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1. CamelliA-based simultaneous imaging of Ca2+ dynamics in subcellular compartments

   https://doi.org/10.1093/plphys/kiac020  

   Guo, Jingzhe ; He, Jiangman ; Dehesh, Katayoon ; Cui, Xinping ; Yang, Zhenbiao ( February 2022 , Plant Physiology)

   As a universal second messenger, calcium (Ca2+) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying the generation of a Ca2+ signature is hampered by limited tools for simultaneously monitoring dynamic Ca2+ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, we have assembled a molecular toolset (CamelliA lines) in Arabidopsis (Arabidopsis thaliana) that enables simultaneous and high-resolution monitoring of Ca2+ dynamics in multiple subcellular compartments through imaging different single-colored genetically encoded calcium indicators. We uncovered several Ca2+ signatures in three types of Arabidopsis cells in response to internal and external cues, including rapid oscillations of cytosolic Ca2+ and apical plasma membrane Ca2+ influx in fast-growing Arabidopsis pollen tubes, the spatiotemporal relationship of Ca2+ dynamics in four subcellular compartments of root epidermal cells challenged with salt, and a shockwave-like Ca2+ wave propagating in laser-wounded leaf epidermis. These observations serve as a testimony to the wide applicability of the CamelliA lines for elucidating the subcellular sources contributing to the Ca2+ signatures in plants.

    

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2. Extracellular ATP plays an important role in systemic wound response activation

   https://doi.org/10.1093/plphys/kiac148  

   Myers, Jr, Ronald J. ; Fichman, Yosef ; Stacey, Gary ; Mittler, Ron ( March 2022 , Plant Physiology)

   Mechanical wounding occurs in plants during biotic or abiotic stresses and is associated with the activation of long-distance signaling pathways that trigger wound responses in systemic tissues. Among the different systemic signals activated by wounding are electric signals, calcium, hydraulic, and reactive oxygen species (ROS) waves. The release of glutamate (Glu) from cells at the wounded tissues was recently proposed to trigger systemic signal transduction pathways via GLU-LIKE RECEPTORs (GLRs). However, the role of another important compound released from cells during wounding (extracellular ATP [eATP]) in triggering systemic responses is not clear. Here, we show in Arabidopsis (Arabidopsis thaliana) that wounding results in the accumulation of nanomolar levels of eATP and that these levels are sufficient to trigger the systemic ROS wave. We further show that the triggering of the ROS wave by eATP during wounding requires the PURINORECEPTOR 2 KINASE (P2K) receptor. Application of eATP to unwounded leaves triggered the ROS wave, and the activation of the ROS wave by wounding or eATP application was suppressed in mutants deficient in P2Ks (e.g. p2k1-3, p2k2, and p2k1-3p2k2). In addition, expression of systemic wound response (SWR) transcripts was suppressed in mutants deficient in P2Ks during wounding. Interestingly, the effect of Glu and eATP application on ROS wave activation was not additive, suggesting that these two compounds function in the same pathway to trigger the ROS wave. Our findings reveal that in addition to sensing Glu via GLRs, eATP sensed by P2Ks plays a key role in the triggering of SWRs in plants.

    

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3. Arabidopsis thaliana CYCLIC NUCLEOTIDE‐GATED CHANNEL2 mediates extracellular ATP signal transduction in root epidermis

   https://doi.org/10.1111/nph.17987  

   Wang, Limin ; Ning, Youzheng ; Sun, Jian ; Wilkins, Katie A. ; Matthus, Elsa ; McNelly, Rose E. ; Dark, Adeeba ; Rubio, Lourdes ; Moeder, Wolfgang ; Yoshioka, Keiko ; et al ( February 2022 , New Phytologist)

   Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca²⁺]_cyt) increase as a second messenger. The downstream PM Ca²⁺channels remain enigmatic. Here, theArabidopsis thalianaCa²⁺channel subunit CYCLIC NUCLEOTIDE‐GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca²⁺]_cytsignalling in roots.

   Extracellular ATP‐induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca²⁺]_cytwere measured with aequorin, and root transcriptional changes were determined by quantitative real‐time PCR. Twocngc2loss‐of‐function mutants were used:cngc2‐3anddefence not death1(which expresses cytosolic aequorin).

   Extracellular ATP‐induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca²⁺dependent, requiring CNGC2 but not CNGC4 (its channel co‐subunit in immunity signalling). Activation of PM Ca²⁺influx currents also required CNGC2. The eATP‐induced [Ca²⁺]_cytincrease and transcriptional response incngc2roots were significantly impaired.

   CYCLIC NUCLEOTIDE‐GATED CHANNEL2 is required for eATP‐induced epidermal Ca²⁺influx, causing depolarization leading to [Ca²⁺]_cytincrease and damage‐related transcriptional response.

    

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4. The Arabidopsis MIK2 receptor elicits immunity by sensing a conserved signature from phytocytokines and microbes

   https://doi.org/10.1038/s41467-021-25580-w  

   Hou, Shuguo ; Liu, Derui ; Huang, Shijia ; Luo, Dexian ; Liu, Zunyong ; Xiang, Qingyuan ; Wang, Ping ; Mu, Ruimin ; Han, Zhifu ; Chen, Sixue ; et al ( September 2021 , Nature Communications)

   Sessile plants encode a large number of small peptides and cell surface-resident receptor kinases, most of which have unknown functions. Here, we report that theArabidopsisreceptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) recognizes the conserved signature motif of SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) fromBrassicaceaeplants as well as proteins present in fungalFusariumspp. and bacterialComamonadaceae, and elicits various immune responses. SCOOP signature peptides trigger immune responses and altered root development in a MIK2-dependent manner with a sub-nanomolar sensitivity. SCOOP12 directly binds to the extracellular leucine-rich repeat domain of MIK2 in vivo and in vitro, indicating that MIK2 is the receptor of SCOOP peptides. Perception of SCOOP peptides induces the association of MIK2 and the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3) and SERK4 and relays the signaling through the cytosolic receptor-like kinasesBOTRYTIS-INDUCED KINASE 1 (BIK1) and AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE 1 (PBL1). Our study identifies a plant receptor that bears a dual role in sensing the conserved peptide motif from phytocytokines and microbial proteins via a convergent signaling relay to ensure a robust immune response.

    

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

   https://doi.org/10.1111/tpj.16656  

   Sowders, Joel M. ; Jewell, Jeremy B. ; Tanaka, Kiwamu ( February 2024 , The Plant Journal)

   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 pathogenBotrytis 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.

    

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