Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment Blackwell Science Ltd 2003? 2003

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1 Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment Blackwell Science Ltd 2003? ? Original Article B and Ca mediated recovery of nodulation and nodule development under salt stress A. El-Hamdaoui et al. Plant, Cell and Environment (2003) 26, Effects of boron and calcium nutrition on the establishment of the Rhizobium leguminosarum pea (Pisum sativum) symbiosis and nodule development under salt stress A. EL-HAMDAOUI, M. REDONDO-NIETO, R. RIVILLA, I. BONILLA & L. BOLAÑOS Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain ABSTRACT The effects of modifying boron (B) and calcium (Ca 2+ ) concentrations on the establishment and development of rhizobial symbiosis in Pisum sativum plants grown under salt stress were investigated. Salinity almost completely inhibited the nodulation of pea plants by Rhizobium leguminosarum bv. viciae This effect was prevented by addition of Ca 2+ during plant growth. The capacity of root exudates derived from salt-treated plants to induce Rhizobium nod genes was not significantly decreased. However, bacterial adsorption to roots was highly inhibited in plants grown with 75 mm NaCl. Moreover, R. leguminosarum 3841 did not grow in minimal media containing such salt concentration. High Ca 2+ levels enhanced both rhizobial growth and adsorption to roots, and increased nodule number in the presence of high salt. Nevertheless, the nodules developed were not functional unless the B concentration was also increased. Because B has a strong effect on infection and cell invasion, these processes were investigated by fluorescence microscopy in pea nodules harbouring a R. leguminosarum strain that expresses green fluorescent protein. Salt-stressed plants had empty nodules and only those treated with high B and high Ca 2+ developed infection threads and exhibited enhanced cell and tissue invasion by Rhizobium. Overall, the results indicate that Ca 2+ promotes nodulation and B nodule development leading to an increase of salt tolerance of nodulated legumes. Key-words: B Ca interaction; infection thread development; nod gene induction; nodulation; nodule invasion; root colonization; salinity. INTRODUCTION Plants differ greatly in their response to salinity (Hasegawa et al. 2000), and most legumes are classified as salt-sensitive crop species (Greenway & Munns 1980). Leguminous plants are singular because they can develop a specialized structure in which the N 2 -fixing process takes place, the root Correspondence: Luis Bolaños, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain. Fax: ; luis.bolarios@uam.es nodule, which is the product of molecular interactions between the host plant and Rhizobium. The effects of salt stress on nitrogen fixation in legumes have been widely reported (for a review: Zahran 1999). High salt can directly impair the interactions between Rhizobium and the host plant by inhibiting nodule formation (Singleton & Bohlool 1984). Salinity can also indirectly affect the symbiosis by reducing the growth of the host plant. One of the major constraints of salt stress is nutrient imbalance (Cramer et al. 1987). Therefore, the study of the interaction among nutrients that are especially required for nodulation, such as boron and calcium, with salt is important to optimize the conditions for salt tolerance of rhizobial-legume symbiosis. Mineral analysis in nodulated Pisum sativum showed that salt-stress leads to a deficiency of nutrients such as potassium, iron, boron and calcium in shoots and roots, and that a supplement of B and Ca recovered the nutrient balance and increased salt-tolerance (El-Hamdaoui et al. 2003). Both nutrients are implicated in the establishment of the symbiosis. Boron deficiency inhibited bacterial plant molecular signalling during early pre-infection events (Redondo-Nieto et al. 2001) and Ca increased the capacity of exudates to induce nod-gene activity in Rhizobium (Richardson et al. 1988). Moreover, B is required for infection thread development and nodule cell invasion, probably due to a B- mediated inhibition of the binding of infection thread matrix material to the cell surface of Rhizobium that promotes endocytosis of bacteria (Bolaños, Brewin & Bonilla 1996). Boron nutrition also affected glycosylation and targeting of glycoproteins during differentiation of endophytic bacteria to N 2 -fixing bacteroids and symbiosome development (Bolaños et al. 2001). Calcium is especially required at the early infection events (Munns 1970). The roles of Ca 2+ include enhancement of bacterial attachment to the root hairs (Smit et al. 1989), and calcium-mediated signalling during bacterial infection (reviewed by Lhuissier et al. 2001). Moreover, the interaction between both nutrients in the development of the N 2 -fixing rhizobiallegume symbiosis has been reported (Bolaños et al. 2002). Nodulation and symbiotic nitrogen fixation is influenced by the level of both nutrients, and a supplement of Ca 2+ was able to alleviate the effects of B deprivation during nodule development Blackwell Publishing Ltd 1003

2 1004 A. El-Hamdaoui et al. Based on those previous reports, the aim of this study was to investigate the effects of different B and Ca 2+ concentrations in the response to salt stress of the establishment and development of the symbiosis between Rhizobium leguminosarum and Pisum sativum plants. MATERIALS AND METHODS Plant growth and inoculation Pea (Pisum sativum cv. Argona) seeds were surfacesterilized with 70% (v/v) ethanol for 1 min and 10% (v/v) sodium hypochlorite for 20 min, soaked for 4 h in sterile distilled water and then germinated on wet filter paper at 25 C. After 4 d, the seedlings were transferred to plastic growth pots and cultivated on perlite with FP medium for legumes (Fahraeus 1957), as the nutrient solution. For salinity conditions, NaCl was added to the nutrient solution at a final concentration of 75 mm. The concentration of B (added as H 3 BO 3 ) was 9.3 mm, and the level of Ca (as CaCl 2 ) was 0.68 mm, for cultures with normal content of B (+B), and Ca (+Ca), with or without salt. The concentrations of B and Ca 2+ were increased up to 55.8 mm (++B) and up to 2.72 mm (++Ca) in the nutrient solutions from the ++B ++Ca treatments of salt-stressed plants. The nutrient solution was changed every 3 d. The ph was always kept between 6.5 and 6.7. One week after transferring to pots, the plants were inoculated with 1 ml per seedling of 10 8 cells ml -1 Rhizobium leguminosarum bv. viciae strain 3841 from an exponential culture in tryptone-yeast extract (TY) medium (Beringer 1974). The inoculated plants were maintained in a growth cabinet at 22 C day/18 C night temperatures with a 16/8 h photoperiod and an irradiance of 190 mmol m -2 s -1. Relative humidity was kept between 60 and 70%. Bacterial cultures To analyse the effects of salt, B and Ca 2+ on R. leguminosarum 3841 growth, bacteria were cultivated in polyethylene flasks containing liquid Y medium (Sherwood 1970) that were vigorously shaken in an orbital shaker, for 48 h at 28 C. Samples were taken every 4 h, and optical density at 600 nm (OD 600nm ) was estimated. Acetylene reduction activity Nitrogenase activity was measured in four plants per treatment from four independent experiments by chromatographic determination of reduced acetylene (ARA) (Postgate 1971). Nodulated roots were incubated for 1 h in a 10% acetylene atmosphere. Samples of 0.5 ml were resolved in a gas chromatograph (Shimadzu GC-8 A; Shimadzu Co., Kyoto, Japan). The activity was determined as ethylene production. Mineral analysis Nodules were collected from plants of each treatment, 3 weeks post-inoculation with Rhizobium, dried at 80 C for 24 h and then oven-ashen and acid-digested (1 M HCl) at 70 C for 30 min. Boron was determined using Azomethine H at ph 5.1 (Wolf 1974) and a Technicon Automatic Analytical System (Martínez et al. 1986). The concentrations of Ca 2+ were analysed by atomic absorption spectrophotometry. Induction of nod gene activity by root exudates For in vitro measurement of nod gene activity, the root exudates were obtained through methanolic extraction according to the method of Maxwell et al. (1989). The analysis of nod gene induction by exudates was made by the determination of b-galactosidase activity, adapted from Miller (1972). The bioassay used a genetically modified strain of R. leguminosarum, strain D24, which contained a transcriptional fusion of the promoter of the nodabcij operon to the lacz gene. The capacity of induction was expressed as b-gal activity units. Methanol (10 ml ml -1 ) was used as negative control (327.6 ± b-gal activity units) and a methanolic solution of 0.5 mm hesperetin, a nod gene-inducing flavone, was used as a positive control ( ± b-gal activity units). Bacterial adsorption to roots Adsorption of rhizobia to pea roots was assayed by the method of Lodeiro et al. (1995). Seedlings of each treatment were incubated in 50 ml of FP medium with about 10 3 cells ml -1 of a late-exponential-phase R. leguminosarum 3841 TY liquid culture at 28 C with rotary shaking at 50 r.p.m. After four washes the roots were plated on TYagar medium. The degree of adsorption was expressed as number of microcolonies developed on roots. When convenient, NaCl, B or Ca 2+ were added to the incubation mixture. Fluorescence microscopy For the study of root hair curling and nodule development with epifluorescence microscopy, pea plants were inoculated with R. leguminosarum strain 3841 carrying phc60, a rhizosphere-stable plasmid that constitutively expresses green fluorescent protein (GFP) (Cheng & Walker 1998). For the acquisition of that plasmid by strain 3841, a three parental conjugation with two Escherichia coli strains carrying phc60 and the helper plasmid prk2013, respectively, was made. Roots and nodules developed were analysed with a fluorescence microscope coupled with reflected light fluorescent attachment (RFC). images of GFP-labelled bacterial cells were obtained by using a filter set consisting of a nm (BP490) band-pass exciter, a 505 nm dicroic filter and a 530 nm long-pass emitter (EO530). Statistical analysis All the experiments were repeated at least four times. All data were statistically analysed by the one-way ANOVA test

3 B and Ca mediated recovery of nodulation and nodule development under salt stress 1005 Table 1. Effects of different concentrations of B (+B, 9.3 mm; ++B, 55.8 mm) and Ca 2+ (+Ca, 0.68 mm; ++Ca, 2.72 mm) on the inhibition by salinity (75 mm NaCl) of nodulation and nitrogenase (acetylene reduction) activity (nmol C 2 H 4 g root -1 h -1 ) of Pisum sativum L. cv. Argona plants 3 weeks post-inoculation with Rhizobium leguminosarum bv. viciae B +Ca +B ++Ca ++B +Ca ++B ++Ca Nodulation (nodules per plant) 6 ± 5 66 ± 21 5 ± 5 82 ± 27 Nitrogenase (% inhibition) 2.7 ± ± ± ± 25.8 Nitrogenase (ethylene production) of control plants (+B +Ca, without NaCl): ± 36.1 nmol C 2 H 4 g root -1 h -1 ; nodulation of control plants: 132 ± 43 nodules per plant. with a sample size n 15. Data in figures and tables are means ± standard deviation. RESULTS The effect of different levels of B (+B, 9.3 mm B; ++B, 55.8 mm B) and Ca 2+ (+Ca, 0.68 mm Ca 2+ ++Ca, 2.72 mm Ca 2+ ) on nodulation (nodules developed per root) and nitrogen fixation (ARA activity) of pea plants 3 weeks after inoculation with R. leguminosarum 3841 under salt stress is summarized in Table 1. The increase of Ca 2+ concentration in the nutrient solution resulted in an increase of nodules developed per root. The supplement of B did not affect nodulation under salt stress. However, ARA activity under salt stress was highly inhibited except in treatments with high Ca 2+ and high B concentrations (++B ++Ca). The measurement of B and Ca 2+ in nodules (Table 2) indicated that the content of both nutrients was diminished by salinity. Increase of Ca 2+ (++Ca) during plant growth in the presence of 75 mm NaCl recovered only partially the content of the nutrients in nodules. Only nodules that developed in salt stress conditions and with a supplement of both nutrients (++B ++Ca treatments) had a content of B and Ca 2+ similar to the control (without salt) nodules. Effects of Ca 2+ and B on early pre-infection events under salt stress As a result of the previous observations, the effects of 75 mm NaCl and different B and Ca 2+ concentrations on Rhizobium pea early interactions were analysed. Figure 1 shows the capacity of exudates derived from roots of plants Table 2. Boron and calcium contents of nodules harvested 3 weeks post-inoculation with Rhizobium leguminosarum bv. viciae 3841 from Pisum sativum L. cv. Argona plants grown with 75 mm NaCl and different concentrations of B (+B, 9.3 mm; ++B, 55.8 mm) and Ca 2+ (+Ca, 0.68 mm; ++Ca, 2.72 mm) +B +Ca +B ++Ca ++B +Ca ++B ++Ca B (mg g -1 dry weight) 14.3 ± ± ± ± 3.8 Ca 2+ (mg g -1 dry weight) 3.1 ± ± ± ± 2.7 Boron in nodules control (+B +Ca, without NaCl), 53.5 ± 4.4 mg B g -1 dry weight; calcium in nodules control, 9.3 ± 2.1 mg Ca 2+ g -1 dry weight. Figure 1. Capacity of methanolic extracts of root exudates derived from Pisum sativum cv. Argona plants growing in the absence or in the presence of 75 mm NaCl and different concentrations of B and Ca 2+ to induce nod genes (measured as b-gal activity in Rhizobium leguminosarum biovar viciae D24 containing a nodabcijp::lacz fusion). Control, NaCl +B +Ca; +B, 9.3 mm B; ++B, 55.8 mm B; +Ca, 0.68 mm Ca 2+ ++Ca, 2.72 mm Ca 2+.

4 1006 A. El-Hamdaoui et al. Figure 2. Rhizobium leguminosarum bv viciae 3841 adsorption capacity to roots from Pisum sativum cv. Argona plants growing in the absence or in the presence of 75 mm NaCl and different concentrations of B and Ca 2+. Roots were incubated for 2 h in FP medium with bacterial suspensions (10 3 cells ml -1 ), washed and plated on TY-agar. Adsorption capacity was measured as bacterial microcolonies developed on roots after 48 h. Control, NaCl +B +Ca; +B, 9.3 mm B; ++B, 55.8 mm B; +Ca, 0.68 mm Ca 2+ ++Ca, 2.72 mm Ca 2+. developed with the different treatments to induce nod gene activity (measured as nodabcij promoter activity). Extracts derived from plants growing with salt did not show an induction capacity that was significantly reduced by salt. The Ca 2+ and B levels did not affect nod gene induction. The results were similar when incubations of bacteria with root exudates were made in TY media containing 75 mm NaCl (data not shown). The capacity of adsorption of bacteria to root was also investigated (Figs 2 & 3). Two kinds of experiments were run. The first experiments (Fig. 2) were designed to study the effect of plant growth conditions on bacterial binding to roots. Roots from plants developed with or without salt and with different B and Ca 2+ concentrations were incubated in FP medium with suspensions of R. leguminosarum 3841 cells (see Material and Methods section). Bacterial attachment to the root surface was clearly inhibited in plants developed with salt. Although the increase of B during the growth of pea plants in the presence of 75 mm NaCl recovered partially bacterial adsorption, only ++ Ca roots showed a bacterial binding similar than control roots. To learn more about the effects of different concentrations of salt, B, and Ca 2+ in the incubation mixture, roots from plants grown in the absence (Fig. 3A) or in the presence (Fig. 3B) of 75 mm NaCl and normal B and Ca 2+ levels were incubated with strain 3841 in FP media with or without salt and different B and Ca 2+ concentrations. The presence of salt during incubations did not alter the adsorption capacity of bacterial cells to the root surface (compare FP + salt with FP bars in Fig. 3a & b). After addition of B but not of Ca 2+ (FP ++B treatments) to the incubation media there were no changes in the phenomenon of adsorption in the presence or in the absence of salt. Only the addition of extra Ca 2+ (FP ++Ca treatments) increased the bacterial attachment to roots, which was similar in roots derived from plants grown with or without salt (highlighted by an asterisk). Figure 3. Effects of the addition to the incubation mixture of salt (75 mm NaCl), 55.8 mm B (++B) or 2.72 mm Ca 2+ (++Ca) on Rhizobium leguminosarum bv viciae 3841 adsorption capacity to roots from Pisum sativum cv. Argona plants grown in the absence (A) or in the presence (B) of 75 mm NaCl and +B (9.3 mm B) + Ca (2.72 mm Ca 2+ ) concentrations. Roots were incubated for 2 h in FP medium with bacterial suspensions (10 3 cells ml -1 ), washed and plated on TY-agar. Adsorption capacity was measured as bacterial microcolonies developed on roots after 48 h. Asterisk highlights a similar Ca 2+ -mediated enhancement of adsorption to plants grown with or without salt.

5 B and Ca mediated recovery of nodulation and nodule development under salt stress 1007 Effects of Ca 2+ and B on rhizobial infection and nodule invasion In addition to the attachment of Rhizobium to the root surface, proliferation of bacteria is needed to infect the root hair and invade nodule tissues. Figure 4B shows that R. leguminosarum 3841 did not grow in Y minimal media containing 75 mm NaCl and 0.68 mm Ca 2+, the concentration of Ca 2+ used for plant growth (+Ca treatments). Bacterial growth was only recovered when Ca 2+ concentration was increased to ++Ca treatments levels (2.72 mm Ca 2+ ). In the absence of salt, bacteria grew with both concentrations of Ca 2+ (Fig. 4A). The level of B did not affect bacterial growth in the presence or in the absence of salt (data not shown). Because the supplement of B is needed for nodule function under salt stress, infection thread development and tissue invasion under salt stress and different Ca 2+ and B concentrations were investigated by epifluorescence microscopy. For the experiments, plants were inoculated with R. leguminosarum 3841 carrying a rhizosphere-stable plasmid that constitutively expresses the GFP. Control (without salt treatments) inoculated roots showed typically curled root hairs with infection threads containing green fluorescent bacteria (Fig. 5A). In contrast, root hairs of salttreated plants with +B +Ca treatments appeared swollen at the tip region (Fig. 5B) and the number of curled root hairs and infection threads developed was highly diminished compared with control treatments (Table 3). The increase of Ca 2+ did not prevent root hair swelling (Fig. 5C) nor infection thread development (Table 3, +B ++Ca treatments). The increase of B could avoid swelling (Fig. 5D) but root hair curling and infection thread development were still inhibited (Table 3, ++B +Ca treatments). Only ++B ++Ca treatments showed infection structures similar to those from control roots (Fig. 5E and Table 3 ++B ++Ca treatment). Similar to the infection process, the phenomenon of endocytosis leading to nodule invasion was completely inhibited by salt. In invaded cells of nodules that developed in the absence of salt, GFP-labelled bacteria were observed in the infected zone (Fig. 5F). In contrast, nodules that developed in the presence of 75 mm NaCl were smaller and appeared to lack normal invaded cells (Fig. 5G). When it appeared, all the green fluorescence was localized in an enlarged network of infection structures inside the nodule (Fig. 5G, arrows). The increase of Ca 2+ or B in salt-treated plants led to larger nodules, that were still uninvaded (Fig. 5H & I). Again, only the supplement of Figure 4. Inhibition by salt (75 mm NaCl) and recovery by Ca 2+ addition (+Ca, 0.68 mm Ca 2+ ++Ca, 2.72 mm Ca 2+ ) of Rhizobium leguminosarum bv viciae 3841 growth on liquid Y minimal medium. (A) growth without salt; (B) growth with 75 mm NaCl. both B and Ca 2+ promoted nodule invasion in salt-stressed plants (Fig. 5J). DISCUSSION There are several reports that demonstrate that salinity is one of the most important stresses affecting rhizobiallegume symbiosis (see Zahran 1999; and refs. therein). Salinity inhibited nodulation and nitrogen fixation in Pisum sativum cv. Argona inoculated with Rhizobium leguminosarum strain 3841 (Table 1), indicating that this symbiosis is extremely sensitive to salt. This study also showed that salt stress altered nodule development at pre-infection (Fig. 2), infection (Fig. 4) and Table 3. Effects of different concentrations of B (+B, 9.3 mm; ++B, 55.8 mm) and Ca 2+ (+Ca, 0.68 mm; ++Ca, 2.72 mm) on root hair curling and infection thread development of Pisum sativum L. cv. Argona plants growing with 75 mm NaCl 3 weeks post-inoculation with Rhizobium leguminosarum bv. viciae B +Ca +B ++Ca ++B +Ca ++B ++Ca Curled root hairs 7 ± 5 16 ± 12 8 ± ± 38 Infection threads 4 ± 4 8 ± 3 5 ± ± 24 Curled root hairs of control plants (+B +Ca, without NaCl), 197 ± 46; infection threads, 152 ± 43.

6 1008 A. El-Hamdaoui et al. Figure 5. Epifluorescence micrographs of root hairs (A E) or nodule sections (F J) from Pisum sativum cv. Argona plants, 6 d and 3 weeks post-inoculation with Rhizobium leguminosarum 3841 carrying the plasmid phc60 that constitutively expresses green fluorescent protein (GFP). (A, F), Plants growing without NaCl and +B +Ca concentrations; (B, G), plants growing with 75 mm NaCl and +B +Ca concentrations; (C, H), plants growing with 75 mm NaCl and +B ++Ca concentrations; (D, I), plants growing with 75 mm NaCl and ++B +Ca concentrations; (F, J), plants growing with 75 mm NaCl and ++B ++Ca concentrations. Arrows indicate green fluorescence inside infection threads (it). m, nodule meristem. +B, 9.3 mm B; ++B, 55.8 mm B; +Ca, 0.68 mm Ca 2+ ++Ca, 2.72 mm Ca 2+. Bar markers: 0.5 mm. invasion (Fig. 5) stages. Legume symbioses have a high requirement for B and Ca 2+ (Carpena et al. 2000), and the levels of both nutrients influence nodulation and nodule function (Bolaños et al. 2002). Given that salinity diminishes the concentration of B and Ca 2+ in nodules, it can be postulated that the addition of external amounts of these nutrients could prevent the negative effects of salt on rhizobial symbioses, as demonstrated in this study. The rhizosphere environment significantly influences the symbiotic interaction between Rhizobium and the host legume. Favourable conditions for the establishment of bacterial populations and for plant growth enhance the inoculum potential and promote the development of a healthy infection site on root hairs, respectively (Tu 1981). Therefore, any factor altering bacterial or plant growth will affect rhizobial infection and nodulation. The results pre-

7 B and Ca mediated recovery of nodulation and nodule development under salt stress 1009 sented here show that 75 mm NaCl inhibited nodulation by almost 100% of the control (Table 1). This is typical for other legume symbiosis such as Vigna radiata (Hafeez, Aslam & Malik 1988) or Cicer arietinum (El-Sheikh & Wood 1990). The addition of Ca 2+ to 2.72 mm increased nodulation to about 60% of the control (Table 1, ++Ca treatments). Calcium was found to be involved in the increase of nodulation by Lowter & Loneragan (1968), and in early infection events in Medicago sativa by Munns (1970). Clarifying the role of Ca 2+ in nodulation, Richardson et al. (1988) reported that exudates obtained from clover seedlings growing with a high Ca 2+ supply had increased nod gene induction activity. However, the capacity of root exudates to induce R. leguminosarum D24 nod genes was not significantly diminished by salt or increased by calcium in our study, and therefore, cannot explain the inhibition of nodulation by salt and recovery by Ca 2+ (Fig. 1). In addition to the exchange of diffusible signals, the adsorption of rhizobia to roots is needed to initiate nodule formation. Data from adsorption experiments (Figs 2 & 3) indicate that the level of bacterial attachment is reduced in roots grown under saline conditions. This could partially explain the reduction of nodulation observed in saltstressed pea plants. The increase of Ca 2+ to 2.72 mm, at either concentration of B, led to roots with a higher adsorption potential (Fig. 2, ++Ca treatments) independent of the presence of salt during growth. This legume Rhizobium attachment is mediated by plant and bacterial components able to use Ca 2+ as a ligand to reinforce the interaction. Calcium increases the secretion of mucilage in roots (Bennet, Breen & Bandu 1990) in which cellulose fibrils can entangle the bacteria. Calcium ions can also strengthen the activity of plant lectins or rhizobial Ca 2+ -dependent ricadhesins (Smit et al. 1989). Moreover, bacterial exopolysaccharide (EPS) can form a gel in the presence of cations as Ca 2+, being a non-specific mechanism for rhizobial attachment (Morris et al. 1989). Given that the content of Ca 2+ in pea roots is also reduced by salt stress (El-Hamdaoui et al. 2003), the absence of this cation on the root surface necessarily will reduce the adsorption capacity of bacteria to roots and hence root colonization. Moreover, the decrease of Ca 2+ content by salt stress can alter the root surface due to a weak package of cell wall components, mainly the pectin fraction corresponding to polygalacturonic acid with capacity to bind to Ca 2+ (Liners et al. 1989). The addition of high concentrations of Ca 2+ (Fig. 3 FP ++Ca) to the mixtures during root bacteria incubation was enough to increase bacterial adsorption, even in plants grown under salt stress and with +Ca levels (Fig. 3B, FP ++Ca). This suggests that the presence of calcium cations on the root surface at the moment of bacterial attachment is imperative to promote the interaction, possibly by the sticking properties that Ca 2+ confers to ricadhesins and/or bacterial EPS. Growth of rhizobia in the presence of +Ca levels was inhibited by salt, and only recovered when Ca 2+ concentration was increased (Fig. 4). The effect of salinity on bacterial cells influenced nodulation in two ways. The outer membrane of rhizobial cells was highly disorganized under salt stress (Zahran et al. 1997), leading to a decrease of the capacity of Rhizobium to attach to root surfaces. Moreover, bacteria attached to the root surface of pea plants growing in the presence of 75 mm NaCl and +Ca treatments cannot proliferate due to growth inhibition. Although the capacity of root exudates to induce nod genes was not reduced by salt, root hairs of inoculated saltstressed pea plants did not show the characteristic effects of Nod factors production, namely root hair deformation and curling (Fig. 5B; Table 3). Most of the root hairs appeared extremely swollen, a typical effect of salt stress also reported by Tu (1981) in soybean plants. Neither Ca 2+ nor B addition prevented this effect (Fig. 5c & d; Table 3). Only the increase of both B and Ca 2+ (++B ++Ca treatments) concentrations was able to revert the effect of salt on root hairs (Fig. 5E; Table 3). Changes in intracellular Ca 2+ in the root hair cytoplasm are early responses of legumes to the application of Nod factors (Cárdenas et al. 1999). This induces rearrangements of the cytoskeleton that lead to root hair curling (Cárdenas et al. 1998) and to polar deposition of wall material to initiate infection thread formation (Lloyd et al. 1987). Therefore, the decreased concentration of Ca 2+ in salt-treated roots must be recovered by the addition of the nutrient to elicit plant response to Nod factors. However, B concentration in pea roots is also diminished by salt, and the absence of the micronutrient might alter the cell wall plasma membrane cytoskeleton continuum. In support of this idea, an increased amount of actin and tubulin in response to B deprivation has been reported (Yu et al. 2001). Therefore, the decrease of B in salt-stressed root hairs could interfere with the cytoskeleton rearrangements during root hair curling and infection thread development. This might explain why not only Ca 2+ but also B addition is needed for Nod factor-induced root hair curling in pea plants growing in the presence of 75 mm NaCl. Following nodule development, salt stress inhibited bacterial invasion and proliferation inside the host cells (Fig. 5G). As 75 mm NaCl inhibited R. leguminosarum 3841 growth in +Ca media (Fig. 4), this effect could be due to a lack of bacterial proliferation after inoculation of plants in the presence of such NaCl concentration. However, although calcium addition was able to enhance 3841 growth in saline media (Fig. 4, ++Ca treatments), nodules were still uninvaded when the concentration of Ca 2+ during plant growth with salt was increased (Fig. 5H). This could be due to the continuing low content of Ca 2+ in these nodules (Table 2). Moreover, boron is required for bacterial plant cell surface interactions that elicit the endocytosis-like invasion process (Bolaños et al. 1996), and B-deficient nodules develop enlarged infection threads that are prematurely aborted prior to bacterial invasion (Redondo-Nieto et al. 2001), showing an appearance similar to salt-stressed pea nodules. However, the increase of B did not enhance invasion either (Fig. 5I). Table 2 shows that, similar to Ca 2+ the content of B in salt-stressed nodules was still low in ++B +Ca treatments, compared with the control. Only the

8 1010 A. El-Hamdaoui et al. addition of both nutrients during plant growth, recovered the content of each nutrient in nodules (Table 2, ++B ++Ca treatments). Therefore, only nodules induced in ++B ++Catreated, salt-stressed plants had enough available B to allow rhizobial invasion and enough Ca 2+ to enhance bacteria proliferation (Fig. 5J). There are several studies that indicate a role of calcium in the adaptation of plants to salt stress (Hasegawa et al. 2000; and refs. therein). Among other reported responses, the increase of Ca 2+ was able to partially re-equilibrate the nutrient balance (i.e. K +, Na + ) altered by salinity (Cramer et al. 1987). However, the decrease of B detected in pea nodules was not counteracted by an increase of Ca 2+. Boron is required at relatively high concentrations for nodule development, as demonstrated by its higher content, about four to five times higher in nodules than in roots (Carpena et al. 2000), and its influence on different stages of nodule development. The inhibitory effects of poor B nutrition on legume development are more evident in plants that rely of symbiotic N 2 -fixation for nitrogen than in plants that received nitrogen in mineral form, due to the lack of nitrogen in addition to B deficiency (Bolaños et al. 1994). Therefore, the results presented herein suggest that the increase of Ca 2+ concentration is not enough to induce tolerance to salinity in legumes when plants grow in symbiosis with Rhizobium, because B is a limiting factor for nodule development under salt stress. Only an increase of B accompanying Ca 2+ concentration would increase the salt tolerance in the establishment and development of the symbiotic relationship. 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