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

Rolle von ABA-abhängigen Ca2+^{2+} Signalen, und der Ca2+^{2+}-gesteuerten Proteinkinase CIPK23, bei der Regulation der Spaltöffnungsbewegungen

Stomata are pores in the leaf surface, formed by pairs of guard cells. The guard cells modulate the aperture of stomata, to balance uptake of CO2 and loss of water vapor to the atmosphere. During drought, the phytohormone abscisic acid (ABA) provokes stomatal closure, via a signaling chain with both Ca2+-dependent and Ca2+-independent branches. Both branches are likely to activate SLAC1-type (Slow Anion Channel Associated 1) anion channels that are essential for initiating the closure of stomata. However, the importance of the Ca2+-dependent signaling branch is still debated, as the core ABA signaling pathway only possesses Ca2+-independent components. Therefore, the aim of this thesis was to address the role of the Ca2+-dependent branch in the ABA signaling pathway of guard cells. In the first part of the thesis, the relation between ABA-induced Ca2+ signals and stomatal closure was studied, with guard cells that express the genetically encoded Ca2+-indicator R-GECO1-mTurquoise. Ejection of ABA into the guard cell wall rapidly induced stomatal closure, however, only in ¾ of the guard cells ABA evoked a cytosolic Ca2+ signal. A small subset of stomata (¼ of the experiments) closed without Ca2+ signals, showing that the Ca2+ signals are not essential for ABA-induced stomatal closure. However, stomata in which ABA evoked Ca2+ signals closed faster as those in which no Ca2+ signals were detected. Apparently, ABA-induced Ca2+ signals enhance the velocity of stomatal closure. In addition to ABA, hyperpolarizing voltage pulses could also trigger Ca2+ signals in wild type guard cells, which in turn activated S-type anion channels. However, these voltage pulses failed to elicit S-type anion currents in the slac1/slah3 guard cells, suggesting that SLAC1 and SLAH3 contribute to Ca2+-activated conductance. Taken together, our data indicate that ABA-induced Ca2+ signals enhance the activity of S-type anion channels, which accelerates stomatal closure. The second part of the thesis deals with the signaling pathway downstream of the Ca2+ signals. Two types of Ca2+-dependent protein kinase modules (CPKs and CBL/CIPKs) have been implicated in guard cells. We focused on the protein kinase CIPK23 (CBL-Interacting Protein Kinase 23), which is activated by the Ca2+-dependent protein CBL1 or 9 (Calcineurin B-Like protein 1 or 9) via interacting with the NAF domain of CIPK23. The CBL1/9-CIPK23 complex has been shown to affect stomatal movements, but the underlying molecular mechanisms remain largely unknown. We addressed this topic by using an estrogen-induced expression system, which specifically enhances the expression of wild type CIPK23, a phosphomimic CIPK23T190D and a kinase dead CIPK23K60N in guard cells. Our data show that guard cells expressing CIPK23T190D promoted stomatal opening, while CIPK23K60N enhanced ABA-induced stomatal closure, suggesting that CIPK23 is a negative regulator of stomatal closure. Electrophysiological measurements revealed that the inward K+ channel currents were similar in guard cells that expressed CIPK23, CIPK23T190D or CIPK23K60N, indicating that CIPK23-mediated inward K+ channel AKT1 does not contribute to stomatal movements. Expression of CIPK23K60N, or loss of CIPK23 in guard cells enhanced S-type anion activity, while the active CIPK23T190D inhibited the activity of these anion channels. These results are in line with the detected changes in stomatal movements and thus indicate that CIPK23 regulates stomatal movements by inhibiting S-type anion channels. CIPK23 thus serves as a brake to control anion channel activity. Overall, our findings demonstrate that CIPK23-mediated stomatal movements do not depend on CIPK23-AKT1 module, instead, it is achieved by regulating S-type anion channels SLAC1 and SLAH3. In sum, the data presented in this thesis give new insights into the Ca2+-dependent branch of ABA signaling, which may help to put forward new strategies to breed plants with enhanced drought stress tolerance, and in turn boost agricultural productivity in the future.Stomata sind Poren in der Blattoberfläche, die von einem Paar von Schließzellen gebildet werden. Die Schließzellen kontrollieren den Öffnungsweite der stomatären Pore, um die Aufnahme von CO2 und den Verlust von Wasserdampf in die Atmosphäre auszubalancieren. Während Trockenperioden bewirkt das Phytohormon Abscisinsäure (ABA) einen Stomaschluss über eine Signalkaskade, welche über Ca2+-abhängige und Ca2+-unabhängige Pfade verfügt. Beide Pfade aktivieren wahrscheinlich Anionenkanäle aus der SLAC1 Familie (Slow Anion Channel Associated 1), welche essentiell sind um den Stomaschluss einzuleiten. Allerdings wird über die Wichtigkeit des Ca2+-abhängigen Pfades noch immer diskutiert, da der ABA-Hauptsignalweg ausschließlich Ca2+-unabhängige Komponenten beinhaltet. Aus diesem Grund war das Ziel dieser Thesis, die Rolle des Ca2+-abhängigen Pfades im ABA-Signalweg aufzulösen. Im ersten Teil der Thesis wurde mit Schließzellen, die den genetisch kodierten Ca2+-Sensor R-GECO1-mTurquoise exprimierten, der Zusammenhang zwischen ABA-induzierten Ca2+ Signalen und dem Stomaschluss untersucht. Die Injektion von ABA in die Zellwand von Schließzellen bewirkte einen schnellen Stomaschluss, jedoch wurde nur bei drei Vierteln der Zellen auch ein zytosolisches Ca2+ Signal erzeugt. Ein kleiner Teil der Stomata (in einem Viertel der Experimente) schloss sich ohne Ca2+ Signal, was zeigt, dass die Ca2+ Signale nicht essentiell für den ABA-induzierten Stomaschluss sind. Es schlossen sich jedoch Stomata schneller, in deren Schließzellen ABA-induzierte Ca2+ Signale detektiert wurden. ABA-induzierte Ca2+-Signale verbesserten also offenbar die Geschwindigkeit des Stomaschlusses. Neben ABA konnten Ca2+ Signale in wildtypischen Schließzellen auch durch hyperpolarisierende Spannungspulse erzeugt werden, welche daraufhin S-Typ Anionenkanäle aktivierten. Diese Spannungspulse konnten jedoch in slac1/slah3 Schließzellen keine S-typischen Anionenströme hervorrufen, was darauf hindeutet, dass SLAC1 und SLAH3 zur Ca2+-aktivierten Leitfähigkeit beitragen. Zusammengefasst deuten unsere Daten darauf hin, dass ABA-induzierte Ca2+ Signale die Aktivität von S-Typ Anionenkanälen verbessern und somit den Stomaschluss beschleunigen. Der zweite Teil der Thesis befasst sich mit dem Signalweg, der den Ca2+-Signalen nachgeschaltet ist. Es wurden zwei Typen Ca2+-abhängiger Proteinkinase-Module (CPKs und CBL/CIPKs) in Schließzellen nachgewiesen. Wir haben uns auf die Proteinkinase CIPK23 (CBL-Interacting Protein Kinase 23) konzentriert, welche von den Ca2+-abhängigen Proteinen CBL1 und CBL9 (Calcineurin B-Like Protein 1 oder 9) über Interaktion mit der NAF Domäne des CIPK23 aktiviert wird. Es konnte bereits gezeigt werden, dass der CBL1/CIPK23 Komplex die stomatäre Bewegung beinflusst, jedoch sind die zugrunde liegenden molekularen Mechanismen bisher weitgehend unbekannt geblieben. Wir haben dieses Thema mit einem Östrogen-induzierten Expressionssystem untersucht, welches spezifisch in Schließzellen die Expression von wildtypischem CIPK23 erhöhte. Hinzu kamen Experimente mit einer phosphomimetischen CIPK23T190D und einer CIPK23K60N mit disfunktionaler Kinasedomäne. Unsere Daten zeigen, dass CIPK23T190D exprimierende Schließzellen eine verbesserte Stomaöffnung aufwiesen, während CIPK23K60N den ABA-induzierten Stomaschluss förderte, was auf eine negativ regulierende Rolle von CIPK23 beim Stomaschluss hindeutet. Elektrophysiologische Messungen zeigten, dass die einwärtsgerichteten K+-Ströme in CIPK23-, CIPK23T190D- oder CIPK23K60N-exprimierenden Schließzellen vergleichbar waren, was darauf hindeutet, dass die Aktivierung von AKT1 durch CIPK23 nicht zur stomatären Bewegung beiträgt. Allerdings führte die Expression von CIPK23K60N, wie auch der Verlust von CIPK23, in Schließzellen zu einer erhöhten S-typischen Anionenkanalaktivität, während eine CIPK23T190D-Expression diese Anionenkanalaktivität inhibierte. Diese Ergebnisse stimmen mit den Beobachtungen zu den gezeigten Veränderungen der stomatären Bewegung überein und deuten daher auf eine regulierende Rolle von CIPK23 für die stomatäre Bewegung durch die Inhibierung von S-Typ Anionenkanälen hin. Insgesamt beweisen unsere Befunde, dass die CIPK23-vermittelte stomatäre Bewegung nicht durch eine Interaktion von CIP23 mit AKT1, sondern durch die Regulierung der S-Typ Anionenkanäle SLAC1 und SLAH3 vermittelt wird. Zusammengefasst ergeben die in dieser Thesis präsentierten Daten neue Einblicke in den Ca2+-abhängigen Pfad des ABA-Signalwegs. Dies könnte in Zukunft helfen neue Strategien zur Zucht von Pflanzen mit verbesserter Trockenstresstoleranz zu entwickeln und somit die agrarwirtschaftliche Produktivität zu erhöhen

A three-stage deadlock prevention strategy for S3PR nets

In this paper we focus on a particular class of Petri nets, called Systems of Simple Sequential Processes with Resources (S3PR). We propose a deadlock prevention strategy consisting of three stages, and based on Mixed Integer Programming (MIP). The advantage of the proposed approach is a usually higher permissiveness and a lower structural and computational complexity with respect to other approaches. Indeed exhaustive siphon enumeration is not required, as well as reachability analysis. Several numerical examples are illustrated to highlight the effectiveness of the approach

SIMULATION OF FLOW BOILING OF NANOFLUID IN TUBE BASED ON LATTICE BOLTZMANN MODEL

In this study, a lattice Boltzmann model of bubble flow boiling in a tube is established. The bubble growth, integration, and departure of 3% Al2O3-water nanofluid in the process of flow boiling are selected to simulate. The effects of different bubble distances and lateral accelerations a on the bubble growth process and the effect of heat transfer are investigated. Results showed that with an increase in the bubble distance, the bubble coalescence and the effect of heat transfer become gradual. With an increase in lateral acceleration a, the bubble growth is different. When a = 0.5e-7 and a = 0.5e-6, the bubble growth includes the process of bubble growth, coalescence, detachment, and fusion with the top bubble and when a = 0.5e-5 and a = 0.5e-4, the bubbles only experience growth and fusion, and the bubbles do not merge with the top bubble directly to the right movement because the lateral acceleration is too large, resulting in the enhanced effect of heat transfer in the tube

THE STUDY OF NATURAL CONVECTION HEAT TRANSFER OF NANOFLUIDS IN A PARTIALLY POROUS CAVITY BASED ON LATTICE BOLTZMANN METHOD

This article used the lattice Boltzmann method to study the heat transmission of natural convective of nanofluids in a 2-D square cavity partially filled with porous medium. The nanoparticles volume fraction of A1203, Cu, and Si02 were 0.5%, 1%, 1.5%, 2%,.5/,06, and 4%, which were mixed with water and 70% of ethylene glycol aqueous solution as the base fluid, and made up six kinds of nanofluids as the research object. Using nanofluids coupled double distribution lattice Boltzmann method model, this paper studied the rules of natural convection heat transfer of different nanofluids with the changing of Rayleigh number and the concentration of the nanoparticles in the 2-D square cavity partially filled with porous medium. The results showed that the average Nusselt number of the hot wall will increase with the increase of Rayleigh number number, and under different heat transfer conditions, there are two different critical Rayleigh numbers. In the case of different concentrations of the same concentration, the critical Rayleigh number is about 105, when Ra > Rae, the average Nusselt number of water is higher; when Ra Rae, there is a slight decreasing in the average Nusselt number with the increasing of concentration. The critical Rayleigh number of water as the base fluid is smaller than that of ethylene glycol as the base fluid

Analysis and Prediction of Flow-Induced Vibration of Convection Pipe for 200 t/h D Type Gas Boiler

This paper is aimed at the analysis and prediction of the fluid-induced vibration phenomenon in the convection tube bundle area caused by Karman vortex street shedding in the background of a 200 t/h large-capacity D-type gas boiler. Based on the numerical simulation of flue heat state flow field and fast Fourier transform, the lift coefficient curve of different monitoring areas and the corresponding Karman vortex street shedding frequency are obtained. The accuracy of the analysis model is validated by comparing Karman vortex shedding frequency with acoustic equipment standing wave frequency. In order to meet the design requirements of the 200 t/h D-type gas boiler for reliable and stable operation, the vibration characteristics and variation rules of a convection tube bundle in a D-type boiler under different working conditions are predicted

Effects of nanoparticle types and size on boiling feat transfer performance under different pressures

The experimental study on the boiling heat transfer performance and visualization of Al2O3-H2O, SiO2-H2O and Al2O3-SiO2-H2O nanofluids with 0.01% mass concentration were carried out. Alumina and silica nanoparticles with average particle diameters of 30 nm and 50 nm were selected. Al2O3-SiO2-H2O was the mixed aqueous nanofluids with a mass ratio of Al2O3 to SiO2 of 1: 1. The results show that the effect of particle size on the boiling heat transfer performance is small. For nanofluids with a particle size of 30 nm, the boiling heat transfer performance of Al2O3-H2O nanofluids is better than that of Al2O3-SiO2-H2O and SiO2-H2O nanofluids under the working pressure of 101 kPa. The critical heat fluxes increased by 7.9% and 22.1% respectively, and the maximum heat transfer coefficients increased by 18.3% and 32.6% respectively. The critical heat flux and the maximum heat transfer coefficient are 162.1 W/cm2 and 7.01W/(m2·K). The working pressure has an important effect on the boiling heat transfer performance of nanofluids. Compared with the boiling heat transfer performance under high pressure conditions, the boiling heat transfer performance of nanofluids is better under low pressure

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

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