Abstract

The circadian clock genes in tomato are key regulators of cold stress adaptation. However, the low-temperature regulatory mechanism of the circadian clock geneSlPCL1remains unclear. In this study, we evaluated the role ofSlPCL1in cold tolerance through low-temperature treatment of transgenic plants. Downstream target genes were identified using RNA-seq, RT-qPCR, yeast-one-hybrid (Y1H), dual-luciferase assays, and electrophoretic mobility shift assay (EMSA), while interacting proteins were characterized using yeast-two-hybrid (Y2H), luciferase complementation imaging (LCI), and pull-down assays, thereby elucidating the molecular mechanism underlyingSlPCL1-mediated low-temperature regulation. We identifiedSlPCL1as a nuclear-localized circadian clock gene with transcriptional repressor activity. Overexpression ofSlPCL1resulted in a cold-sensitive phenotype, whereas virus-induced gene silencing (VIGS)-mediated silencing ofSlPCL1enhanced cold tolerance.SlNPF4.6functions as an abscisic acid (ABA) transporter involved in ABA transport. RNA-seq and RT-qPCR identified the ABA transporterSlNPF4.6as a downstream target. Functional assays confirmed that SlPCL1 binds to the MYB element in theSlNPF4.6promoter to repress its expression. Meanwhile, VIGS-mediated silencing ofSlNPF4.6decreased cold tolerance. Furthermore, the expression levels of the ABA receptorSlPYLsin the silenced lines were significantly reduced, confirming the decrease in intracellular ABA content. SlSUMO1, a ubiquitin-like protein, can influence gene transcription through noncovalent interactions. In addition, SlSUMO1 was found to interact with the SlPCL1 protein, attenuating SlPCL1 transcriptional repression activity. Together, these findings establish an SlSUMO1-mediated fine control mechanism of theSlPCL1-SlNPF4.6regulatory module. This integration of circadian clock regulation uncovers new molecular mechanisms of cold tolerance and supports the development of cold-resistant breeding materials.