A short trans-2-hexenal pulse primes freezing tolerance and a cold-response transcriptome in Arabidopsis thaliana

Volatile organic compounds (VOCs) can prime plants for enhanced stress tolerance, yet how a brief volatile exposure is converted into sustained cold protection remains unclear. We established a sealed-headspace priming assay in Arabidopsis thaliana in which 7-day-old seedlings were exposed to short-chain reactive volatiles for 30 min, allowed to recover for 2 days, and then challenged by acute freezing (-20 °C). Screening green leaf volatiles revealed marked compound specificity: trans-2-hexenal and 4-hexen-3-one conferred strong protection, increasing post-freeze survival from ∼10-15% in controls to ∼55-65%, whereas several structurally related aldehydes were ineffective. Dose-response analyses uncovered a narrow effective window, with maximal survival at intermediate exposure levels and loss of protection at higher doses, consistent with a trade-off between priming and overexposure in a closed headspace. Although multiple volatiles rapidly induced HSFA2, early HSFA2 activation did not correlate with freezing tolerance across treatments. Transcriptome profiling immediately after a 30-min trans-2-hexenal exposure showed rapid induction of proteostasis and stress-related genes, together with enrichment of genes annotated to response to cold; canonical cold-responsive loci such as COR47 and COR413-PM1 were induced before freezing stress. Extending beyond Arabidopsis, a single trans-2-hexenal pretreatment also mitigated chilling injury in japonica rice, reducing necrosis and increasing green leaf area after cold exposure. Collectively, these results identify trans-2-hexenal as a rapid, dose-sensitive volatile signal that primes cold tolerance through coordinated activation of a stress-response transcriptome enriched in cold-response signatures, consistent with a mechanism that accesses cold-protective outputs without prior cold exposure.