Immunochemical studies on barley seed storage proteins

The antigenic relationships between the prolamins of barley, rye and wheat have been studied by examining the specificity of an antibody to C hordein in a quantitative study using a laser nephelometer. The antibody reacts weakly with B hordein and strongly with 75-kdalton and 40-kdalton -secalins from rye and ₃ some -gliadins from wheat. Absorption experiments and immunodiffusion tests indicate that there are shared antigenic determinants for most of the prolamins. All the species with reacting prolamins belong to the sub-family Festucoideae of the Gramineae. The prolamins of maize, pearl millet and sorghum, species of the sub-family Panicoideae, do not react. The results confirm the known lack of homology between the prolamins of the two sub-families and also indicate the presence of relationships not yet established between C hordein, the 75-kdalton and 40-kdalton -secalins and also ₃ gliadin.Abbreviations HMW high molecular weight - PAGE polyacnylamide-gel electrophoresis - PBS phosphate-buffered saline - RLS relative light scattering - SDS sodlum dodecyl sulphate     

Molecular evolution of the seed storage proteins of barley, rye and wheat

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.     

In vitro synthesis of barley storage proteins

Membrane-bound polysomes were isolated from developing endosperms of barley (Hordeum vulgare L.) and shown to support the synthesis of trichloroacetic acid-insoluble material by an in vitro wheat germ protein synthesis system. The mRNA associated with the polysomes was separated from the ribosomes by affinity chromatography on oligo-dT cellulose and was also shown to support in vitro protein synthesis. The poly-A⁺ RNA isolated contained material of between 0.55 and 2.55 kilobases in length with about 6% poly A. The products of in vitro protein synthesis resembled hordeins (the prolamin storage proteins of the barley endosperm) in that they were predominantly soluble in 55% propan-2-ol, contained a low proportion of lysine as compared with leucine and had similar, but not identical, electrophoretic properties. The differences in the electrophoretic behaviour between the products of poly-A⁺ RNA translation and authentic hordeins is suggested to be due to the presence of an extra (leader?) sequence on the former.     

Assembly and transport of seed storage proteins

Plant seeds store nitrogen by accumulating storage proteins in protein bodies within various compartments of the endomembrane system. The prolamin storage proteins of some cereal species are normally retained and assembled into protein bodies within the ER. Yet, these proteins lack a C-terminal KDEL/HDEL signal, suggesting that their retention is regulated by novel mechanisms. Furthermore, in other cereal species, such protein bodies formed within the ER may be subsequently internalized into vacuoles by a special route that does not utilize the Golgi complex. Thus, studies of the routing of seed storage proteins are revealing novel mechanisms of protein assembly and transport in the endomembrane system.     

Long-term storage of collection cultures of actinobacteria

A total of 553 collection cultures of actinobacteria, including 453 reference ISP strains, were studied after long-term storage in a lyophilized state, as soil cultures, and under mineral oil. It was established that their viability reached a near-critical level. A number of methodological approaches to optimization of activation and proliferation of actinobacteria made it possible to restore the viability and major cultural and morphological properties of 65% of actinobacteria stored in a lyophilized state or under mineral oil. The actinobacteria stored as soil cultures almost completely lost their viability. Resuscitated actinobacteria exhibited a high level of genetic instability, which resulted in the emergence of more than three phenotypically different types of colonies. The population spectrum shifted towards an increase in the content of minor phenotypes with low spore-forming capacity. Significant changes in the cultural and morphological properties of a number of resuscitated strains were observed. Desirability of the application of antioxidants or growth-stimulating compounds in order to restore the viability of Actinobacteria cultures and to stabilize them after long-term storage was demonstrated.     

Characterization of olive seed storage proteins

At present little is known about olive seed storage proteins (SSPs). A better understanding of olive SSPs will be important for future biotechnology efforts. In the present study, we first developed a protocol relied on chloroform for preparing protein samples free of lipids from lipid-rich olive seeds. Then, we characterized olive SSPs by SDS-PAGE, N-terminal sequencing and immunoblot. Two smaller subunits (20 and 21.5 kD) of SSPs were purified to homogeneity and used for antibody production or N-terminal sequencing. N-terminal sequencing confirmed that major olive SSPs are 11S globulins. Moreover, the components and size distribution of SSPs are identical among several olive cultivars examined, suggesting that their synthesis is highly conserved in this species. Olive SSPs are soluble in aqueous alcohol, with limited solubility in water and dilute salt. Thus, despite their homology with globulins, olive SSPs are similar in solubility to prolamins and different from globulins in other dicot plants. Finally, the accumulation of olive SSPs during fruit maturation was examined. Our results revealed that the accumulation of SSPs is time-dependent and tissue-specific, and only 105 days after pollination (DAP), did individual components of SSPs synthesize substantially, and accumulate rapidly in large quantities over a short period of time. Our results suggest that a 36 kD protein is the precursor of olive SSPs, and 90–105 DAP seems to be a crucial transition period (from a precursor to mature subunits) for the accumulation of SSPs.     

Secondary structure prediction of seed storage proteins

The comparison of partial primary structure of seed storage proteins leads to show homologies inside of each considered family (Legume seed legumins and cereal prolamins). Predicted secondary structures deduced from the presently known sequences also exhibit considerable homologies, which implies a severe conservatism of these proteins. Short repetitive segments of sequence of 5-20 residues are frequently occurring and give rise to the prediction either of beta-structure (or alpha-helix) bonded by beta-turns or of successive beta-turns. The latter conformation, which would be able to form a helicoidal arrangement, could contribute to a maximal packing of the protein molecules inside of the subcellular organelles (protein bodies) within which they are confined. As the only known function of seed storage proteins is to provide amino acids to the embryo, it is suggested that their ability to occupy a minimal volume is actually a reasonable explanation of their extreme conservatism in the course of evolution.     

Accumulation of seed storage proteins and the taxonomy ofPoaceae

The accumulation of specific seed proteins is a taxonomically valuable feature and can be used to additionally characterize plant taxa. To date, mainly crop proteins have been analysed in thePoaceae. In this investigation seed proteins from 147 species were screened with emphasis on legumin-like proteins and prolamins. The groups resulting from evaluation of the protein profiles correspond with well-known subfamilies and tribes.Panicoideae are clearly separated fromPooideae. WithinPooideae, theBromeae plusTriticeae tribes revealed obvious similarities.Lolium, Festuca andVulpia, generally included in the tribeFestuceae, revealed a protein profile similar to the profile of theBromeae/Triticeae. Legumin-like proteins are accumulated abundantly inBambusoideae andPooideae exceptBromeae/Triticeae, however, only the species included in theAveninae subtribe produce soluble (globulin-type) legumins as already known fromAvena sativa. Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Biochemical and molecular characterization of three barley seed proteins with antifungal properties

We have purified three proteins from barley (Hordeum vulgare L.) seeds which synergistically inhibit the growth of fungi measured in a microtiter well assay. The proteins are a 26-kDa chitinase, a 30-kDa ribosome-inactivating protein, and a 32-kDa (1-3)-beta-glucanase. Full-length cDNAs encoding them were isolated and sequenced to determine the complete primary structures of the proteins. Northern hybridizations with the cDNAs as probes showed that the corresponding mRNAs accumulate differentially during seed development and germination. Chitinase mRNA accumulates to high levels in aleurone cells during late seed development and early germination, while high levels of mRNA encoding the ribosome-inactivating protein accumulate only in the starchy endosperm during late seed development. The glucanase mRNA accumulates to low levels during seed development and to higher levels in aleurone and seedling tissues during germination. Southern hybridizations showed that the three proteins are encoded by small families of three to eight genes. Their biological roles and potential use in genetic engineering studies are discussed.     

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Summary Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different -major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.Contribution no. 55 from the Center of Vegetable Breeding, Portici, Italy     

小麦种子贮藏蛋白质研究进展

小麦醇溶蛋白组成可以作为小麦品种鉴定的指纹图谱,其分离方法有酸性电泳、反相高压液相色谱（RP－HPLC）和毛细管电泳（CE）等手段,3种方法相互补充,而CE分辨率最高。对醇溶蛋白酸性电泳条件的改良和完善仍在进行中,利用最新的分离技术对小麦醇溶蛋白基因进行染色体定位和遗传行为分析是近年来醇溶蛋白研究的另一领域。小麦高分子量麦谷蛋白亚基（HMW－GS）与小麦面包烘烤质量密切相关,关于它的研究目前主要集中在3个方面;对各个迁3移率较近的亚基进行快速,准确分离方法的研究,HMW－GS与小麦面包烘烤质量关系的研究和通过基因工程来改良小麦的品质、提高面粉的加工特性等。低分子量麦保蛋白（LMW－Glutenin)影响小麦面粉的特性,截止目前已经获得了17个该基因的克隆,并对其基因结构进行了描述,有些低分子量麦谷蛋白亚基（LMW－GS）加入碱性面粉后改变了面筋的性质,报道了小麦醇溶蛋白,高分子量麦谷蛋白亚（HMW－GS）、低分子量麦谷蛋白亚基（LMW－GS）3个方面的最新研究进展。     

Extraction and genetic control of two new water-soluble proteins of mature barley seed

This paper describes the genetic control of two new water-soluble proteins in barley. Water-soluble proteins (WSPs) of mature barley seed form part of the albumin/globulin class of seed proteins. They can be extracted from hand-milled grain with water, though some WSPs are more efficiently extracted with a solution of 10 mM dithiothreitol. Polymorphisms for WSPs were detected in isoelectric focusing gels incorporating various ampholine combinations. Two new controlling genes (Wsp4 andWsp5) have been identified and located using wheat/barley chromosome addition lines and barley doubled haploids.Wsp4 is located on chromosome 2 (2H), andWsp5 was found to be tightly linked toWsp2 on the long arm of chromosome 7 (5HL). Segregation of a sixth gene (Wsp6) is also described, but this has not been mapped. The results are discussed with respect to other previously mappedWsp loci.This work was funed by the Scottish Office of Agriculture and Fisheries Department and the Agricultural and Food Research Council.     

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。     

Advances in the molecular biology of plant seed storage proteins

Plant seed storage proteins were among the first proteins to be isolated (20); however, only recently, as a result of using molecular biology techniques, have the amino acid sequences of many of these proteins been determined. With the accumulation of amino acid sequence data for many vicilin-type storage proteins much has been learned concerning the location of conserved amino acid regions and other regions which can tolerate amino acid sequence variation. Combining this knowledge with recent advances in plant gene transfer technologies will allow molecular biologists to correct (by using amino acid replacement mutations) the sulfur amino acid deficiency inherent to bean seed storage proteins. The development of more nutritious soybean and common bean seeds will be of benefit to programs involving human and animal nutrition.     

Species relationships inPhaseolus: Electrophoretic analysis of seed storage proteins

Relationships among storage proteins in seeds from cultivars and primitive accessions of the four economically most important species ofPhaseolus — P. vulgaris, P. coccineus, P. acutifolius andP. lunatus — were studied. Analysis of SDS-polyacrylamide gel electrophoretic patterns of storage seed proteins revealed common characteristics in the major groups of polypeptides forP. vulgaris, P. coccineus andP. acutifolius, while clear differences existed between thesePhaseolus species and P.lunatus.     

Hydrolysis of storage proteins in barley endosperms : analysis of soluble products

Soluble products, released by the hydrolysis of hordeins into the media of barley (Hordeum vulgare cv. Perth) half-seeds were analyzed. Large polypeptide fragments (methanol-insoluble) were identified using the Western immunoblot technique with the antibodies prepared against B and C polypeptides of hordein. A number of hordein IgG-reacting bands were noted in the samples from dry kernels. In samples incubated in the absence of gibberellic acid, polypeptide fragments in the size range of 25 to 30 kilodaltons appeared within 24 hours, and those in the size range of 40 kilodaltons became more prominent. In samples incubated in the presence of gibberellic acid, polypeptide fragments in the size range of 45 to 67 kilodaltons were less apparent and those in the size range of less than 15 kilodaltons were more pronounced. The hordein-related polypeptide fragments were present in low amounts after 72 hours in the presence of gibberellic acid. Methanol-soluble peptides were fractionated, on the basis of size, into two broad peaks. In the absence of gibberellic acid, there was no significant change in their profile over a 72 hour incubation period. In the presence of this growth substance, however, there was a decrease in the proportion of large size peptides (50-70 amino acid residues in length), and an increase in the levels of small peptides (15-35 amino acid residues in length) and amino acids. Our interpretation of the results is that the release of the initial large polypeptide fragments from hordein proteins is mediated by a protease(s) whose appearance is not dependent on the exogenously added gibberellic acid. Further hydrolysis is, however, mediated by proteases induced in the presence of this growth substance.     

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.     

Variation studies on seed storage proteins and phylogenetics of the genus Cucumis

Seed storage proteins were analyzed for variation in polypeptide patterns and proportion of four protein fractions in Cucumis melo and for interrelationships of different taxa of the genus Cucumis. SDS-PAGE of total protein extracts of 11 lines of C. melo representing different geographic regions showed considerable variation in their polypeptides in the range of molecular weights 50–55, 35–39, 20–26 and 16–19 kDa. As compared to one subunit pair reported earlier, two-dimensional gel electrophoresis revealed subunit pairs of molecular weights 53, 52, 50, 42, 39 and 23 kDa, each consisting of a large and a small subunit like those of legumin-like 11S proteins of leguminous seeds. The salt-soluble globulins represented the major protein fraction (44.8–48.1%), followed by glutelins and albumins with prolamins being the lowest in the seed. Based on the protein profiles of seed protein extracts of 28 Cucumis taxa, similarity matrix indices and UPGMA dendrogram showed that eight taxa of C. melo were clustered in two subclusters of four taxa each. Five taxa of C. sativus along with C. zeyheri belonged to another cluster, sister to the third cluster of remaining taxa. In this third cluster, C. anguria var. longaculeatus showed 100% similarity with C. myriocarpus and C. myriocarpus ssp. leptodermis as compared to only 64% similarity with C. anguria; C. myriocarpus ssp. myriocarpus occurred along with C. prophetarum in the same group rather than with C. myriocarpus. The accession PI-299570 representing an undescribed taxon of Cucumis exhibited 100% similarity with C. pustulatus, and C. sagittatus was the most distant from all Cucumis taxa.