# Complex sphingolipid metabolism impacts cell division and plasmodesmal development in the moss Physcomitrium patens

## TL;DR

This study shows that sphingolipid metabolism is crucial for cell division and plasmodesmal development in the moss Physcomitrium patens.

## Contribution

The paper introduces a toolkit for quantifying cell division and plasmodesmal defects in mosses using CRISPR-generated sphingolipid mutants.

## Key findings

- Sphingolipid deficiencies cause abnormal cell division planes and wall depositions in s4h mutants.
- ipcs mutants exhibit incomplete cell divisions and compromised cell autonomy.
- Plasmodesmal structural defects in mutants correlate with impaired macromolecule transport.

## Abstract

Developmental patterning and organ structure are elegantly simple in the moss Physcomitrium patens, which facilitates the cultivation and phenotypic characterization of severe mutant alleles. Essential membrane lipids, such as complex phosphosphingolipids (in plants, glycosyl inositol phosphorylceramides, GIPCs), are difficult to functionally characterize due to non-viable and pleiotropic phenotypes of mutants affected in their synthesis. Following the isolation and biochemical characterization of mutants affected in GIPC synthesis in P. patens, including sphinganine-C4-hydroxylase/sphingoid base hydroxylase (s4h/sbh) and inositol phosphorylceramide synthase (ipcs), we report some of their morphological, histological, and cytological phenotypes. We observed alterations in cell division, expansion, and differentiation. Specifically, the s4h knock-out mutant had abnormal cell division planes, as well as irregular depositions attached to cell walls. Severe ipcs mutant alleles showed frequent incomplete cell divisions, causing compromised cell autonomy as demonstrated by intercellular motility assays. These phenotypes suggest that sphingolipids impact both the orientation and proper formation of the cell plate during cytokinesis. Transmission electron microscopy revealed dramatic plasmodesmal structural defects in ipcs and s4h mutants, and these correlated with a macromolecule transport phenotype in s4h. Our methods can be used as a toolkit for quantifying growth, specifically cell division and plasmodesmal phenotypes in mosses, and our results illuminate key relationships between sphingolipid metabolism and fundamental cell functions. The severity of the observed defects in cell ultrastructure underscores both the resilience and the utility of P. patens as a model for investigating severe mutant phenotypes.

Sphingolipid deficiencies induced by targeted CRISPR mutagenesis produce extensive developmental defects in the moss Physcomitrium patens, impacting cytokinesis and plasmodesmal maturation.