Integrative Small RNA and Transcriptome Analysis Provides Insight Into Key Role of Mir408 Towards Drought Tolerance Response in Cowpea

Abstract Cowpea, a highly nutritious legume crop exhibits high resilience to heat and drought stress. Small RNA and transcriptome sequencing was performed in an elite high-yield Indian cultivar, Pusa Komal. Overall, 233 conserved and 10 novel drought-responsive miRNAs, and around 2995/497 drought-responsive genes were observed in libraries generated from leaf/root tissues of cowpea. Using 5’ RLM-RACE approach, NAC29, auxin response factor 8, copper chaperone for superoxide dismutase, scarecrow-like protein 6, mitochondrial substrate carrier family protein B, and laccase 12 were identified as cleaved targets of differentially expressed miRNAs such as miR167, miR171m, miR398c, miR408, miR1514, novel_792, and novel_796 in cowpea. Moreover, the potential role of differentially up-regulated miR408 in cowpea was exploited by the generation of transgenic cowpea lines overexpressing amiR408a-3p. The transgenic OX-amiR408 cowpea lines displayed enhanced drought and salinity tolerance as compared to control plants and maintained higher relative water content, chlorophyll, and proline content, and lower cellular H2O2 content under drought stress. The SEM analysis revealed reduced stomatal aperture and higher trichome density in transgenic OX-amiR408 lines. Furthermore, genes related to trichome development, transparent testa glabra 1 (TTG1), and glabrous inflorescence stems (GIS3) showed ~1.45-fold change in transgenic lines. Selected DGE for GO enriched terms for biological processes like lipid metabolic processes, hydrogen peroxide catabolic process, and response to oxidative stress, such as LAC3, LAC12, GDSL esterase/lipase CPRD49 and peroxidase 25 were altered in transgenic OX-amiR408 lines.