Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability

A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. Imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.     

Oryzacystatin-II, a cystatin from rice (Oryza sativa L. japonica), is a dimeric protein: possible involvement of the interconversion between dimer and monomer in the regulation of the reactivity of oryzacystatin-II

We examined the biochemical and structural properties of oryzacystatin-II, a phytocystatin in rice (Oryza sativa L. japonica), under heat-stress conditions. The enzyme inhibitory reactivity of oryzacystatin-II was enhanced by heating in a temperature-dependent manner and reached a maximum level by heating at 65 degrees C for 10 min. Size-exclusion chromatography showed that oryzacystatin-II forms a homodimer at ambient temperature and that the enhancement of inhibitory reactivity is due to the conversion of the dimeric to a monomeric form. The monomeric form of oryzacystatin-II reverted to the dimer during storage at 4 degrees C, suggesting that dimerization is an intrinsic property of oryzacystatin-II. The affinity of the monomer for cysteine proteinases was significantly higher than that of the dimer. This is the first paper to describe the noncovalent dimerization for a cystatin under nonstress conditions.     

镉胁迫对玉米幼苗抗氧化酶系统及矿质元素吸收的影响

通过水培实验研究了在10-6、10-5、10-4mol·L-1浓度下,Cd2＋胁迫对玉米幼苗抗氧化酶系统、丙二醛（MDA）含量、Cd积累及矿质元素吸收的影响,同时讨论了Cd2＋毒害的作用机理。结果表明,中、低浓度（10-6、10-5mol·L-1）Cd2＋胁迫下,玉米幼苗MDA含量与对照组相比没有显著变化;高浓度（10-4mol·L-1）Cd2＋胁迫后,MDA含量显著升高（P〈0.05）。Cd2＋胁迫初期,超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性均升高;随着胁迫时间的延长,除叶片中SOD和CAT活性略有上升外,其他抗氧化酶活性逐渐下降,高浓度组抗氧化酶活性下降最明显。植物吸收的Cd主要积累在根部,Cd2＋胁迫能干扰玉米对Fe、Cu、Zn、Mg等养分的吸收。     

Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.     

Enzymatic dehydrogenative polymerization of urushiols in fresh exudates from the lacquer tree, Rhus vernicifera DC

Fresh exudates from the lacquer tree, Rhus vernicifera DC, were extracted with acetone and the solution was chromatographed to isolate monomer, dimer, trimer, and oligomer fractions of urushiols. Constituents of the monomeric and dimeric fractions were then identified by two-dimensional (2D) 1H-13C heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC) NMR spectroscopic techniques. The results showed that the monomeric fraction contained 3-[8'Z,11'E,13'Z-pentadecatrienyl]catechol (1), 3-[8'Z,11'Z,14'-pentadecatrienyl]catechol (2), and 3-pentadecanyl]catechol (3), which was verified by HPLC analysis. The dimeric fraction contained 8'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[9'E,11'E,13'Z-pentadecatrienyl]catechol (4), 14'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecatrienyl]catechol (5), 2-hydroxyl-3- or -6-alkenylphenyl ethyl ether (6), 14'-(3' ',4' '-dihydroxy-2' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadeca-trienyl]catechol (7), 15'-(2' '-hydroxy-3' '- or -6' '-alkenyl)phenyloxy-3-[8'Z,11'Z,13'E)-pentadecatrienyl]catechol (8), 14'-(2' ',3' '-dihydroxy-4' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecantrienyl]catechol (9), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,3'-biphenyl (10), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,4'-biphenyl (11), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-5,4'-biphenyl (12), and 1,2,1'-trihydroxy-6,6'-dialkenyldibenzofuran (13) as constituents. In addition, dimeric ethers and peroxides, such as compounds 14 and 15, were produced by autoxidation of monomeric urushiols in atmospheric air. The possible reaction mechanisms for the dehydrogenative polymerization of urushiols by Rhus laccase present in the fresh raw exudates under the atmospheric oxygen are discussed on the basis of structures identified. This is of primary importance because the use of the urushi exudates as coating materials does not involve organic solvents and is an environmentally friendly process.     

重金属胁迫对水稻不同品种超氧化物歧化酶的影响

用不同浓度的Cd2 + 、Cu2 + 和Hg2 + 处理花期的扬稻 6号 (籼稻 )、武育粳 8号 (粳稻 )和协优 81 8(杂交稻 ) ,测定各器官SOD活性 ,并进行SOD同功酶的电泳分析。结果表明 ,较高浓度重金属显著抑制各品种根系SOD活性 ,也抑制叶片SOD活性 ,但抑制程度因重金属种类和品种类型而异。各浓度均抑制武育粳穗SOD活性 ,但诱导扬稻 6号穗SOD活性增高。协优 81 8穗SOD活性受Cd2 + 抑制 ,但却受高浓度Cu2 + 和Hg2 + 诱导。≥ 2 0ppmCd2 + 、Cu2 + 和Hg2 + 均抑制水稻根系SOD同功酶表达 ,但诱导扬稻 6号和协优 81 8穗SOD同功酶表达。Mn SOD和Cu Zn SOD同功酶对重金属胁迫的敏感性不同 ,重金属对水稻根系Mn SOD的抑制程度明显高于Cu Zn SOD。     

Capability of Lactobacillus plantarum IFPL935 to catabolize flavan-3-ol compounds and complex phenolic extracts

Lactobacillus plantarum IFPL935 was incubated with individual monomeric flavan-3-ols and dimeric A- and B-type procyanidins to identify new metabolites and to determine the effect of compound structural features on bacterial growth and catabolism. Complex extracts rich in A-type proanthocyanidins and phenolic acids from cranberry were also tested. The results showed that L. plantarum IFPL935 exhibited higher resistance to nongalloylated monomeric flavan-3-ols, A-type dimeric procyanidins, and cranberry extract than to (-)-epicatechin-3-O-gallate and B-type dimeric procyanidins. Despite these findings, the strain was capable of rapidly degrading (-)-epicatechin-3-O-gallate, but not A- or B-type dimeric procyanidins. However, it was able to produce large changes in the phenolic profile of the cranberry extract mainly due to the catabolism of hydroxycinnamic and hydroxybenzoic acids. Of most relevance was the fact that L. plantarum IFPL935 cleaved the heterocyclic ring of monomeric flavan-3-ols, giving rise to 1-(3',4'-dihydroxyphenyl)-3-(2″,4″,6″-trihydroxyphenyl)propan-2-ol, activity exhibited by only a few human intestinal bacteria.     

Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity

Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.     

Copper and zinc uptake by celery plants grown on acidic soil amended with biosolids

For trace elements, such as copper (Cu) and zinc (Zn), the bioavailability of these elements, Cu and Zn, in biosolids is important because both are essential elements and both are potential contaminants when biosolids are land applied. A greenhouse study was conducted in factorial experiment based on a completely randomized design (CRD) with four replications on a soil treated with four rates of Cu (0, 50, 100, and 150 mg/kg) and four rates of Zn (0, 150, 300, and 450 mg/kg) on celery plants to investigate the distribution and mobility of these elements as well as growth and antioxidant changes of celery. The results of antioxidant changes were inconclusive due to irregular changes with Zn and Cu applications. However, generally the results show that Cu did not affect superoxide dismutase (SOD) or peroxidase (POD) activities in most of the treatments. On the other hand, Zn stimulated SOD and POD activities in most of the treatments. The photosynthesis rate decreased with the applications of Cu and Zn at the rates above 100 and 300 mg/kg and increased in low Cu concentration (50 mg/kg) compared to S (soil without biosolid).     

硅作用下铜对小麦幼苗生理特性影响研究

通过水培试验,研究了在不同加硅量(0,30,60,90,120,150 mg/L,以SiO2计)作用下,铜(10 mg/L,以Cu计)对小麦幼苗叶绿素含量、抗氧化酶系统活性(SOD、POD、CAT)、脯氨酸含量、可溶性蛋白含量以及MDA含量的影响,结果表明:在加硅量(30,60,90 mg/L,以SiO2计)作用下,小麦幼苗叶绿素a含量、抗氧化酶系统(SOD、POD)活性、脯氨酸含量、可溶性蛋白含量的升高以及MDA含量的降低,减轻了铜对小麦幼苗的毒害作用;在加硅量150 mg/L作用下,小麦幼苗叶片可溶性蛋白含量的降低,MDA含量的升高,加重了铜对小麦幼苗的毒害作用。研究结果表明,加硅处理能够在一定浓度范围内缓解铜对小麦幼苗的毒害作用,从而为铜毒害的小麦区域增施硅肥提供理论依据。     

Detection of Sclerotinia sclerotiorum using a monomeric and dimeric single-chain fragment variable (scFv) antibody

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus capable of causing significant yield losses in numerous crops, including canola, in which the fungus causes sclerotinia stem rot. Immunological detection methods to rapidly determine the presence of S. sclerotiorum on plants may provide growers with a viable diagnostic tool to aid with fungicide use decisions. This paper discusses the generation of a monomeric and dimeric single-chain, variable fragment (scFv) antibody with affinity for S. sclerotiorum using phage display technology. The bacterially expressed and purified scFv is shown to bind S. sclerotiorum with some cross-reactivity with the closely related phytopathogen Botrytis cinerea (Pers.:Fr.). The dimeric scFv displayed improved binding to the fungus as compared to the monomer and could detect the presence of mycelia in inoculated canola petals. To the authors' knowledge, this is the first report of a scFv dimer with affinity for S. sclerotiorum that has the potential for use in the development of a new diagnostic test.     

Dehydroascorbate reductase cDNA from sweet potato (Ipomoea batatas [L.] Lam): expression, enzyme properties, and kinetic studies

A cDNA encoding a putative dehydroascorbate reductase (DHAR) was cloned from sweet potato. The deduced protein showed a high level of sequence homology with DHARs from other plants (67 to approximately 81%). Functional sweet potato DHAR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 12% native PAGE. The protein's half-life of deactivation at 50 degrees C was 10.1 min, and its thermal inactivation rate constant K(d) was 6.4 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 6.0-11.0 and in the presence of 0.8 M imidazole. The K(m) values for DHA and GSH were 0.19 and 2.38 mM, respectively.     

鸡冠花品种间耐热差异与超氧化物歧化酶(SODs)之间的关联

为研究鸡冠花（Celosia cristata）品种间耐热差异与超氧化物歧化酶(SOD)之间的关联,选用鸡冠花耐热品种世纪绿叶和热敏品种世纪铜叶的10叶期幼苗为试材,用二乙基二硫代氨基甲酸钠 (DDTC) 进行48 h预处理之后,在45 ℃、 相对湿度75%~90%,、光照1 500 1x (12 h/d)条件下进行热胁迫处理,研究其形态和生理生化的变化。结果表明,20 mmol/L DDTC预处理能显著抑制幼苗叶片SOD活性,明显降低鸡冠花幼苗的热胁迫耐性,热敏品种受影响程度明显大于耐热品种;经高温梯度胁迫处理后,SOD同工酶条带的亮度变化为先增强后降低,亮度变化最明显的是MnSOD,其次是Cu/ZnSOD。热敏品种在35 ℃诱导产生了1条新的Cu/ZnSOD条带,而耐热品种则在40 ℃时出现此条带;当温度达到45 ℃时,耐热品种留下FeSOD2、微弱的FeSOD3和Cu/ZnSOD1条带,而热敏品种仅剩FeSOD2,其它条带基本消失,表明耐热品种的SOD同工酶在高温下较热敏品种具有更强的活性和稳定性。DDTC预处理抑制了任何与上述热胁迫诱导有关的新的条带产生。由此结果表明,鸡冠花热胁迫耐性与SOD活性相关联,品种间热胁迫耐性差异与FeSOD3和Cu/ZnSOD1活性强度相关联。     

重组甘蔗1-氨基环丙烷-1-羧基(ACC)氧化酶的纯化及其特性

在IPTG诱导下,重组甘蔗(Sacharum sinensis)1-氨基环丙烷-1-羧基(ACC)氧化酶在体外以包涵体蛋白形式表达。包涵体蛋白经Ni2+-NTA金属螯合层析柱纯化和复性后,获得酶活力高达132.58 nmolC2H4/ mg/h 蛋白。该蛋白在pH 5.5 ~ 8.0 和20 ~ 45 ℃温度范围内比较稳定,最适pH 6.7,最适温度33 ℃;米氏常数(Km )61.77 μmol/L,酶动力学参数（Vm）值0.9647 μmol/min,被一定浓度的CO2和底物ACC所激活,受Mg2+、Cu2+、Zn2+和EDTA抑制。     

Soil and plant nutrient management and fruit production of papaya (Carica papaya) in Keaau,Hawaii

Abstract

Variations in papaya yields and fruit quality between papaya growers in Keaau, Hawai’i are mainly caused by differences in nutrient management. The objectives of this study were to (1) identify soil chemical properties and plant mineral compositions and their correlations in commercial papaya orchards; (2) compare soil chemical properties and plant nutrient concentrations between papaya orchards with low, medium and high fruit yields; (3) compare soil chemical properties and plant nutrient composition between orchards with low, medium and high fruit quality; (4) determine the difference in soil chemical properties, plant nutrient composition, and papaya yield and quality between new and long-term papaya land. Between 2016 and 2018, soil and plant tissue samples were collected from 100 commercial papaya orchards. Soil pH, potassium (K), calcium (Ca), and magnesium (Mg) were below and phosphorus (P) was above the recommended range for papaya. Petiole P, K, Ca, and sulfur (S) concentrations were below the recommended range. Fields with low fruit yields and low fruit quality were deficient in P, K, Ca, and zinc (Zn). Long-term papaya land had significantly (p?<?0.05) lower soil pH, total carbon (C), K, Ca, Mg, and sodium (Na), and higher concentrations of soil P than new papaya land. Nitrogen (N), boron (B), zinc (Zn), and copper (Cu) was significantly higher and Ca was significantly lower in petioles from papaya crops grown on long-term papaya land compared to new papaya land.     

Root-zone temperature influences the distribution of Cu and Zn in potato-plant organs.

Root-zone temperatures (RZT) in relation to Cu and Zn uptake and tissue accumulation, and to total biomass, in potato plants (Solanum tuberosum L. var. Spunta) were studied. Using five different plastic mulches (no cover, transparent polyethylene, white polyethylene, coextruded white-black polyethylene, and black polyethylene) resulted in significantly different RZT (16, 20, 23, 27, and 30 degrees C, respectively). These RZT significantly influenced Cu and Zn content (concentrated) and the biomass in various potato organs. Root-zone temperature at 20 degrees C resulted in significantly high Cu content in leaflets, and soluble Cu content in leaflets and stems, whereas 23 and 27 degrees C resulted in significantly high Cu content in roots. However, RZT had no effect on Cu content in tubers or stems or on soluble Cu in roots or tubers. The RZT at 20 degrees C resulted in significantly high Zn and soluble Zn in stems, roots, and tubers; whereas, at 27 degrees C Zn and soluble Zn content were significantly highest in leaflets. The most biomass occurred in roots and tubers at 27 degrees C; whereas in leaves and stems, the RZT influence was highly variable. Total accumulation of both Cu forms was affected by RZT at 20 degrees C, with roots and tubers having significantly the least Cu and stems and leaflets having the most. Total accumulation of both Zn forms by RZT in potato organs was highly variable, but tubers consistently accumulated the most.     

铜对伴矿景天生长及锌镉吸收性的影响

利用植物生长室水培试验和温室土培盆栽试验相结合,研究了Cu对Zn、Cd超积累植物伴矿景天生长及Zn、Cd吸收性的影响。水培试验结果显示,0.31～50μmol/L Cu处理14天对伴矿景天生长及对Zn、Cd吸收性没有显著影响;但100μmol/L Cu处理显著抑制植物生长,降低地上部Zn、Cd及根中Cd浓度,对根中Zn浓度变化没有显著影响。盆栽试验结果发现,在土壤Cu仅为3.61 mg/kg时伴矿景天生长不良,外加Cu显著促进其生长并随Cu浓度升高效应增加;但施用3 mmol/kgEDDS和再次外加250 mg/kg Cu处理使伴矿景天因体内Cu积累量过高而导致明显毒害,地上部Cu最高达1 068 mg/kg。可见低量Cu处理可促进伴矿景天生长,利于植物对土壤Zn、Cd的吸取修复,但土壤中Cu浓度过高将抑制Zn、Cd超积累植物的生长,降低其Zn、Cd吸收能力,在利用该Zn、Cd超积累植物修复高Cu的Zn和Cd污染土壤时应采取适当措施降低Cu毒害效应。     

Metal contamination effects on sunflower (Helianthus annuus L.) growth and protein expression in leaves during development

Metal-ion contamination (Cd, Cu, Pb, and Zn) on sunflower (Helianthus annuus L.) growth and total leaf protein expression were studied in the present work. The height, mass production, and metal distribution (Ca, K, Fe, Mg, Na, and P) in all plant fractions (roots, stems, and leaves) were evaluated. Sunflowers plants contaminated with four metal ions decreases height and mass by 35% and 40%, respectively, compared to control. Significant differences of total protein composition were noted after SDS-PAGE separation. Sunflower proteomics were more affected when 500 mg L(-1) of metal ion was added as contaminant of both zinc and mixed ions solution. In these cases, proteins having a molar mass of 14.5, 34.5, and 54.0 kDa were present at a lower level and alterations in enzymatic activities (SOD and GR) were found. Sunflowers plants contaminated with zinc and the mixed ions solution showed some degree of oxidative stress.