Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F₁ hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T₆ transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.     

Some Properties of Proteolytic Enzymes and Storage Proteins in Recalcitrant and Orthodox Seeds of Araucaria

Araucaria bidwillii Hook. and Araucaria cunninghamii Don D. are two species of conifers whose seeds belong to different physiological categories: A. bidwillii seeds are recalcitrant, while A. cunninghamii seeds are orthodox. The extraction of enzymes and storage proteins was carried out from A. bidwillii and A. cunninghamii megagametophytes. The endopeptidase activities of both species were assayed with azocasein and with haemoglobin; the exopeptidase activities were detected by various N-carbobenzyloxy-dipeptides and L-leucine p-nitroanilide. The use of appropriate proteinase inhibitors, i.e. pepstatin A, ethylenediaminetetraacetic acid and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, showed the presence of aspartic and metallo proteinases and the absence of the cysteine ones both in A. bidwillii and in A. cunninghamii ungerminated seeds. Since the results do not show differences between the types of enzymes in the ungerminated araucarian seeds and those present in some ungerminated angiosperm seeds (barley, wheat, maize, rice, buckwheat), we conclude that their physiological role is similar. The electrophoretical analyses of soluble and insoluble storage proteins of A. cunninghamii showed patterns similar to those found in other gymnosperms, while the storage protein patterns of A. bidwillii seeds were rather atypical.     

中国野生豆科植物资源及豆类蛋白研究概况

豆科植物在世界上分布广泛,有非常重要的经济价值、生态价值、药用价值和观赏价值。豆类蛋白作为植物源蛋白中的优质蛋白,在我国启动“国民营养计划”之双蛋白工程之后,其价值得到重视和开发。我国野生豆科资源丰富,但大部分地区的野生豆科资源没有得到合理的开发和利用。本文结合文献资料,对我国各地区的野生豆科植物资源和价值进行概述,并对豆科植物蛋白质的研究方法进行了系统归纳,以期对野生豆科植物资源及豆类蛋白的应用前景做出展望。     

Improvement of the nutritional value of rye products by solid-state fermentation with fusaria

The use of some cereals in animal nutrition, e.g. rye, is limited because of some constituents. By solid-state fermentation using selected Fusaria it is possible to enhance the nutrition value and to enrich the fermented material with free and essential amino acids and vitamins. The fermentation method is described and the results of analytical assays are discussed.     

Hydrolytic enzyme activities and degradation of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark, and time-course changes in contents of protein fractions, starch, soluble α-amino nitrogen and reducing sugars and in hydrolytic enzyme activities in cotyledons were investigated. In cotyledons of germinated seeds, marked increases in proteolytic (caseolytic, globulytic and gelatin-hydrolyzing) activities and amylolytic activity occurred with concurrent mobilization of storage proteins and starch. Removal of axis organs from seeds at very early stages of germination caused the deteriorated breakdown of storage components and decreased development of proteolytic enzymes in the cotyledons, but this treatment did not significantly affect the appearance of amylolytic activity. The experimental results are discussed in comparison with the hydrolytic enzyme activities of germinating seeds of other leguminous species.     

Functional specialization of <Emphasis Type="Italic">Medicago truncatula</Emphasis> leaves and seeds does not affect the subcellular localization of a recombinant protein

A number of recent reports suggest that the functional specialization of plant cells in storage organs can influence subcellular protein sorting, so that the fate of a recombinant protein tends to differ between seeds and leaves. In order to test the general applicability of this hypothesis, we investigated the fate of a model recombinant glycoprotein in the leaves and seeds of a leguminous plant, Medicago truncatula. Detailed analysis of immature seeds by immunofluorescence and electron microscopy showed that recombinant phytase carrying a signal peptide for entry into the endoplasmic reticulum was efficiently secreted from storage cotyledon cells. A second version of the protein carrying a C-terminal KDEL tag for retention in the endoplasmic reticulum was predominantly retained in the ER of seed cotyledon cells, but some of the protein was secreted to the apoplast and some was deposited in storage vacuoles. Importantly, the fate of the recombinant protein in the leaves was nearly identical to that in the seeds from the same plant. This shows that in M. truncatula, the unanticipated partial vacuolar delivery and secretion is not a special feature of seed cotyledon tissue, but are conserved in different specialized tissues. Further investigation revealed that the unexpected fate of the tagged variant of phytase likely resulted from partial loss of the KDEL tag in both leaves and seeds. Our results indicate that the previously observed aberrant deposition of recombinant proteins into storage organelles of seed tissue is not a general reflection of functional specialization, but also depends on the species of plant under investigation. This discovery will have an impact on the production of recombinant pharmaceutical proteins in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diet ofColobus polykomos on Tiwai Island: Selection of food in relation to its seasonal abundance and nutritional quality

A group of Colobus polykomosat Tiwai, Sierra Leone, demonstrated seasonal flexibility in its diet, with seeds, young leaves, and mature leaves each dominating the diet at different times. Comparison of food consumption with phenological data indicates that seeds are eaten whenever available and are preferred to other foods, while young leaves are preferred to mature leaves. Colobus polykomosalso prefer liane to tree leaves, despite the relatively high quality of mature tree foliage at the Tiwai site. Analysis of protein, fiber, and energy values of foods selected and items available, but not eaten, suggests that preference is related to protein and energy maximization. Leguminous plants, especially Papilionaceae and Mimosaceae, are highlighted as important food sources for C. polykomos;seeds and leaves from these families have a high nitrogen content, and the protein content of leguminous seeds often equals or exceeds that found in leaves. It is predicted, therefore, that colobines living in habitats with a high density of legumes will feed heavily on seeds, subject to constraints such as seasonal availability. Where suitable leguminous species are less common, a mixture of fruits, seeds, and young or mature leaves or both is likely to be selected. The results of this and other recent studies of colobines do not support the notion that colobines are specialist folivores.     

Rice Allergenic Protein and Molecular-genetic Approach for Hypoallergenic Rice

Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley α-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker’s asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic dnaA mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in dnaA mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or dnaA mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of dnaA mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or dnaA membrane preparations. Thus, the dnaA mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

Isolation and biochemical characterization of a novel leguminous defense peptide with antifungal and antiproliferative potency

Leguminous plants have formed a popular subject of research owing to the abundance of proteins and peptides with important biological activities that they produce. The antifungal proteins and peptides have been purified from a number of leguminous species. However, research continues to discover novel antifungal plant-produced peptides and proteins are being needed, specially those novel ones with both antifungal activity and other significant bioactivities. The objective of this study was to isolate a novel peptide from Phaseolus limensis. A 6.8 kDa peptide designated Limyin, with both antifungal and antiproliferative activity, was isolated from the large lima bean (P. limensis) legumes. The isolation procedure consisted of extraction, precipitation, affinity chromatography on Affi-gel blue gel, ion chromatography on SP-Toyopearl, and gel filtration on Superdex 75. Its N-terminal sequence was determined to be KTCENLATYYRGPCF, showing high homology to defensin and defensin precursors from plants. It potently suppressed mycelial growth in Alternaria alternata, Fusarium solani, and Botrytis cinerea. Its antifungal activity was stable up to 80°C. It showed antiproliferative activity towards tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. However, it had no effect on bacteria Staphylococcus aureus and Salmonella. The present findings make a significant addition of the research on leguminous plants.     

Identification of defence proteins from the seed exudates of Cicer arietinum L. and its effect on the growth of Fusarium oxysporum f.sp. ciceri

Germinating seeds tend to release a variety of proteins into their surrounding surfaces; some of which have an inhibitory action against plant pathogens. The aim of this study was to investigate and identify defence proteins present in the exudates from water-imbibed and chitosan-imbibed (0.1% w/v) seeds of chickpea (Cicer arietinum L). Chickpea seeds imbibed in chitosan released a higher amount of proteins in the exudate when compared to the seeds imbibed in water. The obtained exudates were analysed in regard to specific protein activities by enzymatic assays and SDS-PAGE analysis. Results showed that the exude obtained from chickpea seeds imbibed in chitosan solution exhibited a new isoform of chitinase, chitosanase and protease inhibitors. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus, Fusarium oxysporum f.sp. ciceri. Our results suggest that seed exudates protect seeds during their germination from soil pathogens.     

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.     

Estimation of root nodule development by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. Using hydroponic culture

A nitrogen-fixing bacterium isolated from the root nodules of a cultivated leguminous plant, soybean (Glycine max L.), was cultivable and was identified as Rhizobium sp. Bacterial species isolated from root nodules of wild leguminous plants including -bush clover, white dutch clover, wisteria, and false acacia were identified as Burkholderia cepacia, Pseudomonas migulae, Pseudomonas putida, and Flavobacterium sp, respectively, all of which are heterotrophic bacteria that grow in the rhizosphere. Temperature gradient gel electrophoresis (TGGE) 16S-rDNA bands extracted directly from the bacterial population within the root nodules of the wild leguminous plants were identified as Rhizobium sp, Mesorhizobium sp, and Bradyrhizobium sp. none were cultivable. Rhizobium sp. isolated from soybean root nodule generated approximately 48 and 19 mg/L of ammonium in glucose- and starch-defined medium, respectively, during 8 days of growth. The growth rate of Rhizobium sp. was increased by the addition of yeast extract but not by the addition of ammonium. K _m and V _max for starch saccharification measured with the extracellular crude enzyme of Rhizobium sp. were 0.7556 mg/L and 0.1785 mg/L/min, respectively. The inoculation of Rhizobium sp. culture into a hydroponic soybean plant culture activated root nodule development and soybean plant growth. The inoculated Rhizobium sp. survived for at least 4 weeks, based on the TGGE pattern of 16S-rDNA. The 16S-rDNA of Rhizobium sp. isolated from newly developed root nodules was homologous with the inoculated species.     

Bioactive compounds in legumes: pronutritive and antinutritive actions. Implications for nutrition and health

Legume seeds are employed as a protein source for animal and human nutrition not only for their nutritional value (high in protein, lipids and dietary fibre), but also their adaptability to marginal soils and climates. Human consumption of legumes has been increased in recent years, being regarded as beneficial food ingredients. Legume seeds contain a great number of compounds which qualify as bioactive compounds with significant potentials benefits to human health. These compounds vary considerably in their biochemistry and they can be proteins, glycosides, tannins, saponins, alkaloids, etc. Hence, methods for their extraction, determination and quantification are specific of each compound. They do not appear equally distributed in all legumes, and their physiological effects are diverse. Some of these compounds are important in plant defence mechanisms against predators or environmental conditions. Others are reserve compounds, accumulated in seeds as energy stores in readiness for germination. Processing generally improves the nutrient profile of legume seed by increasing in vitro digestibility of proteins and carbohydrates and at the same time there are reductions in some antinutritional compounds. Most antinutritional factors are heat-labile, such as protease inhibitors and lectins, so thermal treatment would remove any potential negative effects from consumption. On the other hand tannins, saponins and phytic acid are heat stable but can be reduced by dehulling, soaking, germination and/or fermentation. New directions in bioactive compounds research in the last decade have led to major developments in our understanding of their role in nutrition. The scientific interest in these compounds is now also turning to studies of their possible useful and beneficial applications as gut, metabolic and hormonal regulators and as probiotic/prebiotic agents.     

沈阳地区北虫草野生与市售菌株人工培育子实体的蛋白质组学比较

【背景】北虫草作为冬虫夏草的代用品,具有与冬虫夏草类似的药理活性,其富含的蛋白质和氨基酸通常作为衡量真菌营养价值的重要指标,从中分离纯化具有潜在临床应用价值的蛋白质或多肽,已成为一个研究热点。【目的】检测沈阳北虫草野生与市售菌株人工培育子实体的蛋白质组成,分析相同培育条件下获得的蛋白种类、数量及其功能的差异,为深入研究鉴定沈阳地区北虫草药用蛋白和针对性驯化提供了蛋白质组学数据基础。【方法】采集沈阳棋盘山野生北虫草菌株,与市售人工栽培北虫草菌株同期分别经组织分离、液体发酵后培育获得子实体,通过蛋白提取、胰酶酶解后,采用非标定量技术液相色谱-质谱联用方法,对野生和市售来源培育的子实体样本进行定量蛋白组的研究。【结果】共鉴定到9 233条特异性肽段和1 923个蛋白,其中含有1 163个可定量蛋白,野生来源培育子实体有214个蛋白表达发生上调,181个蛋白表达发生下调,对这些差异蛋白进行功能富集分析发现,其主要参与能量生产/转换、氨基酸转运/代谢、抗氧化功能。在相同的营养摄取条件下,野生来源培育菌种在各个能量代谢、氨基酸代谢功能中的相关蛋白表达量高于市售来源培育的菌种。野生来源培育菌种的一种抗氧化重要蛋白(Gene Name:ISF_02112)表达量远远高于(Fold Change9)市售来源培育菌种。同时与抗氧化和代谢功能相关的差异蛋白有22个。【结论】沈阳地区北虫草野生菌株经适当人工培育会保留部分优良的生物学特性,2种来源菌株培育的子实体具有丰富及优异抗氧化功能的蛋白,子实体蛋白的抗氧化能力与其整体代谢能力相关。本研究结果为深入研究鉴定北虫草药用蛋白和针对性驯化提供蛋白质组学数据基础。     

Protein Phosphorylation in Polysomes of Pumpkin Cotyledons after Coumarin Treatment

Abstract: The natural compound, coumarin, caused a change in protein pattern and influenced the phosphorylation status of some ribosome-associated proteins of pumpkin seedlings in vivo and in vitro. Low concentrations of coumarin stimulated ribosome-associated protein phosphorylation only in cotyledons but not in roots and stems. Two phosphoproteins whose phosphorylation state was influenced upon coumarin treatment could be isolated and characterized by their relative molecular weight of about 58 and 65 kDa and pl-values at 5.2 and 5.7, respectively. These phosphoproteins are not major constituents of small or large subunits of ribosomes. We did not find any influence of coumarin on phosphorylation of ribosomal proteins S6, LAO and LAI–3.     

Piperidine alkaloids from Piper retrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase

The fruits of Piperretrofractum Vahl. have been used for their anti-flatulent, expectorant, antitussive, antifungal, and appetizing properties in traditional medicine, and they are reported to possess gastroprotective and cholesterol-lowering properties. However, their anti-obesity activity remains unexplored. The present study was conducted to isolate the anti-obesity constituents from P. retrofractum Vahl. and evaluate their effects in high-fat diet (HFD)-induced obese mice. Piperidine alkaloids from P. retrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, were isolated as the anti-obesity constituents through a peroxisome proliferator-activated receptor δ (PPARδ) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPARδ protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300 mg/kg/day for 8 weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. Fat pad mass was reduced in the PRPA treatment groups, as evidenced by reduced adipocyte size. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, total lipid, leptin, and lipase were suppressed by PRPA treatment. PRPA also protected against the development of nonalcoholic fatty liver by decreasing hepatic triglyceride accumulation. Consistent with the in vitro results, PRPA activated AMPK signaling and altered the expression of lipid metabolism-related proteins in liver and skeletal muscle. Taken together, these findings demonstrate that PRPAs attenuate HFD-induced obesity by activating AMPK and PPARδ, and regulate lipid metabolism, suggesting their potential anti-obesity effects.     

Rock-degrading endophytic bacteria in cacti

A plant–bacterium association of the cardon cactus (Pachycereus pringlei) and endophytic bacteria promotes establishment of seedlings and growth on igneous rocks without soil. These bacteria weather several rock types and minerals, unbind significant amounts of useful minerals for plants from the rocks, fix in vitro N₂, produce volatile and non-volatile organic acids, and reduce rock particle size to form mineral soil. This study revealed the presence of large populations of culturable endophytic bacteria inside the seeds extracted from wild plants, from seeds extracted from the guano of bats feeding on cactus fruit, in seedlings growing from these seeds, in the pulp of fruit, and in small, mature wild plants, and are comparable in size to populations of endophytic populations in some agricultural crops. The dominant culturable endophytes were isolates of the genera Bacillus spp., Klebsiella spp., Staphylococcus spp., and Pseudomonas spp. Based on partial sequencing of the 16s rRNA gene, the isolated strains had low similarity to known strains in these genera. However, these strains have higher molecular similarity among endophytes obtained from seeds, endophytes from roots, and some bacterial strains from the rhizoplane. Seedlings developed from seeds with endophytes contain the similar species of endophytes in their shoots, possibly derived from the seeds. This study shows the involvement of endophytic bacteria in rock weathering by cacti in a hot, subtropical desert and their possible contribution to primary colonization of barren rock. This study proposes that cacti capable of acquiring diverse populations of endophytes may give them an evolutionary advantage to gain a foothold on highly uncompromising terrain.