Coronatine elicits phytoalexin production in rice leaves (Oryza sativa L.) in the same manner as jasmonic acid

The phytotoxin coronatine induced the accumulation of the flavonoid phytoalexins sakuranetin and momilactone A in rice leaves. Coronatine-inducible sakuranetin production was under the control of kinetin and ascorbic acid (AsA), as observed with jasmonic acid (JA). The effects of kinetin and AsA on the activity of coronatine indicated that coronatine might elicit sakuranetin production in a manner similar to JA. The similarity of both their structures and the manner of elicitation of coronatine and JA suggest that they might interact at the same active site(s) to lead to phytoalexin production.     

How phenology influences physiology in deciduous forest spring ephemerals

The protein phosphatase inhibitor cantharidin activates defense responses in rice leaves when applied exogenously at concentrations ranging from 100 to 500 μ M . Responses include the accumulation of the major rice phenolic phytoalexin sakuranetin and the lactone phytoalexin momilactone A. Accumulation of sakuranetin was preceded by an induction of phenylalanine ammonia lyase (PAL) activity and an increase in the activity of naringenin 7- O -methyltransferase (NOMT), the key enzyme in sakuranetin biosynthesis. Cantharidin also strongly induced accumulation of the probenazole (PBZ)-inducible protein (PBZ1) and two novel, related proteins named PBZ2 and PBZ3. Endothall, a herbicide and potent protein phosphatase inhibitor, but not its inactive analog (1,4-dimethylendothall) also induced sakuranetin accumulation, increased activity of NOMT and accumulation of the 3 PBZ proteins. In contrast, two other protein phosphatase inhibitors, calyculin A and microcystin LR, did not activate these defense responses. Induction of NOMT and PAL activity, and sakuranetin accumulation, was completely blocked by cycloheximide. Leaf segments treated with cantharidin and endothall showed brownish and orange colored lesions, respectively, similar to the lesion mimic mutants of rice. These results indicate a direct role for protein phosphorylation/dephosphorylation events in the activation of defense responses in rice, in particular on the accumulation of antifungal phytoalexins and the PBZ proteins.     

Jasmonoyl-l-isoleucine is required for the production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice leaves

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl₂-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.     

Involvement of Jasmonic Acid in Elicitor-Induced Phytoalexin Production in Suspension-Cultured Rice Cells

It has been suggested that jasmonic acid (JA) could be an integral part of a general signal transduction system regulating inducible defense genes in plants. It was reported that treatment with an elicitor (N-acetylchitoheptaose) induced production of phytoalexin in suspension-cultured rice (Oryza sativa L.) cells. In this study, the role of JA in the induction of phytoalexin production by N-acetylchitoheptaose was investigated. Exogenously applied ([plus or minus])-JA (10-4 M) clearly induced the production of momilactone A, a major phytoalexin, in suspension-cultured rice cells. On the other hand, in rice cells treated with N-acetylchitoheptaose, endogenous JA was rapidly and transiently accumulated prior to accumulation of momilactone A. Treatment with ibuprofen, an inhibitor of JA biosynthesis, reduced production of momilactone A in the cells treated with N-acetylchitoheptaose, but the addition of ([plus or minus])-JA increased production of momilactone A to levels higher than those in the elicited rice cells. These results strongly suggest that JA functions as a signal transducer in the induction of biosynthesis of momilactone A by N-acetylchitoheptaose in suspension-cultured rice cells.     

Accumulation of salicylic acid,jasmonic acid and phytoalexins in rice, <Emphasis Type="Italic">Oryza sativa</Emphasis>, infested by the white-backed planthopper, <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

In order to clarify the mechanism of induced resistance to blast disease in rice, Oryza sativa, that had been previously infested by the white-backed planthopper, Sogatella furcifera Horváth, we first investigated the accumulation of salicylic acid (SA) and jasmonic acid (JA) in rice plants infested by the planthopper. The results confirmed that infestation of S. furcifera strongly stimulates the production of SA and JA in rice. These results indicate that both salicylate- and jasmonate-mediated pathways (SA and JA pathways), which are involved in the general defense system in plants, were activated in rice infested by S. furcifera. Further results confirmed that S. furcifera infestation induces accumulation of a major rice diterpenoid phytoalexin, momilactone A, and a flavonoid phytoalexin, sakuranetin, which are well known as antimicrobial chemicals, particularly in blast disease caused by the blast fungus, Magnaporthe oryzae B. Couch. All these results strongly suggest the following hypothetical mechanism of induced-resistance to M. oryzae in rice infested by S. furcifera. First, S. furcifera releases some elicitor-active compounds, which might be produced in the salivary glands, into the rice plant during feeding. Next, the defense signal systems, SA- and JA-mediated pathways, are activated by the elicitor. Finally, phytoalexins are induced in rice as antimicrobial compounds mainly through activation of the JA-mediated pathway.     

OsJAR1 Contributes Mainly to Biosynthesis of the Stress-Induced Jasmonoyl-Isoleucine Involved in Defense Responses in Rice

Jasmonate plays key roles in plant growth and stress responses, as in defense against pathogen attack. Jasmonoyl-isoleucine (JA-Ile), a major active form of jasmonates, is thought to play a pivotal role in plant defense responses, but the involvement of JA-Ile in rice defense responses, including phytoalexin production, remains largely unknown. Here we found that OsJAR1 contributes mainly to stress-induced JA-Ile production by the use of an osjar1 Tos17 mutant. The osjar1 mutant was impaired in JA-induced expression of JA-responsive genes and phytoalexin production, and these defects were restored genetically. Endogenous JA-Ile was indispensable to the production of a flavonoid phytoalexin, sakuranetin, but not to that of diterpenoid phytoalexins in response to heavy metal stress and the rice blast fungus. The osjar1 mutant was also found to be more susceptible to the blast fungus than the parental wild type. These results suggest that JA-Ile production makes a contribution to rice defense responses with a great impact on stress-induced sakuranetin production.     

Chitosan activates defense/stress response(s) in the leaves of Oryza sativa seedlings

In this study, we examined the response(s) of rice (Oryza sativa L. japonica-type cv. Nipponbare) seedling leaves treated with a fungal elicitor chitosan (CT). Small brownish necrotic spots (streaks) appeared in the interveinal regions on the leaf surface after treatment by 0.1% CT, over the cut control. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis further revealed strong induction of ascorbate peroxidase, and changes in “phytocystatins” (cysteine proteinase inhibitors). Using two-dimensional polyacrylamide gel electrophoresis, evidence is provided for the accumulation of two major classes of pathogenesis-related (PR) proteins, namely OsPR5 and OsPR10 in the leaves. In parallel, northern analyses revealed potent accumulation of the OsPR5 and OsPR10 mRNAs; a time- and dose-dependent expression, and a requirement for de novo protein synthesis was observed. Furthermore, CT-elicited changes were also accompanied by production of anti-fungal phytoalexins, the flavonoid sakuranetin and the diterpenoid lactone momilactone A, as determined by liquid chromatography-mass spectrometry/mass spectrometry analysis. Present results reveal for the first time the potency of CT in initiating multiple events linked with defense/stress response(s) in the leaves of whole rice plants.     

Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice

Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl(2) treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, we conclude that Os12g0240900 encodes an OsNOMT.     

Possible role of phytocassane,rice phytoalexin,in disease resistance of rice against the blast fungus Magnaporthe grisea

In addition to momilactone, phytocassanes A through E (diterpene phytoalexins) were detected in rice leaves in fields suffering from rice blast. Furthermore, phytocassane accumulation was most abundant at the edges of necrotic lesions, indicating that the phytoalexins prevent subsequent spread of the fungus from the infected site. In pot experiments the pattern of phytocassane accumulation in rice leaves in an incompatible interaction (infection with an avirulent race of Magnaporthe grisea) was more rapidly induced than in a compatible interaction (infection with a virulent race of M. grisea).     

Natural variation in the expression and catalytic activity of a naringenin 7‐O‐methyltransferase influences antifungal defenses in diverse rice cultivars

Phytoalexins play a pivotal role in plant–pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7‐O‐methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested that NOMT is the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, both NOMT expression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed that NOMT induction is correlated with sakuranetin accumulation. In bioassays with Pyricularia oryzae, Gibberella fujikuroi, Bipolaris oryzae, Burkholderia glumae, Xanthomonas oryzae, Erwinia chrysanthemi, Pseudomonas syringae, and Acidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice‐growing environments. In a dendrogram of NOMT genes, those from low‐sakuranetin‐accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice species Oryza rufipogon also exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication.     

Identification of a biosynthetic gene cluster in rice for momilactones

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 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-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.     

Convergent or parallel molecular evolution of momilactone A and B: potent allelochemicals, momilactones have been found only in rice and the moss Hypnum plumaeforme

Plant second metabolites momilactone A and B, which act as potent phytoalexins and allelochemicals, have been found thus far only in rice and the moss Hypnum plumaeforme, although both plants are taxonomically quite distinct. The concentrations of momilactone A and B, respectively, in rice plants were 4.5-140 and 2.9-85 μg/g, and those in H. plumaeforme were 8.4-58.7 and 4.2-23.4 μg/g. Momilactone A and B concentrations in rice and H. plumaeforme plants were increased by UV irradiation, elicitor and jasmonic acid treatments. Rice and H. plumaeforme plants secrete momilactone A and B into the rhizosphere, and the secretion level was also increased by UV irradiation, elicitor and jasmonic acid treatments. In addition, although endogenous concentrations of momilactone A in rice and H. plumaeforme were greater than those of momilactone B, the secretion levels of momilactone B were greater than those of momilactone A in rice and H. plumaeforme, which suggests that momilactone B may be selectively secreted by both rice and H. plumaeforme. As momilactone A and B exert potent antifungal and growth inhibitory activities, momilactone A and B may play an important role in the defense responses in H. plumaeforme and rice against pathogen infections and in allelopathy. The secretion of momilactone A and B into the rhizosphere may also prevent bacterial and fungal infections and provide a competitive advantage for nutrients through the inhibition of invading root systems of neighboring plants as allelochemicals. Therefore, both plants, despite their evolutionary distance, may use same defense strategy with respect to the momilactone A and B production and secretion, which resulting from convergent or parallel evolutionary processes. In the case of parallel evolution, there may be plant species providing the missing link in molecular evolution of momilactones between H. plumaeforme and rice.     

The rice (Oryza sativa) blast lesion mimic mutant, blm, may confer resistance to blast pathogens by triggering multiple defense-associated signaling pathways.

Here we characterized a rice (Oryza sativa L.) blast lesion mimic (blm) mutant, identified previously in an N-methyl-N-nitrosourea-mutagenized population of the cultivar Hwacheong (wild type). The rice blm displayed spontaneous necrotic lesion formation on the leaves during development under long-day condition and temperature shift from 28 to 24 degrees C in the absence of obvious stress/disease, and provided us with a highly reproducible and convenient experimental system in the growth chamber to study blm. The blm phenotype resembled to the cell death of hypersensitive reaction (HR), and subsequent, two-dimensional gel electrophoresis (2-DGE) revealed induction of many leaf proteins; prominent among them were the three pathogenesis-related (PR) marker proteins of class 5 (one spot) and 10 (two spots). Interestingly, the rice blm manifested HR against all races tested of the rice blast fungus (Magnaporthe grisea), providing high resistance in a non-race specific manner. It was also observed that blm was highly resistant to hydrogen peroxide treatment. Using 2-DGE immunoblotting, we identified the presence of 4 new spots cross-reacting with a superoxide dismutase (SOD) antibody, only in blm, suggesting the expression of potentially new SOD protein (isoforms) during lesion formation. In the leaves of blm, autofluorescent compounds accumulated in and around the site of lesion progression. Moreover, enhanced levels of two major rice phytoalexins, sakuranetin and momilactone A were also observed in the leaves of blm. These results indicate that blm confers broad-spectrum resistance to multiple pathogens, and so, it could be hypothesized that the BLM gene product may control the HR-like cell death and its associated multiple defense signaling pathways, as evidenced by induction of known hallmark features (proteins/metabolites) linked with the defense responses, in rice.     

CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochrome P450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNA interference double knock-down of this pair of closely related CYPs reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which was ultimately achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al, and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al, and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene-derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis.     

Role of Jasmonic Acid as a Signaling Molecule in Copper Chloride-elicited Rice Phytoalexin Production

Phytoalexins are antimicrobial secondary metabolites which accumulate in plants against fungal invasion. Their production is triggered not only by fungal invasion, but also by a variety of elicitors. In rice plants, we have shown that CuCl₂ is a potent abiotic elicitor. Jasmonic acid has recently become known to play an important role in secondary metabolite production in plants at the cellular level. This led us to speculate, in CuCl₂-elicited rice leaves, that JA might also play an important role as a signal transducer for phytoalexin production. The endogenous level of JA increased rapidly in CuCl₂-elicited rice leaves, and exogenously applied JA caused a large amount of phytoalexin production in rice leaves. This phytoalexin production by CuCl₂ decreased when rice leaves were treated with JA biosynthesis inhibitors, but that by JA did not. JA is thus suggested to play an important role in the elicitation process leading to phytoalexin production in rice leaves.     

Phytoalexin induction in the sapwood of plants of the Maloideae (Rosaceae): Biphenyls or dibenzofurans

Following fungal inoculation or natural infection, five biphenyl phytoalexins (aucuparin and its 2′ and 4′ oxygenated derivatives) were induced variously in the sapwood of Aronia, Chaenomeles, Eriobotrya, Malus(three spp.) and of Sorbus aucuparia. By contrast, 14 dibenzofuran phytoalexins were induced variously in sapwood of Cotoneaster (7 spp.), Crateagus, Cydonia, Mespilus, Photinia, Pseudocydonia, Pyracantha, Pyrus and two Sorbus spp. (S. chamaemespilum and S. domestica). These were five cotonefurans, three eriobofurans, five pyrufurans and a 2,3,4,7,8-pentaoxygenated dibenzofuran trimethyl ether. No plant has yet been found to produce both types of phytoalexin, although o-hydroxybiphenyls are theoretically precursors of the dibenzofurans. The ability to synthesize either biphenyls or dibenzofurans appears to be genus-specific, except in the case of Sorbus. In 18 of the 38 species tested, these phytoalexins were accompanied by constitutive antifungal phenolics, most of which appeared to be released from bound (glycosidic) forms during the infection process. These were identified variously as hydroquinone, p-hydroxyacetophenone, acetovanillone, 5,7-dihydroxychromone, chrysin, sakuranetin and naringenin. Woody members of the subfamilies Prunoideae and Spiraeoideae failed to yield any phytoalexins on induction, but did contain constitutive antifungal compounds. The limited frequency of the phytoalexin response within the family as a whole is considered in relation to the accumulation of constitutive antifungal agents in these plants.     

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.     

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.