NITROGEN FIXATION UNDER PHYSIOLOGICAL AND SALT-STRESSING CONDITIONS
5. B-Ca RELATIONSHIP IN THE ADAPTATION OF LEGUME SYMBIOSIS TO SALINITY
5. B-Ca RELATIONSHIP IN THE ADAPTATION OF
and Bohlool, 1984). Salinity can also indirectly affect the symbiosis by reducing the growth of the host plant.
5.2. B-Ca interaction in legume-rhizobia symbiosis under salt stress
Osmotic stress or ionic imbalance may cause disorders in almost any physiolog-ical process, and mechanisms of salt-tolerance in plants are genetphysiolog-ically determined (Hasegawa et al., 2000; Zhu, 2001). Therefore, the selection of host legume geno-types that are tolerant to high salt conditions is important to determine the success of the rhizobial symbiosis (Cordovilla et al., 1995; Velagaleti and Marsh, 1989).
Complementarily, the study of the interaction among nutrients that are espe-cially required for nodulation, such as boron and calcium, with salt is important to optimise the conditions for salt tolerance of inoculated legumes. As cited above, Ca2+ deficiency is a typical feature of salt stress, and a reduction of B concentra-tion due to salinity has also been reported (El-Motaium et al., 1994). Therefore, the legume nodule might be especially affected by this nutrient imbalance. The addition of Ca to legumes grown under high NaCl concentrations had positive effects on nitrogen fixation (Akhavan-Kharazian et al., 1991), however, a wide study of the interaction B-Ca related to salt tolerance is still to be developed, in spite the importance of the B/Ca relationship.
5.2.1. Effects of salt stress on growth, nodulation and nitrogen fixation of 5.2.1. symbiotic pea plants
The limiting salt level for pea (Pisum sativum cv. Argona) and its host bacteria Rhizobium leguminosarum bv. viciae strain 3841 was 75 mM NaCl (El-Hamdaoui, 2002). At that salt concentration, the development of plants was severely dimin-ished. Nodulation, measured as number of nodules, and nitrogen fixation, measured as acetylene reduction activity (ARA), were almost completely inhibited by 75 mM NaCl. As a result of low nitrogen fixation, the N-content of salt treated plants was also lower than in control (without salt) plants. These results indicate that nitrogen-fixing pea plants are very salt-sensitive.
Nodules from pea roots developed in the absence of salt presented the typical structure of indeterminate nodules. This included a meristematic tip region with small cells and large regularly shaped cells from the central to the root zone of the nodule. By contrast, nodules developed under salt stress appeared with a very altered structure, without tissue differentiation, and cells were very irregularly shaped.
5.2.2. Effects of B and Ca nutrition on the development and nitrogen fixation of 5.2.2. salt treated symbiotic pea plants
Growth of inoculated pea plants growing under salt stress can be enhanced by an adequate nutrition of B and Ca2+. Increases of Ca concentrations recovered par-tially plant development, as typically reported (LaHaye and Epstein, 1971). However,
combination of increased B and high Ca concentrations produced the best recovery effects on shoot and root development in plants grown in saline media.
Nodulation and N2fixation of plants grown under salinity could also be recov-ered by modifications of B and Ca. The increase of Ca increased the amount of nodules per plant, and that was an effect independent of the concentration of B.
However, Ca was not enough to restore nitrogen fixation, and the addition of extra B was essential to partially recover nodule function.
The study of the structure of nodules developed under high salt and with different B and Ca treatments also shows a beneficial effect of the addition of extra B and extra Ca to the growth media. Compared with salt stressed nodules in media with normal B and Ca levels, the increase of the concentrations of both nutrients resulted in a recovery of the structure of the nodules. Moreover, salt-stressed nodules appeared devoid of rhizobia, and only the addition of both nutrients enhances
Therefore, Ca increases nodulation during growth of plants under salt stress and B is needed to enhance bacterial invasion and differentiation of N2-fixing sym-biosomes.
Figure 11. Effects of salt stress and increased levels of B and Ca on the development of Pisum sativum nodulated plants.
5.2.3. Mineral composition of salt stressed plant grown under different B and 5.2.3. Ca levels
One of the effects of the addition of B and Ca on the increase of salt tolerance of nodulated pea can be due to the maintenance of the nutrient balance. As typically occurs, exposure of plants to salinity led to a massive entry of Na+ and Cl–, which is indicated by a high concentration of both ions in shoots and nodulated roots.
Besides these toxic levels of Na+ and Cl–, one of the major constraints for plant growth on saline substrates is nutrient imbalance.
In nodulated pea, the measurement of B indicated that salinity provokes a defi-ciency of the micronutrient. Although Ca is able to recovers nutrient defidefi-ciency under salt stress (Cramer et al., 1987), it cannot recover the content of B in nodulated roots.
Therefore, the increase of B is imperative. These measurements of nutrient content together with the importance of B and Ca in the development of the symbiosis justifies the alterations by high salt and the increase of salt tolerance in plants growing with a supplement of both nutrients.
Furthermore, not only B and Ca, but also some other nutrients (P, Mg, Mn, Cu . . .) are affected by high salt and recovered by B and Ca. Specially important for symbiotic N2fixation in legumes are potassium and iron. Potassium has a role in plant-water relations; it is the cation with a major contribution to the osmotic potential of cells in nonhalophytic plants (Hsiao and Läuchli, 1986). Functions of K+in higher plants include cell movements, cell extension, nutrient transport, cation-anion balance, and activation or stimulation of a large number of enzymes (see Marschner, 1995). Moreover, it has been shown that the symbiotic systems are more sensitive to low K than are the legumes themselves (Sangakkara et al., 1996), and a depression in the K+ content at high salt levels is also typically detected.
Therefore, the effects of K deficiency in these roots can be in part responsible for the low nitrogenase activity. The addition of Ca, especially at high B treatments, restored the amount of K+ in nodulated plants.
Finally, salinity also reduced the concentration of Fe in nodulated roots, which is particularly recovered by 6B/4Ca treatments. A particular high requirement of iron exists in legumes not only for the nitrogenase complex, but also for the heme component of leghemoglobin and for the cytochrome oxydase of bacteroid electron transport chain (O’Hara et al., 1988). The iron content of roots of all of treatments
Table 6. Effect of different B (+B = 9.3 µM; +6B = 55.8 µM) and Ca2+(+Ca = 0.68 mM; +4Ca = 1.36 mM) concentrations on nitrogen fixation (expressed as nmol C2H2plant–1h–1), nodulation (nodules per plant), and growth of Pisum sativum plants grown in the presence of 75 mM NaCl 4 weeks post-inoculation with Rhizobium leguminosarum.
Control +NaCl +NaCl +NaCl +NaCl
(–NaCl) +B +Ca +B +4Ca +6B +Ca +6B +4Ca
Nitrogenase 217.4 ± 46.1 2.7 ± 1.9 1.8 ± 1.7 6.2 ± 2.4 138.2 ± 33.7
Nodules .4067± 23 0.5± 3 .38± 16 0.4± 4 0.246± 14
g (fw) shoot 002.3± 0.5 0.6 ± 0.2 0.8 ± 0.3 0.7 ± 0.3 001.9± 0.5 g (fw) root 001.7± 0.4 0.4 ± 0.2 0.4 ± 0.1 0.5 ± 0.2 001.6± 0.5
of salt-stressed plants is in the critical range of deficiency, 50–150 µg Fe g–1 dry weight, except nodulated roots of 6B/4Ca treatments.
Therefore, besides the recovery of nodule development by B and Ca, addition of both nutrients can prevent salt stress on nitrogen fixation in legume-rhizobia sym-biosis by counteracting the effects of salt on nutrient balance.
In the system studied of P. sativum cv. Argona inoculated with R. leguminosarum, always a combination of 6 times increase of B and 4 times increase of Ca during plant growth was the best to increase salt tolerance. Other different treatments have only very small increases of tolerance or even inhibited plant and symbiosis development more than salt itself (i.e. B concentrations higher than 6 times normal were very toxic for plant growth). Consequently, as occur under physiological con-ditions, it might be an equilibrated nutritional status regarding B and Ca that produced the highest possible plant growth under salt stress. This status of equi-librium may change when the plant or the stressing-factor that affects plant nutrition changes and the use of a different B/Ca level should allow achieve it and again increase tolerance to the stress.