Terpenoids are one of the large family of natural compounds which are produced in living organisms. The structures of terpenoids are varying from relatively simple linear hydrocarbon chains to complicated ring structures. Among the terpenoids, a variety of cyclic diterpenoids, the plant hormones gibberellins (GAs), phytoalexins, and the constitutively produced antimicrobial compounds oryzalides have been isolated and identified from rice plants. These compounds with unique biological activities have been suggested to be biosynthesized from a common precursor, geranylgeranyl diphosphate (GGDP) via two-step sequential cyclization followed by several oxidation steps. However, some of important biosynthetic enzymes have not been functionally identified, and the details of regulatory mechanisms of their biosynthesis remain to be clarified. In this study, functional analysis of P450s which are involved in biosynthesis of the biologically important rice diterpenoids Gas and phytoalexins was performed, and involvement of cytokinin in induction of production of the diterpenoid phytoalexins in・@rice was investigated.

I. Functional analysis of OsKO2

Gibberellins are biosynthesized from GGDP via the diterpene hydrocarbon precursor ent-kaurene, which is converted into bioactive GAs through several oxidation steps catalyzed by P450s and dioxygenases. Recently, it was shown that some of P450 genes in CYP701A family are involved in the steps from ent-kaurene to ent-kaurenoic acid via ent-kaurenol and ent-kaurenal. Itoh et al. found five CYP70IA genes including ent-kaurene oxidase (KO) gene form a cluster on chromosome 6 of rice (Oryza sativa L. cv. Nipponbare), and indicated that both OsKO1 and OsKO2 complement the dwarf phenotype in the KO-mutant d355nTan-ginbEoxzup.r ession analysis of the genes has shown that OsKO2 is expressed in whole organs, and that OsKO1 expression is restricted to the panicles and roots, suggesting that OsKO2 encodes the main KO that contributes to the GA biosynthesis for vegetative growth in rice. However, the functional identification of OsK02 has not been carried out yet. Therefore, identification of OsKO2 in vitro enzyme activity was attempted using the methylotrophic yeast Pichia pastoris expression system.

The cDNA of OsKO2 and a fungal P450 reductase gene (PhCPR) were introduced into the expression vectors pPICZ and pPIC6 for yeast expression, respectively, both of which were expressed under an alcohol oxidase promoter and as a c-myc epitope tagged fusion protein. After the induction using MeOH, the yeast cells were harvested to prepare the microsomal fraction. Expression of OsKO2-myc and PhCPR-myc was confirmed by western blot analysis. Enzyme assays using ent-kaurene or [2H2]ent-kaurenol as a substrate were carried out. Incubation of the microsomal fraction with [2Hz]ent-kaurenol gave [2H2] ent-kaurenoic acid. When the microsomal fraction was incubated with ent-kaurene, ent-kaurenoic acid was detected as a reaction product. These results indicate that OsKO2 is involved in the 3-step oxidation from ent-kaurene to ent-kaurenoic acid via ent-kaurenol and ent-kaurenal in the GA biosynthetic pathway in rice (Fig. 1).

II. Functional analysis of CYP99A2 and CYP99A3

When plants are attacked by pathogenic microorganisms, they protect themselves by a variety of defense responses including production of the antimicrobial secondary metabolites phytoalexins. In rice, 14 diterpenoid phytoalexins have been identified in suspension-cultured cells treated with biotic elicitors such as a chitin oligosaccharide Nacetylchitooctaose elicitor and/or from leaves that were either infected with the rice blast fungus Magnaporthe oryzae or exposed to UV irradiation. These diterpenoid phytoalexins are classified into four groups based on their basic carbon frameworks: phytocassanes A-E, oryzalexins A-F, momilactones A and B, and oryzalexin S, the major diterpenoid phytoalexins being phytocassanes and momilactones. Concerning enzymes involved in biosynthesis of the diterpenoid phytoalexins, all the six diterpene cyclases catalyzing the conversion of the common precursor GGDP to the four diterpene hydrocarbon precursors via ent- or syn-CDP have been identified. On the other hand, concerning the enzymes catalyzing the downstream oxidation of the diterpene hydrocarbons, involvement of microsomal P450s and/or dehydrogenases has been suggested. However, none of the enzymes involved in the downstream oxidation have been identified. It was previously reported that two diterpene cyclase genes involved in phytocassane biosynthesis, OsKSL7 and OsCPS2, are located in a narrow region on chromosome 2, and that two diterpene cyclase genes involved in momilactone biosynthesis, OsKSL4 and OsCPS4, are located in a narrow region on chromosome 4. In addition, six and two elicitor-inducible P450 genes have been found near the diterpene cyclase genes on chromosomes 2 and 4, suggesting that the phytocassane and momilactone biosynthesis genes are clustered on chromosomes 2 and 4, respectively. To investigate this hypothesis, I focused on the gene cluster on rice chromosome 4 and attempted to perform functional analysis of the two P450 genes CYP99A2 and CYP99A3.

Utilizing the Rice Genome Automated Annotation System (RiceGAAS), it was found that the genes OsCPS4, CYP99A3, the putative dehydrogenase gene AK103462, OsKSL4, and CYP99A2 are linearly arranged within a 168-kb region on chromosome 4. The transcriptional expression of the clustered five genes was up-regulated in suspension-cultured rice cells after treatment with a chitin oligosaccharide elicitor and in rice leaves after UV irradiation. To examine the involvement of CYP99A2 and CYP99A3 in the chitin oligosaccharide elicitor-inducible production of diterpenoid phytoalexins in rice cells, RNAi-mediated knockdown of CYP99A2 and CYP99A3was attempted. Since the ORFs of the two genes share 87% identity at the nucleotide sequence level, RNAi-mediated knockdown of CYP99A2 resulted in production of the double knockdown of the two genes. The accumulation of the major diterpenoid phytoalexins momilactones and phytocassanes in culture media of the double knockdown cell lines after chitin oligosaccharide elicitor treatment was determined by HPLC-MS/MS to indicate that momilactone biosynthesis is specifically suppressed in the double knockdown lines. Functional analysis of the AK103462 protein was performed by one of my colleagues, in parallel with this study, to demonstrate that the AK103462 gene encodes momilactone A synthase (OsMAS), which catalyzes conversion from 3(3-hydroxy-9fA3H- pimara-7,15-dien-19,613-olide to momilactone A. These results strongly suggests that CYP99A2 and/or CYP99A3 are involved in biosynthetic steps between 98H-pimara-7,15-diene and 3f3-hydroxy-9PH-pimara-7,15-dien-19,6(3-olide in the momilactone biosynthetic pathway. It was thus indicated that CYP99A2 and CYP99A3 form a momilactone biosynthetic gene cluster together with the momilactone synthase gene OsMAS and the diterpene cyclase genes OsCPS4 and OsKSL4 on chromosome 4 (Figure 2). In addition, I succeeded in heterologous expression of the recombinant CYP99A2 and CYP99A3 using a baculovirus expression system. Functions of CYP99A2 and CYP99A3 will be able to be determined by enzyme assays when their substrates are available.

III.Involvement of cytokinin in production of diterpenoid phytoalexins in rice

Plant hormones are often involved in the stress responses, which are caused by biotic and abiotic stresses. Defense-associated signaling molecules such as salicylic acid, jasmonic acid, and ethylene have been investigated extensively and suggested to play crucial roles in plant defense mechanisms. In addition, cytokinin is also believed to play important roles in plant defense responses. In fact, cytokinin was suggested to act downstream of a small GTP-binding protein in defense signal transduction pathways in tobacco. Therefore, I investigated the possibility that cytokinin is involved in defense responses, such as phytoalexins production, in rice as well.

Both of the synthetic cytokinin benzyladenine (BA) and the natural cytokinin trans-zeatin (a) induced accumulation of the major diterpenoid phytoalexins phytocassanes and momilactones in culture media of suspension-cultured rice cells and rice leaves in a dose-dependent manner up to 100 μM. In time course study using suspension-cultured rice cells, the levels of diterpenoid phytoalexins increased from 24 h through 96 h in response to BA treatment. Whereas in the chitin oligosaccharide elicitor N-acetylchitooctaose treatment, the levels of diterpenoid phytoalexins began to increase from 8 h and peaked at 72 h. The four diterpene cyclase genes that are involved in phytocassane and momilactone biosynthesis showed strong expression at 8-12 h after chitin oligosaccharide elicitor treatment, whereas in the BA treatment, the expression levels of the genes increased from 8 or 12 h through 96 h after the treatment. It was also confirmed that the expression of four diterpene cyclase genes were induced in rice leaves at 48 and 72 h after the BA treatment. In rice leaf disks, BA also led to increases in the accumulation levels of the diterpenoid phytoalexins and in the expression levels of the four diterpene cyclase genes at 48 h and 72 h after treatment. These effects of BA on accumulation of the diterpenoid phytoalexins in rice leaves were similar to those of exogenously applied JA. It was thus indicated that BA-induced accumulation of the diterpenoid phytoalexins is caused by de novo biosynthesis not only in suspension-culture rice cells but in rice leaves. In rice plants, accumulation of diterpenoid phytoalexins was induced after infection with the pathogen M. oryzae. It was also reported that endogenous cytokinin levels increased in response to infection with M oryzae in rice plants. These results suggest that cytokinin might play a role as a signal molecule in the pathogen-induced production of diterpenoid phytoalexins in rice.

IV Conclusions

In this study, functional analysis of P450s which are involved in biosynthesis of the biologically important rice diterpenoids GAs and phytoalexins was performed, leading to functional identification of OsKO2, a causal gene for the GA-deficient mutant d357511h2o u, and to identification of the momilactone biosynthetic gene cluster. The clustered genes for momilactone biosynthesis exhibit a temporally coordinated expression in suspension-cultured rice cells after elicitor treatment. Such coordinated, stress-inducible clustered gene expression, responsible for the biosynthesis of one particular compound, has not previously been reported in plants. In addition, involvement of cytokinin in pathogen-induced production of the diterpenoid phytoalexins in rice was suggested. This might provide a new insight into plant hormone-dependent defense mechanisms against pathogens in rice.

Figure 1. Involvement of OsK02 in the 3-step oxidation from ent-kaurene to ent-kaurenoic acid in the GA biosynthetic pathway in rice.

Figure 2. Hypothetical momilactone biosynthetic pathway and the possible function of CYP99A2 and/or CYP99A3.

イネにおいては、植物ホルモンであるジベレリンや抗菌性二次代謝産物であるファイトアレキシンなどのユニークな生物活性を有するさまざまなジテルペンが単離同定されてきた。これらのジテルペンは、共通の前駆体であるゲラニルゲラニルニリン酸から二段階の環化とそれに続く数段階の酸化によって生合成されると考えられている。しかしながら、それらの生合成に関与する重要酵素のいくつかについては機能解析が行われておらず、生合成の制御機構についても不明な点が少なくない。そこで、本研究においては、ジベレリンやファイトアレキシンの生合成において重要な機能を果たしているP450酵素の機能解析を行った。また、病原菌誘導のファイトアレキシン生産に関するシグナル伝達系に植物ホルモンであるサイトカイニンが関与する可能性についても追究した。

本研究の背景と目的を述べた第1章に続いて、第2章ではイネのジベレリン生合成経路におけるent-カウレンから研ent-カウレン酸までの3段階の酸化反応を触媒するP450酵素の機能同定について述べている。この酸化反応はCYP701A6(OsKO2)が担うことがイネのd35Tan-ginbozu矮性変異体の研究により示峻されているが、in vitroでの酵索反応による確認はなされていなかった。そこで、イネCYP701A6遺伝子とカビ由来P450reductase遺伝子の両方をメタノール資化性酵母Pichia pastorisの染色体内に導入した形質転換体酵母を作製した。メタノール添加により両タンパク質の生産を誘導した酵母からミクロソーム画分を調製し、ent-カウレン及び[2H2]ent-カウレノールを基質としてin vitroで反応させ、それぞれ、ent-カウレン酸、[2H2]ent-カウレン酸への変換をGC-MSにより確認した。こうして、OsKO2がent-カウレン酸化酵素として機能することを実証し、また、イネのP450酵素の機能解析にP.pastorisが有用なッールとなる可能性を示した。

第3章では、イネのジテルペン型ファイトアレキシンの生合成に関与すると考えられる2種のP450酵素(CYP99A2、CYP99A3)の機能解析を行った。イネの主要なジテルペン型ファイトアレキシンであるモミラクトン類の生合成に関与するジテルペン環化酵素遺伝子であるOg鋸4と0瑚4およびデヒドロゲナーゼ遺伝子(AKl03462)が4番染色体上で近接して存在し、さらにその周辺に2種のP450遺伝子(CYP99A2/CYP99A3)が存在している。また、これらの5種の遺伝子はイネ培養細胞においてキチンオリゴ糖エリシター処理後同調的に発現が誘導されることも示されていた。そこで、`CYP99A2/CYP99A3の二重発現抑制株を作製し、得られたセルラインにおけるエリシター処理後のファイトアレキシン集積量をLC-MSにより定量した。その結果、CYP99A2/CYP99A3二重発現抑制株においては特異的にモミラクトン類の集積が抑制されていることが示され、CYP99A2とCYP99A3の両方、あるいはいずれかがモミラクトン生合成に関与していることが明らかとなった。こうして、4番染色体に4つあるいは5つの遺伝子からなるモミラクトン生合成遺伝子クラスターが存在することを明らかにすることができた。高等植物において単一物質の生合成酵素遺伝子がクラスターを形成している例はいくつか知られているが、いずれも構成的に発現しているものであり、誘導的に発現される遺伝子クラスターはモミラクトン生合成遺伝子クラスターが初めての例と思われる。

第4章では、ジテルペン型ファイトアレキシンの生産誘導に植物ホルモンのサイトカイニンが関与する可能性について追究した。ファイトアレキシンの生産はイネにおける代表的な防御応答の一つであるが、イネのsmallGTP結合タンパク質を発現させたタバコ形質転換体では内生サイトカイニンが増加し、病害抵抗性遺伝子の発現誘導が起こることが報告されている。そこで、本章ではイネのファイトアレキシン生産に対するサイトカイニンの関与を検証するため、ベンジルアデニン(BA)処理後のイネ培養細胞におけるファイトアレキシン生産を調べた。その結果、BA処理により各種ファイトアレキシンの生産誘導、およびその生合成酵素遺伝子群の発現誘導が観察された。またイネ植物体を用いた場合でも同様の結果が得られた。イネいもち病菌が感染したイネにおいてはサイトカイニンの集積量が増加していくことが示されていることを考え合わせると以上の結果は、ファイトアレキシン生産誘導に至るシグナル伝達経路において、サイトカイニンがシグナル分子として機能していることを示唆している。

以上、本論文は、ジベレリンやファイトアレキシンなどのイネのジテルペンの生合成において重要な機能を果たしているP450の機能解析を進めるとともに、ファイ'トアレキシンの生産制御機構に関する新規知見を提供し、ジテルペンの生合成経路の解明とその制御機構の総合的理解に貢献するものであり、学術上、応用上貢献するところが少なくない。よって審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。