The CCCH zinc finger protein PtrTZF10 enhances cold tolerance in association with β-amylase-mediated starch degradation and sugar accumulation in Poncirus trifoliata

Abstract

Cold stress is a major environmental constraint limiting the geographical distribution and productivity of citrus. Poncirus trifoliata, a close cold-hardy relative of citrus, serves as an invaluable reservoir of stress-resistance genes. Here, we functionally characterized PtrTZF10, a cold-responsive CCCH tandem zinc finger gene from P. trifoliata, and demonstrated its positive role in regulating cold tolerance and its association with sugar metabolism. In contrast to the transient induction observed in cold-sensitive lemon, PtrTZF10 was rapidly and persistently induced by cold in P. trifoliata. PtrTZF10 localizes to both the nucleus and plasma membrane, binds the tested DNA and RNA substrates in vitro, and possesses transcriptional activation activity. Ectopic expression of PtrTZF10 in tobacco and lemon significantly enhanced cold tolerance, as reflected by improved photosynthetic efficiency, reduced membrane damage, and lower reactive oxygen species (ROS) accumulation. Conversely, silencing PtrTZF10 in P. trifoliata compromised its freezing tolerance. Transcriptomic profiling under normal growth conditions revealed basal differences in the expression of carbohydrate metabolism-related genes in PtrTZF10-overexpressing lines. Molecular assays further showed that PtrTZF10 associates with and activates the PtrBMY1 promoter, while PtrTZF10 overexpression and silencing were accompanied by corresponding changes in β-amylase activity, starch degradation, and soluble sugar accumulation. Collectively, these findings identify PtrTZF10 as a positive regulator of cold tolerance and support a PtrTZF10-PtrBMY1 regulatory link associated with starch-to-sugar mobilization during the cold response.