Copia and Gypsy retrotransposons activity in sunflower (Helianthus annuus L.)

Background  

Retrotransposons are heterogeneous sequences, widespread in eukaryotic genomes, which refer to the so-called mobile DNA. They resemble retroviruses, both in their structure and for their ability to transpose within the host genome, of which they make up a considerable portion. Copia- and Gypsy-like retrotransposons are the two main classes of retroelements shown to be ubiquitous in plant genomes. Ideally, the retrotransposons life cycle results in the synthesis of a messenger RNA and then self-encoded proteins to process retrotransposon mRNA in double stranded extra-chromosomal cDNA copies which may integrate in new chromosomal locations.     

Effect of perfluidone on metabolism of lipids in maize (Zea mays L.) and sunflower (Helianthus annuus L.)

Treatment of germinating maize seedlings with 0.126 mM of the herbicide Perfluidone (Destun) (1,1,1-trifluoro-4-[phenylsulfonyl]-methanesulfona-o-toluidide) for 2 days in the dark, then 3 days in the light, at 25°C causes decreases in fresh weight, dry weight, shoot length, and in total chlorophyll and carotenoid contents; in contrast, sunflower seedlings seem not to be affected. Perfluidone causes marked decreases in total lipids and in glyco- and phospholipids of maize seedlings. In sunflower cotyledons, total lipids and pigments (chlorophyll, carotenoids) are not affected, but there is an increase in glycolipids at the expense of phospholipids. After Perfluidone treatment, a significant increase in the fatty acid mole ratio (180+181+182)/183 was found for the maize glycolipids, monogalactosyl diacylglycerol (MGD), digalactosyl diacylglycerol (DGD), and sterol glycoside (SG) + esterified sterol glycoside (ESG), and for the phospholipid, phosphatidylcholine (PC). In sunflower seedlings, however, only the fatty acid mole ratio of ESG + SG showed an increase and that of phosphatidylserine (PS) showed a large decrease. The differential response of the two plant species to Perfluidone suggests that the control of linolenic acid biosynthesis may vary depending on plant species and/or on plant tissues.On leave from the Department of Botany, University of London, Royal Holloway College, Egham, Surrey TW20 OEX England     

Characterization of glycolytic initial metabolites and enzyme activities in developing sunflower (Helianthus annuus L.) seeds

Unlike other oilseeds (e.g. Arabidopsis), developing sunflower seeds do not accumulate a lot of starch and they rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Between 10 and 25 days after flowering (DAF), when sunflower seeds form and complete the main period of storage lipid synthesis, the sucrose content of seeds is relatively constant. By contrast, the glucose and fructose content falls from day 20 after flowering and it is always lower than that of sucrose, with glucose being the minor sugar at the end of the seed formation. By studying the apparent kinetic parameters and the activity of glycolytic enzymes in vitro, it is evident that all the components of the glycolytic pathway are present in the crude seed extract. However, in isolated plastids important enzymatic activities are missing, such as the glyceraldehyde-3-phosphate dehydrogenase, involved in the conversion of glyceraldehyde 3-phosphate into 1,3-biphospho-glycerate, or the enolase that converts 2-phosphoglycerate into phosphoenolpyruvate. Hence, phosphoenolpyruvate or one of its derivatives, like pyruvate and malate from the cytosol, may be the primary carbon sources for lipid biosynthesis. Accordingly, the glucose-6-P imported into the plastid is likely to be used in the pentose phosphate pathway to produce the reducing power for lipid biosynthesis in the form of NADPH. Data from crude seed extracts indicate that enolase activity increased during seed formation, from 16 days after flowering, and that this activity was well correlated with the period of storage lipid synthesis. In addition, while the presence of some glycolytic enzymes increased during lipid synthesis, others decreased, remained constant, or displayed irregular temporal behaviour.     

Immunogold labelling indicates high catalase concentrations in amorphous and crystalline inclusions of sunflower (Helianthus annuus L.) peroxisomes

Summary Immunogold labelling and electron microscopy were used to investigate whether catalase was present in peroxisomal inclusions, the composition of which has not yet been determined in plant cells. In the mesophyll cells of sunflower (Helianthus annuus L.) cotyledons, the catalase gold label was confined to peroxisomes. At day 2 of postgerminative growth in darkness, peroxisomes were free of inclusions, and the matrix was homogeneously labelled with gold particles. Thereafter, amorphous inclusions appeared, but by day 5 of growth, conspicuous crystalline inclusions (cores) were the predominant type. This developmental change, first observed in cotyledons grown in continuous light between day 2.5 and 5, also took place in cotyledons kept in permanent darkness. Both amorphous and crystalline inclusions showed a much higher immunogold label than did the peroxisomal matrix, indicating that catalase was a component of both types of peroxisomal inclusions. In contrast to catalase, the immunogold label of glycolate oxidase was almost completely absent from cores and was confined to the peroxisomal matrix. Together with reports on the absence of other enzymes from peroxisomal inclusions in sunflower and other species (Vaughn, 1989) our results suggest that catalase is a major constituent of amorphous and crystalline peroxisomal inclusions in plants.     

向日葵E3泛素连接酶基因克隆及表达分析

该研究从向日葵中克隆了E3泛素连接酶基因HERC2,并进行了生物信息学分析和不同胁迫条件的表达分析。序列分析表明,HERC2(登录号为KT832066)序列的CDS为1 608bp,编码535个氨基酸,预测其分子量131kD,等电点为5.03。HERC2编码的蛋白质为疏水性蛋白质,且为细胞质蛋白;亚细胞定位预测分析表明,向日葵HERC2可能定位在高尔基体中;该蛋白质有5个RCC1保守结构域。向日葵HERC2与已报道的其他植物同源蛋白有相似的保守区域,与醉蝶花亲缘关系最近,而与大豆和野生大豆的亲缘关系最远。与HERC2cDNA对应的gDNA(登录号为KT832067)的ORF长度为3 409bp,与cDNA编码序列比对结果表明,该gDNA由5个外显子和4个内含子组成。实时荧光定量PCR分析表明,向日葵HERC2基因表达受非生物胁迫调节,在不同器官及不同非生物胁迫下存在特异性表达差异。研究认为,HERC2基因应答逆境胁迫具有其特定的表达模式,研究结果为加强对HERC2的利用奠定了基础。     

Mycorrhizal fungi enhance accumulation and tolerance of chromium in sunflower (Helianthus annuus)

Chromium (Cr) is a heavy metal risk to human health, and a contaminant found in agricultural soils and industrial sites. Phytoremediation, which relies on phytoextraction of Cr with biological organisms, is an important alternative to costly physical and chemical methods of treating contaminated sites. The ability of the arbuscular mycorrhizal fungus (AM),Glomus intraradices, to enhance Cr uptake and plant tolerance was tested on the growth and gas exchange of sunflower (Helianthus annuus L.). Mycorrhizal-colonized (AM) and non-inoculated (Non-AM) sunflower plants were subjected to two Cr species [trivalent cation (Cr³⁺) Cr(III) , and divalent dichromate anion (Cr₂O₇⁻) Cr(VI) ]. Both Cr species depressed plant growth, decreased net photosynthesis (A) and increased the vapor pressure difference; however, Cr(VI) was more toxic. Chromium accumulation was greatest in roots, intermediate in stems and leaves, and lowest in flowers. Greater Cr accumulation occurred with Cr(VI) than Cr(III). AM enhanced the ability of sunflower plants to tolerate and hyperaccumulate Cr. At higher Cr levels greater mycorrhizal dependency occurred, as indicated by proportionally greater growth, higherA and reduced visual symptoms of stress, compared to Non-AM plants. AM plants had greater Cr-accumulating ability than Non-AM plants at the highest concentrations of Cr(III) and Cr(VI), as indicated by the greater Cr phytoextraction coefficient. Mycorrhizal colonization (arbuscule, vesicle, and hyphae formation) was more adversely affected by Cr(VI) than Cr(III), however high levels of colonization still occurred at even the most toxic levels. Arbuscules, which play an important role in mineral ion exchange in root cortical cells, had the greatest sensitivity to Cr toxicity. Higher levels of both Cr species reduced leaf tissue phosphorus (P). While tissue P was higher in AM plants at the highest Cr(III) level, tissue P did not account for mycorrhizal benefits observed with Cr(VI) plants.     

油葵HaGPAT1基因的克隆及表达分析

该研究利用RT-PCR技术,从油葵(Helianthus annuus L.)种子中克隆了甘油-3-磷酸酰基转移酶(GPAT)基因(HaGPAT1),对其进行生物信息学分析,并通过实时荧光定量PCR技术(qRT-PCR)检测该基因在不同组织、种子不同发育时期以及不同胁迫条件下的表达特征。结果表明:HaGPAT1基因全长为1 656bp,编码551个氨基酸,相对分子量为62.132kD,等电点为8.84。系统进化树分析表明,HaGPAT1蛋白与高等植物莴苣的GPAT1亲缘关系最近。qRT-PCR分析表明,HaGPAT1基因在油葵花蕊中的表达水平最高,开花后17d的种子中次之;在干旱和盐胁迫条件下,HaGPAT1基因的表达水平均显著上调。研究推测,HaGAT1基因可能在油葵花器官发育中发挥重要作用,并且参与了油葵对干旱和高盐的抗性调节。     

向日葵E3泛素连接酶基因的分析定位和表达鉴定

该研究利用前期获得的向日葵耐盐相关基因E3泛素连接酶基因序列(HERC2),构建瞬时表达载体Cam-35S-HERC2-GFP,采用基因枪法转化洋葱表皮细胞进行亚细胞定位;采用RT-PCR技术,分析盐胁迫下HERC2在耐盐品种P50和盐敏感品种P29根、下胚轴和叶中的表达差异;构建HERC2植物表达载体pPZP221-HERC2,采用农杆菌介导法将HERC2导入烟草,进行耐盐功能验证。结果表明:(1)HERC2蛋白定位在细胞膜、细胞质和细胞核中。(2)受到NaCl胁迫后,HERC2基因在耐盐品种P50和盐敏感品种P29中均上调表达,但耐盐品种中的表达量较高。(3)HERC2基因的表达,能够提高转基因烟草的耐盐性。该研究结果为进一步解析向日葵对盐胁迫的响应机制,以及耐盐新品种的选育奠定了基础。     

Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.)

Peroxidase from date palm (Phoenix dactylifera L.) leaves was purified to homogeneity and characterized biochemically. The enzyme purification included homogenization, extraction of pigments followed by consecutive chromatographies on DEAE-Sepharose and Superdex 200. The purification factor for purified date palm peroxidase was 17 with 5.8% yield. The purity was checked by SDS and native PAGE, which showed a single prominent band. The molecular weight of the enzyme was approximately 55 kDa as estimated by SDS–PAGE. The enzyme was characterized for thermal and pH stability, and kinetic parameters were determined using guaiacol as substrate. The optimum activity was between pH 5–6. The enzyme showed maximum activity at 55 °C and was fairly stable up to 75 °C, with 42% loss of activity. Date palm leaves peroxidase showed K_m values of 0.77 and 0.045 mM for guaiacol and H₂O₂, respectively. These properties suggest that this enzyme could be a promising tool for applications in different analytical determinations as well as for treatment of industrial effluents at low cost.     

油葵HaFAD2-2基因的克隆及表达分析

脂肪酸脱氢酶2(fatty acid desaturase,FAD2)催化油酸生成亚油酸,是植物体内生成多不饱和脂肪酸的关键酶。根据已报道的向日葵(Helianthus annuus L.)FAD2基因序列,设计引物进行RT-PCR,克隆得到油葵FAD2-2基因全长cDNA,命名为HaFAD2-2。该基因开放阅读框为1 152bp,编码383个氨基酸,相对分子质量43.96kD,等电点为8.56。对基因组进行内含子调查发现,该基因在编码区内没有内含子。多序列比对和系统进化分析发现,FAD2-2基因编码蛋白与金盏菊(Calendula officinalis)、斑鸠菊(Vernonia galamensis)等菊科植物具有较近的亲缘关系。qRT-PCR分析表明,HaFAD2-2基因在根、茎、叶、花、子叶和未成熟种子中均有表达,且以叶中的表达量最高,未成熟种子中的表达量最低;低温(5℃、15℃)胁迫处理能显著促进该基因在根中的表达,抑制其在叶中的表达;盐胁迫(300 mmol/L NaCl)处理对其表达也具有抑制作用。该研究结果可为进一步探讨HaFAD2-2基因的功能奠定基础。     

Purification and properties of an aminopeptidase from the mid-gut gland of scallop (Patinopecten yessoensis)

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn²⁺. Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K_m of 32.2 μM and k_cat of 29.5 s⁻¹ with Ala-pNA and a K_m of 11.1 μM and k_cat of 9.49 s⁻¹ with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.     

Purification and characterization of a nitrilase from Fusarium solani O1

An intracellular nitrilase was purified from a Fusarium solani O1 culture, in which the enzyme (up to 3000 U L⁻¹) was induced by 2-cyanopyridine. SDS-PAGE revealed one major band corresponding to a molecular weight of approximately 40 kDa. Peptide mass fingerprinting suggested a high similarity of the protein with the putative nitrilase from Gibberella moniliformis. Electron microscopy revealed that the enzyme molecules associated into extended rods. The enzyme showed high specific activities towards benzonitrile (156 U mg⁻¹) and 4-cyanopyridine (203 U mg⁻¹). Other aromatic nitriles (3-chlorobenzonitrile, 3-hydroxybenzonitrile) also served as good substrates for the enzyme. The rates of hydrolysis of aliphatic nitriles (methacrylonitrile, propionitrile, butyronitrile, valeronitrile) were 14–26% of that of benzonitrile. The nitrilase was active within pH 5–10 and at up to 50 °C with optima at pH 8.0 and 40–45 °C. Its activity was strongly inhibited by Hg²⁺ and Ag⁺ ions. More than half of the enzyme activity was preserved at up to 50% of n-hexane or n-heptane or at up to 15% of xylene or ethanol. Operational stability of the enzyme was examined by the conversion of 45 mM 4-cyanopyridine in a continuous and stirred ultrafiltration-membrane reactor. The nitrilase half-life was 277 and 10.5 h at 35 and 45 °C, respectively.     

Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.     

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)₂) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)_n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)_4–6 and produced (GlcNAc)_2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)₅ to produce (GlcNAc)₂ (79.2%) and (GlcNAc)₃ (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)_n (n = 2–4) was pNp-(GlcNAc)₂ >> pNp-(GlcNAc)₄ > pNp-(GlcNAc)₃. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)_n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.     

Purification and characterization of an aminopeptidase from the chloroplast stroma of barley leaves by chromatographic and electrophoretic methods

Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates. Although some aminopeptidase activities have been found in plant chloroplasts, the identity of these proteins remains unclear. In this work, we report the purification to apparent homogeneity of a soluble aminopeptidase from isolated barley chloroplasts which preferentially degraded alanyl-p-nitroanilide (Ala-pNA). After organelle isolation in a density gradient and precipitation of soluble proteins with ammonium sulfate, the proteins were purified in three consecutive steps including hydrophobic interaction, gel permeation and ion-exchange chromatographies. The purified enzyme appeared as a single band with a M_r of 84 000 in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The M_r of the native enzyme was estimated to be 93 000 by gel permeation chromatography, suggesting that the protein is a monomer. Mass spectrometry analysis of tryptic digests indicates that the primary structure of the protein has not been reported previously. The enzyme was characterized as a metalloprotease as it could be totally inhibited by 1,10-phenanthroline. Strong inhibition could also be observed using the specific aminopeptidase inhibitors amastatin and bestatin. Besides Ala-pNA, the purified protein could also cleave with decreasing activity glycyl-pNA, leucyl-pNA, lysyl-pNA, methionyl-pNA and arginyl-pNA. The possible physiological role of this enzyme in the chloroplast stroma is discussed.     

Purification and partial characterization of an aminopeptidase from mung bean cotyledons

An aminopeptidase (EC 3.4.11.-) was purified to homogeneity, as judged by SDS-PAGE. from mung bean ( Vigna radiata ) cotyledons. The molecular mass of this peptidase was estimated as 75 kDa by gel filtration. When an oligopeptide consisting of 5 amino acid residues was used as substrate, amino acids were released in the order of the N-terminal sequence of the oligopeptide chain. This enzyme apparently requires free sulfhydryl for its activity, as judged by the effects of various proteinase inhibitors. Among aminoacyl- p -nitroanilides examined for the availability as substrates of the enzyme, p -nitroanilides with hydrophobic amino acids were preferred substrates. According to western immunoblot profiles, the enzyme level in cotyledons was high at the early stage of imbibition and declined rapidly after germination.     

Purification and characterization of an enkephalin-degrading aminopeptidase from guinea pig serum

An enkaphalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The amino-peptidase had a pH optimum of 7.0 with K_m values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid β-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.