Integrated physiological, transcriptomic, and metabolomic analysis of exogenous trehalose regulating cold tolerance in grape callus

Abstract

Vitis vinifera is a fruit tree species of great economic value worldwide. Nevertheless, chilling injury induced by cold stress restricts its yield and hinders the development of the grape industry. Based on previous findings of our research group that exogenous trehalose enhances abiotic stress tolerance in grape callus, this study adopted physiological, transcriptomic and metabolomic approaches to explore the potential regulatory relationships associated with exogenous trehalose. In this study, trehalose treatment alleviated chilling injury under 12 °C cold stress and increased the fresh weight of Vitis vinifera ‘Thompson Seedless’ callus. Meanwhile, trehalose application significantly elevated endogenous trehalose and soluble sugar content in grape callus, accompanied by increased activities of POD, SOD, and CAT, as well as reduced levels of MDA and O₂•⁻. KEGG enrichment analysis of transcriptomic data revealed that DEGs were mainly enriched in pathways related to biosynthesis of secondary metabolites, plant hormone signal transduction, starch and sucrose metabolism, and the MAPK signaling pathway in plants. Key DEGs included BAMS, STS, IAA, RLKs, and BGLU, while differentially expressed transcription factors were predominantly distributed in the AP2/ERF-ERF and MYB families. WGCNA identified the brown and turquoise modules as modules putatively correlated with physiological traits in grape callus, from which candidate genes potentially involved in trehalose-mediated cold response, including VQ22, RGLG2, PPR21, CML16, and SPL6, were screened. Combined transcriptomic and metabolomic analysis showed that DEGs and DAMs were jointly notably altered in the metabolic pathways of flavonoids, benzenes and their substituted derivatives, alkanolamines, terpenoids, and alkaloids. Moreover, all DAM-interacting genes identified were DEGs from the transcriptome. RT-qPCR analysis confirmed that the expression patterns of the selected cold-responsive genes were reliable and consistent with the transcriptomic results. Using integrated physiological, transcriptomic and metabolomic approaches, we identified candidate modules and genes related to cold tolerance in grape callus, as well as physiological responses and potential pathways underlying trehalose-mediated cold tolerance. We also characterized candidate genes involved in this regulatory process, providing a theoretical reference for exploring cold tolerance mechanisms in grape callus.