黄瓜叶片胞间隙几丁质酶与抗霜霉菌侵染关系的研究

采用高抗霜霉病的‘津春4号’和易感霜霉病的‘长春密刺’黄瓜的温室穴盘幼苗为材料,研究了接种霜霉菌对黄瓜叶片胞间隙几丁质酶累积变化及幼苗生长的影响.结果显示:(1)与易感品种相比,抗病品种叶片胞间隙液蛋白浓度升高快、含量高;同时几丁质酶活性上升的速度快、幅度大,于接种后第2天达到峰值,且高活性维持至第7天;(2) SDS-PAGE电泳分析发现,抗病品种有一种27 kD的酸性几丁质酶在接种后第2天迅速积累,并在接种后第4、7天呈现出较高的表达量;Western blotting的结果也证实了上述胞间隙几丁质酶积累的变化.(3)与未接种对照相比,接种处理后第4、7天,易感黄瓜幼苗鲜重、干物质积累量均出现显著降低,但在接种后第7天抗病品种的上述生长指标要显著高于易感病品种.研究表明,接种霜霉菌后,抗病黄瓜叶片胞间隙几丁质酶迅速累积且活性快速升高,以减轻霜霉病对幼苗生长的侵害,这可能是黄瓜抗霜霉菌侵染的一种防御机制.     

高温处理防治黄瓜霜霉病的生理生化机制

以黄瓜感病品种‘长春密刺’为试材,通过室内盆栽试验研究高温对已感染霜霉病菌的黄瓜幼苗生理生化特征的影响.结果显示:(1)在黄瓜幼苗接种霜霉菌后8h,用45℃高温处理90min防治效果最明显.(2)与只接种霜霉病菌的黄瓜幼苗相比,接种霜霉菌后进行高温处理可显著提高叶片叶绿素含量,降低丙二醛含量;与对照相比,接种霜霉菌后进行高温处理可显著提高叶片几丁质酶活性,Western blotting验证了几丁质酶的活性变化.(3)SDS-PAGE结果表明,霜霉菌侵染可诱导一种28kD蛋白表达,高温处理后28kD蛋白的表达量降低.研究结果表明高温处理可能在杀死病原菌的同时,还诱导黄瓜幼苗对霜霉病产生了部分抗性.     

Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen.

The expression of PR protein encoding genes and genes involved in the phenylpropanoid metabolism was analysed on grapevine leaves of susceptible and resistant cvs. in response to inoculation with the host-pathogen Plasmopara viticola and the non-host pathogen Pseudoperonospora cubensis, the downy mildew pathogen of cucumber. These experiments were conducted to elucidate whether or not grapevine plants susceptible to downy mildew exhibit an identical defence response after inoculation with the non-host pathogen. Expression analysis of defence-related genes revealed marked differences between the susceptible cultivar "Riesling" (Vitis vinifera) and the resistant cultivar "Gloire de Montpellier" (Vitis riparia). Whereas some genes seem to be expressed constitutively in "Gloire" or induced after an inoculation with both pathogens, expression of defence-related genes in Riesling was influenced mainly after inoculation with the non-host pathogen: PR-2, PR-3, PR-4, a PGIP gene, and especially genes encoding enzymes involved in anthocyanin biosynthesis (DFR, F3H, LDOX) were affected. Therefore, the occurrence of the respective products (flavans and other phenolics) in inoculated leaves was investigated with appropriate histological staining techniques. These stainings revealed a production of catechins and related phenolic compounds within the first 48 hai (hours after inoculation) with Ps. cubensis but not with P. viticola in Riesling, whereas in Gloire no further production was seen, which may be due to the high content of polyphenolics as observed in control leaves. In addition to the staining procedures, sporulation intensity was monitored on leaf discs. Pretreatments of leaf discs with Ps. cubensis led to a reduced browning reaction (as a result of a hypersensitive reaction) in Gloire and significantly reduced the intensity of sporulation in Riesling after a subsequent inoculation with P. viticola.     

Physiology and proteomics of drought stress acclimation in sunflower (Helianthus annuus L.)

An easy and manageable in vitro screening system for drought tolerance of sunflower seedlings based on MS media supplemented with polyethylene glycol 6000 was evaluated. Morphological and physiological parameters were compared between control (-0.05 MPa) and drought-stressed (-0.6 MPa) seedlings of Helianthus annuus L. cv. Peredovick. There was a significant growth deficit in drought-stressed plants compared to control plants in terms of hypocotyl length, and shoot and root fresh mass. Shoot growth was more restricted than root growth, resulting in an increased root/shoot ratio of drought-stressed plants. Accumulation of osmolytes such as inositol (65-fold), glucose (58-fold), proline (55-fold), fructose (11-fold) and sucrose (eightfold), in leaves of drought-stressed plants could be demonstrated by gas-liquid chromatography. Soluble protein patterns of leaves were analysed with two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry. A set of 46 protein spots allowed identification of 19 marker proteins. Quantitative changes in protein expression of drought-stressed versus control plants were detected. In leaves of drought-stressed sunflower seedlings six proteins were significantly up-regulated more than twofold: a putative caffeoyl-CoA 3-O-methyltransferase (4.5-fold), a fructokinase 3 (3.3-fold), a vegetative storage protein (2.5-fold), a glycine-rich RNA binding protein (2.2-fold), a CuZn-superoxide dismutase (2.1-fold) and an unknown low molecular weight protein (2.3-fold). These proteins represent general stress proteins induced under drought conditions or proteins contributing to basic carbon metabolism. The up-regulated proteins are interesting candidates for further physiological and molecular investigations regarding drought tolerance in sunflower.     

干旱期间春小麦叶片多胺含量与作物抗旱性的关系

使用两种抑制剂MCBG(抑制SAMDC活性)和AOA(抑制ACC合成酶活性)研究了干旱期间两个春小麦品种8139(抗旱性较弱)和504(抗旱性较强)叶片多胺(Put、Spd和Spm)含量、RWC水平、SOD和POD活性以及MDA含量的变化,并由此探讨了不同类别多胺与作物抗旱性的关系以及多胺与乙烯在作物对干旱胁迫响应过程中对共同前体SAM的竞争趋向及其生理意义。     

高温诱导黄瓜抗霜霉病机理

研究了高温对黄瓜霜霉病菌致病力的影响以及高温控制霜霉病发生的效果.结果表明,40 ℃高温处理2 h和45 ℃处理1 h对黄瓜霜霉病的诱导抗病性作用最明显,其在接种后4 d时的防效分别为58.40%和45.81%,到接种后6 d时,防效分别下降为39.35%和37.65%.经高温诱导后,过氧化物酶（POD）、苯丙氨酸解氨酶(PAL)、几丁质酶（Cht）、β-1,3-葡聚糖酶（Glu）活性均显著高于对照,与未诱导植株相比,高温诱导后叶片组织的细胞壁表面有大量木质素沉积,表明高温处理后黄瓜表现出对霜霉病的抗病性.     

Antioxidant system and photosynthetic characteristics responses to short-term PEG-induced drought stress in cucumber seedling leaves

The effect of short-term drought stress on the water content, antioxidant system and photosynthetic characteristics was investigated using cucumber (Cucumis sativus L.) seedlings. The results indicated that polyethylene glycol induced water stress reduced water content in shoots of cucumber seedling after treatment of 36 hours, and caused obvious reductions in net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration and transpiration of leaves. In addition, water stress significantly reduced the photosynthetic pigment content and inhibited photochemical activity, including actual photochemical efficiency, maximal quantum yield of photosystem II photochemistry and coefficient for photochemical quenching. Meanwhile non-photochemical quenching increased. As responses to drought stress, significant increases in electrolyte leakage, malondialdehyde, superoxide anion and hydrogen peroxide levels were detected in leaves. The superoxide dismutases, catalase, glutathione reductase and dehydroascorbate reductase activities, protein, ascorbate and glutathione content, all decreased and peroxidases activity increased, while ascorbate peroxidase and monodehydroascorbate reductase activities exhibited different trend under different degree of water stress. Therefore, it can be concluded that water stress strongly disrupted the normal metabolism of leaves and restrained water absorption.     

亚精胺诱导黄瓜幼苗对白粉病抗性的研究

以黄瓜品种‘长春密刺’幼苗为材料,研究了亚精氨(Spd)诱导黄瓜幼苗对白粉病的抗性,并测定Spd处理和白粉菌接种对黄瓜叶片4种防御酶活性及3种防卫基因表达的影响。结果显示:(1)0.2～1.0mmol.L-1 Spd对黄瓜幼苗白粉病抗性均有不同程度的诱抗效果,并以0.8mmol.L-1 Spd处理效果最明显,诱导效率可达55.3%。(2)喷施Spd或接种白粉菌均可提高黄瓜叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶和β-1,3-葡聚糖酶的活性,且诱导并接种处理的植株叶片上述酶活性均比只诱导不接种处理的上升速度更快;同时,Spd处理和接种白粉菌可以提高植株叶片中POX、PAL、PR-1a基因的表达量。研究表明,Spd处理可以诱导防卫基因表达的增强,提高防御酶活性,显著降低病情指数,增强黄瓜幼苗对白粉病的抗性。     

Analysis by high-performance liquid chromatography of free amino acids extracted from needles of drought-stressed and shaded Pinus ponderosa seedlings

Free amino acid concentrations in needles of drought-stressed and shaded seedlings of ponderosa pine (Pinus ponderosa Dougl. ex Laws.) were analyzed to determine the influence of low irradiance on a biochemical response to drought stress. A convenient method was developed for separating and quantitating amino acids in needle extracts using derivatization with dansyl chloride and reversed-phase high-performance liquid chromatography (HPLC). Twenty-one amino acids were separated on an Ultrasphere ODS CIS column and detected with a fluorescence spectrophotometer. As little as 10 pmol of each were detected with good peak separation and reproducible retention times. The results of HPLC analysis showed that drought and shading induced an increase in total amino acid concentrations in needles; shading had the greater effect. Arginine and proline concentrations increased most in needles of drought-stressed seedlings and remained high in unshaded seedlings recovering from drought 48 h after rewatering. Arginine and glutamine increased most in the shaded seedlings, which did not survive severe drought. The large increase in arginine in both drought-stressed and severely shaded seedlings suggests that sequestering and storage of ammonia are important when stress reduces carbon fixation.     

Osmotic solute responses of mycorrhizal citrus (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis>) seedlings to drought stress

This study investigated the accumulation of osmotic solutes in citrus (Poncirus trifoliata) seedlings colonized by Glomus versiforme subjected to drought stress or kept well watered. Development of mycorrhizae was higher under well watered than under drought-stressed conditions. Arbuscular mycorrhizal (AM) seedlings accumulated more soluble sugars, soluble starch and total non-structural carbohydrates in leaves and roots than corresponding non-AM seedlings regardless of soil-water status. Glucose and sucrose contents of well-watered and drought-stressed roots, fructose contents of well-watered roots and sucrose contents of drought-stressed leaves were notably higher in AM than in non-AM seedlings. K⁺ and Ca²⁺ levels in AM leaves and roots were greater than those in non-AM leaves and roots, while AM symbiosis did not affect the Mg²⁺ level. AM seedlings accumulated less proline than non-AM seedlings. AM symbiosis altered both the allocation of carbohydrate to roots and the net osmotic solute accumulations in response to drought stress. It is concluded that AM colonization enhances osmotic solute accumulation of trifoliate orange seedlings, thus providing better osmotic adjustment in AM seedlings, which did not correlate with proline but with K⁺, Ca²⁺, Mg²⁺, glucose, fructose and sucrose accumulation.     

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.     

Effect of Wind-induced Mechanical Stress on Soluble Peroxidase Activity and Resistance to Pests in Cucumber

Wind-induced mechanical stress (MS) significantly increased the soluble peroxidase activity in leaves of cucumber over control levels after 9 days of treatment. In comparison, inoculation with the fungal pathogen Cladosporium cucumerinum induced significant increases in peroxidase after 4 days. Cucurbit anthracnose symptom development caused by Colletotrichum orbiculare was greater on leaves of seedlings exposed to 6 but not to 9 or 12 days of wind-induced MS than on leaves of control plants. In contrast, reproduction of melon aphids was significantly reduced on leaves exposed to 12 days of MS relative to controls. These results indicate that wind-induced MS can induce soluble peroxidase activity in cucumber and have divergent effects on the resistance to insects and pathogens.     

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.     

Comparison of local and systemic induction of acquired disease resistance in cucumber plants treated with benzothiadiazoles or salicylic acid.

The accumulation of chitinase and its involvement in systemic acquired disease resistance was analyzed using acibenzolar-S-methyl and salicylic acid (SA). Resistance against scab (pathogen: Cladosporium cucumerinum) and the accumulation of chitinase were rapidly induced in cucumber plants after treatment with acibenzolar-S-methyl. In contrast, SA protected the plants from C. cucumerinum and the accumulation of chitinase was induced only on the treated leaves. The accumulation of chitinase in response to inoculation with the pathogen was induced more rapidly in cucumber plants previously treated with acibenzolar-S-methyl than in plants pretreated with SA or water. Thus, it appears that a prospective signal(s), that induces systemic resistance, can be transferred from leaves treated with acibenzolar-S-methyl to the untreated upper and lower leaves where systemic resistance is elicited. In contrast, exogenously applied SA is not likely to function as a mobile, systemic resistance-inducing signal, because SA only induces localized acquired resistance.     

Polysaccharide Elicitor from the Endophyte <i>Bionectria</i> sp. Fat6 Improves Growth of Tartary Buckwheat under Drought Stress

Drought can limit the growth and reduce the yield of crops, but the safe and effective bio-approach to improve the drought resistance of crops is very little. We conducted an experiment in which we monitored the effects of polysaccharide from the endophyte Bionectria sp. Fat6 on the growth of Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn) seedlings under control and drought-stressed conditions by determining gas exchange, photosynthesis parameters, photosynthetic pigment contents, and metabolite accumulation. Results indicated that the polysaccharide from endophyte stimulated plant growth and increased the aboveground biomass, root mass, and root/shoot ratio of Tartary buckwheat. Application of the polysaccharide to drought-stressed plants resulted in a significant increase in the net photosynthetic rate, stomatal conductance, and transpiration rate of Tartary buckwheat and decreased the intercellular CO₂ concentration. The contents of chlorophyll a, chlorophyll b, chlorophyll a + b, and carotenoids in leaves were higher in polysaccharide-treated seedlings than that in control. Polysaccharide notably increased the soluble protein and proline content and decreased the malondialdehyde content in Tartary buckwheat leaves. The endophytic polysaccharide may protect Tartary buckwheat against drought by improving leaf gas exchange and photosynthetic capacity, and altering concentrations of protective metabolites. Together, these changes may compensate for the negative impacts of drought stress on the growth of Tartary buckwheat. Thus, the polysaccharide from the endophyte Bionectria sp. Fat6 may be an effective biotic elicitor and a promising bio-approach to improve Tartary buckwheat production worldwide.     

Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid*

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX-95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX-97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.     

萝卜块根中两个具溶菌酶活性的几丁质结合蛋白的纯化及其特性

通过亲和层析和羧甲基一纤维素离子交换层析从萝卜的块根中分离到两个具溶菌酶活性的酶组份：CBP1和CBP2。两者经SDS-PAGE均显示单一蛋白染色条带,其对应的分子量分别为26．9kD和24．8kD。两种蛋白除有溶菌酶活性外,还有几丁质酶活性,但无壳聚糖酶活性。各种类型的几丁质对CBP1和CBP2都有较强的吸附作用,而在还原／非还原的单向SDS-PAGE中却观察不到两者分子中存在二硫键。     

茉莉酸甲酯对花生幼苗生长和抗旱性的影响

经过茉莉酸甲酯处理的花生幼苗,在形态、解剖和生理上都发生变化,其中以１２５ｍｇ／Ｌ处理最显著。处理后的植株幼苗矮化,叶小而厚,叶片贮水细胞大、蒸腾减弱、内源脱落酸和脯氨酸含量增多、过氧化物酶活性加大。由于水分的丧失减少,叶片水分的贮存增加,从而提高幼苗的抗旱性。