Ligand-protein interaction in plant seed thiamine-binding proteins. Binding of various thiamine analogues to the sepharose-immobilized buckwheat-seed protein

Soluble thiamine-binding proteins occur in microorganisms, some animal tissues, and plant seeds. Their representative, the buckwheat-seed protein, was chosen as a model for chemical studies on the mechanism of ligand–protein interaction in these systems. In this work, in order to refine a concept of the chemical topography of the thiamine-binding center, the buckwheat seed protein was immobilized in Sepharose gel and probed with a new set of thiamine-related compounds. In terms of the standard change of Gibbs free energy on the complex formation, the following energetic contributions were specifically assigned to major structural features of the thiamine molecule: (i) 35–45% to the specific electronic structure of planar, unsaturated thiazolium ring with positive charge asymmetrically delocalized, one half of that contribution being attributable to the S(1) atom, (ii) 11–18% to nitrogen atoms and their electronic coupling within the pyrimidine ring, (iii) 15% to the 4-amino group, and (iv) less than 10% to the hydroxyethyl chain.     

Identification of a characteristic antioxidant,anthrasesamone F,in black sesame seeds and its accumulation at different seed developmental stages

Assay-guided fractionation of the methanol extract from black seeds of sesame (Sesamum indicum L.) led to the isolation of an active compound that had a 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. This antioxidant was confirmed to be anthrasesamone F, an anthraquinone derivative previously isolated from different black sesame seeds and biogenetically related to other anthrasesamones in sesame roots. The radical scavenging assay showed that anthrasesamone F had more potent activity than Trolox. The content of anthrasesamone F in different parts and at different developmental stages of black sesame seeds was investigated to clarify the accumulation pattern of this antioxidant in the black seeds. Anthrasesamone F was localized in the seed coat of black seeds and accumulated after the seed coat color changed to black. The content of anthrasesamone F increased gradually with seed maturation and drastically on air-drying, the final stage in sesame cultivation.     

Change of thiamin-binding protein and thiamin levels during seed maturation and germination in sesame

Thiamin-binding proteins (TBPs) occur in many types of plant seeds. The biochemical and structural properties such as subunit structure and affinity for thiamin of the proteins have been characterized. However, the change of TBP and thiamin during seed maturation and germination is little known. Sesame (Sesamum indicum L.) seeds have unique albumin TBPs, because the other TBPs from plant seeds are generally globulins. In this study, we studied the change of the TBP and thiamin levels in sesame seeds. The protein content and thiamin-binding activity of the seeds increased with seed development after flowering. Immunological analysis using an antibody against the TBP of sesame seeds showed that the protein was accumulated in seeds during maturation. The thiamin content of the seeds increased with seed development after flowering. On the other hand, the thiamin-binding activity decreased during seed germination when TBP was degraded. The thiamin content of the seeds decreased during the germination. However, the amount of thiamin phosphate in the seeds during germination was little changed. These results suggested that thiamin was accumulated and stored as a complex with TBP in sesame seeds.     

Genetic variation for in vitro sesame pollen germination and tube growth

  In vitro pollen germination and tube length studies are valuable in elucidating mechanisms (germination capacity and rate, tube growth rate) possibly associated with genetic differences in male transmission. On each of two collection dates, the percentage germination and tube length of the binucleate pollen grains from five diverse sesame (Sesamum indicum L.) genotypes were determined at eight times (30, 60, 90, 120, 150, 180, 240, 300 min) after inoculation on a semisolid medium containing 10% (100 g l^-1) sucrose (C₁₂H₂₂O₁₁), 0.4% (4 g l^-1) purified agar (Fisher Lot 914409), 0.1% (1 g l^-1) calcium nitrate [Ca(NO₃)₂ ⋅ 4H₂O] and 0.01% (100 mg l^-1) boric acid (H₃BO₃). Before heating, the pH of the medium was adjusted to 7.0 with a 0.1 N potassium hydroxide (KOH) solution. Over the five genotypes, 5% germination was found 30 min after inoculation and a maximum of 37% germination 120 min after inoculation with no significant changes thereafter. As indicated by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which germination was initiated and maximum germination attained. Over all five genotypes, the tube length was 91 μm 30 min after inoculation, reaching a maximum of 1000 μm 300 min after inoculation. As shown by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which tube length was observed and the maximum tube length was attained. Little or no relationship between percent germination and tube length was observed among the genotypes. For both percent germination and tube length, the statistical significance of collection date and its interactions with genotype and time after inoculation indicated that environment in the form of collection date was also an influencing factor. These results indicated that genetic differences among genotypes were present for in vitro germination capacity, germination rate and tube growth rate and that these factors singly or in combination could alter male transmission of genetic elements. Received: 5 February 1997 / Accepted: 23 June 1997     

Localization of Thiamine-Binding Protein in Synaptosomes from the Rat Brain

Using preparations of synaptosomes and subsynaptosomal fractions from the rat brain, we studied the localization of thiamine-binding protein (TBP) in the subcellular structures of the neurons. In addition, we studied the distribution in synaptosomes of two types of activity typical of TBP (thiamine triphosphatase and thiamine-binding activities), as well as the effects of factors destroying the plasma membrane of synaptosomes on binding of [¹⁴C]thiamine with the latter. We found that the thiamine-associated activity of TBP was the highest in fractions of the synaptic vesicles and plasma membranes. Hydrolysis of thiamine triphosphate was also most active in these structures. Our results allow us to conclude that TBP is localized mostly in the synaptic vesicles and plasma membranes of synaptosomes.     

Soluble and membrane-bound thiamine-binding proteins from Saccharomyces cerevisiae

Previous communications from this laboratory have indicated that there exists a thiamine-binding protein in the soluble fraction of Saccharomyces cerevisiae which may be implicated to participate in the transport system of thiamine in vivo.In the present paper it is demonstrated that both activities of the soluble thiamine-binding protein and thiamine transport in S. cerevisiae are greatest in the early-log phase of the growth and decline sharply with cell growth. The soluble thiamine-binding protein isolated from yeast cells by conventional methods containing osmotic shock treatment appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of the binding for thiamine was 29 nM which is about six fold lower than the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (0.18 μM) of thiamine transport. The optimal pH for the binding was 5.5, and the binding was inhibited reversibly by 8 M urea but irreversibly by 8 M urea containing 1% 2-mercaptoethanol. Several thiamine derivatives and the analogs such as pyrithiamine and oxythiamine inhibited to similar extent both the binding of thiamine and transport in S. cerevisiae, whereas thiamine phosphates, 2-methyl-4-amino-5-hydroxymethylpyrimidine and $\text{O-}\text{benzoylthiamine}$ disulfide did not show similarities in the effect on the binding and transport in vivo. Furthermore, it was demonstrated by gel filtration of sonic extract from the cells that a thiamine transport mutant of S. cerevisiae (PT-R₂) contains the soluble binding protein in a comparable amounts to that in the parent strain, suggesting that another protein component is required for the actual translocation of thiamine in the yeast cell membrane. On the other hand, the membrane fraction prepared from S. cerevisiae showed a thiamine-binding activity with apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 0.17μM at optimal pH 5.0 which is almost the same with the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the thiamine transport system. The membrane-bound thiamine-binding activity was not only repressible by exogenous thiamine in the growth medium, but as well as thiamine transport it was markedly inhibited by both pyrithiamine and $\text{O-}\text{benzoylthiamine}$ disulfide. In addition, it was found that membrane fraction prepared frtom PT-R₂ has the thiamine-binding activity of only 3% of that from the parent strain of S. cerevisiae.These results strongly suggest that membrane-bound thiamine-binding protein may be directly involved in the transport of thiamine in S. cerevisiae.     

Influence of growth regulators on somatic embryogenesis in sesame

Somatic embryos were induced from hypocotyl-derived callus of sesame (Sesamum indicum Var. TMV 6). The influence of different auxins and cytokinins on somatic embryogenesis was investigated. Among the different auxins tested, 2,4-dichlorophenoxy acetic acid was the most effective and resulted in the highest frequency of responding cultures and highest average number of somatic embryo per responding cultures napthaleneacetic acid, indoleacetic acid, indolebutyric acid and 2,4,5-trichlorophenoxypropionic acid were also effective for embryogenesis, but 2,4,5-trichorophenoxyacetic acid and napthoxyacetic acid were not beneficial. The combined effect of cytokinins with 2,4-d was also studied. Among the four cytokinins tested, 2.2 chμM benzyladenine with 13.6 chμM 2,4-d slightly enhanced embryogenic efficiency; while kinetin, zeatin, 6-γ-γ-dimethylallylaminopurine enhanced the frequency of responding cultures. There was a decrease in the number of somatic embryos per culture in the presence of all cytokinins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins

A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, K_d and B_max values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein.     

Anthrasesamone F from the Seeds of Black Sesamum indicum

A new anthraquinone derivative, named anthrasesamone F, was isolated from the seeds of Sesamum indicum. Its structure was determined to be (Z)-6,7-dihydroxy-2-(6-hydroxy-4-methyl-3-pentenyl)anthraquinone on the basis of spectroscopic evidence.     

The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars

The effect of increasing NaCl concentrations was studied on two different cultivars (cv. Orhangazi and cv. Cumhuriyet) of Sesamum indicum. Seedlings were grown for 40 days in half strength Hoagland solution and after 40 days treated with different NaCl concentrations (0, 50 and 100 mM) for 21 days. Differences in growth parameters, lipid peroxidation, antioxidative enzyme activities and proline accumulation were tested in order to put forward the relative tolerance or sensitivity of the cultivars. Results indicated that both parameters differ according to the cultivar's ability in coping oxidative stress caused by salinity. Constitutive levels of antioxidative enzyme activities were almost the same between the cultivars; however, cv. Cumhuriyet was able to induce antioxidative enzyme activities more efficiently when subjected to salt stress. Growth parameters, lipid peroxidation and proline accumulation results are also in good correlation with supporting this cultivar's being relatively tolerant.     

重要谷类种子贮藏蛋白的特性及改良研究

在成熟谷类种子中,总蛋白约为种子干重的7％～16％,其中大部分为贮藏蛋白。这些贮藏蛋白对于人类和牲畜所需要的营养质量以及在食品加工中都有重要的影响。本文对一些重要谷类种子贮藏蛋白的组成特性以及利用基因工程技术提高谷类种子蛋白质含量和质量进行了全面综述。此外,还简要介绍了在利用基因工程改良谷物种子品质中可能产生的食品安全性问题、如何增加外源基因的表达量和全面提高谷类种子蛋白质含量以及解决的办法。     

厌氧胁迫下芝麻不同耐渍基因型形态、生理及矿质元素变化的比较研究

在部控制氧下比较4种不同耐渍基因型芝麻的形态,生理指数及营养器官矿质元素含量变化。结果表明,耐渍品种种野芝7号与豫芝1号不定根条数增加4－5倍,净光合速率（Ph)下降幅度小,ADH活性增加约2倍,根据Ca,P含量显著升高,K降幅较小,根中其元素以及茎、叶中8种元素呈下降趋势。非耐渍品种丹巴格与遂平小籽黄,不定根条数无明显增加,Pn下降50％－60％,ADH活性可升高5－9倍;根中Ca,P含量亦增高,K下降较多,耐渍基因型有明显的结构适应特征,厌氧代谢的能量补偿作用相对次要,Ca,P在芝麻对厌氧胁迫的生理适应中起重要作用。     

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.     

Water-soluble fractions from defatted sesame seeds protect human neuroblast cells against peroxyl radicals and hydrogen peroxide-induced oxidative stress

Oxidative stress is involved in the development of aging-related diseases, such as neurodegenerative diseases. Dietary antioxidants that can protect neuronal cells from oxidative damage play an important role in preventing such diseases. Previously, we reported that water-soluble fractions purified from defatted sesame seed flour exhibit good antioxidant activity in vitro. In the present study, we investigated the protective effects of white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively) against 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H₂O₂) induced oxidative stress in human neuroblast SH-SY5Y cells. Pretreatment with WS-wsf and GS-wsf did not protect cells against AAPH-induced cytotoxicity, while simultaneous co-treatment with AAPH significantly improved cell viability and inhibited membrane lipid peroxidation. These results suggest that WS-wsf and GS-wsf protect cells from AAPH-induced extracellular oxidative damage via direct scavenging of peroxyl radicals. When oxidative stress was induced by H₂O₂, pretreatment WS-wsf and GS-wsf significantly enhanced cell viability. These results suggest that in addition to radical scavenging, WS-wsf and GS-wsf enhance cellular resistance to intracellular oxidative stress by activation of the Nrf-2/ARE pathway as confirmed by the increased Nrf2 protein level in the nucleus and increased heme oxygenase 1 (HO-1) mRNA expression. The roles of ferulic and vanillic acids as bioactive antioxidants in these fractions were also confirmed. In conclusion, our results indicated that WS-wsf and GS-wsf, which showed antioxidant activity in vitro, are also efficient antioxidants in a cell system protecting SH-SY5Y cells against both extracellular and intracellular oxidative stress.     

Antigenic relationship of legume seed proteins to the 7S seed storage protein of soybean

Extracts enriched for globulin proteins were prepared from the seeds of a large number of legume species and were tested for homology to antisera prepared against the glycosylated 7S seed storage protein of the soybean (Glycine max). Electrophoretic identification and subsequent analysis of proteins precipitated with 7S antisera was useful at relatively short taxonomic distances, particularly within the tribe Phaseoleae, to which G. max belongs. Glycine and most other members of the subtribe Glycininae are unusual within the Phaseoleae in having high molecular weight ($\text{> 70 000}$ dalton) subunit polypeptides. Seeds from other plants representing other subtribes of the Phaseoleae also contained proteins that cross-reacted with the G. max antisera; the molecular weights of these proteins varied from 30 000 to nearly 90 000 daltons. Homology was detected across a wider range of legume tribes within the subfamily Papilionoideae by enzyme-linked immunosorbent assay (ELISA). The results of these experiments suggest both that the 7S proteins of these tribes are evolutionarily related and that at least some features of these apparently rapidly-evolving proteins are under relatively strong selectional constraint.     

Minireview: analysis of rape seed napin structure and potential roles of the storage protein

Structural and functional data on 2S albumins and particularly rape seed napins are reviewed and, based on the coordinates of the three-dimensional structure of napin-like albumin BnIb, are used to model different rape napins. Surprisingly, the modeled napins, despite great sequence homology, differ in tertiary arrangements of the polypeptide chains. It is proposed that these differences in 3D structures of the analyzed rape napins may reflect their functions, which may cover many other potential beneficial purposes besides simple storage.     

Phytoplasmas of sesame and Orosius orientalis are genetically diverse based on 16S rDNA sequencing and PCR-RFLP in Turkey

Sesame phyllody causes significant yield losses mainly in Mediterranean region of Turkey. To detect and identify phytoplasmas of sesame and the cicadellid vector Orosius orientalis, samples were collected during 2011–2013 in Antalya. Nested PCR amplification of the 16S rDNA gave an amplification band of approximately 1246 bp corresponding to the 16Sr F2nR2 region. Identification of the phytoplasmas based on sequence homology revealed presence of the 16Sr groups II (Peanut witches’-broom, PnWB), VI (Clover proliferation, CP) and IX (Pigeon pea witches’-broom, PPWB) in sesame plants. PnWB and CP were detected in O. orientalis. Subgroups were determined by sequencing and PCR-RFLP of the F2nR2 region. 16Sr subgroups II-D, VI-A and IX-C were identified from sesame and II-D and VI-A subgroups from the insect vector. This study shows that phytoplasmas in sesame and O. orientalis are genetically diverse and to the knowledge, VI-A group phytoplasmas from sesame and O. orientalis were characterised molecularly for the first time in Turkey.     

The seed proteins of white spruce and their mobilization following germination

Buffer-soluble proteins that have subunit molecular weights, in the presence of sodium dodecyl sulphate (SDS), of 47, 31 and 27 kilodaltons (kDa) form the major storage proteins in the mature white spruce [ Picea glauca (Moench) Voss] seed. These proteins were found mainly in the megagametophyte. but smaller amounts were identified in the embryo. Following the completion of germination, this reserve was rapidly hydrolyzed in both tissues and probably plays a major nutritional role in the germinated seed. Buffer-insoluble proteins were also found in megagametophytes and embryos from the mature seed. These proteins were soluble in buffer only if SDS was present. Predominant in this class of proteins were several that have a subunit molecular weight and structure that is characteristic of seed crystalloid storage proteins; the subunits were shown to be heterodimers with polypeptide molecular weights in the 33 kDa to 37 kDa and 23.5 kDa to 25 kDa ranges. This reserve was rapidly hydrolyzed in the germinated seed. Storage protein hydrolysis was accompanied by a significant increase in the soluble amino acid pool in both megagametophytes and embryos. Cell-free extracts of mature seed megagametophytes and embryos contained leucine-naphthylamidase (leuNAase) activity. Following germination. this activity was maintained at a constant level in megagametophytes but increased substantially in embryos.     

Effect of drought on biomass, protein content, lipid peroxidation and antioxidant enzymes in two sesame cultivars

The effects of drought on growth, protein content, lipid peroxidation, superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and polyphenol oxidase (PPO) were studied in leaves and roots of Sesamum indicum L. cvs. Darab 14 and Yekta. Four weeks after sowing, plants were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity for next four weeks. Fresh and dry masses, and total protein content in leaves and roots decreased obviously under drought. However, several new proteins appeared and content of some proteins was affected. Measurement of malondialdehyde content in leaves and roots showed that lipid peroxidation was lower in Yekta than in Darab 14. Severe stress increased SOD, POX, CAT and PPO activities in leaves and roots, especially in Yekta. According to the present study Yekta is more resistant to drought than Darab 14.