VaTPS9 from Vitis amurensis Encodes a Trehalose-6-Phosphate Synthase Correlated with Cold Tolerance

<i>Vitis amurensis</i> is a cold-hardy wild grape species and represents valuable germplasm for breeding cold-tolerant grapevines. In this study, we identified a highly expressed gene (<i>VaTPS9</i>) in one-year-old shoots of <i>V. amurensis</i> 'Shuangfeng' during overwintering, but its biological function remained unclear. Temporal and spatial expression analyses revealed distinct expression patterns of <i>VaTPS9</i> among different tissues from June to November, with the highest transcript abundance detected in one-year-old shoots in November. Gene cloning and sequence alignment showed that <i>VaTPS9</i> encoded a type II trehalose-6-phosphate synthase (TPS) and was designated as <i>VaTPS9</i>. Functional analyses demonstrated that overexpression of <i>VaTPS9</i> enhanced cold tolerance in yeast, <i>Arabidopsis thaliana</i>, and <i>V. amurensis</i> callus tissues. Conversely, virus-induced gene silencing (VIGS) of <i>VaTPS9</i> in grapevine plantlets markedly increased cold sensitivity under low-temperature stress. These reciprocal gain- and loss-of-function phenotypes indicate that <i>VaTPS9</i> positively regulates cold tolerance, likely by modulating trehalose metabolism and associated physiological responses, including reactive oxygen species (ROS) homeostasis. Collectively, our findings provide new insights into the molecular basis of cold adaptation in wild grape species and highlight <i>VaTPS9</i> as a promising candidate gene for improving cold tolerance in cultivated grapevine.