Trans-2-hexenal priming supports HSFA2-independent thermotolerance in Arabidopsis thaliana

HSFA2 is a key regulator of acquired thermotolerance after mild-heat priming, but whether volatile-triggered priming depends on this pathway remains unclear. Here, we examined thermotolerance induced by the reactive green leaf volatile trans-2-hexenal (T2H) in Arabidopsis thaliana Col-0 and an hsfa2 T-DNA insertion mutant. Seedlings were primed with mild heat or a 30-min T2H pulse, recovered for 2 days, and challenged with lethal heat. T2H pretreatment improved survival in both genotypes. Survival analysis and image-based quantification of projected green area showed that T2H-supported recovery was retained in hsfa2, whereas recovery after mild-heat priming was reduced. Total chlorophyll content showed the same genotype-dependent pattern: it was lower in hsfa2 than in Col-0 after mild-heat priming but did not differ between genotypes after T2H. Early reverse-transcription quantitative PCR (RT-qPCR) analysis showed that transcript levels of HSFA1A, HSFA1B, HSFA1D, HSFA1E, and HSP101 did not correlate with the observed survival response. In a separate recovery-period analysis, HSP101 transcript abundance in hsfa2 was significantly lower than in Col-0 after both priming treatments, with the larger reduction after T2H despite strong mutant protection. RNA sequencing (RNA-seq) revealed distinct transcriptional responses between heat and volatile priming: mild heat induced broad transcriptome remodeling, whereas T2H induced a more selective stress-associated response enriched in detoxification- and redox-associated, immune-associated, and hypoxia-like transcript categories. In hsfa2, T2H was also associated with increased transcript abundance of WRKY- and MAPK-associated stress-signaling genes relative to heat priming. These findings suggest that reactive volatile priming may support thermotolerance through HSFA2-independent stress-signaling pathways.