过氧化氢参与烟草叶片衰老调节的研究（简报）

叶片衰老是一个复杂的过程．多年来人们对叶片衰老的机制开展了大量的研究。许多研究证明叶片中H2O2的迅速积累与叶片的衰老密切相关^[1-3],然而,H2O2的积累是衰老的结果还是作为信号启动衰老？H2O2作为植物细胞的信号分子,调控一系列重要的生理生化过程,如系统获得抗性（SAR）、细胞衰老与程序化死亡（PCD）、气孔关闭、根的生长、细胞壁的发育等^[4]。H2O2参与脱落酸（A—BA）、甲基茉莉酸（MJ）诱导的水稻叶片衰老,且H2O2产生早于叶片衰老^[2,5]。     

Regulation of Antioxidant Enzymes by Salicylic Acid in Cucumber Leaves

Activities of the antioxidant enzymes involved in superoxide anion (O2-) and hydrogen peroxide (H2O2) metabolism were determined and the contents of O2 and 14202 were also measured. All concentrations of sahcylic acid (SA) tested (0.5, 1.0, 2.5 and 5.0 mmoL/L) significantly enhanced superoxide dismutase (SOD) and peroxidase (POD) activities not only in the first treated true leaf (leaf 1 ) but also in the second untreated true leaf (leaf 2) of Cucumis sativus L. When the leaves were treated with 1 mmol/L SA within 6 to 72 h, the activity of POD increased by 22 % to 67% in the treated leaf 1 and by 14% to 86% in the untreated leaf 2. However, no changes were observed during 3 h after treatment and at 96 h following treatment. Measurement of O2- and H202 showed that there was a significant decrease in 02' content and an increase in H202 content after SA treatment, but catalase (CAT) activity was only slighfiy inhibited and this suggested that the reason of the increase in H2O2 by SA treatment is not due to the inhibition of CAT but rather the increase in SOD activity. It was also found that SA at all concentrations tested could not induce new SOD isozyme but it induced 1 to 2 bands of new POD isozyme within one day after treatment. The results indicate that SA might involve in the regulation of antioxidant enzymes.     

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。     

水稻叶片叶绿素和过氧化氢含量的QTL检测及上位性分析

研究水稻叶片叶绿素和过氧化氢含量的遗传规律,对探讨光合代谢产物遗传规律和开展高产育种具有重要指导意义。利用由日本晴／Kasalath∥日本晴的杂交组合衍生的98个回交重组自交家系(BC1F9)所组成的BIL(backcross inbred lines)群体,在第1、2、3和10染色体上分别检测出5个与叶绿素含量相关的QTL和2个影响剑叶过氧化氢含量的QTL,其中位于第1染色体的RFLP标记C86和C813之间的q-Chll对叶绿素含量的影响最为显著,对表型变异的贡献率达22％,其增效基因来自粳稻品种日本晴;同时在该区间检测到1个与剑叶过氧化氢含量相关的QTL:q-H2O2I,对过氧化氢含量的减效基因来自日本晴品种。上位性分析结果显示影响叶绿素含量及过氧化氢含量的非等位QTL之间存在显著的上位性效应。具有上位性效应的QTL分布于第2、6、11和12染色体上,未检测到与q-Chll或q-H2O2I互作的位点。暗示日本晴品种的RFLP标记C86和C813之间存在1个能够提高叶绿素含量,同时又能降低过氧化氢含量的主效QTL,其加性效应显著而不存在上位性效应。     

Involvement of hydrogen peroxide in leaf abscission signaling, revealed by analysis with an in vitro abscission system in Capsicum plants

Although auxin and ethylene play pivotal roles in leaf abscission, the subsequent signaling molecules are poorly understood. This is mainly because it is difficult to effectively treat the intact abscission zone (AZ) with pharmacological reagents. We developed an in vitro experimental system that reproduces stress-induced leaf abscission in planta. In this system, 1-mm-thick petiole strips, encompassing the AZ, were separated within 4 days of abscission at the AZ through cell wall degradation in an auxin depletion- and ethylene-dependent manner. The system allowed us to show that hydrogen peroxide (H(2)O(2)) is involved in abscission signaling. Microscopic analyses revealed continuous H(2)O(2) production by AZ cells. H(2)O(2) scavengers and diphenylene iodonium, an inhibitor of NADPH oxidase, suppressed in vitro abscission and cellulase expression. Conversely, the application of H(2)O(2) promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission was suppressed by inhibitors of H(2)O(2) production, whereas the expression of ethylene-responsive genes was unaffected by both H(2)O(2) and an H(2)O(2) inhibitor. These results indicated that H(2)O(2) acts downstream from ethylene in in vitro abscission signaling. In planta, salinity stress induced the expression of genes that respond to ethylene and reactive oxygen species, and also induced H(2)O(2) production at the AZ, which preceded leaf abscission. These results indicate that H(2)O(2) has roles in leaf abscission associated with ethylene both in vitro and in planta.     

Hydrogen peroxide concentrations in leaves under natural conditions

While H2O2 has been implicated in numerous plant environmental responses, normal levels and variabilities are poorly established, and estimates of leaf tissue concentrations span more than three orders of magnitude, even in a single species under similar conditions. Here, leaf tissue H2O2 contents under natural conditions are reported after determining (i) that H2O2 in extracts was stable with time, (ii) that H2O2 added to the extract was recovered quantitatively, and (iii) that the H2O2 calibration curve was unaffected (or quantifiably affected) by the extract. The broad applicability of the protocol and variability in leaf concentrations were demonstrated using tissue collected from several habitats in association with three, more extensive, experiments. The first involved nychthemeral studies of the mangrove, Rhizophora mangle L. Lowest H2O2 levels occurred in the early morning and near sunset, with higher levels both at midday and at night. Second, using five temperate species in Spring, concentrations were compared on a warm, sunny day and a cool, cloudy day. Higher concentrations were found on the warm day for Aesculus glabra Willd., Glechoma hederacea L., Plantago major L., and Viola soraria Willd., while there were no differences in Quercus macrocarpa Michx. Finally, the effects of elevated CO2 and ozone were examined in soybean, Glycine max L. Pioneer 93B15 under Free Air gas Concentration Enrichment (FACE) conditions. Both supplements led to elevated H2O2. Overall, mean leaf, midday, and mid-summer H2O2 concentrations ranged from 0.67 micromol (gFW)(-1) in mangrove to 3.6 micromol (gFW)(-1) in A. glabra Willd. Greatest within-species differences were only 2.5-fold in any of the studies.     

马缨丹叶提取液对水葫芦叶片中超氧物歧化酶活性和过氧化氢积累的影响

水葫芦[Eichhornia crassipes（Mart）Solms]是世界上繁殖最快、危害最严重的多年生水生杂草之一。为了避免化学除草剂对水体的污染,生物防治已成为当前水葫芦治理的重要方向。马缨丹（Lantana camara）是马鞭草科的一种植物,其叶片提取物对水葫芦有很强的毒性。研究结果表明：经马缨丹叶提取液处理的水葫芦叶片中,超氧物歧化酶（SOD）活性与H2O2浓度均显著升高,但过氧化氢酶的活性受到抑制,膜脂过氧化程度明显增加。H2O2的组织化学染色结果表明H2O2在气孔细胞中有异常高的积累,H2O2过量产生同时导致水葫芦叶片失绿与细胞死亡。因此,氧胁迫可能是马缨丹提取液对水葫芦毒害的主要原因之一。     

Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins

Increased salinity is a stringent problem to crop production while seed pretreatment can effectively induce salt tolerance in plants. Hydrogen peroxide (H(2)O(2)), a stress signal molecule, was evaluated as seed treatment to produce the metabolic changes, which could lead to improved salt tolerance in wheat. Soaking in 1, 40, 80 and 120 microM H(2)O(2) revealed a low penetration, reaching maximum at 5h (2.58+/-0.23 micro mol g(-1) fresh seeds at 120 microM) and declining thereafter to the level of water control by 8h. This revealed the activation of antioxidants and H(2)O(2) scavenging in seed after 5h. Seeds treated with 1-120 microM H(2)O(2) for 8h and germinated in saline (150 mM NaCl) medium curtailed the mean germination time (MGT) being even less than water controls. Level of H(2)O(2) in seedlings arising from H(2)O(2)-treated seeds grown under salinity was markedly lower than salinized controls, suggesting the operation of antioxidant system in them. These seedlings exhibited better photosynthetic capacity, particularly the stomatal conductance (gs), thus improving the leaf gas exchange due to stomatal component of photosynthesis. Moreover, H(2)O(2) treatment improved leaf water relations and maintained turgor. Although Na(+) and Cl(-) content increased due to salinity, H(2)O(2)-treated seedlings displayed greater tissue K(+), Ca(2+), NO(3)(-) PO(4)(3-) levels and improved K(+):Na(+) ratio. H(2)O(2) treatment enhanced the membrane properties, as revealed from greatly reduced relative membrane permeability (RMP) and less altered ion leakage pattern (comparable to water controls). Seedlings exhibited the expression of two heat-stable (stress) proteins with apparent molecular masses of 32 and 52 kDa. Results suggest that H(2)O(2) signals the activation of antioxidants in seed, which persists in the seedlings to offset the ion-induced oxidative damage. These changes led to the expression of stress proteins and improved physiological attributes, which supported the seedling growth under salinity.     

Enhanced monsoon precipitation and nitrogen deposition affect leaf traits and photosynthesis differently in spring and summer in the desert shrub Larrea tridentata

Leaf-level CO2 assimilation (A(area)) can largely be predicted from stomatal conductance (g(s)), leaf morphology (SLA) and nitrogen (N) content (N(area)) in species across biomes and functional groups. The effects of simulated global change scenarios, increased summer monsoon rain (+H2O), N deposition (+N) and the combination (+H2O +N), were hypothesized to affect leaf trait-photosynthesis relationships differently in the short- and long-term for the desert shrub Larrea tridentata. During the spring, +H2O and +H2O +N plants had lower A(area) and g(s), but similar shoot water potential (Psi(shoot)) compared with control and +N plants; differences in A(area) were attributed to lower leaf N(area) and g(s). During the summer, +H2O and +H2O +N plants displayed higher A(area) than control and +N plants, which was attributed to higher Psi(shoot), g(s) and SLA. Throughout the year, A(area) was strongly correlated with g(s) but weakly correlated with leaf N(area) and SLA. We concluded that increased summer monsoon had a stronger effect on the performance of Larrea than increased N deposition. In the short term, the +H2O and +H2O +N treatments were associated with increasing A(area) in summer, but also with low leaf N(area) and lower A(area) in the long term the following spring.     

Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.     

Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants.

Hydrogen peroxide (H2O2) has been implicated in many stress conditions. Control of H2O2 levels is complex and dissection of mechanisms generating and relieving H2O2 stress is difficult, particularly in intact plants. We have used transgenic tobacco with approximately 10% wild-type catalase activity to study the role of catalase and effects of H2O2 stress in plants. Catalase-deficient plants showed no visible disorders at low light, but in elevated light rapidly developed white necrotic lesions on the leaves. Lesion formation required photorespiratory activity since damage was prevented under elevated CO2. Accumulation of H2O2 was not detected during leaf necrosis. Alternative H2O2-scavenging mechanisms may have compensated for reduced catalase activity, as shown by increased ascorbate peroxidase and glutathione peroxidase levels. Leaf necrosis correlated with accumulation of oxidized glutathione and a 4-fold decrease in ascorbate, indicating that catalase is critical for maintaining the redox balance during oxidative stress. Such control may not be limited to peroxisomal H2O2 production. Catalase functions as a cellular sink for H2O2, as evidenced by complementation of catalase deficiency by exogenous catalase, and comparison of catalase-deficient and control leaf discs in removing external H2O2. Stress analysis revealed increased susceptibility of catalase-deficient plants to paraquat, salt and ozone, but not to chilling.     

Salicylic and jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by Septoria nodorum Berk

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species' (ROS) (superoxide radical O2*- and H2O2) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk. has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O2*- and H2O2 compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.     

植被-大气相互作用中的气孔导度及其尺度转换

气孔导度是衡量植物和大气间水分、能量及CO2平衡和循环的重要指标,探讨气孔导度在叶片、冠层及区域尺度间的尺度转换及累积效应,对更好地认识植被与大气间的水热运移过程,合理评价植被在陆面过程中的地位和作用具有重要意义.本文着重从叶片尺度气孔导度模拟、气孔导度在冠层尺度的累积表现、冠层到区域尺度转换研究及气孔导度累积效应在陆面过程模型中的作用等4个层次总结了近期国内外研究状况,指出其中存在的异质性等问题,并就今后应加强多尺度间的同步观测提出了展望.     

Participation of H2O2 in Enhancement of Cold Chilling by Salicylic Acid in Banana Seedlings

The possible physiological mechanism of enhancement of cold tolerance by salicylic acid (SA) in banana seedlings (Musa acuminata cv. Williams 8188) was explored. Measurements of leakage electrolyte after 2 d of recovery at 30/22 ℃ (day/night) following 3 d of cold stress at 7 ℃ showed that pretreatment with hydroponic solution containing SA 0.3－0.9 mmol/L as foliar spray under normal growth conditions (30/22 ℃) could significantly enhance cold tolerance of banana plants. The highest enhancing effect of SA occurred at 0.5 mmol/L and it showed the lowest leakage rate of electrolyte or smaller leaf wilting area after 2 d of recovery at normal temperature from 3 d of 7 ℃ or 5 ℃ cold stress. Higher concentrations (≥2.5 mmol/L) of SA, however, caused more electrolyte leakage, indicating that they aggravated chilling damage. Enhanced cold tolerance by SA could be related to H2O2 metabolism. Compared with water-treated seedlings (control), SA 0.5 mmol/L treatment inhibited activities of catalase (CAT) and ascorbate peroxidase (APX), increased peroxidase (POX) activity, but did not affect the activity of superoxide dismutase (SOD) under normal growth conditions, and these changes might lead to an accumulation of H2O2, whereas SA pretreatment enhanced the activities of CAT and APX, and reduced the increase in productions of H2O2 and thiobarbituric acid-reaction substances (TBARS) during subsequent 7 ℃ cold stress and recovery periods. Exogenous H2O2 treatments (1.5－2.5 mmol/L) also increased cold tolerance of banana seedlings. Furthermore, pretreatment of banana seedlings with dimethylthiourea (a trap for H2O2) significantly inhibited cold tolerance induced by SA. These results suggested that endogenous H2O2 may be required for SA-enhanced cold tolerance. The significance of the interaction of SA, H2O2 and H2O2-metabolizing enzymes during cold stress has been discussed.     

The role of hydrogen peroxide-producing and hydrogen peroxide-consuming peroxidases in the leaf apoplast of cowpea in manganese tolerance

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.     

Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR

Methylviologen (MV) induces oxidative damages in leaves. In order to understand its mechanism we studied initial biochemical events under light in MV-fed spinach leaves. When isolated chloroplasts were illuminated in the presence of MV, both stromal and thylakoid-bound ascorbate peroxidases (APX) were inactivated rapidly at the same rates, and their inactivation was retarded by ascorbate (AsA) at higher concentrations. Since MV accelerates the photoproduction of O2- in Photosystem (PS) I and simultaneously inhibits the photoreduction of monodehydroascorbate (MDA) to AsA, the inactivation of APX was attributed to the loss of AsA and accumulation of H2O2 in the stroma. Following APX, superoxide dismutase and NADP(+)-glyceraldehyde 3-phosphate dehydrogenase, both of which are vulnerable to H2O2, were inactivated by MV plus light. Dehydroascorbate reductase, monodehydroascorbate reductase, PS II, PS I and ferredoxin-NADP(+) reductase were far less sensitive to the treatment. In the treated leaves, cytosolic APX and guaiacol-specific peroxidase were also inactivated, but slower than chloroplastic APXs were. Catalase was not inactivated. Thus the MV-induced photooxidative damages of leaves are initiated with the inactivation of chloroplastic APXs and develop via the inactivation of other H2O2-sensitive targets. The decay half-life of the MDA signal after a short illumination in the leaves, as determined by in vivo electron spin resonance spectrometry (ESR), was prolonged when the H2O2-scavenging capacity of the leaf cells was abolished by the inactivation of chloroplastic and cytosolic APXs. The measurement of MDA in leaves by ESR, therefore, allows to estimate in vivo cellular capacity to scavenge the photoproduced H2O2.     

Inactivation in vivo of basic peroxidase and increased content of H2O2 in grapevine leaves post treatment with DTT and paraquat

Substantial differences in the in vivo effect of paraquat (Pq) and DTT on basic peroxidase (GBPx) activity and on H2O2 levels were found in grapevine leaves cv. Sultana. GBPx activity decreased and H2O2 levels increased in illuminated Pq treated leaf-discs. Inactivation of GBPx and accumulation of H2O2 depended on the duration and intensity of the illumination to which discs were exposed. Since GBPx was inactivated directly by H2O2 and not by Pq in leaf extracts, and since GBPx are cytosolic isoenzymes and H2O2 is a stable molecule that can easily permeate chloroplast membranes, we concluded that Pq inactivation of GBPx in vivo is mediated by H2O2. In contrast to the effect induced by Pq, DTT directly inactivated GBPx in leaf extracts. In leaf-discs, however, it reduced GBPx activity in the absence of light, although the levels of H2O2 increased only after exposure of the discs to high irradiance, suggesting that under excess of light, a significant fraction of the photosynthetically produced electrons are dissipated through the water-water cycle and H2O2 accumulates as a consequence of GBPx inactivation.     

Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves

Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.     

Hydrogen peroxide is involved in high blue light-induced chloroplast avoidance movements in Arabidopsis

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 (H(2)O(2)), 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 H(2)O(2) is involved in high-fluence BL-induced chloroplast avoidance movements, a laser scanning confocal microscope and a luminescence spectrometer were used to observe H(2)O(2) generation in situ with the assistance of the fluorescence probe dichlorofluorescein diacetate (H(2)DCF-DA). After treatment with high-fluence BL, an enhanced accumulation of H(2)O(2), indicated by the fluorescence intensity of DCF, can be observed in leaf cells of Arabidopsis thaliana. Exogenously applied H(2)O(2) 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 H(2)O(2) generation and the subsequent chloroplast movements can be largely abolished by the administration of the H(2)O(2)-specific scavenger catalase and other antioxidants. In addition, in-depth subcellular experiments indicated that high-fluence BL-induced H(2)O(2) 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 H(2)O(2) generation at both the plasma membrane and the chloroplast, and that the production of H(2)O(2) is involved in high-fluence BL-induced chloroplast avoidance movements.