The role of sucrose synthase in the response of soybean nodules to drought

Experiments were carried out to investigate the effects of droughtstress on enzymatic activities related to carbon and nitrogenmetabolism in soybean nodules. Gradual drought stress, imposedby withholding water nutrients, resulted in declines in thewater potential of leaves and nodules consistent with a significantdecline in N2 fixation. However, the amounts of nitrogenasecomponents 1 and 2 were virtually unaffected by drought stress.Similarly, no significant changes could be detected in aspartateaminotransferase, phosphoenolpyruvate carboxylase, glutaminesynthetase or alkaline invertase activities throughout the experiment.In contrast, sucrose synthase (SS), one of the enzymes involvedin sucrose metabolism in legume nodules, declined dramaticallyin activity and in content within a few days of withholdingwater. Coincident with this decline in SS activity were significantincreases in the nodule contents of sucrose, total free aminoacids and ureides. The amounts of proline, however, did notincrease until some days later. It is suggested that SS mayplay a key role in the regulation of nodule carbon metabolismand, therefore, of nitrogen fixation under drought stress conditions. Key words: Glycine max, soybean, nodule metabolism, drought stress, sucrose synthase     

Continuous CO2 enrichment leads to increased nodule biomass, carbon availability to nodules and activity of carbon-metabolising enzymes but does not enhance specific nitrogen fixation in pea

Recent research has shown that nodule nitrogen fixation is limited under a wide range of environmental constraints by lowered carbon flux within the nodule due to down-regulation of sucrose synthase activity. The aim of this work was to elucidate whether an increase in both carbon flux and activity of enzymes of carbon metabolism in nodules may lead to an increased nitrogen fixation. We report the effects caused by a continuous exposure to atmospheric CO₂ enrichment in nodulated pea plants. CO₂ enrichment led to an enhanced whole-plant growth and increased nodule biomass. Moreover, nodules of plants grown at increased CO₂ showed a higher sugar content as well as enhancement of some activities related to nodule carbon metabolism, such as sucrose synthase, UDP glucose pyrophosphorylase and phosphoenolpyruvate carboxylase. Indeed, acetylene reduction activity, measured by the classical technique, was increased more than four times. However, when specific nitrogen fixation was determined as hydrogen evolution, no significant differences were detected, consistent with the lack of changes of enzymes involved in nitrogen metabolism such as glutamate synthase and aspartate aminotransferase. These results are discussed in the context of the regulation of nitrogen fixation and nodule metabolism.     

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     

Effect of hydro- and osmopriming of chickpea (Cicer arietinum L.) seeds on enzymes of sucrose and nitrogen metabolism in nodules

Three year data on the effect of water- and mannitol (4%) priming of chickpea seeds (12 h at 25°C) showed higher number and biomass of nodules in the plants from primed seeds than from non-primed seeds. The biomass of nodules increased to 75 DAS but decreased by 90 DAS. Activities of sucrose metabolism enzymes (sucrose synthase (SS) and alkaline invertase) and of nitrogen metabolism (glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)) in nodules of primed and non-primed crops during development are reported. SS and alkaline invertase activities increased to 70 DAS and then decreased. In primed plants, the higher SS activity in nodules at 60 and 70 DAS might be responsible for providing more energy and carbon skeleton for nitrogen fixation and for ammonium assimilation in primed plants. At 85 DAS, though the SS activity decreased in comparison with the earlier growth stages, it was still higher in nodules of the primed crops than the non-primed crop. Activity of alkaline invertase was maximum at 70 DAS in the nodules of primed and non-primed crops. Priming increased nodule GS activity at 70 and 85 DAS. GOGAT activity was unaffected by priming but GDH activity was greater in nodules from primed crops at 50 DAS. Elevated SS and GS nodule activities in primed chickpeas might be responsible in increasing nodule biomass and metabolic activity thereby increasing seed fill.     

Anatomy, physiology and biochemistry of root nodules of Sprint-2 Fix-, a symbiotically defective mutant of pea (Pisum sativum L.)

A plant-determined pea mutant Sprint-2 Fix^– and the parentalline Sprint-2 were compared for selected physiological and biochemicalparameters. The Fix^– mutation prevented differentiationof Rhizobium leguminosarum bacteria into bacteroids and producedlarge, white, non-fixing nodules. These lacked nitrogenase-linkedrespiration, but had a background rate of CO₂ evolution similarto the normal Fix⁺ nodules. The cortical structure of the ineffectivenodules suggests the existence of an oxygen diffusion barrierand this was supported by a low oxygen concentration in thecentral region (0.5–3.0%), measured using an O₂ sensitivemicro-electrode. Sucrose and starch contents were similar innormal and ineffective nodules while ononitol content was about15 times lower in the Fix^– nodules. The distribution ofstarch was also different in the two nodule types. The activitiesof glutamine synthetase (GS), sucrose synthase (SS), phosphoenolpyruvatecarboxylase (PEPC) and alanine pyruvate aminotransferase (APAT)were markedly higher in Fix⁺ nodules while the activities ofpyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) andglutamate dehydrogenase (GDH) were higher in Fix^– nodules.The data from immunodetection of host nodule proteins confirmedthe reduced levels of sucrose synthase and the almost completeabsence of glutamine synthetase and leghaemoglobin in mutantnodules. There was no significant difference in the amount ofnitrogenase component 1 extracted from the microsymbiont ofnormal and ineffective nodules, but component 2 was hardly detectablein the Fix^– mutant. Key words: Pisum sativum, Fix^– mutant, nodules     

Cadmium in white lupin nodules: Impact on nitrogen and carbon metabolism

The aims of this work were to investigate the microlocalisation of cadmium (Cd) in Lupinus albus L. cv. Multolupa nodules, and to determine its effects on carbon and nitrogen metabolism. Nodulated white lupin plants were grown in a growth chamber with or without Cd (150 μM). Energy-dispersive X-ray microanalysis showed the walls of the outer nodule cortex cells to be the main area of Cd retention, helping to reduce the harmful effect Cd might have on the amount of N₂ fixed by the bacteroids. Sucrose synthase activity declined by 33% in the nodules of the Cd-treated plants, and smaller reductions were recorded in glutamine synthetase, aspartate aminotransferase, alkaline invertase and NADP-dependent isocitrate dehydrogenase activities. The Cd treatment also sharply reduced nodule concentrations of malate, succinate and citrate, while that of starch doubled, but that of sucrose experienced no significant change. In summary, the present results show that white lupins accumulate significant amounts of Cd in their root nodules. However, the activity of some enzymes involved in ammonium assimilation did decline, promoting a reduction in the plant N content. The downregulation of sucrose synthase limits the availability of carbon to the bacteroids, which might interfere with their respiration. Carbon metabolism therefore plays a primary role in the impaired function of the white lupin root nodule caused by Cd, while N metabolism appears to have a more secondary involvement.     

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 Water Stress on Nodule Physiology and Biochemistry of a Drought Tolerant Cultivar of Common Bean (Phaseolus vulgarisL.)

Plants of EMGOPA-201, a drought tolerant cultivar of commonbean(Phaseolus vulgaris), were maintained either at 90% soilfield capacity (SFC) or stressed by reducing SFC to 70, 50 or30% over a 10 d period. Plant dry weight was not affected byany of these treatments although the number and weight of noduleswas reduced at 50 and 30% SFC. Nitrogenase activity, determinedby the acetylene reduction assay (ARA), was also reduced, ona plant basis, at 50% SFC and was almost stopped at 30% SFC.The latter treatment caused a marked increase in nodule O₂diffusionresistance and induced nodule senescence. A time-course analysisof the 10 d 30% SFC treatment showed a decrease in leaf waterpotential from -0.5 to -0.87 MPa by 8 d, with a cessation ofdry weight increase after 3 d, when leaf water potential was-0.65 MPa. Proteins in the host plant fraction of nodules decreasedto 50% of control values by 10 d and leghaemoglobin (Lb) contentwas also lower at this stage. The activity of sucrose synthase(SS) showed a 76% reduction between 3 and 6 d, whilst glutamatesynthase (GOGAT) activity showed a 40% reduction. The activityof other key enzymes of carbon metabolism was also reduced after10 d. Nodule sucrose content increased to double that of controlnodules by 6 d, before declining back to control levels at 10d. Starch content fell by 3 d and continued to fall throughoutthe stress period. The results are discussed in terms of droughttolerance strategies in relation to growth and metabolism inwhole plants and nodules.Copyright 1999 Annals of Botany Company. Phaseolus vulgaris,common bean, water stress, nitrogen fixation, oxygen diffusion, acetylene reduction, enzyme activity, carbon metabolism.     

Nitrogen transfer in the interface between the symbionts in pea root nodules

Transport mechanisms for transfer of nitrogen from the bacteroid side across the symbiosome membrane of pea (Pisum sativum L.) root nodules were identified by the use of energised bacteroid side-out symbiosome membrane vesicles. Such membrane vesicles were used to study a mechanism with high capacity for transport of ammonium and another mechanism capable of transporting aspartate. Both transport mechanisms are voltage driven and the rate of transport relates positively to the magnitude of the imposed membrane potentials. Competition for transport between ammonium and aspartate was not observed. The ammonium transporter has been identified as a voltage-driven channel whereas the symbiosome membrane aspartate transporter appears to be a H⁺/aspartate symport. The results suggest that nitrogen transfer between the symbionts in pea root nodules involves transfer of amino acids as well as ammonium. In the symbiosome subfraction, which represents the interface between the symbionts, specific aspartate aminotransferase activity was more than four times as high as in the bacteroid cytosol. This finding supports a hypothesis that transamination cycles operating between the symbionts may constitute a component of the transfer of nitrogen between the symbionts.     

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.     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.     

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O₂ limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O₂-limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO₂ fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O₂ regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.     

Comparison of the Protein Composition and Enzymatic Activities during Development between Effective and Plant-Determined Ineffective Nodules in Pea

The protein composition and enzymatic activities during developmentof ineffective nodules, produced by mutant E135 (sym 13) ofpea (Pisum sativum L.), were compared with those of the nitrogen-fixingnodules of the normal parent, the Sparkle cultivar. The proteincomposition of 3-week-old E135 nodules, as determined by SDS-polyacrylamidegel electrophoresis, was quite similar to that of Sparkle nodules.After 4 weeks, however, the intensities of bands of 15-, 38-,and 87-kDa polypeptides were lower in the case of E135 nodules.Western blot analysis using a "nodule-specific" antiserum revealedthat most nodulins could be detected in 3-week-old E135 nodules,but a 35.5-kDa nodulin disappeared after 5 weeks and severalnovel peptides ranging in molecular weight from 26 to 31 kDaappeared after 6 weeks in E135 nodules. The activities of glutaminesynthetase, glutamate synthase, alanine-pyruvate aminotransferase,sucrose synthase, and phosphoenolpyruvate carboxylase increasedduring development of Sparkle nodules, but such increases werenot found in E135 nodules after 5 weeks. These results showthat the nodules of E135 begin to develop normally but differfrom those of Sparkle within 4 weeks, indicating that, duringearly stages of nodule development, the protein compositionand activities of enzymes involved in carbon and nitrogen metabolismare not regulated by the presence or absence of nitrogenaseactivity. (Received February 26, 1993; Accepted May 19, 1993)     

Glutamate Oxaloacetate Transaminase in Pea Root Nodules : Participation in a Malate/Aspartate Shuttle between Plant and Bacteroid

Glutamate oxaloacetate transaminase (l-glutamate: oxaloacetate aminotransferase, EC 2.6.1.1 [GOT]), a key enzyme in the flow of carbon between the organic acid and amino acid pools in pea (Pisum sativum L.) root nodules, was studied. By ion exchange chromatography, the presence of two forms of GOT in the cytoplasm of pea root nodule cells was established. The major root nodule form was present in only a small quantity in the cytoplasm of root cells. Fractionation of root nodule cell extracts demonstrated that the increase in the GOT activity during nodule development was due to the increase of the activity in the cytoplasm of the plant cells, and not to an increase in activity in the plastids or in the mitochondria. The kinetic properties of the different cytoplasmic forms of GOT were studied. Some of the K_m values differed, but calculations indicated that not the kinetic properties but a high concentration of the major root nodule form caused the observed increase in GOT activity in the pea root nodules. It was found that the reactions of the malate/aspartate shuttle are catalyzed by intact bacteroids, and that these reactions can support nitrogen fixation. It is proposed that the main function of the nodule-stimulated cytoplasmic form of GOT is participation in this shuttle.     

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.     

Characterization of a Paracoccus denitrificans mutant pleiotropically impaired in nitrogen metabolism

A Tn5 insertional prototrophic mutant of Paracoccus denitrificans (UBM219) was generated which grew on high (>1 mM) but not low (<0.5 mM) ammonium as sole nitrogen source. It did not utilize nitrate and most amino acids except glutamate and aspartate. UBM219 showed more than 10-fold lower levels of ammonium (methylammonium) transport, aspartate and alanine aminotransferase, but more than 10-fold higher activities of glutamate dehydrogenase and glutamate synthase. This pleiotropy indicates a mutation in a regulatory gene affecting nitrogen metabolism in general. — Ammonia assimilation pathways and regulation in Paracoccus resemble the patterns in enterobacteria with the exception, that alanine is generated by amino transfer from glutamate to pyruvate.Non-standard abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GluDH glutamate dehydrogenase - GPT glutamate/pyruvate aminotransferase - GOT glutamate/oxaloacetate aminotransferase     

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.