Groundnut, cultivated under rain-fed conditions is prone to yield losses due to intermittent drought stress. Drought tolerance is a complex phenomenon and multiple gene expression required to maintain the cellular tolerance. Transcription factors (TFs) regulate many functional genes involved in tolerance mechanisms. In this study, three stress-responsive regulatory TFs cloned from horse gram, (Macrotyloma uniflorum (Lam) Verdc.), MuMYB96, involved in cuticular wax biosynthesis; MuWRKY3, associated with anti-oxidant defense mechanism and MuNAC4, tangled with lateral root development were simultaneously expressed to enhance drought stress resistance in groundnut (Arachis hypogaea L.). The multigene transgenic groundnut lines showed reduced ROS production, membrane damage, and increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzyme activity, evidencing improved antioxidative defense mechanisms under drought stress. Multigene transgenic plants showed lower proline content, increased soluble sugars, epicuticular wax content and higher relative water content suggesting higher maintenance of tissue water status compared to wildype and mock plants. The scanning electron microscopy (SEM) analysis showed a substantial increase in deposition of cuticular waxes and variation in stomatal number in multigene transgenic lines compared to wild type and mock plants. The multigene transgenic plants showed increased growth of lateral roots, chlorophyll content, and stay-green nature in drought stress compared to wild type and mock plants. Expression analysis of transgenes, MuMYB96, MuWRKY3, and MuNAC4 and their downstream target genes, KCS6, KCR1, APX3, CSD1, LBD16 and DBP using qRT-PCR showed a two- to four-fold increase in transcript levels in multigene transgenic groundnut plants over wild type and mock plants under drought stress. Our study demonstrate that introducing multiple genes with simultaneous expression of genes is a viable option to improve stress tolerance and productivity under drought stress.

Cluster bean (Cyamopsis tetragonoloba L.) is one of the multipurpose underexplored crops grown as green vegetable and for gum production in dryland areas. Cluster bean is known as relatively tolerant to drought and salinity stress. To elucidate the molecular mechanisms involved in the drought tolerance of cluster bean cultivar RGC-1025, RNA sequencing (RNA-seq) of the drought-stressed and control samples was performed. De novo assembly of the reads resulted in 66,838 transcripts involving 203 pathways. Among these transcripts, differentially expressed gene (DEG) analysis resulted in some of the drought-responsive genes expressing alpha dioxygenase 2, low temperature-induced 65 kDa protein (LDI65), putative vacuolar amino acid transporter, and late embryogenesis abundant protein (LEA 3). The analysis also reported drought-responsive transcription factors (TFs), such as NAC, WRKY, GRAS, and MYB families. The relative expression of genes by qRT-PCR revealed consistency with the DEG analysis. Key genes involved in the wax biosynthesis pathway were mapped using the DEG data analysis. These results were positively correlated with epicuticular wax content and the wax depositions on the leaf surfaces, as evidenced by scanning electron microscope (SEM) image analysis. Further, these findings support the fact that enhanced wax deposits on the leaf surface had played a crucial role in combating the drought stress in cluster beans under drought stress conditions. In addition, this study provided a set of unknown genes and TFs that could be a source of engineering tolerance against drought stress in cluster beans.