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Herbivorous insects and pathogens cause severe damage to rice tissues, affecting yield and grain quality. Damaged cells trigger downstream defense responses through various signals. Extracellular ATP (eATP), a signaling molecule released during mechanical cell damage, is considered a constitutive damage-associated molecular pattern (DAMP), which is crucial for initiating plant defense responses. Thus, understanding how rice plants cope with DAMPs such as eATP is essential. Here, we found that exogenous ATP affected rice growth and development, cell wall composition, chloroplast development, and cell death. Subsequent global transcriptome analysis revealed that several pathways were involved in the eATP response, including genes related to cell surface receptors, cell wall organization, chlorophyll biosynthesis, heat and temperature stimulation, epigenetic regulation, and reactive oxygen species metabolism. Cell surface receptors, including members of the lectin receptor-like kinases (LecRKs), were found to participate in the eATP response. We further investigated ATP-induced genes in T-DNA activation mutants of OsLecRKs, demonstrating their involvement in eATP signaling in rice. This study confirms a DAMP-mediated transcriptional response in plants and provides novel candidates for advancing resistant rice breeding against insect herbivores and pathogens. 

In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The Kinase Client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2,100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional in vitro and in planta studies, including Integrin-Linked Kinase 4 (ILK4), for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8,000 (8k) peptides based on phosphorylation sites primarily from Arabidopsis thaliana datasets. The 8k peptides are enriched for sites with conservation in other angiosperm plants, with the paired goals of representing functionally conserved sites and usefulness for screening kinases from diverse plants. Screening the 8k library with the active P2K1 kinase domain identified 177 phosphopeptides, including calcineurin B-like protein (CBL9) and G protein alpha subunit 1 (GPA1), which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates CBL9 and GPA1 through in vitro kinase assays. This expanded 8k KiC assay will be a useful tool for identifying novel substrates across diverse plant protein kinases, ultimately facilitating the exploration of previously undiscovered signaling pathways. 

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 furtherin vivoexperiments 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. 

Abstract The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca 2+ -imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism. 

SUMMARY Extracellular ATP (eATP) is known to act as a danger signal in both plants and animals. In plants, eATP is recognized by the plasma membrane (PM)‐localized receptor P2K1 (LecRK‐I.9). Among the first measurable responses to eATP addition is a rapid rise in cytoplasmic free calcium levels ([Ca²⁺]_cyt), which requires P2K1. However, the specific transporter/channel proteins that mediate this rise in [Ca²⁺]_cytare unknown. Through a forward genetic screen, we identified an Arabidopsis ethylmethanesulfonate (EMS) mutant impaired in the [Ca²⁺]_cytresponse to eATP. Positional cloning revealed that the mutation resided in thecngc6gene, which encodes cyclic nucleotide‐gated ion channel 6 (CNGC6). Mutation of theCNGC6gene led to a notable decrease in the PM inward Ca²⁺current in response to eATP. eATP‐induced mitogen‐activated protein kinase activation and gene expression were also significantly lower incngc6mutant plants. In addition,cngc6mutant plants were also more susceptible to the bacterial pathogenPseudomonas syringae. Taken together, our results indicate that CNGC6 plays a crucial role in mediating eATP‐induced [Ca²⁺]_cytsignaling, as well as plant immunity.