Stress memory triggered by combinatorial thermos-drought priming enhances cross-tolerance of Pinus massoniana seedlings: physio-biochemical evidence from multi-stress conditioning

The intensification of climate change has rendered high-temperature and drought stress critical constraints on forest ecosystems. Pinus massoniana Lamb., an ecologically and economically significant conifer species renowned for its abiotic stress resilience, presents an ideal model for investigating stress memory formation in woody perennials. This study establishes a novel priming protocol involving cyclical stress exposure to systematically examine physiological memory formation and subsequent stress tolerance enhancement. Three-month-old seedlings underwent three distinct priming regiments: thermos priming (35/30℃, 14/10 photoperiod), drought priming (~ 25% field capacity), and combinatorial stress (thermos-drought), followed by a 15-day recovery phase before imposing identical combinatorial stress. Comprehensive analyses of growth dynamics, oxidative stress biomarkers, antioxidant systems, and osmoregulatory compounds were conducted through a multi-methodological approach encompassing morphometric measurements, spectrophotometric assays, and enzymatic activity quantification. Principal component analysis and membership function analysis were employed to evaluate the impact of different adversity training treatments on the tolerance of seedlings to combined stress, thereby clarifying the actual effectiveness of adversity training in enhancing the stress resistance. Results indicated that, compared with non-primed controls, those subjected to adversity priming exhibited a reduced inhibitory effect on stem elongation and root collar diameter growth under subsequent stress, along with decrease in lipid peroxidation (malondialdehyde content), reduced relative cell conductance, and enhanced antioxidant capacity. Notably, combinational priming induced synergistic osmoregulatory adaptation, elevating proline accumulation while reducing soluble sugar consumption compared to single-stress priming, suggested the most significant improvements in stress tolerance. The comprehensive evaluation index revealed a stress resilience hierarchy: combinational priming > thermos priming > drought priming > non-primed controls. This study provides the first empirical evidence of stress memory formation in P. massoniana through cross-tolerance mechanisms, demonstrating that cyclical sublethal stress exposure enhances subsequent stress tolerance. The superior efficacy of combinational priming highlights the necessity of multi-stress experimental designs to accurately simulate natural stress regimes. These findings advance our understanding of ecological memory in perennial woody plants and establish practical protocols for developing climate-resilient forestry through targeted stress hardening.