were rewatered c
to 100% FC after 15 days of treatment (grey columns). Mean of four b
replicates. Bars with different letters are significantly different according a a
to l.s.d. test at P < 0.05.
100

Control Stressed Rehydrated

70 0
250
60 c c (c)
bc bc e
b
Electrolyte leakage, %

50 200
Sucrose, µg mg–1 FW

40 d
a a 150
a a
c
30 c c
bc
100 b
20

10 50
a
a
0
Soliman Enfidha 0
Beta vulgaris Beta macrocarpa Soliman Enfidha
Beta vulgaris Beta macrocarpa
Fig. 4. Electrolyte leakage of Beta vulgaris and two provenances in Beta
macrocarpa. Plants were harvested after 1 month of treatment. Control plants Fig. 5. (a) Leaf fructose, (b) glucose and (c) sucrose content in Beta
were irrigated at 100% field capacity (FC) (open columns). Drought-stressed vulgaris and two provenances of Beta macrocarpa. Plants were harvested
plants were irrigated at 25% FC (closed columns). Plants were rewatered after 1 month of treatment. Control plants were irrigated at 100% field
to 100% FC after 15 days of treatment (grey columns). Mean of four replicates. capacity (FC) (open columns). Drought-stressed plants were irrigated at
Bars with different letters are significantly different according to l.s.d. test at 25% FC (closed columns). Plants were rewatered to 100% FC after
P < 0.05. 15 days of treatment (grey columns). Mean of four replicates. Bars
with different letters are significantly different according to l.s.d. test at
P < 0.05.
Rehydration decreased this parameter in B. vulgaris and
in B. macrocarpa Enfidha provenance, whereas no change
occurred in Soliman provenance as compared with stressed (Fig. 6b). The increase in free amino acids content was
plants. Independently of water treatment, total amino acid relatively more pronounced in the provenance Enfidha
content was unchanged in B. vulgaris whereas it was when compared with Soliman provenance (7- and 5-fold,
significantly higher in both provenances of B. macrocarpa respectively, when compared with the Control).
558 Crop & Pasture Science I. Slama et al.

Control Stressed Rehydrated Control Stressed Rehydrated

10 8
(a) (a)
e
Soluble proteins, µg g–1 FW

8
e
c 6

Proline, µmol g–1 FW

d
d
c
6 c b
b
4
a
d
4

2 c
2 bc b
b b a
a
0 0
16 e 0.6
e (b) (b)
e
Total amino acid, µmol g–1 FW

0.5
12
Glycine, µmol g–1 FW
0.4

d
8 0.3
de
d d
c
c c 0.2 c c
4 b b bc
a a a
0.1

0 0
Soliman Enfidha Soliman Enfidha
Beta vulgaris Beta macrocarpa Beta vulgaris Beta macrocarpa

Fig. 6. (a) Leaf soluble proteins, and (b) leaf total amino acids in Beta Fig. 7. (a) Leaf proline and (b) glycine content in Beta vulgaris and two
vulgaris and two provenances of Beta macrocarpa. Plants were harvested provenances of Beta macrocarpa. Plants were harvested after 1 month of
after 1 month of treatment. Control plants were irrigated at 100% field treatment. Control plants were irrigated at 100% field capacity (FC) (open
capacity (FC) (open columns). Drought-stressed plants were irrigated columns). Drought-stressed plants were irrigated at 25% FC (closed
at 25% FC (closed columns). Plants were rewatered to 100% FC after columns). Plants were rewatered to 100% FC after 15 days of treatment
15 days of treatment (grey columns). Mean of four replicates. Bars with (grey columns). Mean of four replicates. Bars with different letters are
different letters are significantly different according to l.s.d. test at P < 0.05. significantly different according to l.s.d. test at P < 0.05.

Proline and glycine content increased to reach a value higher than of the Control in
Under Control conditions, proline was accumulated in B. vulgaris B. vulgaris and B. macrocarpa.
and in B. macrocarpa provenances at low level not exceeding
0.7 mmol g–1 FW (Fig. 7a). The increase of proline content was
Glutamate and glutamine content
higher in Enfidha provenance as compared with Soliman. Leaf
glycine content increased significantly under water deficit A significant increase in leaf glutamate content was recorded in
stress conditions (Fig. 7b). This trend was relatively more both Beta species (Fig. 9a). The highest values were found in
pronounced in Enfidha provenance compared with Soliman Enfidha provenance, since plants growing under water deficit
and to B. vulgaris. stress conditions accumulated ~3 times more glutamate than the
Control. Excepting for B. vulgaris, exposure of plants to water
deficit stress also resulted in an increased content of glutamine
g-aminobutyric acid (GABA) content by ~5- and 12-fold, respectively, as compared with the Control
Under Control conditions B. vulgaris accumulated more GABA in Soliman and Enfidha provenances (Fig. 9b). Enfidha
when compared with the provenances Soliman and Enfidha provenance accumulated the highest content of glutamate and
(8- and 4-fold, respectively) (Fig. 8). In both provenances of glutamine in leaves under water deficit stress conditions when
B. macrocarpa, GABA content was not significantly affected compared with Soliman provenance and to B. vulgaris. The
by water deficit stress, unlike B. vulgaris for which a significant values of glutamate and glutamine content at the recovery
decrease was observed. After rehydration, leaf GABA content period decreased significantly.
Effects of water deficit stress in Beta macrocarpa Crop & Pasture Science 559

Control Stressed Rehydrated Control Stressed Rehydrated

2.5 1.0
f f
(a)

2.0 de
0.8
Glutamate, µmol g–1 FW

GABA, µmol g–1 FW

e
d
1.5 0.6
d
c
bc
1.0 b 0.4
c
b
a ab a
0.5 0.2 a
a a

0 0
Soliman Enfidha
e Beta vulgaris Beta macrocarpa
(b)
1.6 cd Fig. 9. Leaf GABA content in Beta vulgaris and two provenances of
Beta macrocarpa. Plants were harvested after 1 month of treatment.
Glutamine, µmol g–1 FW

c
Control plants were irrigated at 100% field capacity (FC) (open columns).
1.2 Drought-stressed plants were irrigated at 25% FC (closed columns). Plants
were rewatered to 100% FC after 15 days of treatment (grey columns).
Mean of four replicates. Bars with different letters are significantly
0.8 different according to l.s.d. test at P < 0.05.

b
b B. vulgaris. Similarly, it has been reported by Ramanjulu and
0.4 b b Sudhakar (1997) that under drought, the tolerant genotype of
a a
Morus alba L. showed a smaller decrease of RWC than the
sensitive one. (ii) Water deficit stress reduced photosynthesis,
0
Soliman Enfidha as suggested by the high positive correlation (Fig. 11) between
Beta vulgaris Beta macrocarpa shoot growth and net CO2 assimilation (R2 = 0.939). The
decreased photosynthetic CO2 assimilation during water stress
Fig. 8. (a) Leaf glutamate and (b) glutamine content in Beta vulgaris and was also reported (Chaves et al. 2002). Restriction of growth
two provenances of Beta macrocarpa. Plants were harvested after 1 month
can be also due to the impairment of nitrate reduction as a result
of treatment. Control plants were irrigated at 100% field capacity (FC) (open
of decline in nitrate reductase activity during water deficit
columns). Drought-stressed plants were irrigated at 25% FC (closed
columns). Plants were rewatered to 100% FC after 15 days of treatment (Kenis et al. 1994). Water deficit stress had no effect on root
(grey columns). Mean of four replicates. Bars with different letters are growth of the two provenances of B. macrocarpa. However,
significantly different according to l.s.d. test at P < 0.05. the depressive effect of water deficit stress on root growth was
observed in B. vulgaris when compared with the Control. As
P5CS and GDH activities a consequence, water absorption was more limited in the
cultivated species under stressed conditions.
Leaf P5CS activities increased significantly following water Excepting for Enfidha provenance, water deficit stress
deficit stress application (Fig. 10a). However, a relatively decreased strongly sucrose concentration in B. vulgaris and in
higher P5CS activity was recorded in Enfidha provenance. Soliman provenance. Leaf sucrose concentration is influenced
Increased GDH activity was recorded following stress by several factors including the photosynthesis rate, the
application in both B. species (Fig. 10b) especially in the partitioning of photosynthetic carbon between starch and
cultivated species B. vulgaris (8-fold). P5CS and GDH sucrose, the rate of sucrose hydrolysis and the rate of sucrose
activities declined after plant rehydration reaching values export (Huber 1989). In the two studied species, we observed
similar to those observed in Control plants. an increase in leaf hexose (glucose and fructose) concentrations.
Similar results have been observed in other species challenged
Discussion with drought conditions (Lawlor and Cornic 2002). Overall, it is
Our results showed that growth was strongly reduced by water suggested that the starch and sucrose pools in plant leaves are
deficit stress in Soliman provenance and in B. vulgaris. This depleted under drought conditions; in the meantime, the
sensitivity could be explained at several levels: (i) plant water resulting high concentrations of hexose may be involved in
relation was significantly impacted. Indeed, a decline in relative a feedback regulation of photosynthesis (Chaves et al. 2002).
water content was recorded under water deficit stress conditions. Consequently, the total amount of sucrose is significantly
Enfidha provenance (the less sensitive) showed a smaller decreased. In leaves of the studied species, the increase in
decrease when compared with Soliman provenance and hexose concentration under water deficit may be a result of
560 Crop & Pasture Science I. Slama et al.

Control Stressed Rehydrated 18

140
e 16 R 2 = 0.939
(a)
µmol NADPH oxidized min–1 g–1 FW

120 14

µmol CO2 m–2 s–1

12
100 d
d
P5CS activity,

c c 10
80
b 8
b
60
6

40 a 4
a

20 2

0
0 0 1 2 3 4 5

50 Shoot DW, g plant–1

(b)
µmol NAD+ reduced min–1 g–1 FW

45 Fig. 11. Relationship between shoot dry weight and net CO2 assimilation
Glutamate dehydrogenase,

40 in Beta vulgaris and two provenances of Beta macrocarpa. For treatments

refer to captions of Fig. 1.
35 e
d
30
and/or from enhanced stress-induced protein breakdown. In our
25 case amino acid accumulation in B. macrocarpa is due to
bc
20 ab amino acid production rather than protein breakdown indeed
b our results showed an increase in protein levels in leaves of
15
plants submitted to water deficit stress. Although the overall
10 accumulation of amino acids upon stress might indicate cell
a a a
a
5 damage in some species (Widodo et al. 2009), increased levels
of specific amino acids have a beneficial effect during stress
0
Soliman Enfidha acclimation. In B. vulgaris, total amino acid rates in water deficit
Beta vulgaris Beta macrocarpa stress plants remained similar to the well-watered Controls.
Fig. 10. (a) Leaf P5CS, and (b) leaf glutamate activity in Beta vulgaris and
Amino acid accumulation associated to water stress may
two provenances of Beta macrocarpa. Plants were harvested after 1 month actually be a part of an adaptive process contributing to
of treatment. Control plants were irrigated at 100% field capacity (FC) osmotic adjustment. The present study revealed a variation
(open columns). Drought-stressed plants were irrigated at 25% FC (closed in the accumulation of amino acid levels occurs between
columns). Plants were rewatered to 100% FC after 15 days of treatment B. vulgaris and the two provenances of B. macrocarpa. The
(grey columns). Mean of four replicates. Bars with different letters are increase was more marked in Enfidha provenance compared
significantly different according to l.s.d. test at P < 0.05. with B. vulgaris. Varietal variations in the magnitude of
accumulation of amino acids have been suggested as an
increased rates of starch (case of Enfidha provenance) and/or important trait for determining the drought-tolerant potentials
sucrose degradation (B. vulgaris, and Soliman provenance). It of many crop plants (Ramanjulu and Sudhakar 1997). Thus, it
has been reported that in pigeonpea leaves, the activities of seems that the better tolerance of Enfidha to water deficit
enzymes hydrolysing starch (e.g. amylase) and sucrose (e.g. stress can be attributed at least in part to the ability to shift the
acid invertase and sucrose synthase) were increased under metabolic rate, i.e. relatively better RWC coupled with higher
drought stress, which may lead to a depletion of leaf starch amino acid accumulation.
and sucrose and an accumulation of hexose (Keller and Proline is considered to act as an osmolyte and the capacity
Ludlow 1993). Sugars that accumulate in response to stress of plants to accumulate proline has been correlated with stress
can function as osmolytes to maintain cell turgor and to tolerance (Szabados and Savouré 2010). Our results showed a
protect membranes and proteins from stress damage (Kaplan drastic accumulation of proline in Enfidha provenance whereas
et al. 2004). a slight increase was observed in the cultivated species
Apart from disruptions in carbohydrate metabolism, drought B. vulgaris. Proline content in plants is determined by the
may also affect N status and metabolism in plants, and both balance between its biosynthesis and catabolism (Szabados
processes interact with each other (Lawlor and Cornic 2002). and Savouré 2010). Our results showed that water deficit stress
The decreased plant N status under drought conditions may increased P5CS activity, particularly Enfidha provenance and
affect in turn protein synthesis, including the key enzymes for this was concomitant with a high accumulation of proline in
carbon assimilation (photosynthesis) and metabolism. this provenance when compared with the Control and to
Amino acid accumulation has been observed in many studies B. vulgaris.
on plants exposed to abiotic stress (Lugan et al. 2010; Slama It has been suggested that glycine betaine protects
et al. 2015). This increase might arise from amino acid production photosystem II, stabilises membranes, and mitigates oxidative
Effects of water deficit stress in Beta macrocarpa Crop & Pasture Science 561

damage in addition to its role as an osmolyte. This compound Bouyoucos (1983) Les proprieties physiques du sol dependent de sa texture
was accumulated at high level in leaves of Enfidha when et de sa structure. In ‘Les Bases De La Production Végétale. Tome 1.
compared with the Control and to Soliman provenance and to Collection Science et Technique agricoles’. (Ed. D Soltner) pp. 67–87.
B. vulgaris. In Enfidha, proline and glycine betaine could be Bradford MM (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
implied in the protection of the membrane integrity and
binding. Analytical Biochemistry 72, 248–254. doi:10.1016/0003-2697
permeability as shown by the variation of EL (Fig. 3). (76)90527-3
The leaf GDH activities were higher as a response to water Chaves MM, Pereira JS, Maroco J, Rodrigues ML, Ricardo CPP, Osório ML,
deficit stress, and this trend was relatively more marked in the Carvalho I, Faria T, Pinheiro C (2002) How plants cope with water stress
cultivated species B. vulgaris. The changes in the activities of in the field: photosynthesis and growth. Annals of Botany 89, 907–916.
enzymes in response to stress have been frequently reported doi:10.1093/aob/mcf105
(Ramanjulu and Sudhakar 1997). In B. vulgaris, the sharp Dionisio-Sese ML, Tobita S (1998) Antioxidant responses of rice seedlings
increase in GDH activity was concomitant with an important to salinity stress. Plant Science 135, 1–9. doi:10.1016/S0168-9452(98)
reduction in shoot growth and in net CO2 assimilation. GDH 00025-9
was primarily involved in catabolism of glutamate to Dubey RS (1994) Protein synthesis by plants under stressful conditions.
In ‘Hand book of plant and crop stress’. (Ed. Mohammad Pessarkli)
oxoglutarate, thus providing carbon skeletons for the TCA
pp. 277–299. (Marcel Decker Inc.: New York)
cycle, with the concomitant release of ammonia. Robinson Glévarec G, Bouton S, Jaspard E, Riou MT, Cliquet JB, Suzuki A, Limami
et al. (1991) also suggested that the GDH activity is altered in AM (2004) Respective roles of the glutamine synthetase/glutamate
response to shifts in carbon rather than N metabolism, as the synthase cycle and glutamate dehydrogenase in ammonium and amino
elevated activity of GDH is correlated with reduced carbon acid metabolism during germination and post-germination growth in the
availability (Ramanjulu and Sudhakar 1997). GDH activities model legume Medicago truncatula. Planta 219, 286–297. doi:10.1007/
declined after plant rehydration. s00425-004-1214-9
GABA is mainly synthesised from glutamate in the cytosol Goldschmidt EE, Huber SC (1992) Regulation of photosynthesis by end-
by glutamate decarboxylase. A functional GABA shunt is product accumulation in leaves of plants storing starch, sucrose, and
important for stress tolerance. Salt stress enhances the activity hexose sugars. Plant Physiology 99, 1443–1448. doi:10.1104/pp.99.
4.1443
of enzymes involved in GABA metabolism (Renault et al.
Hessini K, Ghandour M, Albouchi A, Soltani A, Koyro HW, Abdelly C
2010). Our result showed that leaf GABA content decline in (2008) Biomass production, photosynthesis, and leaf water relations of
B. vulgaris by 87%. However, in both provenances of Spartina alterniflora under moderate water stress. Journal of Plant
B. macrocarpa, leaf GABA content remained unchanged under Research 121, 311–318. doi:10.1007/s10265-008-0151-2
water deficit stress conditions. The decrease in leaf GABA Hessini K, Lachaal M, Cruz C, Soltani A (2009) Role of ammonium to
content in B. vulgaris could be associated to the relative limit nitrate accumulation and to increase water economy in Wild
sensitivity of the cultivated species to water deficit stress. Swiss Chard. Journal of Plant Nutrition 32, 821–836. doi:10.1080/
As a conclusion the reduction of plant growth under water 01904160902787917
deficit stress may be related to the modifications in several Huber SC (1989) Biochemical mechanism for regulation of sucrose
physiological processes, including altering carbohydrate and/ accumulation in leaves during photosynthesis. Plant Physiology 91,
656–662. doi:10.1104/pp.91.2.656
or N metabolism. Enfidha provenance was more tolerant to
Huber SC, Rogers HH, Mowry FL (1984) Effects of water stress on
water deficit stress when compared with the cultivated species photosynthesis and carbon partitioning in soybean (Glycine max [L.]
B. vulgaris or to Soliman provenance. The relative tolerance of Merr.) plants grown in the field at different CO2 levels. Plant Physiology
Enfidha was associated to a better photosynthesis rate and 76, 244–249. doi:10.1104/pp.76.1.244
leaf water relation and to a relatively higher P5CS activity Kaplan F, Kopka J, Haskell DW, Zhao W, Schiller KC, Gatzke N, Sung DY,
corresponding to higher contents of glutamate; glutamine and Guy CL (2004) Exploring the temperature-stress metabolome of
proline content. Arabidopsis. Plant Physiology 136, 4159–4168. doi:10.1104/pp.104.
Enfidha provenance of the spontaneous species Beta 052142
macrocarpa could be used in the marginal arid ecosystems in Keller F, Ludlow MM (1993) Carbohydrate metabolism in drought-stressed
order to limit the deficit in fodder and to improve the pastoral leaves of pigeonpea (Cajanus cajan). Journal of Experimental Botany
44, 1351–1359. doi:10.1093/jxb/44.8.1351
value of these regions. Besides, this species could serve as a
Kenis JD, Rouby MB, Edelman MO, Silvente ST (1994) Inhibition of
source of genes for genetic improvement to water deficit stress. nitrate reductase by water stress and oxygen in detached oat leaves:
a possible mechanism of action. Journal of Plant Physiology 144,
Acknowledgement 735–739. doi:10.1016/S0176-1617(11)80670-X
Kim JY, Mahe A, Brangeon J, Prioul JL (2000) A maize vacuolar invertase,
This work was supported by the Tunisian Ministry of Higher Education IVR2, is induced by water stress. Organ/tissue specificity and diurnal
and Scientific Research (LR10CBBC02). modulation of expression. Plant Physiology 124, 71–84. doi:10.1104/
pp.124.1.71
Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced
References
metabolic rearrangements and regulatory networks. Journal of
Abdelly C (1997) Mécanismes d’une association de Luzernes spontanées Experimental Botany 63, 1593–1608. doi:10.1093/jxb/err460
et de halophytes pérennes en bordure de sebkha. Thèse de Doctorat, Larcher W (2003) ‘Physiological plant ecology.’ 4th edn. (Springer Verlag:
Faculty of Sciences of Tunis, Tunisia. Berlin, Heidelberg, New York)
Bohnert HJ, Nelson DE, Jensen RG (1995) Adaptation to environmental Lawlor DW, Cornic G (2002) Photosynthetic carbon assimilation and
stresses. The Plant Cell 7, 1099–1111. doi:10.1105/tpc.7.7.1099 associated metabolism in relation to water deficits in higher plants.
562 Crop & Pasture Science I. Slama et al.

Plant, Cell & Environment 25, 275–294. doi:10.1046/j.0016-8025. of GABA transaminase in salt stress tolerance. BMC Plant Biology 10,
2001.00814.x 20. doi:10.1186/1471-2229-10-20
Lugan R, Niogret MF, Leport L, Guegan JP, Larher FR, Savoure A, Kopka J, Robinson SA, Slade AP, Fox GG, Phillips R, Ratcliffe RG, Stewart GR (1991)
Bouchereau A (2010) Metabolome and water homeostasis analysis The role of glutamate dehydrogenase in nitrogen metabolism. Plant
of Thellungiella salsuginea suggests that dehydration tolerance is a Physiology 95, 509–516. doi:10.1104/pp.95.2.509
key response to osmotic stress in this halophyte. The Plant Journal 64, Sambatti JBM, Caylor KK (2007) When is breeding for drought tolerance
215–229. doi:10.1111/j.1365-313X.2010.04323.x optimal if drought is random? New Phytologist 175, 70–80. doi:10.1111/
Lund RE (1989) A User’s Guide to MSUSTAT Statistical Analysis Package j.1469-8137.2007.02067.x
Microcomputer Version 4(12), p. 202. Save R, Castell C, Terradas J (1999) Gas exchange and water relations. In
Parsons IR, Howe TK (1984) Effects of water stress on the water relations ‘Ecology of Mediterranean evergreen ecological studies’. (Eds F Roda’,
of Phaseolus vulgaris and the drought resistant Phaseolus acutifolius. J Retama, A Gracia, J Bellot) pp. 135–147. (Springer: Berlin)
Physiologia Plantarum 60, 197–202. doi:10.1111/j.1399-3054.1984. Slama I, Abdelly C, Bouchereau A, Flowers T, Savouré A (2015) Diversity,
tb04564.x distribution and roles of osmoprotective compounds accumulated
Pelleschi P, Rocher JP, Prioul JL (1997) Effect of water restriction on in halophytes under abiotic stress. Annals of Botany 115, 433–447.
carbohydrate metabolism and photosynthesis in mature maize leaves. doi:10.1093/aob/mcu239
Plant, Cell & Environment 20, 493–503. doi:10.1046/j.1365-3040.1997. Szabados L, Savouré A (2010) Proline: a multifunctional amino acid.
d01-89.x Trends in Plant Science 15, 89–97. doi:10.1016/[Link].2009.11.009
Pottier-Alapetite G (1979) Flore de la Tunisie. Angiospermes-Dicotyledones: Taiz L, Zeiger E (1998) ‘Plant physiology.’ 1st edn. (Sinauer Associates Inc.:
Apetales-Dialypetales. Imprimerie Officielle de la République Massachusetts, London)
Tunisienne/Ministère de l’Enseignement Supérieur et de la Recherche Turner NC (1996) Further progress in crop water relations. Advances in
Scientifique et Ministère de l’Agriculture, Tunis, 651 pp. Agronomy 58, 293–338. doi:10.1016/S0065-2113(08)60258-8
Ramanjulu S, Sudhakar C (1997) Drought tolerance is partly related to Widodo , Patterson JH, Newbigin E, Tester M, Bacic A, Roessner U (2009)
amino acid accumulation and ammonia assimilation: a comparative Metabolic responses to salt stress of barley (Hordeum vulgare L.)
study in two mulberry genotypes differing in drought sensitivity. cultivars, Sahara and Clipper, which differ in salinity tolerance.
Journal of Plant Physiology 150, 345–350. doi:10.1016/S0176-1617 Journal of Experimental Botany 60, 4089–4103. doi:10.1093/jxb/erp243
(97)80131-9 Xue X, Liu A, Hua X (2009) Proline accumulation and transcriptional
Renault H, Roussel V, El Amrani A, Arzel M, Renault D, Bouchereau A, regulation of proline biosynthesis and degradation in Brassica napus.
Deleu C (2010) The Arabidopsis pop2-1 mutant reveals the involvement BMB Reports 42, 28–34. doi:10.5483/BMBRep.2009.42.1.028

[Link]/journals/cp