Abstract
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.
Highlights
Owing to the fluctuating climatic adversities, drought stress serves as a serious threat to plant growth and productivity
A total of 232 pathways were identified for differentially regulated transcripts (DGEs) and the most enriched occurrence was observed for pathways involving chromosome and associated proteins [03036], transporters [02000], transcription factors [03000], exosome [04147], membrane trafficking [04131] and chaperones and folding catalysts [03110] (Fig. 3a, b)
The study focused on the differential expression analysis of miRNAs and their target genes in leaf and root tissues of cowpea under drought stress. miR169b, miR319f, miR408, and novel_27, an isoform of miR408-5p up-regulated, in contrast to miR167, miR171m, miR398c, and miR1514a, observed to down-regulate under drought stress in leaves of cowpea, as detected by sRNA sequencing and validated by northern analysis. miR408, being a universal multi-stress-responsive miRNA, was highly abundant under drought stress, in leaf tissue of cowpea
Summary
Owing to the fluctuating climatic adversities, drought stress serves as a serious threat to plant growth and productivity. Cowpea is a highly nutritious grain legume crop and serves as the primary protein source for millions of people in tropical and sub-tropical regions of Africa, Asia, and other developing countries of the world. Due to its ability to associate with nitrogen-fixing bacteria and vesicular-arbuscular mycorrhizal fungi, cowpea can survive in low fertile soils and grown with cereals in intercropping system. It is a promising crop for cultivation under adverse climatic fluctuations, being highly resilient to heat and drought stress in comparison to other members of the Leguminosae family (Carvalho et al 2017)
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