Proline Betaine Accumulation and Metabolism in Alfalfa Plants under Sodium Chloride Stress. Exploring Its Compartmentalization in Nodules 1

Proline Betaine Accumulation and Metabolism in Alfalfa Plants under Sodium Chloride Stress. Exploring Its Compartmentalization in Nodules1 Jean-Charle...
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Proline Betaine Accumulation and Metabolism in Alfalfa Plants under Sodium Chloride Stress. Exploring Its Compartmentalization in Nodules1 Jean-Charles Trinchant, Alexandre Boscari, Guillaume Spennato, Ghislaine Van de Sype, and Daniel Le Rudulier* Laboratoire de Biologie Ve´ge´tale et Microbiologie, Centre National de la Recherche Scientifique, Formation de Recherche en Evolution 2294, Universite´ de Nice-Sophia Antipolis, Faculte´ des Sciences, Parc Valrose, F–06108 Nice cedex 2, France

The osmoprotectant Pro betaine is the main betaine identified in alfalfa (Medicago sativa). We have investigated the long-term responses of nodulated alfalfa plants to salt stress, with a particular interest for Pro betaine accumulation, compartmentalization, and metabolism. Exposure of 3-week-old nodulated alfalfa plants to 0.2 M NaCl for 4 weeks was followed by a 10-, 4-, and 8-fold increase in Pro betaine in shoots, roots, and nodules, respectively. Isotope-labeling studies in alfalfa shoots indicate that [14C]Pro betaine was synthesized from L-[14C]Pro. [14C]Pro betaine was efficiently catabolized through sequential demethylations via N-methylPro and Pro. Salt stress had a minor effect on Pro betaine biosynthesis, whereas it strongly reduced Pro betaine turnover. Analysis of Pro betaine and Pro compartmentalization within nodules revealed that 4 weeks of salinization of the host plants induced a strong increase in cytosol and bacteroids. The estimated Pro betaine and Pro concentrations in salt-stressed bacteroids reached 7.4 and 11.8 mM, respectively, compared to only 0.8 mM in control bacteroids. Na1 content in nodule compartments was also enhanced under salinization, leading to a concentration of 14.7 mM in bacteroids. [14C]Pro betaine and [14C]Pro were taken up by purified symbiosomes and free bacteroids. There was no indication of saturable carrier(s), and the rate of uptake was moderately enhanced by salinization. Ultrastructural analysis showed a large peribacteroid space in salt-stressed nodules, suggesting an increased turgor pressure inside the symbiosomes, which might partially be due to an elevated concentration in Pro, Pro betaine, and Na1 in this compartment.

Excessive salinity and drought are the most important environmental factors that greatly affect plant growth and productivity worldwide. Osmotic and water stresses cause pleiotopic effects, and stress tolerance is a complex and polygenic trait that involves morphological, physiological, as well as biochemical changes. During osmotic stress, plants induce processes that regulate the osmotic adjustment and maintain sufficient cell turgor for growth to proceed (Zimmermann, 1978). Such adjustment requires the control of intracellular inorganic ions in the cytoplasm, via vacuolar sequestration, and accumulation of organic compounds compartmented mainly in the cytoplasm (Jeschke et al., 1986; Binzel et al., 1988; Bohnert et al., 1995). These organic solutes, termed osmolytes, compatible solutes, or osmoprotectants, are nontoxic low-Mr molecules that raise osmotic pressure and protect some macromolecular structures against denaturation (Timasheff, 1992; Bourot et al., 2000). The main osmoprotectants include polyols and their derivatives (Tarczynski et al., 1993; Wanek and Richter, 1

This work was supported by the Centre National de la Recherche Scientifique of France. * Corresponding author; e-mail [email protected]; fax 33–492– 076–838. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037556.

1997), various sugars (Fouge`re et al., 1991; Kameli and Lo¨sel, 1993), and zwitterions such as amino acids and betaines (Le Rudulier et al., 1984; Csonka and Hanson, 1991). Pro and Gly betaine are the most diversely nitrogenous osmolytes accumulated underosmotic stress conditions in plants (Aspinall and Paleg, 1981; Wyn Jones, 1984; Hasegawa et al., 2000). However, if Gly betaine is produced in large quantity by several families of higher plants, particularly in Chenopodiaceae, Amaranthaceae, and Gramineae (Rhodes and Hanson, 1993), certain flowering plants lack significant amounts of Gly betaine and produce other betaines, like Pro betaine, also known as N,N-dimethylproline or stachydrine. This betaine is found mainly in some species of Plumbaginaceae, Capparidaceae, Rutaceae, Labiatae, Compositae, and Leguminosae (Wyn Jones and Storey, 1981; Rhodes and Hanson, 1993; Hanson et al., 1994). In alfalfa (Medicago sativa), Pro betaine is the major betaine (Robertson and Marion, 1960; Wyn Jones and Storey, 1981), but other betaines, such as pipecolate betaine (homostachydrine), trigonelline, hydroxyproline betaine, and Gly betaine, have also been identified in various alfalfa genotypes (Wood et al., 1991). In addition to alfalfa, other Medicago species, such as Medicago truncatula, Medicago littoralis, Medicago rugosa, and Medicago polymorpha, produce Pro betaine with significant genotypic variations (Wood

Plant Physiology, July 2004, Vol. 135, pp. 1583–1594, www.plantphysiol.org Ó 2004 American Society of Plant Biologists

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et al., 1991; Naidu et al., 1992). Pro betaine, which is released by alfalfa seeds during germination (Phillips et al., 1995), is an inducer of nodulation (nod) genes in Sinorhizobium meliloti, the alfalfa-symbiotic species (Phillips et al., 1992). Interestingly, S. meliloti transports this betaine by a high-affinity Na1-coupled transporter, BetS (Boscari et al., 2002), and via an ABC (ATP-binding cassette) protein-dependent transporter, Hut (Boncompagni et al., 2000). In addition, S. meliloti uses Pro betaine as a carbon and nitrogen source or as an osmoprotectant (Gloux and Le Rudulier, 1989; Goldmann et al., 1991; Burnet et al., 2000). Given the competition among soil bacteria for plant carbon sources, it is interesting to point out that Pro betaine catabolism contributes to rhizobial colonization of seedling roots (Phillips et al., 1998). All these reports ascribe important physiological functions to Pro betaine in alfalfa and in its symbiotic partner. However, if Pro accumulation and metabolism are well documented in higher plants and also in bacteroids from legume nodules (Fouge`re et al., 1991; Kohl et al., 1991; Trinchant et al., 1998), in the case of Pro betaine similar basic questions remain almost unexplored. Thus, the aim of the present study is to gain an understanding of the effects of salinity on Pro betaine pools in shoots, roots, and nodule compartments of alfalfa. Furthermore, in spite of some pioneer efforts to analyze Pro betaine in plants (Essery et al., 1962), nothing is currently known about its catabolism. Thus, we have addressed the question of the effects of salt stress on Pro betaine metabolism in alfalfa plants. The investigation indicates that Pro betaine accumulation in alfalfa represents a long-term response to salinization and, interestingly enough, is associated with Pro accumulation. In vivo isotopelabeling studies show that reduced Pro betaine catabolism under salt stress conditions promotes betaine accumulation. We also provided evidence that Pro betaine transport by purified symbiosomes and isolated bacteroids is slightly increased by salinization of the host plant.

RESULTS

Salt stress was imposed on the nodulated plants 3 weeks after inoculation by adding 0.2 M NaCl to the growth medium, and two physiological parameters, leaf water potential (cL) and acetylene reduction activity (ARA), were measured during the stress period merely to establish the degree of stress that affects the plants. cL and ARA of 3-week-old unstressed plants were 20.6 6 0.1 MPa and 25 6 2.2 mmol ethylene g21 (nodule fresh weight) h21, respectively, and did not vary significantly during the next 4 weeks (Fig. 1, A and B). A linear decrease of cL to 21.8 6 0.2 MPa was observed during the first 2 weeks of stress, followed by a slow decline to 22.1 6 0.2 MPa at the end of the experiment. In parallel, a 40% and 60% reduction in ARA resulted from 2 and 4 weeks of 1584

Figure 1. Effect of salt stress on leaf water potential (A), C2H2 reduction (B), and dry matter (C) of nodulated M. sativa plants. The plants were grown in nitrogen-free mineral SP control medium during 3 weeks after inoculation and maintained on the control medium (d, n) or subjected to 0.2 M NaCl (s, h). Values are the means 6 SE of triplicates from five independent experiments.

salinization, respectively. In terms of growth, measured as dry weight, the inhibition due to salt stress was rather limited during the first 2 weeks: about 10% on roots and approximately 30% in shoots and nodules (Fig. 1C). Then, the reduction of growth was more pronounced, and after 4 weeks of salinization, stressed shoots, roots, and nodules exhibited about one-half of the dry mass of the corresponding organs from the control plants. No foliar injury symptoms were noticed. Plant Physiol. Vol. 135, 2004

Pro Betaine Accumulation in Salt-Stressed Alfalfa Nodules

Accumulation of Pro Betaine and Pro, and Changes in Ion Content in Salt-Stressed Alfalfa Plants

Exposing the roots of alfalfa plants for 2 weeks to 0.2 M NaCl did not raise Pro betaine levels above the values observed in unstressed plants, approximately 3 to 5 mmol g21 (dry weight) in shoots, roots, and nodules (Fig. 2, A–C). In contrast, during the following 2 weeks of stress, Pro betaine content increased more than 10-fold in shoots and about 4- and 8-fold in roots and nodules, respectively. Thus, at the end of the salt treatment, the Pro betaine level was 2.5-fold higher in shoots (approximately 50 mmol g21 dry weight; Fig. 2A) than in roots (Fig. 2B) and nodules (Fig. 2C). Pro betaine itself accounted for 0.7% of shoot dry weight. Pro distribution among alfalfa plant organs did not follow the pattern described for Pro betaine. During the 4-week experimental period, Pro level in unstressed plants was almost constant in shoots and nodules (Fig. 2, D and F), whereas it was slightly decreased in roots (Fig. 2E). Under salt stress, a rapid increase was observed in all organs during the first

3 weeks, and then a plateau value was reached of about 35, 10, and 45 mmol g21 dry weight in shoots, roots, and nodules, respectively. The 2-week delay before Pro betaine accumulation started might be related to the kinetics of Pro accumulation. Indeed, in agreement with the proposed role of Pro as precursor of Pro betaine, any increase in Pro betaine levels should more likely be preceded by a stimulation of Pro biosynthesis. After 4 weeks of salinization, Pro betaine was 2.2- and 1.4-fold more abundant than Pro in roots and shoots, respectively, although Pro reached a 2-fold higher level than Pro betaine in nodules (Fig. 2). Based on a water volume of 0.85 mL g21 fresh-weight nodule tissue, the average Pro betaine and Pro concentrations at the end of the salinization were estimated to be approximately 3.8 and 7.8 mM, respectively. It is noteworthy that alfalfa represents one of the few examples of a plant that accumulates both Pro betaine and Pro. Trigonelline was also detected in the different organs, and salt stress triggered its increase. However, the highest trigonelline level, which was observed in

Figure 2. Pro betaine (A–C) and Pro accumulation (D–F) in nodulated M. sativa plants subjected to salt stress. The plants were grown as described in Figure 1, and maintained on the control medium (n) or subjected to 0.2 M NaCl (s). The levels of Pro betaine and Pro were determined in shoots (A and D), roots (B and E), and whole nodules (C and F). Values are the arithmetic means 6 SE of triplicates from three independent experiments.

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shoots, never represented more than 2% of the Pro betaine content (data not shown). The relationship between ion content in shoots, roots, and nodules and external NaCl concentrations was typical of a sodium excluder, with internal Na1 levels increasing strongly when 0.2 M NaCl was applied. After 2 weeks of stress, the increase in intracellular Na1 was 8-fold in shoots and nodules (Fig. 3, A and C) and about 4-fold in roots (Fig. 3B), with an endogenous content in Cl2 always exceeded that of Na1, except in nodules. As usually observed in glycophytes, the K1 accumulation in roots and shoots was slightly inhibited in response to salinity. However, within the nodules, the magnitude of K1 reduction was quite important, with a 50% decrease. During

the third and fourth weeks of salt stress, increases in Na1 and Cl2 contents of all organs still persisted, but at a much lower rate than before, whereas the decrease in K1 was more pronounced in shoots and roots. Thus, the rapid increases in Na1 and Cl2 contents can be more likely correlated with Pro accumulation rather than with Pro betaine increase, which started after only 2 weeks of salinization. At the end of the salt treatment, the highest ion content variations were observed in nodules, with a 13-fold increase in Na1 and Cl2 contents and a 3-fold decrease in K1 levels (Fig. 3). Similar variations have been observed in nodules of white lupin (Lupinus albus) submitted to salt stress (Fernandez-Pascual et al., 1996). Assuming, as above, that 85% of fresh weight is water, Na1, K1, and Cl2 concentrations in whole nodules were estimated at 15.4, 15.8, and 9.1 mM, respectively. However, because published evidence indicates tissue-specific differences in the concentration of minerals in soybean and lupin nodules (Minchin et al., 1994; Fernandez-Pascual et al., 1996; Streeter, 1998), whether such heterogeneous ion distribution may occur in alfalfa nodules remains to be addressed. Pro Betaine Metabolism in Alfalfa Plants

Figure 3. Effect of salt stress on the ion content of shoots (A), roots (B), and whole nodules (C) of M. sativa. Plant growth conditions were as described in Figure 1. The ion content was measured in 3-week-old nodulated plants maintained on the control medium (light-gray squares), or subjected to 2 (dark-gray squares) or 4 (black squares) weeks of salinization (0.2 M NaCl). Values are the means 6 SE of duplicates from three independent experiments. 1586

The accumulation of Pro betaine in response to salt stress may be due to increased biosynthesis, diminished catabolism, or a combination of both, associated or not with translocation between organs. Information on Pro betaine biosynthesis in alfalfa, and more generally in plants, is rather scarce, and so far only in vivo radiolabeling and tissue culture preliminary data suggest that Pro might be the precursor of Pro betaine (Essery et al., 1962; Sethi and Carew, 1974). Furthermore, if Gly betaine is usually considered as a metabolic end product (Hanson et al., 1985; Rhodes and Hanson, 1993), nothing is currently known about a possible catabolism of Pro betaine in higher plants. Thus, we wanted to test whether (1) Pro betaine biosynthesis from Pro is modulated by salt stress in alfalfa, and (2) Pro betaine can be broken down in control and salinized plants. Since it has been mentioned that Pro betaine biosynthesis occurs only in chlorophyll-containing tissues (Sethi and Carew, 1974), L-[14C]Pro (200 kBq) was supplied directly to alfalfa shoots by dipping their base into the radioactive medium (see ‘‘Materials and Methods’’). During the 15-min incubation period, all the supplied radioactivity was taken up and, during the 3-h chase period, radioactivity was neither detectable in 14CO2 or 14C-volatile bases traps, nor released into the nutrient solution. The amount of radioactivity found in the ethanol-soluble fraction (ESF) was increased during salinization, 1.4- and 2.2-fold in the presence of 0.2 and 0.4 M NaCl, respectively (Table I). At the same time, radioactivity in free Pro was 3 times higher in stressed shoots (0.4 M NaCl) than in control shoots, and this was correlated Plant Physiol. Vol. 135, 2004

Pro Betaine Accumulation in Salt-Stressed Alfalfa Nodules

Table I. Effect of salt stress on labeling of Pro betaine in M. sativa shoots following [14C]Pro supply Fifteen shoots with 6 trifoliate leaves from 4-week-old inoculated plants were incubated in 100 mL of L-[14C]Pro (200 kBq, 20.8 nM) for 15 min and then transferred in no salt (control), 0.2, or 0.4 M NaCl SP medium for 3 h. Radioactivity was measured in ESF, EIF, free Pro, and Pro betaine. Values are the means 6 SE values of duplicates from two independent experiments. Radioactivity

Treatment

ESF

EIF

Pro

Pro Betaine

kBqa

Control 1NaCl 0.2 1NaCl 0.4

catabolism by alfalfa was strongly reduced. Besides Pro betaine, Pro was the most radioactive compound identified in the soluble fraction, which also contained a significant amount of labeling in N-methylPro, Glu, and, to a lesser extent, Asp. These results support the view that Pro is a characteristic product of the Pro betaine catabolic pathway, with N-methylPro as the first intermediate.

M M

41.2 6 2.1 158.8 6 7.92 4.6 6 1.2 2.2 6 0.11 60.6 6 3.0 139.4 6 7.0 44.4 6 2.2 2.4 6 0.12 90.8 6 4.5 109.2 6 5.5 75.4 6 3.8 3.0 6 0.15

Student’s t test analysis showed that the difference in [14C]Pro betaine between 0.4 M NaCl-treated shoots and control shoots was significant at the 2% level. a

with a much lower incorporation of the radiocarbon in the ethanol-insoluble fraction (EIF). Interestingly, [14C]Pro betaine could be identified. However, its radioactivity represented only 1.5% of the total radioactivity taken up by the shoots. Nevertheless, based on Student’s t test, the amount of radioactivity recovered in Pro betaine was significantly higher in shoots subjected to 0.4 M NaCl than in control shoots (P , 0.02). [14C]Pro betaine accounted for 13% of newly synthesized compounds (radioactivity in ESF minus radioactivity in free Pro) in unstressed shoots, whereas it represented 19.5% in shoots subjected to 0.4 M NaCl. Pro betaine biosynthesis in salt-stressed shoots was stimulated (2-fold) upon addition of 1 mM Met to the incubation medium, and radioactivity could be identified in N-methylPro (data not shown). These radiotracer data indicate that Pro betaine is synthesized by methylation of Pro. They also suggest a significant, but limited, effect of salt stress on Pro betaine biosynthesis and the availability of a limited amount of methyl donor(s). To determine whether Pro betaine could be catabolized by alfalfa plants, [14C]Pro betaine was supplied to the roots maintained in no salt or 0.2 M NaCl Sirois and Peterson (SP) medium for 1 d, as described in ‘‘Materials and Methods.’’ Probably due to reduction of transpiration, uptake of [14C]Pro betaine by saltstressed plants was almost 3 times lower than by control plants (Table II). Radioactivity was detectable in the CO2 trap and represented 21% and 6% of the [14C]Pro betaine taken up by control and salt-stressed plants, respectively, demonstrating Pro betaine catabolism and suggesting an inhibitory effect of salt stress on such catabolism. A clear relationship between 14 CO2 formation and 14C incorporation into EIF was observed with almost 3 times more radioactivity in control than in salt-stressed plants. At the same time, radioactivity in Pro betaine was depleted much more rapidly in control plants, with only 15% left after 1 d, compared to 45% in salinized plants. Altogether, these data confirm that, under salinization, Pro betaine Plant Physiol. Vol. 135, 2004

Salt Stress Affects Osmolyte and Ion Nodule Compartmentalization

In order to obtain accurate quantitative determinations of metabolite concentrations in nodule compartments, bacteroids and cytosol were obtained quickly after the nodules were homogenized (see ‘‘Materials and Methods’’). Thus, the endogenous pools of Pro betaine and Pro were unlikely to have changed much. In unstressed nodules, Pro betaine and Pro could be detected in the two compartments, although their levels in bacteroids were extremely low (Fig. 4). Salt stress did enhance Pro betaine content, resulting in a 4-fold increase in bacteroids after 2 weeks and an 8-fold increase in cytosol and bacteroids at the end of 4 weeks of salinization. After determination of the intracellular aqueous volume of bacteroids, as previously described (Fouge`re et al., 1991), Pro betaine concentration in bacteroids from 4-week-stressed nodules was estimated at approximately 7.4 mM. Pro level was also greatly enhanced under salt stress, with a 15-fold increase in cytosol and bacteroids after 4 weeks of salinization. Pro content appeared higher than Pro betaine level in the 2 nodule compartments, and its concentration was estimated at 11.8 mM in bacteroids from 4-week-stressed nodules. In comparison, Pro betaine and Pro concentrations in control nodules of the same age were only 0.8 mM. In contrast to bacteroid isolation, symbiosome preparation was time consuming, and metabolites were more likely exchanged

Table II. Catabolism of [14C]Pro betaine by M. sativa plants Ten 4-week-old inoculated plants were incubated by dipping the roots into 10 mL of no salt (control plants) or 0.2 M NaCl (salinized plants) SP medium containing [14C]Pro betaine (133 kBq, 28.9 nM). After 1 d, radioactivity was measured in CO2 produced as well as in ESF (which includes Pro betaine, Pro, and other soluble compounds), and EIF. Radioactivity is expressed in kBq/10 plants and as percent of [14C]Pro betaine taken up (%). Values are the means 6 SE values of duplicates from two independent experiments. Radioactivity Control Plants kBq

CO2 Pro betaine ESF Pro Others EIF Total absorbed

4.2 3.0 4.0 3.2 5.6 20.0

6 6 6 6 6 6

Salinized Plants %

0.3 0.2 0.3 0.3 0.4 1.5

21 15 20 16 28

kBq

0.4 3.0 1.9 0.7 0.7 6.7

6 6 6 6 6 6

0.03 0.20 0.15 0.05 0.05 0.48

%

6 45 28 11 10

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Figure 4. Effect of salt stress on Pro betaine and Pro content in cytosol (A) and bacteroid fractions (B) of M. sativa nodules. Experimental conditions are as described in Figure 2. Values are the means 6 SE of duplicates from three independent experiments.

between the peribacteroid space (PBS) and the cytosol. Consequently, errors might occur in the determination of Pro betaine and Pro from the PBS because of reallocation or degradation, and, thus, realistic values cannot be presented. Nevertheless, both compounds were identified in this compartment, and their concentrations were enhanced by salinization of the host plant. In terms of ion content in unstressed nodules, K1 was much higher than Na1, 22-fold in cytosol, and about 4-fold in bacteroids (Fig. 5). In response to salt stress, the Na1 level increased in an almost linear manner in cytosol and, after 4 weeks of treatment, it was enhanced by 9-fold. A significant increase (2.2-fold) was also observed within bacteroids from salt-stressed nodules. In parallel, sizable decreases (2.2- to 3.2-fold) in the K1 pool were observed with time. Cl2 levels were differentially affected by salinity; a significant increase (16-fold) was noticed in cytosol, while bacteroids were much less affected (1.2-fold increase). Finally, in 4-week-salinized nodules, the prominent ion was Cl2 in the two compartments, while K1 appeared slightly lower than Na1 in cytosol and bacteroids. In addition, these three ions were also found in substantial amounts in the PBS (data not shown). Uptake of Osmolytes by Isolated Symbiosomes and Free Bacteroids

Because Pro betaine and Pro were detected not only in cytosol but also in PBS and bacteroids, we 1588

were interested in determining whether symbiosomes and bacteroids could transport these compounds, and also in testing to what extent salinization of the plant host could modulate the uptake activities. Thus, the rates of substrate uptake were investigated by incubating purified symbiosomes and free bacteroids with different concentrations (0.5–5 mM) of [14C]Pro betaine or [14C]Pro. Symbiosomes and bacteroids were prepared from nodules exposed to salt stress during 2 weeks, which already showed a large increase in Pro content, and from 4-week-stressed nodules, which presented Pro betaine accumulation. Whatever the age of the nodules, with symbiosomes, as well as with bacteroids, the rate of Pro betaine uptake was linearly enhanced at increasing substrate concentrations (Fig. 6, A and B). There was no indication of a saturable carrier at the investigated substrate concentrations, which were physiologically relevant with the concentrations of Pro betaine and Pro determined in bacteroids from stressed nodules. With preparations from 4-week-stressed nodules, Pro betaine uptake rate was slightly higher in symbiosomes (Fig. 6A) than in bacteroids (Fig. 6B). Salt stress tended to stimulate the uptake rate, both in symbiosomes and bacteroids; a 20% increase was observed at the highest Pro betaine concentration. Pro uptake by both symbiosomes and bacteroids showed a pattern very similar to that of Pro betaine (Fig. 6, C and

Figure 5. Effect of salt stress on the ion content of cytosol (A) and bacteroid fractions (B) of M. sativa nodules. Experimental conditions were as described in Figure 2. Values are the means 6 SE of duplicates from three independent experiments. Plant Physiol. Vol. 135, 2004

Pro Betaine Accumulation in Salt-Stressed Alfalfa Nodules

Figure 6. Pro betaine (A and B) and Pro (C and D) uptake by intact symbiosomes (A and C) and free bacteroids (B and D) from M. sativa nodules. Symbiosomes and bacteroids were purified as described in ‘‘Materials and Methods’’ from nodules of 7-week-old plants grown on the control medium (solid line) or from nodules of plants exposed to 0.2 M NaCl for 4 weeks (dotted line) after inoculation. Values are from 1-min uptake experiments and represent the means 6 SE of duplicates from three independent experiments.

D), with no evidence for carrier-mediated Pro transport, as already reported for symbiosomes from soybean (Glycine max; Udvardi et al., 1990; Pedersen et al., 1996) and fava bean (Vicia faba) L. var minor (Trinchant et al., 1998). Pro uptake was moderately stimulated by salinity. The transport rate of Pro into symbiosomes and bacteroids was not significantly modified between the second and the fourth weeks of salinization, while the uptake rate of Pro betaine was stimulated about 2-fold during the same period (data not shown). Thus, at the end of the salt treatment, both substrates were transported at a similar rate (Fig. 6).

Symbiosome Ultrastructural Changes Associated with Salt Stress

Purification of symbiosomes, which was achieved using a discontinuous Percoll gradient, has revealed differences between preparations from control nodules and nodules from salt-stressed plants. Symbiosomes from unstressed nodules formed a very narrow and dense band at the interface between the 30% and 60% Percoll layers, whereas symbiosomes from saltstressed nodules formed a more diffuse and thick band above the same interface, suggesting that salt stress resulted in a decrease in the average density of symbiosomes, as already observed for symbiosomes from soybean nodules (Pedersen et al., 1996). To find Plant Physiol. Vol. 135, 2004

out whether such modification could be correlated with possible ultrastructural changes in symbiosomes, cytological analysis was conducted. A kinetic study of the salt stress effect was realized with nodules from plants exposed to salt stress during 1 to 4 weeks. Cross-sections of invaded cells from the fixing zone corresponding to zone III according to Vasse et al. (1990) showed a dense cytoplasm filled with bacteroids that were individually enclosed in vesicles formed by the peribacteroid membrane (PBM). However, whereas the PBM was closely tied to the bacteroids in nodules from control plants (Fig. 7, A and C), in 2-week-stressed nodules, the PBS was consistently enlarged (Fig. 7, B and D). Calculations of symbiosome volumes indicated a 2.1-fold increase from 2.1 6 0.3 3 10212 cm3 in control nodules to 4.5 6 0.7 3 10212 cm3 in salt-stressed nodules. Meanwhile, infected cell and bacteroid sizes were not modified by the salt treatment. Similar observations have been reported previously for fava bean nodules subjected to salt stress and have shown a 1.7-fold enlargement of the symbiosome volume (Trinchant et al., 1998). The tight link between this large symbiosome volume and the lowest symbiosome density noticed during purification, before sample preparations for electron microscopy analysis, strengthened the idea that an increase in volume in symbiosomes from salt-treated plants was not due to sample fixation. Since a cause-and-effect relationship between buffer osmolarity and symbiosome volume has already been demonstrated (Ou 1589

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Figure 7. NaCl-induced cytological alterations in nitrogen-fixing zone of M. sativa nodules. Electron micrographs of infected cells from nodules of 5-week-old control plants (A and C) or nodules of plants submitted to 0.2 M NaCl during 2 weeks (B and D). IC, invaded cell; UC, uninvaded cell; B, bacteroid. Black arrows indicate amyloplasts. Bars, 10 mm (A and B) and 1 mm (C and D).

Yang and Day, 1992), the utilization of buffer adjusted at 600 mosmol instead of 450 mosmol would be expected to induce symbiosome plasmolysis and not enlargement. The kinetic analysis revealed that the swelling of symbiosomes was noticeable after 1 week of salt treatment, but the enlargement was much less than after 2 weeks of stress. During the last week of salinization, no further modification in symbiosome volume could be detected. In addition, it is worth mentioning that noninvaded cells from salt-stressed plants contained very large amyloplasts that were not present in the corresponding cells from control plants (Fig. 7, A and C). Obviously, starch accumulation was more pronounced in 4-week-stressed nodules than in 2-week-stressed nodules. Such starch granule accumulation has also been reported in infected cells of the central region and inner cortical cells in hypoxic alfalfa nodules (ArreseIgor et al., 1993). The significance of starch accumulation in stressed nodules remains unclear. However, it has been suggested that modifications in the starch-tosugar ratio due to dysfunction in sugar utilization could modify turgor pressure, leading to alterations in the diffusion barrier for oxygen (Layzell et al., 1990) and, consequently, to nitrogen fixation activity.

DISCUSSION

The first striking feature of the research presented here demonstrated the ability of young nodulated alfalfa plants to accumulate Pro betaine in shoots, 1590

roots, and nodules under salt stress treatment. However, if the well-known salinization-induced increase in Pro concentration started shortly after salt stress application, the initiation of Pro betaine accumulation in the different organs was observed after only 2 weeks of stress (Fig. 2). It seems pertinent to point out that the concentration of Pro betaine in shoots and roots of 4-week-salinized plants exceeded that of Pro. In the context of the generally accepted role of Pro accumulation as a defense mechanism against osmotic challenge by acting as a compatible solute, it is reasonable to believe that Pro betaine likewise could contribute to maintain the turgor pressure essential for continued growth during salinization. Both Pro betaine and Pro accumulation through de novo biosynthesis can be considered as long-term adaptation phenomena, although the kinetics of accumulation was quite different for each compound. Pro betaine could contribute to osmotic adjustment after the primary accumulation of other compounds, such as Pro, has occurred. In bacteria, the osmoprotective role of Pro betaine has been clearly established; in Escherichia coli, when available exogenously, Pro betaine is a more effective osmoprotectant than Pro (Hanson et al., 1994), and in S. meliloti, the beneficial effect of Pro betaine is similar to that of the very potent osmoprotective Gly betaine (Bernard et al., 1986). It is also appropriate to underscore previous physiological studies demonstrating a beneficial effect of an exogenous supply of Pro betaine on nitrogen fixation activity of alfalfa seedlings grown in vitro under sterile conditions and nodulated by S. meliloti (Pocard et al., 1984). Briefly, Plant Physiol. Vol. 135, 2004

Pro Betaine Accumulation in Salt-Stressed Alfalfa Nodules

when such seedlings were submitted to 0.2 M NaCl during 5 d, the ARA of plants maintained in the presence of 10 mM Pro betaine reached 30% to 35% of the ARA from unstressed plants, compared to less than 5% in plants grown in the absence of the betaine. From the results presented here, it is obvious that exogenous Pro betaine is taken by the roots, translocated into the different organs, including nodules, and transported by the bacteroids through the PBM (Table II and Fig. 6). Thus, the availability of a substantial amount of Pro betaine within the nodule can significantly alleviate, directly or indirectly, the negative effect of salt stress on nitrogen fixation. Moreover, since the endogenous pool of Pro was not depleted during Pro betaine accumulation, alfalfa is one of the few plants that accumulates large amounts of both compatible solutes simultaneously. Such a combination might help to explain the relatively high overall osmotic tolerance of alfalfa. Obviously, even if it has been previously shown that elevated intracellular Pro associated with high Gly betaine levels provided a strong noticeable synergistic stimulation of nitrogen fixation activity in Klebsiella pneumoniae subjected to salt stress (Le Rudulier and Bouillard, 1983), it remains to be determined whether a comparable effect between Pro betaine and Pro could also occur in alfalfa nodules. The second pertinent point of this work is to shed some light on Pro betaine metabolism. Radiotracer data presented here provide convincing evidence that [14C]Pro betaine is indeed synthesized in young M. sativa shoots from [14C]Pro (Table I), via N-methylPro. Nevertheless, major unresolved questions remain about Pro betaine biosynthesis, such as the characterization, localization, and regulation of the enzyme(s) involved. In contrast to Gly betaine, which is usually considered a metabolic end product in many plants, including sugar beet (Beta vulgaris), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea; Hanson and Wyse, 1982; Hanson et al., 1985; Nuccio et al., 1998), [14C]Pro betaine supplied to alfalfa roots was efficiently metabolized by unstressed plants. The 14 C-labeling data are consistent with progressive demethylations, as already observed in S. meliloti (Glouxand Le Rudulier, 1989). In this bacterium, two genes are involved in Pro betaine catabolism: stc2 encodes an iron-sulfur monooxygenase of the Rieske type catalyzing the production of N-methylPro (Burnet et al., 2000), and stcD encodes a demethylase that converts N-methylPro to Pro (Phillips et al., 1998). Interestingly, salt stress strongly inhibited the turnover of Pro betaine in alfalfa plants (Table II), as well as in S. meliloti (Gloux and Le Rudulier, 1989). Hence, under salinization, reduced catabolism promotes accumulation of this betaine in alfalfa. This study on Pro betaine in alfalfa is the first, to our knowledge, to address its subcellular compartmentalization in nodules and demonstrate that purified symbiosomes from alfalfa nodules exhibited Pro betaine permeability. Salinization of the host plant strongly enhanced Pro betaine content, particularly in cytosol, Plant Physiol. Vol. 135, 2004

but also in bacteroids (Fig. 4). Pro betaine transport into symbiosomes was slightly stimulated during salinization of the host plant (Fig. 6). Together with the greater pool of Pro betaine in the cytosol (Fig. 4), such activity favored the increase of Pro betaine within the bacteroids. Since investigations with [14C]Pro indicated that S. meliloti did not produce Pro betaine from Pro (data not shown), accumulation of this betaine in bacteroids is unlikely to be due to de novo biosynthesis. Another interesting feature reported here concerns the consequence of salinization of the host plant on the symbiosome volume. In order to avoid burst of the symbiosomes, the buffers used for preparations need to be isotonic with the PBS. It has been accepted that symbiosomes behave as osmometers, and also that the PBS serves as the main compartment to accommodate changes in osmotic pressure (Ou Yang and Day, 1992). In our case, to maintain the integrity of the PBM, the osmolarity of purification buffers needed to be increased, providing convincing evidence that salinization of the host plants induced an increased osmolarity inside the symbiosome. Since the accumulation of Pro betaine and Pro in salt-stressed bacteroids, up to 7.4 and 11.8 mM, respectively, is not carrier mediated, the concentration of these osmolytes in the PBS should also be rather high. Therefore, it seems reasonable to assume that such accumulation contributes to the increased turgor pressure, which might be involved in the enlargement of the symbiosome volume observed on cross-nodule sections (Fig. 7). The occurrence of a high Asn concentration in salt-stressed bacteroids, up to 30 mM (Fouge`re et al., 1991), and a substantial amount of Na1 (Fig. 5) may also be involved in this osmotic adjustment. Indeed, salt stress induced a 2.2-fold increase in Na1 content within bacteroids (Fig. 5), leading to a concentration of about 14.7 mM in Na1, together with 34 mM in Cl2 and 11.5 mM in K1. Such results suggest that the PBM is permeable to these ions when an excess is present within the cytosol, and preliminary experiments using 36 2 Cl have revealed uptake of this anion by purified symbiosomes (data not shown). In this context, it is interesting to note that Udvardi and Day (1997) have already pointed out that Na1 and K1 transport is likely to be important for osmotic balance across the PBM and the inner membrane of bacteroids. However, transport of these ions across symbiotic membranes warrants detailed investigations. Whatever the mechanism, the influx of Na1 and Cl2 into the symbiosome might contribute to the decrease in nitrogen fixation under salt treatment. With regard to this hypothesis, it would be interesting to know whether the negative effect of these ions could be partially overcome in plant genotypes producing increasing amounts of osmolytes under salt stress, as well as in bacteroids showing enhanced capacity for osmolyte transport. We are currently testing the overexpression of the S. meliloti betaine transporter BetS (Boscari et al., 2002) in bacteroids, in order to evaluate its potential role in 1591

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restoration of nitrogen fixation by alfalfa nodules submitted to salt treatment.

MATERIALS AND METHODS Plant Material and Growth Conditions Alfalfa (Medicago sativa L. cv Europe) seeds obtained from the Institut de la Recherche Agronomique (Le Rheu, France) were surface sterilized and germinated for 3 d as previously described (Pocard et al., 1991). Young seedlings were transferred on vermiculite to sand (2:1, v/v) mixture-filled pots moistened with a nitrogen-free nutrient solution (SP medium) containing 1 mM MgSO4, 1 mM CaCl2, 1 mM KH2PO4, 0.5 mM K2HPO4, and trace elements (Sirois and Peterson, 1982). Three days after planting, the young seedlings were inoculated with a bacterial suspension containing 7 3 109 cells mL21 of Sinorhizobium meliloti 102F34 (20 mL suspension/container). S. meliloti was routinely grown overnight, at 30°C, in Luria-Bertani medium (Sambrook et al., 1989) supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 (Boscari et al., 2002). Three weeks after inoculation, the plants were subjected to salt stress by adding NaCl to SP medium (final concentration 0.2 M) or were maintained on NaCl-free medium. Salinization was applied for 4 weeks. All plants were cultured in a growth chamber, at 70% 6 5% air humidity, under a 16-h-light and 8-h-dark cycle, at 24°C 6 1°C and 19°C 6 1°C, respectively, and a photosynthetically active photon flux density of 360 mM m22 s21.

Determination of Growth, Water Potential, and Acetylene Reduction Activity Plants were harvested for analysis 3 weeks after inoculation, before salt treatment, and every week after salinization was applied. The water potential (cL) was measured on the third trifoliate leaf, at mid-day, with a pressure bomb as described by Scholander et al. (1965). The plants were separated into shoots, roots, and nodules, and fresh and dry weights were determined. Nitrogen fixation activity of nodulated alfalfa roots was determined by C2H2 reduction using a gas chromatograph (model 610, ATI-Unicam, Cambridge, UK) equipped with a Porapak T column (80–100 mesh; Hewlett-Packard, Palo Alto, CA), as described by Trinchant and Rigaud (1987). Incubations were made in 5 replicates, at 25°C, in 60-mL rubber cap vials containing O2 (20 kPa) and C2H2 (10 kPa) in argon.

Extraction of Plant Material and Preparation of Symbiosomes and Bacteroids Shoots, roots, and whole nodules of alfalfa (8 g of fresh weight) were crushed in liquid nitrogen in a mortar, and the resulting powder was extracted 3 times in 20 mL of 70% (v/v) ethanol under stirring (10 min). The combined ethanol extracts were evaporated to dryness under vacuum (37°C), and solids were dissolved in 9 mL of ultrapure water. The extracts were kept in 1-mL fractions at 220°C until used. Symbiosomes were isolated aerobically from about 5 to 8 g (fresh weight) of freshly harvested root nodules that were gently crushed in a mortar, at 4°C, in MES-Tris buffer (25 mM, pH 7.2) adjusted with mannitol to 450 or 600 mosmol L21 for control and salt-stressed nodules, respectively (Trinchant et al., 1994). The homogenate was filtered through nylon (200-mm mesh), and the filtrate was centrifuged (40g, 10 min), at 4°C, to remove plant cell debris. Symbiosomes were sedimented by a second centrifugation (500g, 10 min), and purified using a discontinuous Percoll gradient according to Trinchant et al. (1998). They were vigorously vortexed during 5 min to break the PBMs, centrifuged (5,000g, 15 min), and the resulting supernatant represented the PBS fraction. Rapid isolation of bacteroids was essentially as described by Fouge`re et al. (1991). Preparations were conducted aerobically from 1 to 2 g (fresh weight) of nodules homogenized using a mortar and pestle in 10 mL of ice-cold buffer containing 50 mM KH2PO4 (pH 7.4), 10% (w/v) polyvinylpyrrolidone, and mannitol as for symbiosome preparations. The mixture was filtered through nylon and immediately centrifuged (1,500g, 2 min). The supernatant was recovered and centrifuged at 10,000g for 5 min. Solutes from the supernatant (cytosol) and pellets (bacteroids) were instantaneously extracted with 70% (v/v) ethanol as described above for plant organs. Extracts were kept in 0.5 mL at 220°C until used.

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Determination of Osmolyte and Ion Contents Free Pro was determined by the ninhydrin assay as described previously (Trinchant et al., 1998). The red coloration that developed was extracted with toluene, and absorbance of the organic phase determined at 520 nm with a Uvikon 922 spectrophotometer (Kontron, Instruments, Milan, Italy). The calibration curve was linear up to 500 nM of L-Pro in 50-mL samples. Pro betaine and trigonelline contents were determined by HPLC (Waters Associates, Milford, MA), as recommended by Gorham (1984). Separations were performed on a 250 3 5-mm Whatman column packed with Partisil 10-SCX (Waters). Elution was achieved at 25°C, with a 50 mM KH2PO4 buffer (pH 4.6) containing 5% (v/v) methanol, at a flow rate of 1.0 mL min21. Detection was done at 192 nm. Ions were extracted from fresh tissue in 70% (v/v) ethanol and analyzed by HPLC (model AS50/BioLC, Dionex, Vienna) and conductimetric detection. Cation analysis was done from ethanol extracts diluted in 25 mM H2SO4 using a Dionex IonPac CS 12A column (250 3 4 mm) eluted at a flow rate of 1 mL min21 with 25 mM H2SO4:ultrapure water (9:1, v/v), and maintained at room temperature. For anion analysis, the extracts were diluted in acetonitrile (30%, v/v), and separation was obtained on a Dionex IonPac AS 11 column (250 3 4 mm) eluted in 10 min with a linear gradient of 12 to 35 mM KOH. 14

C-Labeling Experiments

14 21 14 21 L-[U- C]Pro (9.62 GBq mmol ) and [ C]Pro betaine (4.6 GBq mmol ) prepared from L-[U-14C]Pro were obtained from the Commissariat a` l’Energie Atomique, Gif sur Yvette, France. [14C]Pro was supplied directly to the shoots by dipping their base into SP medium, or SP medium plus 0.2 or 0.4 M NaCl (200 kBq 100 mL21, 20.8 nM). Plants were maintained under a glass bell connected to two bubble chambers used as CO2 or volatile amines traps, and containing 50 mL of a mixture of ethanolamine to ethanol to water (2:1:7, v/v/v) or 50 mL of 6 M HCl, respectively (Wynn et al., 1973; Pocard et al., 1991). A 10 L h21 air flow was driven through this system by a vacuum pump. The experiments were carried out at 20°C 6 1°C, in a controlled, illuminated environment hood. A 15-min pulse period was followed by a 3-h chase period, and extraction of plant material was conducted as described earlier (Pocard et al., 1991) with 80% ethanol. The ethanolic extracts and the pellets extensively dried over anhydrous calcium chloride constitute the ESF and EIF, respectively. The insoluble fractions were hydrolyzed for 24 h at 105°C in 6 M HCl, in sealed vials, and the radioactivity of each fraction was determined with a liquid scintillation spectrometer (model LS6000SC, Beckman Instruments, Villepinte, France). [14C]Pro betaine (133 kBq 10 mL21, 28.9 nM) was supplied to the nodulated roots dipping into SP medium or SP medium plus 0.2 M NaCl. Plants were kept in the presence of the radioactive substrate for 1 d, as described above for the experiments with [14C]Pro, and immediately extracted. Radioactivity in CO2, ESF, and EIF was measured as above. High-voltage electrophoresis on Whatman 3 MM paper in 0.75 N formic acid for 90 min at 40 V cm21 (Le Rudulier and Bouillard, 1983), followed by paper chromatography developed with methanol to ammonium hydroxyde (9:0.5, v/v) was used to resolve Pro betaine, N-methylPro, and Pro. The same electrophoresis followed by chromatography in n-butanol:acetic acid:water (12:3:5, v/v/v) allowed the separation of Glu and Asp. Chromatograms and electrograms were autoradiographed, and scintillation counting was used to quantify 14C. Pro betaine and N-methylPro were also separated by HPLC, as described above, and the radioactivity of the corresponding fractions quantified by scintillation counting.

Transport Studies Pro betaine and Pro uptake by purified symbiosomes and free bacteroids was measured under aerobic conditions, at 25°C, according to the method described for S. meliloti (Boncompagni et al., 2000). All assays were carried out in 1 mL MES-Tris buffer (25 mM, pH 7.4) containing 3.0 kBq of [14C]Pro betaine or [14C]Pro, at final concentrations from 0.5 to 5 mM. Mannitol was used to adjust the osmolarity of each incubation medium at 450 and 600 mosmol L21 for materials from unstressed and salt-stressed plants, respectively. The uptake was initiated by injecting symbiosome or bacteroid suspensions (0.1 mg protein) into the incubation mixture and stopped after 1 min by rapid filtration through glass microfiber Whatman filters (0.45-mm pore size). Filters were then rinsed with 3 mL of the corresponding cold assay medium, and the radioactivity remaining on the filters was determined with a liquid scintillation counter (LS6000 SC, Beckman Instruments). During the 1-min

Plant Physiol. Vol. 135, 2004

Pro Betaine Accumulation in Salt-Stressed Alfalfa Nodules

incubation period, uptake was linear with time. Reactions were conducted in duplicate, with materials from three independent experiments. Protein contents were estimated according to the Bearden method (Bearden, 1978), with bovine serum albumin as standard.

Electron Microscopy Nodules from control and salt-stressed plants were excised, sliced, and immediately fixed with 2.5% (w/v) glutaraldehyde and 1% (w/v) OsO4 in 0.3 M Na-cacodylate buffer (pH 7.2). Samples were dehydrated through a graded ethanol series (25%–100%, v/v), washed twice with propylene oxide, and embedded in Epon’s resin. Ultrathin sections obtained with a diamond knife in a Reichert Ultracut Ultramicrotome (Vienna) were stained in 7% (w/v) uranyl acetate in methanol and 3.52% (w/v) lead citrate. They were observed on an electron microscope (CM12, Philips, Cambridge, UK). Electron micrographs were digitalized in a rasterized format, and color parameters were adjusted to obtain maximal contrasted images. A single algorithm of neighborhood was applied in order to delimit the symbiosome surfaces, which were then colorized, and the corresponding pixels were counted. An average surface with an acceptable SD of about 16% was calculated from a total of 127 symbiosomes. The symbiosome surface (A) was converted into symbiosome volume (V) by using the formula developed by Lindberg and Vorwerk (1970): V 5 A3/2 3 1.382.

ACKNOWLEDGMENTS We thank Dr. David Tepfer for the generous gift of [14C]Pro betaine, Mr. Alain Gilabert for help in growing the plants, Mr. Olivier Barbier for his advice on ion analysis, and the Centre Commun de Microscopie (Universite´ de Nice-Sophia Antipolis) for technical assistance in the microscopy studies. We are grateful to Dr. Patrick Coquillard for his contribution to the numerical analysis electron micrograph, and we thank Dr. David Day and Dr. John Streeter for valuable discussions and suggestions. Received December 11, 2003; returned for revision March 10, 2004; accepted April 1, 2004.

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