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Changes in specialized metabolites were analyzed in barley (Hordeum vulgare) leaves treated with CuCl 2 solution as an elicitor. LC-MS analysis of the CuCl 2 -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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 CuCl 2 , 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 CuCl 2 . 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)