Zytosolisches Ca2^2+^+, ein zentraler Regulator der vakuolären Ionenleitfähigkeit und der schnellen Auxin-Signaltransduktion in ArabidopsisArabidopsis thalianathaliana

Das Phytohormon Auxin erfüllt wichtige Funktionen bei der Initiierung von pflanzlichen Geweben und Organen, wie auch in der Steuerung des Wurzelwachstums im Zusammenspiel mit äußeren Reizen wie Schwerkraft, Wasser- und Nähstoffverfügbarkeit. Diese Funktionen basieren dabei vor allem auf der Auxin-abhängigen Regulation von Zellteilung und -streckung. Wichtig für letzteres ist dabei die Kontrolle des Zellturgors durch die Vakuole. Als Speicher für Nährstoffe, Metabolite und Toxine sind Vakuolen von essentieller Bedeutung. Vakuolär gespeicherte Metabolite und Ionen werden sowohl über aktive Transportprozesse, als auch passiv durch Ionenkanäle, über die vakuoläre Membran mit dem Zytoplasma ausgetauscht. In ihrer Funktion als second messenger sind Kalziumionen wichtige Regulatoren, aber auch Gegenstand vakuolärer Transportprozesse. Änderungen der zytosolischen Kalziumkonzentration wirken nicht nur lokal, sie werden auch mit einer Signalweiterleitung über längere Distanzen in Verbindung gebracht. Im Rahmen dieser Arbeit wurden elektrophysiologische Methoden mit bildgebenden Methoden kombiniert um Einblicke in das Zusammenspiel zwischen zytosolischen Kalziumsignalen, vakuolärer Transportprozesse und der Auxin-Physiologie im intakten pflanzlichen Organismus zu gewinnen. Kalziumsignale sind an der Regulierung vakuolärer Ionenkanäle und Transporter beteiligt. Um dies im intakten Organismus zu untersuchen wurden im Modellsystem junger Wurzelhaare von Arabidopsis thaliana Messungen mit intrazellulären Mikroelektroden durchgeführt. Mittels der Zwei-Elektroden-Spannungsklemm-Technik konnte bestätigt werden, dass die vakuoläre Membran der limitierende elektrische Wiederstand während intravakuolärer Messungen ist und so gemessene Ionenströme in der Tat nur die Ströme über die vakuoläre Membran repräsentieren. Die bereits bekannte zeitabhängige Abnahme der vakuolären Leitfähigkeit in Einstichexperimenten konnte weiterhin mit einer einstichbedingten, transienten Erhöhung der zytosolischen Kalziumkonzentration korreliert werden. Durch intravakuoläre Spannungsklemmexperimente in Wurzelhaarzellen von Kalziumreporterpflanzen konnte dieser Zusammenhang zwischen vakuolärer Leitfähigkeit und der zytosolischen Kalziumkonzentration bestätigt werden. Die Vakuole ist jedoch nicht nur ein Empfänger zytosolischer Kalziumsignale. Da die Vakuole den größten intrazellulären Kalziumspeicher darstellt, wird seit Langem diskutiert, ob sie auch an der Erzeugung solcher Signale beteiligt ist. Dies konnte in intakten Wurzelhaarzellen bestätigt werden. Änderungen des vakuolären Membranpotentials wirkten sich auf die zytosolische Kalziumkonzentration in diesen Zellen aus. Während depolarisierende Potentiale zu einer Erhöhung der zytosolischen Kalziumkonzentration führten, bewirkte eine Hyperpolarisierung der vakuolären Membran das Gegenteil. Thermodynamische Überlegungen zum passiven und aktiven Kalziumtransport über die vakuoläre Membran legten dabei den Schluss nahe, dass die hierin beschriebenen Ergebnisse das Verhalten von vakuolären H+/Ca2+ Austauschern wiederspiegeln, deren Aktivität durch die protonenmotorische Kraft bestimmt wird. Im Rahmen dieser Arbeit stellte sich weiterhin heraus, dass zytosolisches Kalzium ebenso ein zentraler Regulator eines schnellen Auxin-induzierten Signalweges ist, über den der polare Transport des Hormons reguliert wird. Im gleichen Modellsystem junger Wurzelhaare konnte gezeigt werden, dass die externe Applikation von Auxin eine sehr schnelle, Auxinkonzentrations- und pH-abhängige Depolarisation des Plasmamembranpotentials zur Folge hat. Synchron zur Depolarisation des Plasmamembranpotentials wurden im Zytosol transiente Kalziumsignale registriert. Diese wurden durch einen von Auxin aktivierten Einstrom von Kalziumionen durch den Ionenkanal CNGC14 hervorgerufen. Experimente an Verlustmutanten als auch pharmakologische Experimente zeigten, dass zur Auxin-induzierten Aktivierung des Kalziumkanals die Auxin-Perzeption durch die F-box Proteine der TIR1/AFB Familie erforderlich ist. Durch Untersuchungen der Auxin-abhängigen Depolarisation wie auch des Auxin-induzierten Einstroms von Protonen in epidermale Wurzelzellen von Verlustmutanten konnte gezeigt werden, dass die sekundär aktive Aufnahme von Auxin durch das hochaffine Transportprotein AUX1 für die schnelle Depolarisation verantwortlich ist. Nicht nur die zytosolischen Kalziumsignale korrelierten mit der CNGC14 Funktion, sondern ebenso die AUX1-vermittelte Depolarisation von Wurzelhaaren. Eine unveränderte Expression von AUX1 in der cngc14 Verlustmutante legte dabei den Schluss nahe, dass die Aktivität von AUX1 posttranslational reguliert werden muss. Diese Hypothese erfuhr Unterstützung durch Experimente, in denen die Behandlung mit dem Kalziumkanalblocker Lanthan zu einer Inaktivierung von AUX1 im Wildtyp führte. Die zytosolische Beladung einzelner epidermaler Wurzelzellen mit Auxin hatte die Ausbreitung lateraler und acropetaler Kalziumwellen zur Folge. Diese korrelierten mit einer Verschiebung des Auxin-Gradienten an der Wurzelspitze und unterstützten somit eine hypothetische Kalziumabhängige Regulation des polaren Auxin Transports. Ein Model für einen schnellen, Auxin induzierten und kalziumabhängigen Signalweg wird präsentiert und dessen Bedeutung für das gravitrope Wurzelwachstum diskutiert. Da die AUX1-vermittelte Depolarisation in Abhängigkeit von der externen Phosphatkonzentration variierte, wird die Bedeutung dieses schnellen Signalwegs ebenso für die Anpassung des Wurzelhaarwachstums an eine nicht ausreichende Verfügbarkeit von Phosphat diskutiert.The phytohormone auxin performs important functions in the initiation of plant tissues and organs, as well as in the control of root growth in conjunction with external stimuli such as gravity, water and nutrient availability. These functions are based primarily on the auxin-dependent regulation of cell division and elongation. Important for the latter is the control of the cell turgor by the vacuole. As storage for nutrients, metabolites and toxins, vacuoles are of vital importance. Vacuolar stored metabolites and ions are exchanged across the vacuolar membrane with the cytoplasm via active transport processes as well as passively through ion channels. In their function as second messenger, calcium ions are important regulators but also subject to vacuolar transport processes. Changes in the cytosolic calcium concentration not only act locally, but are also associated with signal transduction over longer distances. In this work, electrophysiological methods were combined with imaging techniques to gain insights into the interaction between cytosolic calcium signals, vacuolar transport processes and auxin physiology in the intact plant organism. Calcium signals are involved in the regulation of vacuolar ion channels and transporters. In order to investigate this in the intact organism, intracellular microelectrode measurements were performed in the model system of bulging Arabidopsis thaliana root hairs. By means of the two-electrode voltage-clamp technique, it could be confirmed that the vacuolar membrane is the limiting electrical resistance during intravacuolar measurements and thus measured ion currents actually represent only the currents across the vacuolar membrane. The already known time-dependent decrease of vacuolar conductivity during intravacuolar experiments could be further correlated with an impalement-related, transient increase of the cytosolic calcium concentration. Intravacuolar voltage-clamp experiments in root hair cells of calcium reporter plants confirmed this relationship between vacuolar conductivity and the cytosolic calcium concentration. However, the vacuole is not just a recipient of cytosolic calcium signals. Since the vacuole represents the largest intracellular calcium reservoir, it has long been argued that it is also involved in the generation of such signals. This could be confirmed in intact root hair cells. Changes in the vacuolar membrane potential affected the cytosolic calcium concentration in these cells. While depolarizing potentials led to an increase of the cytosolic calcium concentration, hyperpolarization of the vacuolar membrane caused the opposite. Thermodynamic considerations of passive and active calcium transport across the vacuolar membrane suggested that the results described herein reflect the behaviour of vacuolar H+/Ca2+ exchangers whose activity is determined by the proton motive force. In addition, cytosolic calcium has been shown to be a key regulator of a rapid auxin-induced signaling pathway that regulates polar transport of the hormone. In the same model system of bulging root hairs it could be shown that the external application of auxin results in a very fast, auxin concentration- and pH-dependent depolarization of the plasma membrane potential. Synchronous with the depolarization of the plasma membrane potential, transient calcium signals were recorded in the cytosol. These were caused by an auxin-activated influx of calcium ions through the ion channel CNGC14. Experiments on loss-of-function mutants as well as pharmacological experiments showed that the auxin-induced activation of the calcium channel requires auxin-perception by the F-box proteins of the TIR1/AFB family. Investigations of auxin-dependent depolarization as well as the auxin-induced influx of protons into epidermal root cells of loss-of-function mutants showed that the secondary active uptake of auxin by the high-affinity transport protein AUX1 is responsible for the rapid depolarization Not only the cytosolic calcium signals correlated with CNGC14 function, but also the AUX1-mediated depolarization of root hairs. An unchanged expression of AUX1 in the cngc14 loss-of-function mutant suggested that the activity of AUX1 must be post-translationally regulated. This hypothesis was supported by experiments in which treatment with the calcium channel blocker lanthanum led to inactivation of AUX1 in the wild type. The cytosolic loading of individual epidermal root cells with auxin resulted in the spread of lateral and acropetal calcium waves. These correlated with a shift of the auxin gradient at the root apex and thus supported a hypothetical calcium-dependent regulation of polar auxin transport. A model for a rapid, auxin-induced and calcium-dependent signaling pathway is presented and its importance for gravitropic root growth is discussed. Since AUX1-mediated depolarization varied with external phosphate concentration, the importance of this rapid signaling pathway is also discussed for the adaptation of root hair growth to an inadequate availability of phosphate

Soil-borne fungi alter the apoplastic purinergic signaling in plants by deregulating the homeostasis of extracellular ATP and its metabolite adenosine

Purinergic signaling activated by extracellular nucleotides and their derivative nucleosides trigger sophisticated signaling networks. The outcome of these pathways determine the capacity of the organism to survive under challenging conditions. Both extracellular ATP (eATP) and Adenosine (eAdo) act as primary messengers in mammals, essential for immunosuppressive responses. Despite the clear role of eATP as a plant damage-associated molecular pattern, the function of its nucleoside, eAdo, and of the eAdo/eATP balance in plant stress response remain to be fully elucidated. This is particularly relevant in the context of plant-microbe interaction, where the intruder manipulates the extracellular matrix. Here, we identify Ado as a main molecule secreted by the vascular fungus Fusarium oxysporum. We show that eAdo modulates the plant's susceptibility to fungal colonization by altering the eATP-mediated apoplastic pH homeostasis, an essential physiological player during the infection of this pathogen. Our work indicates that plant pathogens actively imbalance the apoplastic eAdo/eATP levels as a virulence mechanism

Auxin-Induced Plasma Membrane Depolarization Is Regulated by Auxin Transport and Not by AUXIN BINDING PROTEIN1

Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling

Direct inhibition of phosphate transport by immune signaling in Arabidopsis

Soil availability of inorganic ortho-phosphate (PO 4 3−, P i) is a key determinant of plant growth and fitness. 1 Plants regulate the capacity of their roots to take up inorganic phosphate by adapting the abundance of H +-coupled phosphate transporters of the PHOSPHATE TRANSPORTER 1 (PHT1) family 2 at the plasma membrane (PM) through transcriptional and post-translational changes driven by the genetic network of the phosphate starvation response (PSR). 3–8 Increasing evidence also shows that plants integrate immune responses to alleviate phosphate starvation stress through the association with beneficial microbes. 9–11 Whether and how such phosphate transport is regulated upon activation of immune responses is yet uncharacterized. To address this question, we first developed quantitative assays based on changes in the electrical PM potential to measure active P i transport in roots in real time. By inserting micro-electrodes into bulging root hairs, we were able to determine key characteristics of phosphate transport in intact Arabidopsis thaliana (hereafter Arabidopsis) seedlings. The fast P i-induced depolarization observed was dependent on the activity of the major phosphate transporter PHT1;4. Notably, we observed that this PHT1;4-mediated phosphate uptake is repressed upon activation of pattern-triggered immunity. This inhibition depended on the receptor-like cytoplasmic kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and PBS1-LIKE KINASE 1 (PBL1), which both phosphorylated PHT1;4. As a corollary to this negative regulation of phosphate transport by immune signaling, we found that PHT1;4-mediated phosphate uptake normally negatively regulates anti-bacterial immunity in roots. Collectively, our results reveal a mechanism linking plant immunity and phosphate homeostasis, with BIK1/PBL1 providing a molecular integration point between these two important pathways

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity

Perception of biotic and abiotic stresses often leads to stomatal closure in plants 1,2. Rapid influx of calcium ions (Ca 2+) across the plasma membrane has an important role in this response, but the identity of the Ca 2+ channels involved has remained elusive 3,4. Here we report that the Arabidopsis thaliana Ca 2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca 2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid—a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca 2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca 2+ influx mechanisms in response to different stresses

Auxin-Induced Plasma Membrane Depolarization Is Regulated by Auxin Transport and Not by AUXIN BINDING PROTEIN1

Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling

A voltage‐dependent Ca 2+

Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca$^{2+}$ signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca$^{2+}$ and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca$^{2+}$ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H$^{+}$/Ca$^{2+}$ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca$^{2+}$ homeostat could contribute to calcium signalling when coupled to a recently discovered K$^{+}$ channel-dependent module for electrical excitability of the vacuolar membrane

A voltage-dependent Ca2+^{2+} homeostat operates in the plant vacuolar membrane

Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca$^{2+}$ signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca$^{2+}$ and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca$^{2+}$ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H$^{+}$/Ca$^{2+}$ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca$^{2+}$ homeostat could contribute to calcium signalling when coupled to a recently discovered K$^{+}$ channel-dependent module for electrical excitability of the vacuolar membrane

Voltage-dependent gating of SV channel TPC1 confers vacuole excitability

In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K$^+$-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca$^{2+}$ levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca$^{2+}$, can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K$^{+}$ transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K$^+$-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca$^{2+}$- and voltage-induced electrical excitability to the central organelle of plant cells

Dissection of jasmonate functions in tomato stamen development by transcriptome and metabolome analyses

Background Jasmonates are well known plant signaling components required for stress responses and development. A prominent feature of jasmonate biosynthesis or signaling mutants is the loss of fertility. In contrast to the male sterile phenotype of Arabidopsis mutants, the tomato mutant jai1-1 exhibits female sterility with additional severe effects on stamen and pollen development. Its senescence phenotype suggests a function of jasmonates in regulation of processes known to be mediated by ethylene. To test the hypothesis that ethylene involved in tomato stamen development is regulated by jasmonates, a temporal profiling of hormone content, transcriptome and metabolome of tomato stamens was performed using wild type and jai1-1. Results Wild type stamens showed a transient increase of jasmonates that is absent in jai1-1. Comparative transcriptome analyses revealed a diminished expression of genes involved in pollen nutrition at early developmental stages of jai1-1 stamens, but an enhanced expression of ethylene-related genes at late developmental stages. This finding coincides with an early increase of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in jai1-1 and a premature pollen release from stamens, a phenotype similarly visible in an ethylene overproducing mutant. Application of jasmonates to flowers of transgenic plants affected in jasmonate biosynthesis diminished expression of ethylene-related genes, whereas the double mutant jai1-1 NeverRipe (ethylene insensitive) showed a complementation of jai1-1 phenotype in terms of dehiscence and pollen release. Conclusions Our data suggest an essential role of jasmonates in the temporal inhibition of ethylene production to prevent premature desiccation of stamens and to ensure proper timing in flower development