Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca²⁺ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.

Plants monitor changes in day length to coordinate their flowering time with appropriate seasons. In Arabidopsis, the diel and seasonal regulation of CONSTANS (CO) protein stability is crucial for the induction of FLOWERING LOCUS T ( FT) gene in long days. FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and ZEITLUPE (ZTL) proteins control the shape of CO expression profile antagonistically, although regulation mechanisms remain unknown. In this study, we show that GIGANTEA (GI) protein modulates the stability and nuclear function of FKF1, which is closely related to the stabilization of CO in the afternoon of long days. The abundance of FKF1 protein is decreased by the gi mutation, but increased by GI overexpression throughout the day. Unlike the previous report, the translocation of FKF1 to the nucleus was not prevented by ZTL overexpression. In addition, the FKF1-ZTL complex formation is higher in the nucleus than in the cytosol. GI interacts with ZTL in the nucleus, implicating the attenuation of ZTL activity by the GI binding and, in turn, the sequestration of FKF1 from ZTL in the nucleus. We also found that the CO-ZTL complex presents in the nucleus, and CO protein abundance is largely reduced in the afternoon by ZTL overexpression, indicating that ZTL promotes CO degradation by capturing FKF1 in the nucleus under these conditions. Collectively, our findings suggest that GI plays a pivotal role in CO stability for the precise control of flowering by coordinating balanced functional properties of FKF1 and ZTL.

Xanthomonas oryzae pv. oryzae (Xoo) causes a devastating bacterial leaf blight in rice. Here, the antimicrobial effects of D-limonene, L-limonene, and its oxidative derivative carveol against Xoo were investigated. We revealed that carveol treatment at ≥ 0.1 mM in liquid culture resulted in significant decrease in Xoo growth rate (> 40%) in a concentration-dependent manner, and over 1 mM, no growth was observed. The treatment with D-limonene and L-limonene also inhibited the Xoo growth but to a lesser extent compared to carveol. These results were further elaborated with the assays of motility, biofilm formation and xanthomonadin production. The carveol treatment over 1 mM caused no motilities, basal level of biofilm formation (< 10%), and significantly reduced xanthomonadin production. The biofilm formation after the treatment with two limonene isomers was decreased in a concentration-dependent manner, but the degree of the effect was not comparable to carveol. In addition, there was negligible effect on the xanthomonadin production mediated by the treatment of two limonene isomers. Field emission-scanning electron microscope (FE-SEM) unveiled that all three compounds used in this study cause severe ultrastructural morphological changes in Xoo cells, showing shrinking, shriveling, and holes on their surface. Moreover, quantitative real-time PCR revealed that carveol and D-limonene treatment significantly down-regulated the expression levels of genes involved in virulence and biofilm formation of Xoo, but not with L-limonene. Together, we suggest that limonenes and carveol will be the candidates of interest in the development of biological pesticides.

In this study, we investigated the effects of gamma irradiation on the antioxidant activity and metabolite profiles of Euphorbia maculata calli (PC3012). Gamma irradiation at various doses (0, 0.05, 0.5, and 10 kGy) significantly enhanced the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS⁺) radical scavenging activities of the callus extracts of PC3012 in a dose-dependent manner. High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) analyses revealed that irradiation increased the lysophospholipid content, although no new antioxidant compounds were formed. Furthermore, a PLS-DA analysis revealed evident metabolic differences between non-irradiated and irradiated samples, which were further verified by statistical validation. These findings suggest that gamma irradiation induces specific biochemical modifications that enhance the bioactive properties of PC3012 calli. This technology exhibits potential for utilization in the natural product and food sectors, particularly in the development of functional foods and nutraceuticals with improved health benefits.

This study examined differential regulation of volatile terpenoids produced by rosemary (Rosmarinus officinalis L.) leaves in response to ionizing irradiation. Headspace volatile collection from rosemary leaves by solid-phase microextraction-gas chromatography-mass spectrometry analyses revealed that rosemary leaves biologically synthesized 26 species of volatile terpenoids and their relative ratio were changed after the exposure to γ-irradiation. Relative ratio of oxygenated monoterpenes such as borneol, (-)-verbenone, lemonol, and bornyl acetate (except for 1,8-cineol, linalool, chrysanthenone, camphor, and (+)-verbenone) and sesquiterpene hydrocarbons (i.e., caryophyllene-type) increased when the rosemary leaves were exposed to γ-ray in a dose-dependent manner. On the contrary, the change of the ratio of oxygenated sesquiterpenes such as caryophyllene oxide was negligible, and that of monoterpene hydrocarbons like α-pinene, camphene, myrcene, and limonene was down-regulated. These results indicate that γ-irradiation can modify chemical structures of volatile terpenes, and can, thus, be applied to reconstitute chemical constituents in aromatic plants.