Arabidopsis Thaliana Guard Cells Research Articles

Green Tea Catechins, (-)-Catechin Gallate, and (-)-Gallocatechin Gallate are Potent Inhibitors ofABA-Induced Stomatal Closure.

Stomatal movement is indispensable for plant growth and survival in response to environmental stimuli. Cytosolic Ca2+ elevation plays a crucial role in ABA‐induced stomatal closure during drought stress; however, to what extent the Ca2+ movement across the plasma membrane from the apoplast to the cytosol contributes to this process still needs clarification. Here the authors identify (−)‐catechin gallate (CG) and (−)‐gallocatechin gallate (GCG), components of green tea, as inhibitors of voltage‐dependent K+ channels which regulate K+ fluxes in Arabidopsis thaliana guard cells. In Arabidopsis guard cells CG/GCG prevent ABA‐induced: i) membrane depolarization; ii) activation of Ca2+ permeable cation (I Ca) channels; and iii) cytosolic Ca2+ transients. In whole Arabidopsis plants co‐treatment with CG/GCG and ABA suppressed ABA‐induced stomatal closure and surface temperature increase. Similar to ABA, CG/GCG inhibited stomatal closure is elicited by the elicitor peptide, flg22 but has no impact on dark‐induced stomatal closure or light‐ and fusicoccin‐induced stomatal opening, suggesting that the inhibitory effect of CG/GCG is associated with Ca2+‐related signaling pathways. This study further supports the crucial role of I Ca channels of the plasma membrane in ABA‐induced stomatal closure. Moreover, CG and GCG represent a new tool for the study of abiotic or biotic stress‐induced signal transduction pathways.

Untargeted Metabolomics of Arabidopsis Stomatal Immunity.

Although untargeted metabolomic approaches hold great promise for global identification of low molecular weight metabolites in biological samples, deep coverage and confident identification of the metabolites remains challenging due to the great diversity and number of chemical structures, especially in plants. Additionally, there is a need to employ a cell-specific research approach to many physiological and biological responses to specific environmental stimuli. Here, we report an untargeted metabolomic method using Arabidopsis thaliana guard cell samples during response to systemic signals of pathogen attack. We employed a new Acquire X MSn data acquisition technology, which uses an iterative fragmentation process to increase level-2 identification of unknown metabolites. We were able to increase the number of identified metabolites and thus the metabolome coverage in Arabidopsis guard cells. This method can be applied to studying metabolomes of other cell types and tissues.

Predicting Division Planes of Three-Dimensional Cells by Soap-Film Minimization.

One key aspect of cell division in multicellular organisms is the orientation of the division plane. Proper division plane establishment contributes to normal plant body organization. To determine the importance of cell geometry in division plane orientation, we designed a three-dimensional probabilistic mathematical model to directly test the century-old hypothesis that cell divisions mimic soap-film minima. According to this hypothesis, daughter cells have equal volume and the division plane occurs where the surface area is at a minimum. We compared predicted division planes to a plant microtubule array that marks the division site, the preprophase band (PPB). PPB location typically matched one of the predicted divisions. Predicted divisions offset from the PPB occurred when a neighboring cell wall or PPB was directly adjacent to the predicted division site to avoid creating a potentially structurally unfavorable four-way junction. By comparing divisions of differently shaped plant cells (maize [Zea mays] epidermal cells and developing ligule cells and Arabidopsis thaliana guard cells) and animal cells (Caenorhabditis elegans embryonic cells) to divisions simulated in silico, we demonstrate the generality of this model to accurately predict in vivo division. This powerful model can be used to separate the contribution of geometry from mechanical stresses or developmental regulation in predicting division plane orientation.

Cytosolic malate and oxaloacetate activate S-type anion channels in Arabidopsis guard cells.

Intracellular malate-starch interconversion plays an important role in stomatal movements. We investigated whether malate or oxaloacetate from the cytosolic membrane side regulate anion channels in the plasma membrane of Arabidopsis thaliana guard cells. Physiological concentrations of cytosolic malate have been reported in the range of 0.4-3mM in leaf cells. Guard cell patch clamp and two-electrode oocyte voltage-clamp experiments were pursued. We show that a concentration of 1mM cytosolic malate greatly activates S-type anion channels in Arabidopsis thaliana guard cells. Interestingly, 1mM cytosolic oxaloacetate also activates S-type anion channels. Malate activation was abrogated at 10mM malate and in SLAC1 anion channel mutant alleles. Interestingly, malate activation of S-type anion currents was disrupted at below resting cytosolic-free calcium concentrations ([Ca2+ ]cyt ), suggesting a key role for basal [Ca2+ ]cyt signaling. Cytosolic malate was not able to directly activate or enhance SLAC1-mediated anion currents in Xenopus oocytes, whereas in positive controls, cytosolic NaHCO3 enhanced SLAC1 activity, suggesting that malate may not directly modulate SLAC1. Cytosolic malate activation of S-type anion currents was impaired in ost1 and in cpk5/6/11/23 quadruple mutant guard cells. Together these findings show that these cytosolic organic anions function in guard cell 'plasma membrane' ion channel regulation.

Algae biomass as a precursor for synthesis of nitrogen-and sulfur-co-doped carbon dots: A better probe in Arabidopsis guard cells and root tissues

The challenge of controlling algal blooms and reusing algal biomass remain unsolved worldwide. We introduce a facile method to reuse Nannochloropsis biocrude oil (NBO) for the synthesis of nitrogen and sulfur co-doped carbon dots (N-S-C-dots). N-S-C-dots can pass through the heavily thickened wall of mature Arabidopsis thaliana (A. thaliana) guard cells because of high solubility and excellent biocompatibility. N-S-C-dots exhibit multicolor luminescence and could effectively reduce the interference of autofluorescence in plant cells by changing filters. Bioimaging of root tissues reveals that 2 major factors affect the transmission of N-S-C-dots: high osmotic pressure and intensity of cellular metabolism. This study highlights the potential application of CDs for bioimaging in plant cells and demonstrates the significance of investigating the reuse of algal biomass.

Noncanonical Function of a Small-Molecular Virulence Factor Coronatine against Plant Immunity: An In Vivo Raman Imaging Approach.

Coronatine (1), a small-molecular virulence factor produced by plant-pathogenic bacteria, promotes bacterial infection by inducing the opening of stomatal pores, the major route of bacterial entry into the plant, via the jasmonate-mediated COI1-JAZ signaling pathway. However, this pathway is also important for multiple plant functions, including defense against wounding by herbivorous insects. Thus, suppression of the COI1-JAZ signaling pathway to block bacterial infection would concomitantly impair plant defense against herbivorous wounding. Here, we report additional, COI1-JAZ-independent, action of 1 in Arabidopsis thaliana guard cells. First, we found that a stereoisomer of 1 regulates the movement of Arabidopsis guard cells without affecting COI1-JAZ signaling. Second, we found using alkyne-tagged Raman imaging (ATRI) that 1 is localized to the endoplasmic reticulum (ER) of living guard cells of Arabidopsis. The use of arc6 mutant lacking chloroplast formation was pivotal to circumvent the issue of autofluorescence during ATRI. These findings indicate that 1 has an ER-related action on Arabidopsis stomata that bypasses the COI1-JAZ signaling module. It may be possible to suppress the action of 1 on stomata without impairing plant defense responses against herbivores.

A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization.

The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor.

Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric [CO2] further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO2/HCO3 (-) enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels.

Abscisic acid-responsive guard cell metabolomes of Arabidopsis wild-type and gpa1 G-protein mutants.

Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography-multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼ 350 million guard cell protoplasts from ∼ 30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca(2+)-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1.

Comprehensive Cell-specific Protein Analysis in Early and Late Pollen Development from Diploid Microsporocytes to Pollen Tube Growth

Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development.

Live Cell Imaging of Cytoplasmic Ca2+ Dynamics in Arabidopsis Guard Cells

This protocol describes a classical method for measuring cytoplasmic Ca(2+) dynamics in Arabidopsis thaliana guard cells. The Förster (fluorescence) resonance energy transfer (FRET)-based genetically modified Ca(2+) indicator Yellow Cameleon YC3.6, under the control of the guard cell-specific promoter GC1, is used for Ca(2+) measurements.

Phospholipase dalpha1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis.

We determined the role of Phospholipase Dalpha1 (PLDalpha1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldalpha1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldalpha1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldalpha1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA-ABI1 interaction was required for stomatal closure induced by ABA, H(2)O(2), or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.

Functional proteomics of Arabidopsis thaliana guard cells uncovers new stomatal signaling pathways.

We isolated a total of 3 x 10(8) guard cell protoplasts from 22,000 Arabidopsis thaliana plants and identified 1734 unique proteins using three complementary proteomic methods: protein spot identification from broad and narrow pH range two-dimensional (2D) gels, and 2D liquid chromatography-matrix assisted laser desorption/ionization multidimensional protein identification technology. This extensive single-cell-type proteome includes 336 proteins not previously represented in transcriptome analyses of guard cells and 52 proteins classified as signaling proteins by Gene Ontology analysis, of which only two have been previously assessed in the context of guard cell function. THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1), a myrosinase that catalyzes the production of toxic isothiocyanates from glucosinolates, showed striking abundance in the guard cell proteome. tgg1 mutants were hyposensitive to abscisic acid (ABA) inhibition of guard cell inward K(+) channels and stomatal opening, revealing that the glucosinolate-myrosinase system, previously identified as a defense against biotic invaders, is required for key ABA responses of guard cells. Our results also suggest a mechanism whereby exposure to abiotic stresses may enhance plant defense against subsequent biotic stressors and exemplify how enhanced knowledge of the signaling networks of a specific cell type can be gained by proteomics approaches.

Purification of the functional plant membrane channel KAT1

The inward-rectifying K + channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K + channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a “test set” of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.

ABA‐induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.

AKT2/3 Subunits Render Guard Cell K+ Channels Ca2+ Sensitive

Inward-rectifying K+ channels serve as a major pathway for Ca2+-sensitive K+ influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K+ channels are assembled from multiple K+ channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca2+ sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K+ channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca2+ sensitivity of the K+ inward rectifier. Wild-type channels were blocked by extracellular Ca2+ in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca2+ block, characteristic for the K+ inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca2+ dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca2+ block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca2+, while the dominant guard cell K+ channel KAT2 was Ca2+ insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca2+ sensitivity of the guard cell K+ uptake channel.

OPEN STOMATA1 opens the door to ABA signaling in Arabidopsis guard cells

The plant hormone abscisic acid plays a crucial role in plant responses to drought, salinity and cold. A recent report shows that mutations in the OST1 gene, encoding a serine–threonine protein kinase, render Arabidopsis thaliana guard cells insensitive to abscisic acid, such that stomata remain open in the presence of this phytohormone.

Preparation and applications of Arabidopsis thaliana guard cell protoplasts.

• Guard cells play an important role in the physiology and development of plants. The genetic resources available for Arabidopsis thaliana make it the most favorable plant species for the study of guard cell processes, but it is not easy to isolate highly purified preparations of large numbers of guard cells from this species. Here, we describe methods for isolation of both guard cell and mesophyll cell protoplasts from A. thaliana and their use in the study of unique biochemical and cellular properties of these cell types. • Protocols developed for large- and small-scale preparation of guard cell protoplasts and mesophyll cell protoplasts are described, followed by specific examples of their use in electrophysiological, biochemical and molecular approaches such as patch clamping, enzyme assays, and reverse-transcription polymerase chain reaction. • The protocols described yield millions of highly purified, viable guard cell protoplasts and mesophyll cell protoplasts from A. thaliana. These protoplasts have been used successfully in the study of ion channel properties, assay of ABA activation in phospholipase D activity and comparisons of gene and protein expression levels. • These techniques make it possible to elucidate electrophysiological, biochemical and molecular genetic pathways of guard cell function.

Distinct Molecular Bases for pH Sensitivity of the Guard Cell K+ Channels KST1 and KAT1

Acid-induced potassium uptake through K+ channels is a prerequisite for stomatal opening. Our previous studies identified a pore histidine as a major component of the acid activation mechanism of the potato guard cell K+ channel KST1 (1). Although this histidine is highly conserved among all plant K+ uptake channels cloned so far, the pH-dependent gating of the Arabidopsis thaliana guard cell K+ channel KAT1 was not affected by mutations of this histidine. In both channels, KST1 and KAT1, aspartate mutants in the K+ channel consensus sequence GYGD adjacent to the histidine (KST1-D269N and KAT1-D265N) were inhibited by a rise in the extracellular proton concentration. pH changes affected the half-maximal activation voltage V(1)/(2) of the KST1 mutant, whereas in the mutant channel KAT1-D265N an acid-induced decrease in the maximum conductance gmax indicated the presence of a proton block. In contrast to the wild type KST1, the S4-mutant channel KST1-R181Q exhibited an activation upon alcalization of the extracellular solution. From our electrophysiological studies on channel mutants with respect to the pore histidine as well as the aspartate, we conclude that the common proton-supported shift in the voltage dependence of KST1 and KAT1 is based on distinct molecular elements.

Channel-mediated high-affinity K+ uptake into guard cells from Arabidopsis.

Potassium uptake by higher plants is the result of high- or low-affinity transport accomplished by different sets of transporters. Although K+ channels were thought to mediate low-affinity uptake only, the molecular mechanism of the high-affinity, proton-dependent K+ uptake system is still scant. Taking advantage of the high-current resolution of the patch-clamp technique when applied to the small Arabidopsis thaliana guard cells densely packed with voltage-dependent K+ channels, we could directly record channels working in the concentration range of high-affinity K+ uptake systems. Here we show that the K+ channel KAT1 expressed in Arabidopsis guard cells and yeast is capable of mediating potassium uptake from media containing as little as 10 microM of external K+. Upon reduction of the external K+ content to the micromolar level the voltage dependence of the channel remained unaffected, indicating that this channel type represents a voltage sensor rather than a K+-sensing valve. This behavior results in K+ release through K+ uptake channels whenever the Nernst potential is negative to the activation threshold of the channel. In contrast to the H+-coupled K+ symport shown to account for high-affinity K+ uptake in roots, pH-dependent K+ uptake into guard cells is a result of a shift in the voltage dependence of the K+ channel. We conclude that plant K+ channels activated by acid pH may play an essential role in K+ uptake even from dilute solutions.