An alternate route for cellulose microfibril biosynthesis in plants

Like cellulose synthases (CESAs), cellulose synthase-like D (CSLD) proteins synthesize β-1,4 glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss Physcomitrium patens and used them to investigate CSLD function in the absence of interfering CESA activity. Microscopy and spectroscopy showed that the CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils in P. patens protonemal filaments. They also suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca2+, rather than structural differences in the microfibrils they produce.

Methods
Each set of CSCs was measured by hand. The Polygon selection tool in Fiji (https://fiji.sc/) was used to anchor the outer edge of each lobe without omitting parts of lobes, resulting in a hexagon around the CSC. The included area (A) within the hexagon was used to calculate the estimated long diameter (d), assuming the geometry of a regular hexagon even when small deviations from regularity existed: d = 1.732 x (SQRT (A/2.5982)) (https://rechneronline.de/pi/hexagon.php). The same CSCs were used as input for EMAN2 (https://blake.bcm.edu/emanwiki/EMAN2.