Regulation of Isoflavone Biosynthesis by miRNAs in Two Contrasting Soybean Genotypes at Different Seed Developmental Stages.

Owing to the presence of nutritionally important, health-promoting bioactive compounds, especially isoflavones, soybean has acquired the status of a functional food. miRNAs are tiny riboregulator of gene expression by either decreasing and/or increasing the expression of their corresponding target genes. Despite several works on identification and functional characterization of plant miRNAs, the role of miRNAs in the regulation of isoflavones metabolism is still a virgin field. In the present study, we identified a total of 31 new miRNAs along with their 245 putative target genes from soybean seed-specific ESTs using computational approach. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates metabolism and genetic information processing. Out of that, a total of 5 miRNAs (Gma-miRNA12, Gma-miRNA24, Gma-miRNA26, Gma-miRNA28, and Gma-miRNA29) were predicted and validated for their probable role during isoflavone biosynthesis. We also validated their five target genes using RA-PCR, which is as good as 5'RLM-RACE. Temporal regulation [35 days after flowering, 45, 55, and 65 DAF] of miRNAs and their targets showed differential expression schema. Differential expression of Gma-miR26 and Gma-miRNA28 along with their corresponding target genes (Glyma.10G197900 and Glyma.09G127200) showed a direct relationship with the total isoflavone content. Therefore, understanding the miRNA-based genetic regulation of isoflavone pathway would assist in selection and manipulation to get high-performing soybean genotypes with better isoflavone yield.

A cis-element responsible for cGMP in the promoter of the soybean chalcone synthase gene.

The cyclic nucleotides cGMP and cAMP have been reported to play key roles in the regulation of plant processes and responses. We have previously reported that several genes encoding flavonoid biosynthetic enzymes, including chalcone synthase (CHS) in soybean (Glycine max L.), were induced by cGMP but not cAMP. The soybean genome contains nine CHS gene copies (GmCHS1-9). We investigated the responsiveness of several GmCHS genes to cGMP, cAMP, NO, and white light. Quantitative RT-PCR analysis showed that the transcript levels of GmCHS7 and GmCHS8 were increased by 3.6- and 3.8-fold, respectively, with cGMP whereas the transcript levels of GmCHS2 remained constant. Although cAMP had no effect on the transcript levels of the three genes, NO had an activation effect on all three. White light activated the three genes in a transient manner, with GmCHS2, GmCHS7, and GmCHS8 transcript levels increasing 3-fold after 3 h and decreasing to basal levels after 9 h. The GmCHS8 promoter contains several important cis-elements, including the G-box and H-box forming the Unit-I-like sequence and the MYB binding sequence, a target of the GmMYB176 transcription factor regulating the expression of GmCHS8. A transient gene expression assay revealed the activation of the Unit-I-like sequence, but not of the MYB binding sequence, by cGMP. The combination of G-box and H-box was necessary for cGMP responsiveness. Taken together, these results suggest that the Unit-I-like sequence in the promoters of GmCHS7 and GmCHS8 is a cGMP responsive cis-element in these genes and that NO exerts its effect via cis-elements other than the Unit-I-like sequence.