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.     

植物细胞中蓝光诱导ROS生成的识别和亚细胞定位检测

以2’,7’-二氯二氢荧光素二乙酯(dichlorofluorescein diacetate,H2DCF-DA)为荧光探针孵育拟南芥叶表皮条,利用荧光光谱和激光共聚焦扫描显微技术,对高辐照蓝光诱导下叶肉细胞活性氧(reactive oxygen spe-cies,ROS)的生成,进行了分子识别和亚细胞定位检测。结果表明:植物细胞在蓝光诱导下,可以产生大量的ROS。过氧化氢酶清除实验表明:高辐照蓝光诱导产生的ROS,主要成分是H2O2,并且主要定位在叶绿体和细胞膜上。     

向光素调节植物向光性及其与光敏色素/隐花色素的相互关系

蓝光受体向光素(PHOT1/PHOT2)调节蓝光诱导的植物运动反应, 包括植物向光性、叶绿体运动、气孔运动和叶片伸展等。其中, 向光素介导的植物向光性能够促使植物弯向光源, 确保其以最佳取向捕获光源, 优化光合作用。光敏色素和隐花色素作为光受体也参与植物的向光性调节。该文综述了向光素介导的拟南芥(Arabidopsis thaliana)下胚轴向光弯曲信号转导及其与光敏色素、隐花色素协同作用的分子机制, 以期为改造植物光捕获能力及提高光利用效率提供理论基础。     

Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.     

Integrated role of ROS and Ca<Superscript>+2</Superscript> in blue light-induced chloroplast avoidance movement in leaves of <Emphasis Type="Italic">Hydrilla verticillata</Emphasis> (L.f.) Royle

Directional chloroplast photorelocation is a major physio-biochemical mechanism that allows these organelles to realign themselves intracellularly in response to the intensity of the incident light as an adaptive response. Signaling processes involved in blue light (BL)-dependent chloroplast movements were investigated in Hydrilla verticillata (L.f.) Royle leaves. Treatments with antagonists of actin filaments [2,3,5-triiodobenzoic acid (TIBA)] and microtubules (oryzalin) revealed that actin filaments, but not microtubules, play a pivotal role in chloroplast movement. Involvement of reactive oxygen species (ROS) in controlling chloroplast avoidance movement has been demonstrated, as exogenous H₂O₂ not only accelerated chloroplast avoidance but also could induce chloroplast avoidance even in weak blue light (WBL). Further support came from experiments with different ROS scavengers, i.e., dimethylthiourea (DMTU), KI, and CuCl₂, which inhibited chloroplast avoidance, and from ROS localization using specific stains. Such avoidance was also partially inhibited by ZnCl₂, an inhibitor of NADPH oxidase (NOX) as well as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transport chain (ETC) inhibitor at PS II. However, methyl viologen (MV), a PS I ETC inhibitor, rather accelerated avoidance response. Exogenous calcium (Ca⁺²) induced avoidance even in WBL while inhibited chloroplast accumulation partially. On the other hand, chloroplast movements (both accumulation and avoidance) were blocked by Ca⁺² antagonists, La³⁺ (inhibitor of plasma membrane Ca⁺² channel) and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, Ca⁺² chelator) while LiCl that affects Ca⁺² release from endosomal compartments did not show any effect. A model on integrated role of ROS and Ca⁺² (influx from apolastic space) in actin-mediated chloroplast avoidance has been proposed.     

Abrogation of bleomycin-induced lung fibrosis by nitric oxide synthase inhibitor, aminoguanidine in mice.

The effects of aminoguanidine (AG), a specific inhibitor of inducible nitric oxide synthase, on the bleomycin (BL)-induced lung fibrosis was evaluated in mice. The animals were placed into five groups: saline (SA)-instilled drinking water (SA+H(2)O), saline-instilled drinking water containing 0.5%AG (SA+0.5%AG), BL-instilled drinking water (BL+H(2)O), BL-instilled drinking water containing 0.2%AG (BL+0.2%AG), and BL-instilled drinking water containing 0.5%AG (BL+0.5%AG). The mice had free access to H(2)O or H(2)O containing AG and lab chow ad lib 2 days prior to intratracheal (IT) instillation of BL (0.07U/mouse/100 microL) or an equivalent volume of sterile isotonic saline. The mice in the SA+0.5%AG group consumed the greatest amount of AG without any ill effects than the mice in any other group. There were no differences in any of the measured biochemical determinants between the SA+H(2)O and SA+0.5%AG control groups. The IT instillation of BL in the BL+H(2)O group caused significant increases in the lipid peroxidation, hydroxyproline content, and prolyl hydroxylase activity of lungs and influx of inflammatory cells in the broncheoalveolar lavage fluid (BALF) as compared to both control groups. The intake of aminoguanidine by mice in the BL+0.5%AG group caused significant reductions in the BL-induced increases in all measured biochemical indices of lung fibrosis without any effects on the influx of inflammatory cells in the BALF. In fact, AG in both BL-treated groups additionally increased the total cell counts in the BALF from mice in the BL+0.2%AG and BL+0.5%AG groups as compared to the BL+H(2)O group. Histopathological evaluation of the lungs revealed that the mice in the BL+0.5%AG group had markedly fewer fibrotic lesions than mice in the BL+H(2)O group. These results demonstrate that aminoguanidine minimizes the BL-induced lung fibrosis at both the biochemical and the morphological level and support our earlier hypothesis that the production of nitric oxide plays a significant role in the pathogenesis lung fibrosis caused by BL.     

Phytochrome modulation of blue light-induced chloroplast movements in Arabidopsis

Photometric analysis of chloroplast movements in various phytochrome (phy) mutants of Arabidopsis showed that phyA, B, and D are not required for chloroplast movements because blue light (BL)-dependent chloroplast migration still occurs in these mutants. However, mutants lacking phyA or phyB showed an enhanced response at fluence rates of BL above 10 micromol m-2 s-1. Overexpression of phyA or phyB resulted in an enhancement of the low-light response. Analysis of chloroplast movements within the range of BL intensities in which the transition between the low- and high-light responses occur (1.5-15 micromol m-2 s-1) revealed a transient increase in light transmittance through leaves, indicative of the high-light response, followed by a decrease in transmittance to a value below that measured before the BL treatment, indicative of the low-light response. A biphasic response was not observed for phyABD leaves exposed to the same fluence rate of BL, suggesting that phys play a role in modulating the transition between the low- and high-light chloroplast movement responses of Arabidopsis.     

Changes in ion fluxes during phototropic bending of etiolated oat coleoptiles

BACKGROUND AND AIMS: This work has been conducted to assist theoretical modelling of the different stages of the blue light (BL)-induced phototropic signalling pathway and ion transport activity across plant membranes. Ion fluxes (Ca(2+), H(+), K(+) and Cl(-)) in etiolated oat coleoptiles have been measured continuously before and during unilateral BL exposure. METHODS: Changes in ion fluxes at the illuminated (light) and shadowed (dark) sides of etiolated oat coleoptiles (Avena sativa) were studied using a non-invasive ion-selective microelectrode technique (MIFE). The bending response was also measured continuously, and correlations between the changes in various ion fluxes and bending response have been investigated. For each ion the difference (Delta) between the magnitudes of flux at the light and dark sides of the coleoptile was calculated. KEY RESULTS: Plants that demonstrated a phototropic bending response also demonstrated Ca(2+) influx into the light side approximately 20 min after the start of BL exposure. This is regarded as part of the perception and transduction stages of the BL-induced signal cascade. The first 10 min of bending were associated with substantial influx of H(+), K(+) and Cl(-) into the light (concave) side of the coleoptiles. CONCLUSIONS: The data suggest that Ca(2+) participates in the signalling stage of the BL-induced phototropism, whereas the phototropic bending response is linked to changes in the transport of H(+), K(+) and Cl(-).     

Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis

Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 micromol m(-2) s(-1) of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 micromol m(-2) s(-1) caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 micromol m(-2) s(-1), chloroplasts in pmi2 leaves move to the periclinal walls; 100 micromol m(-2) s(-1) of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.     

Reorganized actin filaments anchor chloroplasts along the anticlinal walls of <Emphasis Type="Italic">Vallisneria</Emphasis> epidermal cells under high-intensity blue light

In epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light (BL) induces the avoidance response of chloroplasts. We examined simultaneous BL-induced changes in the configuration of actin filaments in the cytoplasmic layers that face the outer periclinal wall (P side) and the anticlinal wall (A side). The results clearly showed that dynamic reorganization of the actin cytoskeleton occurs on both sides. Upon BL irradiation, thick, long bundles of actin filaments appeared, concomitant with the directed migration of chloroplasts from the P side to the A side. After 15–20 min of BL irradiation, fine actin bundles on only the A side appeared to associate with chloroplasts that had migrated from the P side. To examine the role of the fine actin bundles, we evaluated the anchorage of chloroplasts by centrifuging living cells. Upon BL irradiation, the resistance of chloroplasts on both the P and A sides to the centrifugal force decreased remarkably. After 20 min of BL irradiation, the resistance of chloroplasts on the A side increased again, but chloroplasts on the P side could still be displaced. The BL-induced recovery of resistance of chloroplasts on the A side was sensitive to photosynthesis inhibitors but insensitive to an inhibitor of flavoproteins. The photosynthesis inhibitors also prevented the fine actin bundles from appearing on the A side under BL irradiation. These results strongly suggest that the BL-induced avoidance response of chloroplasts includes photosynthesis-dependent and actin-dependent anchorage of chloroplasts on the A side of epidermal cells.     

Blue light-induced chloroplast relocation

Chloroplast relocation movement is induced by blue light in most plants tested. Under weak light, chloroplasts move toward a brighter area in a cell (called low-fluence-rate response or accumulation movement), but they avoid strong light and move away from the light (called high-fluence-rate response or avoidance movement). Recently, mutants deficient in the chloroplast avoidance movement were isolated from Arabidopsis thaliana. The results of mutant analyses revealed that the phototropin photoreceptors phot1 and phot2 both control chloroplast accumulation while phot2 alone controls the avoidance movements.     

Pb-Induced Avoidance-Like Chloroplast Movements in Fronds of Lemna trisulca L.

Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed). An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb²⁺, 24 h) in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H₂O₂). In addition, we detected an enhanced accumulation of endogenous H₂O₂ after treatment of plants with lead. Interestingly, H₂O₂-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H₂O₂ can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete) of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the mechanisms of the toxic effect of lead on chloroplasts can include disturbances in their movement and distribution pattern.     

Actin cytoskeleton in Arabidopsis thaliana under blue and red light

BACKGROUND INFORMATION: Actin cytoskeleton is the basis of chloroplast-orientation movements. These movements are activated by blue light in the leaves of terrestrial angiosperms. Red light has been shown to affect the spatial reorganization of F-actin in water plants, where chloroplast movements are closely connected with cytoplasmic streaming. The aim of the present study was to determine whether blue light, which triggers characteristic responses of chloroplasts, i.e. avoidance and accumulation, also influences F-actin organization in the mesophyll cells of Arabidopsis thaliana. Actin filaments in fixed mesophyll tissue were labelled with Alexa Fluor 488-conjugated phalloidin. The configuration of actin filaments, expressed as a form factor (4 pi x area/perimeter(2)), was determined for all actin formations which were measured in fluorescence confocal images. RESULTS: In the present study, we compare form-factor distributions and the median form factors for strong and weak, blue- and red-irradiated tissues. Spatial organization of the F-actin network did not undergo any changes which could be attributed specifically to blue light. Actin patterns were similar in blue-irradiated wild-type plants and phot2 (phototropin 2) mutants which lack the avoidance response of chloroplasts. However, significant differences in the shape and distribution of F-actin formations were observed between mesophyll cells of phot2 mutants irradiated with strong and weak red light. These differences were absent in wild-type leaves. CONCLUSIONS: Actin does not appear to be the main target for the blue-light chloroplast-orientation signal. The modes of actin involvement in chloroplast translocations are different in water and terrestrial angiosperms. The results suggest that co-operation occurs between blue- and red-light photoreceptors in the control of the actin cytoskeleton architecture in Arabidopsis.     

The chloroplast avoidance response decreases internal conductance to CO2 diffusion in Arabidopsis thaliana leaves

The relationship between chloroplast arrangement and diffusion of CO(2) from substomatal cavities to the chloroplast stroma was investigated in Arabidopsis thaliana. Chloroplast position was manipulated by varying the amount of blue light and by cytochalasin D (CytD) treatment. We also investigated two chloroplast positioning mutants. Chloroplast arrangement was assessed by the surface area of chloroplasts adjacent to intercellular airspaces (S(c)). Although it has been previously shown that long-term acclimation to high light is linked with a large S(c), we found that the short-term chloroplast avoidance response reduces S(c). This effect was not apparent in the blue-light-insensitive phot2 mutant, which did not show the avoidance response. As expected, the smaller S(c) induced by the avoidance response was coupled to a similar decrease in internal conductance. This reduction in internal conductance resulted in an increased limitation of the rate of photosynthesis. The limiting effect of S(c) on internal conductance and photosynthesis was also shown in chup1, a mutant with a constant small S(c) as the result of an unusual chloroplast arrangement. We conclude that chloroplast movements in A. thaliana can rapidly alter leaf morphological parameters, and this has significant consequences for the diffusion of CO(2) through the mesophyll.     

Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells

Blue light (BL) receptor phototropins activate the plasma membrane H(+)-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H?-ATPase, mediating stomatal opening. To date, detection of the phosphorylation level of the guard cell H?-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h. Here, we show that BL-induced phosphorylation of guard cell H?-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H?-ATPase. BL-induced phosphorylation of the H?-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant. Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H?-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs. These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H?-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 and ost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants. These results indicate that inhibition of BL-induced phosphorylation of guard cell H?-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.     

Dynamic changes in the actin cytoskeleton during the high-fluence rate response of theMougeotia chloroplast

Summary Since photo-induced orientation movement of a single, ribbon-shaped chloroplast in each cell of the filamentous green algaMougeotia is inhibited in the presence of cytochalasin B, actin is thought to be involved in the process of chloroplast movements. However, this possibility remains to be proved. A specific class of cytoplasmic filaments, which emerge from the advancing front of the moving chloroplast, can be seen by differential interference contrast (DIC) microscopy. However, no one has yet succeeded in defining the nature of these filaments. We have been able to stain the actin filaments (AFs) associated with the moving chloroplast with fluorescein-conjugated phalloidin (FP) after pre-treatment withm-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). No filamentous structures were observed in cells that had been pre-irradiated with low-fluence rate red light. However, transversely oriented fluorescent filaments appeared at the front edge of the moving chloroplast when it began to rotate under irradiation with high-fluence rate white light. These filaments disappeared after completion of the orientation movement, suggesting the simultaneous appearance of AFs and the orientation movement of the chloroplast. Thick cytoplasmic strands connecting the edge of the chloroplast with the parietal cytoplasm were often seen by DIC microscopy before and after completion of the high-fluence rate orientation movement. These thick cytoplasmic strands could not be stained by FP, but were often stained by 3,3-dihexyloxacarbocyanine iodide (DiOC₆(3)), suggesting that they are transvacuolar strands that include endoplasmic reticulum.     

Ion and oxygen fluxes in the unicellular alga Eremosphaera viridis

Plasma membrane fluxes of the large unicellular model algal cell Eremosphaera viridis (De Bary) were measured under various light regimes to explore the role of plasma membrane fluxes during photosynthesis and high light-induced chloroplast translocation. Plasma membrane fluxes were measured directly and non-invasively with self-referencing ion-selective (H(+), Ca(2+), K(+) and Cl(-)) potentiometric microelectrodes and oxygen amperometric microelectrodes. At light irradiances high enough to induce chloroplast migration from the cell periphery to its center, oxygen evolution declined to respiratory net O(2) uptake prior to any significant chloroplast translocation, while net K(+) and Cl(-) influx increased during the decline in photosynthetic activity (and the membrane potential depolarized). The results suggest that chloroplast translocation is not the cause of the cessation of O(2) evolution at high irradiance. Rather, the chloroplast translocation may play a protective role: shielding the centrally located nucleus from damaging light intensities. At both high and low light intensities (similar to ambient growth conditions), there was a strong inverse correlation between H(+) net fluxes and respiratory and photosynthetic net O(2) fluxes. A similar inverse relationship was also observed for Ca(2+) net fluxes, but only at higher light intensities. The net H(+) fluxes are small relative to the buffering capacity of the cell, but are clearly related to both photosynthetic and respiratory activity.     

Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement

The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.     

Ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent hydrogen peroxide synthesis in Vicia faba L

Ultraviolet B (UV-B) radiation is an important environmental signal for plant growth and development, but its signal transduction mechanism is unclear. UV-B is known to induce stomatal closure via hydrogen peroxide (H(2)O(2)), and to affect ethylene biosynthesis. As ethylene is also known to induce stomatal closure via H(2)O(2) generation, the possibility of UV-B-induced stomatal closure via ethylene-mediated H(2)O(2) generation was investigated in Vicia faba by epidermal strip bioassay, laser-scanning confocal microscopy, and assays of ethylene production. It was found that H(2)O(2) production in guard cells and subsequent stomatal closure induced by UV-B radiation were inhibited by interfering with ethylene biosynthesis as well as ethylene signalling, suggesting that ethylene is epistatic to UV-B radiation in stomatal movement. Ethylene production preceded H(2)O(2) production upon UV-B radiation, while exogenous ethylene induced H(2)O(2) production in guard cells and subsequent stomatal closure, further supporting the conclusion. Inhibitors for peroxidase but not for NADPH oxidase abolished H(2)O(2) production upon UV-B radiation in guard cells, suggesting that peroxidase is the source of UV-B-induced H(2)O(2) production. Taken together, our results strongly support the idea that ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent H(2)O(2) generation.