Proteolysis and nitrogen fixation in faba-bean (Vicia faba) nodules under water stress

Nodulated faba-beans ( Vicia faba L. var. minor) exhibiting high rates of N₂ fixation (133 μmol C₂H₄ g⁻¹ dry weight h⁻¹), were subjected to water restriction. A loss of C₂H₂ reduction due to water stress was always associated with a decline of the leghemoglobin content for each of the 4 decreasing values of Ψ_mod. Electron micrographs showed ultrastructural alterations of the fixing tissue, which affected both partners and increased with the severity of water stress. In the nodule cytosol, the alkaline proteolysis approximately doubled when Ψ_mod decreased from −0.55 MPa to −1.55 MPa. Concomitantly, an increase of the nodule intracellular pH from 6.3 to 7.0 was observed. Proteolysis was due to serine proteases, exhibiting a pH-optimum of 8 and which actively degraded purified leghemoglobin in vitro (K_m=100 μ M ). The degradation of leghemoglobin during water stress may contribute to the loss of C₂H₂ reduction and may affect the pattern of recovery upon rewatering.     

Influence of host genotypes on growth,symbiotic performance and nitrogen assimilation in faba bean (Vicia faba L.) under salt stress

Fifteen genotypes of faba bean (Vicia faba L.) were inoculated with salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA 19 in solution culture with 0 (control) and 75 mM NaCl added immediately after transplanting. Genotypes varied in their tolerance of high levels of NaCl. Physiological parameters (dry weight of shoot and root, number and dry weight of nodules) were not affected by salinity in lines VF46, VF64 and VF112. Faba bean line VF60 was sensitive to salt stress. Host tolearance appeared to be a major requisite for nodulation and N₂ fixation under salt stress. Tolerant line VF112 sustained nitrogen fixation under saline conditions. Activity of the ammonium assimilation enzymes glutamine synthetase and glutamate synthase, and soluble protein content, were reduced by salinity in all genotypes tested. Evidence presented here suggests a need to select faba bean genotypes that are tolerant to salt stress.Abbreviations ARA acetylene reduction activity - NADH-GOGAT NADH-dependent glutamate synthase - GS glutamine synthetase     

Nitrate metabolism in roots and nodules of Vicia faba in response to exogenous nitrate

Activities of nitrate reduction enzymes, nitrate reductase activity (NRA) and nitrite reductase activity (NiRA) from roots and nodules of 5 mutant genotypes and one commercial cultivar (Alameda) of faba bean ( Vicia faba L. var. minor) grown in the presence of N₂ alone or with additional NO⁻₃ in the medium have been studied. A naturally occurring mutant (VFM109) with impaired ability to reduce nitrate in its nodules is described. All the other cultivars of V. faba showed nodule NRA, although the range was very wide, from almost negligible (VFM72) up to 2 μmol h⁻¹ (g FW)⁻¹. This activity was entirely of plant origin. Root NRA also ranged widely accross cultivars. However, the level of activity expressed as well as the response of NRA to nitrate followed a pattern opposite to that observed in nodules. Roots and nodules of all cultivars showed very high rates of NiRA, respectively 50 and 150-fold higher than NRA, thus precluding accumulation of nitrite in these tissues. Root enzymes were significantly stimulated by nitrate while negative (NRA) or little effect (NiRA) was found for nodules. Nitrate and nitrite reduction are carried out by inducible enzymes in roots of V. faba and by constitutive enzymes in nodules, indicating that there may be different forms of these enzymes in each tissue. Differences in the plant genotype were a major cause of the variability in nitrate and nitrite reduction by nodulated root systems of V. faba .     

Proline accumulation inside symbiosomes of faba bean nodules under salt stress

A two‐week salt treatment (NaCl, 100 m M ) induced a 50% inhibition of acetylene reduction activity (ARA) of faba bean ( Vicia faba L. var. minor cv. Soravi) nodules, associated with a large increase in the nodule pool of amino acids. The concentration of proline in the different nodule compartments was determined after calculating their respective volumes from their areas on electron micrographs. The proline concentration exhibited a large increase, especially in the cytosol where its amount was 8‐fold enhanced under salt stress, whereas the low proline content of bacteroids was less affected. Increase of proline concentration in faba bean nodules subjected to salt stress was correlated with an enhancement of the cytosolic Δ¹‐pyrroline‐5‐carboxylate synthetase (EC 2.7.2.11 + EC 1.2.1.41; P5CS) activity. Experiments with purified symbiosome preparations showed that the greatest proline content occurred in the peribacteroid space (PBS), where proline was the most abundant amino acid, with a concentration reaching 15.3 m M under salt stress. Proline accumulation in the PBS resulted both from a diffusive transport from the host cell to the symbiosomes through the peribacteroid membrane (PBM) and from the very low rate of uptake by faba bean bacteroids. This accumulation could be partly responsible for the 1.7‐fold enlargement of the symbiosome volume observed in salt‐stressed nodules. In incubations of bacteroids, isolated from salt‐stressed or unstressed plants and supplied with O₂ by purified oxyleghemoglobin, addition of proline stimulated neither O₂ consumption nor ARA. These results were consistent with proline playing a role as osmoticum, rather than energy source for bacteroid N₂ fixation in amide‐exporting legumes such as faba bean.     

The response of Vicia faba to enhanced UV-B radiation under low and near ambient PAR levels

The effects of enhanced UV-B are often overestimated in greenhouse studies due to low levels of photosynthetically active radiation (PAR). For this reason, we studied effects of enhanced UV-B (12 kJ m^–2 d^–1) at low and near ambient PAR levels on young vegetative plants of Vicia faba, in the greenhouse. It was hypothesized that near ambient PAR levels could reduce the negative UV-B effects on growth, due to higher amounts of UV-B absorbing compounds in the leaves and to morphological changes attenuating UV-B damage.We found that effects of enhanced UV-B on the growth were not negative. We found an increase in biomass in response to enhanced UV-B at low and near ambient PAR levels. The increase in biomass was related to increased branching, which leads to a higher interception of PAR. Enhanced irradiance of both PAR and UV-B had similar photomorphogenic effects: thicker and smaller leaves and reduced plant height and internode length. Moreover, the concentration of UV-B absorbing compounds was increased. We conclude that in this study effects of enhanced UV-B were mainly photomorphogenic effects, which were also induced by radiation in the PAR region.     

Ion‐dependent metabolic responses of Vicia faba L. to salt stress

Salt‐affected farmlands are increasingly burdened by chlorides, carbonates, and sulfates of sodium, calcium, and magnesium. Intriguingly, the underlying physiological processes are studied almost always under NaCl stress. Two faba bean cultivars were subjected to low‐ and high‐salt treatments of NaCl, Na₂SO₄, and KCl. Assimilation rate and leaf water vapor conductance were reduced to approximately 25–30% without biomass reduction after 7 days salt stress, but this did not cause severe carbon shortage. The equimolar treatments of Na⁺, K⁺, and Cl^? showed comparable accumulation patterns in leaves and roots, except for SO₄^2? which did not accumulate. To gain a detailed understanding of the effects caused by the tested ion combinations, we performed nontargeted gas chromatography–mass spectrometry‐based metabolite profiling. Metabolic responses to various salts were in part highly linearly correlated, but only a few metabolite responses were common to all salts and in both cultivars. At high salt concentrations, only myo‐inositol, allantoin, and glycerophosphoglycerol were highly significantly increased in roots under all tested conditions. We discovered several metabolic responses that were preferentially associated with the presence of Na⁺, K⁺, or Cl^?. For example, increases of leaf proline and decreases of leaf fumaric acid and malic acid were apparently associated with Cl^? accumulation.     

Heme oxygenase up-regulation under salt stress protects nitrogen metabolism in nodules of soybean plants

The behaviour of enzymes involved in nitrogen metabolism, as well as oxidative stress generation and heme oxygenase gene and protein expression and activity, were analysed in soybean (Glycine max L.) nodules exposed to 50, 100 and 200 mM NaCl concentrations. A significant increase in lipid peroxidation was found with 100 and 200 mM salt treatments. Moreover, superoxide dismutase, catalase and peroxidase activities were decreased under 100 and 200 mM salt. Nitrogenase activity and leghemeoglobin content were diminished and ammonium content increased only under 200 mM NaCl. At 100 mM NaCl, glutamine synthetase (GS) and NADH-glutamate dehydrogenase (GDH) activities were similar to controls, whereas a significant increase (64%) in NADH-glutamate synthase (GOGAT) activity was observed. GS activity did not change at 200 mM salt treatment, but GOGAT and GDH significantly decreased (40 and 50%, respectively). When gene and protein expression of GS and GOGAT were analysed, it was found that they were positively correlated with enzyme activities. In addition, heme oxygenase (HO) activity, protein synthesis and gene expression were significantly increased under 100 mM salt treatment. Our data demonstrated that the up-regulation of HO, as part of antioxidant defence system, could be protecting the soybean nodule nitrogen fixation and assimilation under saline stress conditions.     

The effect of salinity on N fixation and assimilation in Vicia faba

Production of the faba bean in semi-arid and coastal areas maybe limited by the salt sensitivity of faba bean symbiosis. Accordingly,this study was done to analyse the effects of salt on the effectivesymbiosis of faba bean (Vicia faba L. var. minor cultivar Alborea)and salt-tolerant Rhizobium leguminosarum biovar. viciae strainGRA19. After 4 weeks of growth, the nutrient solutions weresupplemented with 50, 75 and 100 mM NaCl for 21 d. Plants wereharvested four times at weekly intervals, beginning at 4 weeks.Vicia faba tolerated low (50 mM NaCl) but not higher levels(75 and 100 mM NaCl) of salt stress. Salinity affected shootgrowth more than root growth. At the end of the culture, thetotal nitrogen content in the shoot was affected more than plantgrowth; conversely, in the root, growth was influenced morethan total nitrogen content. In nodules, nitrogen fixation (acetylenereduction activity) was more sensitive to salinity than ammoniumassimilation (glutamine synthetase and glutamate synthase). Key words: Glutamate synthase, glutamine synthetase, N₂ fixation, Rhizobium leguminosarum, salinity     

Genetic changes under domestication in Vicia faba

Summary The components of variation within each one of two sets of landraces and/or cultivars of Vicia faba, respectively constituted of primitive and advanced morphological types, were studied by means of two sets of 8 × 8 diallel crosses with two repetitions. The results show that primitive and modern forms differ from each other in both the intensity and the kind of selective pressures acting on them, mainly on those characters more modified through the domestication process: i.e., seed morphology and the number of flowers per node. Because of the paramount importance of the additive component in the primitive forms, it is suggested that the most important type of selection on them is the stabilizing one. On the contrary, in the most advanced forms the selection is directional and disruptive : directional towards greater yields, and disruptive separating two morphological types, major and equina. The plant response to these different selective pressures has been to modify the genetic control of different characters: thus the primitive forms generally show only additivity while the most advanced forms show additivity as well as directional and asymmetrical dominance.     

Growth Substances in the Roots of Vicia faba

An investigation has been made into the growth regulators presentin ethanol extracts of the seedling roots of Vicia faba afterseparation on paper partition chromatograms, using segmentsof Avena coleoptiles and mesocotyls and of Pisum sativum.rootsas biological assay material. Acetonitrile purification shows the presence of at least threeauxins running in isobutanol: methanol: water, at Rfs of 0–0·25,0·4–0·6, and 0·65–0·95;the latter may represent two different auxins. A similar, butclearer, picture is shown by the ether-soluble acid fraction.Here an auxin at Rf 0–0·25 also stimulates rootgrowth and could be ‘accelerator ’. A second atRf 0–0·25 is an indole compound which inhibitsroot growth and does not seem to be be IAA. A third at Rf 0·8–1·0is also a root-growth inhibitor and gives no indole reaction.The ‘inhibitor ß’ complex was demonstrated(Rf 0·65–0·85) together with a number ofother inhibitors at lower Rf value. The ether-soluble neutral component also contains auxins orauxin precursors. The water-soluble, ether-insoluble fraction contains four readilyinterconvertible substances with auxin properties. They allappear to inhibit root growth and give no indole reaction.     

Bacteroid oxalate oxidase and soluble oxalate in nodules of faba beans (Vicia faba L.) submitted to water restricted conditions: possible involvement in nitrogen fixation

The inhibitory effect exerted by water stress on acetylene reductionactivity (ARA) by nodulated roots of faba beans (Vicia fabaL.) was correlated with a 40% decline in the organic acid poolof nodule cytosol. Oxalate concentration was lowered (–55%)whereas a stimulation of the bacteroid oxalate oxidase concomitantlyoccurred. This enzyme was characterized by an optimal activityat pH 8 but, as in higher plants, exhibited a K_m for oxalateof 1.4 mM and an inhibition by substrate excess. Oxalate providedto bacteroid incubations supported C₂H₂ reduction up to 2.5mM whereas higher concentrations were strongly inhibitory. Incontrast, purified symbiosomes incubated with oxyleghaemoglobinreduced C₂H₂ in the presence of oxalate concentrations up to10 mM. The peribacteroid membrane (PBM), in controlling theoxalate flux to the bacteroids avoided the substrate inhibitionwhich would limit its efficiency. Thus, oxalate present in highconcentration in faba bean nodules could play a role as complementarysubstrate for bacteroids slowing down the nitrogen fixationdecline induced by water restricted conditions. Key words: Faba bean, water stress, oxalate, acetylene reduction, bacteroid     

Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.     

The influence of nitrate supply on nitrogen fixation during growth of the field bean Vicia faba in sand

Field bean (Vicia faba L.) cv. Maris Bead seeds were inoculated with Rhizobium Catalogue No. 1001, supplied by Rothamsted Experimental Station and grown in sand culture supplied with ¹⁵N-labelled nitrate at two concentrations. Plants were sampled at intervals throughout their growth for ¹⁵N and total N analysis. The rate of nitrate uptake was almost uniform up to pod-fill and was proportional to the nitrate concentration. Nodule weight was slightly depressed by the larger nitrate concentration at all samplings, and there was a corresponding reduction in the amount of atmospheric nitrogen fixed. However, at harvest the bean seeds from plants given most nitrate contained slightly more total N, as the enhanced nitrate uptake outweighed the reduction in fixation.     

Phosphoenolpyruvate carboxylase in root nodules of Vicia faba: Partial purification and properties

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was purified 56-fold from Vicia faba root nodules to a specific activity of 24.8 units mg^-1 protein. Native molecular mass was determined to be 443 kDa by gel permeation chromatography, whereas a molecular mass of 113 kDa was obtained for the subunit by means of SDS-PAGE, indicating that the enzyme is a homotetramer. One peak of activity was obtained by ion-exchange chromatography or gel filtration, and thus there was no evidence of isoenzymes. The effect of pH on PEPC activity was studied, the pH optimum found at 8.25. The effect of substrate (phosphoenolpyruvate, PEP) on the enzyme activity was studied at five different pH values from 6.5 to 9.5. The K_m(PEP) at pH 8.25 proved to be 0.064 m M. Inhibition by malate or activation by glucose-6-phosphate was dependent on the pH of the reaction mixture. Malate behaved as a non-competitive mixed-type inhibitor with a K_i of 0.76 m M , a K_i(s) of 1.15 m M and a K_i(i) of 0.72 m M , at pH 7.0 while at pH 8.25 K_i was about 140 m M. Activation by glucose-6-P was 70% with 4 m M PEP at pH 7, whereas no effect was found at pH 8.25. Experiments with mixed effectors at pH 7 and 1 m M PEP, showed that glucose-6-P can reverse the inhibition caused by L-malate on the PEPC activity.     

Interaction of nitrate uptake and nitrogen fixation in faba beans

An experiment was carried out to determine the relationship between nitrate uptake and nitrogen fixation of faba beans. Therefore inoculated and uninoculated faba beans were grown in nutrient solution with different nitrate concentrations. Nitrate uptake was measured every two days during the growing period. At the end of the experiment the nitrate uptake kinetics were determined with a short time depletion technique and nitrogen fixation was measured with the acetylene reduction method. A limitation of nitrate uptake due to nitrogen fixation was relatively small. Nitrate concentrations of approximately 1 mol m^–3 and 5 mol m^–3 decreased nitrogen fixation to values of 16% and 1% of the control plants which received no nitrate nitrogen. A reduction of nitrogen fixation was mainly due to a decrease of specific nitrogen fixation per unit nodule weight and to a lesser extent due to a reduction of nodule growth. Only the maximum nitrate influx (I_max) seemed to be influenced by nitrogen fixation. Michaelis-Menten constants (K_m) and minimum NO _inf3 ^– -concentrations (C_min) were not significantly influenced by nitrogen fixation.     

Growth, nitrogen fixation and ion distribution in Medicago truncatula subjected to salt stress

This study compared the growth, nodulation, N₂ fixation, and ion distribution in three Medicago truncatula lines, in response to salt in nutrient solution. Two local lines (TN8.20 and TN6.18) and a reference line (Jemalong 6) were inoculated with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl) and grown in a controlled glasshouse with or without 75 mM NaCl. A genotypic variation in tolerance to salt was found: TN6.18 was the most sensitive line whereas TN8.20 was the most tolerant. The relative tolerance of TN8.20 was concomitant with the lowest leaf Na⁺ concentration and the highest nodule biomass production. However, nodule efficiency (amount of nitrogen fixed per g dry weight nodule) decreased in all lines. Results suggest that the tolerance to salt seems to depend on the host plant ability to protect its leaves against an excessive Na⁺ (and Cl^?) accumulation, and its ability to maintain the development of an abundant nodular system, which in turn determines an important rate of nitrogen fixation and allows the plants to conserve their growth potentialities. The loss of the nodular efficiency under salt stress seems to be compensated by a large nodule biomass.     

Cell biological mechanism for triggering of ABA accumulation under water stress in Vicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves of Vicia faba. Mannitol at 890 mmol · kg-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumula