Non-autonomous stomatal control by pavement cell turgor via the K+ channel subunit AtKC1.

Stomata optimize land plants' photosynthetic requirements and limit water vapor loss. So far, all of the molecular and electrical components identified as regulating stomatal aperture are produced, and operate, directly within the guard cells. However, a completely autonomous function of guard cells is inconsistent with anatomical and biophysical observations hinting at mechanical contributions of epidermal origins. Here, potassium (K+) assays, membrane potential measurements, microindentation, and plasmolysis experiments provide evidence that disruption of the Arabidopsis thaliana K+ channel subunit gene AtKC1 reduces pavement cell turgor, due to decreased K+ accumulation, without affecting guard cell turgor. This results in an impaired back pressure of pavement cells onto guard cells, leading to larger stomatal apertures. Poorly rectifying membrane conductances to K+ were consistently observed in pavement cells. This plasmalemma property is likely to play an essential role in K+ shuttling within the epidermis. Functional complementation reveals that restoration of the wild-type stomatal functioning requires the expression of the transgenic AtKC1 at least in the pavement cells and trichomes. Altogether, the data suggest that AtKC1 activity contributes to the building of the back pressure that pavement cells exert onto guard cells by tuning K+ distribution throughout the leaf epidermis.

Adjustment of K+ Fluxes and Grapevine Defense in the Face of Climate Change.

Grapevine is one of the most economically important fruit crops due to the high value of its fruit and its importance in winemaking. The current decrease in grape berry quality and production can be seen as the consequence of various abiotic constraints imposed by climate changes. Specifically, produced wines have become too sweet, with a stronger impression of alcohol and fewer aromatic qualities. Potassium is known to play a major role in grapevine growth, as well as grape composition and wine quality. Importantly, potassium ions (K+) are involved in the initiation and maintenance of the berry loading process during ripening. Moreover, K+ has also been implicated in various defense mechanisms against abiotic stress. The first part of this review discusses the main negative consequences of the current climate, how they disturb the quality of grape berries at harvest and thus ultimately compromise the potential to obtain a great wine. In the second part, the essential electrical and osmotic functions of K+, which are intimately dependent on K+ transport systems, membrane energization, and cell K+ homeostasis, are presented. This knowledge will help to select crops that are better adapted to adverse environmental conditions.

Characterization of the grapevine Shaker K+ channel VvK3.1 supports its function in massive potassium fluxes necessary for berry potassium loading and pulvinus-actuated leaf movements.

In grapevine, climate changes lead to increased berry potassium (K+ ) contents that result in must with low acidity. Consequently, wines are becoming 'flat' to the taste, with poor organoleptic properties and low potential aging, resulting in significant economic loss. Precise investigation into the molecular determinants controlling berry K+ accumulation during its development are only now emerging. Here, we report functional characterization by electrophysiology of a new grapevine Shaker-type K+ channel, VvK3.1. The analysis of VvK3.1 expression patterns was performed by qPCR and in situ hybridization. We found that VvK3.1 belongs to the AKT2 channel phylogenetic branch and is a weakly rectifying channel, mediating both inward and outward K+ currents. We showed that VvK3.1 is highly expressed in the phloem and in a unique structure located at the two ends of the petiole, identified as a pulvinus. From the onset of fruit ripening, all data support the role of the VvK3.1 channel in the massive K+ fluxes from the phloem cell cytosol to the berry apoplast during berry K+ loading. Moreover, the high amount of VvK3.1 transcripts detected in the pulvinus strongly suggests a role for this Shaker in the swelling and shrinking of motor cells involved in paraheliotropic leaf movements.

The Complex Fine-Tuning of K⁺ Fluxes in Plants in Relation to Osmotic and Ionic Abiotic Stresses.

As the main cation in plant cells, potassium plays an essential role in adaptive responses, especially through its involvement in osmotic pressure and membrane potential adjustments. K+ homeostasis must, therefore, be finely controlled. As a result of different abiotic stresses, especially those resulting from global warming, K⁺ fluxes and plant distribution of this ion are disturbed. The hormone abscisic acid (ABA) is a key player in responses to these climate stresses. It triggers signaling cascades that ultimately lead to modulation of the activities of K⁺ channels and transporters. After a brief overview of transcriptional changes induced by abiotic stresses, this review deals with the post-translational molecular mechanisms in different plant organs, in Arabidopsis and species of agronomical interest, triggering changes in K⁺ uptake from the soil, K⁺ transport and accumulation throughout the plant, and stomatal regulation. These modifications involve phosphorylation/dephosphorylation mechanisms, modifications of targeting, and interactions with regulatory partner proteins. Interestingly, many signaling pathways are common to K⁺ and Cl-/NO3- counter-ion transport systems. These cross-talks are also addressed.

The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations.

The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K(+) efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with "phosphorylated" or "dephosphorylated" GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure.

Reduced expression of AtNUP62 nucleoporin gene affects auxin response in Arabidopsis.

BACKGROUND: The plant nuclear pore complex has strongly attracted the attention of the scientific community during the past few years, in particular because of its involvement in hormonal and pathogen/symbiotic signalling. In Arabidopsis thaliana, more than 30 nucleoporins have been identified, but only a few of them have been characterized. Among these, AtNUP160, AtNUP96, AtNUP58, and AtTPR have been reported to modulate auxin signalling, since corresponding mutants are suppressors of the auxin resistance conferred by the axr1 (auxin-resistant) mutation. The present work is focused on AtNUP62, which is essential for embryo and plant development. This protein is one of the three nucleoporins (with AtNUP54 and AtNUP58) of the central channel of the nuclear pore complex. RESULTS: AtNUP62 promoter activity was detected in many organs, and particularly in the embryo sac, young germinating seedlings and at the adult stage in stipules of cauline leaves. The atnup62-1 mutant, harbouring a T-DNA insertion in intron 5, was identified as a knock-down mutant. It displayed developmental phenotypes that suggested defects in auxin transport or responsiveness. Atnup62 mutant plantlets were found to be hypersensitive to auxin, at the cotyledon and root levels. The phenotype of the AtNUP62-GFP overexpressing line further supported the existence of a link between AtNUP62 and auxin signalling. Furthermore, the atnup62 mutation led to an increase in the activity of the DR5 auxin-responsive promoter, and suppressed the auxin-resistant root growth and leaf serration phenotypes of the axr1 mutant. CONCLUSION: AtNUP62 appears to be a major negative regulator of auxin signalling. Auxin hypersensitivity of the atnup62 mutant, reminding that of atnup58 (and not observed with other nucleoporin mutants), is in agreement with the reported interaction between AtNUP62 and AtNUP58 proteins, and suggests closely related functions. The effect of AtNUP62 on auxin signalling likely occurs in relation to scaffold proteins of the nuclear pore complex (AtNUP160, AtNUP96 and AtTPR).

Distinct amino acids in the C-linker domain of the Arabidopsis K+ channel KAT2 determine its subcellular localization and activity at the plasma membrane.

Shaker K(+) channels form the major K(+) conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K(+) channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K(+) rectifying channel1 (AtKC1). These two subunits contribute to K(+) transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch 312TVRAASEFA320 that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K(+)) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.

Molecular mechanisms involved in plant adaptation to low K(+) availability.

Potassium is a major inorganic constituent of the living cell and the most abundant cation in the cytosol. It plays a role in various functions at the cell level, such as electrical neutralization of anionic charges, protein synthesis, long- and short-term control of membrane polarization, and regulation of the osmotic potential. Through the latter function, K(+) is involved at the whole-plant level in osmotically driven functions such as cell movements, regulation of stomatal aperture, or phloem transport. Thus, plant growth and development require that large amounts of K(+) are taken up from the soil and translocated to the various organs. In most ecosystems, however, soil K(+) availability is low and fluctuating, so plants have developed strategies to take up K(+) more efficiently and preserve vital functions and growth when K(+) availability is becoming limited. These strategies include increased capacity for high-affinity K(+) uptake from the soil, K(+) redistribution between the cytosolic and vacuolar pools, ensuring cytosolic homeostasis, and modification of root system development and architecture. Our knowledge about the mechanisms and signalling cascades involved in these different adaptive responses has been rapidly growing during the last decade, revealing a highly complex network of interacting processes. This review is focused on the different physiological responses induced by K(+) deprivation, their underlying molecular events, and the present knowledge and hypotheses regarding the mechanisms responsible for K(+) sensing and signalling.

AtKC1 is a general modulator of Arabidopsis inward Shaker channel activity.

A functional Shaker potassium channel requires assembly of four α-subunits encoded by a single gene or various genes from the Shaker family. In Arabidopsis thaliana, AtKC1, a Shaker α-subunit that is silent when expressed alone, has been shown to regulate the activity of AKT1 by forming heteromeric AtKC1-AKT1 channels. Here, we investigated whether AtKC1 is a general regulator of channel activity. Co-expression in Xenopus oocytes of a dominant negative (pore-mutated) AtKC1 subunit with the inward Shaker channel subunits KAT1, KAT2 or AKT2, or the outward subunits SKOR or GORK, revealed that the three inward subunits functionally interact with AtKC1 while the outward ones cannot. Localization experiments in plant protoplasts showed that KAT2 was able to re-locate AtKC1 fused to GFP from endomembranes to the plasma membrane, indicating that heteromeric AtKC1-KAT2 channels are efficiently targeted to the plasma membrane. Functional properties of heteromeric channels involving AtKC1 and KAT1, KAT2 or AKT2 were analysed by voltage clamp after co-expression of the respective subunits in Xenopus oocytes. AtKC1 behaved as a regulatory subunit within the heterotetrameric channel, reducing the macroscopic conductance and negatively shifting the channel activation potential. Expression studies showed that AtKC1 and its identified Shaker partners have overlapping expression patterns, supporting the hypothesis of a general regulation of inward channel activity by AtKC1 in planta. Lastly, AtKC1 disruption appeared to reduce plant biomass production, showing that AtKC1-mediated channel activity regulation is required for normal plant growth.

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2.

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a 'potassium battery', providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

[The ABC of abscisic acid action in plant drought stress responses]. / Mécanisme moléculaire d'action de l'acide abscissique en réponse à la sécheresse chez les végétaux.

The combined daily consumption of fresh water ranges from 200 to 700 liters per capita per day in most developed countries, with about 70% being used for agricultural needs. Unlike other resources such as the different forms of energy, water has no other alternatives. With the looming prospect of global water crisis, the recent laudable success in deciphering the early steps in the signal transduction of the "stress hormone" abscisic acid (ABA) has ignited hopes that crops can be engineered with the capacity to maintain productivity while requiring less water input. Although ABA was first discovered in plants, it has resurfaced in the human brain (and many other non-plant organisms : sea sponge, some parasites, hydra to name a few), suggesting that its existence may be widespread. In humans, more amazingly, ABA has shown anti-inflammatory and antiviral properties. Even its receptors and key signaling intermediates have homologs in the human genome suggesting that evolution has re-fashioned these same proteins into new functional contexts. Thus, learning about the molecular mechanisms of ABA in action using the more flexible plant model will be likely beneficial to other organisms, and especially in human diseases, which is topical in the medical circle. ABA can accumulate up to 10 to 30-fold in plants under drought stress relative to unstressed conditions. The built up of the hormone then triggers diverse adaptive pathways permitting plants to withstand temporary bouts of water shortage. One favorite experimental model to unravel ABA signaling mechanisms in all of its intimate detail is based on the hormone's ability to elicit stomatal closure - a rapid cellular response of land plants to limit water loss through transpiration. Each microscopic stoma, or pore, is contoured by two specialized kidney-shaped cells called the guard cells. Because land plants are protected by a waxy cuticle impermeable to gas exchange, the stomatal pores are thus the primary portals for photosynthetic CO(2) uptake. Drought, by biasing pathways that lead to rapid closure of these pores, has therefore a negative impact on photosynthesis, and consequently, biomass as well. The stomatal aperture widens and narrows by expansion and contraction, respectively, of these flanking guard cells caused by changes in the intracellular concentrations of ion fluxes. These transport mechanisms most likely share fundamental principles with any excitable cell. These events require coordination of channels, vacuolar and membrane transporters that generate a specific pattern of electrical signals that relay the ABA stimulus. Research on ABA begun in the 1960's has now been crowned by the achievement of having identified the soluble ABA receptor that turns on and off the activities of a kinase/phosphatase pair, as the heart of the signaling complex. Results distilled from the latest structural studies on these ABA receptors, characterized by the so-called START domain, are beginning to tender the most exciting promise for rational design of agonists and antagonists towards modulating stress adaptive ability in plants. This review will chart the recent extraordinary progress that has enlightened us on how ABA controls membrane transport mechanisms that evoke the fast stomatal closing pathway.

Increased functional diversity of plant K+ channels by preferential heteromerization of the shaker-like subunits AKT2 and KAT2.

Assembly of plant Shaker subunits as heterotetramers, increasing channel functional diversity, has been reported. Here we focus on a new interaction, between AKT2 and KAT2 subunits. The assembly as AKT2/KAT2 heterotetramers is demonstrated by (i) a strong signal in two-hybrid tests with intracytoplasmic C-terminal regions, (ii) the effect of KAT2 on AKT2 subunit targeting in tobacco cells, (iii) the complete inhibition of AKT2 currents by co-expression with a dominant-negative KAT2 subunit in Xenopus oocytes, and reciprocally, and (iv) the appearance, upon co-expression of wild-type AKT2 and KAT2 subunits, of new channel functional properties that cannot be explained by the co-existence of two kinds of homotetrameric channels. In particular, the instantaneous current, characteristic of AKT2, displayed new functional features when compared with those of AKT2 homotetramers: activation by external acidification (instead of inhibition) and weak inhibition by calcium. Single channel current measurements in oocytes co-expressing AKT2 and KAT2 revealed a strong preference for incorporation of subunits into heteromultimers and a diversity of individual channels. In planta, these new channels, which may undergo specific regulations, are likely to be formed in guard cells and in the phloem, where they could participate in the control of membrane potential and potassium fluxes.

Regulation of K+ channel activities in plants: from physiological to molecular aspects.

Plant voltage-gated channels belonging to the Shaker family participate in sustained K+ transport processes at the cell and whole plant levels, such as K+ uptake from the soil solution, long-distance K+ transport in the xylem and phloem, and K+ fluxes in guard cells during stomatal movements. The attention here is focused on the regulation of these transport systems by protein-protein interactions. Clues to the identity of the regulatory mechanisms have been provided by electrophysiological approaches in planta or in heterologous systems, and through analogies with their animal counterparts. It has been shown that, like their animal homologues, plant voltage-gated channels can assemble as homo- or heterotetramers associating polypeptides encoded by different Shaker genes, and that they can bind auxiliary subunits homologous to those identified in mammals. Furthermore, several regulatory processes (involving, for example, protein kinases and phosphatases, G proteins, 14-3-3s, or syntaxins) might be common to plant and animal Shakers. However, the molecular identification of plant channel partners is still at its beginning. This paper reviews current knowledge on plant K+ channel regulation at the physiological and molecular levels, in the light of the corresponding knowledge in animal cells, and discusses perspectives for the deciphering of regulatory networks in the future.

Regulated expression of Arabidopsis shaker K+ channel genes involved in K+ uptake and distribution in the plant.

Potassium is the most abundant cation in the cytosol, where it plays a role in basal functions. Rapid uptake and distribution of K+ is therefore required for plant growth. Three members of the so-called Shaker K+ channel gene family (nine genes identified in Arabidopsis) play a role in these transports: AKT1, SKOR and AKT2. The encoded proteins are involved in K+ uptake by the root, K+ secretion into the xylem sap and K+ transport in the phloem tissues, respectively. Using the GUS reporter strategy, we have found that another Shaker channel gene, AtKC1, is expressed in epidermal and cortical cells in roots (supporting the hypothesis of a role in K+ uptake from the soil, together with AKT1), and in trichomes and hydathodes in leaves. These four genes were selected for expression studies, and two-hybrid experiments were performed for channels displaying overlapping expression patterns. The data support the hypothesis that physical interactions could occur in planta between AtKC1 and AKT1, and between AKT1 and AKT2. Potassium deficiency, salt stress and hormonal treatments (ABA, BA, 2,4-D) were found to differentially affect channel mRNA levels, each channel displaying its own regulation pattern. The most prominent effects were (1) a strong induction of AtKC1 transcript accumulation in leaves (hydathodes, trichomes and leaf epidermis) in response to NaCl treatment, suggesting a key role of the protein in adaptation to saline conditions, and (2) a strong decrease in SKOR transcript levels by hormones, supporting the hypothesis that K+ secretion into the xylem sap is under tight hormonal control.

Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA.

The AKT2 K(+) channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, mesophyll, and guard cells. The AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library, we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We further confirmed the interaction by in vitro binding studies. The expression of AtPP2CA (beta-glucuronidase reporter gene) displayed a pattern largely overlapping that of AKT2 and was upregulated by abscisic acid. Coexpression of AtPP2CA with AKT2 in COS cells and Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 current and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis revealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA in planta could allow the control of K(+) transport and membrane polarization during stress situations.