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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.

Understanding molecular mechanisms which underlie transport of cesium (Cs+) in plants is important to limit entry of its radioisotopes from contaminated area to the food chain. The potentially toxic element Cs+, which is not involved in any biological process, is chemically closed to the macronutrient potassium (K+). Among the multiple K+ carriers, the high-affinity K+ transporters family HAK/KT/KUP is thought to be relevant in mediating opportunistic Cs+ transport. On the 13 KUP identified in Arabidopsis thaliana, only HAK5, the major contributor to root K+ acquisition under low K+ supply, has been functionally demonstrated to be involved in Cs+ uptake in planta. In the present study, we showed that accumulation of Cs+ increased by up to 30% in two A. thaliana mutant lines lacking KUP9 and grown under low K+ supply. Since further experiments revealed that Cs+ release from contaminated plants to the external medium is proportionally lower in the two kup9 mutants, we proposed that KUP9 disruption could impair Cs+ efflux. By contrast, we did not measure significant impairment of K+ status in kup9 mutants suggesting that KUP9 disruption does not alter substantially K+ transport in experimental conditions used here. Putative primary role of KUP9 in plants is further discussed.

Early studies have shown that cesium (Cs+) competes with the macronutrient potassium (K+) for uptake by plants. The present study investigates the effect of K+ supply on Cs+ uptake and translocation in Arabidopsis thaliana. Taking advantage of the frequent use of this model plant in previous molecular studies, we discuss the link between functional features described for transporters involved in K+ (and sometimes in Cs+) uptake and results obtained here in both Cs+ influx and accumulation experiments under different K+-treatments. In low K+ condition (10 μM), we observed that roots affinity for Cs+ increased significantly and Cs+ concentration in the external medium clearly affected the efficiency of Cs+ uptake. Our results are consistent with previous molecular studies indicating the role of the high-affinity K+ carrier AtHAK5 in Cs+ uptake under K+-deprivation. Further experiments show that the lack of AtHAK5 has no more effect on Cs+ uptake for external Cs+ concentration above 100 μM. We propose that non-selective cation channels, likely involved in Cs+ uptake under K+-sufficient conditions according to previous studies, could also mediate Cs+ uptake under K+-starvation and high Cs+ concentrations. Finally, evidences for Cs+ translocation mediated by K+ channels are discussed.

International audience; As part of the « post-accidental » management, the DEMETERRES project (RSNR PIA) proposes to develop innovative and environmentally friendly methods for removal of cesium and strontium from soils and liquid matrices in order to rehabilitate them for an agricultural use while minimizing the volume of generated wastes in accordance with the nuclear waste existing processes. Complementary approaches are used: they are based on physico-chemical technologies (such as foams flotation, supercritical CO2 extraction, extractants in fluidized bed reactor …) and biological ones (bioextractants, phytoextraction) which concepts are described. These researches aim to design innovative and performing extractants in term of selectivity and to achieve the pilot reactor phase for each of them. These pilots will group in a network to provide a technological platform lasting the project, to which will be attached an available network of experts. The respective advances of these researches are presented, completed of tests initiated in Japan on contaminated soils through partnerships.

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up-regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro-grafting a pho1 shoot scion onto wild-type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild-type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re-established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, resistance to biotic or abiotic stresses, and ion homeostasis. In Arabidopsis, members of the chloride channel (CLC) family located in intracellular organelles have been shown to be required for nitrate homeostasis or pH adjustment, and previous results indicated that AtCLCc is involved in nitrate accumulation. We investigated new physiological functions of this CLC member in Arabidopsis. Here we report that AtCLCc is strongly expressed in guard cells and pollen and more weakly in roots. Use of an AtCLCc:GFP fusion revealed localization to the tonoplast. Disruption of the AtCLCc gene by a T-DNA insertion in four independent lines affected physiological responses that are directly related to the movement of chloride across the tonoplast membrane. Opening of clcc stomata was reduced in response to light, and ABA treatment failed to induce their closure, whereas application of KNO₃ but not KCl restored stomatal opening. clcc mutant plants were hypersensitive to NaCl treatment when grown on soil, and to NaCl and KCl in vitro, confirming the chloride dependence of the phenotype. These phenotypes were associated with modifications of chloride content in both guard cells and roots. These data demonstrate that AtCLCc is essential for stomatal movement and salt tolerance by regulating chloride homeostasis.

The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the P1B-2 subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2- to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a P1B-2 ATPase in a multicellular eukaryote.

Light activates proton (H(+))-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO(2) to photosynthetic tissues. Light to darkness transition, high CO(2) levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H(+)-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO(2) and darkness. The OST2 gene encodes the major plasma membrane H(+)-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H(+)-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure.

Epidermal cell turgor depends on a K+ channel to regulate stomatal aperture. 26 ème Colloque des Canaux ioniques

ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of “safe-food” strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.

Among the complete set of Arabidopsis ABC proteins, 15 correspond to the soluble or NBD-type (nucleotide binding domains). We have cloned cDNAs coding for three closely homologous NBD-like proteins, At NAP2, At NAP4 and At NAP9 that display a characteristic ABC signature motif. In transitory expression assays, the three At NAP proteins fused to the green-fluorescent protein (GFP) were localized in the cytosol of tobacco and Arabidopsis cell cultures. The expression of these genes is enhanced by 1% sucrose in the culture medium but not by non-assimilable sugars. This result was confirmed with an At NAP2::GUS translational fusion derived from a promoter trap line in which the T-DNA is inserted near the 3′ end of the AtNAP 2 coding sequence. According to the GUS staining, AtNAP 2 is expressed in the elongation zone and meristem of roots of white light-grown seedlings and in the hypocotyl of dark-grown seedlings. We suggest that At NAP2 might be involved in cell elongation processes.

In this review we concentrate on guard cell metabolism and CO2 sensing. Although a matter of some controversy, it is generally accepted that the Calvin cycle plays a minor role in stomatal movements. Recent data emphasise the importance of guard cell starch degradation and of carbon import from the guard cell apoplast in promoting and maintaining stomatal opening. Chloroplast maltose and glucose transporters appear to be crucial to the export of carbon from both guard and mesophyll cells. The way guard cells sense CO2 remains an unresolved question. However, a better understanding of the cellular events downstream from CO2 sensing is emerging. We now recognise that there are common as well as unique steps in abscisic acid (ABA) and CO2 signalling pathways. For example, while ABA and CO2 both trigger increases in cytoplasmic free calcium, unlike ABA, CO2 does not promote a cytoplasmic pH change. Future advances in this area are likely to result from the increased use of techniques and resources, such as, reverse genetics, novel mutants, confocal imaging, and microarray analyses of the guard cell transcriptome.

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H2O2 production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H2O2 production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H2O2 production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.

The stomatal opening in abaxial epidermal strips of Nicotiana glauca is suppressed by the presence of abscisic acid (ABA) or methyl jasmonate (MJ). The role of calcium and related secondary messengers/signaling compounds during the restriction of stomatal opening by ABA or MJ was assessed. External calcium promoted, while EGTA prevented the process of stomatal closure, in case of both ABA and MJ. However, the effect of EGTA was more pronounced in the case of MJ than that of ABA. These observations established that the intracellular calcium is an important factor during stomatal closure induced not only by ABA but also by MJ. Lanthanum, verapamil, ruthenium red (Ca 2+ channel blockers), trifluoperazine and W7 (calmodulin (CaM) antagonists) prevented MJ-induced stomatal closure but had only a partial effect on ABA-mediated stomatal closure. In contrast, U73122, 1-butanol (phospholipase C/D inhibitors) and phorbol myristate acetate (PMA, protein kinase C activator) reversed the stomatal closure caused by ABA but not that by MJ. Thus, the suppression of stomatal opening by ABA appears to be more dependent on phospholipases and protein kinase C than that by MJ. Similarly, the action of MJ on stomata seems to be crucially dependent on CaM and calcium channels of the guard cells. We suggest that MJ could be a useful tool, besides ABA, to analyze the sequence and pattern of signal transduction in stomatal guard cells.

�Summary Because plant wilting has been described as a consequence of cadmium (Cd 2þ ) toxicity, we investigate Cd 2þ effects on plant water losses, gas exchanges and stomatal behaviour in Arabidopsis thaliana L. Effects of 1week Cd 2þ application in hydroponic condition (CdCl2 10–100mM) were analyzed. A 10-lM Cd 2þ concentration had no significant effect on the plant–water relationship and carbon assimilation. At higher Cd 2þ concentrations, a Cd 2þ -dependent decrease in leaf conductance and CO2 uptake was observed despite the photosynthetic apparatus appeared not to be affected as probed by fluorescence measurements. In epidermal strip bioassays, nanomolar Cd2þ concentrations reduced stomatal opening under light in A. thaliana, Vicia faba and Commelina communis. Application of 5mM ABA limited the root-to-shoot translocation of cadmium. However, the Cd 2þ -induced stomatal closure was likely ABA-independent, since a 5-day treatment with 50 lM Cd 2þ did not affect the plant relative water content. Additionally, a similar Cd 2þ induced stomatal closure was observed in the ABA insensitive mutant abi1-1. Interestingly, this mutant displayed a higher transpiration rate than the wild type but did not accumulate more Cd2þ , arguing that Cd 2þ uptake is not dependent only on the transpiration flow. Application of putative calcium channels inhibitors suppressed the inhibitory effect of Cd 2þ in epidermal strip experiments, suggesting that Cd 2þ could enter the guard cell through calcium channels. Patch-clamp studies with V. faba guard cell protoplasts showed that plasma membrane K þ channels were insensitive to external Cd 2þ application whereas Ca 2þ channels were found permeable to Cd 2þ . In conclusion, we propose that Cd 2þ affects guard cell regulation in an ABA-independent manner by entering the cytosol via Ca 2þ channels. Abbreviations: ABA, Abscissic acid; DMSO, dimethyl sulfoxide; DW, dry weight; FW, fresh weight; NPPB, 5-nitro-2,3-phenylpropylaminobenzoic acid; RWC, relative water content.

The RD20 gene encodes a member of the caleosin family, which is primarily known to function in the mobilization of seed storage lipids during germination. In contrast to other caleosins, RD20 expression is early-induced by water deficit conditions and we recently provided genetic evidence for its positive role in drought tolerance in Arabidopsis. RD20 is also responsive to pathogen infection and is constitutively expressed in diverse tissues and organs during development suggesting additional roles for this caleosin. This addendum describes further exploration of phenotypic alterations in T-DNA insertional rd20 mutant and knock-out complemented transgenic plants in the context of early development and susceptibility to a phytopathogenic bacteria. We show that the RD20 gene is involved in ABA-mediated inhibition of germination and does not play a significant role in plant defense against Pseudomonas syringae.

Zn is an essential microelement for all living cells and Zn deficiency is widespread in world's population. At the same time, high Zn concentration and low Cd concentration are toxic to the environment. Both Zn and Cd are transported in planta via Zn/Cd HMA transporters. Engineering of HMAs expression in plants may provide a way for Zn biofortification of food as well as phytoremediation of polluted soils. In the present study we have assessed the impact of Zn/Cd HMAs invalidation/overexpression in Arabidopsis thaliana on Zn and Cd translocation from the roots to the shoots and in Zn grain filling. Overexpression of AtHMA4 had a large impact on Zn and Cd translocation and resulted in a 3-fold higher potential of Cd and Zn extraction from an industrial soil highly contaminated by Zn, Pb and Cd. Despite AtHMA4 overexpressing lines presenting a higher Zn concentration in the shoot, the Zn content in the seeds was found to be lower than in wild type plants. Our results indicate that AtHMA4 overexpression is an efficient tool to increase the root to shoot translocation of Zn and Cd in plants. Concerning biofortification of seeds, this study underlines the need for specific promoters to drive an expression pattern of the transporters in favour of Zn grain filling.

International audience; Faced with declining soil-water potential, plants synthesize abscisic acid (ABA), which then triggers stomatal closure to conserve tissue moisture. Closed stomates, however, also create several physiological dilemmas. Among these, the large CO2 influx required for net photosynthesis will be disrupted. Depleting CO2 in the plant will in turn bias stomatal opening by suppressing ABA sensitivity, which then aggravates transpiration further. We have investigated the molecular basis of how C3 plants resolve this H2 O-CO2 conflicting priority created by stomatal closure. Here, we have identified in Arabidopsis thaliana an early drought-induced spermidine spermine-N(1) -acetyltransferase homolog, which can slow ABA-mediated stomatal closure. Evidence from genetic, biochemical and physiological analyses has revealed that this protein does so by acetylating the metabolite 1,3-diaminopropane (DAP), thereby turning on the latter's intrinsic activity. Acetylated DAP triggers plasma membrane electrical and ion transport properties in an opposite way to those by ABA. Thus in adapting to low soil-water availability, acetyl-DAP could refrain stomates from complete closure to sustain CO2 diffusion to photosynthetic tissues.

Uranium is a natural element which is mainly redistributed in the environment due to human activity, including accidents and spillages. Plants may be useful in cleaning up after incidents, although little is yet known about the relationship between metal speciation and plant response. Here, J-Chess modeling was used to predict U speciation and exposure conditions affecting U bioavailability for plants. The model was confirmed by exposing Arabidopsis thaliana plants to U under hydroponic conditions. The early root response was characterized using complete Arabidopsis transcriptome microarrays (CATMA). Expression of 111 genes was modified at the three timepoints studied. The associated biological processes were further examined by real-time quantitative RT-PCR. Annotation revealed that oxidative stress, cell wall and hormone biosynthesis, and signaling pathways (including phosphate signaling) were affected by U exposure. The main actors in iron uptake and signaling (IRT1, FRO2, AHA2, AHA7 and FIT1) were strongly down-regulated upon exposure to uranyl. A network calculated using IRT1, FRO2 and FIT1 as bait revealed a set of genes whose expression levels change under U stress. Hypotheses are presented to explain how U perturbs the iron uptake and signaling response. These results give preliminary insights into the pathways affected by uranyl uptake, which will be of interest for engineering plants to help clean areas contaminated with U. © 2014 The Royal Society of Chemistry.

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutations abi1--1 and abi2--1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction-of-function allele of ABI2, abi2--1R1. The in vitro phosphatase activity of the abi2--1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2--1R1 plants displayed a wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.

In plants, water availability and CO 2 partial pressure modulate stomatal aperture. Phosphoenolpyruvate carboxylase (PEPCase) is a major enzyme in the pathway leading to malate synthesis which is, with chloride, the main counterion for potassium accumulated in the guard cell vacuole during stomatal opening. Whether phosphorylation of PEPCase could be a major event in guard cell regulation was investigated. Antibodies (APS-lgGs) raised to a synthetic polypeptide of 23 amino acids containing the phosphorylation site (ser-8) of the Sorghum PEPCase recognized the guard cell PEPCase from Commelina communis L. at 110 and 120 kDa. The in vitro phosphorylation of the 110 kDa isoform by PKA was 50% inhibited by APS-lgGs demonstrating that the regulatory phosphorylation site was present and functional in the guard cell enzyme. Phosphorylation by PKA resulted in a 50% increase in the V max of the enzyme (4.2±0.3 compared to 2.8±0.4 pmol h -1 GCP -1 , pH 7.3 and 200 μM PEP) and a reduction in L-malate inhibition (64% compared to 82% inhibition by 1 mM L-malate). In the presence of 1 mM L-malate (pH 7.3) phosphorylation of the enzyme by PKA resulted in a 3-fold increase in the V max . Binding of APS-lgGs to the phosphorylation site of the enzyme led to the highest activity (10.9±2.6 pmol h - 1 GCP -1 ) and to an absence of inhibition by 1 mM L-malate at pH 7.3 and 8.0. These changes in the kinetic properties of the enzyme after phosphorylation should have important consequences in terms of stomatal regulation.

We have investigated the stomatal and phototropic responses to blue light of a number of single and double mutants at various loci that encode proteins involved in blue-light responses in Arabidopsis. The stomatal responses of light-grown mutant plants (cry1, cry2, nph1, nph3, nph4, cry1cry2, andnph1cry1) did not differ significantly from those of their wild-type counterparts. Second positive phototropic responses of etiolated mutant seedlings, cry1, cry2, cry1cry2, andnpq1-2, were also similar to those of their wild-type counterparts. Although npq1 and single and double cry1cry2 mutants showed somewhat reduced amplitude for first positive phototropism, threshold, peak, and saturation fluence values for first positive phototropic responses of etiolated seedlings did not differ from those of wild-type seedlings. Similar to the cry1cry2 double mutants and tonpq1-2, a phyAphyB mutant showed reduced curvature but no change in the position or shape of the fluence-response curve. By contrast, the phototropism mutantnph1-5 failed to show phototropic curvature under any of the irradiation conditions used in the present study. We conclude that the chromoproteins cry1, cry2, nph1, and the blue-light photoreceptor for the stomatal response are genetically separable. Moreover, these photoreceptors appear to activate separate signal transduction pathways.

Carbon dioxide and abscisic acid (ABA) are two major signals triggering stomatal closure. Their putative interaction in stomatal regulation was investigated in well-watered air-grown or double CO 2 -grown Arabidopsis thaliana plants, using gas exchange and epidermal strip experiments. With plants grown in normal air, a doubling of the CO 2 concentration resulted in a rapid and transient drop in leaf conductance followed by recovery to the pre-treatment level after about two photoperiods. Despite the fact that plants placed in air or in double CO 2 for 2 d exhibited similar levels of leaf conductance, their stomatal responses to an osmotic stress (0.16-0.24 MPa) were different. The decrease in leaf conductance in response to the osmotic stress was strongly enhanced at elevated CO 2 . Similarly, the drop in leaf conductance triggered by 1 μM ABA applied at the root level was stronger at double CO 2 . Identical experiments were performed with plants fully grown at double CO 2 . Levels of leaf conductance and carbon assimilation rate measured at double CO 2 were similar for air-grown and elevated CO 2 -grown plants. An enhanced response to ABA was still observed at high CO 2 in pre-conditioned plants. It is concluded that: (i) in the absence of stress, elevated CO 2 slightly affects leaf conductance in A. thaliana; (ii) there is a strong interaction in stomatal responses to CO 2 and ABA which is not modified by growth at elevated CO 2 .

To investigate whether cyclic GMP (cGMP) would mediate, in an intracellular Ca2+ -dependent manner, coupling of auxin to stomatal opening, the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) were compared in epidermal strips of Commelina communis. In this comparison were studied possible effects of intracellular Ca2+ modulators, GTP-binding protein (G-protein) modulators and selective inhibitors of enzymatic reactions which use or generate cGMP. The stomatal response to IBA was almost similarly reversed by the Ca2+ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), the intracellular Ca2+-release inhibitors ruthenium red and procaine, the inactive cGMP analog Rp-8-bromoguanosine 3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPS), the inhibitor of cGMP-producing guanylyl cyclase LY 83583, the G-protein inhibitor mas17 and the G-protein antagonist pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2. Comparison with stomatal opening in response to 8-Br-cGMP, which was almost completely suppressed by either BAPTA, ruthenium red, procaine or Rp-8-Br-cGMPS, strongly suggests that cGMP acts downstream of G-protein activation as a second messenger for IBA signal transduction and that the cGMP pathway likely depends on cytosolic Ca2+signaling.

Stomata1 responses to CO, were investigated in Arabidupsis thaliana wild-type and ABA insensitive mutants (abil-I and u&Z-I). Although stomata from ahi and abi2 mutant plants failed to respond to ABA and were originally wide open, they still remained sensitive to illumination and atmospheric CO+uppression, showing at least 50 %%. of wild-type response. Similar results were obtained studyin g either stomata1 movements in epidermal strip bioassays or changes in leaf conductance in whole plant experiments. The previously reported CO, insensitivity of stomata from ahil and abi2 mutant plants probably has to be ascribed to the experimental conditions used by the authors. Thus, in abil and abi2 mutant plants, total impairment in the response to ABA is accompanied by a limited inhibition in stomata1 response to CO, and light. These results do not support a total convergence of ABA and CO, signalling pathways in guard cells. 0 Elsevier, Paris

Resume Cette note decrit les premiers enregistrements de courants anioniques lents sur des cellules de garde intactes de Commelina communis et d'Arabidopsis thaliana, la plante modele en genetique. Les mesures ont ete accomplies a l'aide de la technique de controle discontinu du potentiel de membrane (dSEVC), en presence d'inhibiteurs de canaux potassiques. Les courants enregistres presentent des caracteristiques d'activation lente lors de la depolarisation de la membrane et de desactivation lente au cours de l'hyperpolarisation. Ces courants sont bloques par le NPPB, un inhibiteur classique de canaux anioniques lents chez Vicia t'aba. Les cinetiques et la reponse pharmacologique de ces courants confortent l'hypothese de leur nature anionique. Ces resultats montrent que les canaux anioniques d'A thaliana peuvent etre aisement etudies sur cellule de garde intacte a l'aide de la technique de dSEVC. Abstract — This note described the first recordings of slow anion currents in intact guard cells of Commelina communis and Arabidopsis thaliana, a species of fundamental genetic interest. Experiments were conducted in the presence of potassium channels inhibitors, using the discontinuous single electrode voltage-clamp (dSEVC) technique. Current recordings display slow activation upon membrane depolarization and slow deactivation during hyperpolarization. These currents were inhibited by NPPB, described as a blocker of slow anion channels in Vicia faba guard cell protoplasts. Kinetic and pharmacological profiles of this current support the hypothesis for an anion flux. These results show that A thaliana anion currents can be easily studied in intact guard cells using the dSEVC technique.

Withering of abscisic acid (ABA)-insensitive mutants in standard watering conditions as well as under drought suggests that ABA would regulate stomatal closing not only under water stress but also under other hydric conditions. Depending on its applied concentration, the ABA closing signal might be transduced through different pathways. This possibility was investigated in epidermal peels of Commelina communis by comparing the effects of different Ca 2+ buffers, protein kinase inhibitors, calmodulin (CaM) antagonists and guanosine-triphosphate-binding protein (G protein) modulators on the stomatal closing responses to 10 nM ABA (ABA 10 ) and 100 nM ABA (ABA 100 ). EGTA specifically suppressed the response to ABA 10 , while the Ca 2+ buffer 1,2-bis( o -aminophenoxy)ethane- N,N,N′,N′ -tetraacetic acid (BAPTA) similarly inhibited the responses to ABA 10 and ABA 100 . The response to ABA 10 was specifically affected by the protein kinase inhibitors KT5926 and 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine (ML-7), the CaM antagonist N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and the phospholipase C inhibitor 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the response to ABA 100 was specifically affected by the G protein antagonist pGlu-GlnD-Trp-Phe-D-Trp-D-Trp-Met-NH 2 (GP Ant-2) and mas 17, an inactive mastoparan analog. These data are consistent with two possible ABA routes, each of them exhibiting specific Ca 2+ -requirements and protein phosphorylations, and being initiated by its own receptor. Furthermore, analogies with animal features suggest that the ABA 10 and ABA 100 closing signals might be extracellularly perceived.

Stomatal responses to light of Arabidopsis thaliana wild-type plants and mutant plants deficient in starch (phosphoglucomutase deficient) were compared in gas exchange experiments. Stomatal density, size and ultrastructure were identical for the two phenotypes, but no starch was observed in guard cells of the mutant plants whatever the time of day. The overall extent of changes in stomatal conductance during 14 h light–10 h dark cycles was similar for the two phenotypes. However, the slow endogenous stomatal opening occurring in darkness in the wild type was not observed in the mutant plants. Stomata in the mutant plants responded much more slowly to blue light (70 μmol m−2 s−1) though the response to red light (250 μmol m−2 s−1) was similar to that of wild-type plants. In paradermal sections, stomatal responses to red light (300 μmol m−2 s−1) were weak for wild-type plants as well as for mutant plants. Stomatal opening was greater under low blue light (75 μmol m−2 s−1) than under red light for the two genotypes. However, in mutant plants, a high chloride concentration (50 mol m−3) was necessary to achieve the same stomatal aperture as observed for the wild-type plants. These results suggest that starch metabolism, via the synthesis of a counter-ion to potassium (probably malate), is required for full stomatal response to blue light but is not involved in the stomatal response to red light.

Commelina communis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by 45 Ca 2+ -overlays. CaM was found to be 3- to 7-fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca 2+ -dependent manner were detected by gold-labelled CaM overlays. Using bioassays with epidermal strips, different CaM-antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 >> ML7 = ML9 >> H7 > KN62. Only the inhibitors with a high specificity for Ca 2+ -CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca 2+ - or a Ca 2+ -CaM-dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.

SummaryPlant water loss and CO2 uptake are controlled by valve-like structures on the leaf surface known as stomata. Stomatal aperture is regulated by hormonal and environmental signals. We show here that stomatal sensitivity to the drought hormone abscisic acid (ABA) is acquired during leaf development by exposure to an increasingly dryer atmosphere in the rosette plant Arabidopsis. Young leaves, which develop in the center of the rosette, do not close in response to ABA. As the leaves increase in size, they are naturally exposed to increasingly dry air as a consequence of the spatial arrangement of the leaves, and this triggers the acquisition of ABA sensitivity. Interestingly, stomatal ABA sensitivity in young leaves is rapidly restored upon water stress. These findings shed new light on how plant architecture and stomatal physiology have coevolved to optimize carbon gain against water loss in stressing environments.

Micromolar concentrations of potassium ferricyanide inhibit light-induced stomatal opening. The extent of the inhibition is dependent on the presence of carbon dioxide and the concentration of potassium ferricyanide needed to obtain 50% inhibition of stomatal opening is 40-fold higher in CO 2 -free air than in normal air. The fungal toxin, fusicoccin (1 μM), overcame the ferricyanide inhibition of stomatal opening indicating that the electron acceptor may interact more or less directly with the activity of the plasma membrane H + -ATPase. Although potassium ferricyanide strongly inhibited stomatal opening, it had only minor effects on stomatal maintaining or stomatal closure due to darkness or ABA

International audience; Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of $PHO1$ in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. $PHO1$ is involved in the export of phosphate into the root xylem vessels and, as a result, the $pho1$ mutant is characterized by low shoot phosphate levels. In leaves, $PHO1$ was found expressed in guard cells and up‐regulated following treatment with ABA. The $pho1$ mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro‐grafting a $pho1$ shoot scion onto wild‐type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. $PHO1$ knockdown using RNA interference specifically in guard cells of wild‐type plants caused a reduced stomatal response to ABA. In agreement, specific expression of $PHO1$ in guard cells of $pho1$ plants complemented the mutant guard cell phenotype and re‐established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of $PHO1$ in the stomatal response to ABA.

With the evolution of high-throughput techniques, performing organism-wide analyses of gene expression, protein content and metabolite profile is becoming easier. This new era of biology opens the way for incredibly promising advances but also extremely challenging difficulties in the aim of integrating these datasets into biologically relevant models. To address questions and unravel networks occurring in cadmium stressed plants, researchers have already taken advantage of such tools. We provide here an overview of the discoveries that have been made regarding plant−cadmium interaction using “omics” methods, and show how their use is relevant to compare species and stresses at the whole plant level. At the end, we propose future breakthroughs to be addressed using these techniques, and discuss the up-coming challenges of data mining and model drawing.

P1B-ATPases are cation transporters found in all organisms. In plants, they are found in almost all membranes including plasma membrane, internal chloroplastic membrane, thylakoids, post-Golgi and tonoplast, where they manage nutrient fluxes (Cu+, Zn2+) as well as detoxification of toxic cations (Co2+, Cd2+, Pb2+, high Zn2+). All P1B-ATPases from Arabidopsis thaliana have now been characterized; they are involved in plant-scale and cell-scale cations fluxes. Orthologs of these genes are being characterized in other species such as rice or soybean, and their expression domain and roles are similar to those of Arabidopsis. Orthologs from hyperaccumulator species, such as Arabidopsis halleri or Thlaspi cearulescens, have been found to be over-expressed due to the presence of strong promoters and gene duplications. They have recently been shown as essential in the hyperaccumulators properties. Our knowledge about plant P1B-ATPases opens new fields of investigation in biotechnology on the use of these transporters in phytoremediation and biofortification strategies. In this chapter, we draw an overview of the present knowledge about plant P1B-ATPases structures, their physiological roles and their phylogeny. Finally, we summarize the potential that P1B-ATPases give us in crop improvement.

UMR BPMP; UMR LEPSE; International audience; Plants overcome water deficit conditions by combining molecular, biochemical and morphological changes. At the molecular level, many stress-responsive genes have been isolated, but knowledge of their physiological functions remains fragmentary. Here, we report data for RD20, a stress-inducible Arabidopsis gene that belongs to the caleosin family. As for other caleosins, we showed that RD20 localized to oil bodies. Although caleosins are thought to play a role in the degradation of lipids during seed germination, induction of RD20 by dehydration, salt stress and ABA suggests that RD20 might be involved in processes other than germination. Using plants carrying the promoter RD20::uidA construct, we show that RD20 is expressed in leaves, guard cells and flowers, but not in root or in mature seeds. Water deficit triggers a transient increase in RD20 expression in leaves that appeared predominantly dependent on ABA signaling. To assess the biological significance of these data, a functional analysis using rd20 knock-out and overexpressing complemented lines cultivated either in standard or in water deficit conditions was performed. The rd20 knock-out plants present a higher transpiration rate that correlates with enhanced stomatal opening and a reduced tolerance to drought as compared with the wild type. These results support a role for RD20 in drought tolerance through stomatal control under water deficit conditions.

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H(+) extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O(2) uptake with a 4H(+) to O(2) ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.

A mass spectrometric method combining (16)O/(18)O and (12)C/(13)C isotopes was used to quantify the unidirectional fluxes of O(2) and CO(2) during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O(2) uptake and CO(2) evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O(2) (61 micromoles of O(2) per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O(2) per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO(2) in darkness at a rate of 27 micromoles of CO(2) per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O(2) evolution and CO(2) fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO(2) evolution by guard cell protoplasts was sharply decreased (37%), while O(2) uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO(2) assimilation and may be dissipated for other purposes such as ion uptake.

Cadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers' yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO(3), As(III), As(V), CuSO(4), or HgCl(2). Abolishment of the catalytic activity by expression of SpHMT1(K623M) mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHMA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans.

Background ABC proteins constitute one of the largest families of transporters found in all living organisms. In Arabidopsis thaliana, 120 genes encoding ABC transporters have been identified. Here, the characterization of one member of the MRP subclass, AtMRP6, is described. Results This gene, located on chromosome 3, is bordered by AtMRP3 and AtMRP7. Using real-time quantitative PCR (RT-Q-PCR) and the GUS reporter gene, we found that this gene is essentially expressed during early seedling development, in the apical meristem and at initiation point of secondary roots, especially in xylem-opposite pericycle cells where lateral roots initiate. The level of expression of AtMRP6 in response to various stresses was explored and a significant up-regulation after cadmium (Cd) treatment was detected. Among the three T-DNA insertion lines available from the Salk Institute library, two knock-out mutants, Atmrp6.1 and Atmrp6.2 were invalidated for the AtMRP6 gene. In the presence of Cd, development of leaves was more affected in the mutants than wild-type plants, whereas root elongation and ramification was comparable. Conclusion The position of AtMRP6 on chromosome 3, flanked by two other MRP genes, (all of which being induced by Cd) suggests that AtMRP6 is part of a cluster involved in metal tolerance, although additional functions in planta cannot be discarded.

At least four genes encoding plasma membrane inward K + channels (K in channels) are expressed in Arabidopsis guard cells. A double mutant plant was engineered by disruption of a major K in channel gene and expression of a dominant negative channel construct. Using the patch-clamp technique revealed that this mutant was totally deprived of guard cell K in channel (GCK in ) activity, providing a model to investigate the roles of this activity in the plant. GCK in activity was found to be an essential effector of stomatal opening triggered by membrane hyperpolarization and thereby of blue light-induced stomatal opening at dawn. It improved stomatal reactivity to external or internal signals (light, CO 2 availability, and evaporative demand). It protected stomatal function against detrimental effects of Na + when plants were grown in the presence of physiological concentrations of this cation, probably by enabling guard cells to selectively and rapidly take up K + instead of Na + during stomatal opening, thereby preventing deleterious effects of Na + on stomatal closure. It was also shown to be a key component of the mechanisms that underlie the circadian rhythm of stomatal opening, which is known to gate stomatal responses to extracellular and intracellular signals. Finally, in a meteorological scenario with higher light intensity during the first hours of the photophase, GCK in activity was found to allow a strong increase (35%) in plant biomass production. Thus, a large diversity of approaches indicates that GCK in activity plays pleiotropic roles that crucially contribute to plant adaptation to fluctuating and stressing natural environments.

Transcriptional regulation in response to cadmium treatment was investigated in both roots and leaves of Arabidopsis, using the whole genome CATMA microarray containing at least 24,576 independent probe sets. Arabidopsis plants were hydroponically treated with low (5 μM) or high (50 μM) cadmium concentrations during 2, 6, and 30 hours. At each time point, Cd level was determined using ICP-AES showing that both plant tissues are able to accumulate the heavy metal. RT-PCR of eight randomly selected genes confirmed the reliability of our microarray results. Analyses of response profiles demonstrate the existence of a regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to cadmium. One of the main response observed in roots was the induction of genes involved in sulfur assimilation–reduction and glutathione (GSH) metabolism. In addition, HPLC analysis of GSH and phytochelatin (PC) content shows a transient decrease of GSH after 2 and 6 h of metal treatment in roots correlated with an increase of PC contents. Altogether, our results suggest that to cope with cadmium, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of GSH for PC biosynthesis. Interestingly, in leaves an early induction of several genes encoding enzymes involved in the biosynthesis of phenylpropanoids was observed. Finally, our results provide new insights to understand the molecular mechanisms involved in transcriptional regulation in response to cadmium exposure in plants.

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H 2 O 2 in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H 2 DCF-DA). Bicarbonate-induced stomatal closure as well as H 2 O 2 production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H 2 O 2 production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H 2 O 2 was quicker and greater with ABA than that with bicarbonate. Such pattern of H 2 O 2 production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H 2 O 2 is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.

The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.

International audience; The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF I and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Delta ycfl strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a wellknown inhibitor of gamma-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of gamma-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Delta ycfl. (c) 2006 Elsevier Masson SAS. All rights reserved.

International audience; Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P1B-type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.

The Arabidopsis thaliana AtHMA4 is a P1B-type ATPase that clusters with the Zn/Cd/Pb/Co subgroup. It has been previously shown, by heterologous expression and the study of AtHMA4 knockout or overexpressing lines in Arabidopsis [1–3], that AtHMA4 is implicated in zinc homeostasis and cadmium tolerance. Here, we report the study of the heterologous expression of AtHMA4 in the yeast Saccharomyces cerevisiae. AtHMA4 expression resulted in an increased tolerance to Zn, Cd and Pb and to a phenotypic complementation of hypersensitive mutants. In contrast, an increased sensitivity towards Co was observed. An AtHMA4::GFP fusion protein was observed in endocytic vesicles and at the yeast plasma membrane. Mutagenesis of the cysteine and glutamate residues from the N-ter degenerated heavy metal binding domain impaired the function of AtHMA4. It was also the case when the C-ter His11 stretch was deleted, giving evidence that these amino acids are essential for the AtHMA4 binding/translocation of metals.

AtHMA4 is an Arabidopsis thaliana P1B-ATPase which transports Zn and Cd. Here, we demonstrate that AtHMA4 is localized at the plasma membrane and expressed in tissues surrounding the root vascular vessels. The ectopic overexpression of AtHMA4 improved the root growth in the presence of toxic concentrations of Zn, Cd and Co. A null mutant exhibited a lower translocation of Zn and Cd from the roots to shoot. In contrast, the AtHMA4 overexpressing lines displayed an increase in the zinc and cadmium shoot content. Altogether, these results strongly indicate that AtHMA4 plays a role in metal loading in the xylem.

The Arabidopsis thaliana AtHMA3 protein belongs to the P(1B)-adenosine triphosphatase (ATPase) transporter family, involved in heavy metal transport. Functional expression of AtHMA3 phenotypically complements the Cd/Pb-hypersensitive yeast strain Deltaycf1, but not the Zn-hypersensitive mutant Deltazrc1. AtHMA3-complemented Deltaycf1 cells accumulate the same amount of cadmium as YCF1-complemented Deltaycf1 cells or wild-type cells, suggesting that AtHMA3 carries out an intracellular sequestration of Cd. A mutant of AtHMA3 altered in the P-ATPase phosphorylation domain did not complement Deltaycf1, suggesting that metal transport rather than chelation is involved. The fusion protein AtHMA3::green fluorescent protein (GFP) is localized at the vacuole, consistent with a role in the influx of cadmium into the vacuolar compartment. In A. thaliana, the mRNA of AtHMA3 was detected mainly in roots, old rosette leaves and cauline leaves. The expression levels were not affected by cadmium or zinc treatments.