Mutation of either arginase structural gene (ARGAH1 or ARGAH2 encoding arginine [Arg] amidohydrolase-1 and -2, respectively) resulted in increased formation of lateral and adventitious roots in Arabidopsis (Arabidopsis thaliana) seedlings and increased nitric oxide (NO) accumulation and efflux, detected by the fluorogenic traps 3-amino,4-aminomethyl-2′,7′-difluorofluorescein diacetate and diamino-rhodamine-4M, respectively. Upon seedling exposure to the synthetic auxin naphthaleneacetic acid, NO accumulation was differentially enhanced in argah1-1 and argah2-1 compared with the wild type. In all genotypes, much 3-amino,4-aminomethyl-2′,7′-difluorofluorescein diacetate fluorescence originated from mitochondria. The arginases are both localized to the mitochondrial matrix and closely related. However, their expression levels and patterns differ: ARGAH1 encoded the minor activity, and ARGAH1-driven β-glucuronidase (GUS) was expressed throughout the seedling; the ARGAH2∷GUS expression pattern was more localized. Naphthaleneacetic acid increased seedling lateral root numbers (total lateral roots per primary root) in the mutants to twice the number in the wild type, consistent with increased internal NO leading to enhanced auxin signaling in roots. In agreement, argah1-1 and argah2-1 showed increased expression of the auxin-responsive reporter DR5∷GUS in root tips, emerging lateral roots, and hypocotyls. We propose that Arg, or an Arg derivative, is a potential NO source and that reduced arginase activity in the mutants results in greater conversion of Arg to NO, thereby potentiating auxin action in roots. This model is supported by supplemental Arg induction of adventitious roots and increased NO accumulation in argah1-1 and argah2-1 versus the wild type.

Warm season N₂-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin. Improved understanding of ureide biochemistry includes two ‘additional’ enzymatic steps in the conversion of uric acid to allantoin in the nodule and the mechanism of allantoin and allantoate breakdown in leaf tissue. Ureide accumulation and metabolism in leaves have also been implicated in the feedback inhibition of N₂-fixation under water limitation. Sensitivity to water deficit differs among soybean cultivars. Manganese supplementation has been shown to modify relative susceptibility or tolerance to this process in a cultivar-dependent manner. A discussion of the potential roles for ureides and manganese in the feedback inhibition of N₂-fixation under water limitation is presented. The existing data are examined in relation to potential changes in both aerial carbon and nitrogen supply under water deficit.

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), classified as Methylobacterium spp., are persistent colonizers of plant leaf surfaces. Reports of PPFM-plant dialogue led us to examine cytokinin production by PPFMs. Using immunoaffinity and high-performance liquid chromatography (HPLC) purification, we obtained 22 to 111 ng of trans -zeatin per liter from culture filtrates of four PPFM leaf isolates (from Arabidopsis , barley, maize, and soybean) and of a Methylobacterium extorquens type culture originally recovered as a soil isolate. We identified the zeatin isolated as the trans isomer by HPLC and by a radioimmunoassay in which monoclonal antibodies specific for trans -hydroxylated cytokinins were used. Smaller and variable amounts of trans -zeatin riboside were also recovered. trans -Zeatin was recovered from tRNA hydrolysates in addition to the culture filtrates, suggesting that secreted trans -zeatin resulted from tRNA turnover rather than from de novo synthesis. The product of the miaA gene is responsible for isopentenylation of a specific adenine in some tRNAs. To confirm that the secreted zeatin originated from tRNA, we mutated the miaA gene of M. extorquens by single exchange of an internal miaA fragment into the chromosomal gene. Mutant exconjugants, confirmed by PCR, did not contain zeatin in their tRNAs and did not secrete zeatin into the medium, findings which are consistent with the hypothesis that all zeatin is tRNA derived rather than synthesized de novo. In germination studies performed with heat-treated soybean seeds, cytokinin-null ( miaA ) mutants stimulated germination as well as wild-type bacteria. While cytokinin production may play a role in the plant-PPFM interaction, it is not responsible for stimulation of germination by PPFMs.

Summary: We describe the identification and functional characterization of two Arabidopsis mitochondrial basic amino acid carriers (BAC), AtmBAC1 and AtmBAC2, which are related to the yeast ornithine (Orn) carrier Ort1p, also known as Arg11p. The arg11 mutant requires arginine (Arg) supplementation because it fails to export sufficient ornithine from the mitochondrion to the cytosol where it is converted to arginine. AtmBAC1 and, to a lesser extent, AtmBAC2 partially replaced the function of Ort1p in yeast arg11. The more efficient putative carrier, AtmBAC1, was expressed in E. coli, purified, and reconstituted into phospholipid vesicles, where it transported the basic l‐amino acids arginine, lysine, ornithine and histidine (in order of decreasing affinity). AtmBAC1 recognized l‐histidine whereas both yeast Ort1p and the mammalian ortholog ORNT1p do not. Also different from ORNT1p, AtmBAC1 did not transport citrulline. AtmBAC1 appeared to be more stereospecific than the yeast and mammalian ornithine carriers, exhibiting greater preference for the l‐forms of arginine, lysine and ornithine. By RT‐PCR, both AtmBAC1 and AtmBAC2 transcripts were detected in stems, leaves, flowers, siliques, and seedlings. Expression of AtmBAC1 in seedlings is consistent with its involvement in Arg breakdown in early seedling development, i.e. delivery of Arg to mitochondrial arginase. The K_m (0.19 mm) for Arg uptake by AtmBAC1 was close to the value we previously determined for the saturable component of Arg uptake into intact mitochondria from soybean seedling cotyledons.

Background: Microarrays are an important tool with which to examine coordinated gene expression. Soybean (Glycine max) is one of the most economically valuable crop species in the world food supply. In order to accelerate both gene discovery as well as hypothesis-driven research in soybean, global expression resources needed to be developed. The applications of microarray for determining patterns of expression in different tissues or during conditional treatments by dual labeling of the mRNAs are unlimited. In addition, discovery of the molecular basis of traits through examination of naturally occurring variation in hundreds of mutant lines could be enhanced by the construction and use of soybean cDNA microarrays. Results: We report the construction and analysis of a low redundancy 'unigene' set of 27,513 clones that represent a variety of soybean cDNA libraries made from a wide array of source tissue and organ systems, developmental stages, and stress or pathogen-challenged plants. The set was assembled from the 5' sequence data of the cDNA clones using cluster analysis programs. The selected clones were then physically reracked and sequenced at the 3' end. In order to increase gene discovery from immature cotyledon libraries that contain abundant mRNAs representing storage protein gene families, we utilized a high density filter normalization approach to preferentially select more weakly expressed cDNAs. All 27,513 cDNA inserts were amplified by polymerase chain reaction. The amplified products, along with some repetitively spotted control or 'choice' clones, were used to produce three 9,728-element microarrays that have been used to examine tissue specific gene expression and global expression in mutant isolines. Conclusions: Global expression studies will be greatly aided by the availability of the sequence-validated and low redundancy cDNA sets described in this report. These cDNAs and ESTs represent a wide array of developmental stages and physiological conditions of the soybean plant. We also demonstrate that the quality of the data from the soybean cDNA microarrays is sufficiently reliable to examine isogenic lines that differ with respect to a mutant phenotype and thereby to define a small list of candidate genes potentially encoding or modulated by the mutant phenotype.