The Defense Mechanism of PpCAD4 in Physcomitrium patens Against Botrytis cinerea

The existence of lignin in <i>Physcomitrium patens</i> has been controversial. However, cinnamyl alcohol dehydrogenase (CAD), the key enzyme in monolignol biosynthesis, has been identified with four gene members in <i>P. patens</i>. Despite the roles of <i>PpCAD1</i> in moss architecture being proven in a previous study, the functions and molecular mechanisms of <i>PpCAD4</i> remain largely unexplored in early terrestrial plants. This study aims to unravel this mystery via a comprehensive analysis of the transcriptome and metabolome of <i>PpCAD4</i>-overexpression (OE) lines compared with wild type (WT) under <i>Botrytis cinerea</i> treatment, firstly. A total of 475 and 1368 significantly differentially expressed genes in <i>PpCAD4</i>-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 <i>PpCAD4</i>-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 <i>PpCAD4</i> 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 <i>B. cinerea</i> assault. Our results demonstrated that the essential roles and potential mechanisms of <i>PpCAD4</i> are essential for defense against pathogens during the adaptation to land in moss.