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.     

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.     

Recent advances regarding diterpene cyclase genes in higher plants and fungi

Cyclic diterpenoids are commonly biosynthesized from geranylgeranyl diphosphate (GGDP) through the formation of carbon skeletons by specific cyclases and subsequent chemical modifications, such as oxidation, reduction, methylation, and glucosidation. A variety of diterpenoids are produced in higher plants and fungi. Rice produces four classes of diterpene phytoalexins, phytocassanes A to E, oryzalexins A to F, oryzalexin S, and momilactones A and B. The six diterpene cyclase genes involved in the biosynthesis of these phytoalexins were identified and characterized. Fusicoccin A was produced by the phytopathogenic Phomopsis amygdali and served as a plant H(+)-ATPase activator. A PaFS, encoding a fungal diterpene synthase responsible for fusicoccin biosynthesis, was isolated. The PaFS is an unusual chimeric diterpene synthase that possesses not only terpene cyclase activity (the formation of fusicoccadiene, a biosynthetic precursor of fusicoccin A), but also prenyltransferase activity (the formation of GGDP). Thus, we identified a unique multifunctional diterpene synthase family in fungi.     

Evolutionary trajectory of phytoalexin biosynthetic gene clusters in rice

Plants frequently possess operon‐like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdane‐related diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent‐copalyl diphosphate or syn‐copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical‐modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane‐related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon‐like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.     

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).     

The relation between growth inhibition and secretion level of momilactone B from rice root

Abstract

The growth inhibitory activity of seven rice (Oryza sativa L.) cultivars and the secretion level of momilactone B from these rice cultivars were determined to understand chemical basis of the interaction of rice with other plant species. All rice cultivars inhibited the growth of hypocotyls and roots of lettuce (Lactuca sativa L.) seedlings when the lettuce was grown together with the rice, and showed different range of the inhibitory activity. These results suggest that all rice cultivars may possess allelopathic activity and the activity may be cultivar dependent. Momilactone B, which is a potent growth inhibitor, was found in root exudates of all rice cultivars, and the momilactone B concentration was also cultivar-dependent. The allelopathic activity of each rice cultivar was closely correlated with momilactone B concentration in the root exudates. The present results suggest that rice cultivars possess various allelopathic activities and their allelopathic activity may depend on the secretion level of momilactone B. Therefore, momilactone B may play an important role in rice allelopathy and in the chemical interactions of rice with other plant species.     

Cholic acid, a bile acid elicitor of hypersensitive cell death, pathogenesis-related protein synthesis, and phytoalexin accumulation in rice

When plants interact with certain pathogens, they protect themselves by generating various defense responses. These defense responses are induced by molecules called elicitors. Since long ago, composts fermented by animal feces have been used as a fertilizer in plant cultivation, and recently, have been known to provide suppression of plant disease. Therefore, we hypothesized that the compounds from animal feces may function as elicitors of plant defense responses. As a result of examination of our hypothesis, an elicitor of rice defense responses was isolated from human feces, and its structure was identified as cholic acid (CA), a primary bile acid in animals. Treatment of rice (Oryza sativa) leaves with CA induced the accumulation of antimicrobial compounds (phytoalexins), hypersensitive cell death, pathogenesis-related (PR) protein synthesis, and increased resistance to subsequent infection by virulent pathogens. CA induced these defense responses more rapidly than did fungal cerebroside, a sphingolipid elicitor isolated from the rice pathogenic fungus Magnaporthe grisea. Furthermore, fungal cerebroside induced both types of rice phytoalexins, phytocassanes and momilactones, whereas CA mainly induced phytocassanes, but not momilactones. In the structure-activity relationship analysis, the hydroxyl groups at C-7 and C-12, and the carboxyl group at C-24 of CA contributed to the elicitor activity. These results indicate that CA is specifically recognized by rice and is a different type of elicitor from fungal cerebroside. This report demonstrated that bile acid induced defense responses in plants.     

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.     

Barnyard grass-induced rice allelopathy and momilactone B

Here, we investigated chemical-mediated interaction between crop and weeds. Allelopathic activity of rice seedlings exhibited 5.3-6.3-fold increases when rice and barnyard grass seedlings were grown together, where there may be the competitive interference between rice and barnyard grass for nutrients. Barnyard grass is one of the most noxious weeds in rice cultivation. The momilactone B concentration in rice seedlings incubated with barnyard grass seedlings was 6.9-fold greater than that in rice seedlings incubated independently. Low nutrient growth conditions also increased allelopathic activity and momilactone B concentrations in rice seedlings. However, the increases in the low nutrient-induced allelopathic activity and momilactone B concentration were much lower than those in barnyard grass-induced allelopathic activity and momilactone B concentration. Root exudates of barnyard grass seedlings increased allelopathic activity and momilactone B concentration in rice seedlings at concentrations greater than 30 mg/L of the root exudates, and increasing the exudate concentration increased the activity and momilactone B concentration. Therefore, barnyard grass-induced allelopathic activity of rice seedlings may be caused not only by nutrient competition between two species, but also by components in barnyard grass root exudates. As momilactone B shows strong allelopathic activities, barnyard grass-induced allelopathic activity of rice may be due to the increased concentration of momilactone B in rice seedlings. The present research suggests that rice may respond to the presence of neighboring barnyard grass by sensing the components in barnyard grass root exudates and increasing allelopathic activity by production of elevated concentration of momilactone B. Thus, rice allelopathy may be one of the inducible defense mechanisms by chemical-mediated plant interaction between rice and barnyard grass, and the induced-allelopathy may provide a competitive advantage for rice through suppression of the growth of barnyard grass.     

The chemical-mediated allelopathic interaction between rice and barnyard grass

Aims

The possible involvement of the chemical-mediated interaction in allelopathy between rice and barnyard grass was investigated.

Methods

Effcts of rice seedlings and rice root exudate on the alleloapthic activity of barnyard grass were determined and a key compound invovled in the allelopathic interaction between rice and barnyard grass was isolated.

Results

Allelopathic activity of barnyard grass was increased by the presence of rice seedlings. Rice root exudates also elevated the allelopahtic activity of barnyard grass. A key compound, which increased the allelopathic activity of barnyard grass, in the rice root exudates was isolated and determined as momilactone B. Momilactone B increased the allelopathic activity of barnyard grass at concentrations greater than 3 μM, and increasing the momilactone B concentration increased the activity.

Conclusions

Momilactone B is known to act as a potent rice allelochemical and to possess strong growth inhibitory activity against barnyard grass. The present research suggests that barnyard grass may response to the presence of neighboring rice by sensing momilactone B in rice root exudates and increase allelopathic activity. Thus, momilactone B may not only act as a rice allelochemical but also play an important role in rice-induced allelopathy of barnyard grass. The induced-allelopathy may provide a competitive advantage for barnyard grass through the growth inhibition of competing plant species including rice. Barnyard grass allelopathy may be one of the inducible defense mechanisms by chemical-mediated plant interaction between rice and barnyard grass. Rice allelopathy was also reported to be increased by the presence of barnyard grass through increasing production and secretion of momilactone B into surrounding environments. During the evolutional process, rice and barnyard grass may have developed the chemical cross talk to activate the defense mechanisms against some biotic stress conditions by detection of certain key compounds.     

Stress-induced allelopathic activity and momilactone B in rice

Allelopathic activity of rice extracts and root exudates against Echinochloa crus-galli increased by heavy metal, cantharidin and jasmonic acid treatments. Since cantharidin (protein phosphatase inhibitor) acts as an elicitor and jasmonic acid is an important signaling molecule regulating inducible defense genes against the pathogen infection, heavy metal stress and pathogen infection may increase alleopathic activity of rice. These treatments also increased the concentrations of momilactone B in rice extracts and root exudates, suggesting that the production of momilactone B in rice and the secretion of momilactone B from rice into the rhizosphere may be enhanced by the treatments. As momilactone B possesses strong phytotoxic and allelopathic activities, the elevated production and secretion of momilactone B of rice by heavy metals, cantharidin and jasmonic acid may contribute to the increasing allelopathic activity of rice. Enhancement of the secretion of momilactone B into the rhizosphere may provide a competitive advantage for root establishment through local suppression of pathogen and inhibition of the growth of competing plant species. Therefore, allelopathy of rice may be one of the inducible defense mechanisms and may be regulated several environmental factors.     

UV-induced momilactone B accumulation in rice rhizosphere

UV-irradiation increased the concentration of momilactone B in shoots and roots of rice seedlings, and increasing the irradiation increased the concentration. The concentration in 90-min UV-irradiated shoots and roots, respectively, was 31.8- and 3.6-fold higher than that in non-irradiated shoots and roots. After UV-irradiation the concentration of momilactone B in rice shoots decreased. There was, however, an accumulation of momilactone B in the medium in which UV-irradiated seedlings were grown. Five days after UV-irradiation, momilactone B in the medium was at a level 2.5 times greater than on day 0, which was 47% of momilactone B in the seedlings, suggesting that rice may actively secrete momilactone B into medium. Therefore, UV-irradiation increased not only production of momilactone B in rice seedlings but also secretion of momilactone B into rice rhizosphere. As momilactone B acts as an antimicrobial and allelopathic agent, secretion of momilactone B into the rhizosphere may provide a competitive advantage for root establishment through local suppression of soil microorganism and inhibition of the growth of competing plant species.     

Momilactone A and B as allelochemicals from moss Hypnum plumaeforme: first occurrence in bryophytes

Momilactones A (1) and B (2), which have been identified as phytoalexins in rice, were isolated from extracts of the moss Hypnum plumaeforme. This is the first isolation and identification of momilactones as allelochemicals from a bryophyte. H. plumaeforme produces considerable amounts of momilactones (isolated yield: 8.4 mg/Kg plant for 1; 4.2 mg/Kg for 2). EtOAc extracts from H. plumaeforme and 2 showed growth inhibitory activity against angiosperms, moss, and liverwort plants. On the other hand, the growth of H. plumaeforme was insensitive to its extract and 2. Our finding suggests that momilactones play an important role as allelochemicals in this moss.