Benzothiadiazole (BTH) Induces Cell-Death Independent Resistance in Phaseolus vulgaris against Uromyces appendiculatus

Benzo‐(1,2,3)‐thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH), trade name Bion^®, was used to induce resistance in bean cultivars Borlotto Nano Lingua di Fuoco (BLF), Borlotto Taylor, Cannellino, Cannellino Montalbano, Saxa and Top Crop, against rust caused by Uromyces appendiculatus. A single 0.3 mm BTH spray 7 days before inoculation was sufficient to fully control the disease in all the examined cultivars. As regards the more susceptible BLF, either a single treatment 14 days before inoculation, or three applications on the third, fifth and seventh day before inoculation, were equally effective to prevent infection. Histochemical and cytochemical investigations showed that BTH causes hydrogen peroxide (H₂O₂) accumulation in the treated tissues. H₂O₂ deposits were localized in situ for the first time in the apoplast of the leaf epidermis. No cell death was detected at BTH concentrations below the phytotoxicity threshold, suggesting that acquired resistance against bean rust is mainly related to the enhanced activity of anionic peroxidases, promoted by H₂O₂ accumulation, thereby leading to cell wall strengthening. This hypothesis is also supported by the long induction phase required to establish complete resistance.     

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system

Summary. The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O₂⁻ and H₂O₂) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O₂⁻ production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl₃ assays, which monitor H₂O₂ production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl₃) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H₂O₂ is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H₂O₂ in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H₂O₂ was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H₂O₂ produced by this in vitro culture system is destined for use during lignin biosynthesis. Correspondence and reprints: Departamento de Biologia Vegetal, Facultad de Biologia, Universidad de Murcia, 30100 Murcia, Spain.     

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.     

Effects of the interaction between ozone and carbon dioxide on gas exchange,ascorbic acid content,and visible leaf symptoms in rice leaves

Tropospheric ozone (O₃) decreases photosynthesis, growth, and yield of crop plants, while elevated carbon dioxide (CO₂) has the opposite effect. The net photosynthetic rate (P _N), dark respiration rate (R _D), and ascorbic acid content of rice leaves were examined under combinations of O₃ (0, 0.1, or 0.3 cm³ m⁻³, expressed as O⁰, O^0.1, O^0.3, respectively) and CO₂ (400 or 800 cm³ m⁻³, expressed as C⁴⁰⁰ or C⁸⁰⁰, respectively). The P _N declined immediately after O₃ fumigation, and was larger under O^0.3 than under O^0.1. When C⁸⁰⁰ was combined with the O₃, P _N was unaffected by O^0.1 and there was an approximately 20 % decrease when the rice leaves were exposed to O^0.3 for 3 h. The depression of stomatal conductance (g _s) observed under O^0.1 was accelerated by C⁸⁰⁰, and that under O^0.3 did not change because the decline under O^0.3 was too large. Excluding the stomatal effect, the mesophyll P _N was suppressed only by O^0.3, but was substantially ameliorated when C⁸⁰⁰ was combined. Ozone fumigation boosted the R _D value, whereas C⁸⁰⁰ suppressed it. An appreciable reduction of ascorbic acid occurred when the leaves were fumigated with O^0.3, but the reduction was partially ameliorated by C⁸⁰⁰. The degree of visible leaf symptoms coincided with the effect of the interaction between O₃ and CO₂ on P _N. The amelioration of O₃ injury by elevated CO₂ was largely attributed to the restriction of O₃ intake by the leaves with stomatal closure, and partly to the maintenance of the scavenge system for reactive oxygen species that entered the leaf mesophyll, as well as the promotion of the P _N.     

山黧豆根系对H2O2诱导氧化胁迫的生理应答

以水培7d苗龄的山黧豆幼苗为材料,向水培溶液中施加不同浓度H2O2处理山黧豆幼苗24h,分析山黧豆根系受氧化胁迫的程度与抗氧化系统的应答特征,以揭示山黧豆对氧化胁迫的耐受机制。结果显示:(1)随外源H2O2处理浓度的不断增加,山黧豆幼苗侧根的数目无显著变化,而其根的鲜重则显著降低。(2)同时,根系组织的内源H2O2染色范围和程度显著增高,但根尖区域始终保持较低水平的H2O2;相反,O-·2染色范围和程度明显减少,根尖区域却始终保持较高水平的O-·2。(3)同期根系抗坏血酸(ASC)含量及过氧化氢酶(CAT)、过氧化物酶(POD)与抗坏血酸过氧化物酶(APX)的活性均表现出了先升高后降低的趋势,而超氧化物歧化酶(SOD)一直表现为持续上升的趋势。研究表明,在外源H2O2胁迫条件下,山黧豆根系O-·2的积累可能与其生长和活力呈正相关,而根系H2O2的积累则与其受氧化胁迫程度呈正相关;低浓度的H2O2处理可以提高山黧豆抗氧化系统对体内活性氧的清除能力。     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Effect of salicylic acid potentiates cadmium-induced oxidative damage in <Emphasis Type="Italic">Oryza sativa</Emphasis> L. leaves

In the present investigation, we studied the possible potentiating effect of salicylic acid (SA) under Cd toxicity in Oryza sativa L. leaves. Cd treatments for 24 h reduced the shoot length, dry biomass and total chlorophyll content followed by high Cd accumulation in shoots. About 16 h presoaking with SA resulted in partial protection against Cd, as observed by minor changes in length, biomass and total chlorophyll. SA priming resulted in low Cd accumulation. Enhanced thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H₂O₂) and superoxide anion (O₂ ⁻) content were seen when Cd was applied alone, while under SA priming the extent of TBARS, H₂O₂ and O₂ ⁻ were significantly low, suggesting SA-regulated protection against oxidative stress. The antioxidant enzymes like Catalase (CAT), guaiacol peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD) showed varied activities under Cd alone. CAT activity increased after Cd treatment, followed by a decline in GPX and GR activity. SOD also declined at the highest concentrations with an initial increase. Under SA-priming conditions, the efficiency of the antioxidant enzymes was significantly elevated. GPx and SOD activity showed significant increase in activity. The ascorbate activity increased after Cd treatment, followed by a decline in glutathione under SA-free condition. SA priming showed gradual increase in these non-enzymic antioxidants. Our results indicate that Cd-induced oxidative stress can be regulated by SA.     

Differential ozone sensitivity interferes with cadmium stress in poplar clones

Information on plant responses to combined ozone and cadmium stresses are scarce and limited to herbaceous species. In this research, two poplar clones (I-214 and Eridano), differently sensitive to O₃, were grown for 5 weeks in pots supplied with 0, 53.5, and 160.5 mg(Cd) kg^?1 (soil d.m.) and then exposed to 15-d O₃ fumigation (0.06 mm³ dm^?3, 5 h a day). The effects of the two stressors, alone or in combination, on Cd, Ca, Fe, and Zn accumulation in above-nad below-ground organs, photosynthesis, leaf pigments, and accumulation of H₂O₂ and NO were investigated. Cadmium induced a reduction in stomatal conductance and a significant accumulation of H₂O₂ and NO in both clones nad negatively affected the carotenoid content in I-214. Ozone, on the other hand, counteracted Cd accumulation in the above-ground organs and significantly increased the xanthophyll de-epoxidation state indicating photoinhibition in O₃-treated plants. Surprisingly, O₃ alone or in combination with Cd decreased H₂O₂ accumulation in I-214. The NO production was generally stimulated by Cd, whereas it decreased following O₃ exposure in I-214. The overall data indicate that Cd and O₃ induced clone specific responses. Moreover, when they were applied in combination, antagonistic rather than synergistic effects were observed.     

The detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy

The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H₂O₂) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H₂O₂ and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H₂O₂ in leaves. In this study, the M strain of Cucumber mosaic virus (M‐CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock‐inoculated leaves, H₂O₂ accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H₂O₂ accumulation was always higher than in healthy leaves. Subcellular H₂O₂ localization observed using CLSM showed that H₂O₂ in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H₂O₂ was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H₂O₂ and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.     

Do anthocyanins function as antioxidants in leaves? Imaging of H2O2 in red and green leaves after mechanical injury

Purified anthocyanin extracts show strong antioxidant properties in vitro, but it is not known whether they can scavenge reactive oxygen in living cells. The oxidative responses in red and green portions of Pseudowintera colorata leaf laminae were compared by the real‐time imaging of H₂O₂ in cells after mechanical injury. An oxidative burst was elicited almost immediately from chloroplasts in the palisade mesophyll, as evidenced using the fluorochromes dichlorofluorescein and scopoletin. H₂O₂ accumulated in green lamina regions for 10 min, and then decreased slowly. By contrast, red regions recovered rapidly, and maintained consistently low levels of H₂O₂. Infusion of cells with N‐acetyl‐l ‐cysteine accelerated the depletion of H₂O₂ from green regions. Wounded leaves ultimately developed a localized necrotic lesion and an intense anthocyanic band. The red regions were enriched in anthocyanins, flavonols, dihydroflavonols, and hydroxycinnamic acids. Only the anthocyanins were suitably located to account for the enhanced rates of H₂O₂ scavenging. The data support the hypothesis that red cells have elevated antioxidant capabilities in vivo.     

罗马直立生菜生长发育及营养指标对臭氧浓度升高和持续熏蒸的响应

环境臭氧(O₃)已成为影响植物生长发育的重要生态因子。为探究地面O₃污染对蔬菜形态学特征及营养指标的影响,选罗马直立生菜(Lactuca sativa var. roman)为实验材料,采用开顶式气室开展熏蒸实验。实验设置4个O₃熏蒸浓度(NF:未过滤的环境空气;NF40:环境空气+40 nmol/mol;NF80:环境空气+80 nmol/mol;NF120:环境空气+120 nmol/mol),每个处理设置3个重复组,分析评价O₃污染对植物造成的可见伤害、生产量、叶片解剖学特征以及食用部位营养指标的影响。研究表明:(1)O₃熏蒸对生菜叶片产生不可逆的可见伤害,叶片出现浅黄色斑点和棕色斑点,且随着熏蒸时间延长,叶片出现黄化,大面积的坏死斑块,衰老加速。(2)高浓度O₃胁迫显著降低了生长阶段的株高(P<0.05)。与NF组相比,NF40、NF80、NF120组分别使生物量下降5.90%、14.99%、39.21%。(3)随着O₃熏蒸浓度升高,气孔密度增加,气孔开度减小。叶片厚度、海绵组织厚度、栅栏组织厚度与O₃暴露剂量AOT40呈显著负相关关系(P<0.05)。(4)高浓度O₃暴露使蔬菜中Ca、Na、Fe、Zn、Mg等元素含量显著降低,脂肪和蛋白质含量增加,生菜的营养指标发生改变。研究表明,罗马直立生菜对环境O₃污染敏感,其生长发育及营养指标在O₃胁迫条件下发生明显变化。目前,关于O₃污染对蔬菜形态学特征影响的研究较少,研究系统探讨蔬菜的叶片厚度、栅栏组织、海绵组织、气孔密度及开度等形态学指标在臭氧污染条件下的变化。蔬菜的品质是关系到"三农"问题的重要方面,研究探讨了臭氧污染对蔬菜的产量及营养指标的影响,可为O₃污染条件下蔬菜的生产提供科学参考。     

Hydrogen peroxide-induced astaxanthin biosynthesis and catalase activity in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) in shake-flask cultures was exposed to 10–20 mmol/L H₂O₂ at various culture stages, and the astaxanthin production was significantly increased by H₂O₂ fed at 0 or 24 h (exponential phase), but only slightly at 48 h (near stationary phase). The astaxanthin production was enhanced most significantly with double feeding of 10 mmol/L H₂O₂ at 0 and 24 h, reaching a cellular content of 1.30 mg/g cell and a volumetric yield of 10.4 mg/L, which were 83 and 65% higher, respectively, than those of the control (0.71 mg/g cell and 6.3 mg/L). The intracellular catalase (CAT) activity was also increased after H₂O₂ treatment. The increases in CAT and astaxanthin of cells could be detected within 4 h of H₂O₂ treatment. The increase in the astaxanthin content of cells was concomitant with a notable decrease in the β-carotene content. The older yeast cells at late culture stage (120 h), due perhaps in part to their higher astaxanthin contents, were more tolerant to H₂O₂ toxicity than the younger cells (24 h). No enhancement of the astaxanthin biosynthesis was attained when H₂O₂ was added to the yeast culture together with a sufficient amount of exogenous CAT. The results suggest that astaxanthin biosynthesis in X. dendrorhous can be stimulated by H₂O₂ as an antioxidative response.     

Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato

The effects of salicylic acid (SA) and hydrogen peroxide (H₂O₂) on freezing tolerance were studied in two potato (Solanum tuberosum) cultivars (Alpha and Atlantic) that differ in cold sensitivity, Alpha being more tolerant to freezing than Atlantic. Lowest freezing survival rates were observed in 4-week-old plants. Freezing treatments consisting of exposure to 6° C for 4 h in the dark were applied 24 h after plants had been transferred from in vitro culture to soil. Catalase activity and H₂O₂ were estimated at the following harvest points: stage (a) 4-week-old in vitro plants treated with either 0.1 mM SA or 5 mM H₂O₂; stage (b) as in (a) but 24 h following transfer to soil prior to freezing treatment; stage (c) as in (b) but measured 15 days after a 4-h freezing treatment. The results show that (1) SA induced freezing tolerance in both cultivars; (2) SA inhibited ascorbate peroxidase activities in both cultivars at all harvest points but inhibited catalase activities in only at stage (a); (3) SA induced H₂O₂ accumulation only in Atlantic at stage (a); (4) H₂O₂ enhanced shoot catalase activities in Atlantic at stages (a) and (b) whereas this treatment had no effect on shoot catalase activities in Alpha; (5) H₂O₂ treatment induced freezing tolerance in Atlantic, even though shoot catalase activities were lower than those of the controls following exposure to freezing temperatures. We conclude that SA does not always lead to H₂O₂ accumulation even though catalase and ascorbate peroxidase activities are decreased as a result of the treatment. Moreover, H₂O₂ accumulation is not always associated with the induction of freezing tolerance, for example at stage (a) where SA-induced tolerance in Alpha was not accompanied by H₂O₂ accumulation. H₂O₂ was able to induce freezing tolerance only in Atlantic, even though H₂O₂ accumulated in both cultivars following this treatment.     

Excess copper induces accumulation of hydrogen peroxide and increases lipid peroxidation and total activity of copper–zinc superoxide dismutase in roots of Elsholtzia haichowensis

The effects of excess copper (Cu) on the accumulation of hydrogen peroxide (H₂O₂) and antioxidant enzyme activities in roots of the Cu accumulator Elsholtzia haichowensis Sun were investigated. Copper at 100 and 300 μM significantly increased the concentrations of malondialdehyde and H₂O₂, and the activities of catalase (E.C. 1.11.1.6), ascorbate peroxidase (E.C. 1.11.1.11), guaiacol peroxidase (GPOD, E.C. 1.11.1.7) and superoxide dismutase (SOD, E.C. 1.15.1.1). Isoenzyme pattern and inhibitor studies showed that, among SOD isoforms, only copper–zinc superoxide dismutase (CuZn–SOD) increased. Excess Cu greatly increased the accumulation of superoxide anion (O₂ ^·−) and H₂O₂ in E. haichowensis roots. This study also provides the first cytochemical evidence of an accumulation of H₂O₂ in the root cell walls as a consequence of Cu treatments. Experiments with diphenyleneiodonium as an inhibitor of NADPH oxidase, 1,2-dihydroxybenzene-3,5-disulphonic acid as an O₂ ^·− scavenger, and N-N-diethyldithiocarbamate as an inhibitor of SOD showed that the source of H₂O₂ in the cell walls could partially be NADPH oxidase. The enzyme can use cytosolic NADPH to produce O₂ ^·−, which rapidly dismutates to H₂O₂ by SOD. Apoplastic GPOD and CuZn–SOD activities were induced in roots of E. haichowensis with 100 μM Cu suggesting that these two antioxidant enzymes may be responsible for H₂O₂ accumulation in the root apoplast.     

Light-enhanced dark respiration in leaves and mesophyll protoplasts of pea in relation to photorespiration,respiration and some metabolites content

The respiration rate of leaves and mesophyll protoplasts of pea (Pisum sativum L.), from plants which were previously kept in darkness for 24 h was doubled following a period of photosynthesis at ambient level of O₂ (21 %), whereas the low level of O₂ (1 % and 4 % for leaves and protoplasts, respectively) reduced this light-enhanced dark respiration (LEDR) to the rate as noted before the illumination. Similarly to respiration rate, the oxygen at used concentrations had no effect on the ATP/ADP ratio in the dark-treated leaves. However, the ATP/ADP ratio in leaves photosynthesizing at 21 % O₂ was higher (up to 40 %, dependence on CO₂ concentration in the range 40–1600 1 dm⁻³) than in those photosynthesizing at 1 % O₂ or darkened at air (21 % O₂). Also, at 1 % O₂ the accumulation of malate was suppressed (by about 40 %), to a value noted for leaves darkened at 21 % O₂. The dark-treatment of leaves reduced the ability of isolated mitochondria to oxidize glycine (by about twofold) and succinate, but not malate. Mitochondria from both the light- and dark-treated leaves did not differ in qualitative composition of free amino acids, however, there were significant quantitative differences especially with respect to aspartate, alanine, glutamate and major intermediates of the photorespiratory pathway (glycine, serine). Our results suggest that accumulation of photorespiratory and respiratory metabolites in pea leaves during photosynthesis at 1 % O₂ is reduced, hence the suppression of postillumination respiration rate.     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

毛白杨细胞质抗坏血酸过氧化物酶基因的克隆及表达分析

利用同源克隆技术得到1个毛白杨细胞质抗坏血酸过氧化物酶基因,命名为PcAPX。该基因编码249个氨基酸残基,预测分子量为33.01kD。采用原核表达技术在大肠杆菌中表达并纯化该蛋白并进行酶活性分析,结果表明:重组PcAPX蛋白对抗坏血酸(AsA)和过氧化氢(H2O2)有很高的活性,其对抗坏血酸的米氏常数(Km)和最大反应速度(Vmax)分别为(0.71±0.03)mmol·L-1和(0.41±0.02)mmol·L-1·min-1·mg-1;对过氧化氢的Km和Vmax分别为(0.60±0.21)mmol·L-1和(0.35±0.12)mmol·L-1·min-1·mg-1,表明PcAPX对AsA和H2O2拥有较高的催化底物的能力和催化效率。利用实时定量RT-PCR分析毛白杨PcAPX基因的表达模式,结果表明其在老叶中表达量高于新叶、韧皮部、形成层和根部。该研究结果将进一步促进毛白杨APX基因家族成员参与植物生长调控的研究。     

Heat shock-mediated H2O2 accumulation and protection against Cd toxicity in rice seedlings

Rice (Oryza sativa L.) seedlings stressed with CdCl₂ (0.5 mM or 50 μM) showed typical Cd toxicity (leaf chlorosis, decrease in chlorophyll content, or increase in H₂O₂ and malondialdehyde contents). Rice seedlings pretreated with heat shock at 45°C (HS) for 2 or 3 h were protected against subsequent Cd stress. Rice seedlings pretreated with HS had similar Cd concentration in leaves caused by CdCl₂ as those non-HS. The content of H₂O₂ increased in leaves 1 h after HS exposure. However, APX and GR activities were higher in HS-treated leaves than their respective control, and it occurred after 2 h of HS treatment. Pretreatment of rice seedlings with H₂O₂ under non-HS conditions resulted in an increase in APX, GR, and CAT activities and protected rice seedlings from subsequent Cd stress. HS-induced H₂O₂ production and protection against subsequent Cd stress can be counteracted by imidazole, an inhibitor of NADPH oxidase complex. Results of the present study suggest that early accumulation of H₂O₂ during HS signals the increase in APX and GR activities, which in turn prevents rice seedlings from Cd-caused oxidative damage.