Identification of a Novel Gene MtbZIP60 as a Negative Regulator of Leaf Senescence through Transcriptome Analysis in Medicago truncatula

Leaves are the primary harvest portion in forage crops such as alfalfa (<i>Medicago sativa</i>). Delaying leaf senescence is an effective strategy to improve forage biomass production and quality. In this study, we employed transcriptome sequencing to analyze the transcriptional changes and identify key <i>senescence-associated genes</i> under age-dependent leaf senescence in <i>Medicago truncatula</i>, a legume forage model plant. Through comparing the obtained expression data at different time points, we obtained 1057 differentially expressed genes, with 108 consistently up-regulated genes across leaf growth and senescence. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the 108 <i>SAGs</i> mainly related to protein processing, nitrogen metabolism, amino acid metabolism, RNA degradation and plant hormone signal transduction. Among the 108 <i>SAGs</i>, seven transcription factors were identified in which a novel bZIP transcription factor MtbZIP60 was proved to inhibit leaf senescence. MtbZIP60 encodes a nuclear-localized protein and possesses transactivation activity. Further study demonstrated MtbZIP60 could associate with MtWRKY40, both of which exhibited an up-regulated expression pattern during leaf senescence, indicating their crucial roles in the regulation of leaf senescence. Our findings help elucidate the molecular mechanisms of leaf senescence in <i>M. truncatula</i> and provide candidates for the genetic improvement of forage crops, with a focus on regulating leaf senescence.