Involvement of cysteine protease PtoCP1 gene in plant hormone-regulated leaf senescence in Populus tomentosa

Cysteine proteases, an important family of proteases, are expressed in different plant tissues and have various functions. This study explored the mechanism underlying the involvement of the cysteine protease PtoCP1 gene in leaf senescence of Populus tomentosa under hormone induction. Initially, poplar leaves were treated with abscisic acid (ABA), methyl jasmonate (MeJA), and ethylene (ACC) for 0, 6 and 12 h, and 1, 2, or 3 d to observe leaf phenotypes, assess chlorophyll content, and measure relative expression of the PtoCP1 gene. The results demonstrated that hormone-induced leaf yellowing was accelerated compared with the control group; the most significant leaf yellowing was observed under ABA treatment, followed by MeJA and ACC treatments. Chlorophyll content was significantly lower in leaves treated with ABA for 3 d compared with the control group; there was no significant difference in chlorophyll content between MeJA and ACC treatments. Furthermore, expression of the PtoCP1 gene was significantly increased at 6 h, 12 h, and 3 d after induction with ABA and MeJA; induction with ACC led to a significant increase in PtoCP1 gene expression at 12 h. Therefore, treatment with exogenous hormones (e.g., ABA, MeJA, and ACC) directly or indirectly regulates the transcription of PtoCP1, indicating its involvement in the induction of plant leaf senescence by exogenous hormones. Next, ptocp1 mutant leaves of P. tomentosa were treated with the same concentrations of hormones. The results demonstrated that the ptocp1 mutant exhibited delayed leaf senescence compared with the wild type, indicating that PtoCP1 can directly or indirectly respond to ABA, MeJA, or ACC induction and participate in leaf senescence in P. tomentosa. Further analysis of PtoCP1 promoter elements revealed the presence of both MeJA response elements and a large number of ABA response elements. Therefore, transgenic Arabidopsis seedlings containing ProPtoCP1-GUS-PBI121 recombinant vector were treated with the corresponding ABA and MeJA hormones. Subsequently, GUS histochemical staining and analysis of the relative expression of the reporter gene GUS were performed to determine whether the promoter had been induced. ABA treatment led to partial enhancement of GUS staining in Arabidopsis, but no significant effect was observed with MeJA treatment. Additionally, ABA treatment led to a significant increase in GUS expression, whereas MeJA treatment did not lead to a significant increase in GUS expression; thus, ABA is directly involved in PtoCP1 gene expression. In conclusion, the PtoCP1 promoter can regulate PtoCP1 gene expression in response to ABA, enabling plants to respond to external stress and initiate leaf senescence. This study enhances the overall understanding of the mechanism by which plant cysteine proteases regulate hormone-induced leaf senescence.