Temperature-Directed Reprogramming of Volatile and Semi-Volatile Metabolism in Ginkgo biloba Microclones Under Cold and Heat Stress

Temperature is a major determinant of plant metabolic plasticity, yet its role in directing volatile and semi-volatile specialized metabolism in Ginkgo biloba remains poorly understood. In this study, we investigated how contrasting low- and high-temperature treatments reshape secondary metabolite contents in G. biloba microclones cultivated in vitro. Plants were exposed to cold (+3 °C) and heat (+30 °C) conditions, and their responses were analyzed using GC–MS profiling, anatomical measurements, chlorophyll fluorescence, and multivariate statistics. Cold treatment selectively increased the abundances of monoterpenes (13.22%) and sesquiterpenes (13.83%), with the strongest accumulation of caryophyllene, eucalyptol, and (1S)-camphor. In contrast, heat treatment reduced ester content to 3.73% and strongly enriched oxy-sesquiterpenes (46.50%) and lactone/ketone/spiroketone (29.54%) contents. The enhanced accumulation of isocalamendiol, isoshyobunone, cyclohexanone derivative, dehydroxy-isocalamendiol, and (+)-2-bornanone was observed under heat. According to the multivariate analysis, control plants were associated with traits reflecting optimal physiological performance, including greater parenchyma, phloem, and xylem thickness, larger vascular bundles, longer stomata, and higher NPQ, qN, Y(NPQ), and Fv/Fm. Cold-treated plants showed thicker epidermis and sclerenchyma, higher stomatal density and width, elevated Y(NO), and an enrichment of esters and terpenoids, whereas heat-treated plants were characterized by thicker adaxial and abaxial epidermis, increased mesophyll thickness, and higher levels of oxygenated metabolites. These findings expand current knowledge beyond terpene trilactones and flavonoids and identify Ginkgo microclones as a useful in vitro model for temperature-guided metabolic reprogramming and targeted metabolite enrichment.