Isolation of a subfamily of genes for R2R3-MYB transcription factors showing up-regulated expression under nitrogen nutrient-limited conditions.

Plant R2R3-MYB transcription factors are encoded by more than 100 copies of genes. In this study, we attempted to isolate some members of the R2R3-MYB superfamily involved in regulation of nitrogen fixation in legumes. A library of 300 recombinant plasmid clones containing the R2R3-MYB fragments of the superfamily was screened by differential hybridization to isolate R2R3-MYB genes whose expression was up-regulated under nitrogen nutrient-limited conditions. Two groups of clones were identified, each of which seemed to represent a gene responsive to nitrogen starvation. The entire coding regions for the genes were further isolated by PCR and were designated LjMYB101 and LjMYB102. By screening a genomic library of Lotus japonicus with a probe derived from LjMYB101, the third gene, LjMYB103, was isolated. In addition, a candidate for the soybean orthologue of LjMYB101 was isolated and designated GmMYB101. Sequence alignment of the genes with members of the plant R2R3-MYB superfamily showed that they all belonged to the subgroup 10 of the superfamily. The expression analysis of the genes showed that the organ-specific and nitrate-regulated expression profile of MYB101 was very similar to that of CHS in Lotus as well as in soybean, suggesting a possible role for MYB101 in regulation of flavonoid biosynthesis in response to nitrate starvation. On the other hand, an interesting relationship, in structure and function, was found between LjMYB101 and LjGln1, suggesting an alternative role for MYB101 in regulation of nitrogen metabolism.