Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor-and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3␤-hydroxy-9␤H-pimara-7,15-dien-19,6␤-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.Plants that are attacked by pathogenic microorganisms respond with a variety of defense reactions. One such reaction is the production of secondary metabolites that serve as plant antibiotics, known as phytoalexins, which are generated through the perception of signal molecules called elicitors, which are mostly derived from pathogens. Fifteen phytoalexin compounds have been identified in suspension-cultured rice cells treated with biotic elicitors such as a chitin oligosaccharide or a cerebroside (1, 2) and/or from rice leaves that were either infected with the rice leaf blast pathogen Magnaporthe grisea or exposed to UV irradiation (3-10). With the exception of the flavonoid sakuranetin, all of these rice phytoalexins are diterpenoids. These compounds have been classified into four structurally distinct types of polycyclic diterpenoid phytoalexins based on the structures of their diterpene hydrocarbon precursors: phytocassanes A to E, oryzalexins A to F, momilactones A and B, and oryzalexin S. The common precursor geranylgeranyl diphosphate is cyclized to ent-copalyl diphosphate (ent-CDP) and then to ent-cassa-12,15-diene and ent-sandaracopimaradiene, leading to phytocassanes A to E and oryzalexins A to F, respectively. Geranylgeranyl diphosphate is also cyclized to syn-CDP and then to 9␤H-pimara-7,15-diene and stemar-13-ene, leading to momilactones A and B and oryzalexin S, respectively. The hypotheti...

Blast fungus-induced accumulations of major rice diterpene phytoalexins (PA), momilactones A and B, and phytocassanes A through E were studied, focusing on their biosynthesis and detoxification. In resistant rice, all PA started to accumulate at 2 days postinoculation (dpi), at which hypersensitive reaction (HR)-specific small lesions became visible and increased 500- to 1,000-fold at 4 dpi, while the accumulation was delayed and several times lower in susceptible rice. Expression of PA biosynthetic genes was transiently induced at 2 dpi only in resistant plants, while it was highly induced in both plants at 4 dpi. Fungal growth was severely suppressed in resistant plants by 2 dpi but considerably increased at 3 to 4 dpi in susceptible plants. Momilactone A treatment suppressed fungal growth in planta and in vitro, and the fungus detoxified the PA in vitro. These results indicate that HR-associated rapid PA biosynthesis induces severe restriction of fungus, allowing higher PA accumulation in resistant rice, while in susceptible rice, failure of PA accumulation at the early infection stage allows fungal growth. Detoxification of PA would be a tactic of fungus to invade the host plant, and prompt induction of PA biosynthesis upon HR would be a trait of resistant rice to restrict blast fungus.

Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-D: -xylulose 5-phosphate synthase (DXS), 1-deoxy-D: -xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5'-diphospho)-2-C-methyl-D: -erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants.

The effect of protein fractionation on the bioavailability of amino acids and peptides and insulin response and whether the protein source influences these effects in humans are poorly understood. This study compared the effects of different sources and degrees of hydrolysis of dietary protein, independent of carbohydrate, on plasma amino acid and dipeptide levels and insulin responses in humans. Ten subjects were enrolled in the study, with five subjects participating in trials on either soy or whey protein and their hydrolysates. Protein hydrolysates were absorbed more rapidly as plasma amino acids compared to nonhydrolyzed protein. Whey protein also caused more rapid increases in indispensable amino acid and branched-chain amino acid concentrations than soy protein. In addition, protein hydrolysates caused significant increases in Val-Leu and Ile-Leu concentrations compared to nonhydrolyzed protein. Whey protein hydrolysates also induced significantly greater stimulation of insulin release than the other proteins. Taken together, these results demonstrate whey protein hydrolysates cause significantly greater increases in the plasma concentrations of amino acids, dipeptides, and insulin.

When plants interact with certain pathogens, they protect themselves by generating various chemical and physical barriers called the hypersensitive response. These barriers are induced by molecules called elicitors that are produced by pathogens. In the present study, the most active elicitors of the hypersensitive response in rice were isolated from the rice pathogenic fungus Magnaporthe grisea, and their structures were identified as cerebrosides A and C, sphingolipids that were previously isolated as inducers of cell differentiation in the fungus Schizophyllum commune. Treatment of rice leaves with cerebroside A induced the accumulation of antimicrobial compounds (phytoalexins), cell death, and increased resistance to subsequent infection by compatible pathogens. The degradation products of cerebroside A (fatty acid methyl ester, sphingoid base, and glucosyl sphingoid base) showed no elicitor activity. Hydrogenation of the 8E-double bond in the sphingoid base moiety or the 3E-double bond in the fatty acid moiety of cerebroside A did not alter the elicitor activity, whereas hydrogenation of the 4E-double bond in the sphingoid base moiety led to a 12-fold decrease in elicitor activity. Furthermore, glucocerebrosides from Gaucher's spleen consisting of (E)-4-sphingenine and cerebrosides from rice bran mainly consisting of (4E,8E)-4,8-sphingadienine and (4E,8Z)-4,8-sphingadienine showed no elicitor activity. These results indicate that the methyl group at C-9 and the 4E-double bond in the sphingoid base moiety of cerebrosides A and C are the key elements determining the elicitor activity of these compounds. This study is the first to show that sphingolipids have elicitor activity in plants.