Characterization of GmSAT1 and related proteins from legume nodules.

Nitrogen is an essential nutrient for plant growth and is normally obtained from the soil medium. Legumes are a unique group of plants that acquired the ability to form a symbiosis with nitrogen-fixing bacteria, called rhizobia, enabling growth in nitrogen-poor soils. GmSAT1, a predicted bHLH transcription factor from soybean, is essential for nitrogen fixation; however the role of this protein remains elusive (Kaiser et al., 1998; Loughlin, 2007). In this work, a further functional characterization of GmSAT1 was undertaken. Using the promoters of known upregulated genes in yeast upon expression of GmSAT1, it was found that purified GmSAT1 directly interacts with DNA. Further, GFP-fusion analysis in onion epidermal cells, found that GmSAT1 localizes to the nucleus, as well as peripheral vesicles, demonstrating that GmSAT1 is a likely a transcription factor. Residues from both the N- and C-termini required for GmSAT1 activity were also identified by exchanging domains with GmSAT2, a protein that arose during the relatively recent whole-genome duplication in soybean. Recently, GmSAT1 was shown to be essential for proper nodule development in soybean (Loughlin, 2007). Therefore, a DNA microarray analysis was conducted to identify transcripts that are differentially expressed after silencing of GmSAT1 by RNA interference (RNAi) in soybean nodules. Of the ninety-five genes downregulated, twelve were associated with the circadian clock, potentially explaining the GmSAT1 RNAi phenotype. Investigations were also initiated in the model legume Medicago truncatula to identify and characterize GmSAT1 orthologues. Two genes, MtSAT1 and MtSAT2 were cloned and \nanalyzed. MtSAT1 and MtSAT2 are expressed in roots and the inner cortex of nodules, similar to GmSAT1. By in planta GFP-fusion analysis, both MtSAT1 and MtSAT2 were found to associate with vesicles and the nucleus. Insertional mutations in either gene alone did not render a phenotype, however downregulating both genes by RNAi disrupted nodule formation. Using the sequence of a newly discovered ammonium channel protein from yeast (ScAMF1), which is activated by GmSAT1, a novel subfamily of major facilitator transporter proteins (MFSs) from plants was identified. Interestingly, members of the MFS gene family are found linked with GmSAT1 loci in soybean, as well as M. truncatula and many sequenced dicots. GmMFS1.3, a representative from soybean, was cloned and characterized. GmMFS1.3 expression was localized to the root stele and the inner cortex of nodules. Further, expression of GmMFS1.3 in yeast induced the uptake of methylammonium. Interestingly, GmMFS1.5 was found to be downregulated in GmSAT1 RNAi nodules. The link between GmSAT1 and the MFS transporters in planta will be the focus of future experiments. A novel receptor-like kinase protein was also characterized from soybean nodules. GmCaMK1 was identified in a protein interaction screen using conserved calmodulin as bait. The calmodulin-binding domain overlaps the GmCaMK1 kinase subdomain XI, however it was found that GmCaMK1 is able to auto-phosphorylate independent of calmodulin. Therefore, calmodulin binding may influence the interaction of GmCaMK1 with its phosphorylation targets. Taken together, these studies have enriched our knowledge of nitrogen fixation, a critically important component of agricultural practice.

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