Biochemical characterization and cellular localization of 11S type storage proteins in olive (Olea europaea L.) seeds

The composition of seed storage proteins (SSPs) in olive endosperm and cotyledon has been analyzed. Precursor forms of these proteins are made up of individual proteins, which have been purified to homogeneity and further named p1-p5 (20.5, 21.5, 25.5, 27.5, and 30 kDa, respectively). N-terminal sequences of p1 and p2 proteins displayed relevant homology to the basic subunit of the 11S family of plant SSPs (legumins). Two-dimensional polyacrylamide gel electrophoresis experiments allowed us to verify the basic character of p1 and p2 and the acidic character of p3, p4, and p5 proteins. In addition, the putative presence of highly similar isoforms or posttranslational modifications of these polypeptides was detected. As a result, a model describing the putative association of p1-p5 proteins into subunits of alpha(acidic)/beta(basic) type has been proposed. Solubility experiments have shown that the majority of these olive seed proteins from the 11S storage protein family are extracted with aqueous alcohol and only partially with water and diluted saline solutions, therefore suggesting their similarity to prolamines. Moreover, no visible differences were found in either subunit composition or 11S proteins mass among six olive cultivars examined. This result suggests that the synthesis of storage proteins is highly conserved in this plant species. By using a rabbit antiserum raised to p1 protein, the proteins have also been immunolocalized in olive seed tissues, showing that they accumulate in conspicuous protein bodies present in both the endosperm and the cotyledon.     

Comparative proteomical analysis of zygotic embryo and endosperm from Coffea arabica seeds

During coffee seed development, proteins are predominantly deposited in cotyledons and in the endosperm. Reserve proteins of the 11S family are the most abundant globulins in coffee seeds, acting as a nitrogen source during roasting and guaranteeing flavor and aroma. The aim of the present study was to compare the protein profiles of endosperm and zygotic embryos of coffee seeds. Proteins were extracted from whole seed as well as from embryo and endosperm, separately. Total proteins were analyzed by two-dimensional electrophoresis (2-DE) followed by identification by mass spectrometry (MS). The most abundant spots observed in the gels of coffee seeds were excised, digested with trypsin, and identified by MS as subunits of the 11S globulin. Spots with identical pI and molecular masses were also observed in the protein profiles of coffee endosperm and embryo, indicating that 11S protein is also highly expressed in those tissues. Peptide sequence coverage of about 20% of the entire 11S globulin was obtained. Three other proteins were identified in the embryo and endosperm 2-DE profiles as a Cupin superfamily protein, an allergenic protein (Pru ar 1), exclusive to the endosperm 2D map, and a hypothetical protein, observed only in the zygotic embryo profile.     

Characterization of Oat Endoproteinases that Hydrolyze Oat Globulins

During the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characterize these endoproteinases. A qualitative PAGE method that used oat globulins as gel‐incorporated substrates was used to determine which enzymes hydrolyzed the globulins. The proteolytic hydrolysis products were studied by hydrolyzing the globulins in vitro with the endoproteinases and analyzing the products by SDS‐PAGE. Class‐specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine‐class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major activity only appeared on the sixth day of germination. Extracts from four‐day germinated oats contained cysteine proteinases that hydrolyzed the globulins in vitro to form a polypeptide of intermediate size (MW ≈34,500). Cysteine proteases from an eight‐day germinated sample totally hydrolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurred quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value where the globulins can be degraded.     

种子萌发过程中呼吸代谢对环境变化的响应

呼吸作用是种子萌发过程中储藏物质分解、代谢不可或缺的途径和能量供给的主要来源,其变化直接影响种子的活力, 进而影响萌发后种子的生长.为系统了解种子萌发过程中, 呼吸代谢对外界环境变化所作出的响应及外界环境变化通过改变呼吸代谢影响种子萌发的内部机理, 本文从干旱、高温、低温、盐渍、紫外辐射增强、重金属与有机污染物及农药污染等环境因子的角度, 概述了近年来国内外相关研究成果, 内容涉及种子萌发时的抗氰呼吸、酶系统、膜系统、激素、内源物质的动员、萌发指数、遗传防护等内容, 总结了种子萌发时呼吸代谢对环境变化的响应机理、外界环境变化影响呼吸作用的途径和结果以及呼吸作用变化对种子萌发的影响, 并提出该领域研究的主流和发展方向, 以及应加强研究的5个方面内容.     

Protein metabolism in rice seedling

The relationship between protein synthesis and degradation in germinating rice seed were studied with protein synthetic inhibitors. Both DNP and 8-AG inhibited the degradation of glutelin, the major storage protein in rice seed, while the inhibitors had no direct effect in the activity of rice seed proteinllse in vitro. The prevention can be partly ascribed to the Inhibition of proteinase synthesis because the inhibitors depressed the increase in proteinase activity during germination. When DNP treatment was started at the onset of germination, the degradation of glutelin in the endosperm was seriously inhibited and the endosperm remained rigid over 9 days of incubation at 30°C. In contrast, the inhibition was less efficient clent when the treatment was started in the later stage. It is suggested that the degradation of the storage protein in rice seed depends on the synthetic process of the hydrolytic enzymes which increase during germination. disintegrate the compartmentation of the endosperm and allow the storage proteins to come in contact with the existing proteinases     

Molecular cloning of 11S globulin and 2S albumin, the two major seed storage proteins in sesame

Insoluble 11S globulin and soluble 2S albumin, conventionally termed alpha-globulin and beta-globulin, are the two major storage proteins and constitute 80-90% of total seed proteins in sesame. Two full-length cDNA clones were sequenced and deduced to encode sesame 11S globulin and 2S albumin precursors, respectively. Deduced amino acid composition reveals that 2S albumin, but not 11S globulin, is a sulfur-rich protein. Three abundant polypeptides of 50-60 kDa were resolved on SDS-PAGE when seed-purified 11S globulin was prepared in nonreducing conditions. Immunological analysis suggests that these three polypeptides are encoded by homologous genes. Immunodetection on the overexpressed protein of the 11S globulin clone in Escherichia coli indicates that this clone encodes the precursor protein of one of the three purified 11S globulin polypeptides.     

四倍体刺槐种子形态及结构变异研究

对四倍体刺槐种子进行观测和解剖,结果证明：四倍体刺槐种子单粒重、有胚率和萌发率分别是二倍体刺槐的26.3%、30.1%和6.3%;单粒重10mg以上的种子（比例4.41%）萌发率达62.28%,小于7mg（比例84.97%）的种子就完全失去了萌发力。四倍体刺槐种胚重量、胚乳重量、种皮重量及三者的构成比例均明显不同于二倍...     

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.     

Thermal Properties of Starch from Selected Maize (Zea mays L.) Mutants During Development

The changes in thermal properties of maize starches during five stages of kernel maturity, (12, 18, 24, 30, and 36 days after pollination [DAP]), from three mutant genotypes, amylose extender (ae), sugary-2 (su₂), and waxy (wx) in an OH43 background, and the OH43 genotype were studied using differential scanning calorimetry (DSC). Within a genotype, DSC values of starches at 24, 30, and 36 DAP were similar to each other and often were significantly different (P < 0.05) from the values at 12 DAP, indicating possible differences in the fine structure of starch during endosperm development. For su₂ starches, the gelatinization onset temperature (T_oG) significantly decreased after 12 DAP and remained low throughout the study. The gelatinization range (R_G) had a similar pattern. For wx starches, T_oG at 18 DAP was significantly lower than at 12 DAP but tended to increase after 18 DAP. The R_G increased significantly after 12 DAP and significantly decreased after 30 DAP. Thus, thermal properties of starches during early development were different from those of their mature counterparts, and differences among the mutant genotypes and the normal starch originated from the earliest endosperm development stage studied (12 DAP).     

Developmental variation of sugars, carboxylic acids, purine alkaloids, fatty acids, and endoproteinase activity during maturation of Theobroma cacao L. seeds

The changes of mono- and oligosaccharides, carboxylic acids, purine alkaloids, and fatty acid composition, and of aspartic endoproteinase activity, were analyzed during seed development in two varieties of cacao (Theobroma cacao). The majority of the components examined either decreased or accumulated steadily in concentration during the second half of bean development. Sucrose is the major sugar in the mature embryo, whereas fructose and glucose are at higher concentrations in the endosperm tissue. Considerable amounts of malate are found in the endosperm, whereas citrate is the dominant carboxylic acid in the embryo. A major change in the fatty acid composition occurs in the young embryo when the proportion of stearic acid increases rapidly at the expense of linoleic acid, which is reduced from about 18 to 3%. Theobromine is the dominant purine alkaloid (ca. 80%), and caffeine appears only toward the end of seed maturity. Aspartic endoproteinase activity increases rapidly during embryo expansion, reaching a maximal activity before final maturity. The results are discussed in conjunction with physiological changes in developing seeds, and the potential contributions of the compounds analyzed for cocoa quality.     

Lipid characterization in vegetative tissues of high saturated fatty acid sunflower mutants

Modifications of the fatty acid composition of plant vegetative tissues produce deficient plant growth. To determine the expression of the seed high-saturated sunflower (Helianthus annuus L.) mutant character during the vegetative cycle, five sunflower mutant lines (three high-stearic and two high-palmitic) have been studied during their germination and vegetative cycle. No significant variations with regard to the control lines were observed in the mutant vegetative tissue lipids; however, during seed germination important differences between lines were found. Although in the early steps of germination the palmitic and stearic acid levels in the respective mutants seedling cotyledons continued being higher than those of the control lines, they decreased and reached values similar to the controls, except in CAS-3. Variations in the cotyledon palmitic acid content with regard to the control line were also observed in high-stearic mutants, suggesting the expression of a modified acyl-ACP thioesterase or recycling of seed fatty acids during seedling development.     

Soil yeasts and their role in seed germination

A collection of yeasts isolated from soils of different types (soddy-podzolic, gray forest, and chernozems) in European Russia was analyzed. Soil yeasts were found to accelerate the development of seeds of common crops (wheat, barley, and rye). Cultures of yeasts from different taxa (representatives of the Rhodotorula, Cystofilobasidium, Sporobolomyces, Metschnikowia, Saccharomyces, Aureobasidium, Debaryomyces, Cryptococcus genera) are shown to stimulate the seed germination. The impact of soil yeasts (and preparations made on their basis) on seed germination is determined by their penetration to seed endosperm (endophytic microflora) followed by the isolation of amylolytic enzymes and physiologically active substances stimulating seed germination. The method of seed treatment is shown to affect greatly the direction of the preparations’ action. When soaking seeds in solutions with the preparation, microorganisms multiply on the seed surface, and when treating seeds using a semi-dry method, seeds themselves and endophytic microorganisms inside them begin to develop. This study can be considered one of the new directions of soil microbiology related to studying the participation of soil microorganisms in the formation of endophytic communities and their role in the germination of vascular plant seeds.     

SPATIAL DISTRIBUTION AND ACCUMULATION OF CALCIUM IN DIFFERENT TISSUES,DEVELOPING SPIKES AND SEEDS OF FINGER MILLET GENOTYPES

□ Total calcium (Ca) content in different tissues, developing spikes (S1, S2, S3 and S4 stages), and distribution sites in seeds of five finger millet genotypes, which were differed in total calcium content, were analyzed. Atomic absorption spectroscopy (AAS) revealed stage-dependent quantitative changes in calcium accumulation from low to high during different stages of developing spikes and also in different tissues and grains. Results of scanning electron microscopic (SEM) energy dispersive X-ray (EDX) analysis showed differential distributions of the calcium in seed components viz. seed coat, aleurone layer and endosperm. Highest calcium content was observed in aleurone layer followed by seed coat while lowest calcium was found in endosperm of all genotypes. Major differences were found amongst genotypes with regard to the calcium distribution in seed components. The differential regulation of transport machinery might be responsible for differential calcium ion delivery and spatial distribution in the seed. A clear understanding of differential accumulation, spatial distribution and the variation of calcium within finger millet genotypes/seeds could be exploited for breeding for their bio-fortification and development of calcium rich functional foods.     

Quality protein maize: a biochemical study of enzymes involved in lysine metabolism.

Quality protein maize (QPM) varieties have been produced by the introduction of opaque-2 modifier genes. Two QPM varieties, BR451 and BR473, a wild type and an opaque-2 variety, have been used to study key enzymes controlling lysine metabolism in the endosperm during development. Aspartate kinase and homoserine dehydrogenase enzymes, which are involved in lysine and threonine biosynthesis, respectively, exhibited identical activity patterns during endosperm development, with a maximum specific activity at 16 days after pollination. The QPM varieties exhibited higher levels of aspartate kinase activity in the endosperm, suggesting an increased rate of lysine biosynthesis when compared to the opaque-2 and wild-type genotypes. Similar results were observed for the lysine ketoglutarate reductase and saccharopine dehydrogenase enzymes, which form a single bifunctional polypetide involved in endosperm lysine degradation. Both enzyme activities were strongly reduced in the opaque-2 maize variety when compared to the wild-type maize, whereas the QPM varieties exhibited even lower levels of lysine ketoglutarate reductase-saccharopine dehydrogenase activities when compared to the opaque-2 variety. The developmental pattern of enzyme activity showed a different profile when compared to the enzymes involved in lysine biosynthesis, with activity being detected only 12-16 days after pollination (DAP) and maximum activities approximately 24 DAP. These results also suggest that the modifier genes have intensified the effect of the opaque-2 mutation on lysine ketoglutarate reductase-saccharopine dehydrogenase. These alterations lead to an increase in soluble lysine in the endosperm of the QPM varieties when compared to the opaque-2 and wild type.     

Cloning, functional expression, and characterization of cystatin in sesame seed

A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K(i) values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination.     

Gene families encoding isoforms of two major sesame seed storage proteins, 11S globulin and 2S albumin

Sesame (Sesamum indicum L.) seed has been recognized as a nutritional protein source owing to its richness in methionine. Storage proteins have been implicated in allergenic responses to sesame consumption. Two abundant storage proteins, 11S globulin and 2S albumin, constitute 60-70 and 15-25% of total sesame proteins, respectively. Two gene families separately encoding four 11S globulin and three 2S albumin isoforms were identified in a database search of 3328 expressed sequence tag (EST) sequences from maturing sesame seeds. Full-length cDNA sequences derived from these two gene families were completed by PCR using a maturing sesame cDNA library as the template. The amino acid compositions of these deduced storage proteins revealed that the richness in methionine is attributed mainly to two 2S albumin isoforms and partly to one 11S globulin isoform. The presence of four 11S globulin and three 2S albumin isoforms resolved in SDS-PAGE was confirmed by MALDI-MS analyses. The abundance of these isoforms was in accord with the occurrence frequency of their EST sequences in the database. A comprehensive understanding of these storage proteins at the molecular level may also facilitate the identification of allergens in crude sesame products that have caused severe allergic reactions increasingly reported in the past decade.     

作物种子胚乳中等位基因多态性表达

本文综述了作物种子胚乳中等位基因表达多态性的研究进展，介绍了基因组印记现象及其对作物种子胚乳性状的表观遗传及种子发育的影响。     

Characterization of Oat Endoproteinases that Hydrolyze Oat Avenins

During oat seed germination, the insoluble storage proteins must be solubilized and transported to the embryo for use by the developing plantlet. We showed earlier that pH 6.2 active serine and metalloproteinases were the predominant gelatin‐hydrolyzing enzymes of oats, while the oat globulins were degraded by pH 3.8 active cysteine proteases. The pH of the endosperms of germinating oats is 6.2. We have continued our characterization of the germinated oat proteinases by determining which hydrolyze avenins, the oat storage prolamins. Avenins of resting seeds were purified and hydrolyzed with proteinases that were extracted from oat seeds that were germinated for various periods. The peptides released were analyzed using SDS‐PAGE. The α‐avenins were hydrolyzed at pH 3.8 by cysteine proteinases from four‐day germinated seeds and the β‐avenins were hydrolyzed by similar enzymes from eight‐day germinated seeds. At pH 6.2 or pH 5.0, the avenins were not degraded by any of the germinated oats endoproteinases. It is probable that some kind of pH compartmentalization occurs within germinating oat seed. After four days of germination, either new proteinases form or some preexisting proteinases are activated. The cysteine proteinases are apparently responsible for the majority of the storage protein hydrolysis that occurs during oat germination.     

作物种子胚乳等位基因多态性表达研究进展

综述了作物种子胚乳中等位基因表达多态性的研究进展,介绍了基因组印记现象及其对作物种子胚乳性状的表观遗传及种子发育的影响。     

苦荞芽期黄酮合成关键酶和MYB转录因子基因的表达分析

苦荞(Fagopyrum tataricum)作为一种药食两用植物,富含以芦丁为主的黄酮类化合物.苦荞芽期芦丁含量较高,其分子机制尚不清楚.本研究选用西荞2号,采用AlCl3法测定了苦荞芽期6～10 d胚轴和子叶中的总黄酮,采用半定量RT-PCR分析其黄酮合成途径中主要关键酶基因苯丙氨酸氨裂解酶基因(Pal)、查尔酮异构酶基因(Chi)和黄酮醇合酶基因(Fls),以及MYB转录因子基因FtMyb1、FtMyb2和FtMyb3的相对表达水平,并对三者之间的相关性进行了统计学分析.结果表明,以相关系数绝对值大于0.75为阈值,子叶中,总黄酮的积累与FtMyb3表达显著正相关(0.9625),与FtMyb2表达显著负相关(-0.8572); Chi与FtMyb2表达显著正相关(0.8468),与FtMyb3表达显著负相关(-0.8010):Pal、Chi和Fls表达彼此显著正相关,相关系数分别为0.9119、0.8920和0.7584.子叶中总黄酮含量在4.58％～5.54％之间,且随芽期递增.Pal、Chi和Fls整体表达趋势相似,均呈现先升高后降低的趋势,且Fls的表达水平明显高于前二者.FtMyb2和FtMyb3整体表达趋势相反,FtMyb2呈下降趋势,FtMyb3呈上升趋势.胚轴中,总黄酮含量与Chi显著负相关(-0.8989); Fls与Chi显著负相关(-0.7498).结果提示,苦荞芽期黄酮合成的分子机制较为复杂,但部分基因表达仍存在显著相关性联系,为进一步选择苦荞分子操作靶位点提供参考.