Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

Suppression of Alternaria brassicicola infection by volatile compounds from spent mushroom substrates.

Most commercially circulating mushrooms are produced via cultivation using artificially produced mushroom substrates. However, after mushroom harvesting, the disposal of spent mushroom substrates (SMSs) is a serious problem for the mushroom industry owing to the need for a disposal site and the cost involved. Thus, in view of the possibility of recycling SMSs as a soil modifier, we examined the effect of soil mixed with SMSs on the infection of Arabidopsis leaves by Alternaria brassicicola, the causal agent of cabbage leaf spot. The mixing of SMSs used for Hypsizygus marmoreus, Pholiota microspora, Lyophyllum decastes, and Auricularia polytricha into culture soil suppressed the lesion formation caused by A. brassicicola. The defense responses of Arabidopsis were not induced by the culturing of these seedlings in soils containing SMSs. Suppressed lesion formation was observed after the seedlings were treated with volatiles emitted from SMSs that were incubated with soil for 7 days and used for H. marmoreus, P. microspora, L. decastes, A. polytricha, Lentinula edodes, and Cyclocybe cylindracea. The volatiles from the SMSs reduced the elongation of A. brassicicola hyphae. GC-MS analyses of extracts from the SMS containing soils led to the detection of various volatile compounds; among these, skatole, 2,4-di-tert-butylphenol, γ-dodecalactone, butyric acid, guaiacol, 6-amyl-2-pyrone, and 1-octen-3-ol were examined for inhibitory activity on A. brassicicola and found to suppress hyphae elongation. These findings indicate that the antifungal volatile compounds emitted by the SMSs suppress A. brassicicola infection.

Identification of methoxylchalcones produced in response to CuCl2 treatment and pathogen infection in barley.

Changes in specialized metabolites were analyzed in barley (Hordeum vulgare) leaves treated with CuCl2 solution as an elicitor. LC-MS analysis of the CuCl2-treated leaves showed the induced accumulation of three compounds. Among them, two were purified by silica gel and ODS column chromatography and preparative HPLC and were identified as 2',3,4,4',6'-pentamethoxychalcone and 2'-hydroxy-3,4,4',6'-tetramethoxychalcone by spectroscopic analyses. The remaining compound was determined as 12-oxo-phytodienoic acid (OPDA), a major oxylipin in plants, by comparing its spectrum and retention time from LC-MS/MS analysis with those of the authentic compound. The accumulation of these compounds was reproduced in leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of the Poaceae species. This inoculation increased the amounts of other oxylipins, including jasmonic acid (JA), JA-Ile, 9-oxooctadeca-10,12-dienoic acid (9-KODE), and 13-oxooctadeca-9,11-dienoic acid (13-KODE). The treatments of the barley leaves with JA and OPDA induced the accumulation of methoxylchalcones, but treatment with 9-KODE did not. These methoxylchalcones inhibited conidial germination of B. sorokiniana and Fusarium graminearum, thereby indicating that these compounds possessed antifungal activity. Consequently, they are considered to be involved in the chemical defense processes as phytoalexins in barley. Accumulation of methoxylchalcones in response to JA treatment was observed in all seven barley cultivars tested, but was not detected in other wild Hordeum species, wheat, and rice, thus indicating that their production was specific to cultivated barley.

Sugars in an aqueous extract of the spent substrate of the mushroom Hypsizygus marmoreus induce defense responses in rice.

Plant defense responses are activated by various exogenous stimuli. We found that an aqueous extract of spent mushroom substrate used for the cultivation of Hypsizygus marmoreus induced defense responses in rice. Fractionation of the spent mushroom substrate extract indicated that the compounds responsible for this induction were neutral and hydrophilic molecules with molecular weights lower than 3 kDa. Compounds with these characteristics, namely glucose, fructose, and sucrose, were detected in the extract at concentrations of 17.4, 3.3, and 1.6 mM, respectively, and the treatment of rice leaves with these sugars induced defense responses. Furthermore, microarray analysis indicated that the genes involved in defense responses were commonly activated by the treatment of leaves with spent mushroom substrate extract and glucose. These findings indicate that the induction of defense responses by treatment with spent mushroom substrate extract is, at least in part, attributable to the sugar constituents of the extract.

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.

Biosynthesis of Phenylamide Phytoalexins in Pathogen-Infected Barley.

Phytoalexins are inducible antimicrobial metabolites in plants, and have been indicated to be important for the rejection of microbial infection. HPLC analysis detected the induced accumulation of three compounds 1-3 in barley (Hordeum vulgare) roots infected by Fusarium culmorum, the causal agent of Fusarium root rot. Compounds 1-3 were identified as cinnamic acid amides of 9-hydroxy-8-oxotryptamine, 8-oxotryptamine, and (1H-indol-3-yl)methylamine, respectively, by spectroscopic analysis. Compounds 1 and 2 had been previously reported from wheat, whereas 3 was an undescribed compound. We named 1-3 as triticamides A-C, respectively, because they were isolated from barley and wheat, which belong to the Triticeae tribe. These compounds showed antimicrobial activities, indicating that triticamides function as phytoalexins in barley. The administration of deuterium-labeled N-cinnamoyl tryptamine (CinTry) to barley roots resulted in the effective incorporation of CinTry into 1 and 2, which suggested that they were synthesized through the oxidation of CinTry. Nine putative tryptamine hydroxycinnamoyl transferase (THT)-encoding genes (HvTHT1-HvTHT9) were identified by database search on the basis of homology to known THT gene sequences from rice. Since HvTHT7 and HvTHT8 had the same sequences except one base, we measured their expression levels in total by RT-qPCR. HvTHT7/8 were markedly upregulated in response to infection by F. culmorum. The HvTHT7 and HvTHT8 enzymes preferred cinnamoyl- and feruloyl-CoAs as acyl donors and tryptamine as an acyl acceptor, and (1H-indol-3-yl)methylamine was also accepted as an acyl acceptor. These findings suggested that HvTHT7/8 are responsible for the induced accumulation of triticamides in barley.

Identification of phenylamide phytoalexins and characterization of inducible phenylamide metabolism in wheat.

Changes in specialized metabolites were analyzed in wheat leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of Poaceae species. HPLC analysis detected the accumulation of six compounds in B. sorokiniana-infected leaves. Of these, we purified two compounds by silica gel and ODS column chromatography and preparative HPLC, and identified them as cinnamic acid amides, N-cinnamoyl-9-hydroxy-8-oxotryptamine and N-cinnamoyl-8-oxotryptamine, by spectroscopic analyses. The remaining four compounds were predicted to be p-coumaric acid amides of hydroxyputrescine, hydroxyagmatine, hydroxydehydroagmatine, and agmatine by mass spectrometry. The accumulation of two cinnamic acid amides was also induced by Fusarium graminearum infection, and by treatment with CuCl2, jasmonic acid, and isopentenyladenine. Antifungal activity of these amides was shown by inhibition of conidial germination and germ tube elongation of F. graminearum and Alternaria brassicicola, indicating that they act as phytoalexins. The accumulation of these amides also detected in barley leaves treated with CuCl2. We examined the accumulation of 25 phenylamides in B. sorokiniana-infected wheat leaves using LC-MS/MS. Hydroxycinnamic acid amides of tryptamine, serotonin, putrescine, and agmatine, were induced after infection with B. sorokiniana. Thus, the induced accumulation of two groups of phenylamides, cinnamic acid amides with indole amines, and p-coumaric acid amides with putrescine and agmatine related amines, represents a major metabolic response of wheat to pathogen infection.

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.

Induction of defense responses by extracts of spent mushroom substrates in rice.

We investigated the effect of treatment with hot water extracts from the spent mushroom substrates (SMSs) of Lentinula edodes and Hypsizygus marmoreus on the resistance of rice leaves to Pyricularia oryzae infection. The spraying of the SMS extracts clearly suppressed the development of lesions caused by Py. oryzae infection. The accumulation of phytoalexins momilactones A and B, oryzalexin A, and sakuranetin was markedly induced by the spraying of extracts. The enhanced expression of defense related genes PR1b and PBZ was also found in leaves sprayed with the extracts. Treatments with the extracts also affected phytohormone levels. The levels of N 6-(Δ2-isopentenyl)adenine and trans-zeatin markedly increased in response to treatment, whereas the levels of salicylic and jasmonic acids were largely unchanged.

Isolation of 6-hydroxy-L-tryptophan from the fruiting body of Lyophyllum decastes for use as a tyrosinase inhibitor.

Tyrosinase is the key enzyme that controls melanin formation. We found that a hot water extract of the lyophilized fruiting body of the fungus Lyophyllum decastes inhibited tyrosinase from Agaricus bisporus. The extract was fractionated by ODS column chromatography, and an active compound was obtained by purification through successive preparative HPLC using an ODS and a HILIC column. Using spectroscopic data, the compound was identified to be an uncommon amino acid, 6-hydroxytryptophan. 6-Hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan were prepared through a Fenton reaction from L-tryptophan and D-tryptophan, respectively. The active compound was determined to be 6-hydroxy-L-tryptophan by comparison of their circular dichroism spectra and retention time on HPLC analysis of the Nα-(5-fluoro-2,4-dinitrophenyl)-L-leuciamide derivative with those of 6-hydroxy-L-tryptophan and 6-hydroxy-D-tryptophan. A Lineweaver-Burk plot of the enzyme reaction in the presence of 6-hydroxy-L-tryptophan indicated that this compound was a competitive inhibitor. The IC50 values of 6-hydroxy-L-tryptophan was 0.23 mM.

Isolation of isolactarane sesquiterpenes from a Phlebia tremellosa culture filtrate and their growth promotion effects on lettuce roots.

The ethyl acetate extract of the culture filtrate of Phlebia tremellosa promoted elongation of the lateral roots of lettuce seedlings at 250 µg/mL. We purified two compounds that promote root elongation by using activity-guided chromatographic fractionation. On the basis of spectroscopic analyses, these compounds were identified to be isolactarane sesquiterpenes derived from the dehydrogenation of merulactone, which was previously isolated from the same species. We named the purified compounds phlelactones A and B. Phlelactones A and B promoted primary root elongation at 100-300 and 10-30 µg/mL and the elongation and formation of lateral roots at 300-1000 and 30-100 µg/mL, respectively.

Identification of antifungal compounds in the spent mushroom substrate of Lentinula edodes.

In view of the possibility that spent mushroom substrates (SMSs) may be used as agricultural materials to prevent crop diseases, we examined the effect of treatment with a hot water extract from the SMS of Lentinula edodes on plant resistance to pathogenic infection. The extract of Le. edodes SMS was sprayed onto the leaves of rice plants, followed by inoculation of the leaves with the conidia of rice blast fungus. The development of lesions was suppressed by treatment with the SMS extract. The extract markedly inhibited the germination of Pyricularia oryzae conidia. We purified compounds 1, 2, and 3, which showed inhibitory effects on conidial germination, from the Le. edodes SMS extract of by successive solvent extraction, column chromatography, and preparative HPLC. Spectroscopic analyses revealed that 1, 2, and 3 were phenolic acids with two carboxyl groups in common.

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.

Novel tyrosinase inhibitors from liquid culture of Neolentinus lepideus.

Tyrosinase is the key enzyme that controls melanin formation in the human skin. We performed a screening of 96 extracts of mushroom cultures and fruiting bodies for examining their inhibitory activity against mushroom tyrosinase. The ethyl acetate extracts of culture filtrate of Neolentinus lepideus exhibited the strongest inhibitory activity. The active compounds 1 and 2 were purified by repeated chromatographic separations from the extract. On the basis of spectroscopic analyses, 1 and 2 were identified to be 1,3-dihydroisobenzofuran-4,5,7-triol and 5-methoxy-1,3-dihydroisobenzofuran-4,7-diol, respectively. Lineweaver-Burk plot of the enzyme reaction in the presence of 1 indicated that 1 was a potent competitive inhibitor. The respective IC50 values of 1 and 2 were 173 and 263 µg/mL. Compound 1 at 15 µg/mL suppressed melanin accumulation stimulated by α-MSH in the murine melanoma B16 cells, as well as the induced accumulation of both tyrosinase transcript and protein without inhibiting cell proliferation.

Evolutionary changes in defensive specialized metabolism in the genus Hordeum.

Plants have developed defensive specialized metabolites over the course of evolution. In the genus Hordeum, which includes the important cereal crop barley, specialized metabolites such as hordatines, benzoxazinones, and gramine have been identified. Hordeum species are classified into four clades, H, Xu, Xa, and I. The presence or absence of defensive specialized metabolites was analyzed in representative Hordeum species that included all of the four clades. In the H clade, Hordeum vulgare accumulated hordatines but not benzoxazinones, whereas H. bulbosum accumulated neither compound. Some accessions in the H clade accumulated gramine. Species in the clades I and Xa accumulated benzoxazinones without hordatines. In H. murinum, a Xu clade species, neither hordatines nor benzoxazinones were detected. Two hitherto undescribed compounds were found to commonly accumulate in H. bulbosum in the H clade and H. murinum in the Xu clade. On the basis of spectroscopic analyses, they were identified as dehydrodimers of feruloylagmatine and were designated murinamides A and B. Radical coupling reactions with feruloylagmatine as a substrate by peroxidase afforded murinamides A and B. These compounds showed antifungal activities against Bipolaris sorokiniana and Fusarium asiaticum, indicating their defensive roles. Because hordatines are also dehydrodimers of hydroxycinnamic acid amides (HCAAs) of agmatine, both the H and Xu clade species are considered to accumulate the same class of compounds. Thus, when the H/Xu clades split from the I/Xa clades during evolution, the defensive metabolites shifted from benzoxazinones to dehydrodimers of agmatine HCAAs plus gramine in the H/Xu clades.

Induced accumulation of tyramine, serotonin, and related amines in response to Bipolaris sorokiniana infection in barley.

The inducible metabolites were analyzed in barley leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of barley. HPLC analysis revealed that B. sorokiniana-infected leaves accumulated 4 hydrophilic compounds. They were purified by ODS column chromatography and preparative HPLC. Spectroscopic analyses revealed that they were tyramine (1), 3-(2-aminoethyl)-3-hydroxyindolin-2-one (2), serotonin (3), and 5,5'-dihydroxy-2,4'-bitryptamine (4). Among these, 2 and 4 have not been reported as natural products. They showed antifungal activity in an assay of inhibition of B. sorokiniana conidia germination, suggesting that they play a role in the chemical defense of barley as phytoalexins. The accumulation of 1-4 was examined also in the leaves of rice and foxtail millet. Rice leaves accumulated 2, 3, and 4, whereas foxtail millet leaves accumulated 3 and 4 in response to pathogen attack, suggesting the generality of accumulation of 3 and 4 in the Poaceae species.

Distribution of the tryptophan pathway-derived defensive secondary metabolites gramine and benzoxazinones in Poaceae.

The Poaceae is a large taxonomic group consisting of approximately 12,000 species and is classified into 12 subfamilies. Gramine and benzoxazinones (Bxs), which are biosynthesized from the tryptophan pathway, are well-known defensive secondary metabolites in the Poaceae. We analyzed the presence or absence of garamine and Bxs in 64 species in the Poaceae by LC-MS/MS. We found that Hordeum brachyantherum and Hakonechloa macra accumulated gramine, but the presence of gramine was limited to small groups of species. We also detected Bxs in four species in the Pooideae and six species in the Panicoideae. In particular, four species in the Paniceae tribe in Panicoideae accumulaed Bxs, indicating that this tribe is a center of the Bx distribution. Bxs were absent in the subfamilies other than Pooideae and Panicoideae. These findings provide an overview of biased distribution of gramine and Bxs in Poaceae species.