Cotton GhABF3-GhAO1 module regulates the cold tolerance via influencing the ascorbic acid metabolism and flavonoid biosynthesis

ABA-responsive element-binding factors (ABFs), as a crucial subfamily within the basic leucine zipper (bZIP) transcription factor family, exert a significant regulatory function in the process of plant responses to stress. However, the genome-wide characteristics and low-temperature response mechanisms of ABFs in cotton remain poorly understood. Herein, through genome-wide analysis across ten Gossypium species, we systematically identified 138 ABF transcription factors characterized by conserved bZIP domains. Evolutionary analysis demonstrated that segmental duplication events, coupled with purifying selection, contributed to family expansion. Cis -element profiling of promoters suggested their involvement in hormonal signaling and abiotic stress responses. Expression analysis displayed that GhABF3 expression level was significantly up-regulated under cold stress. Genetic functional analysis confirmed that GhABF3 -overexpression in A. thaliana enhanced the cold tolerance and virus-induced gene silencing (VIGS)-mediated GhABF3- silencing in cotton compromised cold resistance. Comparative transcriptomic analysis of VIGS-GhABF3 cotton plants indicated that the pathways of hormone signaling, ascorbic acid metabolism, and flavonoid biosynthesis are the significantly enriched pathways by KEGG analysis. Integrated DAP-seq and RNA-seq analyses identified GhAO1 as a direct target of GhABF3 , which was further validated by Y1H and dual-LUC assays. The contents of AsA and flavonoid were significantly reduced in both VIGS-GhABF3 and VIGS-GhAO1 cotton plants. This research conducted a systematic analysis of the evolutionary traits of the cotton ABF family, clarified the function and molecular mechanism underlying GhABF3 -mediated enhancement of low-temperature adaptability, and thus laid a robust basis for further exploration of multi-level regulatory networks and the breeding of cold-tolerant cotton varieties.