Molecular Mechanism Of C2H2-type Zinc Finger Protein MdZAT10 Regulating Leaf Senescence And Drought Sress In Apple

Apple is an important cultivated fruit tree species in China.Premature senescence of leaves seriously affects the nutrition of apple tree,which in turn affects fruit yield and quality.Therefore,it is important to study the mechanism of leaf senescence to regulate plant growth and development and improve yield.Transcription factors play an important role in the regulatory network of plant leaf senescence,however,relatively little research has been done on transcription factors in apple leaf senescence.Apple often suffers from drought stress during growth,which seriously limits tree growth,fruit quality and yield.Transcription factors play an important role in plant response to drought stress.C2H2-type zinc finger proteins are widely exist in plants and play an important functions in regulating plant growth and development and responding to environmental stresses.Previous studies have shown that multiple C2H2-type zinc finger transcription factors are differentially expressed in Arabidopsis senescing leaves,but the molecular mechanism regulating leaf senescence are not clear.In this study,we used molecular biology method to analyze the function of C2H2-type zinc finger transcription factor MdZAT10 in leaf senescence and response to drought in apple.The main results are as follows:1.q RT-PCR results showed that the transcript level of MdZAT10 significantly increased with the natural senescence of leaves.Compared with wild type,overexpression of MdZAT10accelerated leaf senescence in apple and Arabidopsis,while MdZAT10-Anti delayed leaf senescence in apple.Meanwhile,q RT-PCR results showed that overexpression of MdZAT10transgenic calli significantly enhanced the expression levels of senescence-related genes MdSAG29 and MdPAO and downregulated the expression level of negative regulator of leaf senescence MdWRKY70,indicating that MdZAT10 promotes plant leaf senescence.2.The interaction between MdZAT10 and MdABI5 was verified by Y2H,Pull-down and Bi FC assays.The further studies on MdABI5 revealed that overexpression of MdABI5accelerated leaf senescence in apple and Arabidopsis,while MdABI5-Anti apple leaves showed a delayed senescence phenotype.These results indicated that MdABI5 promoted leaf senescence.The dual-luciferase activity detection assay showed that MdABI5 activated the expression of chlorophyll catabolic genes MdNYC1 and MdNYE1.Furthermore,the interaction between MdZAT10 and MdABI5 enhanced the transcriptional activation of MdNYC1 and MdNYE1 by MdABI5.Phenotypic experiments showed that MdZAT10 promoted leaf senescence by enhancing the transcriptional activity of MdNYC1 and MdNYE1 by MdABI5.3.The interaction between MdZAT10 and MdBT2 was verified by Y2H,Pull-down and Bi FC assays.Me JA treatment not only activated the expression of MdZAT10 at the transcriptional level,but also reduced MdZAT10 protein degradation by the 26S proteasome pathway.Phenotypic analysis showed that overexpression of MdZAT10 promoted JA-induced leaf senescence,whereas MdZAT10-Anti apple leaves delayed JA-induced leaf senescence.Me JA treatment promoted the degradation of MdBT2 protein at the posttranslational level.Furthermore,MdBT2 delayed JA-induced leaf senescence.MdBT2 promoted the ubiquitination of MdZAT10 and then MdZAT10 was degraded through the 26S proteasome pathway.Phenotypic analysis showed that overexpression of MdBT2 partially inhibited the MdZAT10-mediated leaf senescence.The results indicated that MdBT2 negatively regulated MdZAT10-mediated leaf senescence.4.qRT-PCR analysis results showed that MdZAT10 could be induced by PEG6000,Na Cl,wounding,SA,and ACC treatments.Phenotypic analysis showed that overexpression of MdZAT10 in apple seedlings,calli,and Arabidopsis reduced the drought tolerance.Under the drought treatment,overexpression of MdZAT10 plants exhibited higher MDA content,H₂O₂content and O₂^-accumulation,and reduced activities of antioxidant enzymes,indicating that MdZAT10 reduced plant resistance to drought.Our study indicated that MdZAT10 can enhance the transcriptional activation of MdNYC1and MdNYE1 by MdABI5 thus promoting leaf senescence.Meanwhile,MdBT2 promoted the ubiquitination and degradation of MdZAT10 protein,thereby inhibiting JA-induced leaf senescence.In addition,MdZAT10 reduced drought tolerance by decreasing ROS scavenging.Therefore,studying the regulatory mechanism of MdZAT10 provides a theoretical basis for the study of plant senescence and drought resistance.