Objectives Zinc oxide nanoparticles (ZnO NPs) have been shown to enhance crop cold tolerance. In this study, we investigated the metabolic pathways by which ZnO NPs improve cold tolerance and developed a ZnO NPs-containing antifreeze agent, aiming to provide theoretical and technical support for the safe overwintering of mangrove forests at the northern limit of their distribution in China.

Methods One-year-old seedlings of the mangrove species Kandelia obovata were planted in pots. The seedlings were first sprayed with water or 200 mg/L ZnO NPs under normal temperature conditions (25^oC/20^oC day/night). The seedlings in each treatment group were divided into two subgroups: one subgroup was maintained under normal-temperature conditions, whereas the other was subjected to low-temperature stress (8^oC /-3^oC day/night) for 3 days, after which chlorophyll fluorescence parameters were measured. Leaves were then harvested for transcriptome sequencing to identify key pathways associated with cold tolerance in K. obovata. Differentially expressed genes (DEGs) were identified using DESeq2 with thresholds of |log₂FC| ≥ 1 and P-value < 0.05. Subsequently, antifreeze-agent experiments were conducted using potted seedlings. The seedlings were divided into three groups: one sprayed with water, one sprayed with conventional antifreeze agent (Antifreeze agent), and the third sprayed with a ZnO NP-containing antifreeze agent of the same formulation. These seedlings were then subjected to cold stress for 4 days, after which photosynthetic parameters and malondialdehyde (MDA) content were measured in a subset of seedlings. The remaining seedlings were transferred to normal-temperature conditions for 7 days, and cold injury symptoms were evaluated.

Results Compared with water-sprayed seedlings maintained under normal temperature conditions, seedlings subjected to low-temperature stress exhibited significant reductions in the actual photochemical quantum yield of PSII (ΦPSII), maximum photochemical efficiency of PSII (Fv/Fm), photochemical quenching coefficient (qP), and maximum efficiency of PSII in the light-adapted state (Fv’/Fm’). Under low-temperature stress, the ΦPSII, Fv/Fm, qP, and Fv’/Fm’ values of ZnO NP-treated seedlings were 2.26, 2.33, 2.40, and 1.71 times those of the water-sprayed low-temperature-stressed seedlings, respectively. A total of 7,840 DEGs were identified between the water-sprayed low-temperature-stressed and water-sprayed normal-temperature groups, including 4,399 up-regulated and 3,441 down-regulated genes. Similarly, a total of 7,856 DEGs were identified between the water-sprayed low-temperature-stressed group and the ZnO NP-sprayed low-temperature-stressed group, including 3,462 up-regulated and 4,394 down-regulated genes. Across all treatments, a total of 36,043 DEGs were identified, and these genes were primarily enriched in photosynthesis, ribosomal-related processes, MAPK signaling, and phenylpropanoid metabolism pathways. Compared with the antifreeze agent treatment group, the ZnO NP-containing antifreeze-agent group showed significant reductions in relative electrolyte conductivity, MDA content, and freezing injury index. This group also exhibited significantly increased net photosynthetic rate, stomatal conductance, transpiration rate, CO₂ saturation point, carboxylation efficiency, and maximum photosynthetic capacity, while showing a decreased CO₂ compensation point, thereby enhancing photosynthetic carbon assimilation and alleviating cold-induced injury. Furthermore, the antifreeze agent alone significantly increased net photosynthetic rate and carboxylation efficiency, reduced MDA content under cold stress, and provided partial protection against cold injury.

Conclusions ZnO NPs stabilized cell membranes and maintained photochemical efficiency and carbon fixation capacity by regulating the expression of genes involved in photosynthesis, ribosomal-related processes, MAPK signaling, and phenylpropanoid metabolism. The ZnO NP-containing antifreeze agent can further enhance the photosynthetic gas exchange and CO₂ fixation capacity of Kandelia obovata seedling leaves under low-temperature stress compared with ordinary antifreeze, and significantly reduce the freezing injury index of Kandelia obovata, consistent with the cold-tolerance mechanisms revealed by transcriptomic analysis of ZnO NP-pretreated seedlings.