The Defense Mechanism of PpCAD4 in Physcomitrium patens Against Botrytis cinerea

The existence of lignin in Physcomitrium patens has been controversial. However, cinnamyl alcohol dehydrogenase (CAD), the key enzyme in monolignol biosynthesis, has been identified with four gene members in P. patens. Despite the roles of PpCAD1 in moss architecture being proven in a previous study, the functions and molecular mechanisms of PpCAD4 remain largely unexplored in early terrestrial plants. This study aims to unravel this mystery via a comprehensive analysis of the transcriptome and metabolome of PpCAD4-overexpression (OE) lines compared with wild type (WT) under Botrytis cinerea treatment, firstly. A total of 475 and 1368 significantly differentially expressed genes in PpCAD4-OE lines compared to the wild type at 6 h and 12 h post-inoculation, which were predominantly enriched in pathways involving flavonoid, phenylpropanoid biosynthesis, and plant hormone signal transduction. Concurrently, metabolomic profiling revealed 160 and 114 differentially accumulated metabolites in PpCAD4-OE at the corresponding time points, with phenolic acids and flavonoids collectively constituting over 45% of these compounds. Furthermore, the MADS-box transcriptional factor PpMC6 negatively regulated PpCAD4 expression by yeast-one-hybrid and dual-luciferase assays. Finally, Catalase isozyme 2 (PpCAT2) and E3 ubiquitin-protein ligase (PpE3) were identified as interactive partners with PpCAD4, respectively, deducing that the increasing of reactive oxygen species might be promoted by PpCAT2 degradation through PpE3 after B. cinerea assault. Our results demonstrated that the essential roles and potential mechanisms of PpCAD4 are essential for defense against pathogens during the adaptation to land in moss.