Arabidopsis Mesophyll Cells Research Articles

Luminal and Cytosolic pH Feedback on Proton Pump Activity and ATP Affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.

Cover Caption: Cytosolic Localization of GLYR1

Journal of Integrative Plant BiologyVolume 54, Issue 3 p. i-i Cover Caption: Cytosolic Localization of GLYR1 First published: 18 April 2012 https://doi.org/10.1111/j.1744-7909.2012.01121.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Glyoxylate reductase isoform 1 (GLYR1) is a key enzyme in the γ-aminobutryate (GABA) pathway that is linked to a remarkable array of biological processes including stress response, signaling, and redox homeostasis. However, previously the intracellular localization of GLYR1 was unclear. Ching et al. (pp 152–168) show that GLYR1 in plant cells is localized to the cytosol and not peroxisomes, as previously proposed, by using Arabidopsis mesophyll cells co-expressing GFP-tagged GLYR1 and a Cherry-tagged peroxisomal protein. Volume54, Issue3March 2012Pages i-i RelatedInformation

Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica

Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice. We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H(2)O(2) treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts. Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H(2)O(2). Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor. OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.

The Arabidopsis aquaporin PIP1;2 rules cellular CO2 uptake

The membrane CO(2) flux into Arabidopsis mesophyll cells was studied using a scanning pH microelectrode. Arabidopsis thaliana mesophyll cells were exposed to photosynthesis-triggering light intensities, which induced cellular CO(2) uptake. Data obtained on a AtPIP1;2 T-DNA insertion line indicated that under these conditions, cellular CO(2) transport was not limited by unstirred layer effects but was dependent on the expression of the aquaporin AtPIP1;2. Complementation of the AtPIP1;2 knockout restored membrane CO(2) transport levels to that of controls. The results provide new arguments for the ongoing debate about the validity of the lipid bilayer model system and the Meyer - Overton rule for cellular gas transport. In conclusion, we suggest a modified model of molecular gas transport mechanisms in living cells.

Effects of Single Walled Carbon Nanotubes on Arabidopsis Mesophyll Cells

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Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells.

Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

The Levels of Male Gametic Mitochondrial DNA Are Highly Regulated in Angiosperms with Regard to Mitochondrial Inheritance

The mechanisms that regulate mitochondrial inheritance are not yet clear, even though it is 100 years since the first description of non-Mendelian genetics. Here, we quantified the copy numbers of mitochondrial DNA (mtDNA) in the gametic cells of angiosperm species. We demonstrate that each egg cell from Arabidopsis thaliana, Antirrhinum majus, and Nicotiana tabacum possesses 59.0, 42.7, and 73.0 copies of mtDNA on average, respectively. These values are equivalent to those in Arabidopsis mesophyll cells, at 61.7 copies per cell. On the other hand, sperm or generative cells from Arabidopsis, A. majus, and N. tabacum possess minor amounts of mtDNA, at 0.083, 0.47, and 1 copy on average, respectively. We further reveal a 50-fold degradation of mtDNA during pollen development in A. majus. In contrast, markedly high levels of mtDNA are found in the male gametic cells of Cucumis melo and Pelargonium zonale (1296.3 and 256.7 copies, respectively). Our results provide direct evidence for mitochondrial genomic insufficiency in the eggs and somatic cells and indicate that a male gamete of an angiosperm may possess mtDNA at concentrations as high as 21-fold (C. melo) or as low as 0.1% (Arabidopsis) of the levels in somatic cells. These observations reveal the existence of a strong regulatory system for the male gametic mtDNA levels in angiosperms with regard to mitochondrial inheritance.

Plant Cell Biology: How to Pattern a Wall

How are the different secondary cell wall deposition patterns generated in the vascular cells of plants? The use of a novel Arabidopsis mesophyll cell culture that transdifferentiates into vascular cells shows a crucial role for a complex of two microtubule-binding proteins.

Functional Differentiation of Brassica napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics

Guard cells are highly specialized cells that form tiny pores called stomata on the leaf surface. The opening and closing of stomata control leaf gas exchange and water transpiration as well as allow plants to quickly respond and adjust to new environmental conditions. Mesophyll cells are specialized for photosynthesis. Despite the phenotypic and obvious functional differences between the two types of cells, the full protein components and their functions have not been explored but are addressed here through a global comparative proteomics analysis of purified guard cells and mesophyll cells. With the use of isobaric tags for relative and absolute quantification (iTRAQ) tagging and two-dimensional liquid chromatography mass spectrometry, we identified 1458 non-redundant proteins in both guard cells and mesophyll cells of Brassica napus leaves. Based on stringent statistical criteria, a total of 427 proteins were quantified, and 74 proteins were found to be enriched in guard cells. Proteins involved in energy (respiration), transport, transcription (nucleosome), cell structure, and signaling are preferentially expressed in guard cells. We observed several well characterized guard cell proteins. By contrast, proteins involved in photosynthesis, starch synthesis, disease/defense/stress, and other metabolisms are preferentially represented in mesophyll cells. Of the identified proteins, 110 have corresponding microarray data obtained from Arabidopsis guard cells and mesophyll cells. About 72% of these proteins follow the same trend of expression at the transcript and protein levels. For the rest of proteins, the correlation between proteomics data and the microarray data is poor. This highlights the importance of quantitative profiling at the protein level. Collectively this work represents the most extensive proteomic description of B. napus guard cells and has improved our knowledge of the functional specification of guard cells and mesophyll cells.

Measurement of Changes in Cytosolic Ca2+ in Arabidopsis Guard Cells and Mesophyll Cells in Response to Blue Light

Phototropins (phot1 and phot2) are blue light (BL) receptors that mediate responses including phototropism, chloroplast movement and stomatal opening, and increased cytosolic Ca(2+). BL absorbed by phototropins activates plasma membrane H(+)-ATPase in guard cells, resulting in membrane hyperpolarization, and drives K(+) uptake and stomatal opening. However, it is unclear whether the phototropin-mediated Ca(2+) increase activates the H(+)-ATPase. Here, we determined cytosolic Ca(2+) concentrations in guard cell protoplasts (GCPs) from Arabidopsis transformed with aequorin. Cytosolic Ca(2+) increased rapidly in response to BL in GCPs from both the wild type and phot1 phot2 double mutants, but was mostly suppressed by an inhibitor of photosynthetic electron flow (DCMU). With depleted external K(+), we observed another slower Ca(2+) increase, which was phototropin- dependent. Fusicoccin, a H(+)-ATPase activator, mimicked the effect of BL. The slow Ca(2+) increase thus appears to result from membrane hyperpolarization. The slow Ca(2+) increase was suppressed by external K(+) and was restored by blockers of inward-rectifying K(+) channels, CsCl and tetraethylammonium, suggesting the preferential uptake of K(+) over Ca(2+). Such efficient K(+) uptake in response to BL was not found in mesophyll cells. Both the fast and the slow Ca(2+) increases were inhibited by Ca(2+) channel blockers (CoCl(2) and LaCl(3)) and a chelating agent (EGTA). These results indicate that the phototropin-mediated Ca(2+) increase was not observed prior to H(+)-ATPase activation in guard cells and that Ca(2+) entered guard cells via Ca(2+) channels through photosynthesis and phototropin-mediated membrane hyperpolarization.

Toward Improvements of Oomycete Transformation Protocols

Some of the most important plant pathogens worldwide are oomycetes, and billions of dollars are expended annually to suppress diseases they cause. More efficient disease suppression technologies will be derived from a better understanding of the basic biology of these organisms, but inefficient transformation currently limits basic molecular investigations. Of the various approaches, transformation of protoplasts using polyethylene glycol/calcium chloride remains most successful, but the frequency of stable transformation remains low and inconsistent. Here we report that modifications of a protocol, previously used for Arabidopsis mesophyll cells, successfully releases protoplasts from four different oomycetes (Phytophthora citricola, Phytophthora infestans, Phytophthora sojae, and Pythium aphanidermatum). The protoplasts of all oomycetes were able to take up DNA and regenerate, with protoplast release as well as regeneration being most efficient in P. aphanidermatum. In addition to a good protoplast production system, more effective transformation vectors may improve stable transformation rates. We constructed, and evaluated 17 novel candidate transformation vectors for their ability to drive transient expression of the beta-glucuronidase (GUS) reporter gene in P. infestans and P. aphanidermatum. Five of the newly constructed vectors were also evaluated in P. sojae and P. citricola, and exhibited a similar pattern of transcriptional activity as in P. infestans and P. aphanidermatum. One of the newly constructed vectors, pDBHAMT35G, containing a chimeric promoter, supported the highest GUS expression in P. infestans and P. citricola, and could potentially be useful for future studies.

Cold transiently activates calcium-permeable channels in Arabidopsis mesophyll cells.

Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore >3.34 A (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants.

Identification of a Vacuolar Sucrose Transporter in Barley and Arabidopsis Mesophyll Cells by a Tonoplast Proteomic Approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.

An Auxilin-Like J-Domain Protein, JAC1, Regulates Phototropin-Mediated Chloroplast Movement in Arabidopsis

The ambient-light conditions mediate chloroplast relocation in plant cells. Under the low-light conditions, chloroplasts accumulate in the light (accumulation response), while under the high-light conditions, they avoid the light (avoidance response). In Arabidopsis (Arabidopsis thaliana), the accumulation response is mediated by two blue-light receptors, termed phototropins (phot1 and phot2) that act redundantly, and the avoidance response is mediated by phot2 alone. A mutant, J-domain protein required for chloroplast accumulation response 1 (jac1), lacks the accumulation response under weak blue light but shows a normal avoidance response under strong blue light. In dark-adapted wild-type cells, chloroplasts accumulate on the bottom of cells. Both the jac1 and phot2 mutants are defective in this chloroplast movement in darkness. Positional cloning of JAC1 reveals that this gene encodes a J-domain protein, resembling clathrin-uncoating factor auxilin at its C terminus. The amounts of JAC1 transcripts and JAC1 proteins are not regulated by light and by phototropins. A green fluorescent protein-JAC1 fusion protein showed a similar localization pattern to green fluorescent protein alone in a transient expression assay using Arabidopsis mesophyll cells and onion (Allium cepa) epidermal cells, suggesting that the JAC1 protein may be a soluble cytosolic protein. Together, these results suggest that JAC1 is an essential component of phototropin-mediated chloroplast movement.

Cyclic Adenosine Monophosphate Regulates Calcium Channels in the Plasma Membrane of Arabidopsis Leaf Guard and Mesophyll Cells

The effect of cAMP on Ca(2+)-permeable channels from Arabidopsis thaliana leaf guard cell and mesophyll cell protoplasts was studied using the patch clamp technique. In the whole cell configuration, dibutyryl cAMP was found to increase a hyperpolarization-activated Ba(2+) conductance (I(Ba)). The increase of I(Ba) was blocked by the addition of GdCl(3). In excised outside-out patches, the addition of dibutyryl cAMP consistently activated a channel with particularly fast gating kinetics. Current/voltage analyses indicated a single channel conductance of approximately 13 picosiemens. In patches where we measured some channel activity prior to cAMP application, the data suggest that cAMP enhances channel activity without affecting the single channel conductance. The cAMP activation of these channels was reversible upon washout. The results obtained with excised patches indicate that the cAMP-activated I(Ba) seen in the whole cell configuration could be explained by a direct effect of cAMP on the Ca(2+) channel itself or a close entity to the channel. This work represents the first demonstration using patch clamp analysis of the presence in plant cell membranes of an ion channel directly activated by cAMP.

A transient RNA interference assay system using Arabidopsis protoplasts.

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.

Light quantity controls leaf-cell and chloroplast development in Arabidopsis thaliana wild type and blue-light-perception mutants.

Plants acclimate to changes in light quantity by altering leaf-cell development and the accumulation of chloroplast components, such that light absorption is favoured under limiting illumination, and light utilisation under non-limiting conditions. Previous evidence suggests an involvement of a high-light photosynthetic redox signal in the down-regulation of the accumulation of the light-harvesting complexes of photosystem 11 (Lhcb) and the expression of the Lhcb genes. and of a blue-light signal in the control of leaf development and in the increase in photosynthetic capacity, as affected by the accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We examined the internal anatomy of leaves, the ultrastructure of chloroplasts and accumulation of light-harvesting complexes and Rubisco in wild-type Arabidopsis thaliana (L.) Heynh. and in mutants in each of the three known blue-light photoreceptors, cryptochrome 1, cryptochrome 2 and phototropin, as well as a mutant in both cryptochromes. Our results indicate an extensive capacity of the Arabidopsis mesophyll cells to adapt to high light fluence rate with an increase in palisade elongation. Under high light, chloroplasts showed increased starch accumulation and reductions in the amount of granal thylakoids per chloroplast, in the proportion of chlorophyll b relative to chlorophyll a, and in the accumulation of the major Lhcb polypeptides. The responses were similar for all four mutants, with respect to their wild types. The results are consistent with either a complete redundancy in function between cryptochromes and phototropin, or their absence of involvement in the light-quantity responses tested. We observed minimal effects of light quantity on Rubisco accumulation over the range of fluence rates used, and conclude that elongation of palisade mesophyll cells and accumulation of Rubisco are controlled separately. This indicates that light acclimation must be the result of a number of individual elementary responses. Quantitative differences in the acclimatory responses were observed between the Landsberg erecta and Columbia wild-type ecotypes used.

Ca(2+)-permeable, outwardly-rectifying K+ channels in mesophyll cells of Arabidopsis thaliana.

Arabidopsis thaliana has become a powerful tool in genetics and molecular biology. In order to use Arabidopsis as a model system for electrophysiological studies on plant cells, a detailed characterization of the transporters present in the plasma and vacuolar membranes of this species is required. We used the patch-clamp technique to study ion channels in the plasma membrane of Arabidopsis mesophyll cells. The most prominent conductance in these cells was a K(+)-selective, voltage-dependent, outwardly-rectifying channel (IK,out). In the whole-cell configuration, IK,out was observed in 100% of the cells assayed. In contrast, inward current was observed in less than 50% of the cells which were bathed in 100 mM K+, and was totally absent from cells bathed in 10 mM K+. The activation kinetics of IK,out were modulated by the external K+ concentration with a faster activation at low external K+. Tail-current analysis revealed that in addition to K+, IK,out is also permeable to Ca2+ and Ba2+. Externally applied Ba2+ also caused a voltage-dependent decrease in current magnitude, indicating that IK,out is also partially blocked by this classic K+ channel blocker. Single channels studied in outside-out patches showed Ca2+ and Ba2+ sensitivity, voltage dependence and time activation similar to that of IK,out in the whole-cell configuration. Given their permeability to Ca2+, these channels may function as an avenue for Ca2+ influx as well as K+ efflux, both of which may affect photosynthesis.

Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls.

This article describes a practical technique for permeabilization of higher plant cell walls, which is usually one of the first steps required for immunolocalization of cellular components (and other cytological methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It replaces the use of wall degrading enzymes or the need to employ laborious sectioning or other mechanical means for providing access of probes to cells. Freeze-shattering retains the integrity of whole tissues and cells surprisingly well and thus is especially useful when used in conjunction with confocal laser scanning microscopy for recording the three-dimensional arrangement of cytoskeletal elements in relation to cell shape. In this article, we demonstrate the effectiveness of this technique for anti-tubulin and anti-actin immunofluorescence and for rhodamine phalloidin labelling of the cytoskeleton in various higher plant tissues including onion root tip and bulb scale epidermis, Tradescantia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells.

Activation of K+ Channels in the Plasma Membrane of Arabidopsis by ATP Produced Photosynthetically.

Light activates a K+ channel and transiently depolarizes the plasma membrane of Arabidopsis mesophyll cells. Genetically or chemically impairing photosynthesis abolished this electrical response to light. These results indicate that illuminated chloroplasts produce a factor that activated K+ channels in the plasma membrane. By patch clamping at the single-channel level, we have obtained evidence that ATP is one such factor. Application of 0.2 to 2 mM ATP to the cytoplasmic side of excised patches of membrane reversibly activated the type of channel that was activated by light in cell-attached patches. In addition, an outward-rectifying K+ channel and an outward-rectifying nonselective cation channel were similarly activated by ATP, whereas a nonselective stretch-activated channel was unaffected by this treatment. This novel mechanism for controlling the permeability of the plasma membrane to K+ may be important to photosynthetic metabolism.