Although common bean (Phaseolus vulgaris) is the most important grain legume in the developing world for human consumption, few genomic resources exist for this species. The objectives of this research were to develop expressed sequence tag (EST) resources for common bean and assess nodule gene expression through high-density macroarrays. We sequenced a total of 21,026 ESTs derived from 5 different cDNA libraries, including nitrogen-fixing root nodules, phosphorus-deficient roots, developing pods, and leaves of the Mesoamerican genotype, Negro Jamapa 81. The fifth source of ESTs was a leaf cDNA library derived from the Andean genotype, G19833. Of the total high-quality sequences, 5,703 ESTs were classified as singletons, while 10,078 were assembled into 2,226 contigs producing a nonredundant set of 7,969 different transcripts. Sequences were grouped according to 4 main categories, metabolism (34%), cell cycle and plant development (11%), interaction with the environment (19%), and unknown function (36%), and further subdivided into 15 subcategories. Comparisons to other legume EST projects suggest that an entirely different repertoire of genes is expressed in common bean nodules. Phaseolus-specific contigs, gene families, and single nucleotide polymorphisms were also identified from the EST collection. Functional aspects of individual bean organs were reflected by the 20 contigs from each library composed of the most redundant ESTs. The abundance of transcripts corresponding to selected contigs was evaluated by RNA blots to determine whether gene expression determined by laboratory methods correlated with in silico expression. Evaluation of root nodule gene expression by macroarrays and RNA blots showed that genes related to nitrogen and carbon metabolism are integrated for ureide production. Resources developed in this project provide genetic and genomic tools for an international consortium devoted to bean improvement.

Soybean (Glycine max L.) is an important source of protein for human and animal nutrition, as well as a major source of vegetable oil. The soybean crop requires adequate water all through its growth period to attain its yield potential, and the lack of soil moisture at critical stages of growth profoundly impacts the productivity. In this study, utilizing 1H NMR-based metabolite analysis combined with the physiological studies we assessed the effects of short-term water stress on overall growth, nitrogen fixation, ureide and proline dynamics, as well as metabolic changes in drought tolerant (NA5009RG) and sensitive (DM50048) genotypes of soybean in order to elucidate metabolite adjustments in relation to the physiological responses in the nitrogen-fixing plants towards water limitation. The results of our analysis demonstrated critical differences in physiological responses between these two genotypes, and identified the metabolic pathways that are affected by short-term water limitation in soybean plants. Metabolic changes in response to drought conditions highlighted pools of metabolites that play a role in the adjustment of metabolism and physiology of the soybean varieties to meet drought effects.

Nitrogen metabolism including nitrate reductase (EC 1.6.6.1), glutamate dehydroge‐nase (EC 1.4.1.2) and glutamate‐oxalacetate aminotransferase (EC 2.6.1.1) activities were studied during growth of petals taken from carnation flowers (Dianthus caryophyllus L. cv. Sir Arthur) together with senescence parameters (lipid hydroper‐oxides, soluble amino acids and permeability). A slight decline in nitrogen percentage on a dry weight basis was found together with a sharp decrease in nitrate reduct‐ase, glutamate‐oxalacetate aminotransferase and glutamate dehydrogenase activities during the maximum growth phase, which was characterized by increase in respiration, dry weight, length, organic nitrogen and DNA per petal. Changes generally associated with senescence, like lipid hydroperoxide and soluble ammo nitrogen accumulation and increases in permeability began to appear already during early growth. The results indicate that permeability and proteolysis may be closely related. The possible significance of the decrease in nitrogen percentage and enzyme activities during growth of petals is discussed.

Sucrose synthase (SS), the key sucrose hydrolytic enzyme (EC 2.4.1.13), plays an important role in N(2)-fixing nodule metabolism. It has also been proposed that N(2) fixation in soybean nodules could be mediated by the potential to metabolize sucrose. The isolation and characterization of a nodule-enhanced SS full-length cDNA clone from the bean Phaseolus vulgaris is reported here. Southern blot analysis indicated that there are at least two SS genes in beans. Using a 3' specific probe from this SS cDNA clone, it was possible to identify a nodule-enhanced SS gene (PvSSn), which is expressed almost exclusively in nodules. A second gene (PvSS), which is expressed in all tissues tested, was detected using a coding region probe. Nodule-enhanced PvSSn transcript levels, but not the enzyme activity or protein amount, is reduced during nodule development. These data indicated that this reduction could be due to a limitation on the carbon availability in the nodule. PvSSn expression is reduced in the asparagine-treated nodules. By contrast, PvSSn transcript levels in nodules increased in the presence of glutamine, allantoin and allopurinol. This result suggests a relationship between ureide transport and SS regulation and could help in understanding why the ureide transport mechanism is activated during nitrogen fixation in bean.

A cDNA clone, designated as PvNAS2, encoding asparagine amidotransferase (asparagine synthetase) was isolated from nodule tissue of common bean (Phaseolus vulgaris cv. Negro Jamapa). Southern blot analysis indicated that asparagine synthetase in bean is encoded by a small gene family. Northern analysis of RNAs from various plant organs demonstrated that PvNAS2 is highly expressed in roots, followed by nodules in which it is mainly induced during the early days of nitrogen fixation. Investigations with the PvNAS2 promoter gusA fusion revealed that the expression of PvNAS2 in roots is confined to vascular bundles and meristematic tissues, while in root nodules its expression is solely localized to vascular traces and outer cortical cells encompassing the central nitrogen-fixing zone, but never detected in either infected or non-infected cells located in the central region of the nodule. PvNAS2 is down-regulated when carbon availability is reduced in nodules, and the addition of sugars to the plants, mainly glucose, boosted its induction, leading to the increased asparagine production. In contrast to PvNAS2 expression and the concomitant asparagine synthesis, glucose supplement resulted in the reduction of ureide content in nodules. Studies with glucose analogues as well as hexokinase inhibitors suggested a role for hexokinase in the sugar-sensing mechanism that regulates PvNAS2 expression in roots. In light of the above results, it is proposed that, in bean, low carbon availability in nodules prompts the down-regulation of the asparagine synthetase enzyme and concomitantly asparagine production. Thereby a favourable environment is created for the efficient transfer of the amido group of glutamine for the synthesis of purines, and then ureide generation.