Integrative small RNA and transcriptome analysis provides insight into key role of miR408 towards drought tolerance response in cowpea

Drought stress response studies and overexpression of vun-miR408 proved it to be essential for abiotic stress tolerance in cowpea. Small RNA and transcriptome sequencing of an elite high-yielding drought-tolerant Indian cowpea cultivar, Pusa Komal revealed a differential expression of 198 highly conserved, 21 legume-specific, 14 less-conserved, and 10 novel drought-responsive microRNAs (miRNAs) along with 3391 (up-regulated) and 3799 (down-regulated) genes, respectively, in the leaf and root libraries. Among the differentially expressed miRNAs, vun-miR408-3p, showed an up-regulation of 3.53-log2-fold change under drought stress. Furthermore, laccase 12 (LAC 12) was identified as the potential target of vun-miR408-3p using 5' RNA ligase-mediated rapid amplification of cDNA ends. The stable transgenic cowpea lines overexpressing artificial vun-miR408-3p (OX-amiR408) displayed enhanced drought and salinity tolerance as compared to the wild-type plants. An average increase of 30.17% in chlorophyll, 26.57% in proline, and 27.62% in relative water content along with lesser cellular H2O2 level was observed in the transgenic lines in comparison with the wild-type plants under drought stress. Additionally, the scanning electron microscopic study revealed a decrease in the stomatal aperture and an increase in the trichome density in the transgenic lines. The expression levels of laccase 3 and laccase 12, the potential targets of miR408, related to lipid catabolic processes showed a significant reduction in the wild-type plants under drought stress and the transgenic lines, indicating the regulation of lignin content as a plausibly essential trait related to the drought tolerance in cowpea. Taken together, this study primarily focused on identification of drought-responsive miRNAs and genes in cowpea, and functional validation of role of miR408 towards drought stress response in cowpea.