The C2H2 zinc-finger protein CaZAT11 positively regulates cold tolerance in pepper and physically interacts with CaSTOP1.

Abstract

Low temperature is a major environmental constraint that challenges pepper growth and productivity. Although C2H2 zinc-finger transcription factors have been implicated in plant responses to abiotic stresses, their roles in pepper cold tolerance remain poorly understood. In this study, we identified the C2H2 zinc-finger protein CaZAT11 as a novel interacting partner of the C2H2-type zinc-finger protein CaSTOP1 through yeast two-hybrid library screening. This interaction was subsequently confirmed by directed yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation assays, with the latter showing that the two proteins interact in the nucleus. In addition, subcellular localization analysis showed that CaZAT11 is localized to the nucleus, and its expression was strongly induced by cold stress. Functional analyses demonstrated that silencing CaZAT11 increased cold sensitivity in pepper, whereas its transient overexpression in pepper and constitutive overexpression of CaZAT11 in Arabidopsis and tomato enhanced cold tolerance. The improved tolerance was accompanied by reduced electrolyte leakage, lower accumulation of reactive oxygen species and malondialdehyde, increased activities of antioxidant enzymes, and elevated expression of conserved cold-responsive genes. Together, these results suggest CaZAT11 as a positive regulator of cold tolerance and reveal it as a previously unrecognized CaSTOP1-interacting component of the pepper cold-responsive network. However, the direct transcriptional targets of CaZAT11 and the precise functional significance of its interaction with CaSTOP1 remain to be determined. Our findings broaden the current understanding of the molecular network underlying cold responses in pepper and provide a basis for future studies on the functional significance of the CaSTOP1-CaZAT11 interaction during cold stress.