Disruption of long-chain base hydroxylation alters growth and impacts sphingolipid synthesis in Physcomitrella patens.

ABSTRACT

Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. In Physcomitrella patens, 4-hydroxysphinganine (phytosphingosine, t180) is the predominant sphingolipid long-chain base (LCB). To assess the functional significance of t180, CRISPR-Cas9 mutagenesis was used to generate mutant lines lacking the sole SPHINGOID BASE HYDROXYLASE (SBH) gene encoding the hydroxylase responsible for converting sphinganine (d180) to t180. Total sphingolipid content in sbh protonemata was 2.4-fold higher than in wild-type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t180 indicated modest alterations in acyl-chain pairing with d180 in GIPCs and ceramides, but dramatic alterations in acyl-chain pairing in glucosylceramides, in which 4,8-sphingadienine (d182) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. The sbh lines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t170, the endogenous sphingolipid content of sbh lines decreased to wild-type levels, and the mutants exhibited phenotypes more similar to wild-type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyte P. patens and angiosperm A. thaliana.