Additive effects of heatwave and water stresses on soybean seed yield is caused by impaired carbon assimilation at pod formation but not at flowering.

Heatwave (HW) combined with water stress (WS) are critical environmental factors negatively affecting crop development. This study aimed to quantify the individual and combined effects of HW and WS during early reproductive stages on leaf and nodule functioning and their relation with final soybean seed yield (SY). For this purpose, during flowering (R2) and pod formation (R4) soybean (Glycine max L. Merr.) plants were exposed to different temperature (ambient[25ºC] versus HW[40ºC]) and water availability (full capacity versus WS[20% field capacity]). HW, WS and their combined impact on yield depended on the phenological stage at which stress was applied being more affected at R4. For gas exchange, WS severely impaired photosynthetic machinery, especially when combined with HS. Impaired photoassimilate supply at flowering caused flower abortion and a significant reduction in final SY due to interacting stresses and WS. On the other hand, at pod formation (R4), decreased leaf performance caused additive effect on SY by decreasing pod setting and seed size with combined stresses. At the nodule level, WS (alone or in combination with HW) caused nodule impairment, which was reflected by lower leaf N. Such response was linked with a poor malate supply to bacteroids and feed-back inhibition caused by nitrogenous compounds accumulation. In summary, our study noted that soybean sensitivity to interacting heat and water stresses was highly conditioned by the phenological stage at which it occurs with, R4 stage being the critical moment. To our knowledge this is the first soybean work integrating combined stresses at early reproductive stages.

Increased Ascorbate Biosynthesis Does Not Improve Nitrogen Fixation Nor Alleviate the Effect of Drought Stress in Nodulated Medicago truncatula Plants.

Legume plants are able to establish nitrogen-fixing symbiotic relations with Rhizobium bacteria. This symbiosis is, however, affected by a number of abiotic constraints, particularly drought. One of the consequences of drought stress is the overproduction of reactive oxygen (ROS) and nitrogen species (RNS), leading to cellular damage and, ultimately, cell death. Ascorbic acid (AsA), also known as vitamin C, is one of the antioxidant compounds that plants synthesize to counteract this oxidative damage. One promising strategy for the improvement of plant growth and symbiotic performance under drought stress is the overproduction of AsA via the overexpression of enzymes in the Smirnoff-Wheeler biosynthesis pathway. In the current work, we generated Medicago truncatula plants with increased AsA biosynthesis by overexpressing MtVTC2, a gene coding for GDP-L-galactose phosphorylase. We characterized the growth and physiological responses of symbiotic plants both under well-watered conditions and during a progressive water deficit. Results show that increased AsA availability did not provide an advantage in terms of plant growth or symbiotic performance either under well-watered conditions or in response to drought.

A novel biosensor to monitor proline in pea root exudates and nodules under osmotic stress and recovery.

BACKGROUND AND AIMS: Plant and bacteria are able to synthesise proline, which acts as a compound to counteract the negative effects of osmotic stresses. Most methodologies rely on the extraction of compounds using destructive methods. This work describes a new proline biosensor that allows the monitoring of proline levels in a non-invasive manner in root exudates and nodules of legume plants. METHODS: The proline biosensor was constructed by cloning the promoter region of pRL120553, a gene with high levels of induction in the presence of proline, in front of the lux cassette in Rhizobium leguminosarum bv. viciae. RESULTS: Free-living assays show that the proline biosensor is sensitive and specific for proline. Proline was detected in both root exudates and nodules of pea plants. The luminescence detected in bacteroids did not show variations during osmotic stress treatments, but significantly increased during recovery. CONCLUSIONS: This biosensor is a useful tool for the in vivo monitoring of proline levels in root exudates and bacteroids of symbiotic root nodules, and it contributes to our understanding of the metabolic exchange occurring in nodules under abiotic stress conditions.

Physiological, Hormonal and Metabolic Responses of two Alfalfa Cultivars with Contrasting Responses to Drought.

Alfalfa (Medicago sativa L.) is frequently constrained by environmental conditions such as drought. Within this context, it is crucial to identify the physiological and metabolic traits conferring a better performance under stressful conditions. In the current study, two alfalfa cultivars (San Isidro and Zhong Mu) with different physiological strategies were selected and subjected to water limitation conditions. Together with the physiological analyses, we proceeded to characterize the isotopic, hormone, and metabolic profiles of the different plants. According to physiological and isotopic data, Zhong Mu has a water-saver strategy, reducing water lost by closing its stomata but fixing less carbon by photosynthesis, and therefore limiting its growth under water-stressed conditions. In contrast, San Isidro has enhanced root growth to replace the water lost through transpiration due to its more open stomata, thus maintaining its biomass. Zhong Mu nodules were less able to maintain nodule N2 fixing activity (matching plant nitrogen (N) demand). Our data suggest that this cultivar-specific performance is linked to Asn accumulation and its consequent N-feedback nitrogenase inhibition. Additionally, we observed a hormonal reorchestration in both cultivars under drought. Therefore, our results showed an intra-specific response to drought at physiological and metabolic levels in the two alfalfa cultivars studied.

Soybean-Nodulating Strains With Low Intrinsic Competitiveness for Nodulation, Good Symbiotic Performance, and Stress-Tolerance Isolated From Soybean-Cropped Soils in Argentina.

Soybean is the most important oilseed in the world, cropped in 120-130 million hectares each year. The three most important soybean producers are Argentina, Brazil, and United States, where soybean crops are routinely inoculated with symbiotic N2-fixing Bradyrhizobium spp. This extended inoculation gave rise to soybean-nodulating allochthonous populations (SNAPs) that compete against new inoculant for nodulation, thus impairing yield responses. Competitiveness depends on intrinsic factors contributed by genotype, extrinsic ones determined by growth and environmental conditions, and strain persistence in the soil. To assess these factors in Argentinean SNAPs, we studied 58 isolates from five sites of the main soybean cropping area. BOX-A1R DNA fingerprint distributed these isolates in 10 clades that paralleled the pHs of their original soils. By contrast, reference Bradyrhizobium spp. strains, including those used as soybean-inoculants, were confined to a single clade. More detailed characterization of a subset of 11 SNAP-isolates revealed that five were Bradyrhizobium japonicum, two Bradyrhizobium elkanii, two Rhizobium radiobacter (formerly Agrobacterium tumefaciens), one Bradyrhizobium diazoefficiens, and one Paenibacillus glycanilyticus-which did not nodulate when inoculated alone, and therefore was excluded from further characterization. The remaining subset of 10 SNAP-isolates was used for deeper characterization. All SNAP-isolates were aluminum- and heat-tolerant, and most of them were glyphosate-tolerant. Meanwhile, inoculant strains tested were sensitive to aluminum and glyphosate. In addition, all SNAP-isolates were motile to different degrees. Only three SNAP-isolates were deficient for N2-fixation, and none was intrinsically more competitive than the inoculant strain. These results are in contrast to the general belief that rhizobia from soil populations evolved as intrinsically more competitive for nodulation and less N2-fixing effective than inoculants strains. Shoot:root ratios, both as dry biomass and as total N, were highly correlated with leaf ureide contents, and therefore may be easy indicators of N2-fixing performance, suggesting that highly effective N2-fixing and well-adapted strains may be readily selected from SNAPs. In addition, intrinsic competitiveness of the inoculants strains seems already optimized against SNAP strains, and therefore our efforts to improve nodules occupation by inoculated strains should focus on the optimization of extrinsic competitiveness factors, such as inoculant formulation and inoculation technology.

Effect of shoot removal on remobilization of carbon and nitrogen during regrowth of nitrogen-fixing alfalfa.

The contribution of carbon and nitrogen reserves to regrowth following shoot removal has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both (15) N2 and (13) C-depleted CO2 on exclusively nitrogen-fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root-derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N2 -fixing machinery, availability of Cnew and Nnew strongly diminished in the plants due to their replacement by C and N assimilated during the post-labelling period. In summary, our study indicated that during the first week of regrowth, root-derived C and N remobilization did not overcome C- and N-limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re-established.

Nodule carbohydrate catabolism is enhanced in the Medicago truncatula A17-Sinorhizobium medicae WSM419 symbiosis.

The symbiotic association between Medicago truncatula and Sinorhizobium meliloti is a well-established model system in the legume-Rhizobium community. Despite its wide use, the symbiotic efficiency of this model has been recently questioned and an alternative microsymbiont, S. medicae, has been proposed. However, little is known about the physiological mechanisms behind the higher symbiotic efficiency of S. medicae WSM419. In the present study, we inoculated M. truncatula Jemalong A17 with either S. medicae WSM419 or S. meliloti 2011 and compared plant growth, photosynthesis, N2-fixation rates, and plant nodule carbon and nitrogen metabolic activities in the two systems. M. truncatula plants in symbiosis with S. medicae showed increased biomass and photosynthesis rates per plant. Plants grown in symbiosis with S. medicae WSM419 also showed higher N2-fixation rates, which were correlated with a larger nodule biomass, while nodule number was similar in both systems. In terms of plant nodule metabolism, M. truncatula-S. medicae WSM419 nodules showed increased sucrose-catabolic activity, mostly associated with sucrose synthase, accompanied by a reduced starch content, whereas nitrogen-assimilation activities were comparable to those measured in nodules infected with S. meliloti 2011. Taken together, these results suggest that S. medicae WSM419 is able to enhance plant carbon catabolism in M. truncatula nodules, which allows for the maintaining of high symbiotic N2-fixation rates, better growth and improved general plant performance.

Split-root systems applied to the study of the legume-rhizobial symbiosis: what have we learned?

Split-root system (SRS) approaches allow the differential treatment of separate and independent root systems, while sharing a common aerial part. As such, SRS is a useful tool for the discrimination of systemic (shoot origin) versus local (root/nodule origin) regulation mechanisms. This type of approach is particularly useful when studying the complex regulatory mechanisms governing the symbiosis established between legumes and Rhizobium bacteria. The current work provides an overview of the main insights gained from the application of SRS approaches to understand how nodule number (nodulation autoregulation) and nitrogen fixation are controlled both under non-stressful conditions and in response to a variety of stresses. Nodule number appears to be mainly controlled at the systemic level through a signal which is produced by nodule/root tissue, translocated to the shoot, and transmitted back to the root system, involving shoot Leu-rich repeat receptor-like kinases. In contrast, both local and systemic mechanisms have been shown to operate for the regulation of nitrogenase activity in nodules. Under drought and heavy metal stress, the regulation is mostly local, whereas the application of exogenous nitrogen seems to exert a regulation of nitrogen fixation both at the local and systemic levels.

Nodule performance within a changing environmental context.

Global climate models predict that future environmental conditions will see alterations in temperature, water availability and CO2 concentration ([CO2]) in the atmosphere. Climate change will reinforce the need to develop highly productive crops. For this purpose it is essential to identify target traits conditioning plant performance in changing environments. N2 fixing plants represent the second major crop of agricultural importance worldwide. The current review provides a compilation of results from existing literature on the effects of several abiotic stress conditions on nodule performance and N2 fixation. The environmental factors analysed include water stress, salinity, temperature, and elevated [CO2]. Despite the large number of studies analysing [CO2] effects in plants, frequently they have been conducted under optimal growth conditions that are difficult to find in natural conditions where different stresses often occur simultaneously. This is why we have also included a section describing the current state of knowledge of interacting environmental conditions in nodule functioning. Regardless of the environmental factor considered, it is evident that some general patterns of nodule response are observed. Nodule carbohydrate and N compound availability, together with the presence of oxygen reactive species (ROS) have proven to be the key factors modulating N2 fixation at the physiological/biochemical levels. However, with the exception of water availability and [CO2], it should also be considered that nodule performance has not been characterised in detail under other limiting growth conditions. This highlights the necessity to conduct further studies considering these factors. Finally, we also observe that a better understanding of these metabolic effects of changing environment in nodule functioning would require an integrated and synergistic investigation based on widely used and novel protocols such as transcriptomics, proteomics, metabolomics and stable isotopes.

A proteomic approach reveals new actors of nodule response to drought in split-root grown pea plants.

Drought is considered the more harmful abiotic stress resulting in crops yield loss. Legumes in symbiosis with rhizobia are able to fix atmospheric nitrogen. Biological nitrogen fixation (SNF) is a very sensitive process to drought and limits legumes agricultural productivity. Several factors are known to regulate SNF including oxygen availability to bacteroids, carbon and nitrogen metabolisms; but the signaling pathways leading to SNF inhibition are largely unknown. In this work, we have performed a proteomic approach of pea plants grown in split-root system where one half of the root was well-irrigated and the other was subjected to drought. Water stress locally provoked nodule water potential decrease that led to SNF local inhibition. The proteomic approach revealed 11 and 7 nodule proteins regulated by drought encoded by Pisum sativum and Rhizobium leguminosarum genomes respectively. Among these 18 proteins, 3 proteins related to flavonoid metabolism, 2 to sulfur metabolism and 3 RNA-binding proteins were identified. These proteins could be molecular targets for future studies focused on the improvement of legumes tolerance to drought. Moreover, this work also provides new hints for the deciphering of SNF regulation machinery in nodules.

Drought stress provokes the down-regulation of methionine and ethylene biosynthesis pathways in Medicago truncatula roots and nodules.

Symbiotic nitrogen fixation is one of the first physiological processes inhibited in legume plants under water-deficit conditions. Despite the progress made in the last decades, the molecular mechanisms behind this regulation are not fully understood yet. Recent proteomic work carried out in the model legume Medicago truncatula provided the first indications of a possible involvement of nodule methionine (Met) biosynthesis and related pathways in response to water-deficit conditions. To better understand this involvement, the drought-induced changes in expression and content of enzymes involved in the biosynthesis of Met, S-adenosyl-L-methionine (SAM) and ethylene in M. truncatula root and nodules were analyzed using targeted approaches. Nitrogen-fixing plants were subjected to a progressive water deficit and a subsequent recovery period. Besides the physiological characterization of the plants, the content of total sulphur, sulphate and main S-containing metabolites was measured. Results presented here show that S availability is not a limiting factor in the drought-induced decline of nitrogen fixation rates in M. truncatula plants and provide evidences for a down-regulation of the Met and ethylene biosynthesis pathways in roots and nodules in response to water-deficit conditions.

Local inhibition of nitrogen fixation and nodule metabolism in drought-stressed soybean.

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.

Is N-feedback involved in the inhibition of nitrogen fixation in drought-stressed Medicago truncatula?

Drought stress is a major factor limiting nitrogen fixation (NF) in crop production. However, the regulatory mechanism involved and the origin of the inhibition, whether local or systemic, is still controversial and so far scarcely studied in temperate forage legumes. Medicago truncatula plants were symbiotically grown with a split-root system and exposed to gradual water deprivation. Physiological parameters, NF activity, and amino acid content were measured. The partial drought treatment inhibited NF in the nodules directly exposed to drought stress. Concomitantly, in the droughted below-ground organs, amino acids accumulated prior to any drop in evapotranspiration (ET). It is concluded that drought exerts a local inhibition of NF and drives an overall accumulation of amino acids in diverse plant organs which is independent of the decrease in ET. The general increase in the majority of single amino acids in the whole plant questions the commonly accepted concept of a single amino acid acting as an N-feedback signal.

Expression and localization of a Rhizobium-derived cambialistic superoxide dismutase in pea (Pisum sativum) nodules subjected to oxidative stress.

Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.

Carbon metabolism and bacteroid functioning are involved in the regulation of nitrogen fixation in Medicago truncatula under drought and recovery.

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.

Absolute quantification of Medicago truncatula sucrose synthase isoforms and N-metabolism enzymes in symbiotic root nodules and the detection of novel nodule phosphoproteins by mass spectrometry.

Mass spectrometry (MS) has become increasingly important for tissue specific protein quantification at the isoform level, as well as for the analysis of protein post-translational regulation mechanisms and turnover rates. Thanks to the development of high accuracy mass spectrometers, peptide sequencing without prior knowledge of the amino acid sequence--de novo sequencing--can be performed. In this work, absolute quantification of a set of key enzymes involved in carbon and nitrogen metabolism in Medicago truncatula 'Jemalong A17' root nodules is presented. Among them, sucrose synthase (SuSy; EC 2.4.1.13), one of the central enzymes in sucrose cleavage in root nodules, has been further characterized and the relative phosphorylation state of the three most abundant isoforms has been quantified. De novo sequencing provided sequence information of a so far unidentified peptide, most probably belonging to SuSy2, the second most abundant isoform in M. truncatula root nodules. TiO(2)-phosphopeptide enrichment led to the identification of not only a phosphorylation site at Ser11 in SuSy1, but also of several novel phosphorylation sites present in other root nodule proteins such as alkaline invertase (AI; EC 3.2.1.26) and an RNA-binding protein.

Use of recombinant iron-superoxide dismutase as a marker of nitrative stress.

Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric oxide with superoxide radical. ONOO(-) is a strong oxidant molecule capable of nitrating peptides and proteins at the phenyl side chain of the tyrosine residues. In the present work, bovine serum albumin (BSA) and recombinant iron-superoxide dismutase from the plant cowpea (Vu_FeSOD) are used as target molecules to estimate ONOO(-) production. The method employs the compound SIN-1, which simultaneously generates *NO and O(2)(-) in aerobic aqueous solutions. First, assay conditions were optimized incubating BSA with different concentrations of SIN-1, and at a later stage, the effect on the tyrosine nitration and catalytic activity of Vu_FeSOD was examined by in-gel activity and spectrophotometric assays. Both BSA and Vu_FeSOD are nitrated in a dose-dependent manner, and, at least in BSA nitration, the reaction seems to be metal catalyzed.

Evidence for transcriptional and post-translational regulation of sucrose synthase in pea nodules by the cellular redox state.

Nitrogen fixation (NF) in legume nodules is very sensitive to environmental constraints. Nodule sucrose synthase (SS; EC 2.4.1.13) has been suggested to play a crucial role in those circumstances because its downregulation leads to an impaired glycolytic carbon flux and, therefore, a depletion of carbon substrates for bacteroids. In the present study, the likelihood of SS being regulated by oxidative signaling has been addressed by the in vivo supply of paraquat (PQ) to nodulated pea plants and the in vitro effects of oxidizing and reducing agents on nodule SS. PQ produced cellular redox imbalance leading to an inhibition of NF. This was preceded by the downregulation of SS gene expression, protein content, and activity. In vitro, oxidizing agents were able to inhibit SS activity and this inhibition was completely reversed by the addition of dithiothreitol. The overall results are consistent with a regulation model of nodule SS exerted by the cellular redox state at both the transcriptional and post-translational levels. The importance of such mechanisms for the regulation of NF in response to environmental stresses are discussed.

The application of ascorbate or its immediate precursor, galactono-1,4-lactone, does not affect the response of nitrogen-fixing pea nodules to water stress.

Nitrogen fixation in legumes is dramatically inhibited by abiotic stresses, and this reduction is often associated with oxidative damage. Although ascorbate (ASC) has been firmly associated with antioxidant defence, recent studies have suggested that the functions of ASC are related primarily to developmental processes. This study examines the hypothesis that ASC is involved in alleviating the oxidative damage to nodules caused by an increase in reactive oxygen species (ROS) under water stress. The hypothesis was tested by supplying 5mM ASC to pea plants (Pisum sativum L.) experiencing moderate water stress (ca. -1 MPa) and monitoring plant responses in relation to those experiencing the same water stress without ASC. A supply of exogenous ASC increased the nodule ASC+dehydroascorbate (DHA) pool compared to water-stressed nodules without ASC, and significantly modulated the response to water stress of the unspecific guaiacol peroxidase (EC 1.11.1.7) in leaves and nodules. However, ASC supply did not produce recovery from water stress in other nodule antioxidant enzymes, nodule carbon and nitrogen enzymes, or nitrogen fixation. The supply of the immediate ASC precursor, galactono-1,4-lactone (GL), increased the nodule ASC+DHA pool, but also failed to prevent the decline of nitrogen fixation and the reduction of carbon flux in nodules. These results suggest that ASC has a limited role in preventing the negative effects of water stress on nodule metabolism and nitrogen fixation.

Reduced carbon availability to bacteroids and elevated ureides in nodules, but not in shoots, are involved in the nitrogen fixation response to early drought in soybean.

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.