Sphingadienine-1-phosphate levels are regulated by a novel glycoside hydrolase family 1 glucocerebrosidase widely distributed in seed plants.

Long-chain base phosphates (LCBPs) such as sphingosine-1-phosphate and phytosphingosine-1-phosphate function as abscisic acid (ABA)-mediated signaling molecules that regulate stomatal closure in plants. Recently, a glycoside hydrolase family 1 (GH1) ß-glucosidase, Os3BGlu6, was found to improve drought tolerance by stomatal closure in rice, but the biochemical functions of Os3BGlu6 have remained unclear. Here we identified Os3BGlu6 as a novel GH1 glucocerebrosidase (GCase) that catalyzes the hydrolysis of glucosylceramide to ceramide. Phylogenetic and enzymatic analyses showed that GH1 GCases are widely distributed in seed plants and that pollen or anthers of all seed plants tested had high GCase activity, but activity was very low in ferns and mosses. Os3BGlu6 had high activity for glucosylceramides containing (4E,8Z)-sphingadienine, and GCase activity in leaves, stems, roots, pistils, and anthers of Os3BGlu6-deficient rice mutants was completely absent relative to that of wild-type rice. The levels of ceramides containing sphingadienine were correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. The levels of LCBPs synthesized from ceramides, especially the levels of sphingadienine-1-phosphate, were also correlated with GCase activity in each rice organ and were significantly lower in Os3BGlu6-deficient rice mutants than in the wild type. These results indicate that Os3BGlu6 regulates the level of ceramides containing sphingadienine, influencing the regulation of sphingadienine-1-phosphate levels and subsequent improvement of drought tolerance via stomatal closure in rice.

A new method for the preparation of a purified glucosylceramide and ceramide from shiitake mushroom.

Ingestion of plant and fungal glucosylceramides is known to reduce colon carcinogenesis and skin barrier damage in mice and humans. However, such effects in animal experiments have not been revealed for plant and fungal ceramides because the content of ceramides contained in plants and fungi is so low that the large amount required for animal experiments is difficult to obtain. Noting that the fungus shiitake mushroom (Lentinula edodes) is rich in a glucosylceramide, (4E,8E)-N-d-2'-hydroxypalmitoyl-1-O-ß-d-glucopyranosyl-9-methyl-4,8-sphingadienine [Glc-d19:2(4E,8E,9Me)-h16:0], we developed a new method to purify this fungal glucosylceramide using ethanol precipitation and high-performance liquid chromatography. We also developed a new method to produce large amounts of a ceramide [d19:2(4E,8E,9Me)-h16:0] from this purified glucosylceramide using human glycoside hydrolase family 30 glucocerebrosidase (imiglucerase). These methods will be useful for elucidating the physiological function by ingestion of fungal ceramides in animal experiments.

Direct LC-ESI-MS/MS analysis of plant glucosylceramide and ceramide species with 8E and 8Z isomers of the long chain base.

Glucosylceramides and ceramides with 8E and 8Z isomers of the long chain base are found in plants. These isomers have been difficult to quantify separately using liquid chromatography-tandem mass spectrometry (LC-MS/MS) because the isomers have the same retention time, their precursor and product ions have the same m/z values, and plant ceramide standards are not commercially available. Here we tested trial separations using various ODS columns and prepared plant ceramide standards generated by human glucocerebrosidase (imiglucerase) using commercially available plant glucosylceramide standards as the substrates. Consequently, we were able to quantify the isomers based on differences in retention times in a TSKgel ODS-120A column (Tosoh, Tokyo Japan) using LC-electrospray ionization-MS/MS (LC-ESI-MS/MS).

Reverse-genetic approach to verify physiological roles of rice phytoalexins: characterization of a knockdown mutant of OsCPS4 phytoalexin biosynthetic gene in rice.

A variety of labdane-related diterpenoids, including phytocassanes, oryzalexins and momilactones, were identified as phytoalexins in rice (Oryza sativa L.). Momilactone B was also isolated as an allelochemical exuded from rice roots. The biosynthetic genes of these phytoalexins have been identified, including six labdane-related diterpene cyclase genes such as OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8 and OsKSL10. Here we identified an OsCPS4 knockdown mutant, cps4-tos, by screening Tos17 mutant lines using polymerase chain reaction. OsCPS4 encodes a syn-copalyl diphosphate synthase responsible for momilactones and oryzalexin S biosynthesis. Because Tos17 was inserted into the third intron of OsCPS4, the mature OsCPS4 mRNA was detected in the cps4-tos mutant as well as the wild type. Nevertheless, mature OsCPS4 transcript levels in the cps4-tos mutant were about one sixth those in the wild type. The cps4-tos mutant was more susceptible to rice blast fungus than the wild type, possibly due to lower levels of momilactones and oryzalexin S in the mutant. Moreover, co-cultivation experiments suggested that the allelopathic effect of cps4-tos against some kinds of lowland weeds was significantly lower than that of the wild type, probably because of lower momilactone content exuded from cps4-tos roots. A reverse-genetic strategy using the cps4-tos mutant showed the possible roles of momilactones not only as phytoalexins but also as allelopathic substances.

Regulation of a proteinaceous elicitor-induced Ca2+ influx and production of phytoalexins by a putative voltage-gated cation channel, OsTPC1, in cultured rice cells.

Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.

Post-exercise whey protein hydrolysate supplementation induces a greater increase in muscle protein synthesis than its constituent amino acid content.

It is well known that ingestion of a protein source is effective in stimulating muscle protein synthesis after exercise. In addition, there are numerous reports on the impact of leucine and leucine-rich whey protein on muscle protein synthesis and mammalian target of rapamycin (mTOR) signalling. However, there is only limited information on the effects of whey protein hydrolysates (WPH) on muscle protein synthesis and mTOR signalling. The aim of the present study was to compare the effects of WPH and amino acids on muscle protein synthesis and the initiation of translation in skeletal muscle during the post-exercise phase. Male Sprague­Dawley rats swam for 2 h to depress muscle protein synthesis. Immediately after exercise, the animals were administered either carbohydrate (CHO), CHO plus an amino acid mixture (AA) or CHO plus WPH. At 1 h after exercise, the supplements containing whey-based protein (AA and WPH) caused a significant increase in the fractional rate of protein synthesis (FSR) compared with CHO. WPH also caused a significant increase in FSR compared with AA. Post-exercise ingestion of WPH caused a significant increase in the phosphorylation of mTOR levels compared with AA or CHO. In addition, WPH caused greater phosphorylation of ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 than AA and CHO. In contrast, there was no difference in plasma amino acid levels following supplementation with either AA or WPH. These results indicate that WPH may include active components that are superior to amino acids for stimulating muscle protein synthesis and initiating translation.

Collagen hydrolysate intake improves the loss of epidermal barrier function and skin elasticity induced by UVB irradiation in hairless mice.

BACKGROUND: Ultraviolet B (UVB) irradiation induces serious damage to the skin. Collagen hydrolysate and collagen-derived peptides have effects on skin function in vivo and in vitro. However, few studies have investigated changes in the epidermal barrier or dermal elasticity caused by UVB. Here, we investigated the loss of epidermal barrier function and skin elasticity induced by UVB irradiation in hairless mice fed collagen hydrolysate. METHODS: Mice were orally administered collagen hydrolysate, in a single dose (20 mJ/cm(2) ) or repeated doses (10-30 mJ/cm(2) , 3 times/week for 6 weeks), and the dorsal skin was exposed to UVB. Skin measurements and histological and analytical studies were performed. RESULTS: In control mice, a single UVB irradiation induced epidermal barrier dysfunction including an increase in transepidermal water loss (TEWL), epidermal hyperplasia, and a decrease in stratum corneum water content. Administration of collagen hydrolysate significantly decreased TEWL and epidermal thickness and increased stratum corneum water content. Repeated UVB irradiation decreased skin elasticity and dermal hyaluronic acid (HA) content in control mice, whereas collagen hydrolysate significantly suppressed both the increase in TEWL and the decrease in stratum corneum water content and improved skin elasticity and dermal HA content. CONCLUSIONS: Collagen hydrolysate administration affects epidermal barrier function and dermal skin elasticity.

Comparison of fructooligosaccharide utilization by Lactobacillus and Bacteroides species.

The utilization of 1-kestose (GF(2)) and nystose (GF(3)), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF(2), whereas L. acidophilus and all the Bacteroides strains utilized both GF(2) and GF(3). Only the strains able to utilize both GF(2) and GF(3) had ß-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher ß-fructosidase activity for GF(2) than for GF(3), whereas those of the Bacteroides strains had higher activity for GF(3) than for GF(2). Furthermore, ß-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF(3) medium was much higher than that of the cells grown in the GF(2) medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF(3) medium was almost the same as that of the cells grown in the GF(2) medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.

Ingestion of Indigestible Cacao Proteins Promotes Defecation and Alters the Intestinal Microbiota in Mice.

Background: In animals, the health effects of ingested cacao proteins are unknown because the proteins are difficult to extract and purify from cacao beans. Objectives: This study aimed to develop an extraction and purification method for cacao proteins and reveal the effect of ingestion of cacao proteins on defecation and intestinal microbiota in mice. Methods: Three groups of mice were fed a control diet (AIN-93 G), a cacao lignin diet (AIN-93 G containing 1.22% cacao lignin), or a cacao protein and lignin diet (AIN-93 G containing 1.97% cacao proteins and 1.22% cacao lignin) by pair-feeding for 8 d. Feces were collected as 2 bulked samples from days 1 to 4 and days 5 to 8 on each diet. The collected feces were weighed and the intestinal microbiota was analyzed by next-generation sequencing-based 16S rRNA. Results: A new extraction and purification method for cacao proteins has been developed, then found that the proteins are resistant to digestive enzymes. However, the cacao protein powder made by this method contained 34.9% of lignin in addition to 56.4% of proteins. Therefore, to reveal the effect by cacao proteins alone, the fecal weight and intestinal microbiota of mice fed the cacao protein and lignin diet were compared with those of mice fed the cacao lignin diet. The fecal weight of mice fed the cacao protein and lignin diet was significantly greater than of mice fed the cacao lignin diet. The relative abundance of Lactococcus and Mucispirillum species in mice fed the cacao protein and lignin diet was significantly higher than in mice fed the cacao lignin diet, but the relative abundance of Anaerotruncus, Oscillospira, and Roseburia species in mice fed the cacao protein and lignin diet was significantly lower than in mice fed the cacao lignin diet. Conclusions: Ingestion of indigestible cacao proteins promoted defecation and altered the intestinal microbiota such as Lactococcus, Mucispirillum, Anaerotruncus, Oscillospira, and Roseburia species in mice.

Regulation of microbe-associated molecular pattern-induced hypersensitive cell death, phytoalexin production, and defense gene expression by calcineurin B-like protein-interacting protein kinases, OsCIPK14/15, in rice cultured cells.

Although cytosolic free Ca(2+) mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca(2+) and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca(2+) sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn(2+)-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.

A new glycosylated dihydrophaseic acid from cacao germs (Theobroma cacao L.).

Cacao beans are composed of cacao nibs and germs. Although numerous chemical and physiological studies on cacao nib compounds have been reported, there is little information on cacao germ compounds. We therefore analyzed an extract from the cacao germ, and found two compounds that were specific to the germ. One of these two compounds was identified as the new glycosylated abscisic acid metabolite, dihydrophaseic acid-4'-O-6″-(ß-ribofuranosyl)-ß-glucopyranoside, and the other as the known compound, dihydrophaseic acid-4'-O-ß-D-glucopyranoside.

OsTGAP1, a bZIP transcription factor, coordinately regulates the inductive production of diterpenoid phytoalexins in rice.

Production of major diterpenoid phytoalexins, momilactones and phytocassanes, is induced in rice upon recognition of pathogenic invasion as plant defense-related compounds. We recently showed that biosynthetic genes for momilactones are clustered on rice chromosome 4 and co-expressed after elicitation, mimicking pathogen attack. Because genes for most metabolic pathways in plants are not organized in gene clusters, examination of the mechanism(s) regulating the expression of such clustered genes is needed. Here, we report a chitin oligosaccharide elicitor-inducible basic leucine zipper transcription factor, OsTGAP1, which is essential for momilactone biosynthesis and regulates the expression of the five genes in the cluster. The knock-out mutant for OsTGAP1 had almost no expression of the five clustered genes (OsCPS4, OsKSL4, CYP99A2, CYP99A3, and OsMAS) or production of momilactones upon elicitor treatment. Inductive expression of OsKSL7 for phytocassane biosynthesis was also largely affected in the ostgap1 mutant, although phytocassane accumulation still occurred. Conversely, OsTGAP1-overexpressing lines exhibited enhanced expression of the clustered genes and hyperaccumulation of momilactones in response to the elicitor. Furthermore, enhanced expression of OsKSL7 and hyperaccumulation of phytocassanes was also observed. We also found that OsTGAP1 overexpression can influence transcriptional up-regulation of OsDXS3 in the methylerythritol phosphate pathway, eventually leading to inductive production of diterpenoid phytoalexins. These results indicate that OsTGAP1 functions as a key regulator of the coordinated transcription of genes involved in inductive diterpenoid phytoalexin production in rice and mainly exerts an essential role on expression of the clustered genes for momilactone biosynthesis.

Contribution of salicylic acid glucosyltransferase, OsSGT1, to chemically induced disease resistance in rice plants.

Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray-based determination of gene expression. Cloning and characterization of the most highly activated probenazole-responsive gene revealed that it encodes UDP-glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of free SA into SA O-beta-glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole-responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly reduced the probenazole-dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice plants.

Phytoalexin accumulation in the interaction between rice and the blast fungus.

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.

Post-exercise carbohydrate plus whey protein hydrolysates supplementation increases skeletal muscle glycogen level in rats.

Recent studies showed that a combination of carbohydrate and protein was more effective than carbohydrate alone for replenishing muscle glycogen after exercise. However, it remains to be unclear whether the source or degree of hydrolysis of dietary protein influences post-exercise glycogen accumulation. The aim of this study was to compare the effect of dietary protein type on glycogen levels in the post-exercise phase, and to investigate the effects of post-exercise carbohydrate and protein supplementation on phosphorylated enzymes of Akt/PKB and atypical PKCs. Male Sprague-Dawley rats, trained for 3 days, swam with a 2% load of body weight for 4 h to deplete skeletal muscle glycogen. Immediately after the glycogen-depleting exercise, one group was killed, whereas the other groups were given either glucose or glucose plus protein (whey protein, whey protein hydrolysates (WPH), casein hydrolysates or branched-chain amino acid (BCAA) solutions. After 2 h, the rats were killed, and the triceps muscles quickly excised. WPH caused significant increases in skeletal muscle glycogen level (5.01 +/- 0.24 mg/g), compared with whey protein (4.23 +/- 0.24 mg/g), BCAA (3.92 +/- 0.18 mg/g) or casein hydrolysates (2.73 +/- 0.22 mg/g). Post-exercise ingestion of glucose plus WPH significantly increased both phosphorylated Akt/PKB (131%) and phosphorylated PKCzeta (154%) levels compared with glucose only. There was a significant positive correlation between skeletal muscle glycogen content and phosphorylated Akt/PKB (r = 0.674, P < 0.001) and PKCzeta (r = 0.481, P = 0.017). Post-exercise supplementation with carbohydrate and WPH increases skeletal muscle glycogen recovery by activating key enzymes such as Akt/PKB and atypical PKCs.

Differential responses of rice to inoculation with wild-type and non-pathogenic mutants of Magnaporthe oryzae.

We analyzed the response of rice to Magnaporthe oryzae infection using two mutant strains deficient in Mgb1 and Mst12, which are essential for the development of appresoria and penetration pegs. Both mutants induced the much lower levels of accumulation of phytoalexins than wild-type, suggesting that the massive production of phytoalexins requires the fungal invasion of rice cells. Intense accumulation of H2O2 in a single whole cell also required fungal penetration. Microarray analysis of rice gene expression revealed mutant-specific gene expression, indicating that signal exchange between rice and M. oryzae commence before fungal penetration of the rice cell. In situ detection of mRNAs for peroxidase and beta-1,3-glucanase showed that expression of these genes also occurs after penetration as observed for phytoalexin production.

Cloning and characterization of cDNAs encoding ent-copalyl diphosphate synthases in wheat: insight into the evolution of rice phytoalexin biosynthetic genes.

In vitro assays using recombinant enzymes enabled three cDNAs encoding ent-copalyl diphosphate synthases to be identified in wheat (Triticum aestivum): TaCPS1, TaCPS2, and TaCPS3. The phylogenetic tree and expression analyses suggest that TaCPS3 is responsible for gibberellin biosynthesis, while TaCPS1 and TaCPS2 are possible functional homologs of diterpene cyclase genes OsCPS2 and OsCPS4 involved in phytoalexin biosynthesis in rice.

Purification and characterization of a new family 45 endoglucanase, STCE1, from Staphylotrichum coccosporum and its overproduction in Humicola insolens.

In the detergent industry, fungal endoglucanases have been used to release microfibrils (defibrillation) from the surface of dyed cellulosic fabrics to enhance color brightness. Although endoglucanases for laundry use must have various properties, such as a neutral or alkaline optimum pH, resistance to anionic surfactants and oxidizing agents (main components in detergents), and high defibrillation activity, all-purpose endoglucanases have not been obtained yet. As a result of screening of endoglucanases, a new family 45 endoglucanase (family 45 glycoside hydrolase), designated STCE1, was obtained and purified to apparent homogeneity from the culture supernatant of Staphylotrichum coccosporum NBRC 31817. The molecular mass of STCE1 was 49 kDa. The optimum pH for the carboxymethyl cellulase activity of STCE1 was 6.0, and the optimum temperature was 60 degrees C. STCE1 was highly resistant to an anionic surfactant and an oxidizing agent. Furthermore, the defibrillation activities on dyed cotton and lyocell fabrics of STCE1 were higher than those of the other representative endoglucanases tested. These results indicate that STCE1 is an all-purpose enzyme for laundry use. A gene encoding STCE1, designated the stce1 gene, was cloned from S. coccosporum, and the complete sequence was determined. STCE1 consisted of three distinct domains: an N-terminal catalytic domain (family 45), a linker domain, and a C-terminal carbohydrate-binding module (family 1). The amino acid sequences of the catalytic domain of STCE1 were phylogenetically close to those of the family 45 endoglucanases EGL3, EGL4, and EGV from a Humicola sp. Hence, the stce1 gene was transferred into Humicola insolens and expressed. As a result, extremely high levels (0.90 mg protein per ml of culture supernatant, 27% of the total proteins) of the recombinant STCE1 were secreted as a mature form in the culture supernatant.

Effects of a bile acid elicitor, cholic acid, on the biosynthesis of diterpenoid phytoalexins in suspension-cultured rice cells.

An elicitor of rice defense responses was recently isolated from human feces and was identified as cholic acid (CA). Pathogen infection in rice leaves induces phytocassanes and momilactones, both of which are major diterpenoid phytoalexins in rice, whereas CA mainly induces phytocassanes. We established a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry protocol for the rapid and accurate quantification of phytocassanes and momilactones. Using this method, we showed that CA preferentially induced the formation of phytocassanes in suspension-cultured rice cells, while a fungal chitin oligosaccharide elicitor induced that of both phytocassanes and momilactones. We further investigated the effects of CA on the expression of diterpene cyclase genes involved in phytoalexin biosynthesis. CA induced the transcription of the genes OsCPS2 (OsCyc2) and OsKSL7 (OsDTC1), which are involved in phytocassane biosynthesis, to a greater extent than the genes OsCPS4 (OsCyc1) and OsKSL4, which are involved in momilactone biosynthesis. OsCPS2 was particularly strongly induced, suggesting that it is one of the main mechanisms by which CA induces high levels of phytocassanes.

Diterpene phytoalexins are biosynthesized in and exuded from the roots of rice seedlings.

Rice (Oryza sativa L.) produces a variety of diterpene phytoalexins, such as momilactones, phytocassanes, and oryzalexins. Momilactone B was previously identified as an allelopathic substance exuded from the roots of rice. We identified in this present study momilactone A and phytocassanes A-E in extracts of, and exudates from, the roots of rice seedlings. The concentration of each compound was of the same order of magnitude as that of momilactone B. Expression analyses of the diterpene cyclase genes responsible for the biosynthesis of momilactones and phytocassanes suggest that these phytoalexins found in roots are primarily biosynthesized in those roots. None of phytocassanes B-E exhibited allelopathic activity against dicot seedling growth, whereas momilactone A showed much weaker allelopathic activity than momilactone B. The exudation of diterpene phytoalexins from the roots might be part of a system for defense against root-infecting pathogens.