Stress-Induced Legume Root Nodule Senescence. Physiological, Biochemical, and Structural Alterations

Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O₂ diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O₂ limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2–4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by γ-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.     

Evidence for carbon flux shortage and strong carbon/nitrogen interactions in pea nodules at early stages of water stress

Symbiotic N₂ fixation in legume nodules declines under a widerange of environmental stresses. A high correlation betweenN₂ fixation decline and sucrose synthase (SS; EC 2.4.1.13) activitydown-regulation has been reported, although it has still tobe elucidated whether a causal relationship between SS activitydown-regulation and N₂ fixation decline can be established.In order to study the likely C/N interactions within nodulesand the effects on N₂ fixation, pea plants (Pisum sativum L.cv. Sugar snap) were subjected to progressive water stress bywithholding irrigation. Under these conditions, nodule SS activitydeclined concomitantly with apparent nitrogenase activity. Thelevels of UDP-glucose, glucose-1-phosphate, glucose-6-phosphate,and fructose-6-phosphate decreased in water-stressed nodulescompared with unstressed nodules. Drought also had a markedeffect on nodule concentrations of malate, succinate, and     

Phloem Glutamine and the Regulation of O2 Diffusion in Legume Nodules

The aim of the present study was to test the hypothesis that the N content or the composition of the phloem sap that supplies nodulated roots may play a role in the feedback regulation of nitrogenase activity by increasing nodule resistance to O2 diffusion. Treating shoots of lupin (Lupinus albus cv Manitoba) or soybean (Glycine max L. Merr. cv Maple Arrow) with 100 [mu]L L-1 NH3 caused a 1.3-fold (lupin) and 2.6-fold (soybean) increase in the total N content of phloem sap without altering its C content. The increase in phloem N was due primarily to a 4.8-fold (lupin) and 10.5-fold (soybean) increase in the concentration of glutamine N. In addition, there was a decline in both the apparent nitrogenase activity and total nitrogenase activity that began within 4 h and reached about 54% of its initial activity within 6 h of the start of the NH3 treatment. However, the potential nitrogenase activity values in the treated plants were not significantly different from those of the control plants. These results provide evidence that changes in the N composition of the phloem sap, particularly the glutamine content, may increase nodule resistance to O2 diffusion and, thereby, down-regulate nodule metabolism and nitrogenase activity by controlling the supply of O2 to the bacteria-infected cells.     

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 m M , 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent ( Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 m M nitrate, but not 5 m M nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 m M nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 m M ) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O₂ limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.     

The Influence of Carbohydrate Reserves on the Response of Nodulated White Clover to Defoliation

A growth-chamber study was carried out to determine whetherthe response of apparent nitrogenase activity (C₂ H₂ reduction)to complete defoliation is influenced by the availability ofcarbohydrate reserves Reserve carbohydrate (TNC) concentrationsof 6-week-old white clover (Trifoliun repens L) plants weremodified by CO₂ pretreatments There was no difference in theresponse of apparent nitrogenase activity to defoliation betweenplants with different TNC concentrations C₂H₂ reduction activitydeclined sharply after defoliation and then recovered similarlyin both high- and low-TNC plants Further experiments were conductedto explain the lack of response of apparent nitrogenase activityto TNC levels Bacteroid degradation was ruled out because invitro nitrogenase activity of crude nodule extracts was stillintact 24 h after defoliation Sufficient carbohydrates appearedto be available to the nodules of defoliated plants becauseadding [¹⁴C]glucose to the nutrient solution did not preventthe decline in apparent nitrogenase activity These conclusionswere supported by the finding that an increase in pO₂ aroundthe nodules of defoliated plants completely restored their C₂H₂reduction activity The comparison of the effects of defoliationand darkness suggested that the decrease in apparent nitrogenaseactivity was not related directly to the interruption of photosynthesisIt appears that lack of photosynthates is not the immediatecause of the decline of nitrogen-fixing activity after defoliation White clover, Trifolium repens L, defoliation, nitrogen fixation, regrowth, reserves, carbohydrates, acetylene reduction, nodule extract     

A reappraisal of nitrate inhibition of nitrogenase in A317, a nitrate reductase-deficient mutant of pea (Pisum sativum)

Symbiotic plants of Pisum sativum L. cv. Juneau and its nitrate reductase-(EC 1. 6. 6. 1)-deficient mutant, A317, were exposed to nitrate for up to 8 days and assessed for nitrate assimilation, nitrogenase activity and nodule carbohydrate status. The mutant, A317, was not impaired in its ability to absorb nitrate over up to 8 days, but was leakier with respect to nitrate reduction ability than previously realized, as 63% of the nitrate absorbed by the plant over 8 days was assimilated (in contrast to 93% in the wild type). After 2 days exposure to 5 m M nitrate, nitrogenase (EC 1.18.2.1) activity was less affected in A317 (84% of initial) than in Juneau (46% of initial): nodule starch reserves were less depleted in A317 (70% of initial) than in Juneau (26% of initial). It was concluded that nitrate reduction is a major cause of nitrate inhibition of nodule activity, and that its effect may be mediated through a decrease in the availability of carbohydrate to the nodules. Longer term (> 4 day) exposure of A317 plants to nitrate resulted in accumulation of nitrate in plant tissues, an associated necrosis of shoot tissue, a marked decrease in nodule starch content and a severe inhibition of nodule activity. This consideration of the effect of the duration of exposure to nitrate is used to resolve a discrepancy between previous reports on the sensitivity to nitrate of nitrogenase activity in nitrate reductase-deficient mutants of pea.     

Symbiotic dinitrogen fixation as affected by short-term application of nitrate to nodulatedPisum sativum L.

Effect of nitrate on the nitrogenase (C₂H₂-reduction) activity, growth of nodule tissue accumulation of nitrate and nitrate reductase activity in 4-weeks-old nodulated peas (Pisum sativum l.) was investigated. A relatively slow decrease of the total nitrogenase activity (μmol C₂H₄ per root per h), as compared with plants cultivated without nitrate, was due to both retardation of further growth of the nodule tissue and to a decrease of their specific nitrogenase activity (μmol C₂H₄ per g_f.wt. per h). However, an absolute and pronounced decrease of both nitrogenase activities occurred only 4 or 7 d after the application of nitrate. The addition of nitrate led to its rapid accumulation in the nodule and leaf tissue with a simultaneous induction of the nitrate reductase activity. The nitrogenase activity was not completely inhibited even after a 7-d cultivation with 280 ppm NO₃ ^?-N in the nutrient medium and after accumulation of up to 180 ppm NO₃ ^?-N_f.wt. in the nodule tissue. The results obtained indicate that the “photosynthate deprivation” reflects competition between assimilation of nitrate and fixation of dinitrogen.     

Nodule protein synthesis and nitrogenase activity of soybeans exposed to fixed nitrogen

Nitrate or ammonium was added to soybean (Glycine max L. Merrill cv Corsoy) plants grown in plastic pouches 10 days after nodules first appeared. By the third day of treatment with 10 millimolar nitrate, nitrogenase specific activity (per unit nodule weight) had decreased to 15% to 25% of that of untreated plants. Longer incubations and higher concentrations of nitrate had no greater effect. In addition, exogenous nitrate or ammonium resulted in slower nodule growth and decreased total protein synthesis in both the bacterial and the plant portion of the nodule (as measured by incorporation of ³⁵S). Two-dimensional gel electrophoresis revealed that the nitrogenase components were not repressed or degraded relative to other bacteroid proteins. In the presence of an optimal carbon source, the nitrogenase specific activity of nodules detached from nitrate-treated plants was equivalent to that of nodules from untreated plants. These results are consistent with models that propose decreased availability or utilization of photosynthate in root nodules when legumes are exposed to fixed nitrogen.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

Induction of Root Nodule Senescence by Combined Nitrogen in Pisum sativum L

Root nodule senescence induced by nitrate and ammonium in Pisum sativum L. was defined by determining nitrogenase activity and leghemoglobin content with the acetylene reduction and pyridine hemochrome assays. Root systems supplied with 100 mm KNO(3) or 100 mm NH(4)Cl exhibited a decrease in nitrogenase activity followed by a decline in leghemoglobin content. Increasing the CO(2) concentration from 0.000320 atm to 0.00120 atm had no effect on the time course of root nodule senescence when 20 mm KNO(3) was supplied to the roots; in vitro nitrate reductase activity was detected in leaves and roots, but not bacteroids. Nitrate appeared in leaves, roots, and the nodule cytosol fraction but not bacteroids when 20 mm KNO(3) was supplied to roots. When nitrate entered through the shoots, however, no root nodule senescence was observed, and no nitrate was detected in root or nodule cytosol fractions although nitrate and nitrate reductase were found in leaves. The results suggest that nitrate does not induce root nodule senescence through competition between nitrate reductase and nitrogenase for products of photosynthesis.     

Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O₂ flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.     

Effect of nitrate on acetylene reduction activity and carbohydrate composition of Phaseolus vulgaris nodules

When Phaseolus vulgaris L. cv. Kentucky Wonder plants were supplied with various levels of nitrate for 34 days, nodule weight (plant)⁻¹, acetylene reduction activity (g nodule)⁻¹, and sugar concentration in nodules were depressed >60% (7.5 m M nitrate vs nil nitrate). Starch concentration in nodules was more than double the sugar concentration and declined only slightly in response to nitrate level. At the highest level of nitrate, sugar concentration in nodules was 50% greater than that in roots and nodule starch was about 6-fold greater than root starch on a fresh weight basis. When plants were grown with 1 m M nitrate and then supplied with 12 m M nitrate for 7 days, the rapid decline in acetylene reduction activity coincided with a decline in sucrose concentration. However, glucose and fructose concentrations declined only after the largest decrease in acetylene reduction had occurred, and the quantitative decrease in glucose and fructose in nodules was small relative to sucrose. Other results showed that the magnitude of the effect of nitrate on some nodule carbohydrate compounds depends on Rhizobium phaseoli strain and on whether plants were grown with or without nitrate prior to experimental treatments. Some of the results are consistent with the carbohydrate-deprivation hypothesis for inhibition of legume nodules by nitrate. However, there are several complications involved in the interpretation of results of this type, and other possible explanations for the results are suggested.     

Possible causes of the physiological decline in soybean nitrogen fixation in the presence of nitrate

Nodulated soybean plants (Glycine max (L.) Merr. cv. Clarke)were supplied with 10 mol m^-3 nitrate at the vegetative stage.This treatment caused a rapid decline in nitrogen fixation (acetylenereduction) activity and a consequent decline in ureides in thexylem sap. However, there was virtually no effect on the nitrogenasecomplex, according to Western blots against components 1 and2. The effect on nitrogen fixation was matched by a decreasein nitrogenase-linked respiration and increases in nodule oxygendiffusion resistance and the carbon cost of nitrogen fixation.The addition of nitrate had little effect on protein contentfrom either nodule plant or bacteroid fractions. Activitiesof nitrate reductase (NR) and nitrite reductase (NiR) from eitherthe plant fraction or the bacteroids were affected in differentways during 8 d of supply. Nodule plant NR and bacteroid NiR were not affected. However,nodule plant NiR increased 5-fold within 2 d of supplying Bacteroid NR only increased after6 d. These results could be interpreted in terms of a restrictednitrate access into the infected region of nodules. However,denitrification was detected within 2 d of nitrate supply insoybean nodules. The results are discussed in relation to possiblecauses of the nitrate-induced decline in nitrogenase activity. Key words: Glycine max, nitrate, nitrogen fixation, nodules     

Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes

The inhibitory effect of nitrate on nitrogenase activity in root nodules of legume plants has been known for a long time. The major factor inducing changes in nitrogenase activity is the concentration of free oxygen inside nodules. Oxygen availability in the infected zone of nodule is limited, among others, by the gas diffusion resistance in nodule cortex. The presence of nitrate may cause changes in the resistance to O2 diffusion. The aim of this paper is to review literature data concerning the effect of nitrate on the symbiotic association between rhizobia and legume plants, with special emphasis on nitrogenase activity. Recent advances indicate that symbiotic associations of Rhizobium strains characterized by a high nitrate reductase activity are less susceptible to inhibition by nitrate. A thesis may be put forward that dissimilatory nitrate reduction, catalyzed by bacteroid nitrate reductase, significantly facilitates the symbiotic function of bacteroids.     

Nitrogenase activity and root nodule metabolism in response to O2 and short-term N2 deprivation in dark-treated Frankia-Alnus incana plants

Inhibition of nitrogenase (EC 1.18.6.1) activity by O₂ has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia‐Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O₂ and short‐term N₂ deprivation (1 h in Ar:O₂). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H₂ evolution in a gas exchange system. Respiration was simultaneously recorded as CO₂ evolution. Dark‐treated plants had lower initial nitrogenase activity in N₂:O₂ (68% of controls), which declined further during a 1‐h period in the assay system in N₂:O₂ at 21 and 17% O₂, but not at 13% O₂. When dark‐treated plants were deprived of N₂ at 21 and 17% O₂ nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark‐treated plants at 13% O₂, and only a smaller and temporary decline in control plants at 21% O₂. When dark‐treated plants were kept at 21% O₂ during 45 min prior to N₂ deprivation at 17% O₂ the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N₂:O₂ at 21% O₂ and during N₂ deprivation was caused by O₂, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N₂ deprivation, suggesting decreased assimilation of NH₄⁺. Contents of ATP and ADP in nodules were not affected by short‐term N₂ deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O₂. It seemed that dark‐treated plants could not adjust their nodule metabolism at higher perceived pO₂ and during cessation of NH₄⁺ production.     

Involvement of Activated Oxygen in Nitrate-Induced Senescence of Pea Root Nodules

The effect of short-term nitrate application (10 mM, 0-4 d) on nitrogenase (N2ase) activity, antioxidant defenses, and related parameters was investigated in pea (Pisum sativum L. cv Frilene) nodules. The response of nodules to nitrate comprised two stages. In the first stage (0-2 d), there were major decreases in N2ase activity and N2ase-linked respiration and concomitant increases in carbon cost of N2ase and oxygen diffusion resistance of nodules. There was no apparent oxidative damage, and the decline in N2ase activity was, to a certain extent, reversible. The second stage (>2 d) was typical of a senescent, essentially irreversible process. It was characterized by moderate increases in oxidized proteins and catalytic Fe and by major decreases in antioxidant enzymes and metabolites. The restriction in oxygen supply to bacteroids may explain the initial decline in N2ase activity. The decrease in antioxidant protection is not involved in this process and is not specifically caused by nitrate, since it also occurs with drought stress. However, comparison of nitrate- and drought-induced senescence shows an important difference: there is no lipid degradation or lipid peroxide accumulation with nitrate, indicating that lipid peroxidation is not necessarily involved in nodule senescence.     

Gas-exchange activity,carbohydrate status,and protein turnover in root nodule subpopulations of field pea (Pisum sativum L. cv. Century)

Root nodule ontogeny was followed in different parts of the root system of field peas (Pisum sativum L. cv. Century) to investigate the contribution to total nitrogen fixation by different nodule subpopulations. Seed-inoculated plants were grown to maturity in controlled-environment growth chambers. In a flow-through system nitrogenase activity (H₂-evolution in air) and nodulated-root respiration (net CO₂-evolution) were measured weekly or biweekly in different parts (top and mid) of the root system. Root nodule extracts were assayed for total soluble cytosolic protein, total heme, proteolytic capacity (at pH 7.0), soluble carbohydrates and starch. Total nitrogenase activity and nodule respiration were higher in the top zone, which was explained by differences in root and nodule mass. Nodule specific nitrogenase activity was similar in both zones, and gradually declined throughout the experiment. No differences were found between nodule subpopulations in the dry-matter specific concentrations of glucose, fructose, sucrose or starch. Neither did nodule concentrations of protein or leghemoglobin differ between the zones. Throughout reproductive growth, no decline was found in total or nodule specific nitrogenase activity, in any of the nodule subpopulations. Growth of the root nodules continued throughout the experiment, though growth of shoot and roots had ceased. The data gives no support for carbohydrate limitation in root nodules during pod-filling, since nodule respiration remained high, the concentration of soluble carbohydrates increased significantly, and the amount of starch was not reduced. We conclude that when this symbiosis is grown under controlled conditions, nitrogenase activity in nodules sub-sampled from the crown part of the root system is representative for the whole nodule population.     

Nitrogenase activity and ureide levels in a supernodulating soybean mutant: Effects of inoculum dose and nitrate treatment

The effects of nitrate on nitrogenase (EC 1.18.2.1) activity of soybean ( Glycine max [L.] Merr) cv. Bragg and its supernodulating mutant derivative, nts382, were compared. A short-term nitrate treatment was used to allow effects on nitrogenase activity to be studied in the absence of effects on nodule growth and a low inoculum dose, which prevented supernodulation of nts382, was employed to test for any interaction between supernodulation and the magnitude of the effect of nitrate on nitrogenase activity. At the usual inoculum dose, nitrogenase activity, per g nocule, of nts382 was lower than that of Bragg and was proportionally less affected by nitrate. Decreasing the inoculum dose increased nitrogenase activity of nts382 and also the proportional decline in response to nitrate. The decline in the ureide conentration in xylem exudate in response to nitrate was proportionally similar to the decline in nitrogenase activity per plant. However, although nitrogenase activity per plant of nts382 was several-fold less than that of Bragg, the ureide flux rate (ureide concentration x xylem sap exudation rate), was not different. At the usual inodulum dose, the ureide content of the nocules, stems plus petioles and leaves of nts382 was greater than that of Bragg. Decreasing the inoculum dose reduced the ureide content of the nodules of nts382 but not of Bragg. Ureide degradative capacity of the leaves was the same for Bragg and nts382. Low activities of 5-phosphoribosyl pyrophosphate amidotransferase (EC 2.4.2.14) and glutamine synthetase (EC 6.3.1.2) in the nodules reflected the low nitrogenase activity of nts382.     

Water-deficit effects on carbon and nitrogen metabolism of pea nodules

Two experiments were carried out to investigate the effects of water-deficit stress on carbon and nitrogen metabolism of Pisum sativum nodules. In the first experiment, leaf w was allowed to reach -1.0 MPa over a period of 14 d whilst in the second experiment -1.5 MPa was reached during the same time period. Nodule activities of phosphoenol pyruvate carboxylase, glutamine synthetase, alkaline invertase, pyruvate decarboxylase, alcohol dehdyrogenase, uridine pyro-phosphorylase, and malate dehydrogenase activities were not affected by water-deficit stress. In the first experiment (-1.0 MPa), sucrose synthase (SS), an enzyme which hydrolyses sucrose to support nodule metabolism, declined by 50% in activity and about 25% in content, according to Western immunoblot data. In the second experiment (-1.5 MPa), SS activity decreased by 75% together with glutamate synthase and aspartate aminotransferase which declined by 60% and 40%, respectively. Coincident with the decline of these activities, a dramatic increase in the nodule content of sucrose and a slight increase in the levels of total free amino acids were found. It has been recently suggested that the decline in SS activity and, therefore, a reduced potential to metabolize sucrose may be an important factor contributing to the overall response of soybean nodules to water stress. These results suggest that this observation may be also correct for temperate legumes with indeterminate nodules. However, in this latter case, the activity of some enzymes involved in nitrogen assimilation (glutamate synthase and aspartate aminotransferase) were also affected by water-deficit stress).Key words: Pisum sativum, water stress, nitrogen metabolism, nodule metabolism, pea, sucrose synthase.