5 October 2025
Youzheng Ning, Bryony C. I. C. Jacobs, Clementine Langlet, Limin Wang, Zhizhong Song, Adeeba M. Dark, Elsa Matthus, Sebastian Eves-van den Akker, Taufiq Rahman, Julia M. Davies - New Phytologist Foundation, 2025
Extracellular ATP (eATP) can function as a damage-associated molecular pattern (DAMP) that signals wounding incurred by microbial ingress and herbivore attacks (Tanaka & Heil, 2021; Yuan et al., 2025). Arabidopsis thaliana root and leaf wounding trigger rapid accumulation of eATP (Dark et al., 2011; Myers et al., 2022). eATP is then sensed by the plasma membrane (PM) co-receptors Does Not Respond to Nucleotide1/P2-type purinoceptor Kinase 1 (DORN1/P2K1) and P2K2 (Choi et al., 2014; Pham et al., 2020). The perception of eATP leads to downstream signalling cascades involving elevation of cytosolic free calcium ([Ca2+]cyt), reactive oxygen species and nitric oxide plus MAP (Mitogen-Activated Protein) kinase activation (Demidchik et al., 2003; Foresi et al., 2007; Chen et al., 2017; Kim et al., 2023). Analyses of eATP-induced transcriptomes point to DORN1/P2K1-dependent regulation of multiple defence-related hormone signalling pathways involving jasmonic acid (JA), ethylene and salicylic acid (Tripathi et al., 2017; Jewell et al., 2019). Accordingly, loss-of-function DORN1/P2K1 and P2K2 mutants show increased susceptibility to a variety of plant pathogens, including fungi (Rhizoctonia solani and Sclerotinia sclerotiorum), oomycetes (Plasmodiophora brassicae, Phytophthora infestans and Phytophthora capsici) and bacteria (Pseudomonas syringae) plus insect herbivores (Pieris rapae, Salix exigua and Spodoptera litura), whilst receptor overexpression lines reduce plant susceptibility (Bouwmeester et al., 2014; Balagué et al., 2017; Chen et al., 2017; Jewell et al., 2022; Kumar et al., 2020; Kundu et al., 2025; Yuan et al., 2025).
[Ca2+]cyt serves as an important second messenger in plant stress and immunity signalling (Dong et al., 2022; Köster et al., 2022). eATP induces a specific biphasic [Ca2+]cyt ‘signature’ in roots through DORN1/P2K1, with the possibility of a relatively minor involvement of P2K2 or as yet unknown receptors (Demidchik et al., 2003; Zhu et al., 2018, 2020; Matthus et al., 2019a,b, 2022; Smith et al., 2021). How eATP receptors initiate this [Ca2+]cyt increase in roots is not fully understood (Sun et al., 2021), but the PM Cyclic Nucleotide-Gated Channel2 (CNGC2) is involved, with the possibility of CNGC4 and CNGC6 also (Duong et al., 2022; Wang et al., 2022). The Arabidopsis CNGC family consists of 20 channel-subunit members, with important functions in signalling (Jarratt-Barnham et al., 2021). CNGC2 has recently been shown to be phosphorylated by DORN1/P2K1 in leaves (Sun et al., 2025) and was reported to mediate the Arabidopsis root’s [Ca2+]cyt response to eATP (Wang et al., 2022). However, the significant residual [Ca2+]cyt increase in the loss-of-function cngc2 mutant indicates that other channels also support the eATP-induced [Ca2+]cyt signal in roots (Wang et al., 2022). Studies have revealed Cyclic Nucleotide-Gated Channel 19 (CNGC19) involvement as a PM Ca2+-permeable channel in generating the [Ca2+]cyt signal and defence signalling pathways induced by herbivore attack on leaves and fungal colonisation of roots (Meena et al., 2019; Jogawat et al., 2020). Both of these challenges can trigger eATP accumulation through wounding (Nizam et al., 2019; Myers et al., 2022). Moreover, CNGC19 and DORN1/P2K1 are co-expressed whilst eATP upregulates DORN1/P2K1-dependent CNGC19 expression specifically in roots (Tripathi et al., 2017; Jewell et al., 2019; Sowders & Tanaka, 2023). Such findings suggest that CNGC19 may be part of the eATP response pathway. Indeed, a recent study by Kundu et al. (2025) has shown that CNGC19 is part of the whole seedling [Ca2+]cyt response to eATP (measured using cytosolic aequorin in the cngc19-2 loss-of-function mutant) and that CNGC19 is a possible interacting partner of DORN1/P2K1 (Kundu et al., 2025). This begs the question of whether CNGC19 operates in the root’s response to eATP.
In this study, CNGC19 was found to make a significant contribution to the Arabidopsis root’s eATP-induced [Ca2+]cyt increase (determined using the intensiometric GCaMP3 [Ca2+]cyt reporter (Vincent et al., 2017) in the cngc19-1 T-DNA insertion loss-of-function mutant). The root transcriptional response to eATP revealed amplification of CNGC19 expression through DORN1/P2K1, but only partially through CNGC2. Transcription of specific eATP-induced defence-related genes was found to require CNGC19, indicative of discrete signalling pathways. These genes included those for the DAMP PM Plant Endogenous Peptide Receptors PEPR1 and PEPR2. As CNGC19 is also needed for Pep1-induced [Ca2+]cyt increase (determined with cngc19-2 mutant seedlings by Meena et al., 2019 and also here with cngc19-1 roots), together the results point to CNGC19 as a common component of these two DAMP pathways.
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