Contamination of oat mesophyll protoplasts by apoplastic polyamine oxidase

Mesophyll protoplasts isolated from peeled oat ( Avena sativa L. cv Victory) leaves with 1% (w/v) Cellulysin in 20 m M KPO₄, pH 5.5 and 0.6 M sorbitol retain about 6% of the polyamine oxidase (PAO, EC 1.4.3.4) activity of the whole peeled leaf. However, more than 99% of the oat leaf PAO activity is apoplastic and can be extracted by vacuum infiltration with 200 m M NaCl and this procedure extracts no activity for the cytoplasmic marker enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49). By these criteria we consider PAO in oat leaves to be totally apoplastic and PAO found in the isolated protoplast to be contamination. The degree of protoplast contamination by PAO depends on the pH and ionic strength of the isolating and washing medium. It can be eliminated by washing protoplasts in 0.6 M sorbitol with 100 m M KPO₄, pH 6.5. Pellets of lysed protoplasts incubated with dialyzed apoplastic enzymes in 5 m M KPO₄, pH 5.5 adsorb about 87% of the added PAO activity but only about 25% of the added peroxidase (EC 1.11.1.7) activity. The adsorbed activity can be solubilized from the pellet by extraction with 1 M NaCl. The results demonstrate that weakly ionically bound cell wall enzymes may contaminate protoplasts isolated and purified by conventional techniques.     

Corn Leaf Isoperoxidase Reaction to Mechanical Injury and Infection with Helminthosporium maydis: Effects of Cycloheximide

Mechanical injury or infection with Helminthosporium maydis race T or O enhanced peroxidase activity in leaves of two corn inbreds which differ in their susceptibility to the fungal race T. Increases in activity were found in the soluble fraction extracted from tissues with 20 mm phosphate buffer (pH 6), and in the ionically bound fraction extracted from wall debris with 0.6 to 1 m NaCl; the covalently bound wall peroxidase fraction was unaffected. Mechanical injury and infection with either race enhanced the same distinctive cathodic isoforms present in the soluble fraction or in both the soluble and ionically bound fractions.     

Peroxidase activities and isoenzyme profiles associated with development of avocado (Persea americana,M.) leaves at different ontogenetic stages

Changes in four peroxidase activity fractions (soluble, membrane-bound, as well as ionically and covalently bound) were studied during development of juvenile and adult avocado leaves. Greater differences were found in the soluble fraction with an increase in total activity at the end of the growth phase. In relation to the ontogenetic stages, there were significant variations in the soluble peroxidase activity of both stages, especially in leaves which have already detained their growth, 263 U/g fresh wt in adult leaves vs. 70 U/g fresh wt juvenile leaves. Moreover, the isozyme profile of this fraction revealed the appearance of an anionic band, R_f 0.35, at much earlier stages in juvenile than in adult leaves. Concerning the other three fractions, there were no marked changes in total activity of either membrane-bound or ionically and covalently bound peroxidases. However, in the isoenzyme profiles of the ionically bound fraction of juvenile leaves, three highly cationic bands appeared at much earlier stages than in adult leaves. In avocado, attempts to use leaf peroxidase activity as marker of ontogenetic age must be taken with caution, since great fluctuations related with developmental stages occur in juvenile and adult leaves.     

Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa L.)

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.     

Cell Wall and Protoplast Isoperoxidases of Corn Leaves in Relation to Cut Injury and Infection with Helminthosporium maydis

Young leaves of two corn (Zea mays) inbreds with normal and Texas male-sterile cytoplasm, which differed in their susceptibility to Helminthosporium maydis Nisikado and Miyake race T, showed no significant qualitative or quantitative differences in their isoperoxidase patterns. Of six cathodic and four anodic isoenzymes present in the soluble fraction, four and two, respectively, comprised the fraction ionically bound to the cell wall. Peroxidase fractions ionically and covalently bound to the wall constituted about 20% of the total peroxidase activity. No new isoperoxidases were detected in either inbred line in response to cutting, infection, or detachment only and exposure to darkness for 40 hours. Three isoperoxidases, all cathodic, mainly reacted to cutting injury as well as fungal infection. One of the isoperoxidases appeared responsible for the increase in the peroxidase activity of the soluble fraction while the other two were responsible for the increase in that of the fraction ionically bound to the walls. The relative increase in the latter fraction was greater for infected leaves than for mechanically injured ones. No significant differences were found between the two inbreds in their peroxidase reactions to cutting injury or infection. Thus, the corn leaf isoperoxidases were distinctive in their distribution in the cell and in their reaction to injury. Changes in their activity induced by infection may result from a nonspecific response to injury.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

水分胁迫下荔枝叶片过氧化物酶和IAA氧化酶活性的变化

以适应山地栽培的抗旱性较强的东刘1号和适应河边栽培的抗旱性较弱的陈紫2年生荔枝（Litchi chinensis Sonn.)实生苗为试验材料,研究了水分胁迫下叶片细胞胞质,与（细胞）壁以离子键结合和壁以共价键结合的过氧化的酶（POD）和IAA氧化酶活性的变化。结果表明：在叶片中POD主要是以与壁以离子键结合的POD存在,占总活性的51．15％－52．15％,其次是细胞胞质POD,占44．20％－44．74％,与壁以共价键结合的POD活性最低,仅占3．44％－3．65％。与POD不同,IAA氧化酶绝大多数存在于细胞胞质中,占总活性的88．93％－89．29％,其次是少量的与壁以离子键结合的IAA氧化酶,占7．32％－7．63％,与壁以共价键结合的IAA氧化酶活性最低,仅占3．39％－3．44％;2个品种间差异不明显。水分胁迫下,叶片细胞胞质以及与壁以离子键和壁以共价键结合的POD和IAA氧化酶（比）活性均上升,抗旱笥较强的品种上升的幅度均大于抗旱性较弱的品种。     

Inducible and constitutive mechanisms of salt stress resistance in <Emphasis Type="Italic">Geum urbanum</Emphasis> L.

The avens (Geum urbanum L.) seedlings were grown for 6 weeks until the expansion of five to six leaves and then exposed to salinity shock (300 mM NaCl in the nutrient medium) or to a gradual (within 4 days) increase in NaCl concentration from 100 to 400 mM. The dynamics of stress-dependent accumulation of Na⁺, Cl^?, proline, and polyamines in leaves and roots was measured, together with activities of antioxidant enzymes, namely, superoxide dismutase (SOD) and guaiacol-dependent peroxidase occurring in soluble, ionically bound, and covalently bound forms. It is shown that avens plants can adapt to gradual salinization by mobilizing stressinducible protective mechanisms (accumulation of proline and spermine) and by activating constitutive enzyme systems (SOD and peroxidase).     

棉花叶片细胞间隙液的提取和鉴定

细胞间隙液中存在许多与植物的生长发育相关的物质,对于研究植物生理生化反应很重要。介绍一种提取棉花叶片的细胞间隙液的方法,操作简单,节省时间和材料。利用改造的对6-磷酸葡萄糖脱氢酶的硝基四氮唑蓝定性法对提取的细胞间隙液进行鉴定,结果显示得到了比较纯的细胞间隙液。     

Peroxidase and Glucose-6-phosphate Dehydrogenase Levels in Cotyledons of Cucumis sativus (L.)

Peroxidase and glucose-6-phosphate dehydrogenase become increasinglyactive in cucumber cotyledons excised from the plant althoughthere is a fall in total protein content. The increases areinhibited by dinitrophenol, D, and L-threo-chloramphenicol,cycloheximide, puromycinj and actinomycin D, and it is concludedthat the enzymes are synthesized de novo. Disc electrophoresisof cotyledon extracts revealed three bands of peroxidase, oneof which becomes much enhanced in excised cotyledons. Citrullineaccumulates markedly in excised cotyledons. If the shoot ofcucumber plants is cut away above the cotyledons, very largeincreases in peroxidase and glucose-6-phosphate dehydrogenaseare observed although yellowing and protein loss are delayed.     

Protein and Peroxidase Modulations in Sunflower Seedlings (Helianthus annuus L.) Treated with a Toxic Amount of Aluminium

The aim of this study is to investigate the effect of aluminium treatment on peroxidases activities and protein content in both soluble and cell-wall-bound fractions of sunflower leaves, stems and roots. Fourteen-day-old seedlings, grown in a nutrient solution, were exposed to a toxic amount of aluminium (500 μM AlNO₃) for 72 h. Under stress conditions, biomass production, root length and leaf expansion were significantly reduced. Also, our results showed modulations on soluble and ionically cell-wall-bound peroxidases activities. In soluble fraction, peroxidases activities were enhanced in all investigated organs. This stimulation was also observed in ionically cell-wall-bound fraction in leaves and stems. Roots showed a differential behaviour: peroxidase activity was severely reduced. Lignifying peroxidases activities assayed using coniferyl alcohol and H₂O₂ as substrates were also modulated. Significant stimulation was shown on soluble fraction in leaves, stems and roots. In ionically cell-wall-bound fraction lignifying peroxidases were enhanced only in stems but severely inhibited in roots. Also, aluminium toxicity caused significant increase on cell wall protein content in sunflower roots.     

Activity of native hydrolytic enzymes and their association with the cell wall of three ectomycorrhizal fungi

The ecological and biogeochemical relevance of hydrolytic enzymes associated with the fungal cell wall has been poorly studied in ectomycorrhizal (ECM) fungi. We used a modified sequential extraction procedure to investigate the activity of various hydrolytic enzymes (β-glucosidase, acid-phosphatase, leucine-aminopeptidase, chitinase, xylanase and glucuronidase) and their association with the cell wall of three ECM fungi (Rhizopogon roseolus, Paxillus involutus and Piloderma croceum). Fungi were grown on C-rich solid medium under three different P concentrations (3.7, 0.37 and 0.037 mM). The sequential extraction procedure classifies enzymes as: (a) cytosolic, (b) loosely bound, (c) hydrophobically bound, (d) ionically bound and (e) covalently bound. Results showed that for the same fungus absolute enzymatic activity was affected by P concentration, whilst enzymatic compartmentalization among the cytosol and the cell wall fractions was not. The association of enzymes with the cell wall was fungus- and enzyme-specific. Our data indicate also that enzymes best known for being either extracellular or cytosolic or both, do act in muro as well. The ecological implications of cell wall-bound enzymes and the potential applications and limitations of sequential extractions are further discussed.     

Antioxidants and Manganese Deficiency in Needles of Norway Spruce (Picea abies L.) Trees

Chlorotic and green needles from Norway spruce (Picea abies L.) trees were sampled in the Calcareous Bavarian Alps in winter. The needles were used for analysis of the mineral and pigment contents, the levels of antioxidants (ascorbate, glutathione), and the activities of protective enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate radical reductase, dehydroascorbate reductase, glutathione reductase). In addition, the activities of two respiratory enzymes (glucose-6-phosphate dehydrogenase, NAD-malate dehydrogenase), which might provide the NADPH necessary for functioning of the antioxidative system, were determined. We found that chlorotic needles were severely manganese deficient (3 to 6 micrograms Mn per gram dry weight as compared with up to 190 micrograms Mn per gram dry weight in green needles) but had a similar dry weight to fresh weight ratio, had a similar protein content, and showed no evidence for enhanced lipid peroxidation as compared with green needles. In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased, whereas the levels of ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, and glutathione were not affected. The ratio of ascorbate to dehydroascorbate was similar in both green and chlorotic needles. These results suggest that in spruce needles monodehydroascorbate radical reductase is the key enzyme involved in maintaining ascorbate in its reduced state. The reductant necessary for this process may have been supplied at the expense of photosynthate.     

Soluble peroxidase in fluid from the intercellular spaces of tobacco leaves

A high proportion of the soluble peroxidase of tobacco (Nicotiana tabacum L. var. Bottom Special) leaves is found in the fluid obtained by centrifugation of a buffer solution previously infiltrated into the intercellular spaces. Only a very small amount of the cytoplasmic enzyme, glucose 6-phosphate dehydrogenase, is present in this fluid. Specific activity data suggest that an active process is responsible for the transfer of soluble peroxidase to the intercellular space and that the intercellular fluid fraction is not simply composed of material moving out of leaf cells by diffusion. The centrifugation method is a satisfactory means of isolating diluted intercellular fluid for biochemical and physiological investigations.     

Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L

Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process.     

Subcellular localization of glucose-6-phosphate dehydrogenase in tobacco mesophyll protoplasts

Subcellular localization of glucose-6-phosphate dehydrogenase (EC 1.1.1.49.) isoenzymes was determined in mesophyll protoplasts prepared from Nicotiana tabacum L. cv. Samsun. Intact chloroplasts and soluble cytosolic proteins were obtained by means of differential centrifugation. The 1000 g pellet contained 97 % of chloroplasts and 16.8 ± 2.1 % of the total activity of glucose-6-phosphate dehydrogenase. The rest of the enzyme was localized in the cytosol which also contained 91 % of the total activity of phosphoenolpyruvate carboxylase.     

Isolation and Expression Analysis of Plastidic Glucose-6-phosphate Dehydrogenase Gene from Rice (Oryza sativa L.)

Glucose-6-phosphate dehydrogenase is a rate-limiting enzyme of pentose phosphate pathway, existing in cytosolic and plastidic compartments of higher plants. A novel gene encoding plastidic glucose-6-phosphate dehydrogenase was isolated from rice (Oryza sativa L.) and designated OsG6PDH2 in this article. Through semiquantitative RT-PCR approach it was found that OsG6PDH2 mRNA was weakly expressed in rice leaves, stems, immature spikes or flowered spikes, and a little higher in roots. However, the expression of OsG6PDH2 in rice seedlings was significantly induced by dark treatment. The complete opening reading frame (ORF) of OsG6PDH2 was inserted into pET30a (+), and expressed in Escherichia coli strain BL21 (DE3). The enzyme activity assay of transformed bacterial cells indicated that OsG6PDH2 encoding product had a typical function of glucose-6-phosphate dehydrogenase.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

Changes in Composition of Soluble Intercellular Proteins Isolated from Healthy and TMV-Infected Nicotiana tabacum L. cv. Xanthi-nc

Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), polyphenoloxidases, peroxidases and proteases activity and PR-proteins composition in leaf tissue and intercellular fluid (ICF) isolated from leaf tissue of healthy and TMV-infected hypersensitive tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants (non-inoculated leaves) were studied. The amount of the proteins and the enzymes of intercellular space was less than 3 % of the total amount of proteins and the enzymes found in homogenate of healthy leaves. The TMV infection did not significantly change this observation. The great increase in the activities of the enzymes was observed in homogenates of the infected leaves, especially of the enzymes involved in biosynthesis of precursors needed for virus multiplication (G6P DH, RNase, PME, PDE). This is in contrast with the activities of the enzymes of ICF, which were only partly increased. The ICF proteins of infected plants were separated by means of ion exchange chromatography on DEAE cellulose. The isozymes of peroxidase, polyphenoloxidase, PME and PDE were identified. Using discontinuous nondenaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions, the detection of isozymes of peroxidases and PR-proteins was performed. By means of SDS-PAGE the molecular masses of PR-proteins were identified: 15 – 16 kDa (group 1), 27 – 28 kDa (group 3: chitinases) and 36 – 40 kDa (group 2a: -1,3-glucanases).