博士(生命科学)

Not only taste but also flavor has a significant impact on the "deliciousness" of food as perceived by people. Flavor characteristics of food have a significant impact on its palatability and influence the qualitative value of the food itself. In this study, the molecular mechanisms of biosynthesis of flavor compounds derived from amino acids in soybean (Glycine max) and fatty acids in mushrooms were elucidated. In Chapter 1, I identified the characteristic aromatic properties of soybean due to sulfur-containing amino acids. Several soybean germplasms, such as Nishiyamahitashi 98-5 (NH) among local varieties in Nagano Prefecture, have an intense seaweed-like flavor after cooking because of their high seed S-methylmethionine (SMM) content. In this study, I compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free L-methionine (L-Met) content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to L-Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, I examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon of about 6 kbp insertion into the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of L-Met to sections of developing soybean seeds resulted in transient increases in L-Met levels, followed by continuous increases in SMM concentrations, which was likely caused by L-Met methyltransferase activity in the seeds. Accordingly, I propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of L-Met, which is converted into SMM to avoid the harmful effects caused by excess free L-Met. In Chapter 2, I studied the biosynthesis mechanism of 1-octen-3-ol, the main volatile component of mushrooms. 1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. As 1-octen-3-ol attracts mosquitoes and flies, its involvement in emitter–receiver ecological communication has been proposed. Although the biosynthetic pathway to form 1-octen-3-ol from linoleic acid through linoleic acid 10(S)-hydroperoxide has been proposed in mushrooms, the enzymes involved in this pathway have not been identified. I determined that the Coprinopsis cinerea dioxygenase 1 and 2 (CcDOX1 and CcDOX2) genes in the mushroom C. cinerea contains an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Through analysis of products formed from linoleic acid by the recombinant CcDOX1 and CcDOX2 proteins, I found that CcDOX1 preferentially catalyzes to form the 10(S)-hydroperoxide of linoleic acid (10S-HPODE), meanwhile CcDOX2 form the 8-hydroperoxide of linoleic acid. Moreover, disruption of Ccdox1 in C. cinerea (ΔCcdox1) mycelia suppressed 1-octen-3-ol synthesis. Administration of the 10S-HPODE to the microsome fraction prepared from mycelia resulted in the efficient production of 1-octen-3-ol. Together, these results indicate that CcDOX1 is essential for the biosynthesis of 1-octen-3-ol as the oxygenase that forms 10S-HPODE, followed by the cleavage enzyme. I studied physiological and ecological significance of 1-octen-3-ol of mushroom. ΔCcdox1 was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the wild-type strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on wild-type mycelia. Thus, 1-octen-3-ol seems to be an attractive compound for some animals that interact with mushrooms.