Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels

Aspartate aminotransferase (AAT) is a key plant enzyme affecting nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C₄ species. To ascertain the molecular genetic characteristics and biochemical regulation of AAT, we have isolated a cDNA encoding the nodule-enhanced AAT (AAT-2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNA expression library with antibodies. Complementation of an Escherichia coli AAT mutant with the alfalfa nodule AAT-2 cDNA verified the identity of the clone. The deduced amino acid sequence of alfalfa AAT-2 is 53 and 47% identical to animal mitochondrial and cytosolic AATs, respectively. The deduced molecular mass of AAT-2 is 50,959 daltons, whereas the mass of purified AAT-2 is about 40 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein's N-terminal domain (amino acids 1-59) contains many of the characteristics of plastid-targeting peptides. We postulate that AAT-2 is localized to the plastid. Southern blot analysis suggests that AAT-2 is encoded by a small, multigene family. The expression of AAT-2 mRNA in nodules is severalfold greater than that in either leaves or roots. Northern and western blots showed that expression of AAT activity during effective nodule development is accompanied by a sevenfold increase in AAT-2 mRNA and a comparable increase in enzyme protein. By contrast, plant-controlled ineffective nodules express AAT-2 mRNA at much lower levels and have little to no AAT-2 enzyme protein. Expression of root nodule AAT-2 appears to be regulated by at least two events: the first is independent of nitrogenase activity; the second is associated with nodule effectiveness.     

PvUPS1, an allantoin transporter in nodulated roots of French bean

Nodulated legumes receive their nitrogen via nitrogen-fixing rhizobia, which exist in a symbiotic relationship with the root system. In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the fixed nitrogen is used for synthesis of the ureides allantoin and allantoic acid, the major long-distance transport forms of organic nitrogen in these species. The purpose of this investigation was to identify a ureide transporter that would allow us to further characterize the mechanisms regulating ureide partitioning in legume roots. A putative allantoin transporter (PvUPS1) was isolated from nodulated roots of French bean and was functionally characterized in an allantoin transport-deficient yeast mutant showing that PvUPS1 transports allantoin but also binds its precursors xanthine and uric acid. In beans, PvUPS1 was expressed throughout the plant body, with strongest expression in nodulated roots, source leaves, pods, and seed coats. In roots, PvUPS1 expression was dependent on the status of nodulation, with highest expression in nodules and roots of nodulated plants compared with non-nodulated roots supplied with ammonium nitrate or allantoin. In situ RNA hybridization localized PvUPS1 to the nodule endodermis and the endodermis and phloem of the nodule vasculature. These results strengthen our prediction that in bean nodules, PvUPS1 is involved in delivery of allantoin to the vascular bundle and loading into the nodule phloem.     

Purification, characterization, and cDNA cloning of profilin from Phaseolus vulgaris.

Profilin from common bean (Phaseolus vulgaris L.) was purified to homogeneity by poly-L-Pro affinity chromatography and gel filtration. The hypocotyl and symbiotic root nodule protein was detected as a single isoform with a 14.4-kD molecular mass and an isoelectric point of 5.3. Partial amino acid and DNA sequencing of a full-length cDNA clone confirmed its identity as profilin. An antibody generated against the purified protein binds to a protein with the same molecular mass in leaves and nodules. Immunolocalization of the protein showed a diffuse distribution in the cytoplasm of hypocotyls and nodules but enhanced staining at the vascular bundles. The strong identity of the sequence among the profilins of birch, maize, and bean suggests that it may play an important role in the signal transduction mechanism of plant cells and plant-bacterial symbioses.     

Cloning, characterization and mRNA expression analysis of PVAS1, a type I asparagine synthetase gene from Phaseolus vulgaris

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.). A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0. The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS. The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes. Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean. Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes.     

Aspartate aminotransferase in alfalfa root nodules : I. Purification and partial characterization

Aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1 [AAT]), a key enzyme in the assimilation of C and N compounds, was purified from the cytosol of alfalfa (Medicago sativa L.) root nodules. Isoforms that increased during nodule development, AAT-2a, AAT-2b, and AAT-2c, were purified greater than 447-fold to apparent homogeneity, and high titer polyclonal antibodies were produced. The native molecular weight of the AAT-2 isoforms was approximately 80 kilodatons with a subunit molecular weight of 40 kilodatons, indicating that the holoenzymes are dimers. The AAT-2 isoforms comprised approximately 0.4% of the total soluble nodule protein. The AAT specific activity was measured in leaf, stem, root, and nodule organs, and zymograms of each were compared. Enzyme activity was 4- to 37-fold greater in effective (nitrogen fixing) nodules than in leaves, stems, and roots. Effective nodule AAT-specific activity was 3- to 8-fold greater than that of plant-controlled ineffective nodules. No differences in K_m were observed between AAT-1 and AAT-2. Antibodies raised against AAT-2 were more selective against AAT-2 than AAT-1. Evidence obtained from zymograms suggests that the expression of alfalfa nodule AAT is controlled at two different gene loci, AAT-1 and AAT-2, resulting in different dimeric isoforms.     

Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L

The ureides allantoin and allantoate are key molecules in the transport and storage of nitrogen in ureide legumes. In shoots and leaves from Phaseolus vulgaris plants using symbiotically fixed nitrogen as the sole nitrogen source, ureide levels were roughly equivalent to those of nitrate-supported plants during the whole vegetative stage, but they exhibited a sudden increase at the onset of flowering. This rise in the level of ureides, mainly in the form of allantoate, was accompanied by increases in allantoinase gene expression and enzyme activity, consistent with developmental regulation of ureide levels mainly through the tissue-specific induction of allantoate synthesis catalysed by allantoinase. Moreover, surprisingly high levels of ureides were also found in non-nodulated plants fertilized with nitrate, at both early and late developmental stages. The results suggest that remobilized N from lower leaves is probably involved in the sharp rise in ureides in shoots and leaves during early pod filling in N(2)-fixing plants and in the significant amounts of ureides observed in non-nodulated plants.     

Evidence for sugar signalling in the regulation of asparagine synthetase gene expressed in Phaseolus vulgaris roots and nodules

A cDNA clone, designated as PvNAS2, encoding asparagine amidotransferase(asparagine synthetase) was isolated from nodule tissue of commonbean (Phaseolus vulgaris cv. Negro Jamapa). Southern blot analysisindicated that asparagine synthetase in bean is encoded by asmall gene family. Northern analysis of RNAs from various plantorgans demonstrated that PvNAS2 is highly expressed in roots,followed by nodules in which it is mainly induced during theearly days of nitrogen fixation. Investigations with the PvNAS2promoter gusA fusion revealed that the expression of PvNAS2in roots is confined to vascular bundles and meristematic tissues,while in root nodules its expression is solely localized tovascular traces and outer cortical cells encompassing the centralnitrogen-fixing zone, but never detected in either infectedor non-infected cells located in the central region of the nodule.PvNAS2 is down-regulated when carbon availability is reducedin nodules, and the addition of sugars to the plants, mainlyglucose, boosted its induction, leading to the increased asparagineproduction. In contrast to PvNAS2 expression and the concomitantasparagine synthesis, glucose supplement resulted in the reductionof ureide content in nodules. Studies with glucose analoguesas well as hexokinase inhibitors suggested a role for hexokinasein the sugar-sensing mechanism that regulates PvNAS2 expressionin roots. In light of the above results, it is proposed that,in bean, low carbon availability in nodules prompts the down-regulationof the asparagine synthetase enzyme and concomitantly asparagineproduction. Thereby a favourable environment is created forthe efficient transfer of the amido group of glutamine for thesynthesis of purines, and then ureide generation. Key words: Asparagine and ureide synthesis, asparagine synthetase, nodules, Phaseolus vulgaris, sugar signalling     

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

Soybean ureide transporters play a critical role in nodule development,function and nitrogen export

Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen‐fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long‐distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins – GmUPS1‐1 (XP_003516366) and GmUPS1‐2 (XP_003518768) – in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1‐1 and GmUPS1‐2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.     

Molecular and whole-plant responses to selection for enzyme activity in alfalfa root nodules: evidence for molecular compensation of aspartate aminotransferase expression

Summary The enzyme aspartate aminotransferase (AAT) plays a key role in the assimilation of fixed-N in alfalfa (Medicago sativa L.) root nodules. AAT activity in alfalfa nodules is due to the activity of two dimeric isozymes, AAT-1 and AAT-2, that are products of two distinct genes. Three forms of AAT-2 (AAT-2a, -2b, and-2c) have been identified. It was hypothesized that two alleles occur at the AAT-2 locus, giving rise to the three AAT-2 enzymes. In a prior study bidirectional selection for root nodule AAT and asparagine synthetase (AS) activities on a nodule fresh weight basis in two diverse alfalfa germ plasms resulted in high nodule enzyme activity subpopulations with about 20% more nodule AAT activity than low enzyme activity subpopulations. The objectives of the study presented here were to determine the inheritance of nodule AAT-2 production and to evaluate the effect of bidirectional selection for AAT and AS on AAT-2 allelic frequencies, the relative contributions of AAT-1 and AAT-2 to total nodule activity, nodule enzyme concentration, and correlated traits. Two alleles at the AAT-2 locus were verified by evaluating segregation of isozyme phenotypes among F₁ and S₁ progeny of crosses or selfs. Characterization of subpopulations for responses associated with selection was conducted using immunoprecipitation of in vitro nodule AAT activity, quantification of AAT enzyme protein by ELISA, and AAT activity staining of native isozymes on PAGE. Results indicate that selection for total AAT activity specifically altered the expression of the nodule AAT-2 isozyme. AAT-2 activity was significantly greater in high compared to low activity subpopulations, and high AAT subpopulations from both germ plasms had about 18% more AAT-2 enzyme (on a nodule fresh weight basis). No significant or consistent changes in AAT-2 genotypic frequencies in subpopulations were caused by selection for AAT activity. Since changes in AAT activity were not associated with changes in AAT-2 genotype, selection must have affected a change(s) at another locus (or loci), which indirectly effects the expression of nodule AAT.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products or vendors that might also be suitable     

Glutathione and homoglutathione synthesis in legume root nodules

High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and gamma-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for gamma-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants.     

Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules

NADH-dependent glutamate synthase (NADH-GOGAT) is a key enzyme in primary ammonia assimilation in Phaseolus vulgaris nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from the nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGAT-I (7.4 kb) and PvNADH-GOGAT-II (7.0 kb), which displayed an 83% homology between them, were isolated using cDNA library screening, 'cDNA library walking' and RT-PCR amplification. Southern analysis employing specific 5' cDNA probes derived from PvNADH-GOGAT-I and PvNADH-GOGAT-II indicated the existence of a single copy of each gene in the bean genome. Both these proteins contain ∼100 amino acid sequences theoretically addressing each isoenzyme to different subcellular compartments. RT-PCR analysis indicated that PvNADH-GOGAT-II expression is higher than PvNADH-GOGAT-I during nodule development. Expression analysis by RT-PCR also revealed that both of these genes are differentially regulated by sucrose. On the other hand, the expression of PvNADH-GOGAT-I , but not PvNADH-GOGAT-II, was inhibited with nitrogen compounds. In situ hybridization and promoter expression analyses demonstrated that the NADH-GOGAT-I and -II genes are differentially expressed in bean root and nodule tissues. In silico analyses of the NADH-GOGAT promoters revealed the presence of potential cis elements in them that could mediate differential tissue-specific, and sugar and amino acid responsive expression of these genes.     

Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.     

Enzymes of ureide synthesis in pea and soybean

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.

The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5′-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5′-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.