Phosphorylation switch helps grapevines weather freezing

Cold snaps can damage grapevine tissues, weaken photosynthesis, and reduce yield stability, yet the molecular relay that converts a temperature drop into cellular protection has remained unclear. This study identifies a calcium-responsive signaling partnership that strengthens grapevine cold tolerance. The calcium-dependent protein kinase VpCDPK13 directly interacts with and phosphorylates calmodulin-binding transcription activator VpCAMTA3 at three major amino-acid sites. This modification improves reactive oxygen species control, protects cell membranes, and sustains photosystem II performance during freezing stress. By showing how a rapid calcium signal is translated into a protective transcriptional response, the work reveals potential molecular targets for developing grapevines and other perennial fruit crops better able to withstand increasingly erratic winter temperatures. 

Low temperature is a major constraint on grape production because grapevines have a relatively narrow freezing-tolerance range. Cold can reduce bud survival, disrupt vegetative and reproductive development, destabilize membranes, and impair photosynthesis across growing seasons. Plants sense sudden temperature changes partly through transient increases in cellular calcium, which are decoded by calcium-dependent protein kinases (CDPKs). Calmodulin-binding transcription activator 3 (CAMTA3) also regulates cold-responsive genes, but phosphorylation can either strengthen or weaken its activity depending on the plant and kinase involved. In grapevine, the connection between CDPK signaling and CAMTA3-mediated protection was not understood. Because of these challenges, deeper investigation is needed into how calcium-dependent phosphorylation controls grapevine adaptation to cold stress.

Researchers from the State Key Laboratory for Crop Stress Resistance and High-Efficiency Production/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A&F University, and the Shandong Institute of Pomology published (DOI: 10.1093/hr/uhag121) the findings online as an accepted manuscript in Horticulture Research on July 24, 2026. The study shows that cold-induced VpCDPK13 phosphorylates VpCAMTA3, activating a protective pathway in wild grapevine, Vitis pseudoreticulata. Combining genetic, biochemical, imaging, and protein-interaction approaches, the team linked this molecular switch to stronger antioxidant capacity, improved membrane stability, and better preservation of photosynthetic function under low-temperature and freezing treatments.

The researchers first found that the VpCDPK13 gene was rapidly induced in leaves exposed to 4°C, while the VpCDPK13 protein accumulated more strongly in the nucleus, positioning it to relay an early cold signal. Grapevines overexpressing VpCDPK13 remained greener after freezing and showed lower electrolyte leakage and malondialdehyde (MDA), reduced hydrogen peroxide and superoxide accumulation, higher peroxidase (POD) activity, and better maximum quantum efficiency of photosystem II (Fv/Fm). Silencing VpCDPK13 produced the opposite pattern.
A yeast two-hybrid assay, split luciferase complementation, co-immunoprecipitation, and bimolecular fluorescence complementation independently confirmed that VpCDPK13 physically associates with VpCAMTA3. Silencing the VpCAMTA3 gene likewise increased oxidative damage and weakened photosynthetic performance. In vitro kinase assays and liquid chromatography–tandem mass spectrometry identified threonine 588, serine 834, and serine 1049 as the main phosphorylation sites. Under cold conditions, a triple phospho-dead VpCAMTA3 mutant retained only about one-fifth of the phosphorylation seen in the wild-type protein. Grapevine leaves carrying a phospho-mimic version of VpCAMTA3 maintained the highest photosystem II efficiency, accumulated less membrane damage and reactive oxygen species (ROS), and showed stronger POD activity. Together, the experiments establish phosphorylation as a positive switch that amplifies VpCAMTA3-mediated cold protection.

The authors said the VpCDPK13–VpCAMTA3 module offers a mechanistic explanation for how grapevine cells turn an early calcium signal into coordinated protection against freezing injury. They said the most important result is not simply that both proteins support cold tolerance, but that phosphorylation links them in a functional sequence that preserves photosynthesis, limits oxidative stress, and stabilizes membranes. The authors added that this response differs from CAMTA3 regulation reported in Arabidopsis, suggesting that perennial fruit crops may have evolved distinct ways to fine-tune cold signaling.

The signaling module could provide candidate genes and phosphorylation sites for breeding or biotechnology programs aimed at improving cold resilience in grapevine. Selection for favorable VpCDPK13 or VpCAMTA3 variants, targeted editing of regulatory regions, or marker-assisted breeding could eventually help reduce winter injury and stabilize production in cooler or increasingly variable climates. The findings may also guide research in other perennial fruit crops where freezing damage threatens long-term productivity. However, the current evidence comes mainly from transgenic plants, transient leaf assays, and controlled cold treatments. Field testing across cultivars, developmental stages, and repeated winter events will be essential before the pathway can be translated into practical crop-improvement strategies.

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References

DOI

10.1093/hr/uhag121

Original Source URL

https://doi.org/10.1093/hr/uhag121

Funding information

Ningxia Hui Autonomous Region Key Research and Development Program, Grant No. 2023BCF01003. The Isotope Laboratory of Northwest A&F University provided technical support.

About Horticulture Research

Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.