Protein phosphorylation is known to play crucial roles in plant tolerance to individual stresses, but how protein phosphorylation is associated with cross-stress tolerance, particularly drought priming-enhanced heat tolerance is largely unknown. The objectives of the present study were to identify phosphorylated proteins and phosphorylation sites that were responsive to drought priming and to determine whether drought priming-enhanced heat tolerance in temperate grass species involves changes in protein phosphorylation. Comparative analysis of phosphoproteomic profiles was performed on leaves of tall fescue (Festuca arundinacea) exposed to heat stress (38/33 °C, day/night) with or without drought priming. A total of 569 differentially regulated phosphoproteins (DRPs) with 1098 phosphorylation sites were identified in response to drought priming or heat stress individually or sequentially. Most DRPs were nuclear-localized and cytosolic proteins. Motif analysis detected [GS], [DSD], and [S..E] as major phosphorylation sites in casein kinase-II and mitogen-activated protein kinases regulated by drought priming and heat stress. Functional annotation and gene ontology analysis demonstrated that DRPs in response to drought priming and in drought-primed plants subsequently exposed to heat stress were mostly enriched in four major biological processes, including RNA splicing, transcription control, stress protection/defense, and stress perception/signaling. These results suggest the involvement of post-translational regulation of the aforementioned biological processes and signaling pathways in drought priming memory and cross-tolerance with heat stress in a temperate grass species.

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