Pectin-modifying enzymes in plant development and interactions with fungi

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The plant cell wall plays a critical role in processes such as morphogenesis and fungal interactions. Pectins (e.g. rhamnogalacturonan I, RG-I) are a particularly dynamic part of the cell wall environment, especially within the middle lamella that forms the interface between adjoining cells. As such, pectins are critical to adhesion and separation of cells (among their other functions). Pectins are remodeled dynamically, including the breakage and rejoining of pectin molecules. In plants, cleavage of the RG-I portion of pectins is believed to mediated by enzymes called RG lyases (RGLs). Our objective is to characterize plant RGL enzyme families, and their role in plant development and interactions with the environment. We will use computational tools to explore the diversity of putative RGLs, by analysis of the genomes and transcriptomes currently available from over 1,200 plant species. This will provide insight into the diversity and evolution of these genes. We will also conduct meta-analyses of existing RNASeq data from a wide range of species to make inferences about RGL functions, including exploring co-expression with other regulators or cell wall; enzymes. Based on the bioinformatic analyses described above, we expect to make new inferences about the evolution of the pectin-modifying genes and regulators, and their roles in plant physiology. Despite their annotations as RGLs, no plant RGL has yet been empirically proven to have RGL activity.  We will characterize the enzymatic activity of selected RGLs in vitro, by purifying them from appropriate hosts, and analyzing their effects on diverse pectic substrates, primarily through a novel capillary-electrophoresis based approach.   To complement the other analyses proposed above, we will also conduct mutational analysis, with a focus on cell adhesion/separation in morphogenesis and plant-microbe interactions. We will use Arabidopsis thaliana and flax (Linum usitatissimum) for these mutational studies. The advantages of arabidopsis as a model system are already well known, and flax offers other benefits because it demonstrates intrusive growth (i.e. of phloem fibers), and associations with both arbuscular mycorrhizal fungi (AMF) and pathogens. We have already established many resources relevant to these studies in both flax and arabidopsis. Loss-of-function RGL mutants will be compared to wild-type using a range of phenotypic assays including: immunohistochemistry, analytical chemistry, and growth and hormone assays, and fungal colonization assays. This research program will provide key insights into the role of one of the least-understood enzymes families in cell wall biology. It will also provide training opportunities for HQP in bioinformatics, gene editing, plant genetics, and analytical chemistry. Finally, the information learned in this research will also facilitate the development of crops (especially flax) with modified pectins for specific industrial end uses.