Arabidopsis Thaliana Mesophyll Cells Research Articles

Ethyl vinyl ketone activates oxidative and calcium burst and CML8-ACA8 participates in calcium recovery in Arabidopsis leaves

Plants produce ethyl vinyl ketone (evk) in response to biotic stress, but the evk's identification and downstream defense response remain unclear. In this paper, it is predicted by docking for the first time that evk can be recognized by RBOH protein and assist the electron transfer of RBOHD/RBOHF by binding to its FAD or NADPH binding site. Surface plasmon resonance (SPR) binding assay shows that evk indeed bind to RBOHD. Here, we show that evk treatment increased H2O2 and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H2O2 signaling functioned upstream of Ca2+ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca2+-ATPASE, ISOFORM 8)–CML8 (CALMODULIN-LIKE 8) interaction promoted Ca2+ efflux to return Ca2+ levels to the resting state.

Mitochondrial movement during its association with chloroplasts in Arabidopsis thaliana

Plant mitochondria move dynamically inside cells and this movement is classified into two types: directional movement, in which mitochondria travel long distances, and wiggling, in which mitochondria travel short distances. However, the underlying mechanisms and roles of both types of mitochondrial movement, especially wiggling, remain to be determined. Here, we used confocal laser-scanning microscopy to quantitatively characterize mitochondrial movement (rate and trajectory) in Arabidopsis thaliana mesophyll cells. Directional movement leading to long-distance migration occurred at high speed with a low angle-change rate, whereas wiggling leading to short-distance migration occurred at low speed with a high angle-change rate. The mean square displacement (MSD) analysis could separate these two movements. Directional movement was dependent on filamentous actin (F-actin), whereas mitochondrial wiggling was not, but slightly influenced by F-actin. In mesophyll cells, mitochondria could migrate by wiggling, and most of these mitochondria associated with chloroplasts. Thus, mitochondria migrate via F-actin-independent wiggling under the influence of F-actin during their association with chloroplasts in Arabidopsis.

Modulation of plant TPC channels by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are powerful modulators of several animal ion channels. It is shown here that PUFAs strongly affect the activity of the Slow Vacuolar (SV) channel encoded by the plant TPC1 gene. The patch-clamp technique was applied to isolated vacuoles from carrot taproots and Arabidopsis thaliana mesophyll cells and arachidonic acid (AA) was chosen as a model molecule for PUFAs. Our study was extended to different PUFAs including the endogenous alpha-linolenic acid (ALA). The addition of micromolar concentrations of AA reversibly inhibited the SV channel decreasing the maximum open probability and shifting the half activation voltage to positive values. Comparing the effects of different PUFAs, it was found that the length of the lipophilic acyl chain, the number of double bonds and the polar head were critical for channel modulation.The experimental data can be reproduced by a simple three-state model, in which PUFAs do not interact directly with the voltage sensors but affect the voltage-independent transition that leads the channel from the open state to the closed configuration. The results indicate that lipids play an important role in co-ordinating ion channel activities similar to what is known from animal cells.

A Protein Phosphatase 2A Catalytic Subunit Modulates Blue Light-Induced Chloroplast Avoidance Movements through Regulating Actin Cytoskeleton in Arabidopsis

Chloroplast avoidance movements mediated by phototropin 2 (phot2) are one of most important physiological events in the response to high-fluence blue light (BL), which reduces damage to the photosynthetic machinery under excess light. Protein phosphatase 2A-2 (PP2A-2) is an isoform of the catalytic subunit of PP2A, which regulates a number of developmental processes. To investigate whether PP2A-2 was involved in high-fluence BL-induced chloroplast avoidance movements, we first analyzed chloroplast migration in the leaves of the pp2a-2 mutant in response to BL. The data showed that PP2A-2 might act as a positive regulator in phot2-mediated chloroplast avoidance movements, but not in phot1-mediated chloroplast accumulation movements. Then, the effect of okadaic acid (OA) and cantharidin (selective PP2A inhibitors) on high-fluence BL response was further investigated in Arabidopsis thaliana mesophyll cells. Within a certain concentration range, exogenously applied OA or cantharidin inhibited the high-fluence BL-induced chloroplast movements in a concentration-dependent manner. Actin depolymerizing factor (ADF)/cofilin phosphorylation assays demonstrated that PP2A-2 can activate/dephosphorylate ADF/cofilin, an actin-binding protein, in Arabidopsis mesophyll cells. Consistent with this observation, the experiments showed that OA could inhibit ADF1 binding to the actin and suppress the reorganization of the actin cytoskeleton after high-fluence BL irradiation. The adf1 and adf3 mutants also exhibited reduced high-fluence BL-induced chloroplast avoidance movements. In conclusion, we identified that PP2A-2 regulated the activation of ADF/cofilin, which, in turn, regulated actin cytoskeleton remodeling and was involved in phot2-mediated chloroplast avoidance movements.

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.

Introducing Aromatic Amino Acid Hydroxylases from Plants

merization domain (reviewed in Fitzpatrick, 2003), these domains were not present in plant AAHs. In addition, the plant AAHs contained a nonconserved sequence in the N terminus that was predicted to be a plastid-targeting peptide. Functional complementation of an Escherichia coli Tyr auxotroph demonstrated that the plant AAHs had Phe hydroxylase activity. Furthermore, analytical size exclusion chromatography showed that, in contrast with animal AAHs, which occur as homotetramers (reviewed in Fitzpatrick, 2003), plant AAHs function as monomers. Treatment with various chelators demonstrated that the activity of plant AAHs, like that of animal AAHs, depends on iron. Next, the authors tested a series of pterins and folates for their ability to act as a cofactor for pine and moss AAHs and identified a folate, 10-formyltetrahydrofolate, as the physiological cofactor of these enzymes. As suggested by the presence of a putative plastid-targeting peptide, targeting assays using full-length green fluorescent protein (GFP)‐tagged pine and moss AAHs in Arabidopsis thaliana mesophyll cells (see figure), and import assays using purified pea (Pisum sativum) chloroplasts

The speed of intracellular signal transfer for chloroplast movement

The photoreceptors for chloroplast photorelocation movement have been known, but the signal(s) raised by photoreceptors remains unknown. To know the properties of the signal(s) for chloroplast accumulation movement, we examined the speed of signal transferred from light-irradiated area to chloroplasts in gametophytes of Adiantum capillus-veneris. When dark-adapted gametophyte cells were irradiated with a microbeam of various light intensities of red or blue light for 1min or continuously, the chloroplasts started to move towards the irradiated area. The speed of signal transfer was calculated from the relationship between the timing of start moving and the distance of chloroplasts from the microbeam and was found to be constant at any light conditions. In prothallial cells, the speed was about 1.0 μm min-1 and in protonemal cells about 0.7 μm min-1 towards base and about 2.3 μm min-1 towards the apex. We confirmed the speed of signal transfer in Arabidopsis thaliana mesophyll cells under continuous irradiation of blue light, as was about 0.8 μm min-1. Possible candidates of the signal are discussed depending on the speed of signal transfer.

Speed of signal transfer in the chloroplast accumulation response

Chloroplast photorelocation movement is important for plants to perform efficient photosynthesis. Phototropins were identified as blue-light receptors for chloroplast movement in Arabidopsis thaliana and in the fern Adiantum capillus-veneris, whereas neochrome functions as a dual red/blue light receptor in the latter. However, the signal transduction pathways involved in chloroplast movement remain to be clarified. To investigate the kinetic properties of signalling from these photoreceptors to the chloroplasts, we deduced the speed of signal transfer using Adiantum capillus-veneris gametophytes. When a region of dark-adapted gametophyte cells was subjected to microbeam irradiation, chloroplasts moved towards the irradiated area even in subsequent darkness. We therefore recorded the movement and calculated the speeds of signal transfer by time-lapse imaging. Movement speeds under red or blue light were similar, e.g., about 1.0 microm min(-1) in prothallial cells. However, speeds varied according to cell polarity in protonemal cells. The speed of signal transfer from the protonemal apex to the base was approximately 0.7 microm min(-1), but roughly 2.3 microm min(-1) in the opposite direction. The speed of signal transfer in Arabidopsis thaliana mesophyll cells was approximately 0.8 microm min(-1) by comparison. Surprisingly, chloroplasts located farthest away from the microbeam were found to move faster than those in close proximity to the site of irradiation both in Adiantum capillus-veneris and A. thaliana.

Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells

Chloroplasts alter their distribution within plant cells depending on the external light conditions. Myosin inhibitors 2,3-butanedione monoxime (BDM), N-ethylmaleimide (NEM), and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) were used to study the possible role of myosins in chloroplast photorelocation in Arabidopsis thaliana mesophyll cells. None of these agents had an effect on the chloroplast high-fluence-rate avoidance movement but all of the three myosin inhibitors blocked the accumulation movement of chloroplasts after a high-fluence-rate irradiation of the leaves. The results suggest that myosins have a role in A. thaliana chloroplast photorelocation.

On the Interaction of Neomycin with the Slow Vacuolar Channel ofArabidopsis thaliana

This study investigates the interaction of the aminoglycoside antibiotic neomycin with the slow vacuolar (SV) channel in vacuoles from Arabidopsis thaliana mesophyll cells. Patch-clamp experiments in the excised patch configuration revealed a complex pattern of neomycin effects on the channel: applied at concentrations in the submicromolar to millimolar range neomycin (a) blocked macroscopic SV currents in a voltage- and concentration-dependent manner, (b) slowed down activation and deactivation kinetics of the channel, and most interestingly, (c) at concentrations above 10 μM, neomycin shifted the SV activation threshold towards negative membrane potentials, causing a two-phasic activation at high concentrations. Single channel experiments showed that neomycin causes these macroscopic effects by combining a decrease of the single channel conductance with a concomitant increase of the channel's open probability. Our results clearly demonstrate that the SV channel can be activated at physiologically relevant tonoplast potentials in the presence of an organic effector molecule. We therefore propose the existence of a cellular equivalent regulating the activity of the SV channel in vivo.

A Mechanosensitive Anion Channel in Arabidopsis thaliana Mesophyll Cells

Mechanosensitive ion channels are expected to play important roles in transducing mechanical stimuli into intracellular signals during the development and morphogenesis of higher plants. We have identified a novel mechanosensitive anion channel in the protoplast of Arabidopsis thaliana mesophyll cells by using the patch-clamp technique. The channel in the outside-out patches could be activated by positive pressure in the pipette while negative pressure had no effect. The amphipathic membrane crenator trinitrophenol, which is supposed to preferentially insert in the outer leaflet of the lipid bilayer of the plasma membrane, synergized with mechanical membrane stretch to activate the channel. These results suggest that the channel activation is mediated by a convex curvature of the plasma membrane. Therefore, activation of this channel may play an important role when cell volume is increasing during cell growth or hypo-osmotic challenge, which is accompanied by membrane stretch with increasingly convex curvature.

Ultrastructural and functional changes of photosynthetic apparatus of arabidopsis thaliana (l.) heynh induced by clinorotation

By the 28 th day of growth upon a slowly rotating horizontal clinostat there had been a rearrangement of chloroplast organisation in the Arabidopsis thaliana mesophyll cells, changes in the native chlorophyll forms and alterations to the composition of the pigment-protein complex.