Physcomitrium (Physcomitrella) patens endo-glucanase 16 is involved in the cell wall development of young tissue

Plants maintain large repertoires of carbohydrate-active enzymes (CAZymes)-comprising between 3% and 10% of their genomes-to synthesize, modify, and degrade the polysaccharide components of the cell wall. We recently identified a unique group of plant endo-glucanases from Glycoside Hydrolase Family 16, viz. EG16 orthologs, which constitute a sister clade to the well-known XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products. Biochemical analysis of EG16 orthologs from poplar (Populus trichocarpa), grapevine (Vitis vinifera), and spreading earthmoss (Physcomitrium patens) has demonstrated that these endo-glucanases are distinctly active on cell wall matrix glycans, mixed-linkage β(1,3);β(1,4)-glucan and xyloglucan (XyG), and that enzyme structure and specificity is highly conserved across diverse plant lineages. However, the physiological role of EG16 orthologs in any species is presently unknown. To shed light on EG16 function in vivo, here we performed reverse genetics and protein localization analyses of the single EG16 ortholog in the model moss P. patens, where this gene is highly expressed in young, expanding tissues, particularly in protonema. Surprisingly, deletion of the PpEG16 gene by homologous recombination led to an increase in growth, as well as accelerated senescence. Notably, the PpEG16 protein was shown to co-localize with XyG in the cell wall of protonema tissue, specifically at cell tips, despite lacking a secretion signal peptide. Although the precise biological role of EG16 orthologs remains elusive, our results implicate these highly conserved glycoside hydrolases in cell wall polysaccharide remodeling and recycling. We anticipate that these foundational results will inform future studies on EG16 function across plant lineages.