Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

Ethanol production from Jerusalem artichoke tubers (Helianthus tuberosus) using Kluyveromyces marxianus and Saccharomyces rosei

This article examines the potential of Jerusalem artichoke as a source for ethanol and single-cell protein SCP. In addition, experimental results are presented on batch fermentation kinetics employing two strains of Kluyveromyces marxianus and one strain of Saccharomyces rosei grown on the extract derived from the tubers of Jerusalem artichoke. Of the three cultures examined, Kluyveromyces marxianus UCD (FST) 55-82 was found to be the best producer of ethanol grown in a simple medium at 35 degrees C. The ethanol production was found to be growth-associated having a mu(max) = 0.41. h(-1) and the ethanol and biomass yields were determined to be Y(p/s) = 0.45 (88% of the theoretical) and Y(x/s) = 0.04 with 92% of the original sugars utilized. On the basis of carbohydrate yields of Jerusalem artichoke reported in the literature and these batch kinetic studies with K. maxxianus, the calculated ethanol yields were found to range from 1400 kg ethanol acre (-1) yr(-1)to a maximum of 2700 kg ethanol acre (-1) yr(-1). The SCP yields for K. marxianus were calculated to range between 130 to 250 kg dry wt cell acre (-1) yr(-1). The potential for developing an integrated process to produce ethanol and SCP is also discussed.     

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58–60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C_α-distance 1.2 Å). However, this peroxidase includes a Mn²⁺ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn²⁺. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.     

植物菌根块菌分子鉴定及菌丝培养基优化的研究

块菌属(Tuber)是由真菌感染其适宜植物根系形成,部分块菌如法国黑孢块菌(T.melanosporum)和中国印度块菌(T.indicum)等为珍稀药食两用菌,具有很高的经济价值.对块菌的过度挖掘造成野生块菌资源匮乏,块菌人工栽培技术则是实现块菌产业可持续性发展的重要基础.目前国内外人工栽培主要采用块菌子实体匀浆制成...     

Growth and Differentiation of Plant Tissue Cultures: I. Changes Accompanying the Growth of Explants from Helianthus tuberosus tubers

Explants derived from artichoke tubers proliferate rapidly whencultured aseptically on a nutrient medium containing sugar,mineral salts, coconut milk, and 2, 4 dichlorophenoxyaceticacid. This rapid growth, which occurs in three distinct stages,is accompanied by massive changes in numbers of cells, freshand dry weight, and pronounced changes in the rates of gaseousexchange. The very short lag phase is followed by a period ofexponential cell division during which the original cellularpattern is partially obliterated. At the end of this growthphase redifferentiation occurs, tracheidea are formed in largenumbers, and distinctive nodular bodies appear which resemblemeristems. Under the conditions of these experiments they donot develop further.     

Plasmalemma fluidity in parenchyma cells from Jerusalem artichoke (Helianthus tuberosus L.) tubers during the break of dormancy

Plasmalemma-enriched fractions were isolated from Jerusalem artichoke tubers along the time course of dormancy break produced by cold treatment. A decrease of membrane fluidity was noted from the 3^rd to the 8^th week of this treatment, as well as a decrease of plasmalemma NADH dehydrogenase activity from the 5^th to the 8^th week. The plasmalemma lipid extracts studied revealed two major phospholipidic components: phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Their respective quantities decreased until the 12^th week, where the phosphatidylcholine level is lower than the phosphatidylethanolamine one. The observed changes are discused in relation to dormant and non-dormant states of tubers and the breaking of dormancy.     

Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers

The adenine nucleotide carrier from Jerusalem artichoke (Helianthus Tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. V _max of the reconstituted ATP/ATP exchange was determined to be 0.53 mol/min per mg protein at 25°C. The half-saturation constant K _m and the corresponding inhibition constant K _i were 20.4 M for ATP and 45 M for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30°C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.     

Stressed Jerusalem artichoke tubers (Helianthus tuberosus L.) excrete a protein fraction with specific cytotoxicity on plant and animal tumour cell

Wounds from Jerusalem artichoke (Helianthus tuberosus L.) tubers excrete bioactive metabolites from a variety of structural classes, including proteins. Here we describe a protein specifically active against tumour cells arising either from human, animal or plant tissues. The non-tumour animal cells or the plant callus cells are not sensitive to these excreta. The active product was only obtained after a wound-drought stress of plant tubers. The cytotoxicity varies according to the tumour cell type. For instance, some human tumour cell lines and especially the human mammary tumour cells MDA-MB-231 were shown to be very susceptible to the active product. The active agent is shown to contain an 18-kDa polypeptide with homology to a superoxide dismutase (SOD). A 28-kDa polypeptide, related to an alkaline phosphatase (AP), was shown to be tightly linked to this 18-kDa polypeptide. The excreted 28-kDa polypeptide also displayed a consensus sequence similar to the group of DING proteins, but with a smaller molecular weight. The superoxide dismutase polypeptide was shown to be involved in the antitumour activity, but the presence of smaller factors (MW<10 kDa), such as salicylic acid, can enhance this activity.     

Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus

Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na⁺/H⁺ antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4 % identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na⁺ and accumulate more K⁺. Expression of HtSOS1 decreased Na⁺ content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte.     

Protein and mineral concentrations in tubers of selected genotypes of wild and cultivated Jerusalem-artichoke (Helianthus tuberosus,asteraceae)

Nineteen wild and cultivated genotypes of Jerusalem-artichoke (Helianthus tuberosus) whose tubers are used as food were analyzed for protein and mineral content at three stages of growth. The protein content of the tubers is comparable to or higher than that of other common root-type crops. Adequate macrominerals of calcium, magnesium, and phosphorus were found in Jerusalem-artichoke. Potassium and sodium concentrations were higher than other root crops. Trace elements (manganes, zinc, and copper) were present in adequate amounts, with iron content higher than several other root crops. Wild and cultivated Jerusalem-artichoke genotypes appear to contain adequate protein and minerals to contribute significantly toward a nutritionally balanced diet.     

菊芋叶片提取物抑菌活性与化学成分的研究

为了开发新型植物源杀菌剂和充分利用菊芋(Helianthus tuberosus L.)资源,本文尝试用石油醚、乙醚、乙酸乙酯和水等4种溶剂对菊芋叶片进行平行提取,采用生长速率法测定菊芋叶片提取物对水稻纹枯菌(Rhizoc-tonia solaniKühn)、小麦赤霉菌[Gibberella zeae(Schw.)Petch]、番茄早疫菌[Alternaria solani(Ellis et Martin)Jones et Grour]和番茄灰霉菌(Botrytis cinerea Pers.)生长量的抑制活性;并通过试管法和滤纸法对菊芋叶片内化学成分进行初步预试。抑菌实验结果表明:(1)菊芋叶片各溶剂提取物处理与对照处理相比差异显著;(2)菊芋叶片水提取物处理与各有机溶剂提取物处理差异也基本显著;(3)菊芋叶片各溶剂提取物同浓度处理对水稻纹枯菌、番茄早疫菌和番茄灰霉菌抑制效果较好;(4)菊芋叶片乙酸乙酯提取物抑菌效果最为显著,浓度为20mg/mL时对番茄早疫菌和番茄灰霉菌已达到完全抑制,对水稻纹枯菌抑制率也达到77.91%。初步化学预试结果说明,菊芋叶片中含有蛋白质、氨基酸、还原糖类、有机酸、酚类和鞣质、黄酮类、内酯类、强心甙以及油脂等化学成分。     

菊芋试管苗的初代培养及愈伤组织不定芽诱导

菊芋（Helianthus tuberosus Linn.）为菊科（Asteraceae）向日葵属（Helianthus Linn.）多年生草本植物,耐寒、耐旱、耐贫瘠、耐盐碱[1];其地下块茎富含菊糖,还可通过发酵生产乙醇,在功能性食用多糖及生物能源方面的开发潜力巨大。菊芋主要通过块茎进行无性繁殖,其种子成活率和发芽率均很低[2],严重阻碍了菊芋的杂交育种。近年来以植物组织培养为基础的一系列现代育种技术为菊芋的种质改良提供了新途径,但由于菊芋的愈伤组织难以诱导不定芽或体胚发生,导致以农杆菌转化为主的转基因育种技术的应用受到限制。     

菊芋类金属硫蛋白基因htMT2的克隆及其表达特征分析

从菊芋 (HelianthustuberosusL .)块茎cDNA文库中得到了一个新的植物类金属硫蛋白基因htMT2的cDNA序列 ,全长 5 0 9bp ,包括 2 4 0bp的开放阅读框、6 2bp的 5′端非翻译区、2 0 7bp的 3′端非翻译区。通过PCR获得了 2个htMT2编码区的部分基因组片段htMTG_1及htMTG_2 ,长度分别为 986bp和 982bp。分析表明两个基因组片段均包含 3个外显子及 2个内含子 ,编码一个由 79个氨基酸残基组成的多肽 ,与从htMT2推测的多肽完全一致 ,该多肽具有植物类金属硫蛋白的典型结构特征 ,N端及C端结构域富含Cys ,分别具有 8个和 7个Cys残基 ,上述两个结构域被一个无Cys的中间区分开。Southern杂交结果表明 ,htMT2在菊芋基因组中以小基因家族的形式存在。Northern杂交结果表明htMT2在叶片、叶柄、茎及块茎中均有表达 ,在茎中有较高水平的表达 ,但在根中未检测到杂交信号。经Cu2 处理后 ,htMT2在茎中的表达量显著降低。与其他 2型金属硫蛋白的序列同源性比较及htMT2对金属离子处理的反应均表明 ,htMT2是一种新的植物类金属硫蛋白基因。     

A micro-batchwise technique method for rapid reconstitution of functionally active mitochondrial ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers

A method for rapid reconstitution of ADP/ATP carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers mitochondria in proteoliposomes is described. The method is based on the well known property of the Amberlite resin to absorb the detergent allowing proteoliposome formation. This has been achieved by a micro-batchwise technique, using a rotating plate stirrer. An evaluation of the optimal conditions, in comparison with the more usual column method is presented. The purified ADP/ATP carrier, incorporated in proteoliposomes by this method, shows a high transport activity and a higher specific activity with respect to proteoliposomes obtained by the column procedure. Furthermore the proteoliposomal preparations are more homogeneous in size, with a diameter ranging from 300 to 350 nm. The method is suitable for the reconstitution of other membrane transport proteins.     

菊芋类金属硫蛋白基因htMT2的克隆及其表达特征分析

从菊芋(Helianthus tuberosus L.)块茎cDNA文库中得到了一个新的植物类金属硫蛋白基因htMT2的cDNA序列,全长509 bp,包括240 bp的开放阅读框、62 bp的5′端非翻译区、207 bp的3′端非翻译区.通过PCR获得了2个htMT2编码区的部分基因组片段htMTG-1及htMTG-2,长度分别为986 bp和982 bp.分析表明两个基因组片段均包含3个外显子及2个内含子,编码一个由79个氨基酸残基组成的多肽,与从htMT2推测的多肽完全一致,该多肽具有植物类金属硫蛋白的典型结构特征,N端及C端结构域富含Cys,分别具有8个和7个Cys残基,上述两个结构域被一个无Cys的中间区分开.Southern杂交结果表明,htMT2在菊芋基因组中以小基因家族的形式存在.Northern杂交结果表明htMT2在叶片、叶柄、茎及块茎中均有表达,在茎中有较高水平的表达,但在根中未检测到杂交信号.经Cu2+处理后,htMT2在茎中的表达量显著降低.与其他2型金属硫蛋白的序列同源性比较及htMT2对金属离子处理的反应均表明,htMT2是一种新的植物类金属硫蛋白基因.