Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine-threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of "moonlighting" receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible

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

Funder: Agence Nationale de la Recherche; Id: http://dx.doi.org/10.13039/501100001665Funder: Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada; Id: http://dx.doi.org/10.13039/501100002790Damage 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, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+ ]cyt signalling in roots. Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+ ]cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-of-function mutants were used: cngc2-3 and defence 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+ ]cyt increase and transcriptional response in cngc2 roots were significantly impaired. CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca2+ influx, causing depolarization leading to [Ca2+ ]cyt increase and damage-related transcriptional response

The Role of Arabidopsis thaliana Cyclic Nucleotide-Gated Channels 2, 6, and 19 in Extracellular ATP Signalling

Calcium (Ca2+) is known as a secondary messenger in plant growth, development, and stress perception. Plants generate stress-specific Ca2+ signals (Ca2+ signatures) to translate extracellular signals into cellular responses. Extracellular Adenosine 5’-triphosphate (eATP) is a crucial signalling molecule in plants but poorly understood. Upon abiotic and biotic stresses, ATP is released into the extracellular matrix, where it is referred as eATP, to regulate plant growth and development, salt tolerance, immunity, and the Damage-Associated Molecular Patterns (DAMP) response. Two plasma membrane (PM) eATP receptors DOES NOT RESPOND TO NUCLEOTIDES1 (DORN1/P2K1) and DORN2/P2K2 have been identified in Arabidopsis thaliana in the past decade. The perception of eATP leads to downstream changes of secondary messengers, including cytosolic free calcium ([Ca2+]cyt) increase, reactive oxygen species (ROS) and nitric oxide (NO) production. Subsequently, [Ca2+]cyt regulates production of defence-related hormones (such as jasmonic acid, JA), plant elicitor peptides (PEPs), and indolic glucosinolates (IG). However, the PM calcium channels underpinning eATP-induced [Ca2+]cyt increase remain poorly studied. Here, my PhD project revealed the role of Cyclic Nucleotide-Gated Channels (CNGC) 2, 6 and 19 as putative calcium channels in eATP signalling. By employing (apo)aequorin as a Ca2+ reporter, CNGC2 was found to be involved in Arabidopsis root’s [Ca2+]cyt responses to both eATP and eADP. Transcriptional analysis showed that the expression of CNGC6 and CNGC19 was upregulated by eATP in a DORN1/P2K1- and CNGC2-dependent manner. The involvement of CNGC6 and CNGC19 in the root’s eATP-induced [Ca2+]cyt increase was then discovered and (for CNGC19) investigated further using GCaMP3 as a [Ca2+]cyt reporter, as a function of phosphate nutrition. Notably, both CNGC2 and CNGC19 were suggested to modulate the root’s transcription of components of eATP-activated defence pathways. As a result, loss-of-function mutants of CNGC19 exhibited increased susceptibility to infection by the parasitic cyst nematode, Heterodera schachtii.Cambridge International & Churchill Pfizer Scholarship, Trinity-Henry Barlow Scholarship, Henry Lester Trust, and Cambridge Philosophical Societ

Phosphate-deprivation and damage signalling by extracellular ATP.

Peer reviewed: TruePhosphate deprivation compromises plant productivity and modulates immunity. DAMP signalling by extracellular ATP (eATP) could be compromised under phosphate deprivation by the lowered production of cytosolic ATP and the need to salvage eATP as a nutritional phosphate source. Phosphate-starved roots of Arabidopsis can still sense eATP, indicating robustness in receptor function. However, the resultant cytosolic free Ca2+ signature is impaired, indicating modulation of downstream components. This perspective on DAMP signalling by extracellular ATP (eATP) addresses the salvage of eATP under phosphate deprivation and its promotion of immunity, how Ca2+ signals are generated and how the Ca2+ signalling pathway could be overcome to allow beneficial fungal root colonization to fulfill phosphate demands. Safe passage for an endophytic fungus allowing root colonization could be achieved by its down-regulation of the Ca2+ channels that act downstream of the eATP receptors and by also preventing ROS accumulation, thus further impairing DAMP signalling

The Complex Story of Plant Cyclic Nucleotide-Gated Channels

Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins

The Complex Story of Plant Cyclic Nucleotide-Gated Channels.

Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins

Heterologous Expression of a Ferritin Homologue Gene PpFer1 from Prunus persica Enhances Plant Tolerance to Iron Toxicity and H2O2 Stress in Arabidopsis thaliana.

Peer reviewed: TrueAcknowledgements: The authors are grateful to grateful to Julia M. Davies, Department of Plant Sciences, University of Cambridge for critical reading and valuable suggestions. The authors are grateful to Irene Murgia, Dipartimento di Biologia, Università degli Studi di Milano, for fer1-2 mutant donation.Publication status: PublishedIn plants, ferritin proteins play an important role in iron (Fe) storage which contributes to plant growth and development. However, the biological functions of ferritins in fruit trees are essentially unknown. In this study, three Ferritin genes were isolated from 'Zhentong No. 3' peach, which were named PpFer1-PpFer3. The expression levels of these genes were different in distinct tissues/organs. Notably, PpFer1 was the most abundantly expressed Ferritin family gene in all tested tissues of 'Zhentong No. 3' peach; its expression levels were significantly enhanced throughout the entire peach seedling under Fe toxicity and H2O2 stress, particularly in the leaves. In addition, over-expression of PpFer1 was effective in rescuing the retarded growth of Arabidopsis fer1-2 knockout mutant, embodied in enhanced fresh weight, primary root length, lateral root numbers, total root length, total leaf chlorophyll, stomatal conductance (Gs), net photosynthetic rate (Pn), transpiration rate, and tissue Fe concentration. This study provides insights into understanding the molecular mechanisms of Fe storage and sequestration in perennial fruit trees

Heterologous Expression of a Ferritin Homologue Gene <i>PpFer1</i> from <i>Prunus persica</i> Enhances Plant Tolerance to Iron Toxicity and H₂O₂ Stress in <i>Arabidopsis thaliana</i>

In plants, ferritin proteins play an important role in iron (Fe) storage which contributes to plant growth and development. However, the biological functions of ferritins in fruit trees are essentially unknown. In this study, three Ferritin genes were isolated from ‘Zhentong No. 3’ peach, which were named PpFer1-PpFer3. The expression levels of these genes were different in distinct tissues/organs. Notably, PpFer1 was the most abundantly expressed Ferritin family gene in all tested tissues of ‘Zhentong No. 3’ peach; its expression levels were significantly enhanced throughout the entire peach seedling under Fe toxicity and H2O2 stress, particularly in the leaves. In addition, over-expression of PpFer1 was effective in rescuing the retarded growth of Arabidopsis fer1-2 knockout mutant, embodied in enhanced fresh weight, primary root length, lateral root numbers, total root length, total leaf chlorophyll, stomatal conductance (Gs), net photosynthetic rate (Pn), transpiration rate, and tissue Fe concentration. This study provides insights into understanding the molecular mechanisms of Fe storage and sequestration in perennial fruit trees

Isolation, heterologous expression, and functional determination of an iron regulated transporter (IRT) gene involved in Fe2+ transport and tolerance to Fe2+ deficiency in Vitis vinifera

AbstractIn plants, iron (Fe) regulated transporters (IRT) play important roles in uptake and transport of Fe that contributes to plant growth and development. However, biological functions of IRT transporters in fruit trees are still unknown. This study isolated 10 VvIRT genes from ‘Marselan’ grape, with varying expression levels across different tissues/organs, particularly enhanced under Fe depletion, especially in roots. Notably, VvIRT7 is the most abundantly expressed IRT gene in grape, beneficially restoring the Fe2+ uptake defect of yeast mutant DEY1453. Furthermore, VvIRT7 showed dominant expression in the roots of irt1/35S::IRT7 complementation lines. Overexpressing of VvIRT7 rescued the retarded growth of irt1 knockout mutant, by increasing the fresh weight, dry weight, total root length, total root surface, lateral root numbers, total leaf chlorophyll, ACO activity, NiR activity, SDH activity, and tissue Fe concentration. This study provides insights for understanding molecular mechanisms of Fe uptake and transport in grape.</jats:p