Differential performance of two forage species, Medicago truncatula and Sulla carnosa, under water-de fi cit stress and recovery
Aida RouachedA,B, Inès SlamaA,C, Walid ZorrigA, Asma JdeyA, Caroline CukierB, Mokded RabhiA, Ons TalbiA, Anis Mohamed LimamiB, and Chedly AbdellyA
ALaboratoire des Plantes Extrêmophiles, BP 901, Centre de Biotechnologie de Borj Cédria, Hammam-Lif 2050, Tunisia.
BUniversity 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.
CCorresponding author. Email: slama_ines@hotmail.fr
Abstract. The response patterns during water deficit stress and subsequent recovery of two forage species,Medicago truncatulaandSulla carnosa, were studied. After germination and pre-treatment, seedlings were individually cultivated for two months under two irrigation modes: 100% and 33% offield capacity. Measured parameters were plant growth, water relations, leaf osmotic potential, lipid peroxidation, and leaf inorganic (Na+and K+) and organic (proline and soluble sugars) solute contents, as well as delta-1-pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH) activities. Our results showed that under control conditions, and in contrast to roots, no significant differences were observed in shoot biomass production between the two species. However, when subjected to water-deficit stress,M. truncatulaappeared to be more tolerant thanS. carnosa(reduction by 50 and 70%, respectively). In the two studied species, water-deficit stress led to an increase in root/shoot ratio and leaf proline and soluble sugar contents, and a decrease in leaf osmotic potential. Enzymatic assay revealed that in the two species, P5CS activity was stimulated whereas that of PDH was inhibited under stress conditions. Despite greater accumulation of proline, sugar, and potassium in leaves ofS. carnosa,M. truncatulawas more tolerant to water deficit. This was essentially due to its capacity to control tissue hydration and water-use efficiency, in addition to its greater ability to protect membrane integrity. Following stress relief,M. truncatulaandS. carnosashowed partial re-establishment of growth capacity.
Additional keywords:legume species, osmolytes, rehydration, tolerance, water relations.
Received 6 February 2013, accepted 4 June 2013, published online 30 July 2013
Introduction
Fodder and grain legumes are characterised by high nutritional value for animals and humans. Their role in symbiotic nitrogen fixation is an advantage that increases productive acreage while improving soil organic fertility and nitrogen economy (Erice et al.2010). This advantage is compromised, however, by the low ability of these species to adapt to adverse environmental conditions such as salinity and drought. Therefore, in arid and semi-arid regions of the world where large areas are salinised and/
or dried, increasing the yield of forage and grain legumes is a priority to meet economic requirements. Assessing the adaptive response of Fabaceae to water-deficit stress using physiological, biochemical, and molecular tools enables the establishment of some pertinent selection criteria in order to improve abiotic stress tolerance in plants.
Drought stress leads to growth inhibition (Bahrani et al.
2010), slows the rate of organ turnover (Peiet al.2013), and affects leaf structure and photosynthetic gas exchange rate (Slama
et al.2007). Plant adaptive response to drought includes stomata closure induced by abscisic acid (ABA) to prevent extensive water loss. As a consequence, stomatal conductance decreases and photosynthetic activity declines following the restriction of CO2diffusion (Chaves1991; Cornic and Fresneau2002). Several changes at the cellular level have been also reported, such as membrane lipid peroxidation (Hernándezet al.2001), disruption of plasma membrane integrity (Ashraf 2009; Noreen et al.
2010), as well as electrolyte leakage increase from cell membranes (Valentovic et al. 2006). The modulation of nitrogen metabolism by water deficit in Medicago truncatula was shown to be partly under ABA control (Planchetet al.2011).
Gene induction of the encoding of delta-1-pyrroline-5- carboxylate synthase (P5CS) is also under ABA control.
However, genes encoding cytosolic glutamine synthetase (GS1b), glutamate dehydrogenase (GDH3), and asparagine synthase (AS) were upregulated in an ABA-independent manner (Planchetet al.2011).
Journal compilationCSIRO 2013 www.publish.csiro.au/journals/cp
CSIROPUBLISHING Crop & Pasture Science
http://dx.doi.org/10.1071/CP13049
Many physiological responses and metabolic changes have been proposed as crucial processes in plant adaptation to drought, because they sustain tissue metabolic activity and enable regrowth upon rewatering (Morgan 1984). The mechanism of osmotic adjustment plays a key role in osmo- tolerance of plants (Yang and Miao 2010). Two types of compounds, inorganic ions and/or organic solutes, play a key role in osmotic adjustment (Slamaet al.2007) by reducing tissue osmotic potential and therefore minimising water loss (Chaves 1991; Chaveset al.2003). Organic solutes, known as compatible osmolytes, include amino acids, glycerol, sugars, and other low- molecular-weight metabolites. However, proline and sugar accumulation could be an indicator of stress. Indeed, Kumar et al.(1984) reported thatCleome gynandra accumulates less sugars and proline and is more resistant to water stress than is C. speciosa. Both species adapted to their native environments with different leaf water status. In addition, the level of solutes indicates the reduction in water status rather than conferring plant tolerance. Potassium is the main inorganic ion involved in osmotic adjustment. Overall, the mechanisms developed by plants to cope with drought are multiple, multidimensional, and closely related to plant species, experimental conditions, and water-deficit stress severity and duration (Zhang and Kirkham 1996). Thus, the development of relevant tools for screening for tolerant plants is difficult. For example, according to several studies, proline is often proposed as relevant tool for selection of plant species and varieties tolerant to osmotic constraint (Cornic and Fresneau 2002).
However, other reports consider that an increased level of free proline is merely a result of stress (Hernándezet al.2001; Ashraf 2009).
Two annual legumes with fodder potential, Sulla carnosa and Medicago truncatula, were considered in the present study. The model plantM. truncatula, a promising species for the improvement of forage production, was compared with S. carnosa, a salt-tolerant legume common in several saline habitats, where it is often subjected to combined effects of salinity and water-deficit stress. Sulla carnosa significantly contributes to primary production of these ecosystems and to their pastoral value. Both species were subjected to water deficit and rewatered in an attempt to simulate natural conditions.
Physiological and biochemical parameters related to photosynthesis, water content, membrane integrity, and organic and mineral solute accumulation are compared and discussed for both species.
Materials and methods Plant material
Our study focussed on two plant species, M. truncatula and S. carnosa. The first species is a small legume native to the Mediterranean region. It has been chosen as a model organism for legume biology. It is also an important forage crop species.
Medicago truncatula line Jemalong A17 was used in this investigation. The second species is a pastoral legume spread across the Mediterranean Basin (Trifi-Farah et al.2002; Choi and Ohashi 2003), well represented in Tunisian central and southern rangelands. Sulla carnosa is of ecological interest since it has been exploited for soil protection and nitrogen
enrichment. Hence, S. carnosa constitutes an important genetic resource and contributes to pastoral production, particularly in semi-arid climates (Trifi-Farah and Marrakchi 2002). Seedling and plant development mode confers on this species an excellent adaptability to arid areas. In its natural biotope, S. carnosa was associated with several halophytic species such as Halocnemum strobilaceum, Arthrocnemum indicum, and Suaeda fruticosa. For this study, seeds of S. carnosa were collected from the saline habitat of Kalbia Sabkha, Tunisia (358480N, 10880E).
Growth conditions and water deficit treatment
Six seeds were sown in 3.5-L plastic potsfilled with limono- sandy soil containing: (cmol kg–1dry soil) 0.23 Na+, 0.94 K+, 0.64 Ca2+, and 0.05 Cl–; and (g kg–1dry soil) 0.23 P2O5and 0.44 total nitrogen. The pH and the electrical conductivity of the aqueous extract (1/10) were 6.68 and 0.05 dS m–1, respectively. The soil field capacity (FC) was determined according to the technique of Bouyoucos (1983) and was 11.5%. One week after sowing, seedlings were thinned to one per pot to achieve homogeneous plants. Following 4 weeks of ample watering (100% FC), plants were divided into two groups: the first group was irrigated with tap water at 100%
FC (control plants), and the second at 33% FC (water-deficit- stressed plants). A preliminary experiment carried out on S. carnosaand M. truncatulawatered with 100, 50, and 25%
FC showed that only 25% FC led to a significant growth decrease. After 5 weeks of treatment, half of water-deficit- stressed plants were rewatered at 100% FC. Regular weighing (every 2 days) enabled to restore the soil moisture at 100 or 33%
FC. For all treatments, tap water was enriched with diluted nutrient solution (Hewitt 1966), and independent of the procedure for watering (100 or 33% FC), plants received the same quantity of nutrients.
The total number of plants was 240 (120 plants per species). In addition, 10 pots without plants were used to monitor evaporative water loss from the soil surface throughout each watering regime. Experiments were carried out in a greenhouse with a 14-h photoperiod. Day/night mean temperature and relative humidity were 3058C/1628C and 555%/905%, respectively. For each species and water regime, plants were harvested after 2 months of treatment.
Growth and water status
Fresh weight (FW) and dry weight (DW) of shoots and roots were determined after counting leaf number. Relative water content (RWC) was measured in the second or third youngest fully expanded leaf harvested weekly in the morning. This parameter was determined using the following equation (Schonfeldet al.1988):
RWCð%Þ ¼100 ðFWDWÞ=TWDWÞ The FW was determined within 2 h after harvest; turgid weight (TW) was obtained after soaking leaves in distilled water in test tubes for 12 h at room temperature (about 208C), under low light conditions. Dry weight was obtained after oven-drying leaf or shoot samples at 608C for 48 h.
B Crop & Pasture Science A. Rouachedet al.
Leaf osmotic potential
Osmotic potential (Ys) was measured as follows. Leaves were quickly collected, cut into small segments, then 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 000G for 15 min at 418C (Martínez-Ballesta et al. 2004). The collected sap was analysed for osmolality (C) estimation. C was assessed with a vapour pressure osmometer (Wescor 5500; Wescor Inc., Logan, UT) and converted from mosmol kg–1to MPa according to the Van’t Hoff equation.
Leaf gas exchange determination
Measurements of net CO2 assimilation (A), transpiration rate (E), and stomatal conductance (gs) were determined on fully expanded leaves of each plant using a portable photosynthesis system (LCi) (ADC BioScientific Inc., Hoddesdon, UK).
Measurement conditions were as follows: photosynthetically active radiation (PAR) 1200mmol m–2s–1, ambient CO2
concentration 378mmol mol–1, and temperature of leaf chamber 2628C. These measurements were made from 12 : 00 to 13 : 00. Data were automatically collected every minute after the stabilisation of photosynthesis rate. Water-use efficiency was expressed asA/Eratio.
Cation assay
Ions K+ and Na+ were assayed by flame emission spectrophotometry after nitric acid extraction (HNO3, 0.5%) of finely ground dry matter.
Lipid peroxidation
The extent of lipid peroxidation was estimated by determining the concentration of malondialdehyde (Draper and Hadley1990).
Leaf material was homogenised in 0.1% (w/v) trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 15 000G for 10 min, and 1 mL of the supernatant was added to 4 mL thiobarbituric acid (TBA) 0.5% (w/v) in 20% (w/v) TCA. The mixture was incubated at 908C for 30 min and the reaction was stopped by placing the reaction tubes in an ice water bath. Samples were centrifuged at 10 000Gfor 5 min and the absorbance of the supernatant was read at 532 nm. The value for non-specific absorption at 600 nm was subtracted. The concentration of malondialdehyde (MDA) was calculated from the extinction coefficient 155 mM–1cm–1.
Electrolyte leakage
To measure electrolyte leakage, 200 mg of leaves was washed with deionised water and cut into 0.5-cm fragments. The leaf fragments were then put in test tubes containing 15 mL deionised water and shaken for 30 min. The initial electrical conductivity (EC1) of the solution was then measured using a conductivity meter (Orion 140; Thermo Scientific, MA, USA). The final electrical conductivity (EC2) of each sample was measured after shaking leaf fragments autoclaved at 1008C for 30 min.
The amount of electrolyte leakage was calculated by using the formula: EL(%) = 100 (EC1)/(EC2).
Proline extraction and analysis by HPLC
Proline was extracted from axes in 96% (v/v) ethanol for 1 h at 48C. After centrifugation (9800G, 48C, 15 min), the ethanol fraction was removed and the same process was repeated with deionised water. The ethanol and water fractions were combined.
After evaporation of the extract under vacuum, organic residues were dissolved in deionised water and extracted with the same volume of chloroform. After centrifugation (12 000G, 48C, 15 min), an aqueous phase containing amino acids was vacuum-dried. The amino acids were then redissolved in deionised water. Samples were passed through a nylon syringe filter then analysed by HPLC. The amino acids were determined by the AccQTag method, which uses AccQFluor reagent (Waters Corp., Milford, MA, USA) to derivatise the amino acids. The reagent is a highly reactive compound (6-aminoquinolyl-N- hydroxysuccinimidyl carbamate) that forms stable derivatives with primary and secondary amino acids, including proline.
Derivatives were separated by reverse-phase (C18 column) HPLC and quantified by fluorescence detection. Each sample was analysed over 1 h.
Sugar extraction and analysis by HPLC
Sugars were extracted by the same method as proline, and they were analysed by HPLC on a Carbopac PA-1 (Dionex Corp., Sunnyvale, CA, USA) using pulsed amperometric detection EC 2000 (Thermo Scientific), Sugars were chromatographed on a CarboPac PA100 4250-mm column (Dionex Corp.) preceded by a guard column (CarboPac PA100, 450 mm). Software Borwin (JMBS Developments, France) was used to collect and analyse data.
Proline dehydrogenase (PDH) and P5CS assays
Frozen leaves were extracted in a pre-chilled mortar with 1% (w/
w) PVP (polyvinylpolypyrrolidone) in 10 mMphosphate buffer (Na2HPO4/KH2PO4, pH 7.5). The homogenate was centrifuged at 15 000Gat 48C for 15 min; the supernatant was used as the crude extract.
Activity of P5CS was measured using 20mL of the crude extract in a reaction mixture containing Tris-HCl buffer (100 mM, pH 7.2) supplemented with MgCl2(20 mM), glutamate (75 mM), and ATP (5 mM). The reaction was initiated by the addition of NADPH (0.4 mM) substrate of the P5CS. The oxidation of NADPH was followed spectrophotometrically at 340 nm every 30 s for 30 min at 308C. Assay of PDH was done by following the NAD+reduction at 340 nm in a 0.15MNa2CO3–HCl buffer (pH 10.3) containing 15 mMproline and 1.5 mMNADP+(Ruiz et al.2002).
Statistical analyses
Significant differences were analysed using Tukey’s parametric or nonparametric tests. All of these tests used anaof 0.05 and were done with XLSTAT software v. 2011 (www.xlstat.com).
Results
Plant growth capacity
Under control conditions, no significant differences were observed in shoot biomass production betweenS. carnosaand M. truncatula(Fig.1a). This parameter was significantly reduced
Differential performance of two forage species Crop & Pasture Science C
under water deficit in the two species; however, the effect was more pronounced inS. carnosathan inM. truncatula.Indeed, shoot growth reduction was 70 and 50%, respectively. Under water deficit (33% FC),M. truncatulaproduced 43% more shoot biomass thanS. carnosa. After rehydration, a partial restoration of growth was established, in whichM. truncatulaandS. carnosa produced 70 and 60% of the biomass of the control, respectively.
Root dry weight (Fig.1b) ofM. truncatulawas not affected under water-deficit conditions compared with the control. In S. carnosaroot dry weight was reduced by 25% under water stress. After rehydration, a total restoration of root dry weight was established inS. carnosa.
Leaf area
Water deficit reduced the total leaf area in both species (Fig.2).
However, the reduction of this parameter was more pronounced in S. carnosa, than in M. truncatula (reduction by 65 and 55%, respectively). Plant rehydration after water-deficit stress increased the total leaf area. This ability to recover leaf
development was more pronounced in S. carnosa(87%) than inM. truncatula(75%) when compared with their controls.
Photosynthetic parameters
Water-deficit stress affected net CO2assimilation rate (A) and stomatal conductance (gs) in both forage species (Table 1).
ParameterAdecreased by 70% inM. truncatula and by 57%
inS. carnosa, andgswas more affected inM. truncatulathan in S. carnosa. Transpiration rate (E) declined significantly in both species.In water-deficit-stressed plants, WUE was significantly decreased in S. carnosaand was unaffected inM. truncatula.
Upon rewatering, the recovery ofAwas partial in both species.
However, in contrast to stomatal conductance ofM. truncatula, that ofS. carnosareached control values.
Water relations
Relative water content was 78% (S. carnosa) and 86%
(M. truncatula) for plants subjected to non-limiting water supply and 63 and 73% for water-deficit-stressed plants (Fig. 3). Thus, a significant decrease was observed in RWC values in the two species under water-deficit stress. For each treatment (control, stressed or rehydrated),M. truncatulaplants exhibited the highest RWC values in its leaves, whereas S. carnosashowed the lowest ones. Under water-deficit stress conditions, RWC was 25% higher in M. truncatula than in S. carnosa. After rewatering, RWC significantly increased and reached the control level inM. truncatula.
Leaf osmotic potential significantly decreased in M. truncatula andS. carnosasubjected to water-deficit stress (Fig. 4). However, this decrease was more pronounced in M. truncatula than in S. carnosa. Rewatering restored this parameter to a level similar to that of control plants in both species.
(b)
Medicago truncatula Sulla carnosa
0.0 0.5 1.0 1.5 2.0 2.5
c b
c
ab
a a
Root dry weight, g plant–1
0 3 6 9
d (a)
a c
d
b c
Shoot dry weight, g plant–1 (0.25)
(0.69) (0.44)
(0.2)
(0.32) (0.23)
Stressed Rehydrated Control
Fig. 1. (a) Shoot and (b) root dry weights in two forage plant species S. carnosaandM. truncatulasubjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Values in parentheses are root/shoot DW ratio of each treatment. Measurements were performed on samples taken from individual plants. Values are means of six replicates and capped lines ares.e. Columns sharing a common letter are not significantly different atP= 0.05.
Leaf area, cm2 plant–1
Sulla carnosa Medicago truncatula 100
300 500 700 900
e
a
e
b c d
Stressed Rehydrated Control
Fig. 2. Leaf area in two forage species S. carnosaandM. truncatula subjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Measurements were performed on samples taken from individual plants. Values are the means of six replicates and capped lines ares.e. Columns sharing a common letter are not significantly different atP= 0.05.
D Crop & Pasture Science A. Rouachedet al.
Membrane integrity
Plant exposure to water-deficit stress significantly increased MDA content in S. carnosa, which accumulated 78% more MDA in its leaves compared with the control. However, this parameter was not significantly affected by water-deficit stress in M. truncatula. Upon rewatering, a total recovery of MDA content was observed only inM. truncatula(Fig.5a).
Under non-limiting watering conditions, leaf electrolyte leakage was higher in S. carnosa than in M. truncatula (Fig. 5b). In stressed plants, electrolyte leakage significantly increased in S. carnosa by 28%. This parameter was not significantly affected by water deficit stress in M. truncatula (Fig. 5b). After rewatering, electrolyte leakage significantly exceeded that of the control and the water-stressed treatment in S. carnosa, whereas it remained unchanged in M. truncatula.
Proline and soluble sugar accumulation
Water-deficit stress led to a significant increase in proline content in leaves (Fig. 6a) of both legumes. This accumulation was higher inM. truncatulathan inS. carnosa, 70 and 185mmol g–1 DW, respectively. Upon rewatering, leaf proline content was similar to that of the control in both species, although significantly higher inM. truncatularelative to its control.
Under water-deficit stress, an increase in leaf soluble sugar content was observed inM. truncatulaandS. carnosa(Fig.6b).
This accumulation was ~5 times the control inS. carnosa, and
~1.5 times the control inM. truncatula. After rewatering and compared with water-deficit-stressed plants, leaf sugar content did not show any reductions, remaining at a high level.
Activities of P5CS and PDH
As shown in Fig.7, water-deficit stress increased the activity of the key enzyme involved in proline biosynthesis (P5CS) in leaves ofM. truncatulaandS. carnosa.In rewatered plants, the activity
Table 1. Net CO2assimilation (A), stomatal conductance (gs), transpiration rate (E), and water-use efficiency (WUE) in two forage speciesSulla carnosaandMedicago truncatulasubjected to three water treatments during 2 months Treatments are control (100% FC), stressed (33% FC), and rewatered. Values are means of six replicatesstandard error. Within
columns, values sharing a common letter are not significantly different atP= 0.05
A(mmol CO2m–2s–1) gs(mol m–2s–1) E(mmol H2O m–2s–1) WUE (mmol CO2mmol–1H2O) Medicago truncatula
Control 15 ± 1.24c 0.13 ± 0.02c 3.06 ± 0.23c 5.16 ± 0.26a
Stressed 4.38 ± 0.54a 0.02 ± 0.007a 0.92 ± 0.21a 5.69 ± 0.044a
Rewatered 9.44 ± 1.26b 0.08 ± 0.007b 1.77 ± 0.20b 5.58 ± 0.33a
Sulla carnosa
Control 14.18 ± 1.18c 0.08 ± 0.009c 1.80 ± 0.24c 7.06 ± 0.87b
Stressed 6.09 ± 0.70a 0.03 ± 0.008a 0.92 ± 0.19a 5.66 ± 0.07a
Rewatered 9.91 ± 0.55b 0.07 ± 003b 1.69 ± 0.12b 5.89 ± 0.91a
Leaf RWC (%)
Sulla carnosa Medicago truncatula 40
50 60 70 80 90 100
c
a b
d d
bc
Stressed Rehydrated Control
Fig. 3. Leaf relative water content in two forage speciesS. carnosaand M. truncatulasubjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Measurements were performed on samples taken from individual plants. Values are the means of six replicates and capped lines ares.e. Columns sharing a common letter are not significantly different atP= 0.05.
Leaf osmotic potential, MPa
–3 –2 –1 0
c
b
c c
c
a Medicago truncatula Sulla carnosa
Stressed Rehydrated Control
Fig. 4. Leaf osmotic potential in two forage plant speciesS. carnosaand M. truncatulasubjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Measurements were performed on samples taken from individual plants. Values are the means of six replicates and capped lines ares.e. Columns sharing a common letter are not significantly different atP= 0.05.
Differential performance of two forage species Crop & Pasture Science E
of this enzyme showed a marked decrease in both species, reaching values close to those of the control (Fig.7a).
Activity of PDH reached about 0.050 and 0.065mmol reduced NAD(P)+mg–1protein h–1 in leaves of control plants inM. truncatulaandS. carnosa, respectively (Fig.7b). Activity of PDH was significantly decreased under water-deficit stress conditions. The decrease was more marked inM. truncatula (reduction by 75%). After rehydration, PDH activity increased in both species and a re-establishment was observed in S. carnosa.
Accumulation of Na+and K+
Water-deficit stress affected ion homeostasis of the two species in different ways. Sodium content (mmol g–1DW) significantly increased in shoots of both species under limiting water conditions (Fig.8a). The significant reduction in water content in response to water deficit led to a greater increase (i.e. more pronounced than content) in leaf Na+ concentration (solute content/water content ratio, expressed in mM), especially in M. truncatula (Fig. 8b). Content and concentration of Na+
decreased in rehydrated plants, reaching values close to those of the control.
In contrast to M. truncatula, which showed a significant decrease in K+ content, water-deficit stress significantly increased leaf K+ content in S. carnosa (Fig. 9a). In S. carnosa, leaf K+concentration largely increased because of the increase in leaf K+ content and of the decrease in tissues hydration (Fig. 9b). However, in M. truncatula, leaf K+ concentration was maintained at values equal to those of control plants. Rehydrated plants showed similar levels in K+ content and concentration compared with the controls. Under water-deficit stress conditions, root Na+(Fig.8d) and K+(Fig.9d) concentration increased significantly in both species
Discussion
This study aimed to evaluate the performance of two plant species M. truncatula(glycophyte) andS. carnosa(halophyte) during water-deficit stress and recovery. Our results showed that under optimal water supply, S. carnosa exhibited the same growth capacity as M. truncatula. Under water deficit, the species were affected differently. The depressive effect of water deficit was more pronounced inS. carnosathan inM. truncatula. Water- MDA, nmol g–1 DW
(a)
30 40 50 60 70 80 90
a c
b b
b
b b
20
Electrolyte leakage (%)
(b)
10 20 30 40 50 60 70
Sulla carnosa Medicago truncatula a
a a
b c
d
Stressed Rehydrated Control
Fig. 5. (a) Malondialdehyde (MDA) content and (b) electrolyte leakage in two forage plant speciesS. carnosaandM. truncatulasubjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Measurements were performed on samples taken from individual plants. Values are the means of six replicates and capped lines ares.e.
Columns sharing a common letter are not significantly different atP= 0.05.
Sugar content, mg g–1 DW (b)
Sulla carnosa Medicago truncatula
c d
d
b
c
0 5 10 15 20 25 30 35 40
a 50 100 150 200 250
(a)
a a
d
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Proline, µmol g–1 DW
Stressed Rehydrated Control
Fig. 6. (a) Proline and (b) sugar contents in leaves of two forage plant speciesS. carnosaandM. truncatulasubjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered.
Measurements were performed on samples taken from individual plants.
Values are the means of six replicates and capped lines ares.e. Columns sharing a common letter are not significantly different atP= 0.05.
F Crop & Pasture Science A. Rouachedet al.
deficit stress induced a series of metabolic, physiological, and morphological changes. It is known that water stress profoundly affects leaf development and plant photosynthesis. In our study, water deficit induced a marked decrease in leaf expansion in both species, the reduction being more pronounced inS. carnosa than in M. truncatula. Leaf area was more affected than the intrinsic photosynthetic capacity estimated by gas exchange parameters in the two species. A considerable decline was observed in net photosynthesis assimilation (A), transpiration rate (E), and stomatal conductance (gs) compared with well- watered plants. This effect was observed by Anjum et al.
(2011a) in maize under drought stress. Similar investigations (Slamaet al.2007; Hesteret al.2001) studied the relationship between growth and net CO2assimilation. However, in several plant species such as Triticum aestivum(Hawkins and Lewis 1993) andOlea europaea(Loretoet al.2003), the correlation between these two parameters was low or absent. After rewatering, a partial re-establishment was observed in the two studied species. This characteristic is very important in forage plants, since in their natural biotopes, they are exposed to alternating dry and rainy periods.
Our results showed that tissue water status was also significantly impaired by water-deficit stress. Several studies reported a decrease in RWC under severe water-deficit stress conditions (Goraiet al.2010; Slamaet al.2011). Both RWC and leaf water potential serve as reliable indicators of plant water status. Plant species are known to differ in their ability to cope with water stress, to maintain different levels of water status, and to differ in the degree of solute accumulation. There is, however, a difference of opinion about the role of accumulated solutes in relation to tolerance (Ashraf2009). Roots have been reported to play a major role in the regulation of water uptake and in the maintenance of plant water balance (Steudle and Peterson 1998). This suggests that optimal growth level ofM. truncatula depends largely on its capacity to maintain suitable tissue hydration and root development in addition to the noticeable decrease in leaf osmotic potential. Moreover,M. truncatulaplants exhibited the capacity to conserve their WUE under stress conditions, in contrast to S. carnosa. McCann and Huang (2008) suggested that inAgrostis stolonifera, cultivars having a better capacity to survive under drought stress conditions were mainly equipped with avoidance mechanisms such as maintaining higher WUE and root viability, elongation, and production under drought stress. These parameters could be used as criteria to select drought-resistant cultivars. In addition,M. truncatula showed a better capacity to conserve membrane integrity and permeability. Indeed, unlikeS. carnosa, leaf MDA content and electrolyte leakage were not affected inM. truncatulaunder water deficit. The low concentration of MDA and the stability of membrane integrity inM. truncatula suggest that this species was more protected against the oxidative stress than S. carnosa. The high accumulation of MDA and H2O2in transgenicM. truncatulamade these plants more susceptible to oxidative damage under the conditions of abiotic stresses (Songet al. 2012). The relationship between oxidative damage and plant growth potential is well documented (Ben Amoret al.2006).
In stressed plants, osmotic potential was lower in M. truncatula than in S. carnosa. Its decrease is generally considered an indicator of osmotic adjustment through the production and/or accumulation of so-called compatible osmolytes (Hessini et al. 2008). Osmotic adjustment in response to water stress was ensured by the accumulation of different types of organic and inorganic solutes in the cytosol—
amino acids (e.g. proline, aspartic acid, and glutamic acid), methylated quaternary ammonium compounds (e.g. glycine betaine and alanine betaine), and carbohydrates (e.g. fructans and sucrose) (Vijn and Smeekens1999; Rathinasabapathi2000;
Hamilton and Heckathorn2001; Sperdouli and Moustakas2012).
Proline accumulation is thefirst response of plants exposed to water-deficit stress in order to reduce cell injury (Anjumet al.
2011a). This accumulation under stress in many plant species has been correlated with stress tolerance, and generally proline concentration is found to be higher in stress-tolerant than in stress-sensitive plants (Anjumet al.2011b). Our results show that proline content was significantly increased in response to water deficit stress inM. truncatulaand inS. carnosa. A drastic increase in proline content was observed inS. carnosasubjected to drought stress. This accumulation may be due to protein degradation and solute concentration after the marked tissue P5CS activity µmol oxidized NADPH (g–1 protein h–1)
0 0.02 0.04 0.06 0.08
Medicago truncatula Sulla carnosa
(b)
PDH activity µmol reduced NAD(P)+ (g–1 protein h–1) 0 0.5 1.0 1.5 2.0 2.5 3.0
(a)
b
a b
a c
b
a b
a a a
c
Stressed Rehydrated Control
Fig. 7. Changes in (a) delta-1-pyrroline-5-carboxylate synthase (P5CS, mmol oxidised NADPH g–1 protein h–1) and (b) proline dehydrogenase (PDH,mmol reduced NAD(P)+g–1protein h–1) in leaves of two forage plant species S. carnosa and M. truncatula subjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Measurements were performed on samples taken from individual plants. Values are the means of six replicates and capped lines ares.e.
Columns sharing a common letter are not significantly different atP= 0.05.
Differential performance of two forage species Crop & Pasture Science G
dehydration, making the osmotic adjustment less effective, because of a high depressive effect of water-deficit stress and a low growth rate. Moreover, in S. carnosa photo-assimilates could be serving more in survival functions such as proline accumulation and its contribution in osmotic adjustment rather than for growth. Also, proline accumulation in plants subjected to water deficit was concomitant with a stimulation of the key enzyme in proline biosynthesis (P5CS) and the inhibition of PDH, involved in this amino acid’s catabolism. Our results are in agreement with those of Slamaet al.(2006). Despite the higher activity of P5CS in M. truncatula leaves, S. carnosa accumulated more proline in its leaf tissue, and this could be related to the stimulation of other pathways of proline biosynthesis (ornithine pathway via d-OAT). Other amino acids such as asparagine and glutamine could play the role of essential amino acids in osmotic adjustment (Hamilton and Heckathorn2001). In our study, asparagine could be involved in osmotic adjustment, conferring on M. truncatula a better tolerance to water-deficit stress; asparagine content was about
3.4 and 4.5mmol g–1 FW in M. truncatula and S. carnosa, respectively, when grown under water deficit conditions (data not shown).
The disturbance of photosynthesis under water deficit can result from stomatal closure induced by ABA. Planchetet al.
(2011) reported that under osmotic stress conditions, the induction of delta-1-pyrroline-5-carboxylate synthase, the key enzyme in proline synthesis, is controlled by ABA in M. truncatula. In many plant species, soluble sugars are a major contributor to osmotic adjustment. Increasing leaf soluble sugars under water-deficit stress could be considered as a criterion of adaptation to drought (Kameli and Lösel 1995). Our results showed a drastic increase in soluble sugar (glucose, fructose, and sucrose) content in S. carnosa and M. truncatula under water-deficit conditions. The accumulation of sugars is accompanied by a reduction of growth in Pistacia atlantica (Azcon-Bieto 1983). Several studies describe the key role of carbohydrates in the regulatory mechanisms implied in the repression of photosynthesis-related
Medicago truncatula 0
100 200 300
(a)
a b
a a
b
a
Shoots
Na+ (µmol g–1 DW)
Sulla carnosa
b b
a
d
c e
0 50 100 150 200
Roots (c)
0 30 60
90 Shoots
a b
a
b c
b (b)
Na+ (mM) (d)
0 10 20 30 40 50
Sulla carnosa Medicago truncatula Roots
a d
b
a c
a
Stressed Rehydrated Control
Fig. 8. Sodium (a) content (mmol g–1DW) and (b) concentration (mM) in leaves and (c,d) roots of two forage speciesS. carnosaandM. truncatulasubjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Measurements were performed on samples taken from individual plants.
Values are the means of six replicates and capped lines ares.e. Columns sharing a common letter are not significantly different atP= 0.05.
H Crop & Pasture Science A. Rouachedet al.
genes (Koch 1996). This can explain the growth decline following sugar accumulation. Although organic compounds are the major constituents of osmoregulation in plant cells during water stress, inorganic ions such as K+ could also contribute to osmotic adjustment (Kameli and Lösel 1995).
Roberts (1998) reported that organic solute synthesis and accumulation consume more energy than inorganic ion uptake.
Potassium accumulation in leaves could be a consequence of its active translocation to photosynthetic organs or of its content increase after a marked restriction of growth (Joneset al.1980).
In our experiment, only S. carnosashowed an increase in K+ concentration in response to water-deficit stress. This species showed a significant relationship between K+concentration and osmotic potential.
In conclusion, our results indicate that water-deficit stress restricted growth capacity in the two forage species. However, M. truncatulawas more tolerant thanS. carnosa. The relative superiority ofM. truncatulawas linked to its capacity to maintain a suitable tissue hydration and to an important decrease in leaf osmotic potential. As found inS. portulacastrum(Slamaet al.
2007), the addition of salt in the culture medium could alleviate the depressive effect of water-deficit stress in the halophytic speciesS. carnosa.
Author contributions
Aida Rouached conducted research, analysed the data, and wrote the paper. Inès Slama participated in the experimental conception and realisation and the manuscript correction. Walid Zorrig and Ons Talbi checked the manuscript structure. Caroline Cukier participated in the biochemical assays and the data analysis. Asma Jdey helped in the experimental work. Mokded Rabhi improved the English of the manuscript. Chedly Abdelly and Anis Mohamed Limami designed the research (project conception, development of overall research plan) and corrected the manuscript.
Acknowledgements
This work was supported by the Tunisian Ministry of Higher Education, Scientific Research a (LR10CBBC10)
c c
a
a
e
b d Roots
(c)
0 200 400 600 800 1000
Sulla carnosa Medicago truncatula K+ (µmol g–1 DW)
a a
200 400 600 800 1000 1200
Shoots (a)
d
b
a c
0
K+ (mM) 0
100 200
Shoots
a a b
b b
b (b)
1400
Medicago truncatula Roots
0 40 80 120 160
Sulla carnosa b
c c
b c
a (d)
Stressed Rehydrated Control
Fig. 9. Potassium (a) content (mmol g–1DW) and (b) concentration (mM) in leaves and (c,d) roots of two forage speciesS. carnosaandM. truncatulasubjected to three water treatments during 2 months: control (100% FC), stressed (33% FC), and rewatered. Measurements were performed on samples taken from individual plants. Values are the means of six replicates and capped lines ares.e. Columns sharing a common letter are not significantly different atP= 0.05.
Differential performance of two forage species Crop & Pasture Science I
References
Anjum SA, Xie X, Wang L, Saleem MF, Man C, Lei W (2011a) Morphological, physiological and biochemical responses of plants to drought stress.African Journal of Agricultural Research9, 2026–2032.
Anjum SA, Wang LC, Farooq M, Khan I, Xue LL (2011b) Methyl jasmonate- induced alteration in lipid peroxidation, antioxidative defense system and yield in soybean under drought.Journal of Agronomy & Crop Science 197, 296–301. doi:10.1111/j.1439-037X.2011.00468.x
Ashraf M (2009) Biotechnological approach of improving plant salt tolerance using antioxidants as markers.Biotechnology Advances 27, 84–93.
doi:10.1016/j.biotechadv.2008.09.003
Azcon-Bieto J (1983) Inhibition of photosynthesis by carbohydrates in wheat leaves.Plant Physiology73, 681–686. doi:10.1104/pp.73.3.681 Bahrani MJ, Bahrami H, Haghighi AAK (2010) Effect of water stress on ten
forage grasses native or introduced to Iran.Grassland Science56, 1–5.
doi:10.1111/j.1744-697X.2009.00165.x
Ben Amor N, Jimenez A, Megdiche W, Lundqvist M, Sevilla F, Abdelly C (2006) Response of antioxidant systems to NaCl stress in the halophyte Cakile maritima. Physiologia Plantarum126, 446–457. doi:10.1111/
j.1399-3054.2006.00620.x
Bouyoucos (1983) Les propriétés physiques du sol dépendent de sa texture et de sa structure. In’les bases de la production végétale’. Tome 1. Collection Sciences et Technique agricoles, pp. 67-87.
Chaves MM (1991) Effect of water deficits on carbon assimilation.Journal of Experimental Botany42, 1–16. doi:10.1093/jxb/42.1.1
Chaves MM, Maroco JP, Pareira JS (2003) Understanding plant responses to drought from genes to the whole plant.Functional Plant Biology30, 239–264. doi:10.1071/FP02076
Choi HB, Ohashi H (2003) Generic criteria and an infrageneric system for Hedysarumand related genera (Papilionoidea-Luguminosea).Taxon52, 567–576. doi:10.2307/3647455
Cornic G, Fresneau C (2002) Photosynthetic carbon reduction and carbon oxidation cycles are the main electron sinks for photosystem II activity during a mild drought.Annals of Botany89, 887–894. doi:10.1093/aob/
mcf064
Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation.Methods in Enzymology186, 421–431. doi:10.1016/
0076-6879(90)86135-I
Erice G, Louahlia S, Irigoyen JJ, Sanchez-Diaz M, Avice JC (2010) Biomass partitioning, morphology and water status of four alfalfa genotypes submitted to progressive drought and subsequent recovery.
Journal of Plant Physiology167, 114–120. doi:10.1016/j.jplph.2009.
07.016
Gorai M, Hachef A, Neffati M (2010) Differential responses in growth and water relationship ofMedicago sativa(L.) cv. Gabès andAstragalus gombiformis(Pom.) under water-limited conditions.Emirates Journal of Food and Agriculture22, 1–12.
Hamilton EW, Heckathorn SA (2001) Mitochondrial adaptation to NaCl.
complex I is protected by antioxidant and small heat shock proteins, whereas complex II is protected by proline and betaine.Plant Physiology 126, 1266–1274. doi:10.1104/pp.126.3.1266
Hawkins HJ, Lewis OAM (1993) Effect of NaCI salinity, nitrogen form, calcium and potassium concentration on nitrogen uptake and kinetics inTriticum aestivumL. cv. Gamtoos.New Phytologist124, 171–177.
doi:10.1111/j.1469-8137.1993.tb03807.x
Hernández JA, Ferrer MA, Jiménez A, Barcelo AR, Sevilla F (2001) Antioxidant systems and O2–/H2O2production in the apoplast of pea leaves, its relation with salt-induced necrotic lesions in minor veins.Plant Physiology127, 817–831. doi:10.1104/pp.010188
Hessini K, Gandour M, Albouchi A, Soltani A, Koyro HW, Abdelly C (2008) Biomass production, photosynthesis, and leaf water relations ofSpartina
alternifloraunder moderate water stress.Journal of Plant Research121, 311–318. doi:10.1007/s10265-008-0151-2
Hester MW, Mendelsohn IA, Mckee KL (2001) Species and population variation to salinity stress inPanicum hemitomon,Spartina patens, and Spartina alterniflora: morphological and physiological constraints.
Environmental and Experimental Botany46, 277–297.
Hewitt EJ (1966) Sand and water culture methods used in the study of plant nutrition. Commonwealth Bureau of Horticulture, Technical Communication22, 431–446.
Jones MM, Osmond CB, Turner NC (1980) Accumulation of solutes in leaves of sorghum and sunflower in reponse to water deficits.Australian Journal of Plant Physiology7, 193–205. doi:10.1071/PP9800193 Kameli A, Lösel DM (1995) Contribution of carbohydrates and other
solutes to osmotic adjustment in wheat leaves under water stress.
Journal of Plant Physiology 145, 363–366. doi:10.1016/S0176-1617 (11)81903-6
Koch KE (1996) Carbohydrate modulated gene expression in plants.Annual Review of Plant Physiology and Plant Molecular Biology47, 509–540.
doi:10.1146/annurev.arplant.47.1.509
Kumar UD, Saraswathy R, Das VSR (1984) Differential performance of Cleome gynandraL. (C4) andC. speciosaL. (C3) under water stress and recovery.Environmental and Experimental Botany24, 305–310.
doi:10.1016/0098-8472(84)90027-3
Loreto F, Centritto M, Chartzoulakis K (2003) Photosynthetic limitations in olive cultivars with different sensitivity to salt stress.Plant Cell and Environment26, 595–604.
Martínez-Ballesta MC, Martínez V, Carvajal M (2004) Osmotic adjustment, water relations and gaz exchange in pepper plants grown under NaCl or KCl.Environmental and Experimental Botany52, 161–174. doi:10.1016/
j.envexpbot.2004.01.012
McCann S, Huang B (2008) Drought responses of Kentucky bluegrass and creeping bentgrass as affected by abscisic acid and trinexapac- ethyl.Journal of the American Society for Horticultural Science133, 20–26.
Morgan JM (1984) Osmoregulation and water stress in higher plants.
Annual Review of Plant Physiology35, 299–319. doi:10.1146/annurev.
pp.35.060184.001503
Noreen Z, Ashraf M, Akram NA (2010) Salt-induced regulation of some key antioxidant enzymes and physio-biochemical phenomena infive diverse cultivars of turnip (Brassica rapaL.).Journal of Agronomy & Crop Science196, 273–285.
Pei F, Li X, Liu X, Lao C (2013) Assessing the impacts of droughts on net primary productivity in China.Journal of Environmental Management 114, 362–371. doi:10.1016/j.jenvman.2012.10.031
Planchet E, Rannou O, Ricoult C, Mercey SB, Grondard AM, Limami AM (2011) Nitrogen metabolism responses to water deficit act through both abscisic acid (ABA)-dependent and independent pathways in M. truncatula during post-germination. Journal of Experimental Botany62, 605–615. doi:10.1093/jxb/erq294
Rathinasabapathi B (2000) Metabolic engineering for stress tolerance, installing osmoprotectant synthesis pathways. Annals of Botany 86, 709–716. doi:10.1006/anbo.2000.1254
Roberts SK (1998) Regulation of K+channels in maize roots by water stress and abscisic acid.Plant Physiology116, 145–153. doi:10.1104/
pp.116.1.145
Ruiz JM, Sánchez E, Garcia PC, Lüpez-Lefebre LR, Rivero RM, Romero L (2002) Proline metabolism and NAD kinase activity in green bean plants subjected to cold-shock. Phytochemistry 59, 473–478. doi:10.1016/
S0031-9422(01)00481-2
Schonfeld MA, Johnson RC, Carver BF, Mornhinweg DW (1988) Water relations in winter wheat as drought resistance indicator.Crop Science28, 526–531.
J Crop & Pasture Science A. Rouachedet al.
Slama I, Messedi D, Ghnaya T, Savouré A, Abdelly C (2006) Effects of water-deficit on growth and proline metabolism in Sesuvium portulacastrum. Environmental and Experimental Botany 56, 231–238. doi:10.1016/j.envexpbot.2005.02.007
Slama I, Ghnaya T, Messedi D, Hessini K, Labidi N, Savouré A, Abdelly C (2007) Effect of sodium chloride on the response of the halophyte species Sesuvium portulacastrum grown in mannitol-induced water stress.Journal of Plant Research120, 291–299. doi:10.1007/s10265- 006-0056-x
Slama I, Tayachi S, Jdey A, Rouached A, Abdelly C (2011) Differential response to water deficit stress in alfalfa (Medicago sativa) cultivars, Growth, water relations, osmolyte accumulation and lipid peroxidation.
African Journal of Biotechnology72, 16 250–16 259.
Song S, Chen Y, Zhao M, Zhang WH (2012) A novel M. truncatula HD-Zip gene, MtHB2, is involved in abiotic stress responses.
Environmental and Experimental Botany 80, 1–9. doi:10.1016/
j.envexpbot.2012.02.001
Sperdouli I, Moustakas M (2012) Interaction of proline, sugars, and anthocyanins during photosynthetic acclimation of Arabidopsis thalianato drought stress.Journal of Plant Physiology169, 577–585.
doi:10.1016/j.jplph.2011.12.015
Steudle E, Peterson CA (1998) How does water get through roots.Journal of Experimental Botany49, 775–788.
Trifi-Farah N, Marrakchi M (2002) Intra- and interspecific genetic variability inHedysarumrevealed by rDNAeRFLP markers.Journal of Genetics &
Breeding56, 1–9.
Trifi-Farah N, Baatout H, Boussaid M, Combes D, Figier J, Hannachi-Salhi A, Marrakchi M (2002) Evaluation des ressources génétiques des espèces du genreHedysarumdans le bassin méditerranéen.Plant Genetic Resources Newsletter130, 1–6.
Valentovic P, Luxova M, Kolarovic L, Gasparikova O (2006) Effect of osmotic stress on compatible solutes content, membrane stability and water relations in two maize cultivars.Plant, Soil and Environment52, 186–191.
Vijn I, Smeekens S (1999) Fructan, more than a reserve carbohydrate.Plant Physiology120, 351–360. doi:10.1104/pp.120.2.351
Yang F, Miao LF (2010) Adaptive responses to progressive drought stress in two poplar species originating from different altitudes.Silva Fennica 44, 23–37.
Zhang J, Kirkham MB (1996) Antioxidant response to drought in sunflower and sorghum seedlings.New Phytologist132, 361–373. doi:10.1111/
j.1469-8137.1996.tb01856.x
Differential performance of two forage species Crop & Pasture Science K
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