Induction of defense-related enzymes in soybean leaves by class IId bacteriocins (thuricin 17 and bacthuricin F4) purified from Bacillus strains

We have recently discovered a new class of bacteriocin (class IId) which stimulates plant growth in a way similar to Nod factors. Nod factors have been shown to provoke aspects of plant disease resistance. We investigated the effects of bacteriocins [thuricin 17 (T17) and bacthuricin F4 (BF4)] on the activities of phenylalanine ammonia lyase (PAL), guaiacol peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and polyphenol oxidase (PPO). Bacteriocin solutions were fed into the cut stems of soybean (Glycine max L. Merr. cv. OAC Bayfield) seedlings at the first trifoliate stage. PAL activity in T17 treated leaves was the highest at 72 h after treatment and was 75.5% greater than the control at that time. At 72 h after treatment POD activities in T17 and BF4 treated leaves increased by 72.7 and 91.3%, respectively, as compared with the control treatment. APX activity was 52.3 and 49.6% respectively, greater than the control in T17 and BF4 treated leaves at 72 h after treatment. SOD activity in T17 treated leaves was the highest at 72 h after treatment and was 26.0% greater than the control at that time. SOD activity was 70.5 and 60.2% greater, respectively, than the control in T17 and BF4 treated leaves, at 72 h. Using PAGE we found that one APX isozyme (28 kDa isoform) showed the strongest induction in all bacteriocin treated leaves at 72 h. Activity of the seven SOD isozymes was increased by both bacteriocins, relative to the control treatment. The 33 kDa PPO isozyme was induced strongly by both bacteriocins, relative to the control treatment. These results indicate that class IId bacteriocins can act as an inducer of plant disease defense-related enzymes and may be acting through mechanisms similar to Nod factors.     

Effect of chitin hexamer and thuricin 17 on lignification-related and antioxidative enzymes in Soybean Plants

Inducers of disease resistance in crop plants have a role in sustainable agriculture. We describe a set of bacteriocins that can potentially improve plant growth by controlling specific pathogens and inducing generalized resistance. Solutions of the bacteriocin thuricin 17 and/or a chitin hexamer (a known inducer and positive control) were applied to leaves of two-week-old soybean plants, and levels of lignification-related and antioxidative enzymes were monitored. Phenyl ammonia lyase (PAL) activity in thuricin 17-treated leaves was highest at 60 h after treatment, being 61.8% greater than the control. PAL activity also was increased 18.1% at 72 h after treatment with the chitin hexamer. Tyrosine ammonia lyase (TAL) activity in leaves was 57.0% higher than the control at 48 h after treatment with thuricin 17, while such activity in chitin hexamer-treated leaves was increased by 23.8% at 72 h. At 36 h after treatment with the chitin hexamer or chitin hexamer + thuricin 17, the total concentration of phenolic compounds was 15.3 or 19.3%, respectively, greater than the control. At 72 h, total phenolic concentrations increased by 23.2 and 19%, respectively, in response to thuricin 17 and chitin hexamer + thuricin 17. POD activity in thuricin 17-treated leaves increased by 74.6 and 81.2% at 48 and 72 h, respectively, whereas SOD activity increased by 24.9 and 79.9%, respectively, in chitin hexamer- and thuricin 17-treated leaves at 48 h. A peroxidase isozyme (31 kDa isomer) was induced in thuricin 17-treated leaves at 60 h, while catalase (59 kDa isomer) was induced in chitin hexamer-treated leaves. PAGE showed that two major SOD bands (Fe-SODs) were produced by both types of treatment. Collectively, these results indicate that the bacteriocin thuricin 17 can act as an inducer of plant disease defenses (i.e., activated lignification-related enzymes, antioxidative enzymes, and related isozymes) and that this induction is similar, but not identical, to that of the chitin hexamer elicitor. Although treatment with thuricin 17 + chitin hexamer also induced those responses, it did not present a clear pattern of additivity or synergy.     

The class IId bacteriocin thuricin-17 increases plant growth

The mechanisms by which many plant growth promoting rhizobacteria (PGPR) affect plants are unknown. We recently isolated a rhizosphere bacterium (Bacillus thuringiensis NEB17), that promotes soybean growth and screened the liquid growth medium in which it grew for plant growth stimulating materials. We have also shown that it produces a bacteriocin (named by us as thuricin-17 and a member of the recently described class IId bacteriocins). Here we show that application of this bacteriocin to leaves (spray) or roots (drench) directly stimulates the growth of both a C₃ dicot (soybean) and a C₄ monocot (corn). This growth stimulation is similar in nature to that previously seen when plants are treated with Nod factors. Strain NEB17 contains three copies of the gene for thuricin 17 that code for identical amino acid sequences. These two lines of evidence suggest that the dual functions of these proteins may have constrained their evolution. This is the first report of direct plant growth enhancement by a bacteriocin.     

Antioxidative responses and proline level in leaves and roots of pea plants subjected to nickel stress

The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST) as well as proline content were studied in leaves and roots of 14 day-old pea plants treated with NiSO₄ (10, 100, 200 μm) for 1, 3, 6 and 9 days. Exposure of pea plants to nickel (Ni) resulted in the decrease in CuZnSOD as well as total SOD activities in both leaves and roots. The activity of APX in leaves of plants treated with 100 and 200 μm Ni increased following the 3rd day after metal application, while in roots at the end of the experiment the activity of this enzyme was significantly reduced. In both organs CAT activity generally did not change in response to Ni treatment. The activity of GST in plants exposed to high concentrations of Ni increased, more markedly in roots. In both leaves and roots after Ni application accumulation of free proline was observed, but in the case of leaves concentration of this amino acid increased earlier and to a greater extent than in roots. The results indicate that stimulation of GST activity and accumulation of proline in the tissues rather than antioxidative enzymes are involved in response of pea plants to Ni stress.     

Sea fennel (<Emphasis Type="Italic">Crithmum maritimum</Emphasis> L.) under salinity conditions: a comparison of leaf and root antioxidant responses

The present study was carried out to compare the effect of NaCl on growth, cell membrane damage, and antioxidant defences in the halophyte Crithmum maritimum L. (sea fennel). Physiological and biochemical changes were investigated under control (0 mM NaCl) and saline conditions (100 and 300 mM NaCl). Biomass and growth of roots were more sensitive to NaCl than leaves. Roots were distinguished from leaves by increased electrolyte leakage and high malondialdehyde (MDA) concentration. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities, ascorbic acid (AA) and glutathione (GSH) concentrations were lower in the roots than in the leaves of control plants. The different activity patterns of antioxidant enzymes in response to 100 and 300 mM NaCl indicated that leaves and roots reacted differently to salt stress. Leaf CAT, APX and glutathione reductase (GR) activities were lowest at 300 mM NaCl, but they were unaffected by 100 mM NaCl. Only SOD activity was reduced in the latter treatment. Root SOD activity was significantly decreased in response to 300 mM NaCl and root APX activity was significantly higher in plants treated with 100 and 300 mM compared to the controls. The other activities in roots were insensitive to salt. The concentration of AA decreased in leaves at 100 and 300 mM NaCl, and in roots at 300 mM NaCl, when compared to control plants. The concentrations of GSH in NaCl-treated leaves and roots were not significantly different from the controls. In both organs, AA and GSH were predominating in the total pool in ascorbic acid and glutathione, under control or saline conditions.     

野牛草克隆分株酶促活性氧清除系统对差异光周期的响应

以盆栽野牛草克隆分株为材料,将克隆分株分别标记为O(姊株)和Y(妹株),设置连接组和断开组两种处理,其中,连接组中O分株和Y分株通过节间子相连,断开组则剪断分株节间子;两组处理的O分株光周期均设置为光照12h/黑暗12h,Y分株光周期均设置为黑暗12h/12h光照(恰好与O分株相反),经过7d的差异光周期处理后进行72h全光照稳定培养,并于全光照条件下在48h内连续测定各分株叶片超氧化物歧化酶(SOD),过氧化物酶(POD),过氧化氢酶(CAT),抗坏血酸过氧化物酶(APX)的活性以及丙二醛(MDA)的含量,探讨野牛草叶片酶促活性氧清除系统对差异光周期的响应特征。结果表明,差异光周期处理1周后,全光照条件下,断开状态的野牛草克隆分株O和Y间叶片中SOD、POD、CAT、APX活性以及MDA含量在24h内基本呈现相反的变化趋势,而野牛草相连克隆分株O和Y间叶片中以上指标在24h内呈现趋于一致的变化规律。研究发现,野牛草酶促活性氧清除系统活性在一天内呈现节律性表达模式,且差异光周期处理下的野牛草相连克隆分株的活性氧清除系统的活性的节律性变化趋于同步。     

Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.     

Changes in Activities of Antioxidant Enzymes and Their Relationship to Genetic and Paclobutrazol-Induced Chilling Tolerance of Maize Seedlings

The potential role of antioxidant enzymes in protecting maize (Zea mays L.) seedlings from chilling injury was examined by analyzing enzyme activities and isozyme profiles of chilling-susceptible (CO 316) and chilling-tolerant (CO 328) inbreds. Leaf superoxide dismutase (SOD) activity in CO 316 was nearly one-half that of CO 328, in which the high activity was maintained during the chilling and postchilling periods. Activity of glutathione reductase (GR) was much higher in roots than in leaves. CO 328 also possessed a new GR isozyme that was absent in roots of CO 316. Ascorbate peroxidase (APX) activity was considerably lower in leaves of CO 328 than in CO 316, and nearly similar in roots. Paclobutrazol treatment of CO 316 induced several changes in the antioxidant enzyme profiles and enhanced their activities, especially those of SOD and APX, along with the induction of chilling tolerance. These results suggest that increased activities of SOD in leaves and GR in roots of CO 328, as well as SOD and APX in leaves and roots of paclobutrazol-treated CO 316, contribute to their enhanced chilling tolerance.     

Alternaria cassiae Alters Phenylpropanoid Metabolism in Sicklepod (Cassia obtusifolia)

Phenylpropaniod metabolism has been implicated in plant defence mechanism(s) against pathogen attack. In this study, phenylpropanoid metabolism was examined over a 72 h time course in the weed sicklepod (Cassia obtusifolia) in relation to pathogenic effects of the fungus Alternaria cassiae. When 3- to 4-week old seedlings were challenged by the pathogen, extrable phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) activity was dramatically increased above that in uninfected plants severalhours after inoculation and exposure to dew. Greatest increases of enzyme activity (3-fold, specific activity basis) occurred at ca 15–23 after treatment with fungal spores. After this peak of activity, PAL activity declined with time in infectedtissue, but remained greater than in uninfected plants through 65 h after treatment. Total methanol-soluble hydroxyphenolic compound levels (PAL products) were higher in shoots (stems and leaves) of infected plants at 48–72 h. Leaves contained a higherconcentration (per gram fresh weight) of hydroxyphenolic compounds than did stems, and infected leaves exhibited a phenolic content greater than that of uninfected leaves at ca 27–72 h. Increased soluble phenolic compound production correlated with the appearance of lesions and necrotic spots on leaves and stems. UV irradiation examination and spectrofluorometric analysis of thin layer chromatographic separations of methanolic exatracts revealed a substantial increase of several components ininfected tissue 48 h after inoculation. Results support the view that PAL activity increases correlate with increased phenolic compound production in this host/pathogen interaction.     

Defense response of resistant and susceptible genotypes of castor (Ricinus communis L.) to wilt disease

The biochemical basis of resistance in castor (Ricinus communis L.) to Fusarium wilt, caused by the pathogen Fusarium oxysporum f. sp. ricini, was investigated. Induction of plant defence against pathogen attack is regulated by a complex network of different signals. Thus changes in various biochemical defenses including antioxidant enzymes, phenolic compounds and pathogenesis related (PR) proteins were investigated in the roots of resistant and susceptible genotypes of castor at 0, 24, 48 and 72 h.a.i. Infection by F. oxysporum significantly increased the superoxide dismutase (SOD) and peroxidase (POX) activities in the roots of susceptible genotypes, while the catalase (CAT) activities were appreciably higher in the roots of resistant genotypes at different stages. Constitutive levels of ascorbate peroxidase (APX) and polyphenol oxidase (PPO) were higher in the resistant genotypes. Also, the activities of phenylalanine ammonia lyase (PAL) and β 1, 3 glucanase significantly increased in the roots of the resistant genotypes after infections. The rate of increment of thiobarbituric acid reactive substances (TBARS) was higher in resistant genotypes after infection. Analysis of isozyme banding pattern of SOD, POX, PPO and esterase on native PAGE electrophoresis revealed that interaction between plant and fungi invoked various isozymes at 48 h of infection. SOD 3 was observed only in resistant genotypes at 24 h.a.i. except Geeta. Similarly induction of POX 5 was observed only in resistant genotypes at 48 h of infection, though the intensity of POX 5 was very less.     

外源茉莉酸及枸杞瘿螨危害诱导的枸杞防御反应

利用植物的诱导抗性防治病虫害越来越受重视, 为寻求枸杞害虫防治的新方法, 探索外源茉莉酸对枸杞的诱导反应及机制, 本研究测定了外源茉莉酸(jasmonic acid, JA)及枸杞瘿螨Aceria pallida Keifer危害对枸杞叶蛋白酶抑制剂(protease inhibitors, PIs)、 超氧化物歧化酶（superoxide dismutase, SOD）、 苯丙氨酸解氨酶（phenylalanine ammonia-lyase, PAL）活性及多糖（polysaccharide）、 木质素（xylogen）含量的变化。结果表明： 外源JA处理或枸杞瘿螨危害均使枸杞叶PIs, SOD和PAL活性及木质素含量显著提高, 却使多糖含量显著降低。胰凝乳蛋白酶抑制剂（chymotrypsin inhibitors, CI)和胰蛋白酶抑制剂(trypsin inhibitor, TI)在喷洒JA或枸杞瘿螨危害的第5 天增幅最大, 且在JA浓度、 瘿螨危害间差异显著(P<0.05), 其中, 0.1 mmol/L JA处理使CI增加了96%, TI增加了128%, 枸杞瘿螨危害使CI增加了94%, TI增加了122%。枸杞叶多糖含量在JA处理或瘿螨危害后显著降低(P<0.05), 且与JA浓度有关, 浓度越高, 降低越明显。 JA或瘿螨危害使枸杞叶木质素增加的最大值出现在处理后的第5 天, 高浓度JA、 瘿螨危害及低浓度JA分别使其增加了70%, 41%和36%, 三者之间差异显著(P<0.05), 第10 天增加幅度减低。 3种处理同样使SOD和PAL活性显著增加(P<0.05), 第5天时增幅最大, 高浓度JA 处理使枸杞叶SOD活性由对照的70.77 U增加到128.98 U, PAL活性由10.91 U增加到20.59 U, 第10天时呈现下降趋势。这些与抗性有关物质的变化及其变化趋势, 说明外源茉莉酸及枸杞瘿螨均可诱导枸杞产生防御反应, 而且反应的程度与茉莉酸浓度及处理后的时间有关。     

钙对水杨酸诱导番茄叶片灰霉病抗性及防御酶活性的影响

以番茄‘L402’品种幼苗为试材,经水杨酸(SA)诱导处理后接种灰霉病菌,再进行外源Ca2+、Ca2+螯合剂和Ca2+抑制剂处理,分析Ca2+和SA处理番茄叶片对灰霉病抗性和主要防御酶系活性的变化,探讨Ca2+和SA对番茄诱导抗病性的影响。结果显示:(1)外源SA可显著提高番茄诱导叶和非诱导叶抗灰霉病能力,Ca2+能进一步增强SA诱导的抗病能力;而Ca2+螯合剂EGTA和质膜钙通道抑制剂LaCl3则不同程度地抑制了SA诱导的番茄灰霉病抗性。(2)外源SA能提高番茄诱导叶和非诱导叶中苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)活性,外源Ca2+亦进一步增强了SA诱导的上述防御酶活性,但缺钙处理则不同程度降低这些防御酶活性。(3)外源补充Ca2+及不同缺钙处理对SA诱导的过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性未发现规律性影响。研究表明,钙对SA诱导番茄抗灰霉病性的增强效应,可能与其提高SA诱导番茄叶片中PAL、PPO和POD等防御酶活性有关。     

核黄素和接种番茄黄化曲叶病毒对番茄叶片防御酶活性的影响

以番茄品种‘1479’为材料,研究了喷施核黄素(Riboflavin)和接种番茄黄化曲叶病毒(TYLCV)对幼苗叶片中过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)3种防御酶活性的影响。结果显示:(1)核黄素能显著降低番茄植株的番茄黄化曲叶病毒病的病情指数,并以2 mmol/L时诱导效果最佳,诱抗效果最高可达41.91%。(2)2mmol/L核黄素处理后96h内,番茄叶片的POD、PPO和PAL活性显著高于对照。(3)接种TYLCV后,核黄素处理和接种TYLCV处理均可诱导番茄叶片中防御酶活性显著增强。研究表明,核黄素处理可诱导POD、PPO和PAL活性的系统增强与番茄对TYLCV的诱导抗性密切相关。     

艳婀珍蝶取食对薇甘菊叶片生理指标的影响

艳婀珍蝶取食后,对薇甘菊叶片的超氧物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、多酚氧化酶(PPO)的活性,总酚含量,有机自由基(DPPH·)清除能力进行分析。结果表明,取食后3h内所测各量即发生变化,但与对照差异不显著。取食4d中,叶片的SOD、POD活性总体上分别高于对照,CAT活性总体上低于对照,说明在艳婀珍蝶胁迫过程中,SOD和POD所起的作用比CAT大。取食叶SOD、POD活性均在48h时达到最大值,之后下降;CAT活性在24h时达到最大值,之后也迅速下降。取食叶PPO活性动态变化程度较大,表现出3个峰值,分别为对照的1·83,1·92倍和2·17倍;总酚含量表现为先上升后下降的趋势;对DPPH·的清除力一直显著性甚至极显著性低于对照。取食叶的SOD与CAT和POD均呈正相关性,且相关性大于对照;PPO与总酚含量在取食叶与对照中也均呈弱正相关性。实验结果表明,薇甘菊的保护酶对艳婀珍蝶胁迫的应激效应是短暂而有限的,艳婀珍蝶的取食破坏了薇甘菊叶片功能,较大程度的干扰了薇甘菊保护酶系统的防御代谢,薇甘菊的总抗氧化能力降低。薇甘菊也不能通过改变酚类物质含量来抵御艳婀珍蝶的取食,艳婀珍蝶取食对薇甘菊有较明显的控制作用。     

Induction of early blight resistance in tomato by <Emphasis Type="Italic">Quercus infectoria</Emphasis> gall extract in association with accumulation of phenolics and defense-related enzymes

Treatment of tomato leaves with aqueous extract (0.5%) of the galls of Quercus infectoria significantly reduced infection from subsequent inoculation with Alternaria solani, the tomato early blight pathogen. When the leaves were challenge-inoculated with A. solani 3 d after application of Q. infectoria gall extract (QIGE), the percent defoliation decreased from 33.6 to 7.3. Two to three day pre-treatment with QIGE reduced the percent defoliation by 77 percent. The biochemical responses of tomato plants to QIGE were also studied. In tomato plants treated with QIGE, phenolic content increased rapidly, reached the maximum at 2 d after treatment. Phenylalanine ammonia-lyase (PAL) activity increased significantly from 1 d after treatment and the maximum enzyme activity was recorded 2 d after treatment at which period a 3-fold increase in PAL activity was observed when compared to the control. Peroxidase (PO) activity was also significantly increased 1 d after treatment and the maximum activity was reached 2 d after treatment. Peroxidase isozyme analysis indicated that PO-1 was increased dramatically in tomato leaves 1 d after treatment and maintained at the same level throughout the experimental period of 6 d. When tomato leaves were treated with QIGE, a two-fold increase in chitinase and β-1,3-glucanase activities was recorded 2 and 3 d respectively, after treatment. The enhanced activities of defense-related enzymes and elevated levels of phenolics in QIGE-treated tomato plants between 1 and 3 d after treatment suggest that these induced biochemical defenses may be involved in the suppression of early blight by QIGE.     

Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.     

Exogenous application of L-phenylalanine and ferulic acid enhance phenylalanine ammonia lyase activity and accumulation of phenolic acids in pea (Pisum sativum) to offer protection against Erysiphe pisi

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of L-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100, 150 ppm) of L-phenylalanine caused increased activity of PAL activity in comparison to control. In pea leaves treated with 50 ppm L-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but it further increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves compared to control. Minimum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt after 24 h at 50 ppm and then increased with time. Treatment with both compounds significantly increased the accumulation of phenolic acids and salicylic acid and reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. Conidial germination on L-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid treated leaves 34% compared to control (46%). Foliar application of different concentrations of L-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum enzyme activity in terms of the accumulation of cinnamic acid (79.3 and 83.5 μg/g fresh wt) was observed following the application of L-phenylalanine after 24 and 48 h respectively. At 50 ppm, cinnamic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt and 74.3 and 86.5 μg/g fresh wt at 100 ppm.     

棉铃虫持续取食对棉花三种防御酶活性的作用

昆虫取食作为一种关键的生物胁迫因子对棉花防御机制产生了重要影响。植物对昆虫取食产生的防御响应,在昆虫与植物的生态关系中具有重要作用。为了明确棉铃虫Helicoverpa armigera取食与棉花防御性之间的动态互作关系,本文研究了棉铃虫持续取食下及停止取食后,棉花中3种防御相关酶活性变化的时间效应。在明确了棉花受损程度与棉铃虫取食时间关系的基础上,分别考察了棉铃虫持续取食2、6、12、18和24h,对棉花中苯丙氨酸解氨酶(PAL)、脂氧合酶(LOX)和多酚氧化酶(PPO)活性的影响。针对棉铃虫持续取食棉叶12h后停止取食,研究了去除虫害胁迫后0、6、12、24和36h,棉花体内PAL、LOX、PPO活性的变化。结果表明:在棉铃虫持续取食棉叶24h内,棉花中3种防御酶的活性响应有所不同,其中,棉铃虫持续取食2和6h对棉花体内PAL活性没有产生显著影响,而持续取食12h显著诱导了PAL活性,持续取食24和36h,均极显著诱导了PAL活性;棉铃虫持续取食2、6、12、和18h均显著诱导了棉花体内LOX活性,持续取食24h极显著诱导了LOX活性;棉铃虫持续取食6h极显著诱导了棉花体内PPO活性,持续取食24h显著诱导了PPO活性。棉铃虫取食12h后停止取食,在去除虫害除胁迫后0、6、12、24和36h,棉花体内PAL活性均显著升高;而LOX活性则呈现出先升高后恢复正常的现象;PPO活性开始无变化,但在胁迫去除后12和24h显著增高,到36h恢复正常。可见,棉花体内PAL、LOX和PPO活性对棉铃虫取食产生的防御响应,与其受虫害持续取食胁迫时间的增长呈正相关,随着取食时间和受危害的程度加大而升高。并且,在虫害胁迫去除后的一定时间内,棉花体内PAL、LOX和PPO活性依然会保持较高的活性状态,而同等程度机械损伤后的棉叶内PAL、LOX、PPO活性均没有发生显著性变化。说明棉花对于棉铃虫取食胁迫的防御与棉花生理生化性质的改变有关,且具有持续性。     

Biochemical alterations in leaves of resistant and susceptible cotton genotypes infected systemically by cotton leaf curl Burewala virus

Leaf curl disease caused by Cotton Leaf Curl Burewala virus (CLCuBuV) has been recognized as serious threat to cotton in Indian subcontinent. However, information about cotton–CLCuBuV interaction is still limited. In this study, the level of phenolic compounds, total soluble proteins, and malondialdehyde (MDA) and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), proteases, superoxide dismutase (SOD), and polyphenol oxidase (PPO) were studied in leaves of two susceptible (CIM-496 & NIAB-111) and two resistant (Ravi and Co Tiep Khac) cotton genotypes. Disease symptoms were mild in the resistant genotypes but were severe in highly susceptible genotypes. The results showed that phenolic compounds, proteins, PAL, POX, CAT, proteases, SOD, PPO, and MDA play an active role in disease resistance against CLCuBuV. The amount of total phenols, proteases, MDA, and PPO was significantly higher in leaves of CLCuBuV-inoculated plants of both resistant genotypes as in non-inoculated plants, and decreased in CLCuBuV-inoculated plants of both susceptible genotypes over their healthy plants. POX, protein content, SOD, and PAL activities showed lower values in resistant genotypes, while they decreased significantly in susceptible genotypes as compared to the noninoculated plants except PAL, which showed non-significant decrease. CAT was found to be increased in both susceptible and resistant genotypes with maximum percent increase in resistant genotype Ravi, as compared to non-inoculated plants. The results showed significantly higher concentrations of total phenols and higher activity of protease, MDA, SOD, and PPO in resistant genotype Ravi after infection with CLCuBuV, suggesting that there is a correlation between constitutive induced levels of these enzymes and plant resistance that could be considered as biochemical markers for studying plant-virus compatible and incompatible interactions.     

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.