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.

Unveiling orphan receptor-like kinases in plants: novel client discovery using high-confidence library predictions in the Kinase-Client (KiC) assay.

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or "clients", remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1's substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

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.

Purinoceptor P2K1/DORN1 Enhances Plant Resistance Against a Soilborne Fungal Pathogen, Rhizoctonia solani.

The purinoceptor P2K1/DORN1 recognizes extracellular ATP, a damage-associated molecular pattern (DAMP) released upon cellular disruption by wounding and necrosis, which in turn, boost plant innate immunity. P2K1 is known to confer plant resistance to foliar biotrophic, hemi-biotrophic, and necrotrophic pathogens. However, until now, no information was available on its function in defense against root pathogens. In this report, we describe the contribution of P2K1 to resistance in Arabidopsis against Rhizoctonia solani, a broad host range, necrotrophic soilborne fungal pathogen. In pot assays, the Arabidopsis P2K1 overexpression line OxP2K1 showed longer root length and a greater rosette surface area than wild type in the presence of the pathogen. In contrast, the knockout mutant dorn1-3 and the double mutant rbohd/f, defective in two subunits of the respiratory burst complex NADPH oxidase, exhibited significant reductions in shoot and root lengths and rosette surface area compared to wild type when the pathogen was present. Expression of PR1, PDF1.2, and JAZ5 in the roots was reduced in dorn1-3 and rbohd/f and elevated in OxP2K1 relative to wild type, indicating that the salicylate and jasmonate defense signaling pathways functioned in resistance. These results indicated that a DAMP-mediated defense system confers basal resistance against an important root necrotrophic fungal pathogen.

DORN1/P2K1 and purino-calcium signalling in plants: making waves with extracellular ATP.

BACKGROUND AND AIMS: Extracellular ATP governs a range of plant functions, including cell viability, adaptation and cross-kingdom interactions. Key functions of extracellular ATP in leaves and roots may involve an increase in cytosolic free calcium as a second messenger ('calcium signature'). The main aim here was to determine to what extent leaf and root calcium responses require the DORN1/P2K1 extracellular ATP receptor in Arabidopsis thaliana. The second aim was to test whether extracellular ATP can generate a calcium wave in the root. METHODS: Leaf and root responses to extracellular ATP were reviewed for their possible links to calcium signalling and DORN1/P2K1. Leaves and roots of wild type and dorn1 plants were tested for cytosolic calcium increase in response to ATP, using aequorin. The spatial abundance of DORN1/P2K1 in the root was estimated using green fluorescent protein. Wild type roots expressing GCaMP3 were used to determine the spatial variation of cytosolic calcium increase in response to extracellular ATP. KEY RESULTS: Leaf and root ATP-induced calcium signatures differed markedly. The leaf signature was only partially dependent on DORN1/P2K1, while the root signature was fully dependent. The distribution of DORN1/P2K1 in the root supports a key role in the generation of the apical calcium signature. Root apical and sub-apical calcium signatures may operate independently of each other but an apical calcium increase can drive a sub-apical increase, consistent with a calcium wave. CONCLUSION: DORN1 could underpin several calcium-related responses but it may not be the only receptor for extracellular ATP in Arabidopsis. The root has the capacity for a calcium wave, triggered by extracellular ATP at the apex.

Early Extracellular ATP Signaling in Arabidopsis Root Epidermis: A Multi-Conductance Process.

Adenosine 5'-triphosphate (ATP) is an important extracellular signaling agent, operating in growth regulation, stomatal conductance, and wound response. With the first receptor for extracellular ATP now identified in plants (P2K1/DORN1) and a plasma membrane NADPH oxidase revealed as its target, the search continues for the components of the signaling cascades they command. The Arabidopsis root elongation zone epidermal plasma membrane has recently been shown to contain cation transport pathways (channel conductances) that operate downstream of P2K1 and could contribute to extracellular ATP (eATP) signaling. Here, patch clamp electrophysiology has been used to delineate two further conductances from the root elongation zone epidermal plasma membrane that respond to eATP, including one that would permit chloride transport. This perspective addresses how these conductances compare to those previously characterized in roots and how they might operate together to enable early events in eATP signaling, including elevation of cytosolic-free calcium as a second messenger. The role of the reactive oxygen species (ROS) that could arise from eATP's activation of NADPH oxidases is considered in a qualitative model that also considers the regulation of plasma membrane potential by the concerted action of the various cation and anion conductances. The molecular identities of the channel conductances in eATP signaling remain enigmatic but may yet be found in the multigene families of glutamate receptor-like channels, cyclic nucleotide-gated channels, annexins, and aluminum-activated malate transporters.

Transcriptomic perspective on extracellular ATP signaling: a few curious trifles.

Extracellular ATP is perceived by the purinoceptor P2K1, leading to induction of defense response in plants. Previously, we described the transcriptomic response to extracellular ATP in wild-type Arabidopsis seedlings and mutants of classical defense hormone signaling pathways (Jewell et al., 2019, Plant Physiol. 179: 1144-58), in which extracellular ATP was found to induce defense-related genes independently and also along with other defense signaling pathways. In the present study, we provide further analysis and discussion of the data that we neglected to describe in the previous transcriptomics report. Briefly, we describe transcriptomic differences between a P2K1 knockout mutant (dorn1) and wild-type seedlings in the absence of exogenous ATP as well as an analysis of genes more responsive to extracellular ATP in a P2K1 overexpression line. Finally, we describe an exaggerated response to extracellular ATP in the ein2 mutant and suggest testable explanations of this phenomenon.

Molecular Mechanism of Plant Recognition of Extracellular ATP.

Adenosine 5'-triphosphate (ATP), a ubiquitously dispersed biomolecule, is not only a major source of biochemical energy for living cells, but also acts as a critical signaling molecule through inter-cellular communication. Recent studies have clearly shown that extracellular ATP is involved in various physiological processes in plants, including root growth, stomata movement, pollen tube development, gravitropism, and abiotic/biotic stress responses. The first plant purinergic receptor for extracellular ATP, DORN1 (the founding member of the P2K family of purinergic receptors), was identified in Arabidopsis thaliana by a forward genetic screen. DORN1 consists of an extracellular lectin domain, transmembrane domain, and serine/threonine kinase, intracellular domain. The predicted structure of the DORN1 extracellular domain revealed putative key ATP binding residues but an apparent lack of sugar binding. In this chapter, we summarize recent studies on the molecular mechanism of plant recognition of extracellular ATP with specific reference to the role of DORN1.