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Nodulation under salt stress of alfalfa lines obtained after in vitro selection for osmotic tolerance
Plant Science 165 (2003) 887 /894 www.elsevier.com/locate/plantsci
Nodulation under salt stress of alfalfa lines obtained after in vitro selection for osmotic tolerance Dimitar Djilianov a,*, Els Prinsen b, Sevgi Oden b, Henry van Onckelen b, Joachim Mu¨ller c,1 a
Abiotic Stress Tolerance and Ecophysiology Group, AgroBioInstitute, 8 Blvd Dragan Tzankov, 1164 Sofia, Bulgaria b Department of Biology, UIA /University of Antwerp, B-2610 Antwerp, Belgium c Friedrich-Miescher-Institute, P.O. Box 2543, CH-4001 Basel, Switzerland Received 4 March 2003; received in revised form 13 June 2003; accepted 13 June 2003
Abstract The culture of pasture legumes is of special interest for the world’s agriculture because it results in the production of high value protein accompanied by improvement of soil fertility, since they undergo symbioses with dinitrogen-fixing bacteria. Alfalfa is the most important forage crop for the arid and semi-arid areas, where increased salinity of irrigated fields is one of the major constraints that limit crop productivity. Recently, using in vitro selection, we obtained alfalfa genotypes with increased tolerance to PEG 6000, an agent that mimics drought. Here, we present studies on salt tolerance with adult plants of these alfalfa genotypes grown either on nitrate or diazotrophically at various non-lethal salt concentrations. Growth parameters and parameters related to nodule physiology and development have been analysed. All R lines had a higher salt tolerance than their explant source genotype (T1) in germination and growth on nitrate-containing medium. Under diazotrophic growth conditions, R1 and R3 were with higher relative salt tolerance than T1. Nodule biomass was depressed at high salt concentrations in all genotypes. This growth decline went in pair with changes in carbohydrate partitioning and in the hormonal balance. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alfalfa; Salt stress; Nodulation; Symbiosis; Soluble carbohydrates; IAA; ABA
1. Introduction Recent problems, such as bovine spongiform encephalitis (BSE) due to food supplements of animal origin, have re-established interest in the use of high value protein from plant sources. The culture of pasture legumes is of special interest, since they undergo symbioses with dinitrogen-fixing bacteria, which improve soil fertility and quality. Alfalfa is the most important forage crop for the arid and semi-arid areas. In these regions, stress caused by the increased salinity of irrigated fields impairs plant growth and is one of the major constraints that limit crop productivity. Classical
* Corresponding author. Tel.: /359-2-9635407; fax: /359-29635408. E-mail address: [email protected] (D. Djilianov). 1 Present address: InnoLife, 44, rue Henner, F-68300 Saint-Louis, France.
breeding methods are too slow and have had limited contribution in improvement of the salt tolerance [1]. To find alternative routes for improvement of alfalfa breeding, plant tissue culture approaches (somaclonal variation, in vitro selection) have been used [2]. In many cases, however, no regeneration from the salt tolerant cell lines has been realized or the regenerated plants showed no inheritance of the trait. Regeneration of salttolerant plants was achieved after single-step selection at cellular level, from non-commercial cultivar Regen-S [1] and genes related to salt tolerance were identified [3]. The expression of such gene (Alfin 1) predominantly in the roots is considered as an important marker for breeders [4]. The gene is essential for root growth and its over-expression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions [5]. Similar results were reported after in vitro selection of somaclone from commercial cultivars [6]. The authors explain the increased tolerance of the
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00291-7
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somaclone by differences in copy number of a gene pA9 presumably coding proline-rich cell wall protein accumulation. As the reaction to most abiotic stresses */drought, high osmotic pressure, a high or low temperature, salt tolerance is considered a quantitative trait. Sometimes, but not always, the tolerance to certain type of environmental stress is positively related to resistance to others [7]. Recently, we obtained alfalfa genotypes with increased tolerance to PEG as selective agent [8]. Induction of somatic embryogenesis was achieved despite the presence of 10% PEG 6000 in the media. Plants were successfully regenerated and potted. Further, detached leaves from plants of the selected lines and the explantsource genotype were subjected to various periods of severe stress with 40% PEG 6000 solution [9]. The lines, resulting from in vitro selection were considered drought tolerant and were able to recover better than the explant-source genotype after osmotic shock, which was related to such ‘recovery’ trends of proline content and ion leakage. From studies with PEG that mimics water stress, it is difficult to extrapolate the reaction to salt stress [10]. Here, we present results concerning salt effects on the growth of nitrate grown plants and on the growth and nodule parameters of diazotrophically grown plants nodulated by Sinorhizobium meliloti . Furthermore, data concerning salt effects on nodule carbohydrate partitioning are presented.
2. Materials and methods 2.1. Plant material For growth and nodulation experiments we used plants, resulting from seeds, obtained after controlled self-pollination of osmotic tolerant genotypes R1 /R4 and of their explant source genotype T1 of alfalfa (Medicago sativa L.) [8,9]. They were surface sterilized by immersion for 10 min in 30% (v/v) H2O2, and transferred to Magenta jars. The upper compartment contained 200 ml of a Perlite/Vermiculite-1:1 (v/v)mixture. The lower part was filled with 200 ml B&Dnutrient solution [11]. It was supplemented with NaCl by adding convenient amounts of a 5 M NaCl stock solution. The final concentrations in the nutrient solution were 0, 37.5, 75 and 150 mM for nodulated plants and 0 and 150 mM in the case of nitrate grown plants. In order to initiate diazotrophic growth, jars were inoculated with 1 ml of a stationary culture of S. meliloti DSM1981 grown in RMM medium [11] after emergence of the seedlings. Other plants were grown without symbionts. In this case, the nutrient solution was amended with 5 mM KNO3. Jars were thinned out to leave only one plant per jar. Plants were grown in a
phytotron under long day conditions (16 h day, 160 mE/ s/m2, 22 8C; night at 18 8C all temperatures 9/2 8C; rH 659/10%). The plants were harvested at the onset of flowering of the T1 plants at 0 mM NaCl. Shoots and nodules were immediately frozen in liquid nitrogen. Shoot biomass was determined after lyophilization of the shoots and nodule biomass was determined after lyophilization of the nodules. Aliquots of the lyophilized nodules were further processed in order to determine their carbohydrate, ABA and IAA contents. 2.2. Analysis of soluble carbohydrates For analysis of soluble carbohydrates, 1/5 mg of lyophilized, powdered plant material were mixed with 10 mg of insoluble polyvinylpyrrolidone (Polyclar AT), suspended in 80% (v/v) methanol and incubated at 70 8C for 10 min. After centrifugation (10 min at 13,000 rpm), the supernatant was removed and the pellet was re-extracted twice. The pellet was dried and conserved for the subsequent analysis of starch. The supernatants were combined, vacuum-dried and resuspended in 0.6 ml ultrapure water (MilliQ, Millipore, Molsheim, F). Some 50 ml of a wet mixed-bed ionexchanger (Serdolit micro blue:red 3:1 v/v) were added in order to remove charged compounds. When setting up the procedure, this ion-exchanger-mix had been checked for hydrolysis of disaccharides. After centrifugation (10 min, 13,000 rpm), the supernatant was removed and the ion-exchanger was washed once with 0.4 ml ultrapure water and centrifuged. The supernatants were combined and lyophilized. Pellets were dissolved in 0.1 ml methanol (50% v/v) and further analyzed by capillary gas chromatography. 2.3. Quantification of starch The insoluble pellets remaining from the carbohydrate extraction were resuspended in 0.2 ml NaOH (0.5 M) and incubated at 60 8C for 1 h. Subsequently, 0.2 ml HCl (0.5 M) was added. After cooling down to room temperature, 0.6 ml acetate/Na/-buffer (pH 4.5, 0.2 M) containing 1 U amyloglucosidase (special quality for starch determination, Roche Diagnostics, Basel, Switzerland) was added and the samples were incubated overnight at 37 8C. The reaction was stopped with 2 min boiling. The samples were centrifuged (10 min at 10,000 /g ) and the supernatants were analyzed for glucose formation using a commercial assay (Roche Diagnostics). 2.4. Capillary gas chromatography For capillary gas chromatography, 50 ml of soluble carbohydrate extracts obtained as described above were completely dried, derivatized as described by Mu¨ller et
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al. [12] and subsequently analyzed using capillary gas chromatography. The gas chromatograph (Carlo Erba Mega 3500, Brechbu¨hler, Zu¨rich, Switzerland) was equipped with a glass column (J&W Scientific capillary DB-17 column, 30 m /0.323 mm; Brechbu¨hler) and with a flame ionization detector (340 8C). After injection (0.3 ml; injector at 320 8C), the column was kept at 70 8C for 2 min and then heated progressively with a rate of 25 8C min 1 to 170 8C, followed by a rate of 7 8C min 1 to 300 8C. The column was kept at 300 8C for 5 min. Carbohydrates were quantified by comparison with an internal standard (mannoheptulose) and nine external standards (arabitol, mannitol, fructose, glucose, myo-inositol, sucrose, trehalose, maltose, raffinose). Chromatograms were integrated and subsequently analyzed using the Maxima software package (Brechbu¨hler). 2.5. Analysis of endogenous IAA and ABA IAA and ABA were analyzed by a combined solid phase extraction procedure based on Prinsen et al. [13]. 13 C6-IAA (Cambridge Isotope Laboratories Inc., Andover, MA; 100 ng) and 18O-ABA (100 ng, prepared following Gray et al. [14]) were used for isotope dilution purposes. After pentafluorobenzyl derivatization, PFBIAA and PFB-ABA were analyzed by negative ion chemical ionization (NICI) GC-SIR-MS (HP 5890 series II coupled to a VG FISONS TRIO 2000 quadrupole mass spectrometer (column 15 m BD-XLB, 0.25 mm i.d. (J&W Scientific), gas phase He, 120 /240 8C; 15 8/min; Tinj.250 8C) [15,16]. 2.6. Statistics All experiments had a two-factorial design (Genotype /Salt). Data were analysed by a two-factorial ANOVA after a convenient transformation of the data if required. Analyses of variance and Student/Newman /Keuls-tests were performed using the software SigmaStat (Jandel Scientific, San Rafael, CA).
3. Results 3.1. Salt effects on growth on nitrate-containing medium In a preliminary experiment (data not shown), we examined the germination of alfalfa seeds under various concentrations of NaCl. As a result, 150 mM NaCl has been chosen as highest sub-lethal concentration for further studies. In a first set of experiments, all genotypes were grown on a nitrate-containing medium without Rhizobia at 0 and 150 mM NaCl. At the onset of flowering of T1 on 0 mM NaCl (6 weeks after sowing), all plants were harvested and their shoot dry
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Fig. 1. Shoot biomass of alfalfa plants grown on B&D-medium supplemented with 5 mM KNO3 and 0 (control) or 150 mM NaCl. The experiments were carried out in three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; ANOVA, followed by Student /Newman /Keuls-test).
weight determined. Under control conditions the shoot dry weight of the R genotypes were slightly, but not significantly lower than that of T1 (Fig. 1). When exposed to salt stress, however the T1 seedlings decreased drastically their growth */ on 150 mM NaCl their dry weight was two times lower than that without salt. On the contrary, no significant differences due to salt treatment appeared in all R genotypes (Fig. 1).
3.2. Salt effects on diazotrophic growth and nodulation parameters In a second set of trials, the alfalfa plantlets were grown without nitrate and in the presence of rhizobia on increasing salt concentrations (from 0 to 150 mM). At the onset of flowering of T1 on 0 mM NaCl, plants were harvested and shoot dry weight and nodule parameters were determined. At no salt, R1, R2 and R3 had significantly lower shoot dry weight than T1 and R4. General and steady decrease in shoot biomass occurred upon exposure to increasing salt concentrations (data shown only for 150 mM NaCl) (Fig. 2). It is important to point out, however that the values of R1 and R3 lines at the highest salt concentration were not significantly different from those on control media. At control conditions there were small insignificant genotype differences in average nodule number per plant (Fig. 3A). A well-defined decrease trend occurred for T1 when the plants were exposed to increased salt concentrations. The reduction reached /200% at high salt. The R lines performed much better with very steady nodule numbers regardless the salt contents. At 150 mM, the R1 and R3 lines had higher nodule numbers than the rest lines.
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Overall, increasing salt concentrations inhibited nodule growth of all alfalfa lines. The inhibition was genotype dependent.
3.3. Non-structural nodule carbohydrates
Fig. 2. Shoot dry weight of alfalfa plants grown on B&D-medium supplemented with 0 or 150 mM NaCl. One week after sowing, plants were inoculated with Sinorhizobium meliloti DSM1981. Plants were harvested at the onset of flowering of genotype T1 at 0 mM NaCl (8 weeks after sowing). Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; ANOVA, followed by Student /Newman /Keuls-test).
In order to analyse the assimilate partitioning in alfalfa nodules, the following parameters were measured: (i) starch, sucrose, glucose and fructose as markers for plant-borne carbohydrates; and (ii) trehalose as a marker for bacteroid borne carbohydrates. The most prominent non-structural carbohydrate in alfalfa nodules was starch. The nodules of R2, R3 and R4 plants were with significantly higher starch contents than the nodules of T1 and R1, although the differences were small on control and low salt media (Fig. 4A). The interaction genotype /salt was significant (P B/0.05). Starch in nodules of all genotypes increased significantly at higher salt concentrations. R3 line plants showed the most prominent increase in starch when exposed to high salt. In alfalfa nodules, the major non-structural soluble carbohydrates (NSC) were sucrose, glucose and fructose. The NSC content varied in relatively narrow limits with small, often insignificant differences between the genotypes. R1 was the only genotype, whose SCH pool sizes were significantly higher when exposed to medium or high salt stress (Fig. 4B). Nodules of T1 plants had the highest trehalose under normal and low salt conditions (Fig. 4C). A drastic decrease occurred when they were exposed to medium and high salt. On the contrary, the R lines responded to medium salt stress with significant increase of trehalose levels. High salt stress resulted in decreased trehalose contents in all genotypes.
3.4. Nodule phytohormones
Fig. 3. Nodule parameters of alfalfa plants grown on various concentrations of NaCl. Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; ANOVA, followed by Student /Newman /Keuls-test). (A) Nodule number per plant; (B) total nodule dry weight (mg per plant).
The exposure to salt resulted in a decrease of total nodule dry weight in most of the genotypes (Fig. 3B). R2 line had relatively steady levels until medium salt concentrations, but suffered a drastic decrease at 150 mM NaCl.
Overall, nodule IAA and ABA contents were not affected by the genotype (P /0.2) but by the salt treatment (P B/0.02). Interactions between genotypes and salt were highly significant (P B/0.01). There were no significant differences in endogenous IAA contents between all lines at control conditions (Table 1). Interestingly, a significant increase of nodule IAA at higher salt levels was found in R1, R2 and R4. The endogenous levels of nodule ABA varied between 3.9 (T1) and 18.5 (R2) nmol/gDW in average in the absence of salt (Table 2). At these conditions, R1 and R2 had significantly higher levels than the other genotypes. Upon salt treatment, a significant decrease of nodule ABA contents was found in genotypes T1 (after an increase at 37.5 mM NaCl), R1 and R2.
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Fig. 4. Non-structural nodule carbohydrates of nodules from alfalfa plants grown on various concentrations of NaCl. Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/ 0.05; ANOVA, followed by Student /Newman /Keuls-test). (A) Starch content; (B) soluble carbohydrates content; (C) trehalose content.
Table 1 Endogenous IAA contents of nodules from alfalfa plants grown on various concentrations of NaCl (as in Fig. 3) NaCl (mM)
IAA (nmol g/DW) T1
R1
R2
R3
R4
0 37.5 75 150
0.399/0.07cd 0.689/0.14c 0.589/0.22c 1.349/0.26bc
0.449/0.03cd 0.459/0.16cd 0.389/0.11cd 2.149/0.35ab
0.389/0.12cd 1.819/0.56ab 0.609/0.18c 1.439/0.50ab
0.449/0.11cd 1.199/0.28bc 1.049/0.39bc 0.599/0.17c
0.369/0.02cd 0.789/0.14c 1.659/0.11b 0.809/0.21c
Effects:
p(Genotype) /0.02 p(NaCl)/0.013
Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; two-way-ANOVA, followed by Student /Newman /Keuls-test).
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Table 2 Endogenous ABA contents of nodules from alfalfa plants grown on various concentrations of NaCl (as in Fig. 3) NaCl (mM)
ABA (nmol/g DW) T1
R1 c
0 37.5 75 150
3.909/1.05 10.019/3.30bc 2.709/0.60c 2.609/0.30c
Effects:
p(Genotype) /0.2 p(NaCl)/0.006
R2 b
12.029/2.00 6.609/2.90bc 2.409/0.60c 3.209/0.20c
R3 a
18.509/2.90 5.309/1.40bc 2.009/0.20c 3.059/0.90bc
R4 c
4.909/0.90 5.009/1.40bc 7.409/1.70b 4.809/0.60bc
3.409/0.90c 4.309/0.90bc 3.809/0.80bc 2.709/0.40bc
Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; two-way-ANOVA, followed by Student /Newman /Keuls-test).
4. Discussion Achieving a tolerance to more than one type of stress is a tempting goal for many breeding programs. A cascade of genes in plants controls tolerance to various abiotic stresses */drought, salt, heat or cold. Recently, transgenic approach made remarkable contributions demonstrating that simple manipulation of gene activity has great potential with regard to plant improvement. In some cases, over-expression of a single transcription factor enhanced induction of other stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other [17]. Other studies stressed on the importance of the promotor showing that the complex of stress-inducible rd29A promoter and the DREB1A cDNA might be useful in improving the stress tolerance of agriculturally important crops by gene transfer [18]. As already mentioned, our alfalfa genotypes were selected as tolerant towards PEG 6000 [8], an agent that mimics drought, but not osmotical stress (see Ref. [10]). In most cases of the present studies, they performed significantly better under salt stress than the explant source line. Studies on nodulation under unfavorable conditions are often focused on isolation of tolerant bacterial strains [19,20]. Under diazotrophic growth conditions, due to the absence of a mineral nitrogen source in the culture medium, growth is strictly dependent on nitrogen fixed by the symbionts. Although fast growing rhizobia have a better salt tolerance (up to 0.5 M NaCl) than slow growing bradyrhizobia, nodulation by these strains is reduced under high salt conditions, even in highly salt tolerant plants, like Prosopis juliflora [21]. Thus, it appears that the plant /symbiont interaction itself is vulnerable to salt stress. Our results confirmed this suggestion. Although all R genotypes were more salt tolerant than T1 on nitrate, this tendency declined under diazotrophic growth conditions. However, the
growth of two R lines (R1 and R3) was not significantly repressed by high salt. Since these lines grow better than T1, they could be the choice for field applications. These two lines fall apart from the other lines with respect to nodulation parameters and to carbohydrate partitioning. Our results indicate that high salt concentrations have major effect on carbohydrate partitioning. Plant borne, non-structural carbohydrates increase in nodules of salt treated plants, especially in the case of R1 (soluble carbohydrates) and R3 (starch). Trehalose is a non-reducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi and invertebrates. Only few plant species, mainly desiccation-tolerant ‘resurrection’ plants, are considered to synthesize trehalose [10,22 /24]. Even exogenously applied to plants, it is considered to be very important as osmoprotectant [25]. In recent studies, stress tolerance of transgenic, trehalose-accumulating rice has been explained not by the primary effect of trehalose as a compatible solute [26], but by effects of trehalose accumulation on assimilates partitioning. In our case, the bacteroid-borne trehalose tends to increase at low and medium salt, then to decrease at high salt concentrations, confirming the results obtained earlier for the same plant species [27]. Interestingly, trehalose contents decreased in nodules of T1-line above 37.5 mM NaCl, whereas in the R lines, they reached their highest levels at 75 mM. A possible explanation could be the eventual shift of carbohydrate allocation from bacteroids to plastids in infected and non-infected cells resulting in large starch pools. According to a generally accepted model, fast ABA accumulation is an important step of the cellular signaling cascade, responsible for the perception of salt- or water deficit in plants [28 /32]. In root nodules, phytohormones have higher steady state-level than in roots of the same plants [33,34]. Studies on ABA levels at long-lasting stress are scarce, especially for nodules. In our case, exposure for more
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than 6 weeks to various salt concentrations led to overall tendency of ABA decrease. This could be regarded as a response to long-term growth on salt, where ABA transport from roots to the shoots and leaves had already taken place [30] In this respect, further studies on the dynamics of ABA accumulation during long exposure to stress in various plant organs are needed. Nodules are known to contain relatively high amounts of IAA [33 /35]. This amount increases even under high salt conditions and genotype-specific differences can be observed. Since IAA has been shown to induce trehalase activity [12], the surprisingly low trehalose contents at high salt concentrations could be indirectly due to the increase of IAA. It is difficult, however, to make any speculations about possible connections between reaction to salt stress and endogenous IAA levels because of the narrow fluctuations of the hormone. As in ABA, further studies on the dynamics of accumulation under stress are needed. In conclusion, our results show that the alfalfa lines originally selected as tolerant to osmotic stress had a higher salt tolerance than their explant source genotype. R1 and R3 were the most promising lines for further studies and practical application. These genotypes showed better germination and growth at high salt concentrations on nitrate as well as under diazotrophic growth conditions. The test system presented here could serve as a good model for further studies on the dynamics of stress response and hormone changes, as well as on the symbiotic interaction between alfalfa and bacteria, a phenomena of considerable practical importance.
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[32] A. Ivanova, D. Djilianov, H. van Onckelen, A. Atanassov, Abscissic acid changes in osmotic stressed leaves of alfalfa genotypes varying in drought tolerance, J. Plant Phys. 150 (1997) 224 /227. [33] Y. Murakamizukami, Y. Yamamoto, S. Yamaki, Analyses of indole-acetic acid and abscisic acid contents in nodules of soybean plants bearing various mycorrhizas, Soil Biol. Biochem. 23 (6) (1991) 291 /298. [34] R.N. Bhattacharyya, P.S. Basu, Phytohormones in root nodules of leguminous climber, Psophocarpus tetragonolobus DC and its rhizobial symbiont, Ind. J. Plant Phys. 38 (1) (1995) 18 /21. [35] K. Thimann, On the physiology of the formation of nodules on legume roots, Proc. Natl. Acad. Sci. USA 22 (1936) 511 /514.
Nodulation under salt stress of alfalfa lines obtained after in vitro selection for osmotic tolerance Dimitar Djilianov a,*, Els Prinsen b, Sevgi Oden b, Henry van Onckelen b, Joachim Mu¨ller c,1 a
Abiotic Stress Tolerance and Ecophysiology Group, AgroBioInstitute, 8 Blvd Dragan Tzankov, 1164 Sofia, Bulgaria b Department of Biology, UIA /University of Antwerp, B-2610 Antwerp, Belgium c Friedrich-Miescher-Institute, P.O. Box 2543, CH-4001 Basel, Switzerland Received 4 March 2003; received in revised form 13 June 2003; accepted 13 June 2003
Abstract The culture of pasture legumes is of special interest for the world’s agriculture because it results in the production of high value protein accompanied by improvement of soil fertility, since they undergo symbioses with dinitrogen-fixing bacteria. Alfalfa is the most important forage crop for the arid and semi-arid areas, where increased salinity of irrigated fields is one of the major constraints that limit crop productivity. Recently, using in vitro selection, we obtained alfalfa genotypes with increased tolerance to PEG 6000, an agent that mimics drought. Here, we present studies on salt tolerance with adult plants of these alfalfa genotypes grown either on nitrate or diazotrophically at various non-lethal salt concentrations. Growth parameters and parameters related to nodule physiology and development have been analysed. All R lines had a higher salt tolerance than their explant source genotype (T1) in germination and growth on nitrate-containing medium. Under diazotrophic growth conditions, R1 and R3 were with higher relative salt tolerance than T1. Nodule biomass was depressed at high salt concentrations in all genotypes. This growth decline went in pair with changes in carbohydrate partitioning and in the hormonal balance. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Alfalfa; Salt stress; Nodulation; Symbiosis; Soluble carbohydrates; IAA; ABA
1. Introduction Recent problems, such as bovine spongiform encephalitis (BSE) due to food supplements of animal origin, have re-established interest in the use of high value protein from plant sources. The culture of pasture legumes is of special interest, since they undergo symbioses with dinitrogen-fixing bacteria, which improve soil fertility and quality. Alfalfa is the most important forage crop for the arid and semi-arid areas. In these regions, stress caused by the increased salinity of irrigated fields impairs plant growth and is one of the major constraints that limit crop productivity. Classical
* Corresponding author. Tel.: /359-2-9635407; fax: /359-29635408. E-mail address: [email protected] (D. Djilianov). 1 Present address: InnoLife, 44, rue Henner, F-68300 Saint-Louis, France.
breeding methods are too slow and have had limited contribution in improvement of the salt tolerance [1]. To find alternative routes for improvement of alfalfa breeding, plant tissue culture approaches (somaclonal variation, in vitro selection) have been used [2]. In many cases, however, no regeneration from the salt tolerant cell lines has been realized or the regenerated plants showed no inheritance of the trait. Regeneration of salttolerant plants was achieved after single-step selection at cellular level, from non-commercial cultivar Regen-S [1] and genes related to salt tolerance were identified [3]. The expression of such gene (Alfin 1) predominantly in the roots is considered as an important marker for breeders [4]. The gene is essential for root growth and its over-expression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions [5]. Similar results were reported after in vitro selection of somaclone from commercial cultivars [6]. The authors explain the increased tolerance of the
0168-9452/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00291-7
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somaclone by differences in copy number of a gene pA9 presumably coding proline-rich cell wall protein accumulation. As the reaction to most abiotic stresses */drought, high osmotic pressure, a high or low temperature, salt tolerance is considered a quantitative trait. Sometimes, but not always, the tolerance to certain type of environmental stress is positively related to resistance to others [7]. Recently, we obtained alfalfa genotypes with increased tolerance to PEG as selective agent [8]. Induction of somatic embryogenesis was achieved despite the presence of 10% PEG 6000 in the media. Plants were successfully regenerated and potted. Further, detached leaves from plants of the selected lines and the explantsource genotype were subjected to various periods of severe stress with 40% PEG 6000 solution [9]. The lines, resulting from in vitro selection were considered drought tolerant and were able to recover better than the explant-source genotype after osmotic shock, which was related to such ‘recovery’ trends of proline content and ion leakage. From studies with PEG that mimics water stress, it is difficult to extrapolate the reaction to salt stress [10]. Here, we present results concerning salt effects on the growth of nitrate grown plants and on the growth and nodule parameters of diazotrophically grown plants nodulated by Sinorhizobium meliloti . Furthermore, data concerning salt effects on nodule carbohydrate partitioning are presented.
2. Materials and methods 2.1. Plant material For growth and nodulation experiments we used plants, resulting from seeds, obtained after controlled self-pollination of osmotic tolerant genotypes R1 /R4 and of their explant source genotype T1 of alfalfa (Medicago sativa L.) [8,9]. They were surface sterilized by immersion for 10 min in 30% (v/v) H2O2, and transferred to Magenta jars. The upper compartment contained 200 ml of a Perlite/Vermiculite-1:1 (v/v)mixture. The lower part was filled with 200 ml B&Dnutrient solution [11]. It was supplemented with NaCl by adding convenient amounts of a 5 M NaCl stock solution. The final concentrations in the nutrient solution were 0, 37.5, 75 and 150 mM for nodulated plants and 0 and 150 mM in the case of nitrate grown plants. In order to initiate diazotrophic growth, jars were inoculated with 1 ml of a stationary culture of S. meliloti DSM1981 grown in RMM medium [11] after emergence of the seedlings. Other plants were grown without symbionts. In this case, the nutrient solution was amended with 5 mM KNO3. Jars were thinned out to leave only one plant per jar. Plants were grown in a
phytotron under long day conditions (16 h day, 160 mE/ s/m2, 22 8C; night at 18 8C all temperatures 9/2 8C; rH 659/10%). The plants were harvested at the onset of flowering of the T1 plants at 0 mM NaCl. Shoots and nodules were immediately frozen in liquid nitrogen. Shoot biomass was determined after lyophilization of the shoots and nodule biomass was determined after lyophilization of the nodules. Aliquots of the lyophilized nodules were further processed in order to determine their carbohydrate, ABA and IAA contents. 2.2. Analysis of soluble carbohydrates For analysis of soluble carbohydrates, 1/5 mg of lyophilized, powdered plant material were mixed with 10 mg of insoluble polyvinylpyrrolidone (Polyclar AT), suspended in 80% (v/v) methanol and incubated at 70 8C for 10 min. After centrifugation (10 min at 13,000 rpm), the supernatant was removed and the pellet was re-extracted twice. The pellet was dried and conserved for the subsequent analysis of starch. The supernatants were combined, vacuum-dried and resuspended in 0.6 ml ultrapure water (MilliQ, Millipore, Molsheim, F). Some 50 ml of a wet mixed-bed ionexchanger (Serdolit micro blue:red 3:1 v/v) were added in order to remove charged compounds. When setting up the procedure, this ion-exchanger-mix had been checked for hydrolysis of disaccharides. After centrifugation (10 min, 13,000 rpm), the supernatant was removed and the ion-exchanger was washed once with 0.4 ml ultrapure water and centrifuged. The supernatants were combined and lyophilized. Pellets were dissolved in 0.1 ml methanol (50% v/v) and further analyzed by capillary gas chromatography. 2.3. Quantification of starch The insoluble pellets remaining from the carbohydrate extraction were resuspended in 0.2 ml NaOH (0.5 M) and incubated at 60 8C for 1 h. Subsequently, 0.2 ml HCl (0.5 M) was added. After cooling down to room temperature, 0.6 ml acetate/Na/-buffer (pH 4.5, 0.2 M) containing 1 U amyloglucosidase (special quality for starch determination, Roche Diagnostics, Basel, Switzerland) was added and the samples were incubated overnight at 37 8C. The reaction was stopped with 2 min boiling. The samples were centrifuged (10 min at 10,000 /g ) and the supernatants were analyzed for glucose formation using a commercial assay (Roche Diagnostics). 2.4. Capillary gas chromatography For capillary gas chromatography, 50 ml of soluble carbohydrate extracts obtained as described above were completely dried, derivatized as described by Mu¨ller et
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al. [12] and subsequently analyzed using capillary gas chromatography. The gas chromatograph (Carlo Erba Mega 3500, Brechbu¨hler, Zu¨rich, Switzerland) was equipped with a glass column (J&W Scientific capillary DB-17 column, 30 m /0.323 mm; Brechbu¨hler) and with a flame ionization detector (340 8C). After injection (0.3 ml; injector at 320 8C), the column was kept at 70 8C for 2 min and then heated progressively with a rate of 25 8C min 1 to 170 8C, followed by a rate of 7 8C min 1 to 300 8C. The column was kept at 300 8C for 5 min. Carbohydrates were quantified by comparison with an internal standard (mannoheptulose) and nine external standards (arabitol, mannitol, fructose, glucose, myo-inositol, sucrose, trehalose, maltose, raffinose). Chromatograms were integrated and subsequently analyzed using the Maxima software package (Brechbu¨hler). 2.5. Analysis of endogenous IAA and ABA IAA and ABA were analyzed by a combined solid phase extraction procedure based on Prinsen et al. [13]. 13 C6-IAA (Cambridge Isotope Laboratories Inc., Andover, MA; 100 ng) and 18O-ABA (100 ng, prepared following Gray et al. [14]) were used for isotope dilution purposes. After pentafluorobenzyl derivatization, PFBIAA and PFB-ABA were analyzed by negative ion chemical ionization (NICI) GC-SIR-MS (HP 5890 series II coupled to a VG FISONS TRIO 2000 quadrupole mass spectrometer (column 15 m BD-XLB, 0.25 mm i.d. (J&W Scientific), gas phase He, 120 /240 8C; 15 8/min; Tinj.250 8C) [15,16]. 2.6. Statistics All experiments had a two-factorial design (Genotype /Salt). Data were analysed by a two-factorial ANOVA after a convenient transformation of the data if required. Analyses of variance and Student/Newman /Keuls-tests were performed using the software SigmaStat (Jandel Scientific, San Rafael, CA).
3. Results 3.1. Salt effects on growth on nitrate-containing medium In a preliminary experiment (data not shown), we examined the germination of alfalfa seeds under various concentrations of NaCl. As a result, 150 mM NaCl has been chosen as highest sub-lethal concentration for further studies. In a first set of experiments, all genotypes were grown on a nitrate-containing medium without Rhizobia at 0 and 150 mM NaCl. At the onset of flowering of T1 on 0 mM NaCl (6 weeks after sowing), all plants were harvested and their shoot dry
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Fig. 1. Shoot biomass of alfalfa plants grown on B&D-medium supplemented with 5 mM KNO3 and 0 (control) or 150 mM NaCl. The experiments were carried out in three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; ANOVA, followed by Student /Newman /Keuls-test).
weight determined. Under control conditions the shoot dry weight of the R genotypes were slightly, but not significantly lower than that of T1 (Fig. 1). When exposed to salt stress, however the T1 seedlings decreased drastically their growth */ on 150 mM NaCl their dry weight was two times lower than that without salt. On the contrary, no significant differences due to salt treatment appeared in all R genotypes (Fig. 1).
3.2. Salt effects on diazotrophic growth and nodulation parameters In a second set of trials, the alfalfa plantlets were grown without nitrate and in the presence of rhizobia on increasing salt concentrations (from 0 to 150 mM). At the onset of flowering of T1 on 0 mM NaCl, plants were harvested and shoot dry weight and nodule parameters were determined. At no salt, R1, R2 and R3 had significantly lower shoot dry weight than T1 and R4. General and steady decrease in shoot biomass occurred upon exposure to increasing salt concentrations (data shown only for 150 mM NaCl) (Fig. 2). It is important to point out, however that the values of R1 and R3 lines at the highest salt concentration were not significantly different from those on control media. At control conditions there were small insignificant genotype differences in average nodule number per plant (Fig. 3A). A well-defined decrease trend occurred for T1 when the plants were exposed to increased salt concentrations. The reduction reached /200% at high salt. The R lines performed much better with very steady nodule numbers regardless the salt contents. At 150 mM, the R1 and R3 lines had higher nodule numbers than the rest lines.
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Overall, increasing salt concentrations inhibited nodule growth of all alfalfa lines. The inhibition was genotype dependent.
3.3. Non-structural nodule carbohydrates
Fig. 2. Shoot dry weight of alfalfa plants grown on B&D-medium supplemented with 0 or 150 mM NaCl. One week after sowing, plants were inoculated with Sinorhizobium meliloti DSM1981. Plants were harvested at the onset of flowering of genotype T1 at 0 mM NaCl (8 weeks after sowing). Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; ANOVA, followed by Student /Newman /Keuls-test).
In order to analyse the assimilate partitioning in alfalfa nodules, the following parameters were measured: (i) starch, sucrose, glucose and fructose as markers for plant-borne carbohydrates; and (ii) trehalose as a marker for bacteroid borne carbohydrates. The most prominent non-structural carbohydrate in alfalfa nodules was starch. The nodules of R2, R3 and R4 plants were with significantly higher starch contents than the nodules of T1 and R1, although the differences were small on control and low salt media (Fig. 4A). The interaction genotype /salt was significant (P B/0.05). Starch in nodules of all genotypes increased significantly at higher salt concentrations. R3 line plants showed the most prominent increase in starch when exposed to high salt. In alfalfa nodules, the major non-structural soluble carbohydrates (NSC) were sucrose, glucose and fructose. The NSC content varied in relatively narrow limits with small, often insignificant differences between the genotypes. R1 was the only genotype, whose SCH pool sizes were significantly higher when exposed to medium or high salt stress (Fig. 4B). Nodules of T1 plants had the highest trehalose under normal and low salt conditions (Fig. 4C). A drastic decrease occurred when they were exposed to medium and high salt. On the contrary, the R lines responded to medium salt stress with significant increase of trehalose levels. High salt stress resulted in decreased trehalose contents in all genotypes.
3.4. Nodule phytohormones
Fig. 3. Nodule parameters of alfalfa plants grown on various concentrations of NaCl. Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; ANOVA, followed by Student /Newman /Keuls-test). (A) Nodule number per plant; (B) total nodule dry weight (mg per plant).
The exposure to salt resulted in a decrease of total nodule dry weight in most of the genotypes (Fig. 3B). R2 line had relatively steady levels until medium salt concentrations, but suffered a drastic decrease at 150 mM NaCl.
Overall, nodule IAA and ABA contents were not affected by the genotype (P /0.2) but by the salt treatment (P B/0.02). Interactions between genotypes and salt were highly significant (P B/0.01). There were no significant differences in endogenous IAA contents between all lines at control conditions (Table 1). Interestingly, a significant increase of nodule IAA at higher salt levels was found in R1, R2 and R4. The endogenous levels of nodule ABA varied between 3.9 (T1) and 18.5 (R2) nmol/gDW in average in the absence of salt (Table 2). At these conditions, R1 and R2 had significantly higher levels than the other genotypes. Upon salt treatment, a significant decrease of nodule ABA contents was found in genotypes T1 (after an increase at 37.5 mM NaCl), R1 and R2.
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Fig. 4. Non-structural nodule carbohydrates of nodules from alfalfa plants grown on various concentrations of NaCl. Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/ 0.05; ANOVA, followed by Student /Newman /Keuls-test). (A) Starch content; (B) soluble carbohydrates content; (C) trehalose content.
Table 1 Endogenous IAA contents of nodules from alfalfa plants grown on various concentrations of NaCl (as in Fig. 3) NaCl (mM)
IAA (nmol g/DW) T1
R1
R2
R3
R4
0 37.5 75 150
0.399/0.07cd 0.689/0.14c 0.589/0.22c 1.349/0.26bc
0.449/0.03cd 0.459/0.16cd 0.389/0.11cd 2.149/0.35ab
0.389/0.12cd 1.819/0.56ab 0.609/0.18c 1.439/0.50ab
0.449/0.11cd 1.199/0.28bc 1.049/0.39bc 0.599/0.17c
0.369/0.02cd 0.789/0.14c 1.659/0.11b 0.809/0.21c
Effects:
p(Genotype) /0.02 p(NaCl)/0.013
Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; two-way-ANOVA, followed by Student /Newman /Keuls-test).
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Table 2 Endogenous ABA contents of nodules from alfalfa plants grown on various concentrations of NaCl (as in Fig. 3) NaCl (mM)
ABA (nmol/g DW) T1
R1 c
0 37.5 75 150
3.909/1.05 10.019/3.30bc 2.709/0.60c 2.609/0.30c
Effects:
p(Genotype) /0.2 p(NaCl)/0.006
R2 b
12.029/2.00 6.609/2.90bc 2.409/0.60c 3.209/0.20c
R3 a
18.509/2.90 5.309/1.40bc 2.009/0.20c 3.059/0.90bc
R4 c
4.909/0.90 5.009/1.40bc 7.409/1.70b 4.809/0.60bc
3.409/0.90c 4.309/0.90bc 3.809/0.80bc 2.709/0.40bc
Mean values9/S.E. are given from three independent replications with four seedlings each, per treatment. Values superscribed by different letters are significantly different (P B/0.05; two-way-ANOVA, followed by Student /Newman /Keuls-test).
4. Discussion Achieving a tolerance to more than one type of stress is a tempting goal for many breeding programs. A cascade of genes in plants controls tolerance to various abiotic stresses */drought, salt, heat or cold. Recently, transgenic approach made remarkable contributions demonstrating that simple manipulation of gene activity has great potential with regard to plant improvement. In some cases, over-expression of a single transcription factor enhanced induction of other stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other [17]. Other studies stressed on the importance of the promotor showing that the complex of stress-inducible rd29A promoter and the DREB1A cDNA might be useful in improving the stress tolerance of agriculturally important crops by gene transfer [18]. As already mentioned, our alfalfa genotypes were selected as tolerant towards PEG 6000 [8], an agent that mimics drought, but not osmotical stress (see Ref. [10]). In most cases of the present studies, they performed significantly better under salt stress than the explant source line. Studies on nodulation under unfavorable conditions are often focused on isolation of tolerant bacterial strains [19,20]. Under diazotrophic growth conditions, due to the absence of a mineral nitrogen source in the culture medium, growth is strictly dependent on nitrogen fixed by the symbionts. Although fast growing rhizobia have a better salt tolerance (up to 0.5 M NaCl) than slow growing bradyrhizobia, nodulation by these strains is reduced under high salt conditions, even in highly salt tolerant plants, like Prosopis juliflora [21]. Thus, it appears that the plant /symbiont interaction itself is vulnerable to salt stress. Our results confirmed this suggestion. Although all R genotypes were more salt tolerant than T1 on nitrate, this tendency declined under diazotrophic growth conditions. However, the
growth of two R lines (R1 and R3) was not significantly repressed by high salt. Since these lines grow better than T1, they could be the choice for field applications. These two lines fall apart from the other lines with respect to nodulation parameters and to carbohydrate partitioning. Our results indicate that high salt concentrations have major effect on carbohydrate partitioning. Plant borne, non-structural carbohydrates increase in nodules of salt treated plants, especially in the case of R1 (soluble carbohydrates) and R3 (starch). Trehalose is a non-reducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi and invertebrates. Only few plant species, mainly desiccation-tolerant ‘resurrection’ plants, are considered to synthesize trehalose [10,22 /24]. Even exogenously applied to plants, it is considered to be very important as osmoprotectant [25]. In recent studies, stress tolerance of transgenic, trehalose-accumulating rice has been explained not by the primary effect of trehalose as a compatible solute [26], but by effects of trehalose accumulation on assimilates partitioning. In our case, the bacteroid-borne trehalose tends to increase at low and medium salt, then to decrease at high salt concentrations, confirming the results obtained earlier for the same plant species [27]. Interestingly, trehalose contents decreased in nodules of T1-line above 37.5 mM NaCl, whereas in the R lines, they reached their highest levels at 75 mM. A possible explanation could be the eventual shift of carbohydrate allocation from bacteroids to plastids in infected and non-infected cells resulting in large starch pools. According to a generally accepted model, fast ABA accumulation is an important step of the cellular signaling cascade, responsible for the perception of salt- or water deficit in plants [28 /32]. In root nodules, phytohormones have higher steady state-level than in roots of the same plants [33,34]. Studies on ABA levels at long-lasting stress are scarce, especially for nodules. In our case, exposure for more
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than 6 weeks to various salt concentrations led to overall tendency of ABA decrease. This could be regarded as a response to long-term growth on salt, where ABA transport from roots to the shoots and leaves had already taken place [30] In this respect, further studies on the dynamics of ABA accumulation during long exposure to stress in various plant organs are needed. Nodules are known to contain relatively high amounts of IAA [33 /35]. This amount increases even under high salt conditions and genotype-specific differences can be observed. Since IAA has been shown to induce trehalase activity [12], the surprisingly low trehalose contents at high salt concentrations could be indirectly due to the increase of IAA. It is difficult, however, to make any speculations about possible connections between reaction to salt stress and endogenous IAA levels because of the narrow fluctuations of the hormone. As in ABA, further studies on the dynamics of accumulation under stress are needed. In conclusion, our results show that the alfalfa lines originally selected as tolerant to osmotic stress had a higher salt tolerance than their explant source genotype. R1 and R3 were the most promising lines for further studies and practical application. These genotypes showed better germination and growth at high salt concentrations on nitrate as well as under diazotrophic growth conditions. The test system presented here could serve as a good model for further studies on the dynamics of stress response and hormone changes, as well as on the symbiotic interaction between alfalfa and bacteria, a phenomena of considerable practical importance.
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