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 a..., 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., , # An alternate route for cellulose microfibril biosynthesis in plants

[https://doi.org/10.5061/dryad.n02v6wx5j](https://doi.org/10.5061/dryad.n02v6wx5j)

## Description of the data and file structure

The data include two .zip files containing annotated digital freeze-fracture electron micrographs from 1) protonemal filaments of CESA-deficient *Physcomitrium patens* and 2) differentiating tracheary elements from *Zinnia elegans*. Original images are typically at 80k magnification, with a few at 70k, always with a 200 nm scale bar. As described in the main text, high confidence rosette CSCs were selected for measurement through steps outlined below. Although rosette CSCs are highly distinctive, other intramembrane proteins can also cluster, making it necessary to critically select the instances for final analysis.

1\) One expert drew circles with Fiji ([https://fiji.sc/](https://fiji.sc/)) around particle clusters that might be CSCs. Fiji merged circles in a few instances of closely tw...