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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. Summary statement: Stomata largely retain a common design across all species of land plants. By contrast, the cells immediately surrounding the guard cells vary widely in nature and origin. Such variety likely reflects a functional diversity in supporting stomatal movements and shaping stomatal kinetics. Yet, our knowledge of the cells surrounding the guard cells in the stomatal complex remains fragmentary at best. [ABSTRACT FROM AUTHOR]

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 Ca2+‐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 Ca2+ in the vacuole, thereby influencing Ca2+ storage and leading to an enhanced Ca2+ 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 Ca2+‐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. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: We uncover a new role for the plasma membrane trafficking SNARE SYP132 in the regulation of PIP2;1 density and function in response to salinity and osmotic stress. Remarkably these pathways appear to intersect with coordinate regulation of AHA1 that is governed by the SYP132 during pathogenesis, thus suggesting that the SNARE is at the centre of an antiparallel association of PIP2 and the H+‐ATPase affected by salinity. [ABSTRACT FROM AUTHOR]

This article discusses the importance of stomata, which are pores on the surfaces of plants that allow for the exchange of CO2 and water vapor. Stomata play a crucial role in photosynthesis and are connected to global carbon and water cycles. The article explores various aspects of stomatal function, including their development, evolution, and the molecular mechanics behind their opening and closing. It also examines the implications of stomatal research for climate change, water availability, and crop production. The article concludes by highlighting the interdisciplinary nature of stomatal research and its significance in understanding plant physiology in the global environment. [Extracted from the article]

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 C4 photosynthesis are usually more resilient than C3 plants to stress, and their stomata operate over a lower dynamic range of CO2 within the leaf. What makes guard cells of C4 plants more responsive than those of C3 plants? We used gas exchange and electrophysiology, comparing stomatal kinetics of the C4 plant Gynandropsis gynandra and the phylogenetically related C3 plant Arabidopsis thaliana. We found, with varying CO2 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 C4 stomatal kinetics. Summary Statement: Compared with Arabidopsis, the outward‐rectifying K+ channels in guard cells of the C4 plant Gynandropsis gynandra incorporate gating that is displaced to favour enhanced K+ efflux and promote K+ influx. These characteristics are likely to underpin the faster stomatal that are observed in the C4 plant. [ABSTRACT FROM AUTHOR]

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 CO2 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. Summary statement: OnGuard3e offers a truly mechanistic approach with which to analyse stomatal gas exchange, whole‐plant carbon assimilation and water‐use efficiency. The software package seamlessly connects whole‐plant gas exchange and photosynthesis with the microscopic mechanics of stomata and is 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. [ABSTRACT FROM AUTHOR]

Summary: 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. See also the Commentary on this article by Chater, 230: 886–888. [ABSTRACT FROM AUTHOR]

Summary: Plants utilising crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C3 and C4 species, CAM stomata open at night for the mesophyll to fix CO2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO2 concentrations within the leaf behind closed stomata for refixation by RuBisCO.CO2 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Plant cells maintain turgor pressure against the cell wall. So vesicle traffic and solute uptake must be tightly coordinated during cell expansion. Secretory vesicle traffic and K+ channel activities are promoted by physical interactions between subsets of vesicle‐trafficking proteins and Kv channels at the plant plasma membrane, but the temporal relationship between these processes has remained unclear. Ion channels open and close rapidly over milliseconds, whereas vesicle fusion requires periods of many seconds. Here, we show that the SNARE binding effects long‐term changes in channel gating kinetics through conformations of the channel voltage sensor that depend, in part, on the leverage afforded by anchoring of the SNARE in the plasma membrane. [ABSTRACT FROM AUTHOR]

Maintaining potassium (K+) nutrition and a robust guard cell K+ inward channel activity is considered critical for plants' adaptation to fluctuating and challenging growth environment. ABA induces stomatal closure through hydrogen peroxide and nitric oxide ( NO) along with subsequent ion channel-mediated loss of K+ and anions. However, the interactions of NO synthesis and signalling with K+ nutrition and guard cell K+ channel activities have not been fully explored in Arabidopsis., Physiological and molecular techniques were employed to dissect the interaction of nitrogen and potassium nutrition in regulating stomatal opening, CO2 assimilation and ion channel activity. These data, gene expression and ABA signalling transduction were compared in wild-type Columbia-0 (Col-0) and the nitrate reductase mutant nia1nia2., Growth and K+ nutrition were impaired along with stomatal behaviour, membrane transport, and expression of genes associated with ABA signalling in the nia1nia2 mutant. ABA-inhibited K+in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for complete stomatal closure in nia1nia2., While NO is an important signalling component in ABA-induced stomatal closure in Arabidopsis, our findings demonstrate a more complex interaction associating potassium nutrition and nitrogen metabolism in the nia1nia2 mutant that affects stomatal function. [ABSTRACT FROM AUTHOR]

GORK is the only outward-rectifying Kv-like K+ channel expressed in guard cells. Its activity is tightly regulated to facilitate K+ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K+ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently-tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K+ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K+ channel blocker Ba2+. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo- and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K+, a characteristic that ensures the channel facilitates only K+ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K+ above 1 m m. We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K+. [ABSTRACT FROM AUTHOR]

Summary The vesicle-trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss-of-function mutant close normally in ABA and high Ca2+. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO2 assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca2+-evoked closure, consistent with a constitutive form of so-called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild-type SYP121 and was phenocopied in wild-type plants in the presence of the vesicle-trafficking inhibitor Brefeldin A. K+ channel current that normally mediates K+ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild-type plants. Evoked stomatal closure was accompanied by internalisation of GFP-tagged KAT1 K+ channels in both wild-type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K+ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole-plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control. [ABSTRACT FROM AUTHOR]

To understand molecular processes in living plant cells, quantitative spectro-microscopic technologies are required. By combining fluorescence lifetime spectroscopy with confocal microscopy, we studied the subcellular properties and function of a GFP-tagged variant of the plasma membrane-bound brassinosteroid receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis seedlings. Shortly after adding brassinolide, we observed BRI1-dependent cell-wall expansion, preceding cell elongation. In parallel, the fluorescence lifetime of BRI1-GFP decreased, indicating an alteration in the receptor's physico-chemical environment. The parameter modulating the fluorescence lifetime of BRI1-GFP was found to be BL-induced hyperpolarization of the plasma membrane. Furthermore, for induction of hyperpolarization and cell-wall expansion, activation of the plasma membrane P-ATPase was necessary. This activation required BRI1 kinase activity, and was mediated by BL-modulated interaction of BRI1 with the P-ATPase. Our results were used to develop a model suggesting that there is a fast BL-regulated signal response pathway within the plasma membrane that links BRI1 with P-ATPase for the regulation of cell-wall expansion. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter ( P UBQ10) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K+ channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P UBQ10 promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P UBQ10 promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots. [ABSTRACT FROM AUTHOR]

In guard cells, activation of anion channels ( Ianion) is an early event leading to stomatal closure. Activation of Ianion has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). However, the dynamics of the action of [Ca2+]i on Ianion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca2+]i dynamics of Ianion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca2+]i using Fura-2 fluorescence imaging. We found that Ianion rises with [Ca2+]i only at concentrations substantially above the mean resting value of 125 ± 13 nm, yielding an apparent Kd of 720 ± 65 nm and a Hill coefficient consistent with the binding of three to four Ca2+ ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of Ianion activity, but without a dependence of the current on [Ca2+]i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca2+]i sensitivity of Ianion, displacing the apparent Kd for [Ca2+]i to 573 ± 38 nm. These findings support previous evidence for different modes of regulation for Ianion, only one of which depends on [Ca2+]i, and they underscore an independence of [Ca2+]i from protein (de-)phosphorylation in controlling Ianion. Most importantly, our results demonstrate a significant displacement of Ianion sensitivity to higher [Ca2+]i compared with that of the guard cell K+ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss. [ABSTRACT FROM AUTHOR]

• The family of voltage-gated potassium channels in plants presumably evolved from a common ancestor and includes both inward-rectifying (Kin) channels that allow plant cells to accumulate K+ and outward-rectifying (Kout) channels that mediate K+ efflux. Despite their close structural similarities, the activity of Kin channels is largely independent of K+ and depends only on the transmembrane voltage, whereas that of Kout channels responds to the membrane voltage and the prevailing extracellular K+ concentration. Gating of potassium channels is achieved by structural rearrangements within the last transmembrane domain (S6). • Here we investigated the functional equivalence of the S6 helices of the Kin channel KAT1 and the Kout channel SKOR by domain-swapping and site-directed mutagenesis. Channel mutants and chimeras were analyzed after expression in Xenopus oocytes. • We identified two discrete regions that influence gating differently in both channels, demonstrating a lack of functional complementarity between KAT1 and SKOR. Our findings are supported by molecular models of KAT1 and SKOR in the open and closed states. • The role of the S6 segment in gating evolved differently during specialization of the two channel subclasses, posing an obstacle for the transfer of the K+-sensor from Kout to Kin channels. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Contents In addition to light, water and CO2, plants require a number of mineral nutrients, in particular the macronutrients nitrogen, sulphur, phosphorus, magnesium, calcium and potassium. After uptake from the soil by the root system they are either immediately assimilated into organic compounds or distributed within the plant for usage in different tissues. A good understanding of how the transport of macronutrients into and between plant cells is adjusted to different environmental conditions is essential to achieve an increase of nutrient usage efficiency and nutritional value in crops. Here, we review the current state of knowledge regarding the regulation of macronutrient transport, taking both a physiological and a mechanistic approach. We first describe how nutrient transport is linked to environmental and internal cues such as nutrient, carbon and water availability via hormonal, metabolic and physical signals. We then present information on the molecular mechanisms for regulation of transport proteins, including voltage gating, auto-inhibition, interaction with other proteins, oligomerization and trafficking. Combining of evidence for different nutrients, signals and regulatory levels creates an opportunity for making new connections within a large body of data, and thus contributes to an integrative understanding of nutrient transport. [ABSTRACT FROM AUTHOR]

Vesicle traffic underpins cell homeostasis, growth and development in plants, and is facilitated by a superfamily of proteins known as SNAREs [soluble NSF ( N-ethylmaleimide-sensitive factor) attachment protein receptors] that interact to draw vesicle and target membrane surfaces together for fusion. Structural homologies, biochemical and genetic analyses have yielded information about the localization and possible roles of these proteins. However, remarkably little evidence is yet available that speaks directly to the functional specificities of these proteins in selected trafficking pathways in vivo. Previously, we found that expressing a cytosolic (so-called Sp2) fragment of one plasma membrane SNARE from tobacco and Arabidopsis had severe effects on growth, tissue development and secretory traffic to the plasma membrane. We have explored this dominant-negative approach further to examine the specificity and overlaps in Sp2 activity by generating a toolbox of truncated SNARE constructs and antibodies for transient expression and analysis. Using a quantitative ratiometric approach with secreted green fluorescent protein (secGFP), we report here that traffic to the plasma membrane is suppressed selectively by Sp2 fragments of plasma membrane SNAREs AtSYP121 and AtSYP122, but not of the closely related SNARE AtSYP111 nor of the SNARE AtSYP21 that resides at the pre-vacuolar compartment (PVC). By contrast, traffic of the YFP-tagged aquaporin fusion protein TIP1;1–YFP to the tonoplast was blocked (leading to its accumulation in the PVC) when co-expressed with the Sp2 fragment of AtSYP21, but not when co-expressed with that of AtSYP121. Export of secGFP was also sensitive to the Sp2 fragment of the novel, plant-specific SNARE AtSYP71 that was recently found to be present in detergent-resistant, plasma membrane fractions. Co-incubation analyses of the plasma membrane SNAREs with the regulatory subdomain included within the Sp2 fragments showed activity in destabilizing protein complexes, but only with the complementary SNAREs. We conclude that the Sp2 fragment action accurately reflects the known specificity and targeting of these SNAREs, implies functional overlaps that are of potential physiological interest, and underscores the use of a dominant-negative strategy in functional studies of a major subfamily of SNAREs in plants. [ABSTRACT FROM AUTHOR]

Vesicle traffic is essential for cell homeostasis, growth and development in plants, as it is in other eukaryotes, and is facilitated by a superfamily of proteins known as soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs). Although SNAREs are well-conserved across phylla, genomic analysis for two model angiosperm species available to date, rice and Arabidopsis, highlights common patterns of divergence from other eukaryotes. These patterns are associated with the expansion of some gene subfamilies of SNAREs, the absence of others and the appearance of new proteins that show no significant homologies to SNAREs of mammals, yeast or Drosophila. Recent findings indicate that the functions of these plant SNAREs also extend beyond the conventional ‘housekeeping’ activities associated with vesicle trafficking. A number of SNAREs have been implicated in environmental responses as diverse as stomata movements and gravisensing as well as sensitivity to salt and drought. These proteins are essential for signal transduction and response and, in most cases, appear also to maintain additional roles in membrane trafficking. One common theme to this added functionality lies in control of non-SNARE proteins, notably ion channels. Other examples include interactions between the SNAREs and scaffolding or other structural components within the plant cell. [ABSTRACT FROM AUTHOR]

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root–shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell . This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an ‘S6 gating domain’ deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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 [Ca2+] ([Ca2+]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 Ca2+ release, and they implicate a target for protein kinase control in ABA signalling that feeds into NO-dependent Ca2+ release. [ABSTRACT FROM AUTHOR]

Summary Vesicular trafficking and membrane fusion are integral to cell growth and development with SNARE proteins, RabGTPases and their associates implicated in membrane fusion and secretion throughout the plant endomembrane system. Although the overall pattern of function is similar to that of animals and yeast, many aspects of endomembrane organization and vesicle trafficking appear unique to plants, for example, the control of cell and vacuolar expansion, asymmetric growth and cell division. However, the dominant membrane trafficking pathways have yet to be defined. Comparative genomics provide important information about vesicle trafficking elements but assigning biological roles based on sequence similarities is extremely difficult. Cellular and genetic approaches are reviewed here that have allowed visualization of vesicle trafficking in plants, including capacitance and dye methods, imaging and marker techniques, protein interactions and reverse genetics. Stomatal guard cells are discussed as cell models for identifying vesicle trafficking pathways and evidence points to a role for vesicle trafficking in stomatal function. For plants generally, kinetic analyses and biochemical studies suggest that several different pools of vesicles, and possibly different mechanisms for delivery, are available for vesicle traffic between endomembrane compartments and the plasma membrane. Characterizing these pathways, their functions and controls provides a major challenge for the future. [ABSTRACT FROM AUTHOR]

Presents a study focusing on the localization and control of expression of the tobacco snare protein known as Nt-Syr1. Methods; Results; Conclusion that Nt-Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress-related signalling pathways.

Eukaryotic cells share a set of secretory pathways for the flux of membrane and protein material. In 1993, ideas about the functioning of three major proteins of the neurosecretory complex were consolidated in the SNARE hypothesis, which proposed that the interaction of these proteins provides both the specificity for vesicle targeting and the molecular machinery for fusion between vesicle and target membranes. Subsequently the organization, molecular mechanics and control of vesicle trafficking have become topics of intense research, and the organization, molecular mechanics and control of visicle trafficking have become topics of intense research, and the hypothesis has evolved to accummodate new discoveries from the analysis of secretion in yeast and mammals. It is likely to be challenged again as more information comes to light about secretory processes in plants. New tools for measuring and manipulating vesicle traffic and secretion are now being used, drawing on in vivo fluorescence and capacitance recording as well as genetic engineering. These new technologies have already begun to yield details wholly unexpected from past studies. Here we focus on recent findings relating to the mechanisms of vesicle trafficking and the background to these developments, highlighting both current understanding of the molecular events of secretion and the gaps therein, as well as discussing emerging themes from work with plants. [ABSTRACT FROM AUTHOR]

Summary: The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein that is membrane anchored near its C-terminus. The protein confers resistance in tomato to races of the fungus Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene confers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the tomato host. One of the earliest responses, both in the native and transgenic hosts, results in K[sup +] salt loss from the infected tissues. However, the mechanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using guard cells as a model. Much detail of guard cell ion channels and their regulation is already known. Measurements were carried out on intact guard cells in epidermal peels, and the currents carried by inward- (I[sub K,in]) and outward-rectifying (I[sub K,out]) K[sup +] channels were characterized under voltage clamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold stimulation of I[sub K,out] and almost complete suppression of I[sub K,in] within 3–5 min. The K[sup +] channel responses were irreversible. They were specific for the Avr9 elicitor, were not observed in guard cells of Nicotiana lacking the Cf-9 transgene and, from kinetic analyses, could be ascribed to changes in channel gating. Both K[sup +] channel responses were found to be saturable functions of Avr9 concentration and were completely blocked in the presence of 0.5 μM staurosporine and 100 μM H7, both broad-range protein kinase antagonists. These results demonstrate the ability of the Cf-9 transgene to couple Avr9 elicitation specifically to a concerted action on two discrete K[sup +] channels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control. [ABSTRACT FROM AUTHOR]

Summary: Chalcone synthase (CHS) is a key enzyme leading to the generation of protective flavonoids in plants under environmental stress. Expression of the CHS gene is strongly upregulated by exposures to UV light, a response also observed in heterotrophic parsley cell cultures. Although there are hints that the stimulus for CHS expression may be coupled to UV-B irradiation through a rise in cytosolic-free Ca[sup 2+] ([Ca[sup 2+]][sub i]), the temporal relationship of these events has never been investigated critically. To explore this question, we have used a CHS promoter/luciferase (CHS/LUC) reporter gene fusion and recorded its expression and [Ca[sup 2+]][sub i] elevation in a transgenic parsley cell culture following millisecond light pulses. Luciferase expression was enhanced maximally seven- (± 2) fold by 30 10 ms flashes of UV-B light. The response was specific to wavelengths of 300–330 nm and could be inhibited in the presence of the Ca[sup 2+] channel blocker nifedipine. In parallel measurements, using Fura-2 fluorescence ratio microphotometry, we found that 10 ms UV-B flashes also evoked a gradual and prolonged rise of [Ca[sup 2+]][sub i] in the parsley cells which was irreversible within the timescale of these experiments, but could be prevented by prior treatment with nifedipine. These, and additional results, indicate a remarkably high temporal sensitivity to, and specificity for, UV-B light in CHS gene expression independent of UV-mediated DNA damage by thymine dimerization. The ability of transient UV-B stimulation to evoke prolonged elevations of [Ca[sup 2+]][sub i] suggests a functional coupling between the initial light stimulus and subsequent gene expression that takes place many tens of minutes later. [ABSTRACT FROM AUTHOR]

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wildtype current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation. [ABSTRACT FROM AUTHOR]

Multiple signalling pathways and their messengers - entailing changes in cytosolic-free Ca2+ ([Ca2+]1), pH (pH1) and protein phosphorylation - underpin K+ and anion channel control during stomatal movements. This redundancy is wholly consistent with the ability of the guard cells to integrate the wide range of environmental and hormonal stimuli that affect stomatal aperture. Signal redundancy effects a spectrum of graded responses by linking pathways to gate signal transmission, and so boosts or mutes the final 'integrated signal' that reaches each ion channel. All evidence supports a role for the ABII protein phosphatase and protein kinase elements in gating K- channel sensitivity to pH and ABA. Changes in [Ca2+]1, in turn, are demonstrably sensitive to pH1. Because each of these signal elements modulate and, in turn, are influenced by the activity of different sets of ion channels, the additional couplings engender a remarkably complex network, layering positive and negative controls with the ion channels that facilitate ion fluxes for stomatal movement. [ABSTRACT FROM AUTHOR]

Summary Protein phosphorylation and cytosolic-free [Ca2+ ] ([Ca2+ ]i ) contribute to signalling cascades evoked by the water-stress hormone abscisic acid (ABA) that lead to stomatal closure in higher-plant leaves. ABA activates an inward-rectifying Ca2+ channel at the plasma membrane of stomatal guard cells, promoting Ca2+ entry by shifting the voltage-sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de-)phosphorylation in plasma membrane patches from Vicia guard cells. Ca2+ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K1/2 , 1.3 ± 0.7 mm) but not the non-hydrolyzable analog ATPγS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A-type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca2+ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage-sensitivities of the Ca2+ current and [Ca2+ ]i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca2+ ]i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane-delimited interaction between 1/2 A-type protein phosphatase(s) and the Ca2+ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca2+ channel gating that leads to [Ca2+ ]i increases in the guard cells. [ABSTRACT FROM AUTHOR]

Summary: A role for cytosolic pH (pH[sub i]) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pH[sub i] are a common consequence of hormonal stimuli in plant cells and contribute to hormonally evoked ion channel control, the origins of these changes remain unknown. To examine a possible role for the tonoplast and vacuolar compartment in these events, pH[sub i] was measured in the presence of auxins and during cytosolic H[sup +] loading with weak acid in vacuolate and evacuolate protoplasts, both from mesophyll and guard cells of Vicia faba L. Evacuolate protoplasts were obtained following ultracentrifugation on Percoll gradients, and pH[sub i] of single protoplasts was recorded in both vacuolate and evacuolate preparations using fluorescence ratio microphotometry and the pH-sensitive dye BCECF. External pH measurements indicated a roughly twofold increase in the rate of net H[sup +] secretion in evacuolate compared with vacuolate protoplasts, and showed that evacuolate protoplasts retained the characteristic stimulation of H[sup +] secretion in the presence of auxin. BCECF fluorescence recording gave resting pH[sub i] values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pH[sub i] were evoked in vacuolate protoplasts by 10 μM concentrations of the auxins 1-naphthalene acetic acid and 3-indoyl-acetic acid, and not by the inactive (anti-auxin) analogue 2-naphthalene-acetic acid. However, auxin treatments failed to evoke a change in pH[sub i] in all but one of 12 experiments with evacuolate protoplasts. Evacuolation also appeared to reduce the transient, dynamic H[sup +] buffering capacity of the protoplasts in the face of acid pH[sub i] loads imposed by adding Na[sup +]-butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short-term pH[sub i] homeostasis and generation of hormonally evoked H[sup +]... [ABSTRACT FROM AUTHOR]

GORK is the only outward-rectifying Kv-like K+ channel expressed in guard cells. Its activity is tightly regulated to facilitate K+ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K+ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently-tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K+ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K+ channel blocker Ba2+. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo- and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K+, a characteristic that ensures the channel facilitates only K+ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K+ above 1 m m. We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K+. [ABSTRACT FROM AUTHOR]

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wildtype current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation. [ABSTRACT FROM AUTHOR]