Professor University of Rhode Island Kingston, Rhode Island
Body of Abstract: Like cellulose synthases (CESAs), cellulose synthase-like D (CSLD) proteins synthesize β-1,4 glucan. CSLDs are important for cell wall deposition during tip growth and cytokinesis in vascular plants. However, it is unknown whether they assemble into multi-protein complexes and produce microfibrillar cellulose. To investigate CSLD function in the absence of interfering CESA activity, we created viable lines of the moss Physcomitrium patens in which all eight CESA genes are inactivated. Freeze fracture transmission electron microscopy of moss lacking CESAs revealed abundant rosette structures in the apical plasma membrane and adjacent to fusing cells plates in protonemal filaments. These rosettes closely resemble CESA-containing cellulose synthesis complexes (CSCs). Cell walls from these moss lines contain microfibrils and x-ray diffraction confirmed the presence of crystalline cellulose. CSLD function in P. patens was further investigated by gene knockout, in locus fluorescent tagging, and live-cell imaging. Double knockout of CSLDs highly expressed in leafy gametophores caused leaf deformities consistent with defects in cell division. Fluorescent tagging of the eight P. patens CSLDs showed that they all localize to cell plates in gametophores and protonemata, and to the apical plasma membrane of tip-growing protonemal cells. Finally, live-cell imaging revealed that CSLDs move in the plasma membrane and that this movement requires catalytic activity. Compared to CESAs, CSLD movements were faster, shorter, less linear, and insensitive to the cellulose synthesis inhibitor isoxaben, and they also failed to track along microtubules. These results indicate that CSLDs assemble as distinct rosette CSCs that are propelled in the plasma membrane as the glucan chains they produce assemble into microfibrils similar to those produced by CESAs. The ancestral functions of CSLDs include both tip growth and cytokinesis, with functional differentiation occurring in the vascular plant lineage. Funded by NSF-IOS awards 2124176 and 2124178.