A comprehensive proteomics and phosphorylation analysis reveals the negative role of VaVIP1 in cold tolerance in grapevine.

Grapevine is an economically important fruit crop worldwide and is susceptible to low temperature stress. Protein post-translational modifications play a crucial role in grapevine cold tolerance. Here, we performed integrated proteomic and phosphoproteomic analyses of Vitis amurensis, identifying widespread phosphorylation alterations across 4797 proteins and 10,104 phosphorylation sites distributed in 56 motifs. Cold treatment induced 165 differential phosphorylation proteins and 635 differential phosphorylation sites. Functional enrichment indicates that these proteins are predominantly involved in metabolic and signaling pathways, such as phenylpropanoid biosynthesis and MAPK cascades. Moreover, the bZIP transcription factor VaVIP1 was differentially phosphorylated in response to cold treatment. VaVIP1 harbours the canonical conserved BRLZ domain. It is localized to both the plasma membrane and nucleus and possesses transcriptional self-activation activity. Overexpression of VaVIP1 exacerbated oxidative injury and membrane damage, attenuated osmotic adjustment, and ultimately reduced grapevine cold tolerance. It suppressed the cold-triggered activation of core CBF pathway genes, revealing a potential molecular mechanism for its function. Collectively, our study not only expands the phosphoproteomic resource for plants but also identifies VaVIP1 as a novel negative regulator of cold tolerance, providing a valuable theoretical foundation for the genetic improvement of low-temperatures tolerance in grapevine.