Chronic ozone exposure preferentially modifies root rather than foliar metabolism of date palm (Phoenix dactylifera) saplings.

In their natural environment, date palms are exposed to chronic atmospheric ozone (O3) concentrations from local and remote sources. In order to elucidate the consequences of this exposure, date palm saplings were treated with ambient, 1.5 and 2.0 times ambient O3 for three months in a free-air controlled exposure facility. Chronic O3 exposure reduced carbohydrate contents in leaves and roots, but this effect was much stronger in roots. Still, sucrose contents of both organs were maintained at elevated O3, though at different steady states. Reduced availability of carbohydrate for the Tricarboxylic acid cycle (TCA cycle) may be responsible for the observed reduced foliar contents of several amino acids, whereas malic acid accumulation in the roots indicates a reduced use of TCA cycle intermediates. Carbohydrate deficiency in roots, but not in leaves caused oxidative stress upon chronic O3 exposure, as indicated by enhanced malonedialdehyde, H2O2 and oxidized glutathione contents despite elevated glutathione reductase activity. Reduced levels of phenolics and flavonoids in the roots resulted from decreased production and, therefore, do not indicate oxidative stress compensation by secondary compounds. These results show that roots of date palms are highly susceptible to chronic O3 exposure as a consequence of carbohydrate deficiency.

[Efficacy of Disease Management Programs Asthma and COPD? Results of a Cross-Sectional Study]. / Wirksamkeit von Disease-Management-Programmen für Asthma und COPD? Ergebnisse einer Querschnittstudie.

BACKGROUND: The efficacy of the German disease management programs (DMP) asthma and chronic obstructive pulmonary disease (COPD) cannot be shown with the legally bound documentations. Studies with control groups are rare. Aim of this work was to investigate in a cross-sectional study whether the disease control differs in participants (DMP+) and non-participants (DMP - ) of the DMPs asthma and COPD. METHODS: The study was a prospective multicenter cross-sectional study. Primary endpoints were the Asthma Control Test™ (ACT) in the asthma part of the study and the COPD Assessment Test™ (CAT) for the COPD part. RESULTS: A total of 1038 asthma patients and 846 COPD patients were included, of whom about 70 % participated in the corresponding DMP. The ACT total score was higher in asthma DMP+ patients than in DMP- patients (mean difference 0.86; 95 % CI: 0.29 - 1.43;p = 0.003), but not clinically relevant. For COPD there was no clinically relevant difference in COPD disease impact (0.52; 95 % CI: - 0.71 - 1.75; p = 0.405). Although DMP patients had to be enrolled in the respective DMP for at least one year, only 60 % of these patients had participated in a structured education. We did not observe a difference in disease control in DMP patients who respectively participated and did not participate in a structured education. DISCUSSION: There was no clinically relevant difference in disease control between DMP+ and DMP- patients. The efficacy of DMPs has been demonstrated internationally in randomized controlled trials. Randomized controlled trials should be conducted in Germany for demonstrating efficacy of DMPs asthma and COPD. REGISTRATION: drks.de, DRKS00007664, Registration date: Jan 15, 2015.

Venus flytrap trigger hairs are micronewton mechano-sensors that can detect small insect prey.

Venus flytraps detect moving insects via highly sensitive, action potential (AP)-producing trigger hairs, which act as high-sensitivity levers, crucial for prey capture and digestion. Controlled stimulation revealed that they can trigger APs for deflections >2.9°, angular velocities >3.4° s-1 and forces >29 µN. Hairs became desensitized and subsequently responded to fast consecutive stimulations; desensitization increased at lower temperatures. Recording of ant trigger hair contact events revealed that even small insects exceed the hairs' sensitivity threshold.

Spatio-temporal Aspects of Ca2+ Signalling: Lessons from Guard Cells and Pollen Tubes.

Changes in cytosolic Ca2+ concentration ([Ca2+]cyt) serve to transmit information in eukaryotic cells. The involvement of this second messenger in plant cell growth as well as osmotic- and water relations is well established. After almost 40 years of intense research on the coding and decoding of plant Ca2+ signals, numerous proteins involved in Ca2+ action have been identified. However, we are still far from understanding the complexity of Ca2+ networks. New in vivo Ca2+ imaging techniques combined with molecular genetics allow visualisation of spatio-temporal aspects of Ca2+ signalling. In parallel, cell biology together with protein biochemistry and electrophysiology are able to dissect information processing by this second messenger in space and time. Here we focus on the time-resolved changes in cellular events upon Ca2+ signals, concentrating on the two best-studied cell types, pollen tubes and guard cells. We put their signalling networks side by side, compare them with those of other cell types and discuss rapid signalling in the context of Ca2+ transients and oscillations to regulate ion homeostasis.

Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the ß-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks.

Gating of the two-pore cation channel AtTPC1 in the plant vacuole is based on a single voltage-sensing domain.

The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembranes. Each subunit consists of two homologous Shaker-like halves, with 12 transmembrane domains in total (S1-S6, S7-S12). In plants, TPC1 channels reside in the vacuolar membrane, and upon voltage stimulation, give rise to the well-known slow-activating SV currents. Here, we combined bioinformatics, structure modelling, site-directed mutagenesis, and in planta patch clamp studies to elucidate the molecular mechanisms of voltage-dependent channel gating in TPC1 in its native plant background. Structure-function analysis of the Arabidopsis TPC1 channel in planta confirmed that helix S10 operates as the major voltage-sensing site, with Glu450 and Glu478 identified as possible ion-pair partners for voltage-sensing Arg537. The contribution of helix S4 to voltage sensing was found to be negligible. Several conserved negative residues on the luminal site contribute to calcium binding, stabilizing the closed channel. During evolution of plant TPC1s from two separate Shaker-like domains, the voltage-sensing function in the N-terminal Shaker-unit (S1-S4) vanished.

Venus Flytrap HKT1-Type Channel Provides for Prey Sodium Uptake into Carnivorous Plant Without Conflicting with Electrical Excitability.

The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na(+)- and K(+)-permeable mutants function as ion channels rather than K(+) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na(+)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.

Vacuoles release sucrose via tonoplast-localised SUC4-type transporters.

Arabidopsis thaliana has seven genes for functionally active sucrose transporters. Together with sucrose transporters from other dicot and monocot plants, these proteins form four separate phylogenetic groups. Group-IV includes the Arabidopsis protein SUC4 (synonym SUT4) and related proteins from monocots and dicots. These Group-IV sucrose transporters were reported to be either tonoplast- or plasma membrane-localised, and in heterologous expression systems were shown to act as sucrose/H(+) symporters. Here, we present comparative analyses of the subcellular localisation of the Arabidopsis SUC4 protein and of several other Group-IV sucrose transporters, studies on tissue specificity of the Arabidopsis SUC4 promoter, phenotypic characterisations of Atsuc4.1 mutants and AtSUC4 overexpressing (AtSUC4-OX) plants, and functional comparisons of Atsuc4.1 and AtSUC4-OX vacuoles. Our data show that SUC4-type sucrose transporters from different plant families (Brassicaceae, Cucurbitaceae and Solanaceae) localise exclusively to the tonoplast, demonstrating that vacuolar sucrose transport is a common theme of all SUC4-type proteins. AtSUC4 expression is confined to the stele of Arabidopsis roots, developing anthers and meristematic tissues in all aerial parts. Analyses of the carbohydrate content of WT and mutant seedlings revealed reduced sucrose content in AtSUC4-OX seedlings. This is in line with patch-clamp analyses of AtSUC4-OX vacuoles that characterise AtSUC4 as a sucrose/H(+) symporter directly in the tonoplast membrane.

Light-induced modification of plant plasma membrane ion transport.

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities.

In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca(2+)-independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca(2+)-sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca(2+)-insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.

Potassium-dependent wood formation in poplar: seasonal aspects and environmental limitations.

Potassium availability and acquisition are pivotal for the generation of biomass and thus wood formation in growing poplar trees. Here, we focus on the role of potassium (K(+)) in wood production, transitions between dormancy and active growth, and limiting environmental conditions. Molecular mechanisms, such as expression and activity of K(+) transporters and channels controlling seasonal changes in wood formation, are discussed.

Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta.

The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state (13)C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40-100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state (13)C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The (13)C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of alpha-chitin than beta-chitin.

Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts.

Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca(2+) signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca(2+) homeostasis and stimulus-induced Ca(2+) signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca(2+)-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca(2+) concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca(2+) concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca(2+) concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca(2+) concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca(2+) concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.

TPK1, a Ca(2+)-regulated Arabidopsis vacuole two-pore K(+) channel is activated by 14-3-3 proteins.

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.

Mechanisms of electrically mediated cytosolic Ca2+ transients in aequorin-transformed tobacco cells.

Cytosolic Ca(2+) changes induced by electric field pulses of 50-micros duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca(2+)-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca(2+) concentration reaching a peak value within <100-200 ms. Peaking of [Ca(2+)](cyt) was followed by a biphasic decay due to removal of Ca(2+) (e.g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of approximately 0.5 and 3-5 s, respectively. Experiments with various external Ca(2+) concentrations and conductivities showed that the fast decay arises from Ca(2+) fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca(2+) fluxes through the tonoplast. The amplitude of the [Ca(2+)](cyt) transients increased with increasing field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical field strength of approximately 450 V/cm, whereas breakdown of the tonoplast required approximately 580 V/cm. The above findings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca(2+) and presumably other vacuolar ingredients.

Foliar water supply of tall trees: evidence for mucilage-facilitated moisture uptake from the atmosphere and the impact on pressure bomb measurements.

The water supply to leaves of 25 to 60 m tall trees (including high-salinity-tolerant ones) was studied. The filling status of the xylem vessels was determined by xylem sap extraction (using jet-discharge, gravity-discharge, and centrifugation) and by (1)H nuclear magnetic resonance imaging of wood pieces. Simultaneously, pressure bomb experiments were performed along the entire trunk of the trees up to a height of 57 m. Clear-cut evidence was found that the balancing pressure (P(b)) values of leafy twigs were dictated by the ambient relative humidity rather than by height. Refilling of xylem vessels of apical leaves (branches) obviously mainly occurred via moisture uptake from the atmosphere. These findings could be traced back to the hydration and rehydration of mucilage layers on the leaf surfaces and/or of epistomatal mucilage plugs. Xylem vessels also contained mucilage. Mucilage formation was apparently enforced by water stress. The observed mucilage-based foliar water uptake and humidity dependency of the P(b) values are at variance with the cohesion-tension theory and with the hypothesis that P(b) measurements yield information about the relationships between xylem pressure gradients and height.

Ion channels meet auxin action.

The regulation of cell division and elongation in plants is accomplished by the action of different phytohormones. Auxin as one of these growth regulators is known to stimulate cell elongation growth in the aerial parts of the plant. Here, auxin enhances cell enlargement by increasing the extensibility of the cell wall and by facilitating the uptake of osmolytes such as potassium ions into the cell. Starting in the late 1990s, the auxin regulation of ion channels mediating K+ import into the cell has been studied in great detail. In this article we will focus on the molecular mechanisms underlying the modulation of K+ transport by auxin and present a model to explain how the regulation of K+ channels is involved in auxin-induced cell elongation growth.

AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner.

The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K(+) channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K(+) currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K(+) transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K(+) channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.

Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress.

The phytohormone abscisic acid (ABA) regulates many stress-related processes in plants. In this context ABA mediates the responsiveness of plants to environmental stresses such as drought, cold or salt. In response to water stress, ABA induces stomatal closure by activating Ca2+, K+ and anion channels in guard cells. To understand the signalling pathways that regulate these turgor control elements, we studied the transcriptional control of the K+ release channel gene GORK that is expressed in guard cells, roots and vascular tissue. GORK transcription was up-regulated upon onset of drought, salt stress and cold. The wilting hormone ABA that integrates responses to these stimuli induced GORK expression in seedlings in a time- and concentration-dependent manner and this induction was dependent on extracellular Ca2+. ABA-responsive expression of GORK was impaired in the ABA-insensitive mutants abi1-1 and abi2-1, indicating that these protein phosphatases are regulators of GORK expression. Application of ABA to suspension-cultured cells for 2 min followed by a 4 h chase was sufficient to manifest transcriptional activation of the K+ channel gene. As predicted for a process involved in drought adaptation, only 12-24 h after the release of the stress hormone, GORK mRNA slowly decreased. In contrast to other tissues, GORK expression as well as K+(out) channel activity in guard cells is ABA insensitive, allowing the plant to adjust stomatal movement and water status control separately.