Functional Analysis of Candidate Genes to Encode Rhamnosyl 3-O-Methyltransferase in the Moss Physcomitrella patens

Studies of cell walls have long contributed to understanding plant function. Relevance to the production of biofuels from biomass has recently added urgency to the study of plant cell walls. Arabinogalactan proteins (AGPs) are highly glycosylated glycoproteins at the plant plasma membrane and cell wall. Consisting of a core polypeptide surrounded by glycans, AGPs contain abundant galactosyl and arabinosyl residues and often some glucuronosyl, rhamnosyl, or other residues. Previous study revealed that AGPs in the moss Physcomitrella patens contain unusual 3-O-methyl-rhamnosyl residues (3-O-Me-Rha) in amounts as high as 15 mole percent. The goals of this dissertation were to identify and evaluate Physcomitrella genes that are candidates to encode the rhamnosyl 3-O-methyltransferase that forms 3-O-Me-Rha. A Mycobacteria gene encoding a rhamnosyl 3-O-methyltransferase and an Arabidopsis gene encoding a glucuronosyl 4-O-methyltransferase were used as queries in bioinformatics searches of the Physcomitrella genome. Eleven candidate genes were identified and, through homologous recombination in Physcomitrella, targeted knockouts of these genes were generated. Based on reductions in 3-O-Me-Rha/Rha content ratio in AGPs of the knockouts, two genes,KO1 and KO9, were selected for further study. A third gene, KO11, was selected for further study because it shares a domain with the Arabidopsis glucuronosyl 4-O-methyltransferase. Further study of KO1 led to revision of its gene model. A phylogenic tree suggested that the revised KO1 protein contains a LpxB domain, which is characteristic of bacterial lipid A synthesis. The Physcomitrella ko1 knockout exhibited phenotypic effects including abnormal growth in protonema and rhizoids, reduced 3-O-Me-Rha/Rha in AGPs, and reduced lignin-like content in cell walls. Heterologous expression of KO1 did not produce detectable 3-O-Me-Rha in tobacco AGPs. Further bioinformatics study of KO9 showed it to be an ortholog of caffeoyl-CoA O-methyltransferase of lignin biosynthesis. The Physcomitrella ko9 knockout did not exhibit altered lignin-like content in its cell walls. Heterologous expression of KO9 in tobacco did not produce detectable 3-O-Me-Rha in tobacco AGPs. The Physcomitrella ko11 knockout exhibited no effect on 3-O-Me-Rha/Rha in AGPs, and no 3-O-Me-Rha was detected in AGPs from transgenic KO11 tobacco. It remains uncertain whether any of the candidate genes encodes rhamnosyl 3-O-methyltransferase in Physcomitrella