Regulation And Preliminary Functional Study Of Arabidopsis Matrix Metalloproteinases (At-MMPs)

Matrix metalloproteinases (MMPs) are a class of highly conserved zinc-dependent endopeptidases that are ubiquitously present in plants and animals. In mammalian, MMP are implicated in the degradation of collagen and other macromolecules of the extracellular matrix and participate in the pathogenesis of metastatic tumor cells and other diseases. However, our understanding of plant MMP is far behind. Mitogen-activated protein kinase (MAPK) cascade is highly conserved signaling modules in eukaryotes that transduce extracellular stimuli into cellular responses to coordinate plant growth, development, and response to environment. There are five members in Arabidopsis At-MMPs gene family. By analyzing the microarray data of the conditional gain-of-function DD plants in which MPK3/MPK6 signaling pathway was activated, we found that the expression of At2-MMP and At3-MMP was greatly enhanced, suggesting that At2-MMP and At3-MMP gene expression may be regulated by this MAPK cascade.In this study, in order to explore the mechanism of gene regulation of Arabidopsis MMP in plant growth, development, and disease resistance, we used real-time quantitative PCR technology to detect At2-MMP and At3-MMP gene expression in a variety of mutant/transgenic backgrounds. We found that At2-MMP and At3-MMP gene expression was regulated by both MPK3 and MPK6, and MPK3 may play a more important role. WRKY33 transcription factor, an important substrate of MPK3/MPK6 in response to pathogen infection, is involved in the up-regulation of gene expression of At2-MMP and At3-MMP. Besides, we have built a PAt2-MMP:GUS reporter system to study the spatiotemporal expression patterns of At2-MMP. GUS staining showed that At2-MMP expresses mainly in the roots, hypocotyls, and vascular tissues. When Arabidopsis is subjected to Botrytis cinerea infection, the gene expression of At2-MMP is activated and the At2-MMP protein accumulates during the infection process.Furthermore, in this study, we screened single mutants of each member of Arabidopsis At-MMPs family and double mutant at2-mmp at3-mmp. We obtained the constitutive and inducible oveerexpression system of At3-MMP by antibiotic selection and immunoblot analysis and built the 35S:At3-MMP-eYFP subcellular localization construction. The establishment of these mutants and overexpression systems provides important materials for exploring the functions of At-MMPs in plant growth, development, and disease resistance.