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.

The usefulness of machine-learning-based evaluation of clinical and pretreatment 18F-FDG-PET/CT radiomic features for predicting prognosis in patients with laryngeal cancer.

OBJECTIVE: To examine whether machine learning (ML) analyses involving clinical and 18F-FDG-PET-based radiomic features are helpful in predicting prognosis in patients with laryngeal cancer. METHODS: This retrospective study included 49 patients with laryngeal cancer who underwent18F-FDG-PET/CT before treatment, and these patients were divided into the training (n = 34) and testing (n = 15) cohorts.Seven clinical (age, sex, tumor size, T stage, N stage, Union for International Cancer Control stage, and treatment) and 40 18F-FDG-PET-based radiomic features were used to predict disease progression and survival. Six ML algorithms (random forest, neural network, k-nearest neighbors, naïve Bayes, logistic regression, and support vector machine) were used for predicting disease progression. Two ML algorithms (cox proportional hazard and random survival forest [RSF] model) considering for time-to-event outcomes were used to assess progression-free survival (PFS), and prediction performance was assessed by the concordance index (C-index). RESULTS: Tumor size, T stage, N stage, GLZLM_ZLNU, and GLCM_Entropy were the five most important features for predicting disease progression.In both cohorts, the naïve Bayes model constructed by these five features was the best performing classifier (training: AUC = 0.805; testing: AUC = 0.842). The RSF model using the five features (tumor size, GLZLM_ZLNU, GLCM_Entropy, GLRLM_LRHGE and GLRLM_SRHGE) exhibited the highest performance in predicting PFS (training: C-index = 0.840; testing: C-index = 0.808). CONCLUSION: ML analyses involving clinical and 18F-FDG-PET-based radiomic features may help predict disease progression and survival in patients with laryngeal cancer. ADVANCES IN KNOWLEDGE: ML approach using clinical and 18F-FDG-PET-based radiomic features has the potential to predict prognosis of laryngeal cancer.

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.

(E)-7-phenyl-2-heptene-4,6-diyn-1-ol from Bidens pilosa as a repellent against isopods.

The development of repellents as alternatives to insecticides has expanded in recent years. However, their use in isopod pest control is limited. To develop an isopod repellent, a plant extract library from wild plants native to the Kochi Prefecture was screened for repellent activity against pillbugs, and 82 samples (87%) exhibited repellent activity. Among them, (E)-7-phenyl-2-heptene-4,6-diyn-1-ol was isolated and identified as a repellent from the root of Bidens pilosa. It had a half-maximal effective concentration of 0.20 µm, with a strong repellency. A study of the structure-activity relationship to (E)-7-phenyl-2-heptene-4,6-diyn-1-ol revealed that the presence of a hydroxyl group and an aromatic at both ends of the length of the seven-carbon chain is important for the expression of repellency. These results can potentially lead to a new repellent of phenylalkyl alcohol.

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.

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.