Molecular Mechanism Of ZAT17-PRL1 Module Regulating Cadmium Stress Response And MiRNA Synthesis In Arabidopsis

Cadmium（Cd）stress and microRNA（miRNA）can significantly affect plant growth and development.Exploring new loci regulating Cd tolerance and miRNA synthesis pathways is of great significance for enhancing the agronomic traits of crops and deepening the understanding of plant growth and development.Transcription factors play a crucial role in regulating plant growth and development,as well as in responding to various stresses.Zinc finger proteins（ZFPs）are a large family of transcription factor proteins in plants that possess finger-like structures and zinc-binding capabilities.ZAT17 belongs to the C1-2i subclass of the ZFP family,which plays a significant role in plant responses to abiotic stresses.Previously,the function of ZAT17 was rarely reported.This study further clarified the role of ZAT17 in the response to Cd stress and the miRNA synthesis pathway.The main results are as follows:（1）The mutant gene of sup8 in the prl1-2 suppressor was ZAT17.It has been previously reported that PRL1 positively regulates Cd stress resistance in Arabidopsis.In this study,we conducted EMS mutagenesis on the mutant prl1-2.We screened its suppressors to identify novel proteins that collectively regulate Cd stress resistance with PRL1 in Arabidopsis.We obtained a group of 11 suppressors of prl1-2（sup）mutants that can restore the developmental defect of prl1-2.Our focus was on the sup8 double mutant.Whole-genome re-sequencing of F2 generation plants backcrossed with the double mutant sup8 and its parent prl1-2 revealed a mutation of nucleotide 131 from cytosine（C）to thymine（T）in ZAT17（AT2G28710）.This mutation resulted in the substitution of proline（Pro）with leucine（Leu）at amino acid position44 in the protein.ZAT17 encodes a nuclear-localized C2H2-type zinc-finger transcription factor,which contains two typical C2H2-type zinc-finger domains and a transcription repressor domain.The amino acid change occurs in the first zinc-finger domain.（2）ZAT17 negatively regulated PRL1-mediated tolerance to Cd stress in Arabidopsis.Under different concentrations of Cd stress,sup8 significantly restored the Cd-sensitive phenotype of prl1-2.RNA-seq analysis and real-time fluorescence quantitative PCR（q RT-PCR）verified that sup8 restored the expression of most of the differentially expressed genes（DEGs）in prl1-2,including the Cd stress response genes.In this study,the transcription level of ZAT17was upregulated under Cd stress.Compared with Col-0,Z-OE1 exhibited increased sensitivity to Cd stress,while Z-Crispr demonstrated improved tolerance to Cd stress,suggesting that ZAT17 negatively regulates resistance to Cd stress.We analyzed the Cd²⁺content and its transfer factors in the above-and below-ground parts of Arabidopsis plants under Cd stress,including Col-0,prl1-2,sup8,Z-OE1,and Z-Crispr.prl1-2 and Z-OE1 could reduce the transport ability of Cd²⁺from roots to shoot parts,leading to a Cd-sensitive phenotype.Since PRL1 functions as a CUL4-DDB1-PRL1 RING E3 ligase,we investigated the ZAT17 protein levels in Z-OE1 under cadmium stress.We observed a decrease in the abundance of the ZAT17-GFP protein as the duration of Cd stress treatment increased,suggesting that Cd stress influenced the stability of the ZAT17 protein.（3）ZAT17 influenced the transcription and alternative splicing（AS）of genes related to the Cd stress signaling pathway.Our RNA-seq analysis of Col-0,Z-OE1,and Z-Crispr Arabidopsis materials revealed that ZAT17 influences the transcription of a significant number of genes.GO enrichment analysis of RNA-seq data revealed that ZAT17 influenced the transcription of genes related to the Cd stress response pathway.The results of RNA-seq were verified by q RT-PCR and yeast one-hybrid experiments,demonstrating that ZAT17 indirectly downregulated the transcription of HMA3 and NRT2.1 under Cd stress.Meanwhile,ZAT17 also affected the transcription levels of IRT1 and PCR1 genes related to Cd detoxification and tolerance.GO analysis of 2010 genes in prl1-2 revealed that PRL1 is implicated in the AS of various cell signaling pathways,involving 78 genes associated with the response to Cd stress.Through intron retention（IR）analysis and RT-PCR experiments,we found that ZAT17 could restore the intronic splicing status of AT5G64250,GGCT2;1,and HEME1 of prl1-2 to the Col-0 level.（4）Previous studies in our laboratory have found that PRL1 is a key protein in the miRNA biosynthesis pathway.This study found that ZAT17 is involved in PRL1-mediated miRNA synthesis in Arabidopsis.ZAT17 restored the transcription of MIR genes,such as MIR158a,MIR167a,MIR167b,MIR171b,MIR172a,and MIR172b,in prl1-2 to the level of Col-0.Using stem-loop q RT-PCR,ZAT17 restored the transcription of miR156,miR167,miR171,and miR173 to the Col-0 level in prl1-2,while some miRNA target genes,such as SPL9,MYB33,MYB65,CUC1,and ARF3 transcripts,were partially restored to Col-0 levels.（5）ZAT17 regulated the accumulation levels of specific miRNAs.To determine the overall effect of ZAT17 on miRNA accumulation levels,we compared the miRNA sequencing results of Z-OE1,Z-Crispr,and Col-0.We found 36 differentially expressed miRNAs（DEMs）in Z-Crispr compared with Col-0.The aforementioned results suggest that ZAT17 regulates the accumulation levels of specific miRNAs,including miR167,miR172,and miR173.（6）ZAT17 regulates the transcription and alternative splicing of specific MIRs.We found that the expression levels of MIR156g,MIR166a,MIR167a,MIR168a,MIR399c,and MIR2111a in Z-Crispr transgenic lines were significantly higher than those in Col-0.However,the expression levels of MIR156g,MIR166a,MIR168a,and MIR2111a in the Z-OE1 transgenic lines were significantly lower than those in Col-0,indicating that ZAT17 negatively regulates the transcription of specific MIR genes.PRL1 belongs to the core components of the MAC splicing complex and promotes pre-m RNA splicing.We used RT-PCR experiments to analyze the IR of MIR genes.We found that the prl1-2 mutant exhibited an AS defect in the MIR171b and MIR172b genes.Interestingly,ZAT17 was able to restore the AS state of MIR genes in prl1-2 to the level observed in Col-0.These results indicate that the ZAT17-PRL1 module is involved in the AS process of MIR genes.（7）ZAT17 interacts with the MAC splicing complex and the miRNA processing complex.Subcellular localization analysis revealed that ZAT17 is localized in the nucleus.Bimolecular fluorescence complementation（Bi FC）and co-immunoprecipitation（Co IP）assays demonstrated that ZAT17 interacts with PRL1 and CDC5,members of the MAC splicing complex,as well as SE,HYL1,and DCL1,members of the miRNA processing complex.In summary,the functional study of the ZAT17 gene established the interaction between the Cd stress response pathway and the miRNA biosynthesis pathway in Arabidopsis.This study has identified a potential locus for the future breeding of Cd-resistant cultivars.It has also advanced the understanding of the molecular mechanisms involved in miRNA processing and synthesis.