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A preface for the June 1999 issue of the "Journal of Experimental Botany" is presented.

Discusses the network of signalling elements in the guard cell ion control of the stomatal aperture that balances transpiration stream and gas exchange for photosynthesis. Role of the hydrogen ion in maintaining low pH levels; Pathways for elevating calcium ion levels; Effect of the phosphorylation state of key regulatory proteins on ion transport.

The evolution of stomata marks one of the key advances that enabled plants to colonise dry land while allowing gas exchange for photosynthesis. In large measure, stomata retain a common design across species that incorporates paired guard cells with little variation in structure. By contrast, the cells of the stomatal complex immediately surrounding the guard cells vary widely in shape, size and count. Their origins in development are similarly diverse. Thus, the surrounding cells are likely a luxury that the necessity of stomatal control cannot do without (with apologies to Oscar Wilde). Surrounding cells are thought to support stomatal movements as solute reservoirs and to shape stomatal kinetics through backpressure on the guard cells. Their variety may also reflect a substantial diversity in function. Certainly modelling, kinetic analysis and the few electrophysiological studies to date give hints of much more complex contributions in stomatal physiology. Even so, our knowledge of the cells surrounding the guard cells in the stomatal complex is far from complete., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Stomata play a crucial role in plants by controlling water status and responding to drought stress. However, simultaneously improving stomatal opening and drought tolerance has proven to be a significant challenge. To address this issue, we employed the OnGuard quantitative model, which accurately represents the mechanics and coordination of ion transporters in guard cells. With the guidance of OnGuard, we successfully engineered plants that overexpressed the main tonoplast Ca 2+ -ATPase gene, ACA11, which promotes stomatal opening and enhances plant growth. Surprisingly, these transgenic plants also exhibited improved drought tolerance due to reduced water loss through their stomata. Again, OnGuard assisted us in understanding the mechanism behind the unexpected stomatal behaviors observed in the ACA11 overexpressing plants. Our study revealed that the overexpression of ACA11 facilitated the accumulation of Ca 2+ in the vacuole, thereby influencing Ca 2+ storage and leading to an enhanced Ca 2+ elevation in response to abscisic acid. This regulatory cascade finely tunes stomatal responses, ultimately leading to enhanced drought tolerance. Our findings underscore the importance of tonoplast Ca 2+ -ATPase in manipulating stomatal behavior and improving drought tolerance. Furthermore, these results highlight the diverse functions of tonoplast-localized ACA11 in response to different conditions, emphasizing its potential for future applications in plant enhancement., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary. Here we show that PIP2;1 occurs in complex with SYNTAXIN OF PLANTS 132 (SYP132) together with the plasma membrane H + -ATPase AHA1 as evidenced through in vivo and in vitro analysis. SYP132 is a multifaceted vesicle trafficking protein, known to interact with AHA1 and promote endocytosis to impact growth and pathogen defence. Tracking native proteins in immunoblot analysis, we found that salinity stress enhances SYP132 interactions with PIP2;1 and PIP2;2 isoforms to promote redistribution of the water channels away from the plasma membrane. Concurrently, AHA1 binding within the SYP132-complex was significantly reduced under salinity stress and increased the density of AHA1 proteins at the plasma membrane in leaf tissue. Manipulating SYP132 function in Arabidopsis thaliana enhanced resilience to salinity stress and analysis in heterologous systems suggested that the SNARE influences PIP2;1 osmotic water permeability. We propose therefore that SYP132 coordinates AHA1 and PIP2;1 abundance at the plasma membrane and influences leaf hydraulics to regulate plant responses to abiotic stress signals., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

If the past century marked the birth of membrane transport as a focus for research in plants, the past 50 years has seen the field mature from arcane interest to a central pillar of plant physiology. Ion transport across plant membranes accounts for roughly 30% of the metabolic energy consumed by a plant cell, and it underpins virtually every aspect of plant biology, from mineral nutrition, cell expansion, and development to auxin polarity, fertilization, plant pathogen defense, and senescence. The means to quantify ion flux through individual transporters, even single channel proteins, became widely available as voltage clamp methods expanded from giant algal cells to the fungus Neurospora crassa in the 1970s and the cells of angiosperms in the 1980s. Here, I touch briefly on some key aspects of the development of modern electrophysiology with a focus on the guard cells of stomata, now without dispute the premier plant cell model for ion transport and its regulation. Guard cells have proven to be a crucible for many technical and conceptual developments that have since emerged into the mainstream of plant science. Their study continues to provide fundamental insights and carries much importance for the global challenges that face us today., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Stomata are microscopic pores at the surface of plant leaves that facilitate gaseous diffusion to support photosynthesis. The guard cells around each stoma regulate the pore aperture. Plants that carry out C 4 photosynthesis are usually more resilient than C 3 plants to stress, and their stomata operate over a lower dynamic range of CO 2 within the leaf. What makes guard cells of C 4 plants more responsive than those of C 3 plants? We used gas exchange and electrophysiology, comparing stomatal kinetics of the C 4 plant Gynandropsis gynandra and the phylogenetically related C 3 plant Arabidopsis thaliana. We found, with varying CO 2 and light, that Gynandropsis showed faster changes in stomata conductance and greater water use efficiency when compared with Arabidopsis. Electrophysiological analysis of the dominant K + channels showed that the outward-rectifying channels, responsible for K + loss during stomatal closing, were characterised by a greater maximum conductance and substantial negative shift in the voltage dependence of gating, indicating a reduced inhibition by extracellular K + and enhanced capacity for K + flux. These differences correlated with the accelerated stomata kinetics of Gynandropsis, suggesting that subtle changes in the biophysical properties of a key transporter may prove a target for future efforts to engineer C 4 stomatal kinetics., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

There are growing doubts about the true role of the common mycorrhizal networks (CMN or wood wide web) connecting the roots of trees in forests. We question the claims of a substantial carbon transfer from 'mother trees' to their offspring and nearby seedlings through the CMN. Recent reviews show that evidence for the 'mother tree concept' is inconclusive or absent. The origin of this concept seems to stem from a desire to humanize plant life but can lead to misunderstandings and false interpretations and may eventually harm rather than help the commendable cause of preserving forests. Two recent books serve as examples: The Hidden Life of Trees and Finding the Mother Tree., Competing Interests: Declaration of interests T. N. declares a conflicting interest as he owns shares in and works part time for the company Arevo AB that develops, produces, and markets organic fertilizers. The other authors have no conflicting interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Gas exchange across the stomatal pores of leaves is a focal point in studies of plant-environmental relations. Stomata regulate atmospheric exchange with the inner air spaces of the leaf. They open to allow CO 2 entry for photosynthesis and close to minimize water loss. Models that focus on the phenomenology of stomatal conductance generally omit the mechanics of the guard cells that regulate the pore aperture. The OnGuard platform fills this gap and offers a truly mechanistic approach with which to analyse stomatal gas exchange, whole-plant carbon assimilation and water-use efficiency. Previously, OnGuard required specialist knowledge of membrane transport, signalling and metabolism. Here we introduce OnGuard3e, a software package accessible to ecophysiologists and membrane biologists alike. We provide a brief guide to its use and illustrate how the package can be applied to explore and analyse stomatal conductance, assimilation and water use efficiencies, addressing a range of experimental questions with truly predictive outputs., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Stomatal pores facilitate gaseous exchange between the inner air spaces of the leaf and the atmosphere. As gatekeepers that balance CO 2 entry for photosynthesis against transpirational water loss, they are a focal point for efforts to improve crop performance, especially in the efficiency of water use, within the changing global environment. Until recently, engineering strategies had focused on stomatal conductance in the steady state. These strategies are limited by the physical constraints of CO 2 and water exchange such that gains in water-use efficiency (WUE) commonly come at a cost in carbon assimilation. Attention to stomatal speed and responsiveness circumvents these constraints and offers alternatives to enhancing WUE that also promise increases in carbon assimilation in the field., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The split-ubiquitin technology was developed over 20 years ago as an alternative to Gal4-based, yeast-two-hybrid methods to identify interacting protein partners. With the introduction of mating-based methods for split-ubiquitin screens, the approach has gained broad popularity because of its exceptionally high transformation efficiency, utility in working with full-length membrane proteins, and positive selection with little interference from spurious interactions. Recent advances now extend these split-ubiquitin methods to the analysis of interactions between otherwise soluble proteins and tripartite protein interactions., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Animal migration is highly sensitised to environmental cues, but plant dispersal is considered largely passive. The common dandelion, Taraxacum officinale , bears an intricate haired pappus facilitating flight. The pappus enables the formation of a separated vortex ring during flight; however, the pappus structure is not static but reversibly changes shape by closing in response to moisture. We hypothesised that this leads to changed dispersal properties in response to environmental conditions. Using wind tunnel experiments for flow visualisation, particle image velocimetry, and flight tests, we characterised the fluid mechanics effects of the pappus morphing. We also modelled dispersal to understand the impact of pappus morphing on diaspore distribution. Pappus morphing dramatically alters the fluid mechanics of diaspore flight. We found that when the pappus closes in moist conditions, the drag coefficient decreases and thus the falling velocity is greatly increased. Detachment of diaspores from the parent plant also substantially decreases. The change in detachment when the pappus closes increases dispersal distances by reducing diaspore release when wind speeds are low. We propose that moisture-dependent pappus-morphing is a form of informed dispersal allowing rapid responses to changing conditions., Competing Interests: MS, OZ, MS, CC, EK, MB, HZ, AB, EM, JB, IV, NN No competing interests declared, (© 2022, Seale et al.)

Stomata of plant leaves open to enable CO 2 entry for photosynthesis and close to reduce water loss via transpiration. Compared with photosynthesis, stomata respond slowly to fluctuating light, reducing assimilation and water use efficiency. Efficiency gains are possible without a cost to photosynthesis if stomatal kinetics can be accelerated. Here we show that clustering of the GORK channel, which mediates K + efflux for stomatal closure in the model plant Arabidopsis, arises from binding between the channel voltage sensors, creating an extended 'sensory antenna' for channel gating. Mutants altered in clustering affect channel gating to facilitate K + flux, accelerate stomatal movements and reduce water use without a loss in biomass. Our findings identify the mechanism coupling channel clustering with gating, and they demonstrate the potential for engineering of ion channels native to the guard cell to enhance stomatal kinetics and improve water use efficiency without a cost in carbon fixation., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Plants acclimate to various types of mechanical stresses through thigmomorphogenesis and alterations in their mechanical properties. Although resemblance between wind- and touch-induced responses provides the foundation for studies where wind influence was mimicked by mechanical perturbations, factorial experiments revealed that it is not always straightforward to extrapolate results induced by one type of perturbation to the other. To investigate whether wind-induced changes in morphological and biomechanical traits can be reproduced, we subjected Arabidopsis thaliana to two vectorial brushing treatments. Both treatments significantly affected the length, mechanical properties and anatomical tissue composition of the primary inflorescence stem. While some of the morphological changes were found to be in line with those induced by wind, changes in the mechanical properties exhibited opposite trends irrespective of the brushing direction. Overall, a careful design of the brushing treatment gives the possibility to obtain a closer match to wind-induced changes, including a positive tropic response., Competing Interests: The authors declare no conflicts of interest., (© The Author(s) 2022.)

Stomatal pores facilitate gaseous exchange between the inner air spaces of the leaf and the atmosphere. The pores open to enable CO 2 entry for photosynthesis and close to reduce transpirational water loss. How stomata respond to the environment has long attracted interest in modeling as a tool to understand the consequences for the plant and for the ecosystem. Models that focus on stomatal conductance for gas exchange make intuitive sense, but such models need also to connect with the mechanics of the guard cells that regulate pore aperture if we are to understand the 'decisions made' by stomata, their impacts on the plant and on the global environment., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Stomata of most plants close to preserve water when the demand for CO 2 by photosynthesis is reduced. Stomatal responses are slow compared with photosynthesis, and this kinetic difference erodes assimilation and water-use efficiency under fluctuating light. Despite a deep knowledge of guard cells that regulate the stoma, efforts to enhance stomatal kinetics are limited by our understanding of its control by foliar CO 2 . Guided by mechanistic modelling that incorporates foliar CO 2 diffusion and mesophyll photosynthesis, here we uncover a central role for endomembrane Ca 2+ stores in guard cell responsiveness to fluctuating light and CO 2 . Modelling predicted and experiments demonstrated a delay in Ca 2+ cycling that was enhanced by endomembrane Ca 2+ -ATPase mutants, altering stomatal conductance and reducing assimilation and water-use efficiency. Our findings illustrate the power of modelling to bridge the gap from the guard cell to whole-plant photosynthesis, and they demonstrate an unforeseen latency, or 'carbon memory', of guard cells that affects stomatal dynamics, photosynthesis and water-use efficiency., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl - transport rate of 442 (±17.7) Cl - /protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl - /protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB 12 PEO 8 ) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl - /photon and 0.28 (±0.04) Cl - /photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl - /protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl - /photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It has been proposed by some plant scientists that plants are cognitive and conscious organisms, although this is a minority view. Here we present a brief summary of some of the arguments against this view, followed by a critique of an article in this same issue of Biochemical and Biophysical Research Communications by Calvo, Baluska, and Trewavas (2020) that cites Integrated Information Theory (IIT) as providing additional support for plant consciousness. The authors base their argument on the assumptions that all cells are conscious and that consciousness is confined to life. However, IIT allows for consciousness in various nonliving systems, and thus does not restrict consciousness to living organisms. Therefore, IIT cannot be used to prove plant consciousness, for which there is neither empirical evidence nor support from other, neuron-based, theories of consciousness., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ferns appear in the fossil record some 200 Myr before angiosperms. However, as angiosperm-dominated forest canopies emerged in the Cretaceous period there was an explosive diversification of modern (leptosporangiate) ferns, which thrived in low, blue-enhanced light beneath angiosperm canopies. A mechanistic explanation for this transformative event in the diversification of ferns has remained elusive. We used physiological assays, transcriptome analysis and evolutionary bioinformatics to investigate a potential connection between the evolution of enhanced stomatal sensitivity to blue light in modern ferns and the rise of angiosperm-dominated forests in the geological record. We demonstrate that members of the largest subclade of leptosporangiate ferns, Polypodiales, have significantly faster stomatal response to blue light than more ancient fern lineages and a representative angiosperm. We link this higher sensitivity to levels of differentially expressed genes in blue-light signaling, particularly in the cryptochrome (CRY) signaling pathway. Moreover, CRYs of the Polypodiales examined show gene duplication events between 212.9-196.9 and 164.4-151.8 Ma, when angiosperms were emerging, which are lacking in other major clades of extant land plants. These findings suggest that evolution of stomatal blue-light sensitivity helped modern ferns exploit the shady habitat beneath angiosperm forest canopies, fueling their Cretaceous hyperdiversification., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

Claims that plants have conscious experiences have increased in recent years and have received wide coverage, from the popular media to scientific journals. Such claims are misleading and have the potential to misdirect funding and governmental policy decisions. After defining basic, primary consciousness, we provide new arguments against 12 core claims made by the proponents of plant consciousness. Three important new conclusions of our study are (1) plants have not been shown to perform the proactive, anticipatory behaviors associated with consciousness, but only to sense and follow stimulus trails reactively; (2) electrophysiological signaling in plants serves immediate physiological functions rather than integrative-information processing as in nervous systems of animals, giving no indication of plant consciousness; (3) the controversial claim of classical Pavlovian learning in plants, even if correct, is irrelevant because this type of learning does not require consciousness. Finally, we present our own hypothesis, based on two logical assumptions, concerning which organisms possess consciousness. Our first assumption is that affective (emotional) consciousness is marked by an advanced capacity for operant learning about rewards and punishments. Our second assumption is that image-based conscious experience is marked by demonstrably mapped representations of the external environment within the body. Certain animals fit both of these criteria, but plants fit neither. We conclude that claims for plant consciousness are highly speculative and lack sound scientific support.

Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic "currency" of the membrane. The dynamics of membrane voltage-so-called action, systemic, and variation potentials-have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here, we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport-an electrical "substrate"-and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signaling via second messengers that rely on ion flux, including cytosolic free Ca2+, H+, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plants are known to exhibit a thigmomorphogenetic response to mechanical stimuli by altering their morphology and mechanical properties. Wind is widely perceived as mechanical stress and in many experiments its influence is simulated by applying mechanical perturbations. However, it is known that wind-induced effects on plants can differ and at times occur even in the opposite direction compared with those induced by mechanical perturbations. In the present study, the long-term response of Arabidopsis thaliana to a constant unidirectional wind was investigated. We found that exposure to wind resulted in a positive anemotropic response and in significant alterations to Arabidopsis morphology, mechanical properties, and anatomical tissue organization that were associated with the plant's strategy of acclimation to a windy environment. Overall, the observed response of Arabidopsis to wind differs significantly from previously reported responses of Arabidopsis to mechanical perturbations. The presented results suggest that the response of Arabidopsis is sensitive to the type of mechanical stimulus applied, and that it is not always straightforward to simulate one type of perturbation by another., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Activation of plasma membrane (PM) H+-ATPase activity is crucial in guard cells to promote light-stimulated stomatal opening, and in growing organs to promote cell expansion. In growing organs, SMALL AUXIN UP RNA (SAUR) proteins inhibit the PP2C.D2, PP2C.D5, and PP2C.D6 (PP2C.D2/5/6) phosphatases, thereby preventing dephosphorylation of the penultimate phosphothreonine of PM H+-ATPases and trapping them in the activated state to promote cell expansion. To elucidate whether SAUR-PP2C.D regulatory modules also affect reversible cell expansion, we examined stomatal apertures and conductances of Arabidopsis thaliana plants with altered SAUR or PP2C.D activity. Here, we report that the pp2c.d2/5/6 triple knockout mutant plants and plant lines overexpressing SAUR fusion proteins exhibit enhanced stomatal apertures and conductances. Reciprocally, saur56 saur60 double mutants, lacking two SAUR genes normally expressed in guard cells, displayed reduced apertures and conductances, as did plants overexpressing PP2C.D5. Although altered PM H+-ATPase activity contributes to these stomatal phenotypes, voltage clamp analysis showed significant changes also in K+ channel gating in lines with altered SAUR and PP2C.D function. Together, our findings demonstrate that SAUR and PP2C.D proteins act antagonistically to facilitate stomatal movements through a concerted targeting of both ATP-dependent H+ pumping and channel-mediated K+ transport., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The mechanical properties of plants are important for understanding plant biomechanics and for breeding new plants that can survive in challenging environments. Thus, accurate and reliable methods are required for the determination of mechanical properties such as stiffness and Young's modulus of elasticity. Much attention has been paid to the application of static methods to plants, while dynamic methods have received considerably less attention. In the present study, a dynamic forced vibration method for mechanical characterisation of Arabidopsis inflorescence stems was developed and validated against the conventional three-point bending test. Compared to dynamic tests based on free vibration, the current method allows to determine simultaneously more than one natural frequency, thus increasing the overall accuracy of the results. In addition, this method can be applied to the top parts of the stems that are more flexible, and where application of the three-point bending test is often limited. To demonstrate one of the potential applications of this method, it was applied to evaluate the influence of turgor pressure on the mechanical properties of Arabidopsis stems. Overall, the new dynamic testing approach has been shown to provide reliable data for the local mechanical properties along the Arabidopsis inflorescence stem., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Plants utilising crassulacean acid metabolism (CAM) concentrate CO 2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C 3 and C 4 species, CAM stomata open at night for the mesophyll to fix CO 2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO 2 concentrations within the leaf behind closed stomata for refixation by RuBisCO. CO 2 may contribute to stomatal closure but additional mechanisms, plausibly including Mal activation of anion channels, ensure closure in the light. In the CAM species Kalanchoë fedtschenkoi, we found that guard cell anion channel activity, recorded under voltage clamp, follows KfSLAC1 and KfALMT12 transcript abundance, declining to near zero by the end of the light period. Unexpectedly, however, we found that extracellular Mal inhibited the anion current of Kalanchoë guard cells, both in wild-type and RNAi mutants with impaired Mal metabolism. We conclude that the diurnal cycle of anion channel gene transcription, rather than the physiological signal of Mal release, is a key factor in the inverted CAM stomatal cycle., (© 2020 University of Glasgow New Phytologist © 2020 New Phytologist Trust.)

Vesicle exocytosis underpins signaling and development in plants and is vital for cell expansion. Vesicle tethering and fusion are thought to occur sequentially, with tethering mediated by the exocyst and fusion driven by assembly of soluble NSF attachment protein receptor (SNARE) proteins from the vesicle membrane (R-SNAREs or vesicle-associated membrane proteins [VAMPs]) and the target membrane (Q-SNAREs). Interactions between exocyst and SNARE protein complexes are known, but their functional consequences remain largely unexplored. We now identify a hierarchy of interactions leading to secretion in Arabidopsis ( Arabidopsis thaliana ). Mating-based split-ubiquitin screens and in vivo Förster resonance energy transfer analyses showed that exocyst EXO70 subunits bind preferentially to cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distribution and suppressed vesicle traffic similarly to the dominant-negative truncated protein SYP121 ΔC , which blocks secretion at the plasma membrane. These phenotypes are consistent with the epistasis of exo70A1 in the exo70A1 syp121 double mutant, which shows decreased growth similar to exo70A1 single mutants. However, the exo70A1 vamp721 mutant showed a strong, synergy, suppressing growth and cell expansion beyond the phenotypic sum of the two single mutants. These data are best explained by a hierarchy of SNARE recruitment to the exocyst at the plasma membrane, dominated by the R-SNARE and plausibly with the VAMP721 longin domain as a nexus for binding., (© 2020 American Society of Plant Biologists. All rights reserved.)

Starch in Arabidopsis ( Arabidopsis thaliana ) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and β-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H + -ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H + , K + , and Cl - transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport., (© 2020 The authors.)

Plant membrane transport, like transport across all eukaryotic membranes, is highly non-linear and leads to interactions with characteristics so complex that they defy intuitive understanding. The physiological behaviour of stomatal guard cells is a case in point in which, for example, mutations expected to influence stomatal closing have profound effects on stomatal opening and manipulating transport across the vacuolar membrane affects the plasma membrane. Quantitative mathematical modelling is an essential tool in these circumstances, both to integrate the knowledge of each transport process and to understand the consequences of their manipulation in vivo. Here, we outline the OnGuard modelling environment and its use as a guide to predicting the emergent properties arising from the interactions between non-linear transport processes. We summarise some of the recent insights arising from OnGuard, demonstrate its utility in interpreting stomatal behaviour, and suggest ways in which the OnGuard environment may facilitate 'reverse-engineering' of stomata to improve water use efficiency and carbon assimilation., (© 2020 The Author(s).)

Cell expansion requires that ion transport and secretory membrane traffic operate in concert. Evidence from Arabidopsis ( Arabidopsis thaliana ) indicates that such coordination is mediated by physical interactions between subsets of so-called SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which drive the final stages of vesicle fusion, and K + channels, which facilitate uptake of the cation to maintain cell turgor pressure as the cell expands. However, the sequence of SNARE binding with the K + channels and its interweaving within the events of SNARE complex assembly for exocytosis remains unclear. We have combined protein-protein interaction and electrophysiological analyses to resolve the binding interactions of the hetero-oligomeric associations. We find that the RYxxWE motif, located within the voltage sensor of the K + channels, is a nexus for multiple SNARE interactions. Of these, K + channel binding and its displacement of the regulatory protein SEC11 is critical to prime the Qa-SNARE SYP121. Our results indicate a stabilizing role for the Qbc-SNARE SNAP33 in the Qa-SNARE transition to SNARE complex assembly with the R-SNARE VAMP721. They also suggest that, on its own, the R-SNARE enters an anomalous binding mode with the channels, possibly as a fail-safe measure to ensure a correct binding sequence. Thus, we suggest that SYP121 binding to the K + channels serves the role of a primary trigger to initiate assembly of the secretory machinery for exocytosis., (© 2019 The author(s).)

Models of guard cell dynamics, built on the OnGuard platform, have provided quantitative insights into stomatal function, demonstrating substantial predictive power. However, the kinetics of stomatal opening predicted by OnGuard models were threefold to fivefold slower than observed in vivo. No manipulations of parameters within physiological ranges yielded model kinetics substantially closer to these data, thus highlighting a missing component in model construction. One well-documented process influencing stomata is the constraining effect of the surrounding epidermal cells on guard cell volume and stomatal aperture. Here, we introduce a mechanism to describe this effect in OnGuard2 constructed around solute release and a decline in turgor of the surrounding cells and its subsequent recovery during stomatal opening. The results show that this constraint-relaxation-recovery mechanism in OnGuard2 yields dynamics that are consistent with experimental observations in wild-type Arabidopsis, and it predicts the altered opening kinetics of ost2 H + -ATPase and slac1 Cl - channel mutants. Thus, incorporating solute flux of the surrounding cells implicitly through their constraint on guard cell expansion provides a satisfactory representation of stomatal kinetics, and it predicts a substantial and dynamic role for solute flux across the apoplastic space between the guard cells and surrounding cells in accelerating stomatal kinetics., (© 2019 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.)

SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion. Much of vesicle traffic at the Arabidopsis ( Arabidopsis thaliana ) plasma membrane is subject to the Sec1/Munc18 protein SEC11, which, along with plasma membrane K + channels, selectively binds with the SNARE SYP121 to regulate its assembly in complex. How SEC11 binding is coordinated with the K + channels is poorly understood, as both SEC11 and the channels are thought to compete for the same SNARE binding site. Here, we identify a second binding motif within the N terminus of SYP121 and demonstrate that this motif affects SEC11 binding independently of the F 9 xRF motif that is shared with the K + channels. This second, previously unrecognized motif is centered on residues R 20 R 21 of SYP121 and is essential for SEC11 interaction with SYP121. Mutation of the R 20 R 21 motif blocked vesicle traffic without uncoupling the effects of SYP121 on solute and K + uptake associated with the F 9 xRF motif; the mutation also mimicked the effects on traffic block observed on coexpression of the dominant-negative SEC11 Δ149 fragment. We conclude that the R 20 R 21 motif represents a secondary site of interaction for the Sec1/Munc18 protein during the transition of SYP121 from the occluded to the open conformation that leads to SNARE complex assembly., (© 2019 The author(s). All Rights Reserved.)

Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K + channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K + conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii , and the moss Physcomitrella patens Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The photoreceptor UVR8 has a pivotal role in mediating plant responses to UV-B wavelengths. Dimeric UVR8 dissociates into monomers following UV-B photoreception, and there is evidence that this process is accompanied by conformational changes that may facilitate interaction of UVR8 with other proteins to initiate signaling. Hence monitoring UVR8 dimer/monomer status and conformation is key to understanding UVR8 action. Here we have used Fluorescence Resonance Energy Transfer (FRET) to study these processes in both wild-type and mutant UVR8 proteins in vivo. UVR8 was fused to GFP and mCherry at the C- and N-termini, respectively and both the FRET efficiency and loss of GFP fluorescence after photobleaching were measured. In addition, measurements were made for UVR8 fused to either GFP or mCherry to eliminate intra-molecular FRET signals. The results indicate that dissociation of UVR8 dimer to monomer principally accounts for the loss of FRET signal for wild-type UVR8 and there is little evidence of a contribution from conformational change in vivo. Examination of plants expressing UVR8W285F and UVR8D96N,D107N are consistent with these mutant proteins being constitutively dimeric and monomeric, respectively. The methods employed here will be valuable for monitoring UVR8 dimer/monomer status in vivo in relation to signaling, and will facilitate characterization of dimer/monomer status and conformation of further UVR8 mutants.

SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion and contribute to homoeostasis, pathogen defense, cell expansion, and growth in plants. In Arabidopsis ( Arabidopsis thaliana ), two homologous Qa-SNAREs, SYNTAXIN OF PLANTS121 (SYP121) and SYP122, facilitate the majority of secretory traffic to the plasma membrane, and the single mutants are indistinguishable from wild-type plants in the absence of stress, implying a redundancy in their functions. Nonetheless, several studies suggest differences among the secretory cargo of these SNAREs. To address this issue, we conducted an analysis of the proteins secreted by cultured wild-type, syp121 , and syp122 mutant Arabidopsis seedlings. Here, we report that a number of cargo proteins were associated differentially with traffic mediated by SYP121 and SYP122. The data also indicated important overlaps between the SNAREs. Therefore, we conclude that the two Qa-SNAREs mediate distinct but complementary secretory pathways during vegetative plant growth., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Vesicle traffic is tightly coordinated with ion transport for plant cell expansion through physical interactions between subsets of vesicle-trafficking (so-called SNARE) proteins and plasma membrane Kv channels, including the archetypal inward-rectifying K + channel, KAT1 of Arabidopsis. Ion channels open and close rapidly over milliseconds, whereas vesicle fusion events require many seconds. Binding has been mapped to conserved motifs of both the Kv channels and the SNAREs, but knowledge of the temporal kinetics of their interactions, especially as it might relate to channel gating and its coordination with vesicle fusion remains unclear. Here, we report that the SNARE SYP121 promotes KAT1 gating through a persistent interaction that alters the stability of the channel, both in its open and closed states. We show, too, that SYP121 action on the channel open state requires SNARE anchoring in the plasma membrane. Our findings indicate that SNARE binding confers a conformational bias that encompasses the microscopic kinetics of channel gating, with leverage applied through the SNARE anchor in favour of the open channel., (© 2018 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.)