Anatomical, Physiological, and Transcriptome Analyses Revealing Pod Shattering of Medicago ruthenica Associated with Pericarp Lignin Biosynthesis.

Background: Medicago ruthenica, a perennial legume forage valuable for ecological restoration and improved breeding, suffers significant harvest losses due to pod shattering. Pod shattering is a trait not only linked to not only pod ventral suture, but also pericarp properties. In this study, we aimed to (1) elucidate the role of pericarp in explosive pod shattering by comparing shattering-susceptible (SPD) and shattering-resistant (RPD) M. ruthenica genotypes, and (2) identify key regulatory genes and pathways underlying this mechanism. Methods: We conducted comparative analyses of pericarp anatomy and physiological traits (pericarp components such as water content, cellulose, hemicellulose, pectin, and lignin; and the activities of enzymes such as cellulose synthase A (CesA), phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) in SPD and RPD pods). Transcriptome of pod pericarps identified differentially expressed genes (DEGs) for the selection of candidates functional genes. Promoter analysis was performed on candidate functional genes to identify specific regulated factors. The functional role of auxin signaling was validated through exogenous auxin application and the assessment of pod shattering rates and gene expression. Results: SPD pod pericarps exhibited significantly higher lignification of endocarp, lignin, cellulose, hemicellulose and pectin content, but lower water content than RPD. Principal component analysis identified that lignin contributes the highest loading value (0.727) contributor to pod shattering. The activities of five cell wall biosynthesis enzymes were higher in SPD pod pericarps than RPD. Transcriptome analysis identified more than 3419 DEGs in SPD pericarps. KEGG enrichment highlighted "phenylpropanoid biosynthesis" as the most significant pathway. A total of 57 lignin-biosynthesis-related DEGs were upregulated in SPD, including 15 PODs. Promoters of 11 POD genes contained MYB-binding motifs and 8 contained auxin-responsive elements, a total of 76 MYB transcription factors (mostly upregulated) and 9 auxin biosynthesis genes (mostly downregulated) were differentially expressed in SPD. Exogenous auxin application significantly reduced SPD pod shattering to 23.6% and concurrently downregulated PODs expression. Conclusions: This study establishes that enhanced lignification within the pericarp endocarp by the upregulation of lignin biosynthetic genes (particularly PODs), coupled with upregulation by MYB transcription factors and downregulation by auxin, is a core mechanism of explosive pod shattering in M. ruthenica. The identified DEGs, especially MYBs, PODs, and auxin pathway genes, provide gene information for breeding shattering-resistant M. ruthenica varieties through molecular design or marker-assisted selection.