JXB Advance Access originally published online on June 11, 2008
Journal of Experimental Botany 2008 59(10):2891-2901; doi:10.1093/jxb/ern147
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RESEARCH PAPER |
Hydrogen peroxide is involved in high blue light-induced chloroplast avoidance movements in Arabidopsis
MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China
* To whom correspondence should be addressed. E-mail: xingda{at}scnu.edu.cn
Received 6 April 2008; Revised 29 April 2008 Accepted 30 April 2008
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Abstract |
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One of the most important functions of blue light (BL) is to induce chloroplast movements in order to reduce the damage to the photosynthetic machinery under excess light. Hydrogen peroxide (H2O2), which is commonly generated under various environmental stimuli, can act as a signalling molecule that regulates a number of developmental processes and stress responses. To investigate whether H2O2 is involved in high-fluence BL-induced chloroplast avoidance movements, a laser scanning confocal microscope and a luminescence spectrometer were used to observe H2O2 generation in situ with the assistance of the fluorescence probe dichlorofluorescein diacetate (H2DCF-DA). After treatment with high-fluence BL, an enhanced accumulation of H2O2, indicated by the fluorescence intensity of DCF, can be observed in leaf cells of Arabidopsis thaliana. Exogenously applied H2O2 promotes the high-fluence BL-induced chloroplast movements in a concentration-dependent manner within the range of 0–10–4 M, not only increasing the degree of movements but also accelerating the start of migrations. Moreover, the high-fluence BL-induced H2O2 generation and the subsequent chloroplast movements can be largely abolished by the administration of the H2O2-specific scavenger catalase and other antioxidants. In addition, in-depth subcellular experiments indicated that high-fluence BL-induced H2O2 generation can be partly abolished by the addition of diphenyleneiodonium (DPI), which is an NADPH oxidase inhibitor, and the blocker of electron transport chain dichlorophenyl dimethylurea (DCMU), respectively. The results presented here suggest that high-fluence BL can induce H2O2 generation at both the plasma membrane and the chloroplast, and that the production of H2O2 is involved in high-fluence BL-induced chloroplast avoidance movements.
Key words: Blue light, chloroplast avoidance movements, hydrogen peroxide, RL transmittance
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Introduction |
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Plants have developed a series of highly sophisticated short- and long-term mechanisms that help them to adapt to changing environmental conditions. For example, when exposed to low-fluence blue light (BL), the chloroplasts accumulate perpendicular to the incident light, along the periclinal cell walls. In response to high-fluence BL, the chloroplasts move parallel to the incident light, along the anticlinal cell walls (Zurzycki, 1955). This BL-induced relocation of the chloroplasts is regarded as an adaptive mechanism to improve the efficiency of photosynthesis and to avoid any damage by strong light (Seitz, 1972).
It is known that phototropin1 (phot1) and phot2, as plant-specific BL receptors, can mediate BL-induced chloroplast movements (Briggs et al., 2001). Phot1 was first identified as a BL receptor responsible for phototropism by using an Arabidopsis mutant that showed impaired phototropic bending in response to BL (Christie et al., 1998). Later, a homologue of phot1 was also isolated from an Arabidopsis mutant defective in the avoidance response and was renamed phot2 (Jarillo et al., 1998; Briggs and Christie, 2002). In general, phot2 functions under a relatively high intensity of BL. For example, it can, as a photoreceptor mediating the photoavoidance response of chloroplasts, prevent strong light from damaging the photosynthetic apparatus (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Recent molecular characterization has shown that both phototropins contain a Ser/Thr protein kinase domain located within their C terminus. Furthermore, the N-terminal region of phot1 and phot2 both contain a repeated motif of 110 amino acids (aa), designated LOV1 and LOV2 (Christie et al., 1999; Christie and Briggs, 2001). LOV1 and LOV2 belong to the Per–Arnt–Sim (PAS) domain superfamily, which are found in a variety of proteins. They have been reported to mediate protein–protein interactions and to function as internal sensors of oxygen, light, and redox changes in the electron transport system or overall cellular redox status (Taylor and Zhulin, 1999; Briggs and Christie, 2002).
An increasing body of evidence has shown that redox seems to intervene in the regulation of multiple processes functional at virtually every stage of plant development (Buchanan and Balmer, 2005). Therefore, tight control of redox homeostasis is fundamental in cell metabolism. Although plant cells possess an array of antioxidant mechanisms to cope with high rates of reactive oxygen species (ROS) production, most forms of biotic or abiotic stimulus could result in the enhanced production of ROS, which inevitably disrupts the balance of the cellular redox status (Mou et al., 2003). It has been widely reported that hydrogen peroxide (H2O2), as a major member of ROS, acts at subtle levels as a second messenger molecule in biological processes such as development and stress perception (Levine et al., 1994; Orozco-Cárdenas et al., 2001). In higher plants, H2O2 can be generated by several different pathways, including a plasma membrane localized NADPH oxidase, glycollate oxidase, and the Mehler reaction in the chloroplasts. The H2O2 signal can be transmitted through alterations in Ca2+ fluxes and the cellular redox state, both of which are considered to be very early events that follow the rise in H2O2 levels (Rentel and Knight, 2004).
Although it has been reported that strong light could change the cellular redox state and trigger the xanthophyll cycle, which in turn plays a role in modulating the BL-dependent chloroplast movements (Tlalka et al., 1999), it is still not clear whether the H2O2 signal is directly involved in BL-induced chloroplast movements, and what is the source of the H2O2 generation. To address these questions, the changes in chloroplast movement in response to H2O2 were therefore examined using Arabidopsis. In this work, evidence is provided that H2O2, which was generated at both the cell membrane and the chloroplast by prolonged exposure to high-fluence BL, may be involved in the high-fluence BL-induced chloroplast avoidance movements. The mechanism whereby H2O2 was perceived to be underlying the BL-induced chloroplast movements is also discussed.
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Materials and methods |
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Chemicals
Dichlorofluorescein diacetate (H2DCF-DA; Molecular Probes, Eugene, OR) was dissolved in DMSO to produce a 100 mM stock solution, which was aliquoted. Catalase (CAT, bovine liver), diphenyleneiodonium (DPI), ascorbic acid (AsA), and dichlorophenyl dimethylurea (DCMU) were from Sigma (St Louis). Unless stated otherwise, the remaining chemicals were of analytical grade from Chinese companies.
Plant material
Seeds of Arabidopsis were sown in water-soaked Scott's Plug or Metro mix and incubated at 4 °C in darkness for 3–4 d. Seedlings were grown in soil in a plant growth chamber (Conviron, model E7/2, Winnipeg, Canada) under a 12/12 h light/dark cycle, a photon fluence rate of 100 µmol m–2 s–1, and a day/night temperature cycle of 22±0.5 °C. All the experiments were performed on the leaves from 4–5-week-old seedlings and repeated at least three times.
Transmittance measurements
In plants, chlorophyll is the main absorber of red light. Due to the migration of the chloroplasts to the anticlinal walls the chlorophyll distributed at the cell surface will be reduced, leading to an increase in red light (RL) transmittance. Upon migration of the chloroplasts to the periclinal cell walls, the majority of chlorophyll will be distributed at the cell surface, leading to a decrease in RL transmittance (DeBlasio et al., 2003, 2005). Therefore the increase in RL transmittance is used to indicate the avoidance movement and the decrease in RL transmittance to indicate the accumulation movement. In our experiments, RL transmittance was measured using the ultraviolet-visible spectrometer (Lambda 35, Perkin-Elmer, UK) according to the protocol described by DeBlasio et al. with some modifications (DeBlasio et al., 2003). Leaves from 4–5-week-old Arabidopsis plants were excised and dark-acclimated for 9–15 h in the 50 mM KCl/10 mM TRIS-MES (pH 6.1, loading buffer). The dark-adapted leaves were incubated with different concentrations of H2O2 (0–10–1 M) for 1 h in the dark, and were then directly exposed to different photon flux density of BL for different times. The different photon flux density of BL (0.3, 5, 15, 30, or 60 µmol m–2 s–1) was obtained by changing the height between the leaf and a set of light-emitting diodes (LED) (450±25 nm), which were mounted perpendicular to the leaf surface. Photon flux density was measured using a quantum meter (Li-Cor 250; Li-Cor, Lincoln, NE, USA) equipped with a light sensor (Li-Cor 190SA; Li-Cor). After BL or/and H2O2 treatments for the indicated times, the leaves that had been gently sandwiched between two glass slides were immediately inserted into the solid sample holder of the ultraviolet-visible spectrometer for measuring RL transmittance. To characterize the recovery dynamics of chloroplast movements, the changes in RL transmittance was determined at the indicated time after the BL source was turned off and H2O2 was removed by washing five times with the loading buffer. For each leaf, change in percentage RL transmittance was calculated as:
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Dye loading
Freshly prepared abaxial leaf strips were first incubated for 30 min in 10 ml loading buffer to eliminate any background ROS that may be produced during the preparation of the leaf strips. The leaf strips were then placed in a small Petri dish containing 2.97 ml loading buffer and incubated with 30 µl of H2DCF-DA from a 100 mM stock solution in DMSO for 10–15 min in the dark before the different treatments.
Luminescence spectrometer
The dye-loaded leaf strips were transferred to a dish containing fresh buffer to wash off excess dye and subsequently irradiated with BL, RL, or green light (GL) of 30 µmol m–2 s–1 for different times.
At the indicated times, a leaf strip was placed flat onto a plastic holder and fixed at both ends with silicon grease and the DCF fluorescence emitted from the leaf strip was recorded with a luminescence spectrometer (LS55, Perkin-Elmer, UK). The luminescence spectrometer was set to an excitation of 488 nm and an emission of 525 nm, with a slit width of 2.5 nm. To determine the effects of antioxidants on ROS production, the final concentration of 100 units ml–1 catalase, 10 µM DCMU, 200 µM AsA, 50 µM DPI or 50 mM mannitol was added into the dish just before light irradiation.
Laser scanning confocal microscope
In situ detection of ROS production was performed using a commercial laser scanning microscope (LSM510/ConfoCor2) combination system (Zeiss, Jena, Germany). For excitation, a blue argon-ion laser (488 nm) set on 3% power was used; the emission fluorescence was collected by the 500–550 nm bandpass for DCF fluorescence. The viability of the cells within the mesophyll tissue under these media conditions was >80%, as tested by fluorescein diacetate staining (data not shown).
To analyse the effects of exogenous H2O2 on chloroplast movements in living mesophyll cells, fresh leaves were gently cut into 1 mm sections and then mounted under a coverslip on a microscope slide with loading buffer. Micrographs of cross-sections from each treatment were captured at x20 magnification using the commercial laser scanning confocal microscope.
For calculating the numbers of chloroplasts located along the anticlinal and periclinal walls, a thin temporary section was prepared from the leaves after treatment with BL or H2O2. Then, leaf sections were mounted under a coverslip on a microscope slide with loading buffer. The optical images of temporary sections with a 0.5 µm pinhole were taken at x20 magnification by using the same confocal system described above, and the numbers of chloroplasts in the anticlinal and periclinal walls were calculated.
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Results |
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The effects of exogenous H2O2 on BL-induced chloroplast movements
The effects of the exogenously applied H2O2 on high-fluence BL-induced chloroplast avoidance movements were investigated first. It is clear that the chloroplasts of mesophyll cells of dark-acclimated Arabidopsis leaves after a 60 min treatment with BL at 30 µmol m–2 s–1 move to the anticlinal walls, parallel to the incident light (Fig. 1A, B). Moreover, the chloroplast movements induced by high-fluence BL are more marked in the presence of 100 µM H2O2 than in the absence of H2O2 (Fig. 1C, D). Analysis of chloroplast position showed that, in leaves exposed to high-fluence BL for 30 min, approximately 60% of the chloroplasts were located along the anticlinal walls (Fig. 1I), whereas under the same light conditions, approximately 85% of the chloroplasts were along the anticlinal walls in the presence of exogenous H2O2 (Fig. 1J). With the extension of the exposure time to 60 min, only 8% of the chloroplasts were located at the periclinal walls in the presence of exogenous H2O2, although 20% of the chloroplasts could still be observed in the periclinal walls without H2O2 treatment.
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Effects of different concentrations of H2O2 on BL-induced changes in RL transmittance
It has been proved that the changes in light transmittance and absorption through intact leaves could be used as a reliable indicator of chloroplast movements (DeBlasio et al., 2005; Luesse et al., 2006). Chloroplast migration to the periclinal cell walls in response to low-fluence rates of light results in a decrease in RL transmittance through leaves due to greater surface area coverage by chloroplasts, whereas under high-light conditions, chloroplasts migrate to the anticlinal walls, and RL transmittance increases as the amount of surface area covered by chloroplasts decreases. To confirm the enhanced effects of H2O2 on BL-induced chloroplast movements further, the change in percentage RL transmittance of leaves was examined in the presence of H2O2. Exogenous application of H2O2 increased the RL transmittance of leaves in a dose-dependent manner at a concentration of H2O2
10–4 M in the presence of 30 µmol m–2 s–1 BL, although it has no effects on the RL transmittance of leaves in the darkness (Fig. 2). Moreover, when BL was removed, the RL transmittance of leaves could recover to the original level. Within the concentration range of 0–10–4 M, the maximum promotion of the RL transmittance was observed after BL for 30 min. For example, the RL transmittance of leaves was increased to 196% of the original level by H2O2 of 10–4 M. However, high concentrations of H2O2 (10–1–10–3 M) appeared to have a harmful effect on the normal function of the cell because neither changes nor full recovery in the RL transmittance could be found (see insert of Fig. 2).
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Different intensity of BL-induced changes in RL transmittance in the presence of H2O2
Figures 3 and 4 show a time-course of the change in RL transmittance in Arabidopsis leaves in response to sequential treatments of low-, intermediate-, and high-intensity BL (0.3, 5, 15, 30, and 60 µmol m–2 s–1, respectively). Upon exposure to 15, 30, and 60 µmol m–2 s–1 BL, RL transmittance through Arabidopsis leaves increased by approximately 0.6%, 0.77%, and 1.0%, respectively. However, a more prominent increase in the RL transmittance (0.8%, 1.5%, and 1.8%, respectively) could be observed in leaves after being treated simultaneously with high-fluence BL and 10–4 M H2O2 (Fig. 3). In addition, regardless of the presence or absence of H2O2, irradiation with 0.3 or 5 µmol m–2 s–1 BL for 1 h decreased RL transmittance through Arabidopsis leaves by about 0.4% compared with the dark-acclimated phase (Fig. 4). Specifically, upon exposure to 5 µmol m–2 s–1 continuous BL, transmittance increased for about 15 min before decreasing to a value lower than that measured before the BL treatment. However, the time of increasing transmittance was extended from 15 min to 40 min in the presence of H2O2 (Fig. 4). These data indicated that exogenous H2O2 could not only increase the degree of high fluence BL-induced chloroplast movements but also accelerate the trigger of the chloroplast migration induced by both low and high BL.
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High-fluence BL induces H2O2 production in mesophyll cells
Having established that exogenous H2O2 was involved in the promotion of chloroplast avoidance movements induced by high-fluence BL in the above leaf bioassay and RL transmittance analysis experiments (Figs 1–4
), it was then examined whether high-fluence BL might increase the level of H2O2 in mesophyll cells. In this study, the change in intracellular H2O2 level was directly measured by a sensitive fluorophore dichlorofluorescein (H2DCF). The non-polar diacetate ester (H2DCF-DA) of H2DCF enters the cell and is hydrolysed to the more polar, non-fluorescent compound H2DCF, which is therefore trapped. Subsequent oxidation of H2DCF by H2O2, catalysed by peroxidases, yields the highly fluorescent DCF (Allan and Fluhr, 1997; Cathcart et al., 1983). Figure 5 shows the dynamics of H2O2 generation indicated by the relative fluorescence intensity of DCF in the abaxial leaf strip during exposure to 0.3 µmol m–2 s–1 BL or 30 µmol m–2 s–1 light (BL, RL, and GL). It is clear that the intensity of DCF fluorescence in leaf strips after treatment with high-fluence BL increased more significantly than with RL. In addition, only very low fluorescence intensity could be detected after exposure to 0.3 µmol m–2 s–1 BL, whereas no fluorescence occurred after GL treatment compared with the control (Fig. 5A). The experiments performed on leaf strips using phototropin mutants show that the DCF fluorescence caused by intracellular H2O2 increased more significantly in wild type and the phot1 single mutant than in the phot2 single mutant and the phot1phot2 double mutant after treatment with high-fluence BL (Fig. 5B). This suggested that the phot2 photoreceptor is related to the generation of H2O2 induced by high-fluence BL. A single cell assay performed using laser scanning confocal microscopy also illustrated that 30 µmol m–2 s–1 BL induced a significant increase in DCF fluorescence intensity in mesophyll cells, and the accumulated H2O2 was observable at both the chloroplast and the plasma membrane of mesophyll cells after treatment with high-fluence BL for 30 min (Fig. 6A–F). The result clearly showed that a large quantity of H2O2 had been generated in the presence of phot2 during the process of high-fluence BL-induced chloroplast avoidance movements.
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The effects of scavengers on H2O2 generation and chloroplast avoidance movements
To investigate the role of intracellular H2O2 in high-fluence BL-induced chloroplast avoidance movements, scavengers of ROS were used to abolish the intracellular H2O2. As shown in Fig. 7, the increases in DCF fluorescence intensity in abaxial leaf strips induced by both 30 min and 60 min exposure to high-fluence BL were abolished by 100 units ml–1 CAT, a specific scavenger of H2O2. A single cell assay also demonstrated that, after treatment with CAT, H2O2 generation was remarkably scavenged in high BL-treated mesophyll cells of Arabidopsis leaves (Fig. 8A–H). Other scavengers of ROS (such as AsA and mannitol) have a similar effect on H2O2 generation induced by high-fluence BL (data not shown).
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The effects of scavengers of ROS on high-fluence BL-induced chloroplast avoidance movements, indicated by a change in the percentage RL transmittance of the leaves, are shown in Fig. 9. Apparently these scavengers could, to a different extent, inhibit the increase in RL transmittance of leaves induced by high-fluence BL. This indicated that intracellular H2O2 may be necessary for high-fluence BL-induced chloroplast avoidance movements.
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The effects of DCMU and DPI on H2O2 generation
It was noted that H2O2 induced by high-fluence BL occurred at both the chloroplast and plasma membrane of mesophyll cells (Fig. 6A–F). In order to determine how H2O2 is generated at these two subcellular sources, the changes in DCF fluorescence intensity of leaves induced by high-fluence BL were further examined in the presence of DCMU, an inhibitor of photosynthetic electron transport or DPI, a specific inhibitor of NADPH oxidase (Gray and Cresswell, 1984; Schneider et al., 2003). As shown in Fig. 10, addition of 10 µM DCMU before high-fluence BL treatment partly abolished the increases in DCF fluorescence intensity in Arabidopsis leaves. This suggested that the photosynthetic process is responsible for the generation of H2O2 induced by high-fluence BL at the chloroplast. In a parallel experiment, 50 µM DPI could also partly abolish high-fluence BL-induced DCF fluorescence caused by intracellular H2O2. This implied that the reduction of fluorescence intensity is due to the blocking of H2O2 generation through NADPH oxidase by DPI.
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Discussion |
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Under various environmental and developmental stimuli, ROS are commonly generated in many biological systems (Potikha et al., 1999). It has been widely confirmed that ROS appear to play a crucial role in the physiological and pathological processes of plants. ROS generated by light could serve as factors to control the redox state, functioning as controlling factors for redundant pathways of light signal transduction. For example, H2O2, a form of ROS, is implicated as an intermediary messenger in the BL-induced stomatal movement through mediating the activity of an H+ pump in the plasma membrane (Zhang et al., 2004).
Here, new evidences is provided that H2O2 is involved in high fluence BL-induced chloroplast avoidance movements in Arabidopsis mesophyll cells. The following results support this conclusion: (i) exogenously applied H2O2 might accelerate the trigger for chloroplast migration and enhance high-fluence BL-induced chloroplast avoidance movements, (ii) high-fluence BL could induce H2O2 production at both the chloroplast and the cell membrane, (iii) scavenging of endogenous H2O2 blocked the high-fluence BL-induced chloroplast avoidance movements, (iv) the phot2 mutant with impaired chloroplast avoidance movements showed much less H2O2 generation relative to the phot1 mutant, (v) the changes in H2O2 level were consistent with chloroplast avoidance movements.
It has been established that low and intermediate light at fluence rates less than 16 µmol m–2 s–1 cause chloroplast accumulation, while high light at fluence rates above 16 µmol m–2 s–1 induces the chloroplast avoidance movements (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). In an experiment using a variety of BL fluence rates, we also found two similar chloroplasts movements without H2O2 (Figs 3, 4). Moreover, microscope imaging and statistical analysis demonstrated that exogenous application of 100 µM H2O2 could prominently promote high-fluence BL-induced chloroplast avoidance movements (Fig. 1). Further RL transmittance assays confirmed that, within the range of 5–60 µmol m–2 s–1 BL, the extent of the chloroplast avoidance movements was greater in the presence of 100 µM H2O2 than in the absence of it (Fig. 3). This is compatible with the fact that H2O2 is a versatile signal molecule in a variety of biological responses (Zhang et al., 2001; Yoshida and Hasunuma, 2004). Although H2O2 could accelerate the trigger of the low BL-induced chloroplast migration as well as in the high BL-induced chloroplast avoidance movements, it had no effect on the degree of low BL-induced chloroplast accumulation movements (Fig. 4), indicating that the H2O2 effect may be specific to high-fluence BL-induced chloroplast avoidance movements but not the low light accumulation response. This is consistent with the finding that low BL could not cause the generation of endogenous H2O2 as compared to the control (Fig. 5A), implying that H2O2 is not necessary for the low BL-induced chloroplast accumulation movements. As for the accelerated effects of H2O2 on the initiation of the low BL-induced chloroplast migration, it is possible that exogenously applied H2O2 provoked the change in this chloroplast movement with no physiological significance. However, the underlying mechanism by which H2O2 accelerated the low BL-induced accumulation movement may be interesting and needs to be explored further.
Using H2DCF-DA as a probe of H2O2, it was demonstrated that a large quantity of H2O2 was induced by high-fluence BL at the levels of the cell and leaf (Figs 5A, 6), which is consistent with the result obtained by Chandrakuntal et al. (2004). Analysis of the time-courses of H2O2 generation and chloroplast avoidance movements revealed that the migration of chloroplasts induced by high-fluence BL exhibited dynamics similar to the production of H2O2 within 60 min (Figs 1, 3–5). Furthermore, high-fluence BL-induced chloroplast avoidance movements could be largely reduced by using CAT to scavenge the intracellular H2O2 (Figs 7–9). AsA and mannitol also have a similar inhibitory effect on high-fluence BL-induced chloroplast avoidance movements (Fig. 9). This result clearly demonstrated that H2O2 might function as an intermediate in high-fluence BL signalling in chloroplast avoidance movements.
How does H2O2 mediate the high-fluence BL-induced chloroplast avoidance movements? This question might be answered by applying the fact that ROS can alter the cellular redox state. In Arabidopsis plants, phototropin contained two LOV domains, which are relatively conserved in a variety of proteins and are thought to function as internal sensors of oxygen, redox potential, and light (Huala et al., 1997; Taylor and Zhulin, 1999). For example, ROS may control the reversibility of the adduct formation between the chromophore and cysteine residue, which could be modified by ROS, within the LOV domain in phototropin (Crosson and Moffat, 2001). It has also been reported that intracellular ROS may change the redox state of the LOV domain in WC-1 protein and thus regulate the protein signal transduction in photomorphogenesis (Yoshida and Hasunuma, 2004). In our experiment, H2O2 was produced in the presence of high-fluence BL (Figs 5, 6). Hence, the H2O2 generated might act directly on phot2 by changing the redox state of the LOV domain of phot2 and mediating BL signalling transduction in chloroplast avoidance movements.
In the washout experiments, at concentrations lower than 10–4 M, the effects of H2O2 on chloroplast migration were reversible, whereas at concentrations higher than 10–4 M, the effects were irreversible (Fig. 2). This suggested that at low concentrations the effects of H2O2 could be attributed to the activation of a signalling cascade in governing chloroplast movements, whereas at high concentrations H2O2 might damage cellular function. In fact, a similar dual role of H2O2 in regulating stomatal movements depending on its concentration has been described in previous work (Zhang et al., 2001). The evidence that BL-induced H2O2 production enhanced chloroplast migration in Arabidopsis leaves exposed to BL, and that the BL-induced chloroplast movements was reversible by washout and could be decreased by H2O2 removal, indicate that BL-induced H2O2 does not damage plant cells, and it can act as a modulator of BL-induced chloroplast movements.
In higher plants, ROS can be generated by several different pathways in a diverse range of physiological response (Allan and Fluhr, 1997; Bolwell et al., 1998). These pathways may include a cell wall localized peroxidase (Bolwell et al., 1995), amine oxidases (Allan and Fluhr, 1997), NADPH oxidases (Van Gestelen et al., 1997; Xing et al., 1997), and nuclei (Ashtamker et al., 2007). Furthermore, the highly energetic reactions of photosynthesis and an abundant oxygen supply make the chloroplast a particularly rich potential source of ROS. It has been suggested that when NADPH availability is limited, O2 competing for electrons from photosystem I leads to the generation of ROS through the Mehler reaction (Munekage et al., 2004).
The subcellular source and possible molecular events involved in H2O2 generation in mesophyll cells and leaf strips treated with high-fluence BL have been investigated. A single cell assay (Fig. 6) showed that the plasma membrane and the chloroplasts might be the main regions of H2O2 production. Results from experiments applying DPI and DCMU suggested that the plasma membrane localized NADPH oxidases and photosynthetic electron transport are responsible for the H2O2 generation at the plasma membrane and chloroplasts, respectively (Fig. 10). It has been reported that the activity of NADPH oxidases could be activated by the increase in the concentration of cytosolic Ca2+ ([Ca2+]cyt) to catalyse NADPH and O2 to generate ROS in plant development and environmental defence (Lamb and Dixon, 1997; Sagi and Fluhr, 2001). Moreover, a number of studies showed that the increase in [Ca2+]cyt induced by BL could be commonly observed in many experimental systems (Sato et al., 1999; Tlalka and Fricker, 1999). Mutants in phot2 show a lower H2O2 production under the same BL conditions (Fig. 5B), indicating that phototropin2 is required for the possible generation of H2O2 induced by high-fluence BL. From the DPI and phot2 mutant experimental results, we can thus speculate that the high-fluence BL-induced increase in [Ca2+]cyt via phototropin2 may activate the activity of NADPH oxidase to generate H2O2 at the plasma membrane, and in turn the H2O2 generated may act on phototropin2 to promote the chloroplast movements. This speculation is consistent with the concept that phototropin2 is responsible for avoidance movements (Sakai et al., 2001). However, it should also be noted that DPI has been demonstrated to inhibit blue light-induced phosphorylation of phototropin in Vicia faba (Kinoshita et al., 2003). In future studies, it might be intriguing to determine whether the inhibition of phototropin phosphorylation could lead to the decrease in H2O2 production. On the other hand, BL, like RL, as photosynthetically active radiation energy, could also be absorbed by photosystems to produce H2O2 through the Mehler reaction (Fig. 5A). But H2O2 generated from photosynthesis seems not to be associated with chloroplast movements in our present study. Further studies are needed to clarify how the photoreceptors responsible for chloroplast movements perceive H2O2 from different sources and whether H2O2 from the plasma membrane and the chloroplasts are both required for high-fluence BL-induced chloroplast avoidance movements.
In summary, the accumulated evidence suggests that H2O2 is generated parallel to chloroplast avoidance movements induced by high-fluence BL, and that H2O2 itself cannot alter chloroplast position but it can promote chloroplast avoidance movements in the presence of BL, providing a new insight into the ROS and BL signal network. These results not only suggest a new role of H2O2 in high-fluence BL-induced chloroplast avoidance movements, but also provides a possible explanation of the interaction between ROS and LOV domains. At present, the relationship between H2O2 and other intracellular signal molecules (Ca2+, phot1, and phot2) is unknown and needs further study.
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Acknowledgements |
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We thank T Sakai (RIKEN Yokohama Institute, Yokohama, Japan) for kindly providing seeds of the Arabidopsis phot1 single mutant, phot2 single mutant, and phot1phot2 double mutant. We appreciate the helpful advice and technical assistance of Mrs YH Tang from our laboratory. We especially appreciate the conversation with the members of our group in developing some of the ideas presented in this study. This research is supported by the National Natural Science Foundation of China (30470494) and the National High Technology Research and Development Program of China (863 Program) (2007AA10Z204).
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Abbreviations |
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aa, amino acid; AsA, ascorbic acid; BL, blue light; CAT, catalase; DCMU, dichlorophenyl dimethylurea; DPI, diphenyleneiodonium; GL, green light; H2DCF-DA, dichlorofluorescein diacetate; H2O2, hydrogen peroxide; LOV domains, light, oxygen, and voltage domains; phot, phototropin; RL, red light; ROS, reactive oxygen species.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Allan AC, Fluhr R. Two district sources of elicited reactive oxygen species in tobacco epidermal cells. The Plant Cell (1997) 9:1559–1572.[Abstract]
Ashtamker C, Kiss V, Sagi M, Davydov O, Fluhr R. Diverse subcellular locations of cryptogein-induced reactive oxygen species production in tobacco Bright Yellow-2 cells. Plant Physiology (2007) 143:1817–1826.
Bolwell GP, Butt VS, Davies DR, Zimmerlin A. The origin of the oxidative burst in plants. Free Radical Research (1995) 23:517–532.[Web of Science][Medline]
Bolwell GP, Davies DR, Gerrish C, Auh C-K, Murphy TM. Comparative biochemistry of the oxidative burst produced by rose and French bean cells reveals two distinct mechanisms. Plant Physiology (1998) 116:1379–1385.
Briggs WR, Beck CF, Cashmore AR, et al. The phototropin family of photoreceptors. The Plant Cell (2001) 13:993–997.
Briggs WR, Christie JM. Phototropins 1 and 2: versatile plant blue-light receptors. Trends in Plant Science (2002) 7:204–210.[CrossRef][Web of Science][Medline]
Buchanan BB, Balmer Y. Redox regulation: a broadening horizon. Annual Review of Plant Biology (2005) 56:187–220.[CrossRef][Medline]
Cathcart R, Schwiers E, Ames BN. Detection of picomol levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Analytical Biochemistry (1983) 134:111–116.[CrossRef][Web of Science][Medline]
Chandrakuntal K, Kumar PG, Laloraya M, Laloraya MM. Direct involvement of hydrogen peroxide in curvature of wheat coleoptile in blue-light-treated and dark-grown coleoptiles. Biochemical and Biophysical Research Communications (2004) 4:1190–1196.
Christie JM, Briggs WR. Blue light sensing in higher plants. Journal of Biological Chemistry (2001) 276:11457–11460.
Christie JM, Reymond P, Powell GK, Bernasconi P, Raibekas AA, Liscum E, Briggs WR. Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism. Science (1998) 282:1698–1701.
Christie JM, Salomon M, Nozue K, Wada M, Briggs WR. LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proceedings of the National Academy of Sciences, USA (1999) 96:8779–8783.
Crosson S, Moffat K. Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction. Proceedings of the National Academy of Sciences, USA (2001) 98:2995–3000.
DeBlasio SL, Luesse DL, Hangarter RP. A plant-specific protein essential for blue-light-induced chloroplast movements. Plant Physiology (2005) 139:101–114.
DeBlasio SL, Mullen JL, Luesse DL, Hangarter RP. Phytochrome modulation of blue-light-induced chloroplast movements in Arabidopsis. Plant Physiology (2003) 133:1471–1479.
Gray GM, Cresswell CF. The effect of inhibitors of photosynthetic and respiratory electron transport on nitrate reduction and nitrite accumulation in excised Z. mays L. leaves. Journal of Experimental Botany (1984) 35:1166–1176.
Huala E, Oeller PW, Liscum E, Han I-S, Larsen E, Briggs WR. Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science (1997) 278:2120–2123.
Jarillo JA, Ahmad M, Cashmore AR. PHR2: a novel Arabidopsis gene related to the blue-light photoreceptor/photolyase family. Plant Physiology (1998) 117:719.
Jarillo JA, Gabrys H, Capel J, Alonso JM, Ecker JR, Cashmore AR. Phototropin-related NPL1 controls chloroplast relocation induced by blue light. Nature (2001) 410:952–954.[CrossRef][Medline]
Kagawa T, Sakai T, Suetsugu N, Oikawa K, Ishiguro S, Kato T, Tabata S, Okada K, Wada M. Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response. Science (2001) 291:2138–2141.
Kinoshita T, Emi T, Tominaga M, Sakamoto K, Shigenaga A, Doi M, Shimazaki K-I. Blue-light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in stomatal guard cells of broad bean. Plant Physiology (2003) 133:1453–1463.
Lamb C, Dixon RA. The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology (1997) 48:251–275.[CrossRef][Web of Science]
Levine A, Tenhaken R, Dixon RA, Lamb C. H2O2 from the oxidative burst orchestrates the plant hypersensitive response. Cell (1994) 79:583–593.[CrossRef][Web of Science][Medline]
Luesse DR, DeBlasio SL, Hangarter RP. Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis. Plant Physiology (2006) 141:1328–1337.
Mou Z, Fan W, Dong X. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell (2003) 113:935–944.[CrossRef][Web of Science][Medline]
Munekage Y, Hashimoto M, Miyake C, Tomizawa K, Endo T, Tasaka M, Shikanai T. Cyclic electron flow around photosystem I is essential for photosynthesis. Nature (2004) 429:579–582.[CrossRef][Medline]
Orozco-Cárdenas ML, Narváez-Vásquez J, Ryan CA. Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. The Plant Cell (2001) 13:179–191.
Potikha TS, Collins CC, Johnson DI, Delmer DP, Levine A. The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers. Plant Physiology (1999) 119:849–858.
Rentel MC, Knight MR. Oxidative stress-induced calcium signaling in Arabidopsis. Plant Physiology (2004) 135:1471–1479.
Sagi M, Fluhr R. Superoxide production by plant homologues of the gp91phox NADPH oxidase: modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiology (2001) 126:1281–1290.
Sakai T, Kagawa T, Kasahara M, Swartz TE, Christie JM, Briggs WR, Wada M, Okada K. Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. Proceedings of the National Academy of Sciences, USA (2001) 98:6969–6974.
Sato Y, Kadota A, Wada M. Mechanically induced avoidance response of chloroplasts in fern protonemal cells. Plant Physiology (1999) 121:37–44.
Schneider B, Mutel V, Pietri M, Ermonval M, Mouillet-Richard S, Kellermann O. NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells. Proceedings of the National Academy of Sciences, USA (2003) 100:13326–13331.
Seitz K. Primary process controlling light induced movement of chloroplasts. Acta Protozoologica (1972) 11:225–235.[Medline]
Taylor BL, Zhulin IB. PAS domains: internal sensors of oxygen, redox potential, and light. Microbiology and Molecular Biology Reviews (1999) 63:479–506.
Tlalka M, Fricker M. The role of calcium in blue-light-dependent chloroplast movement in Lemna trisulca L. The Plant Journal (1999) 20:461–473.[CrossRef][Web of Science][Medline]
Tlalka M, Runquist M, Fricker M. Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in Lemna trisulca L. The Plant Journal (1999) 20:447–459.[CrossRef][Web of Science][Medline]
Van Gestelen P, Asard H, Caubergs RJ. Solubilization and separation of a plant plasma membrane NADPH-O
synthase from from other NAD(P)H oxidoreductases. Plant Physiology (1997) 115:543–550.[Abstract]
Xing T, Higgins VJ, Blumwald E. Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells. The Plant Cell (1997) 9:249–259.[Abstract]
Yoshida Y, Hasunuma K. Reactive oxygen species affect photomorphogenesis in Neurospora crassa. Journal of Biological Chemistry (2004) 279:6986–6993.
Zhang X, Zhang L, Dong FC, Gao JF, Galbraith DW, Song CP. Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiology (2001) 126:1438–1448.
Zhang X, Wang HB, Takemiya A, Song CP, Kinoshita T, Shimazaki K. Inhibition of blue light-dependent H+ pumping by abscisic acid through hydrogen peroxide-induced dephosphorylation of the plasma membrane H+-ATPase in guard cell protoplasts. Plant Physiology (2004) 136:4150–4158.
Zurzycki J. Chloroplast arrangement as a factor in photosynthesis. Acta Societatis Botanicorum Poloniae (1955) 24:27–63.
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