FaCPK19-FaTT1 axis decodes heat-induced Ca2+ signals and enhances thermotolerance in tall fescue.

Ca2+ signals play a crucial role in heat stress (HS) resistance. However, how Ca2+ signals are decoded and regulate the downstream targets of HS signaling remains unclear in plants. Cool-season grasses are sensitive to high-temperature conditions and should evolve distinctive heat stress response mechanisms. Here, we identified the calcium-dependent protein kinase 19 (FaCPK19), which decoded the unique Ca2+ signal triggered by HS in cool-season grass tall fescue (Festuca arundinacea Schreb.). The FaCPK19 was activated upon HS and positively regulated thermotolerance in tall fescue. FaCPK19 interacted with the thermo-tolerance 1 (FaTT1), which encodes an α2 subunit of the 26S proteasome, and phosphorylated its Thr172 (T172) residue. Under HS, overexpressing FaTT1T172A (FaTT1T172A-OE, phospho-deficient) grasses resulted in significantly lower 20S and 26S proteasome activity and reduced expression of downstream HS-responsive genes compared to gain-function transgenic grasses (FaTT1-OE). The Facpk19 mutant was weakened in 20S and 26S proteasome activity under HS. Loss of T172 phosphorylation in FaTT1T172A-OE showed a weakened heat-tolerance phenotype than FaTT1-OE. T172 residue at TT1 is evolutionarily conserved from plants to animals, indicating it could be widely used for thermotolerance breeding in grasses and crops. Our findings reveal a Ca2+-FaCPK19-FaTT1 axis by which an HS-induced Ca2+ signal is decoded with enhanced thermotolerance in tall fescue.