Tylosema esculentum (marama bean) is a drought tolerant legume native to the Kalahari Desert and widely distributed across Southern Africa. However, the molecular mechanisms underlying its drought tolerance remain largely unexplored. This study employs a de novo transcriptome analysis to identify key genes and molecular pathways associated with T. esculentum tolerance to water deficit. Plants were grown under normal and water deficit conditions in a controlled greenhouse experiment. Transcriptomic profiling identified 144 differentially expressed genes, with 91 showing increased expression and 53 showing decreased expression in response to water deficit. Gene Ontology enrichment analysis showed that overexpressed genes in T. esculentum are involved in key biological processes including response to water deficit, regulation of biological quality, regulation of DNA-templated transcription and chloroplast organization process. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that T. esculentum tolerance to water deficit is associated with activation of key pathways involved in thiamine and riboflavin metabolism, cysteine and methionine metabolism and MAPK signalling. To validate the RNA-seq data, six drought-responsive genes were analyzed using quantitative real-time PCR (RT-qPCR). The RT-qPCR results were consistent with RNA-seq data, confirming the reliability of the de novo transcriptome profiling. Notably, phospholipase C, purple acid phosphatase (PAP and PAP17), phosphatase 2C isoforms, and aldehyde dehydrogenase (ALDH) were upregulated, highlighting their central roles in signal transduction, phosphate remobilization, ABA signaling and oxidative stress mitigation in T. esculentum under water deficit. Our findings provide important information on the genetic basis of drought tolerance in T. esculentum, serving as a foundation for future studies aimed at enhancing drought tolerance in T. esculentum and potentially other crops.
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