The SlWRKY54‐SlZF61 Module Synergistically Regulates SLDLO2 to Enhance Cold Tolerance in Tomato Under Low‐Temperature Stress

ABSTRACT Low‐temperature stress affected plant growth, while WRKY transcription factors could regulate downstream genes to enhance the resistance of plants. In this study, SlWRY54 overexpression enhances seedling low‐temperature tolerance by promoting chlorophyll synthesis and photosynthetic efficiency, while knockout exacerbates membrane damage and reduces photosynthetic capacity, increasing sensitivity to low temperatures. Transcriptome analysis indicated induction of photosynthesis‐related pathway genes in slwrky54 plants under low‐temperature stress. Y1H, EMSA, and Dual‐LUC experiments confirmed that SlWRKY54 specifically binds to and activates the SlDLO2 promoter; silencing SlDLO2 attenuated the low‐temperature tolerance conferred by SlWRKY54 overexpression. Additionally, SlZF61 interacts with SlWRKY54, enhancing its regulatory effect on SlDLO2 . Overexpression of SlZF61 promoted chlorophyll synthesis and enhanced photosynthetic efficiency to improve low temperature tolerance. In contrast, knockout of SlZF61 reduced photosynthetic capacity, which increased sensitivity to low temperatures. In summary, SlWRKY54 binds to the SlDLO2 promoter, and SlZF61 interacts with SlWRKY54 to form a protein complex, enhancing this binding. They synergistically activate SlDLO2 transcription to improve seedling low‐temperature tolerance by promoting chlorophyll synthesis and photosynthetic efficiency. This coordinated regulatory mechanism provides a new theoretical basis and practical insights for enhancing tomato low‐temperature tolerance and ensuring stable production under low‐temperature stress conditions.