Differential effect of NaCl and polyethylene glycol on the ultrastructure of chloroplasts in rice seedlings

Ionic and osmotic effects of salinity on the ultrastructure of chloroplasts in salt-treated rice seedlings were investigated. After rice seedlings were grown in hydroponic culture for three weeks, they were treated with NaCl and polyethylene glycol (PEG) 4000 both at a water potential of -1.0 MPa for 3 days. The most notable difference in ultrastructural change between NaCl and PEG treatment was observed in the damage in chloroplast membranes. NaCl induced swelling of thylakoids and caused only a slight destruction of the chloroplast envelope. PEG caused severe destruction of the chloroplast envelope compared with NaCl, however thylakoids did not swell. Our observations suggested that in salt-treated rice plants, the ionic effects induced swelling of thylakoids and the osmotic effects caused the destruction of chloroplast envelope.     

Deposition pattern of hydrogen peroxide in the leaf sheaths of rice under salt stress

Deposition pattern of hydrogen peroxide (H₂O₂) under salt stress (100 mM NaCl) was examined cytochemically in rice (Oryza sativa L. cv. Pokkali) through the reaction of H₂O₂ with cerium chloride (CeCl₃) to produce electron dense precipitates of cerium perhydroxide. The distribution pattern of cerium perhydroxide precipitates in leaf sheath was considerably different from other parts of rice under salinity stress. Cerium perhydroxide precipitates were mainly accumulated on the tonoplast of leaf sheath under salinity, although they were localized on the cell wall and plasma membrane in all other tissues such as leaf blade and root.     

H2O2 destruction by ascorbate-dependent systems from chloroplasts

Washed lamellae from isolated spinach chloroplasts exhibited peroxidative activity with 3,3′-diaminobenzidine or ascorbate as electron donors. By heat treatment or by incubation of the chloroplasts with pronase a heat-labile enzymic activity (system A) and a heat-stable non-enzymic peroxidative activity (system B) could be differentiated.System A is membrane-bound, reacts with 3,3′-diaminobenzidine and with ascorbate as electron donors, shows a sharp pH optimum between 7.5 and 8.0 with both substrates and is inhibited competitively by cyanide.The heat-stable factor can be extracted from the chloroplast lamellae by heat treatment, reacts only with ascorbate as electron donor, shows increasing activity with higher pH values but no optimum and is not inhibited by cyanide.Both peroxidative systems in connection with a relatively high concentration of ascorbate in chloroplasts should represent an important tool for the detoxification of H₂O₂ which is produced in these organelles by photosynthetic O₂ reduction.     

Effects of salt-tolerant rootstock grafting on ultrastructure,photosynthetic capacity,and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-scavenging system in chloroplasts of cucumber seedlings under NaCl stress

Plant growth, photosynthetic parameters, chloroplast ultrastructure, and the ascorbate-glutathione cycle system in chloroplasts of self-grafted and rootstock-grafted cucumber leaves were investigated. Grafted plants were grown hydroponically and were exposed to 0, 50, and 100 mM NaCl concentrations for 10 days. Under NaCl stress, the hydrogen peroxide (H₂O₂) content in cucumber chloroplasts increased, the chloroplast ultrastructure was damaged, and the gas stomatal conductance, intercellular CO₂ concentration, as well as shoot dry weight, plant height, stem diameter, leaf area, and leaf relative water content were inhibited, whereas these changes were less severe in rootstock-grafted plants. The activities of ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2), and dehydroascorbate reductase (DHAR EC 1.8.5.1) were higher in the chloroplasts of rootstock-grafted plants compared with those of self-grafted plants under 50 and 100 mM NaCl. Similar trends were shown in leaf net CO₂ assimilation rate and transpiration rate, as well as reduced glutathione content under 100 mM NaCl. Results suggest that rootstock grafting enhances the H₂O₂-scavenging capacity of the ascorbate–glutathione cycle in cucumber chloroplasts under NaCl stress, thereby protecting the chloroplast structure and improving the photosynthetic performance of cucumber leaves. As a result, cucumber growth is promoted.     

Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.     

Shade light interaction with salicylic acid in regulating growth of sun (alpine) and shade (prairie) ecotypes of Stellaria longipes

Magnesium (Mg) deficiency in plants is a widespread problem, affecting productivity and quality in agriculture. The mechanism of Mg deficiency inducing antioxidant enzyme activities has not been elucidated in rice. We examined the relationship among abscisic acid (ABA), H₂O₂, and antioxidant enzymes in the leaves of rice seedlings grown under conditions of Mg deficiency. The expression of OsRab16A, an ABA responsive gene, was used to determine the content of ABA. Mg deficiency resulted in increased ABA content in leaves of rice seedlings. The production of H₂O₂ was examined by 3,3-diaminobenzidine staining and a colorimetric method. Mg deficiency also induced H₂O₂ production in leaves, which was blocked by dipehnyleneiodonium chloride (DPI), an NADPH oxidase inhibitor. Tungstate (Tu), an ABA biosynthesis inhibitor, was effective in reducing Mg deficiency-increased ABA content, as well as Mg deficiency-induced H₂O₂ production. Both Tu and DPI were effective in reducing Mg deficiency-induced activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in the leaves. Mg deficiency-induced ABA accumulation may trigger increased production of H₂O₂, which may involve plasma-membrane NADPH oxidase, and, in turn, up-regulates the activities of antioxidant enzymes in leaves of rice seedlings.     

Signal regulation of proline metabolism in callus of the halophyte Nitraria tangutorum Bobr. grown under salinity stress

Callus of the halophyte Nitraria tangutorum Bobr. was used to investigate proline metabolism and its signal regulation under salinity stress. Enhanced levels of proline and hydrogen peroxide (H₂O₂) were observed in calli exposed to salinity stress, and elevated levels of calcium (Ca) were detected in early responses to 75?mM NaCl treatment. Additionally, NaCl treatment induced significant elevation of ornithine-??-aminotransferase (OAT) activity, but notable decreases occurred in the activities of glutamyl kinase (GK) and proline dehydrogenase (PDH). H₂O₂ scavenger dimethylthiourea and pyruvate inhibited the accumulation of proline and the stimulation of OAT in salinity-stressed calli. Moreover, the utilization of Ca chelator EGTA and Ca channel blocker verapamil abolished the enhancement of proline level induced by 75?mM NaCl treatment for 3?days. These results suggest that the accumulation of proline is correlated to the increase of OAT activity and the decrease of PDH activity in response to salinity, and that elevated Ca signal during the early stage of NaCl treatment and the excitation of OAT activity resulting from the increase of H₂O₂ generation are essential for proline accumulation in salinity-stressed calli.     

Salinity induced the changes of root growth and antioxidative responses in two wheat cultivars

This study aimed to investigate the inhibitory mechanism of root growth and to compare antioxidative responses in two wheat cultivars, drought-tolerant Ningchun and drought-sensitive Xihan, exposed to different NaCl concentrations. Ningchun exhibited lower germination rate, seedling growth, and lipid peroxidation than Xihan when exposed to salinity. The loss of cell viability was correlated with the inhibition of root growth induced by NaCl stress. Moreover, treatments with H₂O₂ scavenger dimethylthiourea and catalase (CAT) partly blocked salinity-induced negative effects on root growth and cell viability. Besides, the enhancement of superoxide radical and H₂O₂ levels, and the stimulation of CAT and diamine oxidase (DAO) as well as the inhibition of glutathione reductase (GR) were observed in two wheat roots treated with salinity. However, hydroxyl radical content increased only in Xihan roots under NaCl treatment, and the changes of soluble peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and cell-wall-bound POD activities were different in drought-tolerant Ningchun and drought-sensitive Xihan exposed to different NaCl concentrations. In conclusion, salinity might induce the loss of cell viability via a pathway associated with extracellular H₂O₂ generation, which was the primary reason leading to the inhibition of root growth in two wheat cultivars. Here, it was also suggested that increased H₂O₂ accumulation in the roots of drought-tolerant Ningchun might be due to decreased POD and GR activities as well as enhanced cell-wall-bound POD and DAO ones, while the inhibition of APX and GR as well as the stimulation of SOD and DAO was responsible for the elevation of H₂O₂ level in drought-sensitive Xihan roots.     

Intracellular location of cytochrome oxidase active in vivo in the cyanophytes,Synechocystis sp. PCC6714 andAnacystis nidulans Tx20 and R2, grown under various conditions

Summary Intracellular location of cytochrome oxidase (cytoxidase) active in vivo was studied cytochemically with four strains of cyanophytes, using 3,3-diaminobenzidine (DAB) oxidation. DAB was oxidized in the dark bySynechocystis sp. PCC6714 and two strains ofAnacystis nidulans (Tx20 and R2) grown under photoautotrophic, heterotrophic and salt-stressed conditions, respectively. Electron microscopic observations showed that DAB-oxidation in the dark occurred in the thylakoids, but was insignificant on or around the cytoplasmic membrane. However, deposition of DAB-oxidation product around the cytoplasmic membrane was observed with cells of the thylakoid-less cyanophyteGlaeobacter violaceus ATCC29082. All DAB oxidations observed with the four strains were inhibited completely by cyanide, the inhibitor of cyt-oxidase, but not by 3-amino-1,2,4-triazole, the inhibitor of peroxidase. The results show that (1) DAB was oxidized by the cyt-oxidase functioning in the respiratory system, and that (2) cyt-oxidase in thylakoids was active in vivo.Abbreviations AT 3-amino-1,2,4-triazole - Cyt oxidase cytochrome oxidase - ETS electron transport system - DAB 3,3-diaminobenzidine     

Exogenous ethanol treatment alleviates oxidative damage of Arabidopsis thaliana under conditions of high-light stress

Abiotic stresses, such as high light and salinity, are major factors that limit crop productivity and sustainability worldwide. Chemical priming is a promising strategy for improving the abiotic stress tolerance of plants. Recently, we discovered that ethanol enhances high-salinity stress tolerance in Arabidopsis thaliana and rice by detoxifying reactive oxygen species (ROS). However, the effect of ethanol on other abiotic stress responses is unclear. Therefore, we investigated the effect of ethanol on the high-light stress response. Measurement of chlorophyll fluorescence showed that ethanol mitigates photoinhibition under high-light stress. Staining with 3,3′-diaminobenzidine (DAB) showed that the accumulation of hydrogen peroxide (H₂O₂) was inhibited by ethanol under high-light stress conditions in A. thaliana. We found that ethanol increased the gene expressions and enzymatic activities of antioxidative enzymes, including ASCORBATE PEROXIDASE1 (AtAPX1), Catalase (AtCAT1 and AtCAT2). Moreover, the expression of flavonoid biosynthetic genes and anthocyanin contents were upregulated by ethanol treatment during exposure to high-light stress. These results imply that ethanol alleviates oxidative damage from high-light stress in A. thaliana by suppressing ROS accumulation. Our findings support the hypothesis that ethanol improves tolerance to multiple stresses in field-grown crops.     

Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48℃ in the dark for 30 min or gradually elevated temperature from 30℃ to 80℃ at 0.5℃/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of Fo-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3℃ lower in the Chl b-less mutants (48℃) than in the wild type (51℃). At temperature up to about 45℃, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastruc-ture in the wild type showed no obvious alteration. After 55℃ exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chl _b-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a cer-tain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the in-ternal oxidative stress produced at severely high temperature.     

Thermostability of photosynthesis in two new chlorophyllb-less rice mutants

Leaves of the two new chlorophyllb-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48°C in the dark for 30 min or gradually elevated temperature from 30°C to 80°C at 0.5°C/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H₂O₂ accumulation. There were different patterns of F_o-temperature curves between the Chlb-less mutants and the wild type plant, and the temperature of F_o rising threshold was shifted 3°C lower in the Chlb-less mutants (48°C) than in the wild type (51°C). At temperature up to about 45°C, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chlb-less mutants, but chloroplast ultrastructure in the wild type showed no obvious alteration. After 55°C exposure, the disordered thylakoid and significant H₂O₂ accumulation in leaves were found in the two Chlb-less mutants, whereas in the wild type plant, less H₂O₂ was accumulated and the swollen thylakoid still maintained a certain extent of stacking. A large extent of the changes in qP, NPQ and F_v/F_m was consistent with the Pn decreasing rate in the Chlb-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chlb-less mutants showed a tendency for higher thermosensitivity, and loss of Chlb in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the internal oxidative stress produced at severely high temperature.     

Effect of NaCl stress on H2O2 metabolism in rice leaves

The effect of NaCl stress on H₂O₂ metabolismin detached rice leaves was studied. NaCl (200 mM)treatment did not cause the accumulation ofH₂O₂ and resulted in no increase in lipidperoxidation and membrane leakage of leaf tissues. The activities of peroxidase, ascorbate peroxidase,superoxide dismutase, and glutathione reductase wereobserved to be greater in NaCl-stressed rice leavesthan in control leaves. However, glycolate oxidasewas lower in NaCl-treated rice leaves than in thecontrol leaves. There was no difference in catalaseactivity between NaCl and control treatments. Theseresults suggest that some antioxidant enzymes can beactivated in response to oxidative stress induced byNaCl.     

NaCl和Na2SO4胁迫下两种刺槐叶肉细胞叶绿体超微结构

二倍体刺槐(diploid Robinia pseudoacacia)是我国水土保持林的先锋树种,具有较强的适应性和抗逆性,对改善生态环境、防治水土流失、调节水文状况有重要作用。四倍体刺槐(tetraploid Robinia pseudoacacia)是二倍体刺槐的加倍品种,也称多倍体刺槐,由韩国引进,具有速生、耐盐碱、耐干旱和耐烟尘等特点。目前,关于四倍体刺槐的研究,主要集中于栽培技术和繁殖技术方面,而关于四倍体刺槐叶片超微结构与其耐盐性的关系尚缺乏报道。比较了二倍体刺槐和四倍体刺槐在NaCl和Na₂SO₄胁迫下,叶片叶绿体超微结构的变化特点,一方面可以对二者的耐盐性进行鉴定,同时也可以探讨不同盐分胁迫条件下的作用机制。利用NaCl和Na₂SO₄进行20d的盐胁迫处理,观察叶绿体超微结构的变化特点,发现:NaCl处理前,二者叶肉细胞叶绿体为梭形、形态饱满、结构完整,NaCl处理后10d时,二倍体刺槐的叶绿体出现变形、膜模糊、基粒片层松散、类囊体解体、脂质球增多等现象,NaCl处理后20d时,叶绿体肿胀、变形,基粒片层断裂,膜系统解体。Na₂SO₄处理后10d时,二倍体刺槐的叶绿体肿胀,膜模糊,基粒片层松散、类囊体解体,Na₂SO₄盐胁迫处理后20d时,膜系统全部解体,结构破坏更为严重。总体来说,四倍体刺槐在盐胁迫后叶绿体结构变化不明显,只是在Na₂SO₄处理20d时,四倍体刺槐的叶绿体出现中空、基粒片层松散、膜边缘模糊现象。在处理前,两种刺槐的叶绿体均紧贴细胞壁,分布于细胞壁边缘。在NaCl处理后10d时,二倍体刺槐的叶绿体仍呈有序排列,紧贴细胞壁,但在处理后20d时,大部分叶绿体脱离细胞壁,呈随机分布。在Na₂SO₄处理后10d时,二倍体刺槐部分叶绿体脱离细胞壁,位于细胞中央。在Na₂SO₄处理后20d时,二倍体刺槐叶绿体大部分与细胞壁脱离。四倍体刺槐在两种盐胁迫处理前后叶绿体的排列变化不明显,均分布于细胞壁边缘,紧贴细胞壁。所以在盐胁迫下,耐盐植物叶片的叶绿体表现为结构完整,基粒片层清晰,类囊体结构完整,而不耐盐植物则表现为叶绿体超微结构松散、变形,基粒片层模糊,破坏严重时基粒片层扭曲,叶绿体解体,失去完整结构。     

水稻磷脂氢谷胱甘肽过氧化物酶在蛋白质水平上的组织和诱导表达特征

蛋白质是生命活动的主要承担分子,了解蛋白质在有机体中的时空分布对于正确解析蛋白质的功能十分重要．磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是目前发现的唯一能够直接还原膜上脂类过氧化物的抗氧化酶,在保护生物膜免受过氧化损伤方面有着重要作用．采用Western blot技术,分析了水稻PHGPx (OsPHGPx) 在水稻不同组织以及多种胁迫条件下的蛋白质表达特征．结果表明,OsPHGPx在成熟水稻植株内主要分布于叶组织中,以旗叶中含量最高,而在水稻幼苗中则在茎及叶组织中均检测到较强的杂交信号．OsPHGPx在幼苗中的表达受到H₂O₂和NaCl的强烈诱导,但植物激素对其表达的影响较弱．H₂O₂和NaCl的诱导效果呈现出时间及剂量的相关性,当用0.5 mmol/L H₂O₂处理12 h或用500 mmol/L NaCl处理24 h,此时OsPHGPx表达量达到最大值．对H₂O₂清除剂二甲基硫脲处理的水稻幼苗,外源H₂O₂的再处理并不能诱导OsPHGPx的表达,而NaCl的诱导效果并不受影响,说明H₂O₂可能并不介导NaCl诱导OsPHGPx的表达．这些结果为进一步研究OsPHGPx在水稻中生物学功能奠定了基础．     

Hydrogen peroxide is involved in the regulation of rice (Oryza sativa L.) tolerance to salt stress

In the present study, we investigated the salt tolerance mechanism of two rice cultivars (Zhenghan-2 and Yujing-6), which show different tolerance to drought and disease. NaCl induced higher extent of lipid peroxide and ion leakage in Yujing-6 roots than those in Zhenghan-2 roots. H₂O₂ accumulation in Zhenghan-2 roots was lower than that in Yujing-6 roots under salt stress. Comparatively, NaCl treatment did not increase O₂ ^? contents in both rice roots, however, O₂ ^? level in Yujing-6 roots was higher than that in Zhenghan-2 roots under both control and salt stress conditions. Ascorbate peroxidases (APX) activity increased more significantly in Zhenghan-2 roots than that in Yujing-6 roots. The activity of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glucose-6-phosphate dehydrogenase (G6PDH) was similarly enhanced in both rice roots under salt stress; however, they showed higher levels in Zhenghan-2 roots than in Yujing-6 roots. Exogenous H₂O₂ could enhance APX, CAT, POD, SOD and G6PDH activities in a concentration-dependent manner in both rice roots. Diphenylene iodonium (DPI), a plasma membrane (PM) NADPH oxidase inhibitor, which counteracted the NaCl-induced H₂O₂ accumulation, markedly decreased the activity of above enzymes. Moreover, ion leakage increased dramatically in Zhenghan-2 roots and reached to the similar level of Yujing-6 roots under NaCl+DPI treatment. Taken together, H₂O₂, which is mainly generated from PM NADPH oxidase, is involved in Zhenghan-2 rice tolerance to salt stress by enhancing the cellular antioxidant level.     

Subcellular Adaptation to Salinity and Irradiance in Dunaliella salina

Dunaliella salina V-63 was cultivated in different concentrations of NaCl (0.5, 1.0, 2.5, 3.0, or 4.0 M) and at two irradiances (170 or 220 μmol m⁻²s⁻¹). Concentration-dependent suppression of growth was observed above 1 M NaCl, and elevated salinity induced formation of salt-containing vacuoles. However, the changes in the chloroplast ultrastructure following changes in salinity and irradiance (increase of invaginations and protuberances, numerous grana with low number of thylakoids, less number of starch grains, etc.) appeared to be of primary importance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromotoxic effects of exogenous hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) in barley seeds exposed to salt stress

The effect of exogenous hydrogen peroxide (H₂O₂) on mitotic activity and chromosomal aberrations in root tip meristems of barley (Hordeum vulgare L. var. Tokak 157/37) germinated under salinity was analyzed. The inhibitory effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration (0.00 control, 0.35, 0.40, 0.45 M, molal NaCl). The frequency of chromosomal aberrations of seeds germinated in medium with 0.40 M NaCl after pretreatment with H₂O₂ (30 μM, micromolal) was significantly higher than the control group. The highest concentration of NaCl (0.45 M) together with H₂O₂ caused total inhibition of germination. In this study, the intention was to determine the performance of H₂O₂ in alleviating detrimental effect of salt stress on mitotic activity and chromosomal aberrations. However, H₂O₂ did not reduce the detrimental effect of NaCl on these parameters. Also, it caused higher chromotoxic effect compared to those of control groups.     

High Resolution Imaging of Temporal and Spatial Changes of Subcellular Ascorbate,Glutathione and H2O2 Distribution during Botrytis cinerea Infection in Arabidopsis

In order to study the mechanisms behind the infection process of the necrotrophic fungus Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H₂O₂) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H₂O₂ accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H₂O₂ in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts (e.g. decrease of chloroplast number, decrease of starch and thylakoid contents, increase of plastoglobuli size), chlorosis and necrosis of the leaves.