FERONIA orchestrates P2K1-driven purinergic signaling in plant roots.

Extracellular ATP (eATP) orchestrates vital processes in plants, akin to its role in animals. P2K1 is a crucial receptor mediating eATP effects. Immunoprecipitation tandem mass spectrometry data highlighted FERONIA's significant interaction with P2K1, driving us to explore its role in eATP signaling. Here, we investigated putative P2K1-interactor, FERONIA, which is a versatile receptor kinase pivotal in growth and stress responses. We employed a FERONIA loss-of-function mutant, fer-4, to dissect its effects on eATP signaling. Interestingly, fer-4 showed distinct calcium responses compared to wild type, while eATP-responsive genes were constitutively upregulated in fer-4. Additionally, fer-4 displayed insensitivity to eATP-regulated root growth and reduced cell wall accumulation. Together, these results uncover a role for FERONIA in regulating eATP signaling. Overall, our study deepens our understanding of eATP signaling, revealing the intricate interplay between P2K1 and FERONIA impacting the interface between growth and defense.

CPK28 is a modulator of purinergic signaling in plant growth and defense.

Extracellular ATP (eATP) is a key signaling molecule that plays a pivotal role in plant growth and defense responses. The receptor P2K1 is responsible for perceiving eATP and initiating its signaling cascade. However, the signal transduction mechanisms downstream of P2K1 activation remain incompletely understood. We conducted a comprehensive analysis of the P2K1 interactome using co-immunoprecipitation-coupled tandem mass spectrometry, leading to the identification of 121 candidate proteins interacting with P2K1. In silico analysis narrowed down the candidates to 47 proteins, including Ca2+-binding proteins, ion transport-related proteins, and receptor kinases. To investigate their involvement in eATP signaling, we employed a screening strategy based on changes in gene expression in response to eATP in mutants of the identified interactors. This screening revealed several Ca2+-dependent protein kinases (CPKs) that significantly affected the expression of eATP-responsive genes, suggesting their potential roles in eATP signaling. Notably, CPK28 and CPK6 showed physical interactions with P2K1 both in yeast and plant systems. Calcium influx and gene expression studies demonstrated that CPK28 perturbed eATP-induced Ca2+ mobilization and some early transcriptional responses. Overexpression of CPK28 resulted in an antagonistic physiological response to P2K1-mediated eATP signaling during both plant growth and defense responses to the necrotrophic pathogen Botrytis cinerea. Our findings highlight CPK28, among other CPKs, as a modulator of P2K1-mediated eATP signaling, providing valuable insights into the coordination of eATP signaling in plant growth and immunity.

The intrinsically disordered C-terminus of purinoceptor P2K1 fine-tunes plant responses to extracellular ATP.

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.

A histochemical reporter system to study extracellular ATP response in plants.

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 ß-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.

Extracellular ATP Shapes a Defense-Related Transcriptome Both Independently and along with Other Defense Signaling Pathways.

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 ATP-induced 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 calmodulin-binding transcription activators are nuclear transducers of P2K1-mediated extracellular ATP signaling.