MpDWF5A-Encoded Sterol Δ7-Reductase Is Essential for the Normal Growth and Development of Marchantia polymorpha.

Sterols are essential components of 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 the enzymes responsible for sterol biosynthesis in M. polymorpha. We further focused on characterizing two genes, MpDWF5A and MpDWF5B, which showed high homology with A. thaliana DWF5, encoding Δ5,7-sterol Δ7-reductase (C7R). Functional analysis using a yeast expression system revealed that MpDWF5A converted 7-dehydrocholesterol to cholesterol, indicating that MpDWF5A is a C7R. Mpdwf5a-knockout (Mpdwf5a-ko) lines were constructed using CRISPR/Cas9-mediated genome editing. Gas chromatography-mass spectrometry 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 castasterone or 6-deoxocastasterone, a bioactive brassinosteroid (BR), 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-ko defect is due to the deficiency of typical phytosterols and, in part, a BR-like compound derived from phytosterols.

Fungal-Type Terpene Synthases in Marchantia polymorpha Are Involved in Sesquiterpene Biosynthesis in Oil Body Cells.

The liverwort Marchantia polymorpha possesses oil bodies in idioblastic oil body cells scattered in its thallus. Oil bodies are subcellular organelles in which specific sesquiterpenes and bisbibenzyls are accumulated. Therefore, a specialized system for the biosynthesis and accumulation of these defense compounds specifically in oil bodies has been implied. A recent study on M. polymorpha genome sequencing revealed 10 genes that shared high similarities with fungal-type terpene synthases (TPSs). Eight of these fungal-type TPS-like genes in M. polymorpha (MpFTPSL1-6, -9 and -10) are located within a 376-kb stretch on chromosome 6 and share similarities of over 94% at the nucleotide level. Therefore, these genes have likely originated from recent gene duplication events. The expression of a subset of MpFTPSLs was induced under non-axenic growth on vermiculite, which increased the amounts of sesquiterpenes and number of oil bodies. The tdTomato fluorescent protein-based in-fusion reporter assay with MpFTPSL2 promoter revealed fluorescent signals specifically in oil body cells of the thallus, indicating that MpFTPSL2 functions in oil body cells. Recombinant MpFTPSL2 expression in Escherichia coli led to sesquiterpene synthesis from farnesyl pyrophosphate. Moreover, suppression of a subset of MpFTPSLs through RNA interference reduced sesquiterpene accumulation in thalli grown on vermiculite. Taken together, these results suggest that at least a subset of MpFTPSLs is involved in sesquiterpene synthesis in oil body cells.

Molecular cloning and functional characterization of an O-methyltransferase catalyzing 4'-O-methylation of resveratrol in Acorus calamus.

Resveratrol and its methyl ethers, which belong to a class of natural polyphenol stilbenes, play important roles as biologically active compounds in plant defense as well as in human health. Although the biosynthetic pathway of resveratrol has been fully elucidated, the characterization of resveratrol-specific O-methyltransferases remains elusive. In this study, we used RNA-seq analysis to identify a putative aromatic O-methyltransferase gene, AcOMT1, in Acorus calamus. Recombinant AcOMT1 expressed in Escherichia coli showed high 4'-O-methylation activity toward resveratrol and its derivative, isorhapontigenin. We purified a reaction product enzymatically formed from resveratrol by AcOMT1 and confirmed it as 4'-O-methylresveratrol (deoxyrhapontigenin). Resveratrol and isorhapontigenin were the most preferred substrates with apparent Km values of 1.8 µM and 4.2 µM, respectively. Recombinant AcOMT1 exhibited reduced activity toward other resveratrol derivatives, piceatannol, oxyresveratrol, and pinostilbene. In contrast, recombinant AcOMT1 exhibited no activity toward pterostilbene or pinosylvin. These results indicate that AcOMT1 showed high 4'-O-methylation activity toward stilbenes with non-methylated phloroglucinol rings.