Responses of leaf growth and gas exchanges to salt ...

Responses of leaf growth and gas exchanges to salt stress during reproductive stage in wild wheat relative Aegilops geniculata Roth. and wheat (Triticum durum Desf.) Khaled Mguis, Ali Albouchi, Mejda Abassi, Ayda Khadhri, Mbarka YkoubiTej, et al. Acta Physiologiae Plantarum ISSN 0137-5881 Acta Physiol Plant DOI 10.1007/s11738-012-1185-6

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Author's personal copy Acta Physiol Plant DOI 10.1007/s11738-012-1185-6

ORIGINAL PAPER

Responses of leaf growth and gas exchanges to salt stress during reproductive stage in wild wheat relative Aegilops geniculata Roth. and wheat (Triticum durum Desf.) Khaled Mguis • Ali Albouchi • Mejda Abassi • Ayda Khadhri Mbarka Ykoubi-Tej • Asma Mahjoub • Nadia Ben Brahim • Zeineb Ouerghi

•

Received: 21 May 2012 / Revised: 4 December 2012 / Accepted: 11 December 2012 Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2013

Abstract In order to investigate the effect of salinity on the growth and photosynthesis of the wild wheat and wheat, three accessions of Aegilops geniculata from Ain Zana, Zaghouan and Sbitla and one variety of durum wheat (Triticum durum) were grown in the INRAT greenhouse and treated with different salinity levels. The growth of leaves, water status and gas exchange parameters have been measured at the reproductive stage. The flag leaf length, total leaf dry weight, water status, CO2 assimilation rate, stomatal conductance, intercellular CO2 and transpiration for the three Ae. geniculata accessions and wheat variety significantly decreased with increasing salt. The decline in photosynthesis measured in response to salt stress was proportionally greater than the declines in transpiration, resulting in a reduction of water-use efficiency, at both the leaf and whole-plant levels. Among the factors inhibiting photosynthetic activity, those of a stomatal nature had a greater effect. This study has shown a Communicated by W. Filek. K. Mguis A. Albouchi M. Abassi Unite´ d’Agrosylvopastoralisme, INRGREF, Rue Hedi Karray, 2080 Ariana, Tunisia K. Mguis A. Mahjoub N. B. Brahim Laboratoire de Botanique, INRAT, Rue Hedi Karray, 2080 Ariana, Tunisia K. Mguis (&) Z. Ouerghi Unite´ de Physiologie et Biochimie de la tole´rance aux sels des plantes, De´partement de Biologie FST, FST, Universite´ de Tunis El Manar, 2092 Tunis, Tunisia e-mail: [email protected] A. Khadhri M. Ykoubi-Tej De´partement de biologie FST, FST, Universite´ de Tunis El Manar, Campus Universitaire, 1060 Tunis, Tunisia

high degree of variation of these characters mainly related to geographical origin. It was observed also that Sbitla accession was less affected by the imposed salt stress than all the others while Ain Zana was the most affected one. Keywords Aegilops geniculata Growth Gas exchanges Salinity Triticum durum

Introduction Salt stress limits wheat (Triticum durum) production in vast area worldwide, and the problem is ever increasing because of irrational acts causing secondary salinization as well as because of global warming with the consequent rise in sea level and increase in storm incidence particulary in coastal area (Peltier and Tushingham 1989; Pessarakli and Szabolcs 1999; Wassmann et al. 2004). Salt stress can affect physiological processes from seed germination to plant development, resulting in reduced growth and yield (Ashraf 2004). The complexity of the plant responses to salt stress can be partially explained by the fact that salinity imposes both ionic and osmotic stress as well as nutritional imbalance (Munns et al. 2006) and salt exclusion from photosynthetic tissues was considered an important mechanism associated with salt tolerance in monocots (Yeo et al. 1990; Davenport et al. 2005; Mguis 2010). Stomatal closure is often a rapid initial response to salt stress. In wheat, James et al. (2002) showed that a stress-induced reduction in stomatal conductance was seen when the leaf emerged, but after some time there was a further decline probably caused by salt toxicity, as the concentrations of Na? and Cl- in the leaf tissue increase further. Photosynthesis is a key metabolic pathway in plants. Maintaining good photosynthetic rate leads to maintenance of growth

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under salt stress. The decline in net photosynthesis under salt stress could be due to stomatal or non-stomatal limitations, or both (Dubey 2005). Most of the world’s crop species are glycophytes; thus, they do not grow under high soil salinity (Kao et al. 2006). However, with increasing food demand from the growing human population, the need to develop salt-tolerant crop varieties is unavoidable. To develop salt-tolerant crops, it is necessary to identify the degree of salinity tolerance within crops, it is necessary to identify the degree of salinity tolerance within crops and their wild-type relatives. Durum wheat represents only 8 % of total wheat production but 80 % is growing under Mediterranean climates (Monneveux et al. 2000). In these regions, salinity limits considerably yield, together with drought and heat. Special efforts should consequently be made to increase the tolerance abiotic stresses in this species (Monneveux et al. 2000). The genus Aegilops is closely related to Triticum (Van Slageren 1994). Interest has developed in recent years in exploiting Aegilops spp. as important genetic resources for wheat improvement (Farooq et al. 1996; Zaharieva et al. 2001). Aegilops geniculata Roth. (= Ae. ovata L.) is an annual, selfing allo-tetraploid species (2n = 4x = 28) with MU genome (Van Slageren 1994). This species grows in Mediterranean regions characterized by a dry summer season with high temperature and high irradiance. As with other wild species, it can acclimate to these constraints by escape, avoidance and tolerance (Colmer et al. 2006). Aegilops geniculata Roth. (UUMM) has been pursued by Farooq (2004) as a potential source for improvement of salt tolerance in wheat. So, a better understanding of the adaptive features of this species may promote its use for wheat genetic improvement. In the present study, physiological factors associated with sensitivity to salinity were evaluated of Ae. geniculata accessions (Ain Zana, Zaghouan and Sbitla) and durum wheat (T. durum) variety (Chili). The objective of the present study is, first, to evaluate growth, water status and gas exchange of leaves of Ae. geniculata accessions (Ain Zana, Zaghouan and Sbitla) and durum wheat (T. durum) variety (Chili), secondly to select tolerant accession to saltiness in order to improve wheat. This study forms a part of a large wheat improvement programme in Tunisia, focusing on the use of Ae. geniculata to expand genetic variability, develop alternate plant types and physiological processes, and increase salt and drought stresses and high temperature tolerance in durum wheat (T. durum Desf.).

Materials and methods Plant material Three A. geniculata accessions originated from Ain Zana, Zaghouan and Sbitla (Table 1) were evaluated in this

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Table 1 Accessions of Aegilops geniculata Roth. and characteristics of sites of origin Code of accessions

Site

Province

Altitude (m)

Rainfall (mm)

Bioclimatic area

AZ

Ain Zana

Kroumirie

641

700

Sub-humid

Z

Zaghouan

Dorsale

400

496

Upper semi arid

S

Sbitla

Centre

670

320

Upper arid

study. They were collected on a large range of climatic conditions and in different sites altitude of Tunisia. Durum wheat variety ‘‘Chili’’ provided by the Cereal Laboratory of INRAT (National Institute of Agronomic Research of Tunisia) is an old and precocious variety, it has as highly average 120 cm, it is late (20 days after modern one) and low yielding. Furthermore, it is susceptible to lodging days to heading (Maamouri et al. 2000) and it is one of the most ancient salt-tolerant varieties (MallekMaalej and Ben Salem 2002). Growing conditions Experiments were carried during 2010–2011, in a greenhouse at National Institute of Agronomic Research of Tunisia (INRAT) in Ariana (Northeast, and elevation 10 m above sea level). The mean annual temperature was 25 C with monthly means ranging from 15 C in January to 35 C in August. The seedling was achieved in pots, of 28 cm of diameter and 25 cm of height. No pesticides were applied and weeds were manually eliminated. The pots were filled by the substratum of culture formed by a mixture 50 % loam (organic matter content 2 %, pH 7.8) and 50 % sand. Each pot contained 10 kg of soil a layer of 5 cm of gravel was placed in the bottom of every pot to guarantee a good drainage. After replenishment of the pots, 10 seeds per pot were sowed in a homogeneous way to a depth of 2–3 cm, and after emergence (7 days), the seedlings were thinned to six plants per pot. During the 7 first days, pots were watered daily with tap water while adding the necessary volume to bring soil to its capacity in the field. Thereafter, pots were watered twice a week. The average temperature in greenhouse was 23.9 C (range 18.5–28.3 C) during the day and 14.2 C (range 10.3–17.9 C) during night. Salt treatment started when the seedling had approximately three to four leaves (4 weeks after seedling). Sodium chloride was added in four concentrations of 50, 100, 150, and 200 mM. To avoid osmotic shock, saline treatment was progressively imposed; increasing the concentration, by 50 mM, every second irrigation until the final concentration (200 mM NaCl) was reached. Prior salt treatment, nutrient solution was supplied instead of water. The nutrient solution was prepared


according to Maas et al. (1986) and contained 2 mM Ca(NO3)2, 2 mM KH2PO4, 2 mM MgSO4, 1 mM KNO3, 0.1 mM Fe-EDTA, 0.02 H3BO4, 0.005 mM CuSO4 and 0.01 mM H24Mo7N6O24 4H2O. Pots were irrigated at each stage of plant development (tillering, shooting, heading, flowering and grain filling (milk and dough stage) with nutrient solution. The experiment was set up as completely randomized design of five salt levels, four populations (three Aegilops accessions and one durum wheat variety) and four replicates. Each pot (six seedlings) was considered as one replicate. Each treatment contained 24 seedlings per accession or variety and the total number of plant was evaluated to 480. Measurement Beginning in the heading stage (16 weeks after emergence) and continuing during the flowering stage (2 weeks), measurements were made between 10:00 a.m. and 12:00 a.m. under conditions of photosynthetically active radiation (830 \ PAR \ 1,070 lmol quanta m-2 s-1). Growth and water status The flag leaves length (LL) of the tagged tillers of Aegilops accessions and durum wheat variety was measured in ‘‘cm’’ and the plants were harvested, total leaves were separated and immediately weighted (FW). The samples were dried at 70 C for dry-weight (DW) determination. Water content (WC) was calculated according to the following equation: WC = (FW-DW)/FW. The indication of sensitivity (IS) was calculated according to the following equation (Slama 1982): IS = (DWNaCl - DWcontrol) 9 100/DWcontrol Measurements were taken from eight plants (two plants per replicate) from each population per treatment.

efficiency (WUE) and stomatal limitation (Ls) were calculated from the ratios A/E and (1-Ci/Ca), respectively (Berry and Downton 1982). Statistical analysis The experiment was a complete randomized design consisting of five salinity treatments, four populations (three Ae. geniculata accessions and one durum wheat variety) and four replicates. The data were analysed using appropriate procedures of the SAS software 6.12 (Khawarizmi Center, Tunis version 1998). Analysis of variance (ANOVA) was performed with the statistical programme Minitab (Minitab Inc.; College Park, PA), involving two levels of classification (salinity and population) with interactions. A Duncan’s multiple range test was carried out to determine if significant (P \ 0.05) difference occurred between accessions and treatments.

Results Variance analysis revealed a highly significant species; population and treatment effects (Table 2). Population effect was significant in all traits (growth, water status and gas exchange treatments). The expression of 9 out of 10 traits showed significant difference (P \ 0.05) between Aegilops accessions and wheat variety indicating a high level of genetic variability (Table 2). This included three growth and water status (LL, DW and WC) and six gas exchange parameters measured (PN, gs Ci, g’I, WUE and Ls). No significant population effects were detected for E. The 10 parameters were significantly affected by the salt treatments (P \ 0.05). Population 9 treatment interactions were also detected for all traits, indicating variable performance of populations in different salt levels.

Gas exchange Growth and water status Gas exchange parameters were measured on the flag of the tagged tillers using an open portable system ADC model LCA-4 infrared gas analyser (Analytical Development Co., Hoddesdon, UK), leaf temperature 32 ± 2 C, relative humidity was from 40 to 50 %, and ambient CO2 (Ca) concentration 365 ± 5 cm3 m-3. CO2 assimilation rate (PN), stomatal resistance (rs), transpiration rate (E) and intercellular CO2 (Ci) were automatically recorded by the machine (about 2 min) and three sub-measurements were made on four plants (one plant per replicate) in each treatment. The stomatal conductance of (g0 s) was calculated from the ratio 1/(1.6rs) (Be´jaoui 2006). The internal conductance of CO2 (g0 i = PN/Ci) affected the CO2 between the intercellular spaces and carboxylation sites in chloroplast and Rubisco activity (El Aouni 1980). Water-use

For all saline levels, the length of flag leaf for the three Aegilops populations and durum wheat variety showed variation (Table 3). Wheat plants present the most elevated leaves length compared to the Aegilops plants. Aegilops Sbitla accessions presented the most elevated leaves length and Ain Zana population displayed each value. The leaf length decreases progressively with increasing stress (Table 3). However, At 200 mM NaCl, flag leaf length was reduced by 68, 50 and 55 % for Ain Zana, Zaghouan, Sbitla and wheat variety (Chili), respectively, compared with the control plants. Total leaf growth of all three Aegilops accessions and durum wheat variety were reduced to a similar by salinity (Fig. 1a). Total leaf biomass production in absence of

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Author's personal copy Acta Physiol Plant Table 2 Summary of analysis of variance for 10 growth, water status and gas exchange traits of three Ae. geniculata Roth. accessions ‘Ain Zana, Zaghouan and Sbitla’ and durum wheat variety ‘Chili’ grown under different salinity levels (0, 50, 100, 150, 200 mM) at Ariana, Tunisia Parameters

Abbr.

Sources of variation Population (Pop)

Treatment (Tr)

Pop and Tr

LL

704.93***

210.7***

738.99***

DW

4.31***

31.45***

0.45***

WC

1.60***

68.58***

1.95***

Growth parameters 4th leaves length Total leaves dry weight Water status Water content Gas exchange parameters Photosynthesis rate

A

35.808***

2,398.26***

6.05***

Stomatal conductance

gs

0.0063***

0.327***

0.0012***

Transpiration

E

0.135 ns

179.619***

0.639***

Intercellular CO2

C

2,143.1***

89,191.1***

601.24***

Internal conductance

gi

0.017*

0.879***

0.011***

Water-use efficiency

WUE

4.078***

18.7373***

0.164*

Stomatal limitation

Ls

0.0152***

0.67***

0.0047***

F-probabilities are indicated by symbols ns non-significant differences * Significant difference at P \ 0.05 *** Significant difference at P \ 0.001

Table 3 Fourth leaf length (LL) of three Ae geniculata Roth. accessions ‘Ain Zana, Zaghouan, Sbitla’ and one durum wheat variety ‘Chili’ grown under salt stress (0, 50, 100, 150, 200 mM) conditions Salinity levels (mM)

Ae Ain Zana

Ae Zaghouan

Ae Sbitla

Wheat

0

9.21

9.57

9.54

31.45

50

6.50

7.59

8.51

27.64

100

4.92

6.39

7.04

22.01

150

3.72

5.04

5.84

19.39

200

2.99

3.96

4.58

14.28

salinity differed between wheat and Ae geniculata, and Aegilops accessions. Salt strongly inhibited plant growth (Fig. 1b). Salt tolerance is shown in a more conventional way in (Fig. 1b), as the indication of sensitivity (IS). Increasing NaCl reduced significantly leaf water content (Fig. 2). However, the reduction depended on the NaCl level. Indeed, at 200 mM water content was reduced by 90, 83, 74 and 82 % for Ain Zana, Zaghouan, Sbitla and wheat variety (Chili), respectively, compared with the control. Gas exchange parameters Photosynthesis rate (PN) of the flag leaf of all Aegilops accession and wheat variety was reduced to a similar extent by salinity. PN in the control was higher in the wheat variety than in Aegilops accessions (Fig. 3a). However, the

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effect of salinity on PN was similar in the tow species. An increasing NaCl treatment led to a decrease PN. Under salt stress, Sbitla accession had the highest photosynthetic rate, followed by Zaghouan and wheat and Ain Zana had the least PN. At 200 mM, PN decreased with 96, 88, 86 and 92 % for Ain Zana, Zaghouan, Sbitla and wheat variety (Chili), respectively, compared with the control. Stomatal conductance (g0 s) in flag leaf was similar among the three Aegilops accessions in the control, but g0 s in this treatment was higher in the Wheat variety ‘Chili’. g0 s decreased progressively with increasing salt concentration (Fig. 3b). Indeed, at 200 mM these decreases of g0 s were 89, 82, 77 and 85 % for Ain Zana, Zaghouan, Sbitla and wheat variety ‘Chili’, respectively. However, the reductions of A, g0 s of Aegilops accessions and wheat variety under salt stress were associated with the increase of Ci (Fig. 3c) and the reduction of g0 i (Fig. 3d). Indeed, at 200 mM, Ci was increased by 30, 24, 21 and 23 % and g0 i was decreased by 97, 92, 87 and 94 % for Ain Zana, Zaghouan, Sbitla and wheat variety ‘Chili’, respectively, compared with the control. Salinity strongly inhibited transpiration (E) (Fig. 3e). For instance, 200 mM NaCl caused 80, 83, 77 and 85 % reduction for Ain Zana, Zaghouan, Sbitla and wheat variety ‘Chili’, respectively, compared with the control. The water use efficiency (WUE = PN/E) in leaves of all Aegilops accession and durum wheat variety ‘Chili’ decreased with increasing treatment NaCl concentration


(Table 4), with the greatest increment in Sbitla, the least in Ain Zana, but stomatal limitation (Ls) increased with increasing salt treatment, with the greatest increment in Ain Zana, and the least in Sbitla (Table 4). Strong relationships between PN and g0 s (Fig. 4a) and between PN and g0 i (Fig. 4b) for the plants studied were showed.

Discussion

Fig. 1 a Total leaves dry weight, b induction of sensitivity of three Ae. geniculata Roth. accessions ‘Ain Zana., Zaghouan and Sbitla’ durum Wheat variety ‘Chili’ grown under different salinity levels (0, 50, 100, 150, 200 mM NaCl). The data are means values of eight measurements and vertical bars are LSD0.05

Fig. 2 Water leaves content (WC) (C) of three Ae. geniculata ‘Ain Zana, Zaghouan and Sbitla’ and one durum wheat Variety ‘Chili’ grown under different salinity levels (0, 50, 100, 150 and 200 mM). The data are mean values of eight measurements, and vertical bars are LSD0.05

Substantial variation was observed in our study for traits related to the adaptation of Ae. geniculata Roth. to salt stress. The variation among species and accessions for growth leaves and gas exchange treatments was mainly explained by geographical origin, suggesting that those traits are mainly constitutive and result from natural selection pressure exerted by the climatic constraints (Table 2). Although data were obtained from plants grown in pots, the result can be related to in situ performance. Sbitla population, which is better adapted to naturally occurring salt stress, was more tolerant to experimentally imposed salt stress than Ain Zana and Zaghoaun accessions. Ain Zana population was the most sensitive to the salt stress. Growth of both species (wheat variety and the three Ae. geniculata Roth. accessions) decreased with increasing salt stress, as indicated by flag leaves length and total leaves dry weight (Table 3; Fig. 1a, b). This was observed in other species, such as in barley (Royo et al. 2000), in wheat (Mahar et al. 2003; Munns and James 2003). Indeed, in the wheat variety and the three Ae. geniculata Roth. accessions, investigated in this study, the saltiness appears by a depressive effect on the growth apparent since the first days of installation of the saline constraint (Mguis et al. 2008). The results reported by Munns and Rawson (1999) in wheat showed that saline stress generally appears by a weak growth, a reduction of surface and number of leaves and an acceleration of senescence of the mature leaves. Also, Cramer and Quarrie (2002) in maize noted that salt stress reduced the development of the aerials parts by inhibition of the apparition of new leaves. The observed reduction in plant biomass might be due to a combination of slow growth and development as a result of osmotic stress (Shani and Ben-Gal 2005). Leaf water content decreased significantly with increasing salt stress (Fig. 2), implying that there was an accumulation of Na? and Cl- concentrations. This accumulation of these ions in the transpiring leaves exceeded the capacity of cell compartmentalization in vacuoles resulting in salt apoplastic accumulation and ultimately cellular hydration and that plant suffered from restricted water availability to cells (Munns et al. 2000; Netondo et al. 2004a). Salinity may directly or indirectly inhibit cell division and enlargement in the plant’s growing point and chloride

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Fig. 3 a CO2 assimilation rate (PN), b stomatal conductance of CO2 (g0 s), c intercellular of CO2 (Ci), d internal conductance of CO2 (g0 i) and e Transpiration rate of three Ae. geniculata Roth. accessions ‘Ain Zana, Zaghouan and Sbitla’ durum Wheat variety ‘Chili’ grown under

Table 4 Water-use efficiency (WUE) and limit stomatal (Ls) of three Ae. geniculata Roth. accessions ‘Ain Zana., Zaghouan and Sbitla’ and one durum Wheat variety ‘Chili’ grown under salt stress (0, 50, 100, 150, 200 mM) conditions Salinity levels (mM)

Ae Ain Zana

Ae Zaghouan

Ae Sbitla

Wheat

WUE (lmol CO2 mmol-1 H2O) 0

3.19

3.22

3.51

3.52

50

2.92

3.06

3.43

3.34

100

2.29

2.77

3.35

3.14

150

1.58

2.02

2.40

2.14

200

1.07

1.60

2.07

1.69

0

0.45

0.46

0.46

0.42

50

0.56

0.55

0.54

0.51

100 150

0.69 0.76

0.67 0.74

0.63 0.72

0.63 0.72

200

0.86

0.78

0.78

0.81

Ls

induces elongation of the palisade cells, which leads to leaves becoming succulent. Reduction in plants growth with increasing saltines in this study reflects the increased metabolic energy cost and reduced carbon gain, which are associated with salt adaptation (Kao et al. 2006). It also reflects salt on tissues, in photosynthetic rate per unit of

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different salinity levels (0, 50, 100, 150, 200 mM NaCl) the data are mean values of four replications with three measurements per replicate and vertical bars are LSD0.05

leaf area (Netondo et al. 2004b). Indeed, at reproduction stage, salinity stress markedly inhibited PN, g0 s, g0 i and E on the flag leaf (Fig. 3a, b, d, e). Sbitla accession maintained the highest photosynthetic CO2 (PN) fixation rate under salt stress, and this was associated with higher g0 s (Fig. 3a, b) than Zaghouan accession and wheat variety. Similar results were founded with Triticum aestivum and Hordeum vulgare (Sharma and Hall 1991), also, in flag leaves of barely (Belkhodja et al. 1999). In addition, saltsensitive rice cultivars salinity caused a substantial reduction in PN and g0 s (Dionisio-Ses and Tobita 2000), whereas leaf gas exchange was infected by high salinity (100–400 mM) in the halophyte Suaeda salsa (Lu et al. 2003). At the three of accession Ae geniculata and wheat the decrease in PN, Ls, and the strong PN–g0 s relationship (Fig. 4a) may indicate that stomata were imposing a larger limitation on PN under salt stress conditions. Possible reasons for this include stomatal closure, feedback inhibition due to reduced sink activity, decreased efficiency of Rubisco, displacement of essential cations from the endomembrane structure (leading to changes in permeability), and swelling and disorganization of the grana (Flowers and Yeo 1981), or due to the direct effects of salt on stomatal conductance via a reduction in guard cell and intercellular CO2 partial pressure (Dionisio-Ses and Tobita 2000). Many studies have reported that stomatal and non-stomatal


Fig. 4 Relationships a between assimilation rate (PN) and stomatal conductance of CO2 (g0 s), and b between assimilation rate (PN) and internal conduction of CO2 (g0 i) for three Ae. geniculata accessions A. zana open triangle, Zaghouan filled diamond, Sbitla filled square and wheat variety open circle grown at different salinity levels. Data points represent individual measurement

components are responsible for decrease in PN (Tezara et al. 2003). In the present study, the increase of Ci attest that some non-stomatal limitation can influence the photosynthesis. Indeed, the variation of Ci is consequence of CO2 flux from stomata and binding sites in the cytoplasm. A slight increase of Ci and in favour of a slightly stronger internal limitation on PN (Be´jaoui 2006). Similarly, the strong positive PN–g0 i relationships (Fig. 4b) expresses that some non-stomatal factors were responsible for limiting PN of the plants studies. In addition, the results showed that the internal concentration CO2 reported depending on the intensity of the stress and population. Under 50 mM NaCl, it is rather the stomatal limitation acting on the photosynthesis of plants studied because there was not significant increase in Ci. This is in concordance with various studies indicating that under moderate salt stress stomatal closure often predominate (Cronic and Massacci 1996). From 100 mM NaCl, salt induced the development of non-stomatal factors limiting photosynthesis evidenced by the average value of Ci than those of control and varying

from 20 % at 100 mM and 25 % at 200 mM. Non-stomatal inhibition of photosynthesis by salinity has been reported in several species (Kao et al. 2006; Abassi 2009; Mguis 2010). It can result from large malfunction in the chloroplast (Warran et al. 2004) by inhibition photochemical process and/or incorporation of CO2 (Terashima and Ono 2002). It may also be due to reduce of chlorophyll concentration of the leaves of plants grown at NaCl concentrations higher than 100 mM and decrease of the concentrations of essential ions such as Ca2? and Mg2? in the mesophyll cells (Netondo et al. 2004a). At reproductive stage, the plants studied showed the same behaviour at the limiting factors in PN. Indeed, the three accession of Ae. geniculata and wheat cultivar, the reduction of PN was accompanied by reduction of g0 s and g0 i attesting to the simultaneous effect of stomatal and nonstomatal components on PN. Similar results with salt stress have been reported in G. tabanica (Kao et al. 2006) and P. nigra (Abassi 2009). However, the similar correlation PN–g0 s (Fig. 4a) and PN–g0 i (Fig. 4b), translated stomatal and internal limitations to photosynthesis. WUE was higher for exposed to higher salinities (Table 4), suggesting an adjustment within the plant, such as stomatal control of water losses to conserve water (Ayala and O’Leary 1995). Zhang et al. (2008) reported that the transpiration rate generally tends to decline with increasing rhizospheric salinity and this could be attributed to lower water potential in roots and the transport of abscisic acid (ABA) from root to shoot to induce stomatal closure. In wheat, James et al. (2002) observed that reduction in stomatal conductance occurred in salt stress before an apparent decline in leaf water potential, and argued that chemical signals are likely to cause decrease in stomatal conductance. The A. geniculata Roth. accessions provides germplasm that has potential for crop improvement; it belongs to the wild species. Wild-related species have been considered until now much more as sources of resistance to pests and diseases than as sources of diversity permitting deep modification of architecture and physiology of the cultivated species. There are indications that Ae. geniculata Roth. can contribute useful genes for salt tolerance (Monneveux et al. 2000). Systematic approaches to increase the level of a biotic stress tolerance require the evaluation of genetic variability in Ae. geniculata Roth. gene pool, both within and among accessions. In this study, a large variation for salt-stress tolerance was found within accessions. Single population such as Sbitla or Ain Zana or Zaghouan that exhibit rich growth and photosynthesis diversity may provide valuable resources for traits of agronomic importance. The salt-stress tolerant identified in the Ae. geniculata Roth. accessions and wheat variety ‘Chili’ may be combined with breeding lines exhibiting high yield potential. Genotypic analysis in the combination

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with physiological studies is required to establish whether the salt-stress tolerant lines are similar, i.e., the same mechanisms. If different mechanisms are identified, there will be potential for recombining these for further improvement. Author contribution Prof. Ali Albouchi provided all the necessary equipment (pots, growing medium, nutrient solution, nursery…) for this study and he proposed and corrected experimental protocol. Dr. Mejda Abassi has measured the gas exchange parameters were using an open portable system ADC model LCA-4 infrared gas analyser. Dr. Ayda khadhri performed the statistical analyses. Prof. Nadia Ben Brahim and Dr. Asma Mahjoub have carried out with me, 15 accessions of Ae. geniculata in various bioclimatic and ecological conditions of northern and central Tunisia. Dr. Tej-Mbarka Ykoubi measured the physicochemical composition of the substrate and determined the composition of the nutrient solution. Prof. Zeineb Ouerghi corrected the manuscript.

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