SAL1-PAP retrograde signaling orchestrates photosynthetic and extracellular reactive oxygen species for stress responses

Cellular responses to abiotic stress involve multiple signals such as reactive oxygen species (ROS), Ca²⁺, abscisic acid (ABA), and chloroplast-to-nucleus retrograde signals such as 3′-phosphoadenosine 5′-phosphate (PAP). The mechanism(s) by which these messengers intersect for cell regulation remain enigmatic, as do the roles of retrograde signals in specialized cells. Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate chloroplast and plasma membrane ROS production. Contrary to its role in upregulating processes leading to quenching of ROS in foliar tissue, we show that in guard cells, PAP induces chloroplast ROS accumulation via photosynthetic electron transport and apoplast ROS via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins. Both subcellular ROS sources are necessary for stress hormone ABA-mediated stomatal closure, as well as PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD instead of RBOHF. Three calcium-dependent protein kinases (CPKs) transcriptionally induced by PAP, namely CPK13, CPK32, and CPK34, concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two SLAC1 serine (S) residues, including S120, which is also targeted by the ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify chloroplast retrograde signals as critical nodes in cellular stress response networks of guard cells.