A hierarchical ScMYB11L–ScZAT1–ScDREB1A module defines a derepression pathway for cold-response regulation in sugarcane

• ScZAT1 is a sugarcane C2H2-ZFP and transcriptional repressor of cold tolerance. • ScZAT1 suppresses CBF/DREB signaling by targeting key promoter regions. • ScZAT1 negatively modulates cold-induced redox and ROS homeostasis. • ScMYB11L inhibits ScZAT1, forming a novel “repressor-of-a-repressor” module. • This repression is transcriptional, unlike AtMYB15’s post-translational regulation. Cold stress is a major constraint on sugarcane ( Saccharum spp.) productivity, yet the transcriptional regulatory mechanisms governing its cold adaptation remain poorly characterized. Here, we identify the C2H2-type zinc finger protein ScZAT1 as a transcriptional repressor associated with restrained activation of the CBF pathway during cold stress. Overexpression of ScZAT1 in Arabidopsis increased freezing sensitivity, leading to reduced survival, impaired photosynthetic capacity, elevated reactive oxygen species accumulation, and reduced antioxidant enzyme activities. Mechanistically, ScZAT1 binds directly to the promoters of core cold-responsive genes, including AtCBF1 and its sugarcane DREB/CBF-type homolog ScDREB1A , and represses their transcription. This repression is alleviated by the upstream MYB transcription factor ScMYB11L, which directly targets and represses the ScZAT1 promoter under cold stress, supporting a hierarchical ScMYB11L–ScZAT1–ScDREB1A regulatory module. In transgenic sugarcane with altered ScMYB11L expression under chilling treatment, ScMYB11L overexpression suppressed ScZAT1 and was accompanied by enhanced ScDREB1A induction, whereas RNAi-mediated silencing of ScMYB11L showed the opposite trend. Furthermore, expression profiling of sugarcane cultivars with contrasting cold tolerance revealed that the cold-tolerant FN39 tended to exhibit higher ScMYB11L and ScDREB1A expression with relatively lower ScZAT1 expression compared to the susceptible cultivar XTT22. Our findings define a novel derepression module for cold resilience in sugarcane, highlighting its potential as a target for genetic improvement.