Deciphering Regulatory Mechanism(s) Underlying Isoflavone Biosynthesis and Accumulation in Soybean (Glycine max L.) Seeds

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 miRNAs during secondary metabolites biosynthesis in plants, 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. In addition, a total of 9 existing miRNAs and their 10 corresponding target genes were also identified by manual search the public databases. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates metabolism and genetic information processing. Out of 31 newly identified miRNAs, a total of 5 miRNAs (GmamiRNA12, 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 50 RLMRACE. In addition, 9 existing miRNAs and their 10 corresponding target genes were also validated in soybean for their probable role during isoflavonoid biosynthesis and accumulation. Temporal expression [35 days after flowering, 45, 55, and 65 DAF] study of all the miRNAs and their targets (newly identified as well as existing) in two contrasting soybean genotypes (NRC37: high isoflavone; and NRC7: low isoflavone) showed differential expression schema across all the stages studied. Furthermore, we also quantified various aglycosylated forms of isoflavones in above mentioned genotypes and stages to establish a correlation among the expression pattern of miRNA-target pair and total isoflavone accumulation. Result of temporal expression profiling of miRNAs and their corresponding target genes showed differential expression behaviour across all the stages and genotypes under study. Differential expression of Gma-miR26-Glyma.10G197900, Gma-miRNA28-Glyma.09G127200, Gma-miR159-MYB65, Gma-miRNA1534-MYB96, Gma-miRNA5030-MYB176, Gma-miRNA156-SPL9, Gma-miRNA319-TCP3, TCP4, Gma-miRNA162-WD40, Gma-miRNA396-UDP-glucose: flavonoid 3-O-glucosyltransferase and GmamiRNA5434-CHI3 showed an important relationship with the total isoflavone content in both the soybean genotypes across all the stages studied. In addition, in order to understand the role of cytosine methylation during isoflavonoid biosynthesis and accumulation, we performed cytosine methylation analysis in the coding region of IFS1 and IFS2 gene. The results indicated that there is increased 5-mC in both the isoforms in NRC37 compared to NRC7 soybean genotype signifying the positive role of 5-mC in the coding region of the gene which could contribute towards enhanced expression. Taken together the expression data of miRNAs-target gene pair, cytosine methylation along with the differential accumulation pattern of different forms of isoflavones, we for the first time report the role of miRNAs and cytosine methylation during isoflavone biosynthesis and accumulation in soybean. Therefore, the finding of the present work would certainly increase our understanding of the miRNA and cytosine methylation based genetic regulation of isoflavone pathway which would assist in selection and manipulation to get high-performing soybean genotypes with better isoflavone yield. सोयाबीन (ग्लाइिसन मकै्स एल.) के बीज म अन्तिनर् िहत आइसोफ्लोवोन जवैसं ेषण एवं सचंय के िनयामक तंतर् की गूढ़ाथर्ना