抗坏血酸对花生原生质体分离过程中膜损伤的保护作用

酶解处理使花生叶肉细胞原生质体膜脂质过氧化产物丙二醛(MDA)积累,添加抗坏血酸(ASA)能降低MDA的积累。未添加ASA时,酶解初期,过氧化物酶(POD)、过氧化氢酶(CAT)活性上升,说明原生质体存在各种抵御不良变化的机制,但酶解时间加长,POD活性下降。添加ASA能使超氧化物歧化酶(SOD)活性明显上升。试验结果说明:在原生质体分离过程中会导致膜损伤,细胞自身存在一个响应的防御机制,添加ASA能降低酶解过程对原生质膜的损伤。     

Biochemical response between insects and plants: an investigation of enzyme activity in the digestive system of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and leaves of Coffea arabica (Rubiaceae) after herbivory

Plant defence mechanisms can reduce the digestive enzyme activity of insect pests. The aim of this study was to determine the relationship between the production of proteinase inhibitors, lipoxygenase and polyphenol oxidase activity in Coffea arabica (Catuai IAC 15) plants, and the digestive enzyme activity in the pest Leucoptera coffeella (Lepidoptera: Lyonetiidae) after feeding on the plant. The production of proteinase inhibitors was evaluated with L‐BApNA as a substrate. We studied lipoxygenase activity with linoleic acid and polyphenol oxidase activity with catechol substrates, in coffee plants damaged (T1) and not damaged (T2) by L. coffeella. L. coffeella digestive enzyme activity was verified by trypsin‐like (substrate l ‐BApNA and l ‐TAME), chymotrypsin‐like (BTpNA and ATEE), cysteine proteases (l ‐BApNA) and total protease (azocasein). Proteinase inhibitor production and lipoxygenase and polyphenol oxidase activity in C. arabica increases (P ≤ 0.05) with L. coffeella damage. Our results provide important information that these enzymatic activities may play a role in plant defence processes in C. arabica. Trypsin‐like activity increases, whereas chymotrypsin‐like and cysteine protease activity decrease in the midgut of L. coffeella, which acts as a defence mechanism.     

Stimulation and attenuation of induced resistance by elicitors and inhibitors of chemical induction in tomato (Lycopersicon esculentum) foliage

Elicitors and inhibitors of chemical induction were used to manipulate the activities of several putative defense-related proteins in leaves of the tomato, Lycopersicon esculentum Mill. The four presumptive defenses manipulated were proteinase inhibitors, polyphenol oxidase, peroxidase, and lipoxygenase. The elicitors used were jasmonic acid, methyl jasmonate, ultraviolet light, and feeding by larvae of the noctuid, Helicoverpa zea Boddie; the inhibitors used were salicylic acid and acetylsalicylic acid. These chemical manipulations were combined with short-term growth assays using larvae of the generalist noctuid, Spodoptera exigua Hubner, in order to assess the relative roles of the proteins in induced resistance to S. exigua. When activities of proteinase inhibitors and/or polyphenol oxidase in leaf tissue were high (e.g., in damaged or elicited plants), growth rates of larvae of S. exigua were low; when activities of polyphenol oxidase and proteinase inhibitors were low (e.g., in undamaged or damaged, inhibited plants), growth rates of larvae were high. In contrast, high activities of peroxidase and lipoxygenase were not associated with decreases in suitability of leaf tissue for S. exigua. The association of high levels of proteinase inhibitors and polyphenol oxidase with resistance to S. exigua – irrespective of the presence or absence of damage – strongly implicates these proteins as causal agents in induced resistance to S. exigua.     

Role of oxidative stress in cereal protoplast recalcitrance

Cereal leaf protoplasts are often extremely unstable in culture and usually lyse within 24 hours. Using the thiobarbituric acid test and the ferrous thiocyanate test we have shown that corn (Zea mays L. cv. Market Beauty) and wheat (Triticum aestivum L. cv. Benito) leaf protoplasts accumulate peroxides and peroxide degradation products during culture. This increase correlated with an increase in lipoxygenase activity. On the other hand, enzymes involved in detoxification of peroxides such as catalase and peroxidase decreased during culture. The occurrence of lipid peroxidation in leaf protoplasts is likely to be a consequence of a temporary imbalance in the enzymes involved in oxygen metabolism. It has previously been shown that the lipoxygenase inhibitor n-propyl gallate stabilizes the protoplasts in culture and so peroxidation is likely to be the cause of leaf protoplast instability. Protoplasts obtained from suspension cultures are stable in culture and do not undergo lipid peroxidation. This stability is due to a decrease in lipoxygenase activity and increases in catalase and peroxidase activity after protoplast isolation.Abbreviations MDA malonaldehyde - 2,4-D 2,4-dichlorophenoxyacetic acid - PVP polyvinylpolypyrrolidone Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

两种污泥连续施用对潮土重金属含量及酶活性的影响

采用盆栽土培试验,研究了工业污泥(化工厂底泥)及城市污水处理厂剩余污泥中重金属存在的形态与含量,以及两种污泥连续施用对潮土重金属含量及酶活性的影响。结果表明,两种污泥中的重金属主要是以非交换态存在,城市污水处理厂剩余污泥中总的重金属含量比工业污泥低,而重金属的有效性比工业污泥高;污泥的施用能增加潮土中脲酶的活性,多酚氧化酶及中性磷酸酶的活性与污泥的施用量有一定相关性,并与土壤中交换态Zn、Cu含量呈一定负相关,土壤多酚氧化酶及过氧化氢酶活性可作为土壤中重金属Zn污染的指示指标;污泥的施用有提高潮土中交换态Cu、Zn及Pb含量的趋势。     

Techniques for enhanced release of leaf protoplasts in Populus

Microscopic examination of Populus leaf tissue following enzyme treatment revealed two factors contributing to low protoplast yields: (1) poor penetration of the enzymes into the tissue, and (2) entrapment of protoplasts in leaf debris during protoplast purification procedures. A simple combination of rapid grinding of the tissue in an Omni-mixer prior to enzyme treatment and forceful washing of leaf-debris after digestion provided high exposure of the cells, uniform digestion, and high yields of protoplasts of two Populus clones. Protoplasts exhibited cell wall regeneration and long-term viability in culture. The relative yield advantages of the techniques varied with the inherent digestibility of each clone but could produce up to 600 percent greater protoplast yields in a woody plant species in which protoplast isolation was previously limited. The techniques were also applicable to an herbaceous species, Solanum etuberosum.Abbreviations BA benzyladenine - NAA naphthalene acetic acid - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - (NC-XXXX) North Central Forest Experiment Station accession number assigned to Populus hybrid clones     

花椒叶浸提液对土壤微生物数量和土壤酶活性的影响

通过用花椒叶浸提液浇灌盆栽花椒幼苗,研究浸提液对土壤酶和土壤微生物的影响.结果表明, 花椒叶浸提液使根际土中细菌、真菌和放线菌数量以及微生物总数均有不同程度的减少,根际土中真菌和放线菌的数量变化呈降-升-降-升的趋势.20、60和80 g·L^-1浓度的叶浸提液使非根际土中细菌的数量显著增加21.59%、107.55%和8.62%,而40 g·L-1浓度的叶浸提液则使非根际土中细菌数量显著降低22.56%.叶浸提液使根际土蛋白酶、蔗糖酶和酸性磷酸酶活性明显低于非根际土相应的酶活性,而过氧化氢酶和多酚氧化酶活性则显著升高.土壤的蛋白酶活性与蔗糖酶活性呈显著正相关,与土壤放线菌数量呈显著负相关;多酚氧化酶活性与蔗糖酶活性呈显著负相关,与细菌、真菌、放线菌以及微生物总数呈显著正相关;放线菌只与蛋白酶、多酚氧化酶、蔗糖酶3种酶活性及真菌呈显著相关,与过氧化氢酶、酸性磷酸酶以及细菌和微生物总数的相关性均不显著.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

Antioxidant Enzyme Activities in Arbuscular Mycorrhizal (<Emphasis Type="Italic">Glomus intraradices</Emphasis>) Fungus Inoculated and Non-inoculated Maize Plants Under Zinc Deficiency

A greenhouse experiment was conducted to examine the changes in antioxidant enzyme activities of arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck and Smith inoculated (M+) and non-inoculated (M−) maize (Zea mays L.) plants (variety COHM5) under varying levels of zinc (0, 1.25, 2.5, 3.75 and 5.0 mg kg⁻¹). Roots and shoots sampled at 45 days after sowing (DAS) were estimated for its antioxidant enzymes (superoxide dismutase, peroxidase) IAA oxidase, polyphenol oxidase, acid phosphatase and nutritional status especially P and Zn concentrations. Mycorrhizal inoculation significantly (P ≤ 0.01) increased all the four antioxidant enzymes in both roots and shoots at 45 DAS regardless of Zn levels. All enzyme activities except SOD increased progressively with increasing levels of Zn under M+ and M− conditions. The SOD activity got decreased in roots and shoots at 2.5 and 3.75 mg Zn kg⁻¹. Acid phosphatase activity in M+ roots and shoots were higher in all levels of Zn but the values decreased with increasing levels of Zn particularly in roots. Mycorrhizal fungus inoculated plants had higher P and Zn concentrations in both stages in comparison to non-inoculated plants. Our overall data suggest that mycorrhizal symbiosis plays a vital role in enhancing activities of antioxidant enzymes and nutritional status that enables the host plant to sustain zinc deficient conditions.     

Changes in the activities of catalase, peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Factors influencing soil enzyme activity in China’s forest ecosystems

Enzyme activity (EA) mediates soil organic matter (SOM) degradation, transformation, and mineralization, thereby maintaining the biogeochemical cycles and energy flow of ecosystems. To determine the main factors explaining EA variations in China’s forest ecosystems, we created a database of soil EAs and relevant variables using data from the literature and analysed relationships between EAs and both climatic and edaphic variables. Catalase, phenol oxidase, acid (alkaline) phosphatase, and protease activities differed significantly among different types of forests. Catalase and urease activities were generally higher in primosols, cambisols, and argosols than in ferrosols. EA largely decreased with soil depth and increased with SOM. Phenol oxidase and urease activities were negatively correlated with mean annual temperature (MAT); in contrast, catalase, invertase, and protease activities first decreased (< 2.5 °C), increased (2.5–17.5 °C), and then decreased (> 17.5 °C) with increasing MAT. Although protease activity was slightly positively correlated with mean annual precipitation (MAP), catalase, phenol oxidase, and urease activities were all negatively related to MAP. Catalase, invertase, acid (alkaline) phosphatase, urease, and protease activities first increased (< 2000 m.a.s.l.) and then decreased (2000–4100 m.a.s.l.) with increasing elevation. Principal component analysis revealed most EAs to be correlated with climate conditions and soil pH. These findings suggest that climatic and edaphic variables directly and indirectly correlate with forest type and greatly impact soil EA.     

华西雨屏区苦竹林土壤酶活性对模拟氮沉降的响应

2007年11月-2009年5月,对华西雨屏区苦竹人工林进行了模拟氮沉降试验,氮沉降水平分别为：对照(0 g N·m^-2·a^-1)、低氮(5 g N·m^-2·a^-1)、中氮(15 g N·m^-2·a^-1)和高氮(30 g N·m^-2·a^-1).在氮沉降进行半年后,每月采集各样方0～20 cm土壤样品,测定其土壤酶活性,连续测定1年.结果表明：苦竹人工林样地中6种土壤酶活性的季节变化较明显,蔗糖酶、纤维素酶和酸性磷酸酶活性的高峰期出现在春季,脲酶活性高峰期出现在秋季,而过氧化物酶和多酚氧化酶活性高峰期出现在冬季;氮沉降增加了苦竹林土壤中木质素分解酶和碳、氮、磷分解相关酶（多酚氧化酶、蔗糖酶、酸性磷酸酶和脲酶）的活性,抑制了纤维素酶活性,而对过氧化物酶的影响不显著;苦竹林生态系统处于一种氮限制状态,氮沉降刺激了微生物-酶系统对土壤有机质的分解.     

施用生物炭6年后对稻田土壤酶活性及肥力的影响

利用田间定位试验,研究0(BC₀)、7.5(BC₁)、15(BC₂)和22.5(BC₃)t·hm^-2水稻秸秆生物炭及3.75 t·hm^-2水稻秸秆(STR)一次性施加6年后对稻田土壤肥力及酶活性的影响.结果表明: 施用生物炭6年后土壤有机碳、有效磷和速效钾含量显著增加,增幅分别为34.6%、12.4%和26.2%,土壤pH值和容重显著降低,但对土壤全氮含量无显著影响.土壤脲酶和酸性磷酸酶的活性显著增加,土壤荧光素二乙酸酯酶(FDA水解酶)和芳基硫酸酯酶的活性受到不同程度的抑制,其中,BC₂处理的土壤脲酶活性增加量最大,增幅为36.5%.土壤酸性磷酸酶活性随着生物炭施加量的增加而增加,与土壤速效磷含量呈显著正相关关系;土壤FDA水解酶和脲酶主要与土壤速效钾含量有关;酸性磷酸酶和芳基硫酸酯酶与土壤容重呈显著正相关.施用生物炭6年后土壤脱氢酶和多酚氧化酶活性明显升高,增幅分别为48.8%和27.5%,而过氧化氢酶活性逐渐下降,且显著低于对照BC₀.STR处理显著增加了土壤脲酶、FDA水解酶、脱氢酶、酸性磷酸酶和芳基硫酸酯酶的活性,降低了过氧化氢酶和多酚氧化酶的活性,降幅分别为23.4%和15.9%.     

Activity Changes in Selected Enzymes from Soybean Leaves Following Ozone Exposure

Soybeans [Glycine max(L.) Merr.] were harvested at various time periods after a 2-h exposure to either 0 or 0.5 μ1/1 ozone to determine the effects of ozone on selected enzymes. Carbohydrate metabolism was modified by a depression of glyceraldehyde 3-phaosphate dehydrogenase and an activation of glucose 6-phosphate dehydrogenase. Ozone did not alter the levels of RNase, protease, acid phosphatase or esterase as might be expected if ozone enhanced leaf senescence. The activities of phenylalanine ammonia lyase, polyphenol oxidase and peroxidase were initially depressed and then stimulated following the ozone exposure. The reactions of soybeans to an acute ozone stress were more nearly akin to those elicited in response to other stresses than to the process of senescence.     

Oxidative Enzymes of Ceratocystis fimbriata Isolates Differing in Host Specificity

Eight isolates of Ceratocystis fimbriata differing in pathogenicity on sweet potato roots were compared in vitro for peroxidase, catalase and polyphenol oxidase activities. Pathogenic isolates were generally lower in specific activities of peroxidase and catalase than the nonpathogenic isolates. Cultures of pathogenic isolates were distinguished by black culture liquid compared to the yellow color of nonpathogenic isolates. No consistent differences among the isolates were apparent when the specific activity of polyphenol oxidase preparations were compared on various phenolic substrates. The peroxidase of C. fimbriata had an approximate molecular weight of 81,000 when compared with known protein standards on a column of Sephadex G-100 dextran gel. Its substrate specificity differed from horseradish peroxidase in that it was nonreactive with guaiacol.     

Cellular Location of Degradative Enzymes in Staphylococcus aureus

Staphylococus aureus, ATCC 6538P, was fractionated into protoplast membranes, mesosomal vesicles, periplasm, and cytoplasm. These fractions and the culture fluid were then assayed for various degradative enzyme activities. They were not restricted to a single fraction nor dispersed homogeneously, but were distributed predominantly (on the basis of specific activity) as follows: nuclease in the culture fluid; alkaline phosphatase, 5'-nucleotidase, and acid phosphatase in the periplasm; adenosine triphosphatase in the protoplast membrane; and protease (low levels) in mesosomal vesicles. No significant esterase nor cell wall hydrolytic activity was found in any fraction. S. aureus 80/81 was studied for penicillinase activity after induction with benzyl penicillin; this enzyme was localized in the mesosomal vesicles. Electron microscopy did not reveal any ultrastructural changes associated with secretion of the extracellular fraction. Overall, these studies demonstrate that degradative enzymes are located in several surface compartments and that, therefore, the mesosome does not function as a prototype lysosome in S. aureus.     

模拟氮沉降对太岳山油松林土壤酶活性的影响

为研究土壤酶活性对氮沉降增加的响应,以山西太岳山油松人工林和天然林为研究对象,于2009年8月开始实施模拟氮沉实验,试验设置对照(CK,0 kg N hm-2a-1);低氮(LN,50 kg N hm-2a-1);中氮(MN,100 kg N hm-2a-1);高氮(HN,150 kg N hm-2a-1)4种氮处理,自2012年起每年5、7、9月在各处理样方采集表层0—20 cm土壤,测定土壤酶活性(过氧化物酶、多酚氧化酶、纤维素酶、蔗糖酶、脲酶、中性磷酸酶)。研究结果表明:施氮处理下的脲酶与中性磷酸酶活性均有所提高,而低氮处理下天然林中的多酚氧化酶与人工林中的蔗糖酶显著低于对照,中氮、高氮处理下过氧化物酶、多酚氧化酶、天然林中的纤维素酶以及人工林中的蔗糖酶显著降低。总的来说,人工模拟氮沉降促进了土壤中脲酶和中性磷酸酶的活性,抑制了过氧化物酶和多酚氧化酶的活性,并降低了天然林土壤中的纤维素酶活性和人工林中的蔗糖酶活性,但对天然林中蔗糖酶和人工林中的纤维素酶无影响。主导木质素降解的多酚氧化酶活性与纤维素酶、蔗糖酶活性显著相关,纤维素酶与蔗糖酶活性的下降可能是由木质素降解受到抑制,土壤微生物可利用碳源减少所引起。另外,受到天然林土壤含氮量较高的影响,与人工林相比,天然林的多酚氧化酶活性对模拟氮沉降更敏感。由于被抑制的酶均与土壤有机质降解密切相关,氮沉降增加将减缓山西油松林土壤有机质的降解,有利于有机质在土壤中的积累。     

铜对三叶草-土壤酶系统的影响

通过盆栽实验研究了重金属Cu污染对植物(三叶草)-土壤酶(脲酶、蔗糖酶、过氧化氢酶和多酚氧化酶)系统的影响.结果表明,随着Cu浓度增加,脲酶、蔗糖酶、过氧化氢酶和多酚氧化酶活性均逐渐减小,与Cu浓度有高度相关性,蔗糖酶>多酚氧化酶>脲酶>过氧化氢酶.在处理浓度不变情况下,酶活性随时间而变化,且呈现低Cu浓度(<00 mg·kg^-1)时4种酶活性均有所上升,而Cu浓度增高(00～3 000 mg·kg^-1)时各酶活性逐渐下降的趋势.统计分析表明,在每一梯度浓度上,4种酶在Ⅰ、Ⅱ、Ⅲ组内均存在显著差异性(P<0.01),与植物受重金属Cu污染时的生长情况一致.随着Cu浓度增加,土壤pH值逐渐下降,而电导率上升;同一Cu浓度下的pH值和电导率均随时间呈缓慢上升趋势,统计分析显示,二者在Ⅰ、Ⅱ、Ⅲ组内均存在显著差异性(P<0.01).土壤pH值和电导率与4种土壤酶活性有高度相关性,多酚氧化酶>蔗糖酶>过氧化氢酶>脲酶.这4种酶同时可作为检测土壤环境质量的指标.     

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

Seasonal activity of non-specific acid and alkaline phosphatases in beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) leaves

The activities of acid and alkaline phosphatases along with phosphorus content in leaves of European beech (Fagus sylvatica L.) were studied for a period from April to October. The phosphorus content of beech leaves was highest in April, at the beginning of the vegetation period; from May to October it was twofold lower than in April. Acid phosphatase activity (per unit fresh weight) in leaves collected from the middle part of the crown decreased significantly in May and July compared to the enzyme activity in April. In both the low and middle parts of the crown, the acid phosphatase activity had a peak in August, and thereafter decreased in September and October. No correlations between acid phosphatase activity and phosphorus concentrations were found. Alkaline phosphatase activity was very low and in some cases near the detection limit during the whole observation period.