High temperatures are a major threat to plant growth and development, leading to yield losses in crops. Calcium-dependent protein kinases (CPKs) act as critical components of Ca2+ sensing in plants that transduce rapid stress-induced responses to multiple environmental stimuli. However, the role of CPKs in plant thermotolerance and their mechanisms of action remain poorly understood. To address this issue, tomato (Solanum lycopersicum) cpk28 mutants were generated using a CRISPR-Cas9 gene-editing approach. The responses of mutant and wild-type plants to normal (25°C) and high temperatures (45°C) were documented. Thermotolerance was significantly decreased in the cpk28 mutants, which showed increased heat stress-induced accumulation of reactive oxygen species (ROS) and levels of protein oxidation, together with decreased activities of ascorbate peroxidase (APX) and other antioxidant enzymes. The redox status of ascorbate and glutathione were also modified. Using a yeast two-hybrid library screen and protein interaction assays, we provide evidence that CPK28 directly interacts with cytosolic APX2. Mutations in APX2 rendered plants more sensitive to high temperatures, whereas the addition of exogenous reduced ascorbate (AsA) rescued the thermotolerance phenotype of the cpk28 mutants. Moreover, protein phosphorylation analysis demonstrated that CPK28 phosphorylates the APX2 protein at Thr-59 and Thr-164. This process is suggested to be responsive to Ca2+ stimuli and may be required for CPK28-mediated thermotolerance. Taken together, these results demonstrate that CPK28 targets APX2, thus improving thermotolerance. This study suggests that CPK28 is an attractive target for the development of improved crop cultivars that are better adapted to heat stress in a changing climate.
