Functional Analysis And Regulation Of PpCAD4 Gene In Physcomitrium Patens

Cinnamoyl alcohol dehydrogenase(CAD)serves as a critical enzyme in the lignin synthesis pathway,significantly influencing physiological processes such as plant morphogenesis,growth,development,and resistance to pathogenic bacterial infections.Evolutionary analysis revealed that PpCAD4 was distantly related to its counterparts.While Pp CAD1 has been confirmed to synthesize lignin monomers,the function of PpCAD4 remains unreported.And under the stress of Botrytis cinerea,the expression level of CAD4 increased most significantly.Thus,this study investigates the biological role of PpCAD4 in P.patens and Arabidopsis thaliana and examines the regulatory expression mechanism of PpCAD4 using a yeast hybridization system.The following findings were obtained:1.PpCAD4 affects plants growth and development.PpCAD4 gene affects the formation of P.patens.Notably,the Ppcad4-ko plants displayed more compact phyllid and blade arrangements,suggesting a potential connection to plant water transportation.And PpCAD4 impacts Arabidopsis thaliana growth,the overexpressed plants exhibited a 55%reduction in root length and increase in silique length compared to WT.2.PpCAD4 participates in stress of P.patens.Under the stress of Botrytis cinerea,overexpression of PpCAD4 slowed down the invasion rate of B.cinerea,PpCAD4-OE resulted in symptoms later than WT and Ppcad4-ko,and the content of phenols and flavonoids in this plant was higher than WT.Under salt and cold stress,Ppcad4-ko plants are prone to death;PpCAD4-OE plants are more tolerant to salt and cold.Therefore,the PpCAD4 maybe participates in stress of plants.3.Possible regulatory mechanisms of PpCAD4 gene.Through yeast one-hybrid assays,it was demonstrated that MADS5 can directly bind to the PpCAD4 promoters in vitro,suggesting that MADS5 may bind to the PpCAD4 promoters to regulate plant growth and development.Multiple proteins interacting with PpCAD4 were identified in the yeast two-hybrid experiments.With one-to-one verification revealing that both Pp CAT2 and Pp E3 could interact with PpCAD4.It is speculated that PpCAD4 may interact with Pp CAT2 and Pp E3 to modulate plant adaptation to stress.In summary,PpCAD4 affects plant growth and development,MADS5 may bind to the PpCAD4 promoters governs plant growth and development;It is preliminarily determined that PpCAD4 participate in the defense response of P.patens;PpCAD4 may interaction with Pp CAT2 and Pp E3,enhancing the plant’s stress resistance capabilities.