Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types. The authors show that RALF22 has a dual role in cell wall assembly in root hairs: as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

BioelectricityVol. 5, No. 1 Guest EditorialFree AccessBioelectricity in Plants: From So Simple a BeginningJosé A. FeijóJosé A. FeijóAddress correspondence to: José A. Feijó, PhD, Department of Cell Biology and Molecular Genetics, University of Maryland, 0118 Bioscience Research Building, 4066 Campus Dr., College Park, MD 20742-5815, USA E-mail Address: jfeijo@umd.eduDepartment of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.Search for more papers by this authorPublished Online:18 Mar 2023https://doi.org/10.1089/bioe.2023.0011.editorialAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail In the heady days of the Victorian era, a forward thinking British scientific community formulated, tested, and contested new scientific concepts with remarkable vitality. The "Origin of the species" and many other groundbreaking books had been recently published, primary scientific societies were increasingly opened to the public, and their proceedings reported in the popular press. Electricity had become a public and scientific fascination, and physicochemical discoveries were strongly linked to the dizzying pace of the industrial revolution. Overall, there was a generalized wonder at the power of science and what it could achieve.In this climate, physiological botany emerged as a scientific pursuit with a primary emphasis on the possibility of mechanistic similarities between animals and plants. Although still somewhat zoocentric in nature, as a full appreciation of the scale of evolutionary history and the archaic origins of species required time to develop, the exploration of physiological commonalities was pursued by many of the notable biologists of the period. Darwin himself discussed the plant digestion, sensitivity, and other "animal-like" properties in his 1875 book Insectivorous Plants. J. Burdon-Sanderson, a contemporary of Darwin's son Francis and eventually a Regius professor at the University of Oxford, was intrigued by these observations. In the period from 1873 to 1880 Darwin and Burdon-Sanderson met frequently, exchanged materials through his son Francis and exchanged over 60 catalogued and curated1 letters on the subject, right until Darwin's death in 1882.Although in most of this correspondence the main theme was digestion, on August 13, 1873, Burdon-Sanderson drifted from the main subject and wrote, "…It has occurred to me that it would be very interesting to show that when the Drosera leaf is excited the same electrical changes occur as in muscle & nerve. If you think so, it would not be difficult to plan an experiment. The fact if it can be demonstrated, would afford additional evidence of the identity of the processes." Two days after (August 15) Darwin responded: "I should think that it would be extremely interesting to ascertain whether there is any electrical change in the leaves of Drosera when they are excited; but I should think Dionæa would be much better for the purpose" (Fig. 1a). Sanderson worked quickly, and 3 months later communicated to the Royal Society his results on the Venus flytrap, including the observations:2FIG. 1. Left: Facsimile of a letter from Darwin to Burdon-Sanderson, suggesting the usage of Dionea (venus flytrap) to test the hypothesis that plants could have an electrically stimulated mechanism of motion as animals do. Right: Facsimile of the 1873 publication of Burdon-Sanderson in the Proceedings of the Royal Society, reporting the first evidence of a bioelectric phenomenon in plants (Ref.2). Color images are available online."(…) Negative variation—a. If, the leaf being so placed on the electrodes that the normal leaf-current is indicated by a deflection, a fly is allowed to creep into it, it is observed that the moment the fly reaches the interior (so as to touch the sensitive hairs on the upper surface of the lamina), the needle swings to the right, the leaf at the same time closing on the fly. b. The fly having been caught does not remain quiet in the leaf; each time it moves, the needle again swings to the right, always coming to rest in a position somewhat further to the left than before, and then slowly resuming its previous position. c. The same series of phenomena present themselves if the sensitive hairs of a still expanded leaf are touched with a camel-hair pencil." And so Burdon-Sanderson described the first plant bioelectric phenomenon in the form of what we would today call an action potential3 when describing a bioelectric response that evolved in carnivorous plants to capture animal preys.It is appropriate that on the 150th anniversary of this accomplishment, we revisit the state of the art of bioelectricity in plants. It is especially relevant that it comes in the form of a special issue within a journal that emerged from the necessity to provide a conceptual space for the discussions of the paradigms inherent to BioElectricity. These processes that intrigued and fascinated Darwin and Burdon-Sanderson, keep us busy today as they are among the most intriguing biological phenomena still requiring a full comprehension.The Long and Winding Road to Contemporary Plant BioelectricityWarranting justice to Darwin's suggestion to examine Dionaea 150 years ago, many of the principles at stake in these early discussions are still being experimentally addressed using the Dionaea system due to its unique electrophysiological properties and adaptations. The genome has been described and experimentation with the system has revealed how action potentials are generated by different channels,4,5 as well as how these mechanism interface with the digestive processes of digestion that so fascinated Darwin.6But like in many other areas of biology, new experimental approaches required appropriate instrumentation. Crucial for Burden-Sanderson were the instruments invented by German pioneer Emil du Bois-Reymond, considered the father of electrophysiology. Among his inventions, the German scientist created neutral means of coupling instruments to tissue, invented the "magneto-electrometer" (AC generator), the "rhecord" (potentiometer) and the galvanometer, which was sensitive enough to record his results. With these devices, du Bois-Reymond was able to detect the first action currents in frog muscles in 1843.7Following du Bois-Reymond footsteps, Jagadish Chandra Bose, a polymath credited for exceptional discoveries and insight in many areas (e.g., microwave, radio, and physics of materials) invented many instruments adapted for various plant physiology applications (Fig. 2). By the end of the 19th century, Bose had invented specialized tools to measure many features of plants (e.g., the "crescograph" to measure growth), and had explored multiple species that exhibited animal-like "irritability" (e.g., Mimosa pudica). Bose produced an unrivalled body of literature on plant bioelectricity (Fig. 2). His most outstanding contribution to the field of biophysics is considered to be the demonstration of the electrical nature of the conduction of various stimuli (e.g., wounds and chemical agents).FIG. 2. Top: Facsimiles of two of the most prominent magnum opuses of Sir Jagadish Chandra Bose that revolutionized our knowledge of bioelectricity in plants and continue to inspire successive generations ever since. Bottom: Facsimiles of various devices that Bose invented and developed to record bioelectric or growth patterns in plants. Counterclockwise, apparatus for determination of latent period and velocity of transmission of excitation in Mimosa, and respective trace; device to record electric variations between the pulvinus and the second indifferent point on the stem of Mimosa, and respective record of the excitation patterns in the form of action potentials; the "electro-magnetic phytograph" that records temporal series of contractile responses of parts of a plant, namely leaves.Umrath subsequently found the filamentous algae Nitella to be the best model organism for the first ever demonstration of an action potential by means of an intracellular electrode in 19308 (Fig. 3), two decades before a similar achievement was reported for animals.9 The first measurements of resting membrane potentials in roots10 would inaugurate an extremely proficuous decade for plant physiology, with many of the hydric and mineral relationships in plants being elucidated by means of electrophysiological experimentation.FIG. 3. Top: Diagram of the "electrometer" used by Umrath to impale filaments of the green algae Nitella and record the first intracellular action potentials of any cell, shown in the middle and bottom traces (adapted from Ref.8).The 1970s witnessed the rising of the new patch-clamp methods invented by Erwin Neher and Bert Sakmann in Germany, which ultimately led them to the Nobel prize. Up until today, this method is the single most relevant to the study of individual channels and membrane biology. Its application to plant cells was nevertheless delayed by the nature of plant cell walls. The requirement for direct attachment of the electrode to the plasma membrane implies the cell wall as a serious limitation to the application of patch-clamp in plants. This hurdle was finally circumvented by two teams in Germany and the United States, which almost simultaneously showed the feasibility of patch-clamping after enzymatically removing the cell wall to produce protoplasts (cell-wall naked plant cells).11,12These demonstrations effectively paved the way to the utilization of many of the methods developed for neurobiology and have supported the development of new paradigms in plant bioelectricity field making this area vibrant and thriving. Interestingly, plants have much more negative membrane potentials, an enormous dependence on electric-based phenomena associated to the transport of ions from the soil, their translocation through the plant body and the regulation of gas traffic by stomata, raising unique conceptual questions. Vindicating Darwin and Burdon-Sanderson curiosity about the "animal-like" properties of plants, plants have been shown to propagate defensive signals through long-distance slow electric potential waves13–15 (Fig. 4) many of which are dependent on ionotropic glutamate receptors, the very same hallmark of neurons and neural communication.16FIG. 4. Top: Temporal series of propagation along the whole plant, leaf by leaf, of a glutamate induced (arrow) wave of electric potential as reported by a Ca2+ sensor. Bottom: The same experiment but using plants mutated for the Arabidopsis glutamate receptors 3.3 and 3.6, showing no propagation of the signal (adapted from Ref.15).In This Issue on Bioelectricity in PlantsThis issue covers a wide range of topics and levels of organization, from electron transfer biology during photosynthesis, to organismic competition and mutualism during plant biotic interactions.Man and colleagues explore oxygenic photosynthesis mechanisms, where light and chlorophyll interaction result in quantum phenomena not completely understood. The energy from these interactions leads to the breakdown of the water molecule, releasing the oxygen that feeds all respiratory phenomena of life in this planet, and two bioelectric, membrane-based, phenomena. On the one hand, electrons are released to energize a compact set of proteins and lipids, where electrons are transferred down ladders of redox potentials to produce Reduced Nicotinamide Adenine Dinucleotide Phosphate. On the other hand, protons are partitioned between two compartments into a Mitchell canonical chemosmotic system to generate Adenosine TriPhosphate (ATP).These two molecules transfer the photonic energy to the production of carbon polymers that feed earth's ecosystems. The chapter by Nixon's group provides a textbook approach to these processes, systematically highlighting the unknown aspects and caveats of the current understanding of photosynthesis, as well as new approaches that might be employed to optimize productivity. These are hot topics, in times where completely synthetic methods of generating food from electricity generated by photovoltaic panels start to constitute alternative proof of principle for the "green" photosynthesis.17Brownlee and Wheeler write an authoritative review of the field of Ca2+ signaling in plants and algae, with a focus on cell polarity and tip growth, and a wide coverage of all channels and mechanisms that may be involved in these processes. Brownlee was for decades the director of the prestigious Marine Biology Association Laboratory of Plymouth and a leader in experimentation with algal membrane biology and Ca2+ signaling. Among others he has studied algae from different groups namely Fucus, Chlamydomonas, and more recently Coccolithophores and diatoms. The diatoms would be the organism used to one of his latest major breakthroughs, the finding that these organisms possess functional equivalents to the Ca2+/Na+ four-domain channel proteins that underpin many of the fast-gating properties behind the properties of the animal nervous system.18No land plant possesses an equivalent system, thus lacking the possibility of generating millisecond-like action potentials. These channels were thought to have evolved on the animal lineage alone, but Brownlee and his team showed that a one-domain channel with similar fast-gating properties evolved in diatoms, and is used for fast locomotion, relaunching the whole debate of when and why fast channels supporting millisecond action potentials may have initially evolved. These and many aspects related to the anatomy of ion transport and signaling, the inherent basis of bioelectric phenomena, are described by one of the more relevant names in the area for decades.Next, Corti and colleagues extend the discussion of bioenergetic endo-symbiotic organelles to mitochondria, specifically addressing the elusive functional cross talk between mitochondria and chloroplasts. Team leader Ildiko Szabo has been long associated with the description and characterization of one of the most relevant ion channels for mitochondria function, the elusive mitochondrial calcium uniporter.19 She has also been involved in the characterization of plant glutamate receptor phenotypes associated with Ca2+.20Now the team builds on these and other pieces of evidence to propose a coordinated action of Ca2+ and reactive oxygen species as a way of synchronizing the response of plant cells to environmental signals. Both mitochondria and plastid have highly energized membrane systems, with membrane potentials compatible to their functions, and inherently these mechanisms should bear the existence of bioelectric patterns in the form of specific choreographies of membrane potential and ionic concentration that Corti and colleagues now propose to integrate in a new functional model.Nunes and Feijó introduce readers to the pollen tube with a new perspective. Pollen tubes have long been known to generate the largest extracellular electric fields around any cell,21,22 and in the past decades this group and others have systematically deconstructed the various ionic components that underlie this electric field, and their molecular mechanisms. Here the authors go a step forward, by revisiting a previous hypothesis that the polarized distribution of channels and pumps could generate a cytosolic standing electric field.23 The detailed knowledge of localization of various transport proteins, activities, and phenotypes (e.g., the AHA-type H+-ATPases24), and the quantification of the conspicuous apical gradients of various ions (Ca2+, H+, Cl−, and possibly K+) allow for better supported predictions of membrane voltage gradients and eventual cytosolic electric fields. If these hypotheses receive full experimental validation, they could have relevant impact in our current understanding of cell polarity and formation of intracellular domains.In the only experimental article of this collection, Li and colleagues also explore the uniqueness of the pollen tube system to dissect the differential choreographies of cytosolic H+ and Ca2+ variations, arguably the two most relevant ionic second messengers. For this purpose, they use a novel dual Ca2+/pH probe (CapHensor) designed by leading author Kai Konrad.25 The specificities of this probe, which expresses two probes on equimolar concentration and balanced fluorescence, enable high temporal and spatial sensitivity. Li and colleagues explored these properties to generate detailed maps and correlations between the activities of these two second messenger ions, and opens the door to new interpretations of macroscopic phenomena by synchronization of waves and oscillations associated to ion activity and electric potential.Morato and Sena provide a discussion on the provocative field of bioelectric interaction and putative communication between plant roots and microbes and then move to insect–flower interactions. The authors make an extensive revision of diverse fields and provide a detailed account of various levels of organization and interactions where physiological electric fields may have a functional impact in plant biology. Among others, mycorrhization and nodulation in the root, as well as electric interactions during pollination are the focus of special attention.The pièce de résistance of the present issue comes in the form of an autobiographic account by Dale Sanders. The format is the one that the journal has been establishing as a trademark closing article for special issues, "My experiments in BioElectricity." Active for more than three decades, Sanders has been an international reference and leader in all-things ion transport and related physiological consequences. Picking up on all the excitement inherent to the knowledge about channels that patch-clamp brought, gearing much of the research in the 1980s, Sanders built a career from doctoral and postdoctoral training on some of the best laboratories, before landing at a position in the University of York.There, he helped establishing what would be some of the highest standards in the fields of plant membrane biology and electrophysiology. His account on how some of these discoveries unfolded is well revealing of the finesse of his intellectual method. Many of his associates became leaders on his own. He edited and reviewed and became a driving force behind the series of tri-annual International Workshops in Plant Membrane Biology (this year in its 19th iteration in Taipei26), the most representative meeting for people involved in researching plant bioelectricity, directly or indirectly. Among many accolades, he became a fellow of the Royal Society, and his laboratory quickly became known both by the quality of the work and by an amazing productivity, during many years putting out a solid article every month on average.Later in his life Dale became the director of the prestigious John Innes Research Center and, in difficult conditions, helped keeping the John Innes for years as the number one BioMedical Institution in the world in terms of research indicators. Although surely that implied a major constraint in terms of focus in practicing science, he continued to collaborate and leading many high-impact research projects, such as the elucidation of plant signaling after aphid attack,27 or the discovery of the elusive Ca2+ channel that promotes the Ca2+ nuclear oscillations that trigger nodulation in the roots of legumes.28Although this special issue does not have the ambition of a thorough coverage of everything happening in the field of plant BioElectricity, we feel confident that it makes for a good showcase for the many directions to where the field is evolving. In the year in which we commemorate the 150 years of the pioneering studies of Burdon-Sanderson, we hope that this collection may be inspiring and contribute to the visibility of a field that gave so much to science in general and in Plant Biology in particular.AcknowledgmentsSpecial thanks to Angus Murphy, Custodio Nunes, Colin Brownlee, and Marta Rufino for the careful and swift revision of the article that greatly improved its content and readability.Author Disclosure StatementNo competing financial interests exist.Funding InformationJ.A.F. lab is supported by the National Institute of Health (R01-GM131043) and the National Science Foundation (MCB-1930165).References1. The Darwin Correspondence project. Available from: https://www.darwinproject.ac.uk/letter/?docId=letters/DCP-LETT-8988.xml [Last accessed: March 7, 2023]. Google Scholar2. Burdon-Sanderson J. Note on the Electrical Phenomena which accompany irritation in the leaf of Dionea muscipula. Proc R Soc London 1873;21:495–496. Crossref, Google Scholar3. Schuetze SM. The discovery of the action potential. Trends Neurosci 1983;701:215–222. Google Scholar4. Scherzer S, Bohm J, Huang S, et al. A unique inventory of ion transporters poises the Venus flytrap to fast-propagating action potentials and calcium waves. Curr Biol 2022;32:4255–4263. Crossref, Medline, Google Scholar5. Brownlee C. Plant physiology: The anatomy of a plant action potential. Curr Biol 2022;32:1000-R1002. Crossref, Google Scholar6. Hedrich R, Neher E. Venus Flytrap: How an excitable, carnivorous plant works. Trends Plant Sci 2018;3:220–234. Crossref, Google Scholar7. Du Bois Reymond E. Vorlaufiger Abriss einer Untersuchung über den sigenannten Froschstrom und umber die elektromotorischen Fische. Annl Phys 1843;58:1–30. Google Scholar8. Umrath K. Untersuchungen über Plasma 1 und Plasmastr.mung an Characeen.2 Protoplasma 1930;9:576–597. Google Scholar9. Nastuk WL, Hodgkin AL. The electrical activity of single muscle fibers. J Cell Comp Physiol 1950;35(1):39–73. Crossref, Google Scholar10. Etherton B, Higinbotham N. Transmembrane potential measurements of cells of higher plants as related to salt uptake. Science 1960;131:409–410. Crossref, Medline, Google Scholar11. Schroeder J, Hedrich R, Fernandez JM. Potassium-selective single channels in guard cell protoplast of Vicia faba. Nature 1984;3112:361–362. Crossref, Google Scholar12. Moran N, Ehrenstein G, Iwasa K, et al. Ion channels in plasmalemma of wheat protoplasts. Science 1984;226:835–838. Crossref, Medline, Google Scholar13. Wildon DC, Thain JF, Minchin PEH, et al. Electrical signalling and systemic proteinase inhibitor induction in the wounded plant. Nature 1992;360:62–65. Crossref, Google Scholar14. Mousavi SA, Chauvin A, Pascaud F, et al. Glutamate receptor-like genes mediate leaf-to-leaf wound signalling. Nature 2013;500:422–426. Crossref, Medline, Google Scholar15. Toyota M, Spencer D, Sawai-Toyota S, et al. Glutamate triggers long-distance, Ca2+-based plant defense signaling. Science 2018;361:1112–1115. Crossref, Medline, Google Scholar16. Simon AA, Retamal-Navarro C, Feijó JÁ. Merging signaling with structure: Functions of plant glutamate receptor ion channels. Annu Rev Plant Biol 2023. In Press; doi: 10.1146/annurev-arplant-070522-033255 Crossref, Medline, Google Scholar17. Cai T, Sun H, Qiao J, et al. Cell-free chemoenzymatic starch synthesis from carbon dioxide. Science 2021;373:1523–1527. Crossref, Medline, Google Scholar18. Helliwell KE, Chrachri A, Koester JA, et al. Alternative mechanisms for fast Na+/Ca2+ signaling in eukaryotes via a novel class of single-domain voltage-gated channels. Curr Biol 2019;29:1503–1511. Crossref, Medline, Google Scholar19. De Stefani D, Raffaello A, Teardo E, et al. A forty-kilodalton protein of the inner membrane is the mitochondrial Ca2+ uniporter. Nature 2011;476:336–340. Crossref, Medline, Google Scholar20. Teardo E, Carraretto L, De Bortoli S, et al. Alternative splicing-mediated targeting of the arabidopsis glutamate receptor 3.5 to mitochondria affects organelle morphology. Plant Physiol 2014;167: 216–227. Crossref, Medline, Google Scholar21. Weisenseel M, Nuccitelli R, Jaffe LF. Large electrical currents traverse growing pollen tubes. J Cell Biol 1975;66:556–567. Crossref, Medline, Google Scholar22. Jaffe LF, Nuccitelli R. Electrical controls of development. Annu Rev Biophys Bioeng 1977;7(10):16–19. Google Scholar23. Michard E, Alves F, Feijó JA. The role of ion fluxes in polarized cell growth and morphogenesis: The pollen tube as an experimental paradigm. Int J Dev Biol 2009;53(8–10):1609–1622. Crossref, Medline, Google Scholar24. Hoffmann RD, Portes MT, Olsen LI, et al. Plasma membrane H+-ATPases sustain pollen tube growth and fertilization. Nat Commun 2020;11(1):1–15. Crossref, Medline, Google Scholar25. Li K, Prada J, Damineli DSC, et al. A novel genetically encoded method for simultaneous Ca2+- and pH ratio imaging. New Phytol 2021;230:2292–2310. Crossref, Medline, Google Scholar26. International Workshop on Plant Membrane Biology. Available from: http://iwpmb2023.imb.sinica.edu.tw/index [Last accessed: March 11, 2023]. Google Scholar27. Vincent TR, Avramova M, Canham J, et al. Interplay of plasma membrane and vacuolar ion channels, together with BAK1, elicits rapid cytosolic calcium elevations in Arabidopsis during aphid feeding. Plant Cell 2017;29(6):1460–1479. Crossref, Medline, Google Scholar28. Charpentier M, Sun J, Martins TV, et al. Nuclear-localized cyclic nucleotide–gated channels mediate symbiotic calcium oscillations. Science 2016;352:1102–1105. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 5Issue 1Mar 2023 InformationCopyright 2023, Mary Ann Liebert, Inc., publishersTo cite this article:José A. Feijó.Bioelectricity in Plants: From So Simple a Beginning.Bioelectricity.Mar 2023.1-6.http://doi.org/10.1089/bioe.2023.0011.editorialPublished in Volume: 5 Issue 1: March 18, 2023PDF download

Bioelectricity has been studied since the 18th century in every branch of the tree of life. Bioelectricity is involved in cell growth, proliferation, and behavior, which at a tissue-scale level translates in dramatic tissue rearrangements during embryology and somatic development. Although ion fluxes can be measured in single cells, it is not unanimous that a single cell may create and sustain different bioelectrical states within itself by means of electrochemical nonequilibrium phenomena. We address this possibility, with a focus on the pollen tube as a biological model. Pollen tubes are the subject of intense research given its unique properties, evolutionary streamlined to very fast apical growth and sensitivity to external cues that affect chemotropic responses. Pollen tube's functions rely on conspicuous tip-focused ions dynamics, involving the formation of steep ion gradients, with ion concentration differences over an order of magnitude when compared with the shank cytosol. These gradients are thought to be based on the spatial segregation of ion transporters, channels, and pumps along the cell, creating distinct electrochemical environments at the tip and the shank. But how polarity is generated and maintained is still a matter of debate. In the past we hypothesized that opposing electrochemical forces of depolarization at the tip and hyperpolarization in the shank could create a membrane potential gradient spanning from the tip to the shank that would be part of feedback mechanisms essential to cell polarity in these and likely other cell types. In this study, we review the latest progress on understanding apical growth from the perspective of bioelectricity-driven morphogenesis.

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.

Summary Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides which, curiously, bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSINs (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signaling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 (LLG1) and FERONIA (FER), triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a signaling molecule that regulates this process. This mechanism may also underlie wall assembly and expansion in other plant cell types.

Cell polarity is a macroscopic phenomenon established by a collection of spatially concentrated molecules and structures that culminate in the emergence of specialized domains at the subcellular level. It is associated with developing asymmetric morphological structures that underlie key biological functions such as cell division, growth, and migration. In addition, the disruption of cell polarity has been linked to tissue-related disorders such as cancer and gastric dysplasia. Current methods to evaluate the spatiotemporal dynamics of fluorescent reporters in individual polarized cells often involve manual steps to trace a midline along the cells' major axis, which is time consuming and prone to strong biases. Furthermore, although ratiometric analysis can correct the uneven distribution of reporter molecules using two fluorescence channels, background subtraction techniques are frequently arbitrary and lack statistical support. This manuscript introduces a novel computational pipeline to automate and quantify the spatiotemporal behavior of single cells using a model of cell polarity: pollen tube/ root hair growth and cytosolic ion dynamics. A three-step algorithm was developed to process ratiometric images and extract a quantitative representation of intracellular dynamics and growth. The first step segments the cell from the background, producing a binary mask through a thresholding technique in the pixel intensity space. The second step traces a path through the midline of the cell through a skeletonization operation. Finally, the third step provides the processed data as a ratiometric timelapse and yields a ratiometric kymograph (i.e., a 1D spatial profile through time). Data from ratiometric images acquired with genetically encoded fluorescent reporters from growing pollen tubes were used to benchmark the method. This pipeline allows for faster, less biased, and more accurate representation of the spatiotemporal dynamics along the midline of polarized cells, thus advancing the quantitative toolkit available to investigate cell polarity. The AMEBaS Python source code is available at: https:// github.com/badain/amebas.git

Plant glutamate receptor-like (GLR) genes encode ion channels with demonstrated roles in electrical and calcium (Ca2+) signaling. The expansion of the GLR family along the lineage of land plants, culminating in the appearance of a multiclade system among flowering plants, has been a topic of interest since their discovery nearly 25 years ago. GLRs are involved in many physiological processes, from wound signaling to transcriptional regulation to sexual reproduction. Emerging evidence supports the notion that their fundamental functions are conserved among different groups of plants as well. In this review, we update the physiological and genetic evidence for GLRs, establishing their role in signaling and cell-cell communication. Special emphasis is given to the recent discussion of GLRs' atomic structures. Along with functional assays, a structural view of GLRs' molecular organization presents a window for novel hypotheses regarding the molecular mechanisms underpinning signaling associated with the ionic fluxes that GLRs regulate. Newly uncovered transcriptional regulations associated with GLRs-which propose the involvement of genes from all clades ofArabidopsis thaliana in ways not previously observed-are discussed in the context of the broader impacts of GLR activity. We posit that the functions of GLRs in plant biology are probably much broader than anticipated, but describing their widespread involvement will only be possible with (a) a comprehensive understanding of the channel's properties at the molecular and structural levels, including protein-protein interactions, and (b) the design of new genetic approaches to explore stress and pathogen responses where precise transcriptional control may result in more precise testable hypotheses to overcome their apparent functional redundancies.

Plant glutamate receptor-like channels (GLRSs) control many plant-specific functions, including sperm chemotaxis in moss, pollen tube growth and biotic responses. Despite their physiological significance, GLRs molecular properties, such as ones behind ion permeation and ligand gating are not fully understood. We have evidence of a uniquely poor ionic selectivity, characterized by a strong anion coupled to weaker calcium permeability, and of possible roles for heteromerization in GLR regulation. In particular, AtGLR3.7 behaves as an obligatory heteromer that affects the ion permeation and gating of heteromers when it associates with other GLRs. The recent structural data obtained for two members of Arabidopsis thaliana GLRs(10.1016/j.str.2020.09.006, 10.1016/j.molcel.2021.05.025) increased the robustness of modeling approaches for exploring GLR molecular function. Using protein modeling and molecular dynamics simulation, we inspected the region at the intersection of 4 GLR subunits that forms the ion permeation pathway and selectivity filter (SF) of AtGLR3.7 and AtGLR3.3. As a homomer, AtGLR3.7 possesses 8 positively charged residues in this region, pointing directly to the permeation pathway of the channel. We observed a high instability of AtGLR3.7 SF, which is stabilized by the application of an electrical field forcing anions to move across the permeation pathway. We conclude that positive residues present in the ion permeation pathway and SF of AtGLR3.7 pull anions toward the SF and stabilize it. This phenomenon was not observed in the AtGLR3.3 homomer (that does not display charged residue in SF vicinity), while it was pronounced in the AtGLR3.7/3.3 heteromer, suggesting that heterodimerization stabilizes the SF conformation by enhancing the movement of anions. We discuss the mechanism of GLR function regulation and diversification by heterodimerization.

Physiological oscillations (or rhythms) pervade all spatiotemporal scales of biological organization, either because they perform critical functions or simply because they can arise spontaneously and may be difficult to prevent. Regardless of the case, they reflect regulatory relationships between control points of a given system and offer insights as read-outs of the concerted regulation of a myriad of biological processes. Here we review recent advances in understanding ultradian oscillations (period < 24h) in plant cells, with a special focus on single-cell oscillations. Ion channels are at the center stage due to their involvement in electrical/excitabile phenomena associated with oscillations and cell-cell communication. We highlight the importance of quantitative approaches to measure oscillations in appropriate physiological conditions, which are essential strategies to deal with the complexity of biological rhythms. Future development of optogenetics techniques in plants will further boost research on the role of membrane potential in oscillations and waves across multiple cell types.

Wounding is a trigger for both regeneration and defense in plants, but it is not clear whether the two responses are linked by common activation or regulated as trade-offs. Although plant glutamate-receptor like proteins (GLRs) are known to mediate defense responses, here, we implicate GLRs in regeneration through dynamic changes in chromatin and transcription in reprogramming cells near wound sites. We show that genetic and pharmacological inhibition of GLR activity increases regeneration efficiency in multiple organ repair systems in Arabidopsis and maize. We show that the GLRs work through salicylic acid (SA) signaling in their effects on regeneration, and mutants in the SA receptor NPR1 are hyper-regenerative and partially resistant to GLR perturbation. These findings reveal a conserved mechanism that regulates a trade-off between defense and regeneration, and they also offer a strategy to improve regeneration in agriculture and conservation.

Wounding is a trigger for both regeneration and defense in plants, but it is not clear if the two responses are linked by common activation or regulated as tradeoffs. While plant glutamate-like receptors (GLRs) are known to mediate defense, here we implicate GLRs in regeneration through dynamic changes in chromatin and transcription in reprogramming cells near wound sites. We show that genetic and pharmacological inhibition of GLR activity increases regeneration efficiency in multiple organ repair systems and plant species. Perturbation of GLR-mediated function speeds cell division and re-specification of the stem cell niche while dampening defense responses. We show that the GLRs work through salicylic acid (SA) signaling in regeneration, with mutants in the SA receptor NPR1 partially resistant to GLR perturbation and hyper regenerative. These findings reveal a conserved mechanism that regulates a tradeoff between defense and regeneration and offer new strategies to improve regeneration in agriculture and conservation.

Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports of their biological functions continue to expand; however, attaining quantitative estimates with high spatiotemporal resolution in single cells poses a major research challenge. Here, we detail the use of the genetically encoded pH-sensitive pHluorin reporter expressed in Arabidopsis thaliana pollen tubes to assess cytosolic measurements with calibration to provide actual pH values. This technique enabled us to identify critical phenotypes and establish the importance of tip-focused pH gradient for pollen tube growth, although it can be adapted to other experimental systems.

Whereas the role of calcium ions (Ca2+) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca2+ and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca2+- and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca2+-dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca2+. In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca2+-increases and cytosolic acidification by c. 2 min, suggesting a Ca2+/pH-independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca2+- and pH-responses. We propose close interrelation in Ca2+- and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.

BioelectricityVol. 3, No. 4 Calls for PapersCall for Special Issue Papers: Bioelectricity in Plant MorphogenesisDeadline for Manuscript Submission: September 1, 2022Guest Editors: Dr. Giovanni Sena and Prof. José A. FeijóGuest Editors: Dr. Giovanni SenaImperial College, LondonSearch for more papers by this author and Prof. José A. FeijóUniversity of MarylandSearch for more papers by this authorPublished Online:16 Dec 2021https://doi.org/10.1089/bioe.2021.29028.cfpAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Call for Special Issue Papers: Bioelectricity in Plant Morphogenesis." Bioelectricity, 3(4), pp. 239–240FiguresReferencesRelatedDetails Volume 3Issue 4Dec 2021 InformationCopyright 2021, Mary Ann Liebert, Inc., publishersTo cite this article:Guest Editors: Dr. Giovanni Sena and Prof. José A. Feijó.Call for Special Issue Papers: Bioelectricity in Plant Morphogenesis.Bioelectricity.Dec 2021.239-240.http://doi.org/10.1089/bioe.2021.29028.cfpPublished in Volume: 3 Issue 4: December 16, 2021Online Ahead of Print:December 2, 2021PDF download

The ion-selective vibrating probe has been used to detect and quantify the magnitude and direction of transmembrane fluxes of several ions in a wide range of biological systems. Inherently non-invasive, vibrating probes have been essential to access relevant electrophysiological parameters related to apical growth and morphogenesis in pollen tubes, a highly specialized cell where spatiotemporal tuning of ion dynamics is fundamental. Of relevance, crucial processes to the cell physiology of pollen tubes associated with protons and anions have been elucidated using vibrating probes, allowing the identification of diverse molecular players underlying and regulating their extracellular fluxes. The use of Arabidopsis thaliana as a genetic model system posed new challenges given their relatively small dimensions and difficult manipulation in vitro. Here, we describe protocol optimizations that made the use of the ion-selective vibrating probe in Arabidopsis pollen tubes feasible, ensuring consistent and reproducible data. Quantitative methods like this enabled characterizing phenotypes of ion transporter mutants, which are not directly detectable by evident morphological and reproductive defects, providing valuable insights into molecular and cellular mechanisms. The protocol for quantifying extracellular proton and anionic fluxes detailed here can be adjusted to other systems and species, while the sample preparation can be applied to correlated techniques, facilitating the research of pollen tube growth and development.

Glutamate receptor-like channels (GLRs) play important roles in numerous plant physiological processes. GLRs are homologous to ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in vertebrates. Here we determine crystal structures of Arabidopsis thaliana GLR3.2 ligand-binding domain (LBD) in complex with glycine and methionine to 1.58- and 1.75-Å resolution, respectively. Our structures show a fold similar to that of iGluRs, but with several secondary structure elements either missing or different. The closed clamshell conformation of GLR3.2 LBD suggests that both glycine and methionine act as agonists. The mutation R133A strongly increases the constitutive activity of the channel, suggesting that the LBD mutated at the residue critical for agonist binding produces a more stable closed clamshell conformation. Furthermore, our structures explain the promiscuity of GLR activation by different amino acids, confirm evolutionary conservation of structure between GLRs and iGluRs, and predict common molecular principles of their gating mechanisms driven by bilobed clamshell-like LBDs.

Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.