Hexane extracts from Tephrosia vogelii Hook. f. protect stored maize against the weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

Powdered dried leaves of Tephrosia vogelii (Hook) (Leguminosae: Papilionoideae) were extracted using hexane, acetone, and ethanol. The extracts were tested for their ability to protect stored maize from damage by Sitophilus zeamais Motschulsky, the major maize weevil in Africa. The acetone and ethanol extracts were ineffective, but the hexane extract had a relatively high efficacy, producing within 7 days a slow reduction in adult survival, reduced numbers of eggs laid and reduced numbers of F₁ progeny, resulting in seed damage averaging 8.8% compared with 98.6% in the untreated control after one generation. This hexane extract provided control of S. zeamais at a higher level compared with neem (the botanical control) but at a lower level compared with pirimiphos‐methyl (the synthetic control). These findings are discussed in the context of T. vogelii containing rotenoids.     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

Efficacy of cashew kernel extracts in the control of the maize weevil,Sitophilus zeamais (Coleoptera: Curculionidae)

Six different solvents namely: methanol, ethanol, water, acetone, pet-ether or n-hexane was used to extract the essential oils from cashew kernel. The extracted oils were evaluated on Sitophilus zeamais (Motschulsky 1855) for mortality, oviposition and adult emergence effects. The long-term storage and water absorption capacity of the treated maize grains was also investigated. Results showed that the steam distillate was most lethal on S. zeamais. The least potent extracts on S. zeamais were those extracted with methanol and ethanol. The extracts drastically reduced oviposition in S. zeamais. Maize grains treated with steam distillate extract had the lowest water absorption capacity, while the highest water absorption capacity was obtained in grains treated with ethanol and methanol. Extracts of steam distillate, n-hexane, petroleum ether and acetone completely prevented infestation and subsequence damage of the treated maize grains for a period of three months. In the grains treated with the methanol and ethanol extracts, damage of 28.61% and 6.24%, respectively were obtained. The results obtained reveal that extracts of cashew kernel are effective in controlling S. zeamais and could serve as an alternative to synthetic insecticides.     

西花蓟马蛋白的提取及双向电泳体系的建立

【目的】蛋白样品的制备是获得良好双向凝胶电泳(2-DE)图谱的前提,建立合理的西花蓟马蛋白的双向电泳体系,获得分辨率较高、重复性较好的图谱,能够为后续的研究提供有力支撑。【方法】实验以西花蓟马成虫为实验材料,对比了饱和酚法、TCA/丙酮法和直接裂解法3种蛋白提取方法,从中选出最适宜双向电泳分析的一种蛋白提取方法。【结果】3种方法蛋白提取率差异显著,直接裂解法蛋白提取率最高,饱和酚法的蛋白提取率最低;3种方法的SDS-PAGE条带数差异不明显;TCA/丙酮法的双向凝胶图谱效果最好,蛋白点最多。【结论】TCA/丙酮法能够有效去除西花蓟马蛋白中的干扰物质,是最适合西花蓟马双向凝胶电泳的蛋白提取方法,为后续西花蓟马在蛋白组学方面的研究奠定了基础。     

DNA array profiling of gene expression changes during maize embryo development

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)⁺RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)⁺ RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6. Electronic Publication     

Extracts from Tephrosia vogelii for the protection of stored legume seeds against damage by three bruchid species

Aliquots of powdered dried leaves of Tephrosia vogelii (Leguminosae – Papilionoideae) were extracted with hexane, acetone and ethanol. The extracts were tested for their potential to protect stored legume seeds from damage by the bruchids Acanthoscelides obtectus, Callosobruchus maculatus and Callosobruchus chinensis (Coleoptera – Bruchidae). While the acetone and ethanol extracts were ineffective except for a significant but low effect on A. obtectus, the hexane extract had a relatively high efficacy, providing similar control of all three bruchid species. This effect was manifested in slowly‐reduced adult survival within 7 days and in reduced numbers of eggs laid and of F₁ progeny produced, resulting in seed damage averaging 7.1% compared to 99.8% in the untreated controls after one generation. These findings are discussed in the context of previously identified constituents of T. vogelii, the rotenoids rotenone, tephrosin and deguelin.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Proteomic analysis of seed viability in maize

To identify specific proteins related to maize seed viability, seeds of Zhengdan 958 (one of the high-yield maize hybrids in China) were sorted based on viability evaluation with triphenyltetrazolium chloride (TTC) assay and used for comparative proteomic analysis. After TTC staining, embryos of high-viability seeds were deep red (R type), while embryos of dead seeds were white (W type). Proteomic analysis revealed that 28 protein spots identified were differently expressed significantly between R and W embryos, of which 20 were up-regulated and 8 down-regulated in R embryos. Among them were proteins involved in stress response, protein folding, and stabilization, as wells as proteins related to nutrient reservoir and metabolism. Prominently, small heat shock proteins, late embryogenesis abundant (LEA) proteins, and antioxidant enzymes were highly up-regulated, while two proteases were highly down-regulated in R embryos compared to W embryos. One of LEA proteins was EMB564, which declined in abundance during artificial aging of seeds. Our results suggested an association of EMB564 with maize seed viability. It would be of interest to use these small proteins to develop quick tests for seed quality.     

Enzyme Inhibition and Antioxidant Activities of Asparagus officinalis L. and Analysis of Its Phytochemical Content by LC/MS/MS

In the study, water, ethanol, methanol, dichloromethane, and acetone extracts of Asparagus officinalis L. were obtained by maceration. DPPH⋅, ABTS⋅⁺, FRAP, and CUPRAC methods determined the antioxidant capacities of all extracts. Moreover, the in vitro effects of extracts on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carbonic anhydrase (CA)-I, CA-II and α-Glycosidase were investigated. At a 10 μg/ml concentration, the extract with the highest Fe³⁺ reduction capacity was ethanol (AE), and the extract with the highest Cu²⁺ reduction capacity was acetone (AA). AE for AChE (IC₅₀=21.19 μg/ml) and α-Glycosidase (IC₅₀: 70.00 μg/ml), methanol (AM) for BChE (IC₅₀=17.33 μg/ml), CA−I and II (IC₅₀=79.65 and 36.09 μg/ml, respectively) showed the most potent inhibition effect. The content analysis of acetone extract was performed with LC/MS-MS, the first three phytochemicals found most were p-Coumaric acid, rutin, and 4-hydroxybenzoic acid (284.29±3.97, 135.39±8.19, and 102.06±5.51 μg analyte/g extract, respectively).     

Mitochondrial biogenesis during germination in maize embryos

Mitochondrial biogenesis and metabolism were investigated during maize (Zea mays) seed germination. Mitochondria from dry and imbibed seed exhibited NADH-dependent O(2) uptake that was completely inhibited by KCN and antimycin A. Mitochondria in the dry seed had a lower rate of succinate-dependent O(2) uptake relative to that measured in imbibed and germinated seed. The activities of the tricarboxylic acid (TCA) cycle enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, NAD-malic enzyme, and citrate synthase, are similarly low in mitochondria from dry seed and this correlates with a lower relative abundance of the mitochondrial matrix-located citrate synthase and pyruvate dehydrogenase complex E1alpha-subunit polypeptides. Electron microscopy revealed that mitochondria in the dry seed have a poorly developed internal membrane structure with few cristae; following 24 h of germination the mitochondria developed a more normal structure with more developed cristae. The mitochondria from maize embryos could be fractionated into two subpopulations by Suc density gradient centrifugation: one subpopulation of buoyant density equivalent to 22% to 28% (w/w) Suc; the other equivalent to 37% to 42% (w/w) Suc. These two subpopulations had different activities of specific mitochondrial enzymes and contained different amounts of specific mitochondrial proteins as revealed by western-blot analysis. Both subpopulations from the dry embryo were comprised of poorly developed mitochondria. However, during imbibition mitochondria in the heavy fraction (37%-42% [w/w] Suc) progressively acquired characteristics of fully functional mitochondria found in the germinated seedling in terms of structure, enzymic activity, and protein complement. In contrast, mitochondria in the light fraction (22% to 28% [w/w] Suc) show no significant structural change during imbibition and the amounts of specific mitochondrial proteins decreased significantly during germination.     

Determination of phosphinothricin acetyltransferase in genetically transformed canola seed by a two-antibody sandwich enzyme immunoassay

We report a quantitative, two-antibody sandwich immunoassay for phosphinothricin acetyltransferase (PAT), the selectable marker protein. The method yielded a standard curve with a working range of 0 to 5 ng PAT per mL extract. Replicate absorbance values for standards within a single assay showed a coefficient of variation typically less than 5 percent. Over three separate assays, the coefficient of variation for the slope and y-intercept of the standard curve was 3.8 and 3.7 percent, respectively. TransgenicBrassica napus L. seed was used to demonstrate the utility of the assay. Non-transgenic seed extracts did not show a positive immunoassay signal. Determinations of the PAT enzyme conducted on spiked non-transgenic seed extracts repeated in three separate assays fell in the acceptable range of 80 to 120 percent recovery. Transgenic canola seed, analyzed in three separate assays, showed a mean PAT enzyme content of 403 ng/g with a standard error of 19 ng/g and a coefficient of variation of 8.1 percent. The method has also been applied to several other tissues and processed products of canola, maize, and tobacco.     

Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii NCIMB 8052

Mixed sugars from tropical maize stalk juice were used to carry out butanol fermentation with Clostridium beijerinckii NCIMB 8052. Batch experiments employing central composite design (CCD) and response surface methodology (RSM) optimization were performed to evaluate effects of three factors, i.e. pH, initial total sugar concentration, and agitation rate on butanol production. Optimum conditions of pH 6.7, sugar concentration 42.2 g/L and agitation rate 48 rpm were predicted, under which a maximum butanol yield of 0.27 g/g-sugar was estimated. Further experiments demonstrated that higher agitation facilitated acetone production, leading to lower butanol selectivity in total acetone–butanol–ethanol (ABE). While glucose and fructose are more preferable by C. beijerinckii, sucrose can also be easily degraded by the microorganism. This study indicated that RSM is a useful approach for optimizing operational conditions for butanol production, and demonstrated that tropical maize, with high yield of biomass and stalk sugars, is a promising biofuel crop.     

Seed Transmission of Maize Dwarf Mosaic Virus in Sweet Corn

Sweet corn seed from several maize dwarf mosaic virus (MDMV)-infected hybrids grown in the field were tested for transmission of MDMV through the seed. Seeds collected in 1979, 1980, 1981, and 1982, were germinated in the greenhouse the following winters. Only one seedling of 22,189 was MDMV-infected During the last three years, seed were dissected at different maturities and the seed parts tested for the presence of MDMV by both infectivity and enzyme-linked immunosorbent assay (ELISA). At 21 days after pollination, MDMV always was detected in the pericarp, but rarely in the endosperm or embryo. No MDMV was detected in the embryo of mature kernels, but virus occasionally was detected in the endosperm and pericarp. MDMV was regularly detected in unfertilized kernels and whole silks, but not in pollen by infectivity, ELISA or serological specific electron microscopy. MDMV was detected in glumes and whole anthers.     

Adult response of the almond seed wasp,Eurytoma amygdali, to chemicals from its host and certain nonhosts

Females of the almond seed wasp,Eurytoma amygdali Enderlein (Hymenoptera, Eurytomidae), responded in an olfactometer positively to odours from almond flowers and unripe fruits, but not to almond leaf odours and odours from flowers and unripe fruits of certain other nonhostPrunus species. Males responded to none of these odours. Extracts of undamaged unripe almond fruits (using ethanol, methanol, acetone, hexane, dichloromethane, or petroleum ether) stimulated female aggregation on glass surfaces treated with these extracts; in addition, certain fruit extracts (ethanol, methanol, or acetone) stimulated oviposition. Extracts of undamaged almond leaves (ethanol, methanol, or acetone) and flowers (ethanol or methanol) also stimulated female aggregation and oviposition. Aggregation and oviposition in response to an ethanol extract of almond fruits was intense in females aged 5 to 14 days and from 12∶00 to 18∶00h (photophase between 06∶00 and 20∶00). Certain almond fruit (ethanol, methanol, acetone or hexane) and flower extracts (ethanol or methanol) also provoked female response in the olfactometer. The results strongly suggest that certain chemical stimuli emanating from parts of the almond tree play a major role in host selection and oviposition. Some of the extracts tested may be a good source for the isolation, identification and synthesis of compounds stimulating attraction, aggregation and oviposition in nature. *** DIRECT SUPPORT *** A3414024 00003     

The role of seed reserves in arbuscular mycorrhizal formation and growth of Leucaena leucocephala (Lam.) de Wit. and Zea mays L.

We show here that seed reserves in Leucaena leucocephala (Lam.) de Wit. and Zea mays L. (maize) are important for mycorrhizal formation and seedling growth. Seed reserve removal reduced mycorrhizal formation markedly in Leucaena but not in maize, except at 15 and 45 days after seed reserve removal. Partial or total removal of seed reserves decreased plant growth and tissue nutrient concentrations in both hosts. Nodule number in Leucaena, which was related positively to plant biomass and mycorrhizal infection levels, was reduced when one or both cotyledons were severed. Leucaena seedlings without or with partial seed reserves had higher nutrient use efficiencies throughout seedling growth. But such an effect was observed only initially in maize. Seed reserve removal increased the specific absorption rates of nutrients in both hosts. Phosphorus absorption rate was significantly and positively related to root infection levels in both Leuceana and maize. Though the growth rates of plants without seed reserves were low initially, these plants had higher growth rates during later stages. We conclude that seed reserves are not only important for seedling growth, but also for mycorrhizal formation and nodulation. Received: 15 July 1999 / Accepted: 6 December 1999     

Diversity and seasonal phenology of aboveground arthropods in conventional and transgenic maize crops in Central Spain

One of the major concerns regarding the release of Bt maize is its potential negative impact on non-target organisms present in this crop. In this paper, we compare the temporal phenology and community structure of the aboveground arthropods in commercial Bt maize fields in Central Spain with those of conventional maize crops, with or without an insecticide (imidacloprid) seed treatment, over a period of three years. Spiders, harvestmen, centipedes, ground beetles, rove beetles, carrion beetles, click beetles, earwigs and damsel bugs were captured in pitfall traps every year in sufficient number to provide meaningful phenological data. One predator spider and three omnivorous species of ground beetles have been consistently present in the maize fields: Pardosa occidentalis, Poecilus cupreus, Pseudophonus rufipes and Pseudophonus griseus, respectively. Rove beetles were caught to a lesser extent, with three dominant species: Acrotona aterrima, Philonthus varians and Platystethus nitens. The variability in activity–density patterns of the aboveground fauna was mainly influenced by the year, but no detrimental effects could be attributed to Bt maize. The only exception being the changes detected in rove beetles, although these differences were transitory and varied from year to year. No changes in species richness and diversity indices for spiders and ground beetles resulted from treatments. However, imidacloprid-treated maize caused a reduction in species richness of rove beetles, even though the abundance of the main species was not reduced. Our results suggest that Bt maize could be compatible with natural enemies that are common in maize fields in Spain.     

Glycinebetaine increases chilling tolerance and reduces chilling-induced lipid peroxidation in Zea mays L.

Chilling tolerance was increased in suspension‐cultured cells and seedlings of maize (Zea mays L. cv ‘Black Mexican Sweet’) grown in media containing glycinebetaine (GB). A triphenyl tetrazolium chloride (TTC) reduction test indicated that after a 7 d chilling period at 4 °C, cells treated with 1 mm GB at 26 °C for 1 d had a survival rate (30%) that was twice as high as that of untreated controls. The addition of 2·5 m M GB to the culture medium resulted in maximum chilling tolerance (40%). The results of a cell regrowth assay were consistent with viability determined by the TTC method. In suspension‐cultured cells supplemented with various concentrations of GB, accumulation of GB in the cells was proportional to the GB concentration in the medium and was saturated at a concentration of 240 μ mol (g DW) ^{? 1}. The degree of increased chilling tolerance was positively correlated with the level of GB accumulated in the cells. The increased chilling tolerance was time‐dependent; i.e. it was first observed 3 h after treatment and reached a plateau after 14 h. Feeding seedlings with 2·5 m M GB through the roots also improved their chilling tolerance, as evidenced by the prevention of chlorosis after chilling for 3 d at 4 °C/2 °C. Lipid peroxidation, as expressed by the production of malondialdehyde, was significantly reduced in GB‐treated cells compared with the untreated controls during chilling. These results suggest that increased chilling tolerance may be due, in part, to the reduction of lipid peroxidation of the cell membranes in the presence of GB.     

Distribution of Cadmium,Lead, Nickel,and Strontium in Imbibing Maize Caryopses

Seed germination is tolerant to heavy metals apparently because the seed coat is impermeable to metal ions. However, it is not clear whether the seed coat is a universal barrier for all metals. In addition, depending on their physical and chemical properties, a distribution of various metals may differ within an imbibing caryopsis, and therefore they produce dissimilar effects on seed germination. The toxic effects of Cd(NO₃)₂, Pb(NO₃)₂, Ni(NO₃)₂, and Sr(NO₃)₂ were estimated from the germination rates of maize (Zea mays L.) caryopses following two-day incubation with these salts. The distribution of heavy metals and Sr was studied by histochemical methods based on the formation of colored complexes with dithizone (Cd and Pb), dimethylglyoxyme (Ni), and sodium rhodizonate (Sr). Although the metals under study did not affect maize radicle protrusion, they inhibited seed germination in the following order: Cd > Ni ≈ Pb > Sr. Cd and Pb accumulated mainly in the seed coat cells, but Sr and Ni in the embryo cells and in the cells of endosperm (Sr) and scutellum (Ni). Although Cd was found only in the seed coat, it was the strongest inhibitor of seed germination. Apparently, due to high toxicity, Cd exerted its inhibitory effect at the concentrations too low for histochemical assay. In spite of easy translocation across the seed coat of imbibing caryopses, Sr did not considerably inhibit radicle protrusion and seed germination, apparently because of its low toxicity and predominant localization in the apoplast of embryo and endosperm cells.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 635–640.Original Russian Text Copyright © 2005 by Seregin, Kozhevnikova.