Cold-induced SlPAL3 confers cold tolerance in tomato through interacting with putative transcription factor SlMYB58.

Abstract

Cold stress severely restricts tomato growth, yield, and geographical distribution. However, the molecular mechanisms governing phenylpropanoid-mediated cold tolerance remain incompletely understood, especially the upstream regulators of specific phenylalanine ammonia-lyase (PAL) isoforms. In this study, we functionally characterized SlPAL3 and identified its putative upstream transcriptional activator SlMYB58 in tomato. Using phylogenetic analysis, subcellular localization, and the generation of CRISPR-Cas9 knockout (CR) and overexpression (OE) transgenic lines, we demonstrated that cytoplasm-localized SlPAL3 positively regulates cold tolerance. Under 4 °C treatment, OE-SlPAL3 plants exhibited enhanced phenotypes, including significantly higher peroxidase activity (2.2~3.1-fold), proline accumulation (2.1~2.6-fold), and lignin content (1.6~2.0-fold) compared to wild-type plants. In contrast, CR lines showed severe wilting and compromised physiological responses. Notably, SlPAL3 overexpression substantially reduced the accumulation of superoxide anion and hydrogen peroxide, as visualized by NBT and DAB staining, and upregulated cold-responsive markers, including SlSGR, SlABI1, and SlSOD. Furthermore, yeast one-hybrid and dual-luciferase assays revealed that putative SlMYB58, a conserved R2R3-MYB transcription factor, directly binds to and activates the SlPAL3 promoter. Collectively, these findings establish the SlMYB58-SlPAL3 module as a novel regulatory axis that integrates transcriptional activation with phenylpropanoid metabolism under cold stress. This study provides a mechanistic framework for understanding cold-induced metabolic reprogramming in tomato and identifies a strategic genetic target for breeding cold-tolerant tomato cultivars.