ORCID IDs: 0000-0002-1265-0962 (J.B.J.); 0000-0002-0558-1575 (J.M.S.); 0000-0002-8743-966X (D.R.G.); 0000-0001-5045-560X (K.T.).ATP is not only an essential metabolite of cellular biochemistry but also acts as a signal in the extracellular milieu. In plants, extracellular ATP is monitored by the purinergic receptor P2K1. Recent studies have revealed that extracellular ATP acts as a damage-associated molecular pattern in plants, and its signaling through P2K1 is important for mounting an effective defense response against various pathogenic microorganisms. Biotrophic and necrotrophic pathogens attack plants using different strategies, to which plants respond accordingly with salicylate-based or jasmonate/ethylene-based defensive signaling, respectively. Interestingly, defense mediated by P2K1 is effective against pathogens of both lifestyles, raising the question of the level of interplay between extracellular ATP signaling and that of jasmonate, ethylene, and salicylate. To address this issue, we analyzed ATP-induced transcriptomes in wild-type Arabidopsis (Arabidopsis thaliana) seedlings and mutant seedlings defective in essential components in the signaling pathways of jasmonate, ethylene, and salicylate (classic defense hormones) as well as a mutant and an overexpression line of the P2K1 receptor. We found that P2K1 function is crucial for faithful ATPinduced transcriptional changes and that a subset of genes is more responsive in the P2K1 overexpression line. We also found that more than half of the ATP-responsive genes required signaling by one or more of the pathways for the classical defense hormones, with the jasmonate-based signaling being more critical than others. By contrast, the other ATP-responsive genes were unaffected by deficiencies in signaling for any of the classical defense hormones. These ATP-responsive genes were highly enriched for defense-related Gene Ontology terms. We further tested the ATP-induced genes in knockout mutants of transcription factors, demonstrating that MYCs acting downstream of the jasmonate receptor complex and calmodulinbinding transcription activators are nuclear transducers of P2K1-mediated extracellular ATP signaling.

When cells experience acute mechanical distress, they release ATP from their cellular compartment into the surrounding microenvironment. This extracellular ATP (eATP) can then act as a danger signal—signaling cellular damage. In plants, cells adjacent to damage detect rising eATP concentrations through the cell-surface receptor kinase, P2K1. Following eATP perception, P2K1 initiates a signaling cascade mobilizing plant defense. Recent transcriptome analysis revealed a profile of eATP-induced genes sharing pathogen- and wound-response hallmarks—consistent with a working model for eATP as a defense-mobilizing danger signal. To build on the transcriptional footprint and broaden our understanding of dynamic eATP signaling responses in plants, we aimed to i) generate a visual toolkit for eATP-inducible marker genes using a β-glucuronidase (GUS) reporter system and ii) evaluate the spatiotemporal response of these genes to eATP in plant tissues. Here, we demonstrate that the promoter activities of five genes, ATPR1, ATPR2, TAT3, WRKY46, and CNGC19, were highly sensitive to eATP in the primary root meristem and elongation zones with maximal responses at 2 h after treatment. These results suggest the primary root tip as a hub to study eATP-signaling activity and provide a proof-of-concept toward using these reporters to further dissect eATP and damage signaling in plants.

P2K1 is a plant‐specific purinoceptor that perceives extracellular ATP (eATP), a signaling molecular implicated in various physiological processes. Interestingly, P2K1 harbors a C‐terminal intrinsically disordered region (IDR). When we overexpressed a truncated P2K1 (P2K1t) lacking the IDR, primary root growth completely ceased in response to eATP. We investigated the functional roles of the IDR in P2K1 using a combination of molecular genetics, calcium imaging, gene expression analysis, and histochemical approaches. We found that the P2K1t variant gave rise to an amplified response to eATP, through accumulation of superoxide, altered cell wall integrity, and ultimate cell death in the primary root tip. Together, these observations underscore the significant involvement of the C‐terminal tail of P2K1 in root growth regulation.