CsWRKY33: A Crucial Transcription Factor in the MAPK Pathway Inhibited by Trypsin, Participating in the Suppression of Cucumis sativus Senescence

Abstract

Background The Mitogen-Activated Protein Kinase (MAPK) cascade pathway extensively participates in the physiological regulation processes of plants. Trypsin, as an inducer of resistance, significantly enhanced the storage quality of Cucumis sativus (C. sativus). However, the mechanism by which trypsin regulated the synthesis of phenylpropanoid compounds through the MAPK pathway to influence fruit resistance remained to be elucidated. Methods To investigate the molecular mechanism underlying trypsin-induced C. sativus resistance, we conducted a combined transcriptomic and widely targeted metabolomic analysis, validated through Virus-Induced Gene Silencing (VIGS). Results Transcriptomic results revealed that a total of 83 differentially expressed genes (DEGs)were enriched in the MAPK pathway, with 48 genes significantly downregulated and 35 genes significantly upregulated. GSEA analysis further identified the WRKY33 transcription factor from the leading edge subset. Bioinformatics analysis indicated that CsWRKY33 shared high homology with WRKY22 in Arabidopsis. The combined analysis of transcriptomics and widely targeted metabolomics demonstrated significant upregulation in the synthesis of compounds such as vanillin, dihydrocharcone-4'-O-glucoside, and 2-hydroxy-3-phenylpropanoic acid. Co-expression network analysis showed that these key metabolites were negatively regulated by CsWRKY33. VIGS results showed that silencing CsWRKY33 enhanced fruit resistance and extended storage time. Conclusion This study revealed that trypsin could downregulate the expression of CsWRKY33 and promote the synthesis of compounds with high antioxidant and antibacterial activity, such as vanillin, dihydrocharcone-4'-O-glucoside, and 2-hydroxy-3-phenylpropanoic acid. This enhancement led to increasing fruit disease resistance and delayed senescence and decay.