Identification and evolution of a diterpenoid phytoalexin oryzalactone biosynthetic gene in the genus Oryza.

The natural variation of plant-specialized metabolites represents the evolutionary adaptation of plants to their environments. However, the molecular mechanisms that account for the diversification of the metabolic pathways have not been fully clarified. Rice plants resist attacks from pathogens by accumulating diterpenoid phytoalexins. It has been confirmed that the composition of rice phytoalexins exhibits numerous natural variations. Major rice phytoalexins (momilactones and phytocassanes) are accumulated in most cultivars, although oryzalactone is a cultivar-specific compound. Here, we attempted to reveal the evolutionary trajectory of the diversification of phytoalexins by analyzing the oryzalactone biosynthetic gene in Oryza species. The candidate gene, KSLX-OL, which accounts for oryzalactone biosynthesis, was found around the single-nucleotide polymorphisms specific to the oryzalactone-accumulating cultivars in the long arm of chromosome 11. The metabolite analyses in Nicotiana benthamiana and rice plants overexpressing KSLX-OL indicated that KSLX-OL is responsible for the oryzalactone biosynthesis. KSLX-OL is an allele of KSL8 that is involved in the biosynthesis of another diterpenoid phytoalexin, oryzalexin S and is specifically distributed in the AA genome species. KSLX-NOL and KSLX-bar, which encode similar enzymes but are not involved in oryzalactone biosynthesis, were also found in AA genome species. The phylogenetic analyses of KSLXs, KSL8s, and related pseudogenes (KSL9s) indicated that KSLX-OL was generated from a common ancestor with KSL8 and KSL9 via gene duplication, functional differentiation, and gene fusion. The wide distributions of KSLX-OL and KSL8 in AA genome species demonstrate their long-term coexistence beyond species differentiation, suggesting a balancing selection between the genes.

Natural variation of diterpenoid phytoalexins in rice: Aromatic diterpenoid phytoalexins in specific cultivars.

Rice (Oryza sativa L.) plants accumulate antimicrobial compounds known as phytoalexins in response to pathogen attack. To date, more than 20 compounds have been isolated as phytoalexins from rice, mostly diterpenoids. However, the quantitative analysis of diterpenoid phytoalexins in various cultivars has revealed that the cultivar 'Jinguoyin' does not accumulate these compounds at detectable concentrations. Therefore, in this study, we attempted to detect a new class of phytoalexins from Bipolaris oryzae infected leaves of 'Jinguoyin'. We detected five compounds in the leaves of the target cultivar, whereas these compounds were not detected in the leaves of 'Nipponbare' or 'Kasalath', which are representative cultivars of the japonica and indica subspecies. Subsequently, we isolated these compounds from ultraviolet (UV)-light-irradiated leaves and determined their structures by spectroscopic analysis and the crystalline sponge method. All the compounds were diterpenoids containing a benzene ring and were detected from the pathogen-infected rice leaves for the first time. Because the compounds showed antifungal activity against B. oryzae and Pyricularia oryzae, we propose that they function as phytoalexins in rice and named them abietoryzins A-E. The abietoryzins tended to accumulate at high concentrations in cultivars that accumulated low levels of known diterpenoid phytoalexins after UV-light irradiation. Of the total of 69 cultivars in the WRC, 30 cultivars accumulated at least one of the abietoryzins, and, in 15 cultivars, the amounts of some abietoryzins were the highest among those of the analyzed phytoalexins. Therefore, abietoryzins are a major phytoalexin group in rice, although their presence has, to date, been overlooked.

A nonproteinogenic amino acid, ß-tyrosine, accumulates in young rice leaves via long-distance phloem transport from mature leaves.

Oryza sativa L. ssp. japonica cv. Nipponbare produces a nonproteinogenic amino acid (3R)-ß-tyrosine from l-tyrosine by tyrosine aminomutase (OsTAM1). However, physiological and ecological function(s) of ß-tyrosine have remained obscure. Often an improved understanding of metabolite localization and transport can aid in design of experiments to test physiological functions. In the current study, we investigated the distribution pattern of ß-tyrosine in rice seedlings and found that ß-tyrosine is most abundant in the youngest leaves. Based upon observations of high TAM1 activity in mature leaves, we hypothesized that ß-tyrosine is transported from mature leaves to young leaves. Patterns of predominant mature synthesis and young leaf accumulation were supported by stable isotope studies using labeled ß-tyrosine and the removal of mature leaves. Stem exudate analyses was also consistent with ß-tyrosine transport through phloem. Thus, we identify young leaves as a key target in efforts to understand the biological function(s) of ß-tyrosine in rice.

Evaluation of antixenosis in soybean against Spodoptera litura by dual-choice assay aided by a statistical analysis model: Discovery of a novel antixenosis in Peking.

The method for evaluating soybean (Glycine max) antixenosis against the common cutworm (Spodoptera litura) was developed based on a dual-choice assay aided by a statistical analysis model. This model was constructed from the results of a dual-choice assay in which Enrei, a soybean cultivar susceptible to S. litura, was used as both a standard and a test leaf disc for 2nd-5th instar larvae. The statistical criterion created by this model enabled the evaluation of the presence of antixenosis. This method was applied to four soybean varieties, including Tamahomare (susceptible), Himeshirazu (resistant), IAC100 (resistant), and Peking (unknown), as well as Enrei. Subsequently, the degrees of antixenosis were also compared by F-test, followed by maximum likelihood estimation (MLE). According to the results, the antixenosis of Tamahomare, Himeshirazu, and IAC100 was statistically reevaluated and Peking exhibited a novel antixenosis, which was stronger for 3rd-5th instar larvae than for 2nd instar.

Isopentylamine is a novel defence compound induced by insect feeding in rice.

Plants produce a broad variety of defensive metabolites to protect themselves against herbivorous insects. Although polyamines have been implicated in various responses to abiotic and biotic stress, there have been no studies focused on amines in response to insect herbivory. By screening for bioactive amines, we identified isopentylamine as a novel type of herbivory-induced compound in rice leaves, which was derived from the amino acid leucine in stable isotope labelling experiments. Accumulation of isopentylamine increased during herbivory by the brown planthopper (Nilaparvata lugens, BPH) and the rice-feeding armyworm (Mythimna loreyi), as well as in response to treatment with the plant hormone, jasmonic acid. Likewise, isopentylamine accumulation was compromised in rice jasmonate biosynthesis mutants, hebiba and Osjar1. In bio-assays, BPH insects feeding on rice seedlings submerged in 50 mg/L isopentylamine solution had a higher mortality compared with BPH feeding on seedlings submerged in water. Notably, the rice leaves submerged in 50 mg/L solution showed the endogenous concentrations of isopentylamine similar to that induced by BPHs. These results suggest that isopentylamine functions as a new type of plant defence metabolite that is rapidly induced by herbivore attack and deters insect herbivores in rice.

Molecular Basis Underlying Common Cutworm Resistance of the Primitive Soybean Landrace Peking.

The common cutworm (CCW; Spodoptera litura) is one of the major insect pests of soybean in Asia and Oceania. Although quantitative trail loci related to CCW resistance have been introduced into leading soybean cultivars, these do not exhibit sufficient resistance against CCW. Thus, understanding the genetic and metabolic resistance mechanisms of CCW as well as integrating other new resistance genes are required. In this study, we focused on a primitive soybean landrace, Peking, which has retained resistances to various pests. We found a resistance to CCW in Peking by the detached-leaf feeding assay, and subsequently determined the genetic and metabolic basis of the resistance mechanism using chromosome segment substitution lines (CSSLs) of Peking. Several characteristic metabolites for Peking were identified by the metabolomic approach using liquid chromatography/mass spectrometry combined with a principle component analysis. The structure of seven metabolites were determined by nuclear magnetic resonance (NMR) analysis. The genomic segments of Peking on chromosome 06 (Chr06) and Chr20 had a clear association with these metabolites. Moreover, a line possessing a Peking genomic segment on Chr20 inhibited growth of the CCW. The genetic factors and the metabolites on Chr20 in Peking will be useful for understanding mechanisms underlying CCW resistance and breeding resistant soybean cultivars.

Natural variation of diterpenoid phytoalexins in cultivated and wild rice species.

Rice (Oryza sativa) leaves accumulate phytoalexins in response to pathogen attack. The major phytoalexins in rice are diterpenoids such as momilactones, phytocassanes, and oryzalexins. We analyzed the abundance of momilactones A and B and phytocassanes A and D in UV-light-irradiated leaves of cultivars from the World Rice Core Collection (WRC). Both types of phytoalexins were detected in most cultivars; however, their accumulated amounts varied greatly from cultivar to cultivar. The amounts of momilactones A and B tended to be higher in japonica cultivars than those in indica cultivars. However, the accumulated amounts of phytocassanes were not related to differences in subspecies. In addition, variation in phytoalexin content was observed for seven wild rice species. During the analysis of momilactone A in cultivars from the WRC, two unknown compounds were detected in'Jaguary' and 'Basilanon'. We isolated these compounds from UV-light-irradiated leaves and determined their structures. The compound isolated from 'Jaguary' was an isomer of momilactone A that had an abietane skeleton, while that from 'Basilanon' was di-dehydrogenated phytocassane A; these compounds were denoted as oryzalactone and phytocassane G. Oryzalactone accumulated in only three cultivars, whereas phytocassane G accumulated in almost all of the cultivars from the WRC. These findings indicate the existence of large natural variation in the phytoalexin composition in rice.

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.

Allelic Differentiation at the E1/Ghd7 Locus Has Allowed Expansion of Rice Cultivation Area.

The photoperiod-insensitivity allele e1 is known to be essential for the extremely low photoperiod sensitivity of rice, and thereby enabled rice cultivation in high latitudes (42-53° north (N)). The E1 locus regulating photoperiod-sensitivity was identified on chromosome 7 using a cross between T65 and its near-isogenic line T65w. Sequence analyses confirmed that the E1 and the Ghd7 are the same locus, and haplotype analysis showed that the e1/ghd7-0a is a pioneer allele that enabled rice production in Hokkaido (42-45° N). Further, we detected two novel alleles, e1-ret/ghd7-0ret and E1-r/Ghd7-r, each harboring mutations in the promoter region. These mutant alleles alter the respective expression profiles, leading to marked alteration of flowering time. Moreover, e1-ret/ghd7-0ret, as well as e1/ghd7-0a, was found to have contributed to the establishment of Hokkaido varieties through the marked reduction effect on photoperiod sensitivity, whereas E1-r/Ghd7-r showed a higher expression than the E1/Ghd7 due to the nucleotide substitutions in the cis elements. The haplotype analysis showed that two photoperiod-insensitivity alleles e1/ghd7-0a and e1-ret/ghd7-0ret, originated independently from two sources. These results indicate that naturally occurring allelic variation at the E1/Ghd7 locus allowed expansion of the rice cultivation area through diversification and fine-tuning of flowering time.

Variation of diterpenoid phytoalexin oryzalexin A production in cultivated and wild rice.

Rice (Oryza sativa) leaves accumulate phytoalexins in response to pathogen attack. The major phytoalexins in rice are diterpenoids such as oryzalexins, momilactones, and phytocassanes. We measured the amount of oryzalexin A in leaves irradiated by UV light, treated with jasmonic acid, or inoculated with conidia of Bipolaris oryzae in the japonica cultivar Nipponbare and the indica cultivar Kasalath. Nipponbare leaves accumulated oryzalexin A at a high concentration, but Kasalath leaves did not. The locus responsible for this difference was mapped using backcrossed inbred lines and chromosome substitution lines. A region on Chr. 12 containing the KSL10 gene was responsible for the deficiency in oryzalexin A in the Kasalath cultivar. The amount of KSL10 transcript increased in Nipponbare leaves but not in Kasalath leaves in response to UV light irradiation, indicating that the suppressed expression of KSL10 caused the deficiency of oryzalexin A in Kasalath. We analyzed oryzalexin A accumulation in UV light-irradiated leaves of cultivars in the world rice core collection. There were cultivars that accumulated oryzalexin A and those that did not, and both of these chemotypes were found in japonica and indica subspecies. Furthermore, these chemotypes were found in the wild rice species Oryza rufipogon. The phylogenetic relationship of KSL10 sequences was not correlated to oryzalexin A chemotypes. These findings suggested that the biosynthesis of oryzalexin A was acquired by a common ancestor of O. rufipogon and was lost multiple times during the evolutionary process.

An easy, inexpensive, and sensitive method for the quantification of chitin in insect peritrophic membrane by image processing.

Chitin, poly (ß-(1→4)-N-acetyl-d-glucosamine), is an important biopolymer for insects that is utilized as a major component of peritrophic membrane. The chitin content in peritrophic membrane is of expedient interest from a pest control perspective, although it is hard to quantify chitin. In this study, we establish a facile method for the quantification of chitin in peritrophic membrane by image processing. In this method, chitin was indirectly quantified using chitosan-I3- complex, which exhibited a specific red-purple color. A calibration curve using a chitosan solution showed good linearity in a concentration range of 0.05-0.5 µg/µL. We quantified the amount of chitin in peritrophic membrane of Spodoptera litura (Lepidoptera: Noctuidae) larvae using this method. Throughout the study, only common inexpensive regents and easily attainable apparatuses were employed. This method can be easily applied to the sensitive quantification of the amounts of chitin and chitosan in materials by wide range of researchers. Abbreviations: LOD: limit of detection; LOQ: limit of quantification; ROI: region of interest; RSD: relative standard deviation.

Accumulation of 9- and 13-KODEs in response to jasmonic acid treatment and pathogenic infection in rice.

The inducible metabolites in rice leaves treated with 1 mM jasmonic acid (JA) were analyzed using HPLC. We detected an increase in the levels of two compounds, 1 and 2. Based on the comparison with mass spectra and chromatographic behavior with authentic compounds, 1 and 2 were identified as 13-oxooctadeca-9,11-dienoic acid (13-KODE) and 9-oxooctadeca-10,12-dienoic acid (9-KODE), respectively, which have not been detected in rice to date. The accumulation of these compounds was also induced by an infection by Bipolaris oryzae. Treatment of rice leaves with KODEs induced the accumulation of defensive secondary metabolites, sakuranetin, naringenin, and serotonin, suggesting that KODEs may play a role in the elicitation of defense responses. The compounds that have an α, ß-unsaturated carbonyl group similar to KODEs did not reproduce the response of accumulation of defensive secondary metabolites, suggesting that additional structural factors such as long hydrophobic carbon chain are needed to elicit defense responses.

Biosynthesis and accumulation of GABA in rice plants treated with acetic acid.

Rice seedlings (Oryza sativa) that have died from drought cannot be rescued by watering afterward, but pre-treatment with exogenous acetic acid enabled the plants to produce shoots again after being watered (hereinafter referred to as "drought resilience"). To elucidate the metabolism of acetic acid, we treated rice plants with 13C-labeled acetic acid and traced 13C-labeled metabolites using LC-MS and 13C-NMR techniques. The LC-MS and 13C-NMR spectral data of the root extracts indicated that the acetic acid treatment was absorbed into the plants and then was metabolized to gamma-aminobutyric acid (GABA) by glutamic acid decarboxylase (GAD). GABA accumulation in the roots took place in advance of that in the shoots, and the survival rate against drought stress increased in proportion to the amount of GABA accumulated in the shoots. Therefore, GABA accumulation in shoots may be a key step in drought resilience induced by the acetic acid treatment.

QTLs underlying the genetic interrelationship between efficient compatibility of Bradyrhizobium strains with soybean and genistein secretion by soybean roots.

Soybean plants establish symbiotic relationships with soil rhizobia which form nodules on the plant roots. Nodule formation starts when the plant roots exudate isoflavonoids that induce nod gene expression of a specific Bradyrhizobium. We examined the specific indigenous rhizobia that form nodules with the soybean cultivars Peking and Tamahomare in different soils. PCR-RFLP analysis targeted to the 16S-23S rRNA gene internal transcribed spacer (ITS) region of the bacterial type of each root nodule showed that Bradyrhizobium japonicum (USDA110-type) and Bradyrhizobium elkanii (USDA94-type) had high compatibility with the Tamahomare and Peking cultivars, respectively. We grew 93 recombinant inbred lines (RIL) of soybean seeds derived from the cross between Peking and Tamahomare in three different field soils and identified the indigenous rhizobia nodulating each line using the same PCR-RFLP analysis. QTL analysis identified one QTL region in chromosome-18 with a highly significant additive effect that controls compatibility with both B. japonicum USDA110 and B. elkanii USDA94. We also measured the amount of daidzein and genistein secretion from roots of the 93 RILs by HPLC analysis. QTL analysis showed one QTL region in chromosome-18 controlling genistein secretion from roots and coinciding with that regulating compatibility of specific indigenous rhizobia with soybean. The amount of genistein may be a major regulatory factor in soybean-rhizobium compatibility.

A fragmentation study of isoflavones by IT-TOF-MS using biosynthesized isotopes.

To aid in the identification and quantification of biologically and agriculturally significant natural products, tandem mass spectrometry can provide accurate structural information with high selectivity and sensitivity. In this study, diagnostic fragmentation patterns of isoflavonoids were examined by liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). The fragmentation scheme for [M+H-2CO]+ ions derived from isoflavones and [M+H-B-ring-CO]+ ions derived from 5-hydroxyisoflavones, were investigated using different isotopically labeled isoflavones, specifically [1',2',3',4',5',6',2,3,4-13C9] and [2',3',5',6',2-D5] isoflavones. Specific isotopically labeled isoflavones were prepared through the biosynthetic incorporation of pharmacologically applied 13C- and D-labelled L-phenylalanine precursors in soybean plants following the application of insect elicitors. Using this approach, we empirically demonstrate that the [M+H-2CO]+ ion is generated by an intramolecular proton rearrangement during fragmentation. Furthermore, [M+H-B-ring-CO]+ ion is demonstrated to contain a C2H moiety derived from C-ring of 5-hydroxyisoflavones. A mechanistic understanding of characteristic isoflavone fragmentation patterns contributes to the efficacy and confidence in identifying related isoflavones by LC-MSn.

Lineage-specific gene acquisition or loss is involved in interspecific hybrid sterility in rice.

Understanding the genetic basis of reproductive barriers between species has been a central issue in evolutionary biology. The S1 locus in rice causes hybrid sterility and is a major reproductive barrier between two rice species, Oryza sativa and Oryza glaberrima The O. glaberrima-derived allele (denoted S1g) on the S1 locus causes preferential abortion of gametes with its allelic alternative (denoted S1s) in S1g/S1s heterozygotes. Here, we used mutagenesis and screening of fertile hybrid plants to isolate a mutant with an allele, S1mut, which does not confer sterility in the S1mut/S1g and S1mut/S1s hybrids. We found that the causal mutation of the S1mut allele was a deletion in the peptidase-coding gene (denoted "SSP") in the S1 locus of O. glaberrima No orthologous genes of SSP were found in the O. sativa genome. Transformation experiments indicated that the introduction of SSP in carriers of the S1s allele did not induce sterility. In S1mut/S1s heterozygotes, the insertion of SSP led to sterility, suggesting that SSP complemented the loss of the functional phenotype of the mutant and that multiple factors are involved in the phenomenon. The polymorphisms caused by the lineage-specific acquisition or loss of the SSP gene were implicated in the generation of hybrid sterility. Our results demonstrated that artificial disruption of a single gene for the reproductive barrier creates a "neutral" allele, which facilitates interspecific hybridization for breeding programs.

Induced phenylamide accumulation in response to pathogen infection and hormone treatment in rice (Oryza sativa).

Rice plants accumulate various specialized metabolites, including phenylamides, in response to pathogen attack. We prepared 25 phenylamides, and developed a method of analyzing them by multiple reaction monitoring with liquid chromatography coupled with tandem mass spectrometry. We analyzed phenylamides in rice leaves infected with Cochliobolus miyabeanus and Xanthomonas oryzae. The phenylamides induced included benzoyltryptamine, cinnamoyl-, p-coumaroyl-, feruloyl-, and benzoylserotonins, cinnamoyl and benzoyltyramines, feruloylagmatine, and feruloylputrescine. Some of the phenylamides exhibited antimicrobial activity against C. miyabeanus and X. oryzae, indicating that they are phytoalexins. Treatment with jasmonic acid, salicylic acid, 6-benzylaminopurine, and ethephone also induced phenylamide accumulation. The compositions of the induced amides varied depending on the plant hormone used, and cinnamoyltryptamine, cinnamoylserotonin, and cinnamoyltyramine were not induced by the plant hormones. These findings suggest that several plant hormones and additional factors are involved in phenylamide accumulation in response to pathogen infection in rice.

Identification of environmentally stable QTLs controlling Saponin content in Glycine max.

Saponins are secondary metabolites that are widely distributed in plants. There are two major saponin precursors in soybean: soyasapogenol A, contributing to the undesirable taste, and soyasapogenol B, some of which have health benefits. It is important to control the ratio and content of the two major saponin groups to enhance the appeal of soybean as a health food. The structural diversity of saponin in the sugar chain composition makes it hard to quantify the saponin content. We measured the saponin content in soybean by removing the sugar chain from the saponin using acidic hydrolysis and detected novel quantitative trait loci (QTLs) for saponin content. Major QTLs in the hypocotyl were identified on chromosome 5 near the SSR marker, Satt 384, while those in the cotyledon were on chromosome 6 near Sat_312, which is linked to the T and E1 loci. Our results suggest that saponin contents in the hypocotyl and cotyledon are controlled by different genes and that it is difficult to increase the beneficial group B saponin and to decrease the undesirable group A saponin at the same time.

Inducible De Novo Biosynthesis of Isoflavonoids in Soybean Leaves by Spodoptera litura Derived Elicitors: Tracer Techniques Aided by High Resolution LCMS.

Isoflavonoids are a characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. For example, in soybean (Glycine max: Fabaceae) multiple isoflavones are induced and accumulate in leaves following attack by Spodoptera litura (Lepidoptera: Noctuidae) larvae. To quantitatively examine patterns of activated de novo biosynthesis, soybean (Var. Enrei) leaves were treated with a combination of plant defense elicitors present in S. litura gut content extracts and L-α-[13C9, 15N]phenylalanine as a traceable isoflavonoid precursor. Combined treatments promoted significant increases in 13C-labeled isoflavone aglycones (daidzein, formononetin, and genistein), 13C-labeled isoflavone 7-O-glucosides (daidzin, ononin, and genistin), and 13C-labeled isoflavone 7-O-(6″-O-malonyl-ß-glucosides) (malonyldaidzin, malonylononin, and malonylgenistin). In contrast levels of 13C-labeled flavones and flavonol (4',7-dihydroxyflavone, kaempferol, and apigenin) were not significantly altered. Curiously, application of fatty acid-amino acid conjugate (FAC) elicitors present in S. litura gut contents, namely N-linolenoyl-L-glutamine and N-linoleoyl-L-glutamine, both promoted the induced accumulation of isoflavone 7-O-glucosides and isoflavone 7-O-(6″-O-malonyl-ß-glucosides), but not isoflavone aglycones in the leaves. These results demonstrate that at least two separate reactions are involved in elicitor-induced soybean leaf responses to the S. litura gut contents: one is the de novo biosynthesis of isoflavone conjugates induced by FACs, and the other is the hydrolysis of the isoflavone conjugates to yield isoflavone aglycones. Gut content extracts alone displayed no hydrolytic activity. The quantitative analysis of isoflavone de novo biosynthesis, with respect to both aglycones and conjugates, affords a useful bioassay system for the discovery of additional plant defense elicitor(s) in S. litura gut contents that specifically promote hydrolysis of isoflavone conjugates.

The Tyrosine Aminomutase TAM1 Is Required for ß-Tyrosine Biosynthesis in Rice.

Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-ß-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with ß-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated ß-tyrosine production in Nipponbare. Rice cultivars that do not produce ß-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although ß-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant ß-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 µM. As ß-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice.