Identification of 6-Deoxocastasterone and Brassinolide from a Liverwort, Marchantia polymorpha

Abstract

Brassinosteroids (BRs) are steroidal plant hormones which play essential roles in plant growth and morphogenesis. [1][2][3][4] The presence of BRs in lower plants has been already demonstrated, [5][6][7] but biosynthesis of BRs in lower plants has not been well known yet. To get information on biosynthesis of BRs in lower plants, we have previously investigated endogenous BRs and their biosynthetic precursors in a liverwort, Marchantia polymorpha, resulting in identification of castasterone (CS) and its two biosynthetic precursors, campesterol (CR) and campestanol (CN) in the lower plant.8,9 CS, CR and CN are members of the early and late C6-oxidation pathway ( Fig. 1) in higher plants, 10~15 which suggested that the liverwort contained the early and/or late C6-oxidation pathway to produce BRs. To verify that, we attempted to identify other BRs included in the pathway with a large amount of M. polymorpha in the study.Naturally-grown M. polymorpha (5.3 Kg) collected in early July, 2001, at Banpo area, Seoul, was extracted with 80% methanol. After concentrating to aqueous phase in vacuo, 200 ng of deuterium labeled ([26, 28-2 H 6 ]) teasterone (TE), typhasterol (TY), 6-deoxocastasterone (6-deoxoCS), CS and brassinolide (BL) were added to the extract as internal standards for quantitative analysis. By guidance of the rice lamina inclination assay, 16 the extract was reextracted, solvent-partitioned and column-chromatographed using silica gel, Sephadex LH-20 and ODS (RP-18). The resulting active fractions were combined, and further purified by a reversed phase HPLC.After HPLC, BR-like activities were detected in several fractions (Fig. 2). To get hint for the presence of BRs in the active fractions, authentic TE, TY, 6-deoxoCS, CS and BL were analyzed by the same HPLC condition, providing that the authentic BRs were eluted in the fraction 36, 41, 46, 20 and 14-15, respectively. Therefore, it was thought that fraction I, II, III, IV and V may contain [ 2 H 6 ]-BL, -CS, -TE, -TY and -6-deoxoCS added as internal standard possibly accompanied with deuterium unlabeled ([ 2 H 0 ]) endogenous BL, CS, TE, TY and 6-deoxoCS, respectively. To confirm that, the fractions were derivatized to a bismethaneboronates (BMBs) or monomethaneboronate (MB)-trimethylsilyl (TMSi) ethers, and analyzed by GC-MS and/or GC-selected ion monitoring (SIM).In the GC-SIM, BMB of an active compound in fraction I gave mass ions at m/z 534 and 161 which are a molecular and a base peak for [ 2 H 6 ]-BL BMB at the same retention time as that of authentic sample (Table 1). In addition, the other active compound in the same fraction showed ions at m/z 528 and 155 which were 6 mass less than those derived from