Possible involvement of momilactone B in rice allelopathy

Since residues and extracts of rice plants were known to inhibit the germination and growth of several plant species, the possible involvement of a growth inhibitor, momilactone B, in rice allelopathy was discussed. Momilactone B was found in shoots and roots of rice plants over their entire life cycle. The level of momilactone B in shoots and roots increased with rice plant growing until flowering initiation, and then decreased. The highest level of momilactone B in the shoots and roots at the day of flowering initiation was 245 and 64.1 nmol g(-1) fresh weight, respectively. Thus, 1 kg of rice shoots and roots, respectively, may be able to release 245 and 64.1 micromol of momilactone B into the soil or neighboring environment by decomposition of their residues, which may be sufficient to cause growth inhibition of their neighboring or successional plants. The growth inhibitory activity of momilactone B and the occurrence of momilactone B in rice plants suggest that momilactone B may contribute the growth inhibitory effect of rice residues and extracts, indicating that momilactone B may have an important role in the rice allelopathy.     

Isolation and identification of a potent allelopathic substance in rice root exudates

A search for growth inhibitors in rice root exudates was undertaken in order to clarify the allelopathic system in rice ( Oryza sativa L.). Rice seedlings inhibited the growth of cress ( Lepidium sativum L.) and lettuce ( Lactuca sativa L.) seedlings when the cress and lettuce were grown with rice seedlings. The putative compound causing the inhibitory effect of rice seedlings was isolated from their culture solution, and the chemical structure of the inhibitor was determined by spectral data as momilactone B. Momilactone B inhibited the growth of cress and lettuce seedlings at concentrations greater than 3 and 30 µ M , respectively. The concentration of momilactone B was 3.4 and 1.1 nmol per seedling in the culture solutions of husked and non-husked rice seedlings, respectively. These results suggest that rice seedlings may release momilactone B into the environment and the stress caused by the husk-treatment may increase the amount of momilactone B released. Thus, momilactone B may play an important role in rice allelopathy.     

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.     

Rice seedlings release momilactone B into the environment

Since the growth inhibitor momilactone B was found recently in root exudates of rice (Oryza sativa L.), 3-day-old rice seedlings were transferred to hydroponic culture and the level of momilactone B released into the environment from the seedlings was measured. At day 15 after transfer, the level of momilactone B in the culture solution was 1.8 nmol per seedling compared with endogenous levels of 0.32 and 0.63 nmol per root and shoot, respectively, suggesting that rice seedlings actively releases momilactone B into the culture solution. This release must occur from the roots because only rice roots were immersed in the culture solution. Momilactone B inhibited the growth of ten cress (Lepidium sativum L.) seedlings at concentrations greater than 3 microM. Ten rice seedlings were incubated with ten cress seeds in a Petri dish containing 1 ml of medium, the medium contained 18 nmol of momilactone B, which came to 18 microM. This level of momilactone B was enough to reveal growth inhibition of the cress seedlings. Release level of momilactone B and its effectiveness as a growth inhibitor suggest that it may play an important role in rice allelopathy.     

Concentration and release level of momilactone B in the seedlings of eight rice cultivars

The release levels of a growth inhibitor, momilactone B, from rice (Oryza sativa L.) seedlings of eight cultivars were compared with the endogenous concentrations of momilactone B in their seedlings. All rice cultivars contained momilactone B in the seedlings, and their concentrations differed between the cultivars. Momilactone B was also found in all culture solutions in which these rice seedlings were grown, and the concentrations differed between the cultivars. The momilactone B concentrations in the culture solutions were reflected in the momilactone B concentrations in the seedlings. These results suggest that all rice cultivars may produce momilactone B and release momilactone B into the culture solutions. In addition, the release level of momilactone B may depend on the production level of momilactone B in the seedlings, which may affect allelopathic potential of these rice cultivars because as a growth inhibitor, momilactone B is able to act as an allelochemical.     

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.     

Secretion of momilactone A from rice roots to the rhizosphere

The secretion levels of momilactone A from rice (Oryza sativa L.) seedlings of eight cultivars into the rhizosphere were compared with the endogenous momilactone A concentrations in their shoots and roots. All rice cultivars contained momilactone A in the shoots and roots, and concentrations differed among the cultivars. Momilactone A was also found in all culture solutions in which the rice seedlings were grown, and the concentrations differed among the cultivars. The momilactone A concentrations in the culture solutions were reflected in the momilactone A concentrations in the shoots. These results suggest that all rice cultivars may produce momilactome A and secrete momilactone A into the culture solutions. The secretion levels of momilactone A may be more dependent on their capacities for momilactone A production in the shoots than on their capacities for momilactone A transportation from the shoots into the environment via the roots. As momilactone A acts as an antimicrobial and allelopathic agent, the secretion of momilactone A into the rice rhizosphere may provide a competitive advantage for root establishment through local suppression of soil microorganisms and inhibition of the growth of competing plant species.     

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.     

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 cross talk between rice and barnyardgrass

The chemical cross talk between rice and barnyardgrass which is one of the most noxious weeds in rice cultivation was investigated. Allelopathic activity of rice was increased by the presence of barnyardgrass seedlings or barnyardgrass root exudates. Rice allelochemical, momilactone B, concentration in rice seedlings and momilactone B secretion level from rice were also increased by the presence of barnyardgrass seedlings or barnyardgrass root exudates. As momilactone B possesses strong growth inhibitory activity and acts as an allelochemical, barnyardgrass-induced rice allelopathy may be due to the increased momilactone B secretion. These results suggest that rice may respond to the presence of neighboring barnyardgrass by sensing the chemical components in barnyardgrass root exudates and increase allelopathic activity by elevated production and secretion levels of momilactone B. Thus, rice allelopathy may be one of the inducible defense mechanisms by chemical-mediated plant interaction between rice and barnyardgrass and the induced-allelopathy may provide a competitive advantage for rice through suppression of the growth of barnyardgrass.Key words: allelopathy, Echinochloa, chemical interaction, induced-allelopathy, momilactone, Oryza sativaThe chemical cross talk between host and symbiotic or parasitic plants is an essential process for the development of physical connections in symbiosis and parasitism.^1–3 Barnyardgrass is one of the most common and noxious weeds in rice paddy fields.⁴ Although barnyardgrass is adapted rice production system due to its similarity in growth habit, the reason why barnyardgrass so often invades into the rice paddy fields is unknown. There might be some special interactions between both plant species.Plants are able to accumulate phytoalexins around infection sites of pathogens soon after sensing elicitors of pathogen origin. This accumulation of phytoalexins can protect the plants from further pathogen infection.^5,6 Plants are also able to activate defense mechanisms against attacking herbivores by sensing volatile compounds, such as methacrolein and methyl jasmonate, released by herbivore-attacked plant cells. The volatile-sensed plants increase the production of phenolics, alkaloids, terpenes and defense proteins, which reduce herbivory attacks.^7,8 Therefore, plants are able to elevate the defense mechanisms against several biotic stress conditions by detection of various compounds.Allelopathy is the direct influence of organic chemicals released from plants on the growth and development of other plants.^9–11 Allelochemicals are such organic chemicals involved in the allelopathy.^12,13 Allelochemicals can provide a competitive advantage for host-plants through suppression of soil microorganism and inhibition of the growth of competing plant species because of their antibacterial, antifungal and growth inhibitory activities.^3,14,15Rice has been extensively studied with respect to its allelopathy as part of a strategy for sustainable weed management, such as breeding allelopathic rice strains. A large number of rice varieties were found to inhibit the growth of several plant species when these rice varieties were grown together with these plants under the field or/and laboratory conditions.^16–20 These findings suggest that rice may produce and release allelochemicals into the neighboring environments and may inhibit the growth of the neighboring plants by the allelochemicals.Potent allelochemical, momilactone B, was isolated from rice root exudates.²¹ Momilactone B inhibits the growth of typical rice weeds like barnyardgrass and Echinochloa colonum at concentrations greater than 1 µM and the toxicity of momilactone B to rice itself was very low.²² In addition, rice plants secrete momilactone B from the roots into the rhizosphere over their entire life cycle.²² The observations suggest rice allelopathy may be primarily dependant on the secretion levels of momilactone B from the rice seedlings.^22,23Allelopathic activity of rice exhibited 5.3- to 6.3-fold increases when rice and barnyardgrass seedlings were grown together. Root exudates of barnyardgrass seedlings also increased allelopathic activity and momilactone B concentration in rice seedlings. The increasing the exudate concentration increased the allelopathic activity and momilactone B concentration in rice.²⁴ Thus, the chemical components in barnyardgrass root exudates may affect gene expressions involved in momilactone B biosynthesis. However, effects of the barnyardgrass root exudates on the secretion level of mimilactone B from rice has not yet reported.Rice seedlings were incubated in the medium containing barnyardgrass root exudates for 10 d, and secretion level of momilactone B by rice was determined (Fig. 1). The root exudates increased the secretion level significantly at concentrations greater than 30 mg/L of barnyardgrass root exudates, and increasing the concentration increased the secretion level. At concentrations of 300 mg/L of the root exudates, the secretion level was 10-fold greater than that in control (0 mg of root exudate). There was no significant difference in the osmotic potential between the medium contained barnyardgrass root exudates and control medium (all about 10 mmol/kg), and pH value of the medium was maintained at 6.0 throughout the experiments.²⁵ These results suggest that unknown chemical components in the barnyardgrass root exudates may induce the secretion of momilactone B from rice. As momilactone B possesses strong phytotoxic and allelopathic activities,^21–23,25 the elevated production and secretion of momilactone B in rice may provide a competitive advantage for root establishment through local suppression of pathogens and inhibition of the growth of competing plant species including barnyardgrass. Thus, barnyardgrass-induced rice allelopathy may be caused by the chemical components in the barnyardgrass root exudates.Open in a separate windowFigure 1Effects of barnyardgrass root exudates on momilactone B secretion level in rice. Rice seedlings were incubated in the medium containing barnyardgrass root exudates for 10 d, and secretion level of momilactone B was determined as described by Kato-Noguchi.²⁴ The experiment was repeated six times with three assays for each determination. Different letters show significant difference (p < 0.01) according to Tukey''s HSD test.Although mechanisms of the exudation are not well understood, it is suggested that plants are able to secrete a wide variety of compounds from root cells by plasmalemma-derived exudation, endoplasmic-derived exudation and proton-pumping mechanisms.^3,15 Through the root exudation of compounds, plants are able to regulate the soil microbial community in their immediate vicinity, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species.^3,14,15 The present research suggests that rice may be aware of the presence of neighboring barnyardgrass by detection of certain key in barnyardgrass root exudates, and this sensorial function may trigger a signal cascade resulting in increasing rice allelopathy through increasing production of momilactone B and secretion of momilactone B into the rhizosphere. Therefore, rice allelopathy may potentially be an inducible defense mechanism by chemical-mediated plant interactions between rice and barnyardgrass.     

Allelochemicals released by rice roots and residues in soil

A few rice (Oryza sativa L.) varieties or rice straw produce and release allelochemicals into soil in which interfere with the growth of neighboring or successive plants. Allelopathic rice PI312777 and Huagan-1 at their early growth stages released momilactone B, 3-isopropyl-5-acetoxycyclohexene-2-one-1, and 5,7,4′-trihydroxy-3′,5′-dimethoxyflavone into soil at phytotoxic levels, but non-allelopathic rice Huajingxian did not. Both allelopathic and non-allelopathic rice residues released momilactone B and lignin-related phenolic acids (p-hydroxybenzoic, p-coumaric, ferulic, syringic and vanillic acids) into the soil during residue decomposition to inhibit successive plants. The results indicated that allelochemicals involved in rice allelopathy from living and dead plants are substantially different. Interestingly, the concentrations of the allelochemicals released from the allelopathic rice seedlings in soil increased dramatically when they were surrounded with Echinochloa crus-galli. The concentrations of the allelochemicals were over 3-fold higher in the presence of E. crus-galli than in the absence of E. crus-galli. However, the same case did not occur in non-allelopathic Huajingxian seedlings surrounded with E. crus-galli. In addition to allelochemical exudation being promoted by the presence of E. crus-galli, allelopathic rice seedlings also increased allelochemical exudation in response to exudates of germinated E. crus-galli seeds or lepidimoide, an uronic acid derivative exuded from E. crus-galli seeds. These results imply that allelopathic rice seedlings can sense certain allelochemicals released by E. crus-galli into the soil, and respond by increased production of allelochemicals inhibitory to E. crus-galli. This study suggests that rice residues of both allelopathic and non-allelopathic varieties release similar concentrations and types of allelochemicals to inhibit successive plants. In contrast, living rice plants of certain allelopathic varieties appear to be able to detect the presence of interspecific neighbors and respond by increased allelochemicals.     

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.     

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.     

Absorption of momilactone A and B by Arabidopsis thaliana L. and the growth inhibitory effects

Although most allelochemicals can potentially cause growth inhibition in receiver plants, there is little information available about the absorption of these allelochemicals by the receiver plants. The present research describes the absorption of momilactone A and B by Arabidopsis thaliana (L.) and effects of the absorption on Arabidopsis growth. Exogenously applied momilactone A and B inhibited the growth of Arabidopsis hypocotyls and roots at concentrations greater than 10 and 1μmol/L, respectively. The levels of momilactone A and B in Arabidopsis hypocotyls were approximately 3.2 and 2.4% of momilactone A and B, respectively, in the medium and those in Arabidopsis roots were about 3.9-3.4%, respectively. The absorption rates of momilactone A and B by Arabidopsis were not significantly different. The present research suggests that momilactone A and B may be absorbed in proportion to their applied levels, and the growth inhibitory effects of momilactone A and B may also correlated with their endogenous levels. However, the effectiveness of momilactone B on growth inhibition was much greater than that of momilactone A, and the sensitivities of hypocotyls to momilactone A and B were greater than those of roots. This is the first report describing the absorption of potent rice allelochemicals, momilactone A and B by receiver plants.     

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.     

Rice allelopathy and the possibility for weed management

In attempts to control weeds in rice, much effort has been focused on rice allelopathy research for more than 30 years. Among screening methods that have been developed, some estimate the allelopathic potential of various rice cultivars in a limited time and space, which is less costly and can be conducted year round. Rice allelopathy activity is variety dependent and origin dependent, where Japonica rice shows greater allelopathic activity than Indica and Japonica–Indica hybrids. Allelopathic characteristics in rice are quantitatively inherited and several allelopathy‐involved traits have been identified. Numerous phytotoxins such as cytokinins, diterpenoids, fatty acids, flavones, glucopyranosides, indoles, momilactones (A and B), oryzalexins, phenols, phenolic acids, resorcinols and stigmastanols have been identified and determined as growth inhibitors in rice. However, the fate and actual modes of action of these compounds as well as other potent rice phytotoxins in nature are not well understood. The question of which compounds play a major role in rice allelopathy has remained obscure; however, rice allelopathy might be attributable to the interaction of all present allelochemicals. Despite locating genes determining or involving allelopathy in rice having attracted much effort, the introduction of these genes into target rice cultivars has not yet been achieved. Success in breeding new rice cultivars having good weed‐suppressing ability would benefit farmers in rice‐cultivating countries and play an important role in sustainable agricultural production.     

Secretion of momilactone A and B by the moss Hypnum plumaeforme

Nevertheless both plants are taxonomically quite distinct, momilactone A and B have been found only in rice and the moss, Hypnum plumaeforme which often forms large pure colonies. But biological meanings of momilactone A and B in H. plumaeforme is unknown. UV-irradiation induced a 15- and 16-fold increase in the secretion level of momilactone A and B, respectively, by H. plumaeforme into the growth medium. Jasmonic acid and the protein phosphatase inhibitor, cantharidin, also increased the momilactone A and B secretion levels by 12- to 15-fold. Cantharidin acts as an elicitor, jasmonic acid is an important signaling molecule regulating inducible defense genes against the pathogen infections. Therefore, elicitor and/or pathogen attacks may increase the secretion of momilactone A and B. As momilactone A and B are phytoalexic and allelopathic, the increasing secretion of momilactone A and B may be associated with the activation of the defense responses of H. plumaeforme in the rhizosphere where plants must compete with invading root systems of neighboring plants and prevent from bacteria and fungi infections. Momilactone A and B may be able to prevent H. plumaeforme from pathogen infections and help competition with neighboring plants resulting in the formation of pure colonies.Key words: defense mechanism, growth inhibitor, momilactone, musci, pathogen, phytoalexin, rhizosphereAlthough rice and the moss Hypnum plumaeforme Wils are taxonomically quite distinct, momilactone A and B have so far been found only in rice and H. plumaeforme.^1–4 Momilactone A and B in rice plants are known to be synthesized as a part of defensive responses and exhibit antibacterial and antifungal activities.^5–7 Rice plants were also found to secrete momilactone A and B from their roots into the rhizosphere and exhibit alleloapthic activities against weed plants.^1,8–10 The plant rhizosphere is a densely populated area in which plant roots must compete with invading root systems of neighboring plants for space, water and mineral nutrients, and with other soil-bore organisms including bacteria and fungi.^11–14 Therefore, momilactone A and B probably play an important role in rice defense mechanism in the rhizosphere as antimicrobial and allelopathic agents. However, it has not clear that biological meanings of momilactone A and B in H. plumaeforme. H. plumaeforme is often dominative in plant communities and forms large pure colonies.^15,16H. plumaeforme was grown on MS growth medium and the concentrations of momilactone A and B in the medium were determined as the secretion levels of momilactone A and B from H. plumaeforme. The secretion levels of momilactone A and B were 4.0 and 6.3 µg g⁻¹ dry weight of H. plumaeforme, respectively (4 Thus, the secretion levels of momilactone A and B, respectively, were 6.8 and 27% of momilactone A and B concentrations in H. plumaeforme. Therefore, although the endogenous concentration of momilactone A in H. plumaeforme was greater than that of momilactone B, the secretion level of momilactone B was much greater than that of momilactone A, which suggests that momilactone B may be selectively secreted into the medium than momilactone A. In addition, biological activity of momilactone B was much greater than that of momilactone A.¹⁰

Table 1

Effects of UV-irradiation, cantharidin and jasmonic acid on the secretion of momilactone A and B from H. plumaeforme

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.