Cell-free Conversion of Castasterone in Cultured Cells of Phaseolus vulgaris and Marchantia polymorpha

Abstract

Brassinosteroids (BRs) are steroidal plant hormones, which are required for the normal growth and development of plants. Castasterone (CS) and brassinolide (BL) are the most frequently identified BRs in the plant kingdom. Feeding experiments using isotope-labeled substrates revealed that BL is biosynthesized from CS by 7-oxalactonation. However, many plants which possess a fair amount of CS contain very low levels of BL. In addition, in plants which can convert CS to BL, the conversion rate is extremely low, yielding high levels of CS in the plants. Because CS, as well as BL, is known to induce feedback regulation of earlier steps in BR biosynthesis pathways, accumulation of CS may limit or alter BR biosynthesis in plants. Therefore, endogenous levels of CS should be reduced, after BL production, to a level below that at which feedback regulation can occur. This is a difficult proposition, as little is yet known about the catabolism of CS. This dearth of available data prompted us to investigate the catabolism of CS in cultured Phaseolus vulgaris and Marchantia polymorpha cells, in which the presence of CS and BL and conversion of CS to BL have been demonstrated. Cultured cells (5 g) of P. vulgaris and M. polymorpha were homogenized and centrifuged at 8, 000 × g for 10 minutes, and the resulting supernatants were re-centrifuged at 20,000 × g for 30 min. Cold acetone was then added to the obtained supernatants (final volume 40%), and the acetone precipitates were re-suspended in 0.1 M Na phosphate buffer (pH 7.4) containing 1.5 mM 2-mercaptoethanol and 30% glycerol for crude enzyme solutions. Non-labeled CS and NADPH (4.8 mM) were added to the enzyme solutions as a substrate and a cofactor, respectively, to examine catabolism of CS in the plants. After incubating at 37 C for 30 minutes, the assay mixtures were extracted with ethyl acetate (1.2 mL × 3). The obtained ethyl acetate soluble fractions were loaded on a Sep-Pak C18 cartridge eluted with 50%, and 100% methanol (5 mL each). The 100% methanol fractions were further purified by reversed phase HPLC (Nova Pak, C18, 8 × 100 mm) and eluted with 40% acetonitrile at a flow rate of 1 mL min−1. Fractions were collected every min, and analyzed by a preparative TLC (Merck, HPTLC F254) developed with a 6 : 1 mixture of chloroform and methanol. Besides fraction 19-21, which contained CS (added as the substrate), fraction 13-15 exhibited a BR-like blue-purplish spot at Rf 0.30. The metabolite in the fractions was analyzed by GC-MS/-SIM after methaneboronation. In GC-MS, bismethaneboronate (BMB) of the metabolite showed a molecular ion at m/z 498 and the most abundant ion at m/z 141, due to the fission of C20/C22, which was reduced in mass compared with CS BMB. The mass reduction suggests that a methyl in CS was eliminated in the metabolite. Therefore, the metabolite was proposed to be either 26-norCS or 28-norCS. In GC-MS, BMB of 26-norCS and 28-norCS showed basically the same mass spectrum, but their retention times on GC were clearly different. As shown in Table 1, GC retention time of BMB of the metabolite was equal to that of 26-norCS BMB. Consequently, the metabolite was characterized as 26-norCS. 26-NorCS showed approximately one-tenth the level of activity of CS, indicating that 26-norCS is a catabolite of CS in plant cells. Because isotope-labeled 26-norCS is not available, activity for the enzyme catalyzing the conversion of CS to 26-norCS, namely CS C-26 demethylase, was measured by GC-SIM based quantification method, yielding 0.90 and 0.31 ng mg−1