MpDWF5A-encoded sterol Δ7-reductase is essential for the normal growth and development of Marchantia polymorpha

Sterols are the essential components of the eukaryotic cell membranes. However, studies on sterol biosynthesis in bryophytes are limited. This study analyzed the sterol profiles in the bryophyte model plant Marchantia polymorpha L. The thalli contained typical phytosterols such as campesterol, sitosterol, and stigmasterol. BLASTX analysis of the M. polymorpha genome against the Arabidopsis thaliana sterol biosynthetic genes confirmed the presence of all of the enzymes responsible for sterol biosynthesis in M. polymorpha. In this study, we focused on characterizing two genes, MpDWF5A and MpDWF5B, which showed high homology with A. thaliana DWF5, encoding Δ5,Δ7-sterol Δ7-reductase. Functional analysis using a yeast expression system revealed that MpDWF5A converted 7-dehydrocholesterol to cholesterol, indicating that MpDWF5A is a Δ5,Δ7-sterol Δ7-reductase. Mpdwf5a-knockout lines (Mpdwf5a-ko) were constructed using CRISPR/Cas9 mediated genome editing. GC-MS analysis of Mpdwf5a-ko revealed that phytosterols such as campesterol, sitosterol, and stigmasterol disappeared, and instead, the corresponding Δ7-type sterols accumulated. The thalli of Mpdwf5a-ko grew smaller than those of the wild type, and excessive formation of apical meristem in the thalli was observed. In addition, the gemma cups of the Mpdwf5a-ko were incomplete, and only a limited number of gemma formations were observed. Treatment with 1 µM of castesterone or 6-deoxocastasterone, a bioactive brassinosteroid, partly restored some of these abnormal phenotypes, but far from complete recovery. These results indicate that MpDWF5A is essential for the normal growth and development of M. polymorpha and suggest that the dwarfism caused by the MpDWF5A defect is due to the deficiency of typical phytosterols and, in part, a brassinosteroid-like compound derived from phytosterols.