The application of a moderate water deficit (water potential of ؊1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (؊1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, ␤-carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD ؉ , NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N 2 ase) activity. Exposure of plants to a moderate drought (leaf water potential of 21.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N 2 ase activity (243%), accumulation of succinate (136%) and Suc (158%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (22.1 MPa) decreased N 2 ase (282%) and SS (230%) activities and increased malate (140%), succinate (168%), and Suc (1435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe-and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (.64%) of N 2 ase, a complete recovery of Suc, and a decrease of malate (248%) relative to control. The increase in O 2 diffusion resistance, the decrease in N 2 ase-linked respiration and N 2 ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N 2 ase activity in alfalfa nodules.

Drought is one of the environmental factors most affecting crop production. Under drought, symbiotic nitrogen fixation is one of the physiological processes to first show stress responses in nodulated legumes. This inhibition process involves a number of factors whose interactions are not yet understood. This work aims to further understand changes occurring in nodules under drought stress from a proteomic perspective. Drought was imposed on Medicago truncatula 'Jemalong A17' plants grown in symbiosis with Sinorhizobium meliloti strain 2011. Changes at the protein level were analyzed using a nongel approach based on liquid chromatography coupled to tandem mass spectrometry. Due to the complexity of nodule tissue, the separation of plant and bacteroid fractions in M. truncatula root nodules was first checked with the aim of minimizing cross contamination between the fractions. Second, the protein plant fraction of M. truncatula nodules was profiled, leading to the identification of 377 plant proteins, the largest description of the plant nodule proteome so far. Third, both symbiotic partners were independently analyzed for quantitative differences at the protein level during drought stress. Multivariate data mining allowed for the classification of proteins sets that were involved in drought stress responses. The isolation of the nodule plant and bacteroid protein fractions enabled the independent analysis of the response of both counterparts, gaining further understanding of how each symbiotic member is distinctly affected at the protein level under a water-deficit situation.

Regulation of symbiotic nitrogen fixation (SNF) during drought stress is complex and not yet fully understood. In the present work, the involvement of nodule C and N metabolism in the regulation of SNF in Medicago truncatula under drought and a subsequent rewatering treatment was analyzed using a combination of metabolomic and proteomic approaches. Drought induced a reduction of SNF rates and major changes in the metabolic profile of nodules, mostly an accumulation of amino acids (Pro, His, and Trp) and carbohydrates (sucrose, galactinol, raffinose, and trehalose). This accumulation was coincidental with a decline in the levels of bacteroid proteins involved in SNF and C metabolism, along with a partial reduction of the levels of plant sucrose synthase 1 (SuSy1). In contrast, the variations in enzymes related to N assimilation were found not to correlate with the reduction in SNF, suggesting that these enzymes do not have a role in the regulation of SNF. Unlike the situation in other legumes such as pea and soybean, the drought-induced inhibition of SNF in M. truncatula appears to be caused by impairment of bacteroid metabolism and N(2)-fixing capacity rather than a limitation of respiratory substrate.

Drought stress is a major factor limiting symbiotic nitrogen fixation (NF) in soybean crop production. However, the regulatory mechanisms involved in this inhibition are still controversial. Soybean plants were symbiotically grown in a split-root system (SRS), which allowed for half of the root system to be irrigated at field capacity while the other half remained water deprived. NF declined in the water-deprived root system while nitrogenase activity was maintained at control values in the well-watered half. Concomitantly, amino acids and ureides accumulated in the water-deprived belowground organs regardless of transpiration rates. Ureide accumulation was found to be related to the decline in their degradation activities rather than increased biosynthesis. Finally, proteomic analysis suggests that plant carbon metabolism, protein synthesis, amino acid metabolism, and cell growth are among the processes most altered in soybean nodules under drought stress. Results presented here support the hypothesis of a local regulation of NF taking place in soybean and downplay the role of ureides in the inhibition of NF.

Abbreviations -APX, ascorbate peroxidase; C i , internal CO 2 concentration; GPX, guaiacol peroxidase; MnSOD, FeSOD, CuZnSOD, superoxide dismutases containing Mn, Fe, or Cu plus Zn as metal cofactors; Ψ w , water potential. 4 IntroductionWater stress has profound effects on crop production. Even plants with an optimum water supply experience transient water shortage periods, where water absorption cannot compensate for water loss by transpiration (Kramer and Boyer 1997). In addition, many other environmental stresses, such as cold, salinity and high temperature, have a water stress component. At the mole cular and cellular levels, drought and other adverse conditions induce oxidative stress in plant tissues (Thompson et al. 1987, Smirnoff 1993, which can be diagnosed by the accumulation of lipid peroxides, oxidized proteins, or modified DNA bases (Moran et al. 1994, Halliwell and Gutteridge 1999).Superoxide dismutases (SODs) are ubiquituous metalloenzymes that catalyze the dismutati on of superoxide radical to H 2 O 2 and O 2 . The superoxide radical is a potential precursor of the highly oxidizing hydroxyl radical and, therefore, SODs are a critical defense of plants, other aerobic organisms, and some anaerobes against oxidative stress (Halliwell and Gutteridge 1999).Three classes of SODs, differing in their metal cofactor, are known in plants. All three SODs are nuclear-encoded but localized in different subcellular compartments. Typically, CuZnSODs are in the cytosol and chloroplasts, MnSODs in the mitochondria and peroxisomes, and FeSODs in the chloroplasts (Bowler et al. 1994(Bowler et al. , del R o et al. 1998. Transgenic plants overexpressing SODs in the chloroplasts, mitochondria, and cytosol have been generated (Bowler et al. 1991, Van Camp et al. 1996. In some cases, transgenic plants showed superior tolerance to oxidative stress induced by incubation of leaf disks with methylviologen or by exposure of plants to ozone (Bowler et al. 1991, Sen Gupta et al. 1993. In other cases, but using the same stress inducers, no beneficial effects were found (Tepperman and Dunsmuir 1990, Pitcher et al. 1991). These contradictory results were ascribed to differences in the SOD constructs, in the methodology used to analyze the transformants, and in the growth conditions of the plants (Slooten et al. 1995, Allen et al. 1997).The above-mentioned studies were performed mainly with tobacco, but important crop legumes are now amenable for transformation (Christou 1994). In these plants, SOD and ascorbate peroxidase (APX) play critical protective roles in nodule activity (Puppo and Rigaud 1986, Dalton et al. 1998). Conceivably then, overexpression of antioxidant enzymes in legumes 5 could provide additional protection to the process of N 2 fixation, especially during senescence and under stress conditions. In a previous study, we have analyzed the SOD composition of several transgenic lines of alfalfa and characterized three of them at the molecular level (Rubio et al. 2001). Using the three selected lines (1-10...

Nitrogen fixation (NF) in soybean (Glycine max L. Merr.) is highly sensitive to soil drying. This sensitivity has been related to an accumulation of nitrogen compounds, either in shoots or in nodules, and a nodular carbon flux shortage under drought. To assess the relative importance of carbon and nitrogen status on NF regulation, the responses to the early stages of drought were monitored with two soybean cultivars with known contrasting tolerance to drought. In the sensitive cultivar ('Biloxi'), NF inhibition occurred earlier and was more dramatic than in the tolerant cultivar ('Jackson'). The carbon flux to bacteroids was also more affected in 'Biloxi' than in 'Jackson', due to an earlier inhibition of sucrose synthase activity and a larger decrease of malate concentration in the former. Drought provoked ureide accumulation in nodules of both cultivars, but this accumulation was higher and occurred earlier in 'Biloxi'. However, at this early stage of drought, there was no accumulation of ureides in the leaves of either cultivar. These results indicate that a combination of both reduced carbon flux and nitrogen accumulation in nodules, but not in shoots, is involved in the inhibition of NF in soybean under early drought.

Symbiotic N2 fixation in legume nodules declines under a wide range of environmental stresses. A high correlation between N2 fixation decline and sucrose synthase (SS; EC 2.4.1.13) activity down-regulation has been reported, although it has still to be elucidated whether a causal relationship between SS activity down-regulation and N2 fixation decline can be established. In order to study the likely C/N interactions within nodules and the effects on N2 fixation, pea plants (Pisum sativum L. cv. Sugar snap) were subjected to progressive water stress by withholding irrigation. Under these conditions, nodule SS activity declined concomitantly with apparent nitrogenase activity. The levels of UDP-glucose, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate decreased in water-stressed nodules compared with unstressed nodules. Drought also had a marked effect on nodule concentrations of malate, succinate, and alpha-ketoglutarate. Moreover, a general decline in nodule adenylate content was detected. NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42) was the only enzyme whose activity increased as a result of water deficit, compensating for a possible C/N imbalance and/or supplying NADPH in circumstances that the pentose phosphate pathway was impaired, as suggested by the decline in glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) activity. The overall results show the occurrence of strong C/N interactions in nodules subjected to water stress and support a likely limitation of carbon flux that might be involved in the decline of N2 fixation under drought.