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Stomatal pores facilitate gaseous exchange between the inner air spaces of the leaf and the atmosphere. The pores open to enable CO2 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.

Stomata of most plants close to preserve water when the demand for CO2 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 CO2. Guided by mechanistic modelling that incorporates foliar CO2 diffusion and mesophyll photosynthesis, here we uncover a central role for endomembrane Ca2+ stores in guard cell responsiveness to fluctuating light and CO2. Modelling predicted and experiments demonstrated a delay in Ca2+ cycling that was enhanced by endomembrane Ca2+-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.

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.

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., The Plant Cell, 32 (7), ISSN:1040-4651, ISSN:1531-298X, ISSN:1532-298X

Stomata of plant leaves open to enable CO

Stomata of most plants close to preserve water when the demand for CO

Serial membranes that operate through a common compartment, the cytosol for transport at the plasma membrane and tonoplast, are intrinsically connected and communicate through this pool of solutes.

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., Models of stomatal function build on the OnGuard platform and our deep knowledge of guard cell transport and metabolism have provided quantitative insights into stomatal function. However, the kinetics of stomatal opening predicted by OnGuard models have consistently failed to match the accelerated opening observed in vivo, thus highlighting a missing component in model construction. Incorporating explicitly a solute flux‐based mechanism of constraint relaxation and recovery between cells surrounding the stoma and the stomatal guard cells accommodates the accelerated stomatal opening kinetics observed in vivo and accurately predicts the slowed kinetics of stomatal opening in guard cell transport mutants.

Significance The projected increase in drought severity and duration worldwide poses a significant threat to the health of terrestrial ecosystems. We reveal that unique genetic features of desiccation sensing and protection in streptophyte algae not only distinguish them from chlorophyte algae, but also represent a crucial evolutionary step that may have facilitated colonization and subsequent diversification of terrestrial habitats. We demonstrate the evolutionary significance of a molecular mechanism underlying how plants sense drought stress via the coordination of chloroplast retrograde signaling to trigger the closure of stomata, protecting vital photosynthetic tissue. Our findings constitute a significant step forward in understanding the evolution of plant drought tolerance, contributing to the diversification of terrestrial plant communities through past global climate transitions., 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.

If we want to understand how the environment has shaped the appearance and behavior of living creatures we need to compare groups of individuals that differ in genetic make-up and environment experience. For complex phenotypic features, such as body posture or facial expression in humans, comparison is not straightforward because some of the contributing factors cannot easily be quantified or averaged across individuals. Therefore, computational methods are used to reconstruct representative prototypes using a range of algorithms for filling in missing information and calculating means. The same problem applies to the root system architecture (RSA) of plants. Several computer programs are available for extracting numerical data from root images but they usually do not offer customized data analysis or visual reconstruction of RSA. We have developed Root-VIS, a free software tool, which facilitates the determination of means and variance of many different RSA features across user-selected sets of root images. Furthermore, Root-VIS offers several options to generate visual reconstructions of root systems from the averaged data to enable screening and modeling. We have confirmed the suitability of Root-VIS, combined with a new version of EZ-Rhizo, for the rapid characterization of genotype-environment interactions and gene discovery through genome-wide association studies in Arabidopsis (Arabidopsis thaliana).

The characteristics of transport across plant membranes, like all eukaryotic membranes, is highly non-linear. The complexity inherent to such characteristics defies intuitive understanding and, in these circumstances, quantitative mathematical modelling is essential as a tool, both to integrate the detailed knowledge of individual transporters and to extract the properties emerging from their interactions. As the first, fully-integrated and quantitative modelling environment for the study of ion transport dynamics in a plant cell, the OnGuard platform offers a unique tool for examining such emergent properties associated with guard cell metabolism and ion transport at the plasma membrane and tonoplast. The OnGuard platform has already yielded details guiding phenotypic and mutational studies. These advances represent key steps towards ‘reverse-engineering’ of stomatal physiology to improve water use efficiency and carbon assimilation, based on rational design and testing in simulation. The newly expanded platform, OnGuard2, bridges the micro-macro gap in stomatal models, coupling whole-plant transpiration to the molecular functionalities of the guard cell. Here we set out guidelines for use of OnGuard2 and outline a standardized approach that will enable users to advance quickly in applying the platform in classroom and laboratory situations.

Stomatal movements depend on the transport and metabolism of osmotic solutes that drive reversible changes in guard cell volume and turgor. These processes are defined by a deep knowledge of the identities of the key transporters and of their biophysical and regulatory properties, and have been modeled successfully with quantitative kinetic detail at the cellular level. Transpiration of the leaf and canopy, by contrast, is described by quasilinear, empirical relations for the inputs of atmospheric humidity, CO

It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell.

The physical requirement for charge to balance across biological membranes means that the transmembrane transport of each ionic species is interrelated, and manipulating solute flux through any one transporter will affect other transporters at the same membrane, often with unforeseen consequences. The OnGuard systems modeling platform has helped to resolve the mechanics of stomatal movements, uncovering previously unexpected behaviors of stomata. To date, however, the manual approach to exploring model parameter space has captured little formal information about the emergent connections between parameters that define the most interesting properties of the system as a whole. Here, we introduce global sensitivity analysis to identify interacting parameters affecting a number of outputs commonly accessed in experiments in Arabidopsis (

Abscisic acid (ABA)-driven stomatal regulation reportedly evolved after the divergence of ferns, during the early evolution of seed plants approximately 360 million years ago. This hypothesis is based on the observation that the stomata of certain fern species are unresponsive to ABA, but exhibit passive hydraulic control. However, ABA-induced stomatal closure was detected in some mosses and lycophytes. Here, we observed that a number of ABA signaling and membrane transporter protein families diversified over the evolutionary history of land plants. The aquatic ferns Azolla filiculoides and Salvinia cucullata have representatives of 23 families of proteins orthologous to those of Arabidopsis (Arabidopsis thaliana) and all other land plant species studied. Phylogenetic analysis of the key ABA signaling proteins indicates an evolutionarily conserved stomatal response to ABA. Moreover, comparative transcriptomic analysis has identified a suite of ABA-responsive genes that differentially expressed in a terrestrial fern species, Polystichum proliferum. These genes encode proteins associated with ABA biosynthesis, transport, reception, transcription, signaling, and ion and sugar transport, which fit the general ABA signaling pathway constructed from Arabidopsis and Hordeum vulgare. The retention of these key ABA-responsive genes could have had a profound effect on the adaptation of ferns to dry conditions. Furthermore, stomatal assays have shown the primary evidence for ABA-induced closure of stomata in two terrestrial fern species P. proliferum and Nephrolepis exaltata. In summary, we report, to our knowledge, new molecular and physiological evidence for the presence of active stomatal control in ferns.

Grasses began to diversify in the late Cretaceous Period and now dominate more than one third of global land area, including three-quarters of agricultural land. We hypothesize that their success is likely attributed to the evolution of highly responsive stomata capable of maximizing productivity in rapidly changing environments. Grass stomata harness the active turgor control mechanisms present in stomata of more ancient plant lineages, maximizing several morphological and developmental features to ensure rapid responses to environmental inputs. The evolutionary development of grass stomata appears to have been a gradual progression. Therefore, understanding the complex structures, developmental events, regulatory networks, and combinations of ion transporters necessary to drive rapid stomatal movement may inform future efforts towards breeding new crop varieties.

Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K+ uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H+ load on the cytosol and promoted Ca2+ influx to elevate cytosolic pH (pHi) and free cytosolic Ca2+ concentration ([Ca2+]i), in turn regulating the K+ channels. We have confirmed these predictions, measuring pHi and [Ca2+]i in vivo, and report that experimental manipulation of pHi and [Ca2+]i is sufficient to recover K+ channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K+ channels. Additionally, these findings underscore the importance of H+-coupled anion transport for pHi homeostasis.

Stomatal guard cells play a key role in gas exchange for photosynthesis while minimizing transpirational water loss from plants by opening and closing the stomatal pore. Foliar gas exchange has long been incorporated into mathematical models, several of which are robust enough to recapitulate transpirational characteristics at the whole-plant and community levels. Few models of stomata have been developed from the bottom up, however, and none are sufficiently generalized to be widely applicable in predicting stomatal behavior at a cellular level. We describe here the construction of computational models for the guard cell, building on the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis. The OnGuard software was constructed with the HoTSig library to incorporate explicitly all of the fundamental properties for transporters at the plasma membrane and tonoplast, the salient features of osmolite metabolism, and the major controls of cytosolic-free Ca2+ concentration and pH. The library engenders a structured approach to tier and interrelate computational elements, and the OnGuard software allows ready access to parameters and equations ‘on the fly’ while enabling the network of components within each model to interact computationally. We show that an OnGuard model readily achieves stability in a set of physiologically sensible baseline or Reference States; we also show the robustness of these Reference States in adjusting to changes in environmental parameters and the activities of major groups of transporters both at the tonoplast and plasma membrane. The following article addresses the predictive power of the OnGuard model to generate unexpected and counterintuitive outputs.

The dynamics of stomatal movements and their consequences for photosynthesis and transpirational water loss have long been incorporated into mathematical models, but none have been developed from the bottom up that are widely applicable in predicting stomatal behavior at a cellular level. We previously established a systems dynamic model incorporating explicitly the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis. Here we describe the behavior of the model in response to experimentally documented changes in primary pump activities and malate (Mal) synthesis imposed over a diurnal cycle. We show that the model successfully recapitulates the cyclic variations in H+, K+, Cl−, and Mal concentrations in the cytosol and vacuole known for guard cells. It also yields a number of unexpected and counterintuitive outputs. Among these, we report a diurnal elevation in cytosolic-free Ca2+ concentration and an exchange of vacuolar Cl− with Mal, both of which find substantiation in the literature but had previously been suggested to require additional and complex levels of regulation. These findings highlight the true predictive power of the OnGuard model in providing a framework for systems analysis of stomatal guard cells, and they demonstrate the utility of the OnGuard software and HoTSig library in exploring fundamental problems in cellular physiology and homeostasis.

To date the use of fluorescent reporter constructs in analysing membrane transport has been limited primarily to cell lines expressing stably either the tagged transporter protein(s) or markers to identify lineages of interest. Strategies for transient expression have yet to be exploited in transport analysis, despite their wide application in cellular imaging studies. Here we describe a Gateway-compatible, bicistronic vector, incorporating the constitutive Ubiqutin-10 gene promoter of Arabidopsis that gives prolonged expression after transient transformation and enables fluorescence marking of cells without a fusion construct. We show that Arabidopsis root epidermal cells are readily transformed by co-cultivation with Agrobacterium and are tractable for quantitative electrophysiological analysis. As a proof of principle, we transiently transformed Arabidopsis with the bicistronic vector carrying GFP as the fluorescent marker and, separately, the integral plasma membrane protein SYP121 essential for the inward K+ channel current. We demonstrate that transient expression of SYP121 in syp121 mutant plants is sufficient to rescue the K+ current in vivo. The combination of transient expression and use of the bicistronic vector promises significant advantages for studies of membrane transport and nutrient acquisition in roots

The SNARE (for soluble N-ethylmaleimide–sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K+ channel subunit KC1 and forms a tripartite complex with a second K+ channel subunit, AKT1, to control channel gating and K+ transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein–protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K+ channel gating when heterologously expressed, and that rescue of KC1-associated K+ current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE–ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.

In guard cells, activation of anion channels (I(anion)) is an early event leading to stomatal closure. Activation of I(anion) has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). However, the dynamics of the action of [Ca(2+)](i) on I(anion) has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca(2+)](i) dynamics of I(anion) in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca(2+)](i) using Fura-2 fluorescence imaging. We found that I(anion) rises with [Ca(2+)](i) only at concentrations substantially above the mean resting value of 125 +/- 13 nm, yielding an apparent K(d) of 720 +/- 65 nm and a Hill coefficient consistent with the binding of three to four Ca(2+) ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I(anion) activity, but without a dependence of the current on [Ca(2+)](i). The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca(2+)](i) sensitivity of I(anion), displacing the apparent K(d) for [Ca(2+)](i) to 573 +/- 38 nm. These findings support previous evidence for different modes of regulation for I(anion), only one of which depends on [Ca(2+)](i), and they underscore an independence of [Ca(2+)](i) from protein (de-)phosphorylation in controlling I(anion). Most importantly, our results demonstrate a significant displacement of I(anion) sensitivity to higher [Ca(2+)](i) compared with that of the guard cell K(+) channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.

The root system is essential for the growth and development of plants. In addition to anchoring the plant in the ground, it is the site of uptake of water and minerals from the soil. Plant root systems show an astonishing plasticity in their architecture, which allows for optimal exploitation of diverse soil structures and conditions. The signalling pathways that enable plants to sense and respond to changes in soil conditions, in particular nutrient supply, are a topic of intensive research, and root system architecture (RSA) is an important and obvious phenotypic output. At present, the quantitative description of RSA is labour intensive and time consuming, even using the currently available software, and the lack of a fast RSA measuring tool hampers forward and quantitative genetics studies. Here, we describe EZ-Rhizo: a Windows-integrated and semi-automated computer program designed to detect and quantify multiple RSA parameters from plants growing on a solid support medium. The method is non-invasive, enabling the user to follow RSA development over time. We have successfully applied EZ-Rhizo to evaluate natural variation in RSA across 23 Arabidopsis thaliana accessions, and have identified new RSA determinants as a basis for future quantitative trait locus (QTL) analysis.

Highlights • A model scaling stoma behaviour at leaf level is proposed. • The model is tested by reproducing natural variations of light intensity. • Stomatal aperture and photosynthesis decrease as a function of sugar accumulation. • Leaf anatomy influences the rapidity of stomatal conductance. • The model dissects intrinsic water use efficiency into traits of interest., Intrinsic water use efficiency (Wi), the ratio of net CO2 assimilation (A) over stomatal conductance to water vapour (gs), is a complex trait used to assess plant performance. Improving Wi could lead in theory to higher productivity or reduced water usage by the plant, but the physiological traits for improvement and their combined effects on Wi have not been clearly identified. Under fluctuating light intensity, the temporal response of gs is an order of magnitude slower than A, which results in rapid variations in Wi. Compared to traditional approaches, our new model scales stoma behaviour at the leaf level to predict gs and A during a diurnal period, reproducing natural fluctuations of light intensity, in order to dissect Wi into traits of interest. The results confirmed the importance of stomatal density and photosynthetic capacity on Wi but also revealed the importance of incomplete stomatal closure under dark conditions as well as stomatal sensitivity to light intensity. The observed continuous decrease of A and gs over the diurnal period was successfully described by negative feedback of the accumulation of photosynthetic products. Investigation into the impact of leaf anatomy on temporal responses of A, gs and Wi revealed that a high density of stomata produces the most rapid response of gs but may result in lower Wi.

Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

Recent work has indicated that nitric oxide (NO) and its synthesis are important elements of signal cascades in plant-pathogen defence, and are a prerequisite for drought and abscisic acid (ABA) responses in Arabidopsis thaliana and Vicia faba guard cells. NO regulates inward-rectifying K + channels and Cl∼ channels of Vicia guard cells via intracellular Ca 2+ release. However, its integration with related signals, including the actions of serine-threonine protein kinases, is less well defined. We report here that the elevation of cytosolic-free [Ca 2+ ] ([Ca 2+ ] i ) mediated by NO in guard cells is reversibly inhibited by the broad-range protein kinase antagonists staurosporine and K252A, but not by the tyrosine kinase antagonist genistein. The effects of kinase antagonism translate directly to a loss of NO-sensitivity of the inward-rectifying K + channels and background (Cl - channel) current, and to a parallel loss in sensitivity of the K + channels to ABA. These results demonstrate that NO-dependent signals can be modulated through protein phosphorylation upstream of intracellular Ca 2+ release, and they implicate a target for protein kinase control in ABA signalling that feeds into NO-dependent Ca 2+ release.

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca 2+ -sensitive ion channels of Vicia guard cells by promoting Ca 2+ release from intracellular stores to raise cytosolic-free [Ca 2+ ]. NO-sensitive Ca 2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca 2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca 2+ channels and Ca 2+ -insensitive K + channels, and NO scavengers failed to block the activation of these K + channels evoked by ABA. These results place NO action firmly within one branch of the Ca 2+ -signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.

Transport across eukaryotic cell membranes, including those of plant cells, is so complex that it defies intuitive understanding. Quantitative systems modelling is essential in these circumstances, to test our understanding of the system through the experimental scrutiny of predictions. OnGuard is the first, fully integrated and quantitative modelling package developed for the study of cellular homoeostasis in guard cells and its relevance to stomatal aperture dynamics. It offers a unique tool for exploring the properties arising from interactions between plasma membrane and tonoplast transporters with the processes involved in the control of pH, ionic and neutral osmolite concentrations, membrane potentials and ion buffering in cytoplasm and vacuole. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, opening the way towards ‘reverse-engineering’ of stomatal guard cells with the aim of improving water-use efficiency. We focus here on the fundamental insights OnGuard models offer for understanding the physiological phenomena of oscillations in membrane voltage, cytosolic-free Ca2+ concentration, and their roles in stomatal movements. OnGuard models faithfully reproduce differences in stomatal closure with oscillation frequency, much as observed in vivo and including an optimal cycle period. Analysis shows that these oscillations arise from the balance of transport activities at the plasma membrane and tonoplast to generate a range of resonant frequencies. The lowest frequencies are of sufficient duration to permit substantial changes in cytosolic-free Ca2+ concentration. Thus, we demonstrate the oscillations as an emergent property of the system of transport at the two dominant membranes of the guard cell.

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) by means of Ca 2+ entry from outside and release from intracellular stores. The mechanism(s) for Ca 2+ flux across the plasma membrane is poorly understood. Because [Ca 2+ ] i increases are voltage-sensitive, we suspected a Ca 2+ channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA. We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba 2+ as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 ± 0.4 pS and a high selectivity for Ba 2+ (and Ca 2+ ) over K + and Cl − . Unlike other Ca 2+ channels characterized to date, these channels rectified strongly toward negative voltages with an open probability ( P o ) that increased with [Ba 2+ ] outside and decreased roughly 10-fold when [Ca 2+ ] i was raised from 200 nM to 2 μM. Adding 20 μM ABA increased P o , initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P o with periods near 50 s. A similar, but delayed response was observed in 0.1 μM ABA. These results identify a Ca 2+ -selective channel that can account for Ca 2+ influx and increases in [Ca 2+ ] i triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca 2+ channel control.

The discovery of the START family of abscisic acid (ABA) receptors places these proteins at the front of a protein kinase/phosphatase signal cascade that promotes stomatal closure. The connection of these receptors to Ca2+ signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive pyr1/pyl1/pyl2/pyl4 quadruple mutant. One difficulty is that flux through plasma membrane Ca2+ channels and Ca2+ release from endomembrane stores coordinately elevate cytosolic free Ca2+ concentration ([Ca2+]i) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca2+ channels at the plasma membrane of intact guard cells of Arabidopsis (Arabidopsis thaliana). We have used this method to resolve the loss of ABA-evoked Ca2+ channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca2+]i increases in vivo. The basal activity of Ca2+ channels was not affected in the mutant; raising the concentration of Ca2+ outside was sufficient to promote Ca2+ entry, to inactivate current carried by inward-rectifying K+ channels and to activate current carried by the anion channels, both of which are sensitive to [Ca2+]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca2+ channels and trigger stomatal closure in the mutant. These results offer direct evidence of PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca2+ channels through ROS, thus affecting [Ca2+]i and its regulation of stomatal closure.

The conformation of peptides when bound to different HLA class II molecules is of interest in the study of specificity and function of responding T cells. Here, we report the investigation of the HLA-DR binding profiles of an immunodominant and HLA-promiscuous mycobacterial peptide, p38G. Its binding affinities were found to be high for DR1, moderate for DR2, DR7 and DR8, low for DR4, DR5, DR6 and DR9, and below detection for DR3. The minimum peptide length required for binding was, in the majority of cases, nine residues and 11 in two instances (DR2 and DR4). Peptide binding to DR2 was attributed to the DRB5*0101 and not to the DRB1*1501 gene product. Substitution analysis of the amino acid residues involved in binding to DR1 and DRB5*0101 identified F-354 as the common primary contact residue (P1), while allele-specific differences were found in positions P4, P6 and in the C-terminal anchor residue (valine at P9 for DR1 or lysine at P10 for DRB5*0101). Computer-assisted evaluation of these empirical data produced a molecular model, suggesting that the peptide binds to DR1 in an elongated conformation, similar to that of other peptide MHC class II complexes. In contrast, the DRB5*0101 bound peptide is likely to be kinked, which so far was considered characteristic only for peptides within MHC class I complexes. The different conformations imposed on the same peptide by distinct HLA alleles may represent an important mechanism for the control of T cell responses.

The structure of tetrahydridobis[1,2-bis(diphenylphosphino)ethane-P,P']molybdenum(IV), [MoH 4 (dppe) 2 ] (dppe = Ph 2 PCH 2 CH 2 PPh 2 ), is reported as the tetrahydrofuran solvate. The molecule has dodecahedral geometry with the four P atoms in the B sites and the hydrides in the A sites. Mean Mo-P and Mo-H distances are 2.420 (3) and 1.67 (2) A, respectively

Voltage clamp is one of the key techniques for the dissection, identification, and monitoring of ion transporters in plant cells. Voltage clamp-based research work on salinity stress in plants enables the characterization of many plant ATP-dependent pumps, ion channels, and ion-coupled carriers through heterologous expression in Xenopus laevis oocytes and in vivo measurements in salt-tolerant and salt-sensitive giant green algae such as Chara and many plant species. We have modified and developed a reliable set of procedures for voltage clamp analysis in intact guard cells and root epidermal cells from Arabidopsis thaliana with potentially broad applications in the salinity response of plants. These procedures greatly extend the duration of measurements and scope for analysis of the predominant K(+) and anion channels.

Genetic resources available forArabidopsis thalianamake this species particularly attractive as a model for molecular genetic studies of guard cell homeostasis, transport and signalling, but this facility is not matched by accessible tools for quantitative analysis of transport in the intact cell. We have developed a reliable set of procedures for voltage clamp analysis of guard cells fromArabidopsisleaves. These procedures greatly simplify electrophysiological recordings, extending the duration of measurements and scope for analysis of the predominant K+and anion channels of intact stomatal guard cells to that achieved previously in work withViciaand tobacco guard cells.

Voltage clamp is one of the key techniques for the dissection, identification, and monitoring of ion transporters in plant cells. Voltage clamp-based research work on salinity stress in plants enables the characterization of many plant ATP-dependent pumps, ion channels, and ion-coupled carriers through heterologous expression in Xenopus laevis oocytes and in vivo measurements in salt-tolerant and salt-sensitive giant green algae such as Chara and many plant species. We have modified and developed a reliable set of procedures for voltage clamp analysis in intact guard cells and root epidermal cells from Arabidopsis thaliana with potentially broad applications in the salinity response of plants. These procedures greatly extend the duration of measurements and scope for analysis of the predominant K(+) and anion channels.

Reaction of cyanoguanidine, NCNC(NH 2 ) 2 , with trans -[ReCl(CNMe)(dppe) 2 ] (dppe = Ph 2 PCH 2 CH 2 PPh 2 ) in CH 2 Cl 2 in the presence or absence of Tl[BF 4 ], forms trans -[Re(NCNC(NH 2 ) 2 )(CNMe)(dppe) 2 ]A [A  BF 4 ( Ia ) or Cl ( Ib )]. The crystal structure of Ia has been determined by an X-ray diffraction analysis. Ib can also be obtained by using cyanamide, NCNH 2 , instead of cyanoguanidine, in refluxing solvent. Treatment of Ia with KO t BU affords trans -[ReNCNC(NH)NH 2 (CNMe)(dppe) 2 ] ( II ), trans -[Re(NCNCN)(CNMe)(dppe) 2 ] ( III ) or trans -[Re(NCNHCN)(CNMe) (dppe) 2 [BF 4 ] ( IV ). The redox properties of these complexes have been studied by cyclic voltammetry and controlled potential electrolysis at Pt electrodes in aprotic media, and the P L ligand parameter has been estimated for the cyanoguanidine and derived ligands. Moreover, cathodic reduction of I or IV partially generates II or III , respectively.

Vanadium nitrido complexes are generally not accessible by routes successful in rhenium and molybdenum chemistry. The vanadium–nitrogen triple bond is reactive, and easily forms phosphiniminato complexes, two of which [V(NPMe2Ph)Cl3(PMe2Ph)2] and [V(NPMe2Ph)Cl3(PMe2Ph)2]·H2O, have been structurally characterised. The compound [V(NMe)Cl3(py)2](py = pyridine) and related alkylimido species have been synthesised. They can apparently be converted by base to alkylideneamido complexes, but the only material unequivocally and structurally characterised from such systems was [VOCl2(py)2], formed by hydrolysis.

The X-ray crystal structure of trans-[FeBr2(depe)2](depe = Et2PCH2CH2PEt2) has been determined, and its catalysis of the reactions between MgBrEt and RBr (R = Et, Prn, Bun, PhCH2 or H2CCHCH2) to give alkanes and alkenes investigated. Detailed analysis of the products and the time courses of the reactions demonstrate that the hydrido-species trans-[FeH(Br)(depe)2]n+(n= 0 or 1) are not intermediates in the reaction, but are products formed in the termination steps. It is shown that the catalysis can proceed by one of two pathways, depending on the alkyl halide. One pathway is proposed to involve the intermediacy of iron(I) species, and the other of iron(III) complexes. Both catalytic cycles contain the same fundamental reaction: the abstraction of a β-hydrogen atom from a [FeEt(Br)(depe)2]n+ species by alkyl radicals to give the alkane and ethylene. The X-ray crystal structure of trans-[FeH(Br)(depe)2]BPh4 is reported.

Reactive oxygen species (ROS) are essential for development and stress signalling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration and influence nutrient uptake and partitioning. Although both Ca2+ and K+ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K+ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H2O2. We show that H2O2 rapidly enhanced current amplitude and activation kinetics of heterologouslyexpressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H2O2 and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H2O2-sensitive site on the SKOR protein which is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, C168 located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H2O2. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify C168 as a critical target for H2O2, and implicate ROSmediated control of the K+ channel in regulating mineral nutrient partitioning within the plant. Fil: Garcia-Mata, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina Fil: Wan, Jianwen. Soochow University; China Fil: Gajdanowicz, Pawel. Universität Potsdam; Alemania Fil: Gonzalez, Wendy. Universidad de Talca; Chile Fil: Hills, Adrian. Fil: Donald, Naomi. Fil: Riedelsberger, Janin. Fil: Amtmann, Anna. Universität Potsdam; Alemania Fil: Dreyer, Ingo. Universität Potsdam; Alemania Fil: Blatt, Michael R..

There is now growing evidence that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling in plants. Work from this laboratory first demonstrated that a soluble, inhibitory (dominant-negative) fragment of the SNARE NtSyp121 blocked K+ and Cl- channel responses to the stress-related hormone abscisic acid (ABA), but left open a question about functional impacts on signal intermediates, especially on Ca2+-mediated signalling events. Here, we report one mode of action for the SNARE mediated directly through alterations in Ca2+ channel gating and its consequent effects on cytosolic-free [Ca2+] ([Ca2+]i) elevation. We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor, SNAP, which promotes [Ca2+]i elevation independently of the plasma membrane Ca2+ channels. Consistent with these observations, Ca2+ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca2+]i, and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca2+]i transients. These observations offer primary evidence for the functional coupling of the SNARE with Ca2+ channels at the plant cell plasma membrane and, because [Ca2+]i plays a key role in the control of K+ and Cl- channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.

The complexes [VX2L][L =N,N′-ethylenebis(salicylideneiminate)(salen) or N,N′-1,2-phenylenebis-(salicylideneiminate)(salphen), X = Cl or Br] have been prepared by established routes, but attempts to prepare the iodo-analogue led to other products, including [(salen)V(µ-O)VO(salen)][I5]. It is not possible to prepare hydrazine or hydrazide complexes directly from the dihalides, but [V(NH2NHPh)2(salen)]I was synthesised by an indirect route, and its structure determined by X-ray analysis. The phenylhydrazines are bound end-on. Several other new vanadium-(III) and (IV) species are described.

Deprotonation of the methylimide trans-[Mo(NMe)Cl(dppe)2]+(dppe = Ph2PCH2CH2PPh2) gives the reactive methyleneamide trans-[Mo(NCH2)Cl(dppe)2]A; oxidation of A at a platinum anode or by iodine gives trans-[Mo(CN)Cl(dppe)2]B, the structure of which has been determined by X-ray crystallography. Carbon-13 labelling studies suggest that the rearrangement of the MoNC framework to MoCN is intramolecular. Compound A reacts with chalcogens to give heterocumulene complexes trans-[Mo(NCX)Cl(dppe)2]C(X = O, S or Se), and the X-ray crystal structure of the sulphur derivative shows that the NCS ligand is N-bonded as an isothiocyanate. The electrochemistry of compound B is extensive: reduction under N2 gives trans-[Mo(N2)(CN)(dppe)2]– and in the presence of phenol affords the isolable aminocarbyne trans-[Mo(CNH2)Cl(dppe)2].

The oxidation of [VO(salen)][salen =N,N′-ethylenebis(salicylideneiminate)] with iodine produces a mixed vanadium(IV)–vanadium(V) species containing [(salen)VOVO(salen)]+ and the counter ion I5–, as determined by X-ray crystal-structure analysis. The oxidation of related vanadyl Schiff-base complexes produces similar species. Magnetic and spectroscopic properties of these new complexes are described.

Reaction of Bu t CCH with trans -[Mo(N 2 ) 2 (dppe) 2 ] (dppe = Ph 2 PCH 2 CH 2 PPh 2 ) gives [MoH 3 (CCBu t )(dppe) 2 ], whose X-ray structure is reported.

[N(C 2 H 5 ) 4 ][FeCl 4 ] cristallise dans P6 3 mc avec a=8,212, c=13,201 A, Z=2, affinement jusqu'a R=0,055. [H(C 2 H 5 ) 4 ][FeBrCl 3 ] cristallise dans P6 3 mc avec a=8,230, c=13,242 A, Z=2, affinement jusqu'a R=0,055. Les cristaux sont isostructuraux dans les deux cas les cations sont desordonnes autour d'un site de symetrie 3 m. L'ion [FeCl 4 ] − est bien resolu dans une symetrie 3m (et presque parfaitement tetraedrique). L'ion [FeBrCl 3 ] − est desordonne avec le Br remplacant le Cl dans l'un ou l'autre de ses deux sites

The treatment of [FeCl2(diphosphine)2] with sodium borohydride in ethanol produces the hydrides [FeH3(diphosphine)2]+ in high yield. These complexes react with a variety of small molecules including N2 to give further complexes. CO2 and CS2 insert into the iron hydride bond to yield formato- and dithioformato-complexes, respectively, and carboxymethyl acetylene and phenyl acetylene yield a cyclic alkenyl complex and an acetylide, respectively.