Abstract
Increasing global temperatures pose a growing risk to the growth, productivity, and metabolite stability of Cannabis sativa L., highlighting the need to understand its heat stress responses. However, the temporal dynamics of its defense mechanisms and their direct impact during prolonged heat exposure remain largely unknown. In this study, a cannabidiol-rich hemp variety (C. sativa cv. Pink Pepper) was exposed to 45 °C for 40, 80, and 160 h (HS40, HS80, and HS160) to characterize physiological responses and end-point molecular responses to heat stress. Reactive oxygen species accumulation and membrane damage were assessed by quantifying hydrogen peroxide (H2O2) and malondialdehyde (MDA), which increased sharply by HS40 to 7.79- and 7.00-fold relative to the control, respectively, and remained similarly elevated through HS160. Antioxidant capacity showed increased superoxide dismutase (SOD) activity from HS40. From HS80 onward, catalase (CAT) activity was significantly but modestly reduced, whereas non-enzymatic antioxidant defenses, including total phenolics and total flavonoids, increased 9.89-fold and 8.79-fold, respectively. The osmoprotectant proline and the osmotic-regulating pool of total soluble carbohydrates increased 5.04-fold and 4.25-fold, indicating a delayed protective response. In contrast, the major cannabinoids, total Δ9-tetrahydrocannabinol and total cannabidiol declined from HS80, consistent with the downregulation of cannabinoid biosynthesis genes. Transcriptome analysis at HS160, representing the longest heat stress exposure in this study, revealed a broad transcriptional response strategy of C. sativa. Genes associated with heat shock proteins, protein folding, ribosome biogenesis, and protein processing were strongly upregulated, whereas pathways related to photosynthesis, carbohydrate metabolism, and cannabinoid biosynthesis were downregulated. This study provides a detailed perspective on the distinct temporal patterns of oxidative, antioxidant, and metabolic responses in C. sativa under heat stress, addressing a significant gap in the current understanding of its adaptation mechanisms. Collectively, these findings elucidate key biochemical and transcriptomic mechanisms underlying heat adaptation in C. sativa and provide a foundation for developing climate-resilient cultivars.