Littérature scientifique sur le sujet « Plant glutamate receptor-like channel »

Plant glutamate receptor-like channels (GLRs) are the homologues of ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in mammals, and they play important roles in various plant-specific physiological processes, such as pollen tube growth, sexual reproduction, root meristem proliferation, internode cell elongation, stomata aperture regulation, and innate immune and wound responses. Notably, these biological functions of GLRs have been mostly linked to the Ca2+-permeable channel activity as GLRs can directly channel the transmembrane flux of Ca2+, which acts as a key second messenger in plant cell responses to both endogenous and exogenous stimuli. Thus, it was hypothesized that GLRs are mainly involved in Ca2+ signaling processes in plant cells. Recently, great progress has been made in GLRs for their roles in long-distance signal transduction pathways mediated by electrical activity and Ca2+ signaling. Here, we review the recent progress on plant GLRs, and special attention is paid to recent insights into the roles of GLRs in response to environmental stimuli via Ca2+ signaling, electrical activity, ROS, as well as hormone signaling networks. Understanding the roles of GLRs in integrating internal and external signaling for plant developmental adaptations to a changing environment will definitely help to enhance abiotic stress tolerance.

Animals require rapid, long-range molecular signaling networks to integrate sensing and response throughout their bodies. The amino acid glutamate acts as an excitatory neurotransmitter in the vertebrate central nervous system, facilitating long-range information exchange via activation of glutamate receptor channels. Similarly, plants sense local signals, such as herbivore attack, and transmit this information throughout the plant body to rapidly activate defense responses in undamaged parts. Here we show that glutamate is a wound signal in plants. Ion channels of the GLUTAMATE RECEPTOR–LIKE family act as sensors that convert this signal into an increase in intracellular calcium ion concentration that propagates to distant organs, where defense responses are then induced.

The genome of Arabidopsis thaliana (L. Heynh.) contains 20 coding sequences for homologues of animal ionotropic glutamate receptors. These glutamate receptor-like receptors act as sensors and mediators of a multitude of exogenous as well as endogenous signals and are found in all analysed plant species. Their molecular structure clearly indicates a function as integral membrane proteins with a ligand-gated ion channel activity. Altered gene expressions and the occurrence of mRNA splice variants confer a high flexibility on the gene as well as on the RNA level. An individual glutamate receptor of A. thaliana is able to bind two different ligands (most probable amino acids and their derivatives), whereas a functional receptor complex is likely to consist of four single proteins. These features enable an immense number of sensitivities against various local and temporal stimuli. This review encompasses the last 15 years of research concerning glutamate signalling and glutamate receptors in plants. It is aimed at summarising their major characteristics and involvements to obtain a broader and farer reaching perspective of these fundamental components of plant signal transduction.

Plants defend against herbivores and nematodes by rapidly sending signals from the wounded sites to the whole plant. We investigated how plants generate and transduce these rapidly moving, long-distance signals referred to as systemic wound signals. We developed a system for measuring systemic responses to root wounding in Arabidopsis thaliana. We found that root wounding or the application of glutamate to wounded roots was sufficient to trigger root-to-shoot Ca2+ waves and slow wave potentials (SWPs). Both of these systemic signals were inhibited by either disruption of both GLR3.3 and GLR3.6, which encode glutamate receptor–like proteins (GLRs), or constitutive activation of the P-type H+-ATPase AHA1. We further showed that GLR3.3 and GLR3.6 displayed Ca2+-permeable channel activities gated by both glutamate and extracellular pH. Together, these results support the hypothesis that wounding inhibits P-type H+-ATPase activity, leading to apoplastic alkalization. This, together with glutamate released from damaged phloem, activates GLRs, resulting in depolarization of membranes in the form of SWPs and the generation of cytosolic Ca2+ increases to propagate systemic wound signaling.

Binding of specific microbial epitopes [MAMPs (microbe-associated molecular patterns)] to PRRs (pattern recognition receptors) and subsequent receptor kinase activation are key steps in plant innate immunity. One of the earliest detectable events after MAMP perception is a rapid and transient rise in cytosolic Ca2+ levels. In plants, knowledge about the signalling events leading to Ca2+ influx and on the molecular identity of the channels involved is scarce. We used a transgenic Arabidopsis thaliana line stably expressing the luminescent aequorin Ca2+ biosensor to monitor pharmacological interference with Ca2+ signatures following treatment with the bacterial peptide MAMPs flg22 and elf18, and the fungal carbohydrate MAMP chitin. Using a comprehensive set of compounds known to impede Ca2+-transport processes in plants and animals we found strong evidence for a prominent role of amino acid-controlled Ca2+ fluxes, probably through iGluR (ionotropic glutamate receptor)-like channels. Interference with amino acid-mediated Ca2+ fluxes modulates MAMP-triggered MAPK (mitogen-activated protein kinase) activity and affects MAMP-induced accumulation of defence gene transcripts. We conclude that the initiation of innate immune responses upon flg22, elf18 and chitin recognition involves apoplastic Ca2+ influx via iGluR-like channels.

Glutamate receptors (GLRs) are involved in multiple functions during the plant life cycle through affecting the Ca2+ concentration. However, GLRs in Brassica species have not yet been reported. In this study, 16 glutamate receptor-like channels (GLR) belonged to two groups were identified in the Brassica rapa (B. rapa) genome by bioinformatic analysis. Most members contain domains of ANF_receptor, Peripla_BP_6, Lig_chan, SBP_bac_3, and Lig_chan_Glu_bd that are closely related to glutamate receptor channels. This gene family contains many elements associated with drought stress, low temperature stress, methyl jasmonate (MeJA), salicylic acid (SA), and other stress resistance. Gene expression profiles showed that BraGLR genes were expressed in roots, stems, leaves, flowers, and siliques. BraGLR5 expression was elevated after drought stress in drought-sensitive plants. BraGLR1, BraGLR8, and BraGLR11 expression were significantly upregulated after salt stress. BraGLR3 expression is higher in the female sterile-line mutants than in the wild type. The expression levels of BraGLR6, BraGLR9, BraGLR12, and BraGLR13 were significantly higher in the male sterile-line mutants than in the wild type. The expression of most BraGLRs increased after self-pollination, with BraGLR9 exhibiting the greatest increase. These results suggest that BraGLRs play an important role in abiotic stress tolerance and sexual reproduction.

Nel corso della loro vita le piante, essendo organismi sessili, sono continuamente soggette a cambiamenti ambientali che necessitano di essere accuratamente percepiti, a cui devono seguire appropriate risposte sia a livello locale che sistemico che ne garantiscano la sopravvivenza. Il calcio (Ca2+) è uno ione che agisce come importante secondo messaggero in tutti gli esseri viventi, in grado di accoppiare la percezione di uno stimolo extracellulare a peculiari risposte intracellulari. La specificità di trasduzione del segnale basata sul Ca2+ è ottenuta grazie alla generazione di transienti incrementi della sua concentrazione citosolica ([Ca2+]cyt), specifici nella loro evoluzione spaziale e temporale, alla quale ci si riferisce come “Ca2+ signatures”. La decodifica delle “Ca2+ signatures” da parte di proteine capaci di legare il Ca2+ permette la messa in atto di appropriate risposte fisiologiche. Nelle piante, è stato documentato che transienti variazioni citosoliche di Ca2+ sono coinvolte in svariati processi fisiologici che includono lo sviluppo della radice, lo sviluppo del tubetto pollinico e il processo di fecondazione, la risposta a stress abiotici, la regolazione dell’interazione pianta-microbo. Transienti incrementi nella [Ca2+]cyt con caratteristica intensità, frequenza, dinamica e durata sono generati dalla azione orchestrata di sistemi di influsso ed efflusso del Ca2+, che includono canali, pompe e scambiatori del Ca2+ che sono localizzati a livello delle membrane cellulari. Data l’importanza e l’universalità della trasduzione del segnale basata sul Ca2+, risulta essere di primaria importanza l’identificazione degli attori molecolari che governano la generazione dei segnali Ca2+. In questo contesto, lo studio delle dinamiche del Ca2+ in vivo rappresenta un potente strumento investigativo. Nel corso del mio dottorato di ricerca, ho esplorato il meraviglioso mondo del “Ca2+ imaging” utilizzando il vasto universo di Biosensori fluorescenti per il Ca2+ geneticamente codificati. Ho appreso e affinato tecniche per produrre immagini di alta qualità rappresentative di dinamiche del Ca2+ in vivo, sia a livello di intero organismo che a singola cellula. Le competenze che ho acquisito mi hanno permesso di contribuire a vari progetti tutti accomunati da quello che è un comune denominatore, ovvero il ruolo cardine del Ca2+ nella regolazione di svariati processi di trasduzione del segnale. Mi sono avventurato nello studio di vari aspetti legati alla segnalazione del Ca2+ tra cui: (i) gli aumenti della [Ca2+]cyt indotti nelle cellule dell’apice radicale in risposta a differenti amminoacidi, contribuendo a definire i determinanti molecolari sottostanti a tali risposte (Alfieri et al., 2020); (ii) la caratterizzazione dei transienti incrementi della [Ca2+]cyt indotti da auxine naturali e da molecole analoghe dell’auxina, e la decifrazione del ruolo di alcuni attori molecolari coinvolti nella genesi delle risposte [Ca2+]cyt indotte da auxina (Wang, Himschoot, Grenzi et al., 2022); (iii) lo sviluppo di un nuovo biosensore per il Ca2+ geneticamente codificato per indagare il ruolo del reticolo endoplasmico nella modellazione delle “Ca2+ signatures” in processi di sviluppo, così come in risposta a vari stimoli (Resentini, Grenzi et al., 2021); (iv) l’effetto di modulazione che alcuni composti chimici hanno sulle oscillazioni spontanee nella [Ca2+]cyt delle cellule di guardia che governano l’apertura e la chiusura degli stomi. Ho inoltre contribuito alla scrittura di reviews legate al mondo del “Ca2+ signalling” in pianta. Tutti i lavori pubblicati, così come i lavori in preparazione, sono allegati al termine di questa dissertazione, alla quale rimando gentilmente il lettore. Qui presenterò il lavoro portato avanti nel contesto del mio progetto di dottorato, il quale si è focalizzato principalmente alla comprensione dei meccanismi rapidi di segnalazione a lunga distanza. Le risposte sistemiche sono governate da eventi di segnalazione a lunga distanza che richiedono l’attività dei Recettori del Glutammato (GLRs). I GLRs sono proteine omologhe ai Recettori del Glutammato animali (iGluRs), ovvero canali ionici attivati da ligando presenti nel sistema nervoso centrale. Nonostante negli animali è chiaro che gli iGluRs mediano il passaggio di ioni a seguito del loro legame con il L-Glutammato, il meccanismo attraverso cui i GLRs sono attivati in pianta è ancora largamente discusso. Ad esempio, non si è ancora a conoscenza se il legame dell’aminoacido ai GLRs sia realmente necessario per la loro attivazione. In questo lavoro di tesi, analizzando i dati della struttura cristallografica del Dominio di Legame all’ amminoacido (LBD) del AtGLR3.3 di Arabidopsis thaliana, abbiamo identificato dei residui amminoacidici coinvolti nel legame del glutammato. Abbiamo dunque introdotto mutazioni puntiformi nella sequenza genomica del AtGLR3.3 per inficiare o abolire la sua abilità di legare il ligando amminoacidico, e con i costrutti ottenuti abbiamo eseguito una complementazione genetica dei mutanti knock out per il gene GLR3.3 (glr3.3-1 e glr3.3-2). Combinando analisi di imaging, genetica, e bioelettronica, dimostriamo che un danno a livello della foglia, come una ferita o una bruciatura, e l’applicazione di uno stress ipoosmotico alla radice, inducono l’aumento sistemico nella concentrazione apoplastica di L-Glutammato che attiva i GLRs attraverso il legame al loro LBD. Inoltre, il nostro lavoro supporta l’evidenza che gli eventi di segnalazione a lunga distanza siano governati da cambiamenti nello stato di turgore della pianta e che i GLRs siano a valle di essi.
Throughout their life plants, being sessile organisms, are continuously exposed to environmental challenges that need to be properly perceived and that require appropriate local and systemic responses. Calcium ion (Ca2+) is a key second messenger in all living beings that couples the perception of extracellular stimuli to characteristics intracellular responses. The specificity of the Ca2+-based signalling is achieved through the generation of specific spatial and temporal transient elevations in the cytosolic Ca2+ concentration [Ca2+]cyt, which are referred to as “Ca2+ signatures”. The interplay of Ca2+ signatures with a toolkit of cognate Ca2+-binding proteins that decode these increases allow the plant to implement a series of tailored physiological responses (e.g., gene expression, metabolism, developmental reprogramming) to withstand the stress. In plants, transient increases in the [Ca2+]cyt have been documented to be involved in several physiological processes including root or pollen tube growth and fertilization, abiotic stress responses, plant-microbe interaction. Ca2+ transients with unique magnitude, frequency, shape, and duration are generated by the orchestrated action of Ca2+ influx and efflux systems that include Ca2+ channels, pumps, and exchangers located at different cellular membranes. Given the importance and universality of Ca2+-based signalling, the identification of actors of the molecular machinery that govern the generation of Ca2+ signals is of primary importance. In this context, the study of Ca2+ dynamics in vivo represents a powerful tool. In the frame of my PhD, I explored the marvellous world of Ca2+ imaging using some of the instruments made available from a vast universe of genetically encoded fluorescent Ca2+ biosensors. I learned and refined techniques to produce high-end images of in vivo Ca2+ dynamics both at the entire organism and single-cell level. The expertise that I acquired allowed me to contribute to different projects, all unified by the common denominator that is the master regulatory role of Ca2+ in many signalling processes. I therefore contributed to the study of: (i) the [Ca2+]cyt responses of root tip cells in response to different amino acids, helping to define the molecular determinants involved in the process (Alfieri et al., 2020); (ii) the characterization of [Ca2+]cyt transients induced by the administration of natural auxins and auxin analogues, and the deciphering of the role of molecular actors involved in the genesis of the auxin-induced [Ca2+]cyt response (Wang, Himschoot, Grenzi et al., 2022); (iii) the development of a novel genetically encoded Ca2+ biosensors to unravel the role of the endoplasmic reticulum in the shaping of the Ca2+ signature in developmental processes, as well as in response to various stimuli (Resentini, Grenzi et al., 2021); (iv) the modulatory effects of chemicals on the spontaneous [Ca2+]cyt oscillations of guard cells that govern the opening and closing of stomata. I also contributed to the preparation of reviews linked to the field of Ca2+ signalling. All the published manuscripts, as well as works in preparation, are attached at the end of this dissertation, to which I kindly redirect the readers. Here, I am presenting the main work of my PhD project which focused on the understanding of how local damages can trigger inducible defence mechanisms in systemic organs and tissues. Systemic responses are mediated by long-distance signalling that requires the activity of Glutamate Receptor-Like channels (GLRs). GLRs are homologs of animal Ionotropic Glutamate Receptors (iGluRs) which are ligand-gated cation channels in the central nervous system. Even though iGluRs are gated through the binding with the L-Glutamate, the mechanism throughout GLRs are activated in planta is poorly understood. As an example, we still do not know if the GLRs binding of amino acids is necessary for their activity. In this PhD thesis, we took the advantage of the recently obtained crystal structure of the Arabidopsis thaliana AtGLR3.3 Ligand Binding Domain (LBD) to identify residues involved in the amino acid-binding. We, therefore, introduced single point mutations in the genome sequence of the AtGLR3.3 gene to prevent or abolish its amino acid-binding, and with the obtained constructs we complemented the glr3.3 KO. By combining high-end imaging, genetics, and bioelectronics we prove that leaf injury, such as wound and burn, and root-applied hypo-osmotic stress induce the systemic apoplastic increase of L-Glutamate that activates GLR channels through their LBD. In addition, our work supports the evidence that long-distance signalling is governed by a systemic change in the turgor state and that GLRs are downstream of it.