Proteome changes in leaves of Brassica napus L. as a result of Sclerotinia sclerotiorum challenge

Sclerotinia stem rot, caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, is a serious disease of canola (Brassica napus L.). To increase the understanding of the B. napus- S. sclerotiorum interaction, proteins potentially involved in mediating this interaction were identified and characterized. Upon infection of canola leaves by S. sclerotiorum, necrosis of host leaves was observed by 12 h and rapidly progressed during the later time points. These morphological observations were supported by microscopic study performed at different time points after pathogen challenge. Leaf proteins were extracted and analyzed by 2-DE, which revealed the modulation of 32 proteins (12 down- and 20 up-regulated). The identities of these proteins were established by ESI-q-TOF MS/MS and included proteins involved in photosynthesis and metabolic pathways, protein folding and modifications, hormone signaling, and antioxidant defense. Gene expression analysis of selected genes was performed by qRT-PCR, whereas the elevated levels of the antioxidant enzymes peroxidase and superoxide dismutase were validated by enzyme assays. To the authors' best knowledge, this is the first proteomics-based investigation of B. napus-S. sclerotiorum interaction, and the roles of many of the proteins identified are discussed within the context of this pathosystem.     

Seasonal variation of defense-related gene expression in leaves from Bois noir affected and recovered grapevines

Although Bois noir is one of the main phytoplasma diseases of grapevine, the gene expression and enzyme activities that underlie physiological changes occurring in symptomatic and recovered (with spontaneous or induced symptom remission) plants are mostly unknown. Bois noir symptomatic leaves (September 2006, 2007) and symptomless leaves from infected symptomatic plants (September 2007) of Sangiovese (moderately susceptible) and Chardonnay (highly susceptible) cultivars were collected. Moreover, leaves from infected symptomless plants of both cultivars were harvested in June 2007. Leaves from recovered plants were also collected in the same periods. In recovered plants of both cultivars, class III chitinase and almost every time phenylalanine ammonia-lyase and chalcone synthase expression were increased for all collection periods. In symptomatic leaves of both cultivars, the expressions of the same genes were up-regulated and also those of β-1,3-glucanase and flavanone 3-hydroxylase. The activities of chitinase, phenylalanine ammonia-lyase, β-1,3-glucanase, and superoxide dismutase generally correlated with gene expression. For the moderately susceptible Sangiovese, the defense genes were generally up-regulated in both symptomatic and symptomless leaves (for all collection periods). This behavior was not observed in the highly susceptible Chardonnay, in which changes in gene expression were linked to evident symptom display. Therefore, the physiological response of the plants to this pathogen infection appear to be the reason for the resistance of the cultivar to the disease.     

Responses of Components of Antioxidant System in Moongbean Genotypes to Cadmium Stress

Four genotypes (Pusa 9531, Pusa 9072, Pusa Vishal, PS‐16) of moongbean [Vigna radiata (L.) Wilczek] grown in earthen pots were treated with cadmium at 0, 25, 50, and 100 mg kg^?1 soil. Cadmium tolerance (CdT), the ability of a plant to maintain growth at high levels of cadmium (Cd), was calculated as the ratio of dry‐matter production in the untreated and the Cd‐treated soils. The moongbean genotypes showed a differential response to Cd concentrations; Pusa 9531 was identified as Cd tolerant, whereas PS 16 was Cd susceptible. To find out the physiological basis of these differences, we investigated the possible role of antioxidant (enzymatic and nonenzymatic) defense systems. Activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), and glutathione reductase (EC 1.6.4.2) and the amounts of ascorbate and glutathione were monitored in the Cd‐tolerant and Cd‐sensitive moongbean genotypes. The results revealed the presence of a strong antioxidant defense system in the Cd‐tolerant genotype (Pusa 9531) for providing adequate protection against oxidative stress caused by Cd.     

Variation in resistance to the cabbage aphid ( Brevicoryne brassicae) between and within wild and cultivated Brassica species

Seven Brassica species were evaluated for their resistance to the cabbage aphid, Brevicoryne brassicae, in a series of field experiments. Four wild Brassica species, two 8 chromosome species with similarities to the B genome of Brassica nigra (Brassica fruticulosa and Brassica spinescens) and two 9 chromosome species containing the C genome (Brassica incana and Brassica villosa) were identified as possessing consistently high levels of antibiosis mediated resistance to B. brassicae. None of the species were shown to possess consistently high levels of antixenosis resistance. In more detailed glasshouse experiments one B-like genome species, B. fruticulosa, showed considerable variation between accessions collected from different sites for resistance to B. brassicae. In addition, individual accessions of one A genome species (Brassica rapa) and one C genome species (Brassica alboglabra) were shown to be highly variable in their resistance to B. brassicae, some plants of each accession being highly resistant and others very susceptible. The implications of the variability in resistance to B. brassicae within wild Brassica species for exploitation in Brassica breeding programmes are discussed.     

植物防御酶与桉树对焦枯病抗性的关系

为筛选鉴定桉树对焦枯病(CylindrocladiumqulnqueSeptatumMorgan)抗性的生化指标,对福建省11个桉树主栽种系接种焦枯病菌前后关键防御酶系中的过氧化物酶(POD)和超氧岐化酶(SOD)活性及其同工酶变化进行了研究.结果表明:无论是桉树健叶还是接种后的病叶,体内POD、SOD活性均表现为抗病种系>中抗种系>中感种系>感病种系,呈规律性变化;接种前后POD同工酶谱带数与桉树对焦枯病的抗性间不呈规律性变化,而SOD谱带数与抗病性成正比.从抗病育种角度出发可将接种前后桉树POD、SOD活性和接种后SOD同工酶谱带作为桉树对焦枯病抗性早期鉴定的生化指标.     

铵硝比例对不同基因型小白菜硝酸盐积累影响机理研究

本研究以前期工作中筛选出的硝酸盐积累量存在显著差异的2个不同基因型小白菜品种为材料,在人工气候箱水培条件下研究了不同铵硝比例对小白菜硝酸盐积累量、硝酸还原酶活性（NRA）和硝酸盐吸收基因NRT1和NRT2的表达量的影响。结果表明,不同铵硝比例对小白菜硝酸盐积累量有显著影响,且存在基因型差异。四月慢对硝酸盐吸收、积累及同化利用的能力都强于华冠青梗菜,尤其是在高NO3- 比例处理时。与华冠青梗菜相比,四月慢对NO3- 的同化利用的能力更不易受铵硝比例的影响。NRT1和NRT2主要在根部表达,且NRT1的表达量显著高于NRT2,NRT1和NRT2的表达量变化规律只能在一定程度上解释小白菜不同基因型间硝酸盐积累量的差异,小白菜不同基因型品种间硝酸盐积累量差异的机理还需要进一步研究。     

山东石榴品种对枝条干腐病的抗性评价及其病原菌鉴定

为明确山东省主要石榴品种对枝条干腐病的抗性及引起枝条干腐病的病原菌种类,本研究对山东省枣庄市的20个主要石榴品种进行田间抗性评价。通过组织分离法对病原菌进行分离,利用柯赫氏法则验证其致病性,并通过显微形态特征观察及分子生物学分析开展病原菌鉴定。结合形态学、致病性测定和分子生物学分析结果,将石榴枝条干腐病病原菌鉴定为单间座壳菌(Diaporthe eres)。抗性评价结果显示,白皮酸、碧榴表现为近免疫;大青皮、秋艳、岗榴1号、大马牙、峄城三白、冰糖籽、黑美人表现为抗病;枣庄玛瑙、墨石榴、九洲红、青皮软籽、黄金榴、枣庄软仁表现为中抗;冠榴、谢花甜、红皮马牙表现为感病;大红袍和泰山红表现为高感。筛选得到的近免疫、抗病品种可作为石榴抗性育种和种质资源改良提供研究材料。本研究结果为山东石榴枝条干腐病的抗病机理研究和系统性防控提供了参考。     

Physiological and biochemical effects of salicylic acid on Pisum sativum exposed to isoproturon

In the present study, attenuation of isoproturon (IPU) toxicity by salicylic acid (SA) was observed. Seven-day-old seedlings of pea (Pisum sativum L. cv. Azad P-1) were treated with 10 mM IPU. IPU influenced physiological and biochemical parameters. IPU significantly inhibited growth variables like shoot and root height, fresh and dry biomass of the pea. The contents of carotenoids, chlorophylls, protein and activity of nitrate reductase were inhibited significantly. IPU enhanced the accumulation of H₂O₂, ion leakage and lipid peroxidation due to induction of oxidative stress in pea. The activities of antioxidant enzymes, namely superoxide dismutase, catalase and ascorbate peroxidase increased while the activities of guaiacol peroxidase decreased. However, exogenous SA regulated the toxic effects of IPU. The indices of oxidative stress appeared to be alleviated by SA. Pigment content and activities of enzymes increased approximately up to the level of control. IPU caused non-target phytotoxicity to P. sativum. The natural growth regulator/allelochemical has potential to overcome the adverse effects caused by IPU.

Abbreviations: CAT: catalase; EL: electrolyte leakage; IPU: isoproturon; LP: lipid peroxidation; MDA: malondialdehyde; NR: nitrate reductase; POD: guaiacol peroxidase; SOD: superoxide dismutase; TCA: trichloroacetic acid     

Phytotransformation of Phorate by <Emphasis Type="BoldItalic">Brassica juncea</Emphasis> (Indian Mustard)

Over 5 days, Brassica juncea removed 54% of the highly toxic insecticide phorate from the medium with the formation of phorate sulfoxide in small quantity. The loss of phorate from the medium followed first-order kinetics. The half-life of phorate disappearance from water decreased by ~4.5-fold in the presence of B. juncea. Mild phorate phytotoxicity was evident from the elevated activities of the antioxidative enzymes like glutathione-disulfide reductase, glutathione S-transferase, superoxide dismutase, and catalase in the plants. Nevertheless, the ubiquitous antioxidative peroxidase was not significantly increased, nor the total glutathione content, due to phorate exposure. Phosphotriester bond hydrolysis and glutathione S-transferase-mediated conjugation seemed to be the key reactions for phorate metabolism by B. juncea. From the limited information available, for the first time, a tentative mapping of phytotransformation pathways was performed.     

Calcium uptake and resistance to bacterial wilt of mutually grafted tomato seedlings

Bacterial wilt of tomato (Lycopersicon esculentum Mill.) caused by Ralstonia solanacearum Smith is a serious disease in Japan. We previously reported that calcium (Ca) nutrition in tomato significantly affected the resistance to the disease, and that highly resistant cultivars were characterized by a high Ca uptake. We examined the relationship between the Ca uptake and resistance using mutually grafted seedlings of tomato cultivars differing in their resistance. A susceptible (‘Ponderosa’) or moderately resistant (‘Zuiei’) cultivar (scion) was grafted on the rootstock of a susceptible, moderately resistant, or highly resistant cultivar (‘Hawaii 7998’). Roots or petioles of the grafted seedlings were inoculated with the pathogen, and the development of bacterial wilt was observed. Although Ca uptake by shoot increased by grafting on the rootstock of a highly resistant cultivar, the development of the disease was not influenced by the difference in Ca uptake, and depended on the resistance of the cultivar to which the inoculated part of the graft belonged. It was concluded that the differences in Ca uptake of the shoot of the grafted tomato seedlings might not be related to the expression of the resistance to bacterial wilt.     

Sensitivity of soft red winter wheat cultivars to chlorate‐induced toxicity

The use of chlorate as a nitrate analogue to screen soft red winter wheat (Triticum aestivum L.) cultivars for differences in nitrate reductase activity (NRA) was studied by adding potassium chlorate to a hydroponic nutrient solution in which wheat seedlings were growing. After 14 days, leaf symptoms indicating chlorate‐induced toxicity were rated. It was hypothesized that wheat plants which were susceptible to chlorate‐induced toxicity reduced chlorate and nitrate more rapidly than did resistant plants. In experiments testing the potential of this assay, wheat and barley (Hordeum vulgare L.) cultivars previously reported to have low NRA were less susceptible to chlorate‐induced toxicity than were cultivars reported to have high NRA. The assay was used to screen 15 soft red winter wheat cultivars for differences in sensitivity to chlorate‐induced toxicity. Variable toxic reactions were observed both among and within the cultivars. To determine whether the within‐cultivar variation was environmental or genetic, single plant selections for contrasting chlorate response were made, and bulked progeny were rescreened. In eight of 15 cultivars, the contrasting selections were different for chlorate‐induced toxic response, indicating heterogeneity for this trait within these eight cultivars. These chlorate‐selected lines may also be near‐isogenic lines for NRA. Seedling screening of wheat for chlorate response may be useful for identification of high NRA breeding lines.     

Drought stress-induced oxidative stress and antioxidative responses in four wheat (Triticum aestivum L.) varieties

Drought stress was imposed on four varieties of wheat (Triticum aestivum L.), Mohan Wonder (MW), Kedar (K), Gayetri (GY) and Gandhari (GN), for 3, 6 and 9 days. The activities of all five tested antioxidative enzymes, peroxidase, ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase, were enhanced initially in varieties K and GN, whereas in MW and GY, catalase and superoxide dismutase showed a decrease in activity at all periods of drought stress. Peroxidase and glutathione reductase activities increased even on the ninth day of stress in K and GN, but all other activities showed a decrease after 3 days of stress. H₂O₂ accumulation increased with drought stress, but in K and GN there was decrease during prolonged drought stress. Lipid peroxidation increased significantly due to drought stress, which was higher in the case of MW and GY. Proline, phenol and ascorbate content increased with period of drought stress. Carotenoid accumulation also increased initially. Total chlorophylls showed a general decrease during drought stress. The results of this study indicate that two of the varieties, MW and GY, are susceptible to drought stress, whereas the other two, K and GN, are tolerant, with peroxidase and glutathione reductase being most important in conferring tolerance.     

磷对薄皮甜瓜生理代谢和产量形成影响的研究

为探究磷素对甜瓜生理代谢和产量形成的影响,以金妃甜瓜为材料,采用基质培养方法,研究不同浓度的磷素处理后,甜瓜果实横径、纵径、叶绿素含量、硝态氮含量、硝酸还原酶活性、可溶性糖含量、过氧化氢酶活性、超氧化物歧化酶活性、单果重和产量的变化规律。结果表明,在结果期,0.9~1.2mmol·L~(-1)范围内的磷用量对甜瓜果实生长促进作用最强,叶片中生理活性物质含量和保护性酶活性提高,进而提高甜瓜光合能力和生理代谢水平,增加果实横径和纵径,提高甜瓜产量。在授粉后30 d,0.9~1.2 mmol·L~(-1)范围内的磷用量显著提高了果实横径、果实纵径和产量,与0.3 mmol·L~(-1)(T1)磷用量相比,分别增加21.38%~26.00%、12.62%~34.09%和36.62%~46.72%;同时,在授粉后20~25 d,与T1相比,该范围内的磷用量显著提高了叶片叶绿素含量、硝态氮含量、可溶性糖含量以及硝酸还原酶、过氧化氢酶、超氧化物歧化酶活性,分别增加21.23%~29.99%、102.55%~128.59%、51.10%~56.51、24.91%~39.92%、7.46%~9.26%和13.23%~18.62%。而当磷用量低于0.6 mmol·L~(-1)(T2)或高于1.5 mmol·L~(-1)(T5)时,各项指标均显著下降。因此,0.9~1.2 mmol·L~(-1)范围内的磷素量施用可以提高甜瓜植株生理活性物质含量和保护性酶活性,促进甜瓜产量形成,但甜瓜植株的生理特性和产量指标在不同磷浓度下有显著差异。本研究结果为甜瓜生产合理施用磷肥提供了理论依据和实践指导。     

Water stress and accumulation of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid in grass pea (Lathyrus sativus)

Grass pea seedlings were grown in an irrigated field. Roots of 15-day-old seedlings were treated with PEG, and leaves were studied. With the duration of PEG treatment, changes in the lipid peroxidation and activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase as well as contents of hydrogen peroxide and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP) were assayed. The results indicate that with the duration of PEG treatment, activities of superoxide dismutase, peroxidase, and catalase decreased, whereas contents of hydrogen peroxide and ODAP, extent of lipid peroxidation, and activity of glutathione reductase increased. Both diethyldithiocarbamate and aminotriazole strongly inhibit activities of superoxide dismutase and catalase, respectively. At same time, the extent of lipid peroxidation was obviously increased. However, mannitol decreased the extent of lipid peroxidation. Diethyldithiocarbamate, aminotriazole, and mannitol do not affect the accumulation of ODAP. The observations suggest that there is no direct relationship between the accumulation of ODAP and the metabolism of free radicals. In addition, the relationship between water stress and ODAP accumulation in grass pea is discussed.     

An Oxidative Mechanism for the Inhibition of Iodothyronine 5′-Monodeiodinase Activity by Lead Nitrate in the Fish,Heteropneustes fossilis

An oxidative mechanism in the lead-induced inhibition of thyroid function with special reference to iodothyronine 5′- monodeiodinase (5′-ID) activity has been identified in the fish, Heteropneustes fossilis. Lead treatment (2.5, 5.0 and 10.0 ppm of lead nitrate/day for 30 days) enhanced tissue lipid peroxidation (LPO) and decreased the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). In addition, serum triiodothyronine (T3) concentration and hepatic 5'-ID activity were markedly decreased by 10 ppm of lead nitrate. These findings support the view that higher concentrations of lead nitrate inhibit the extrathyroidal conversion of thyroxine (T4) to T3. Moreover, oxidative stress from lead intoxication could be responsible for inhibiting 5′-ID activity.     

锰硼营养对黄瓜产量及抗氧化酶活性的影响

采用田间试验、植株生理生化测定方法,研究了Mn、B营养对黄瓜产量与抗氧化酶活性的影响。结果表明,Mn、B营养具有明显的互作效应,适宜的Mn、B配施可提高黄瓜产量和叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(cAT)、硝酸还原酶(NR)活性以及叶绿素、抗坏血酸(ASA)含量,降低丙二醛(MDA)含量。黄瓜生产能力与超氧化物歧化酶、过氧化物酶、过氧化氢酶、硝酸还原酶活性显著正相关,与丙二醛含量呈显著负相关。     

Nitrate Reductase-Dependent Nitric Oxide Production Is Involved in Microcystin-LR-Induced Oxidative Stress in Brassica rapa

Histochemical and biochemical approaches were used to investigate the phytotoxicity induced by microcystin-LR (MC-LR) in the shoots of Brassica rapa seedlings. MC-LR exposure was able to induce oxidative stress by triggering the over-generation of reactive oxygen species (ROS) including superoxide anion radical (O₂ ^{?? ?} ) and hydrogen peroxide (H₂O₂) in the shoots of B. rapa. MC-LR exposure led to the significant increase in the concentration of endogenous nitric oxide (NO) in B. rapa. However, such increase was completely suppressed by the treatment with nitrate reductase (NR) inhibitor NaN₃, while l-NMMA, a NO synthase (NOS) inhibitor, had only slight effect on the content of endogenous NO in MC-LR-treated plant. These data suggested that NR-dependent pathway was the main source for endogenous NO generation under MC-LR stress. Afterwards, treatment with NaN₃ reduced the ROS generation, lipid peroxidation, and loss of membrane integrity in MC-LR-treated plant. MC-LR stress induced the increase in the expression of superoxide dismutase, ascorbate peroxidase, and catalase. However, such an effect could be reversed by the treatment with NaN₃. These results indicate that NR-dependent NO production mediates MC-LR-induced oxidative stress by triggering the over-generation of ROS in B. rapa.     

Do reactive oxygen species (ROS) induced by NaCl contribute to ammonium accumulation in Spartina alterniflora?

Growth, activity of antioxidant enzymes viz. glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX), and some metabolic processes related to ammonium metabolism were investigated in a salt‐tolerant Spatina alterniflora. In comparison to 0 mM–NaCl treatment, growth of S. alterniflora plant increased significantly at 200 mM NaCl, but was highly inhibited at 500 mM NaCl. Ammonium concentration in the leaves and roots increased 2.1–3.4 times when plants were treated with 500 mM NaCl. Under 200 mM NaCl, antioxidant‐enzyme activities increased, however, at 500 mM the antioxidant system was unable to compensate reactive oxygen species induced by NaCl. At this high level of salinity, ammonium production through nitrate reductase (NR) was inhibited, but no significant changes in the activities of glutamine synthetase (GS) or glutamate dehydrogenase (GDH) were found. We conclude that the accumulation of ammonium under high salt stress was not due to inhibition of the assimilatory activities of GS or GDH. Ammonia accumulation under high salinity may result from amino acid and protein catabolism activated by reactive oxygen species (ROS) and/or a lack of carbon skeletons to incorporate ammonium into organic molecules due to a decrease in photosynthetic activity in salt‐stressed plants.     

Effect of different fertilizers on nitrogen isotope composition and nitrate content of Brassica campestris

The effect of different fertilizers on the δ(15)N value, nitrate concentration, and nitrate reductase activity of Brassica campestris and the δ(15)N value of soil has been investigated through a pot experiment. The δ(15)N mean value of B. campestris at the seedling stage observed in the composted chicken treatment (+8.65‰) was higher than that of chemical fertilizer treatment (+5.73‰), compost-chemical fertilizer (+7.53‰), and control check treatment (+7.86‰). There were significantly different δ(15)N values (p < 0.05) between B. campestris cultivated with composted chicken manure treatment and with chemical fertilizer treatment. The similar results were also found at the middle stage and the terminal stage. The variation of δ(15)N value in soil for different treatments was smaller than that of B. campestris, which was +6.71-+8.12‰, +6.83-+8.24‰, and +6.85-8.4‰, respectively, at seedling stage, middle stage, and terminal stage. With the growth of B. campestris, the nitrate content decreased in all treatments, and the nitrate reductase activity in B. campestris increased except for the CK. Results suggested that the δ(15)N values of B. campestris and soil were more effected by the fertilizer than by the dose level, and the δ(15)N value analysis could be used as a tool to discriminate the B. campestris cultivated with composted manure or chemical fertilizer.