Legumin and vicilin,storage proteins of legume seeds

The structure, location in the seed and distribution of the storage protein of legume seeds are described. Methods which have been employed for the extraction, purification and characterisation of seed globulins are reviewed in relation to modern biochemical practice. The physical, chemical and immunological characteristics of the classical legumin and vicilin preparations from Pisum sativum are summarised and the distributions of proteins with sedimentation coefficients and/or immunological determinants similar to those of legumin and vicilin, are tabulated. The structure and composition of various purified legumin and vicilin-type proteins from a variety of legumes, are compared.     

Legumin of Vicia faba major: accumulation in developing cotyledons,purification, mRNA characterization and chromosomal location of coding genes

Summary Experiments were carried out on Vicia faba major involving (1) determination of the pattern of legumin accumulation during seed development, (2) protein purification from mature cotyledons, (3) the characterization of legumin mRNA, and (4) the chromosomal localization of the genes coding for legumins. In developing cotyledons the synthesis of legumin begins 28 days after petal desiccation (DAPD), and 4 days after initiation of vicilin synthesis. The two subunits (_A and _A) of legumin A appear 2 days earlier than those (_B and _B) of legumin B. While the accumulation of vicilin peaks on the 30th DAPD, that of legumin continues during further seed development, and the synthesis of legumin mRNA peaks on the 37th DAPD. Northern blot hybridizations using two DNA plasmids containing cDNA inserts with sequence homology to the A- and B-type legumin genes, respectively, indicated that legumin mRNAs extracted from cotyledons 36 DAPD band below the 18S RNA band. In addition, a faint band below that of the 25S RNA band can be observed in legumin mRNAs extracted from cotyledons at an earlier developmental stage (30 DAPD). By means of polyacrylamide gel electrophoresis in the presence or absence of SDS and 2-mercaptoethanol, two fractions could be eluted after zonal isoelectric precipitation of the globulins from mature seeds: one fraction contains mainly vicilin, the other, legumin. In situ hybridization showed that legumin genes are arranged in two clusters: the genes coding for legumin A are located in the longer arm of the one between the two shortest subtelocentric chromosome pairs whose centromere is in a less terminal position; those coding for legumin B are located in the non-satellited arm of the longer submetacentric pair.     

Legumin- and vicilin-like proteins from spores of the fern Matteuccia struthiopteris

Legumin- and vicilin-like proteins have been isolated from spores of the fern Matteuccia struthiopteris. Their relationship with seed legumin and vicilin was demonstrated by cross-reactivities of antibodies directed against respective storage globulins from Vicia faba as evidenced by Western blotting. The Matteuccia legumin-like protein was characterised as a 300-340 kDa holoprotein preferentially consisting of a 32 kDa alpha-chain and a 24 kDa beta-chain. Patterns of limited proteolysis of the spore legumin-like protein and seed legumins were similar as well. In contrast to seed legumins, the Matteuccia legumin-like protein is devoid of disulfide bridges between alpha- and beta-chains. A 52 kDa polypeptide of the Matteuccia vicilin-like protein, first detected by SDS gel electrophoresis, is probably encoded by a vicilin-like gene specifically expressed in Matteuccia struthiopteris spores (Shutov et al. 1998). The vicilin-like holoprotein was found to form a complex of 600 kDa apparent molecular mass, presumably composed of four vicilin-like trimers.     

Processing Pisum sativum seed storage protein precursors in vitro

The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea(Pisum sativum) is complex,resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin,the major storage globulins.Translation in vitro of mRNAs hybridselected from mid-maturation pea seed RNAs by defined vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed;the derived polypeptides were of comparable sizes to those observed in vivo.The feasibility of transcribing mRNA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone,providing a method for determining polypeptide products of an expressed sequence.This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.     

Legumin-like and vicilin-like seed storage proteins: Evidence for a common single-domain ancestral gene

Legumin-like 11S and vicilin-like 7S globulins are the main storage proteins of most angiosperms and gymnosperms. The subunits of the hexameric legumin are synthesized as a precursor comprising a N-terminal acidic - and a C-terminal basic -chain. The trimeric vicilin molecule consists of subunits composed of two symmetrical N- and C-terminal structural domains.In a multiple alignment we have compared the N-terminal and C-terminal domains of 11 legumns and seven vicilins of several dicot, monocot, and gymnosperm species. The comparisons using all six possible pairwise combinations reveal that the N-terminal and C-terminal domains of both protein families are similar to each other. These results together with data on the distribution of variable and conserved regions, on the positions of susceptible sites for proteolytic attack, as well as on the published 7S protein tertiary structure suggest that both protein families share a common single-domain ancestor molecule and lead to the hypothesis that a triplication event has occurred during the evolution of a putative legumin/vicilin ancestor gene.Moreover, the comparison of the intron/exon pattern reveals that at least three out of five intron positions are precisely conserved between the genes of both protein families, further supporting the idea of a common evolutionary origin of recent legumin and vicilin encoding genes. Correspondence to: H. Bäumlein     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

Heterogeneity of sulphur content in the storage proteins of pea cotyledons

By means of crossed immunoelectrophoresis of the cotyledonary storage proteins of Pisum sativum L. it was shown that reduced accumulation of the legumin fraction, resulting from severe sulphur deficiency during growth, is accompanied by relative suppression of a quantitatively minor storage protein (Peak 3) shown previously by subunit analysis to be related to the vicilin series of holoproteins. The pattern of isotopic labelling of the storage proteins after injection of [³⁵S]methionine into the pedicel during seed development under normal nutritional conditions indicated that Peak-3 protein, like legumin, has a relatively high content of sulphur amino-acids. Like certain of the vicilin molecules carrying the determinants responsible for Peak-4, Peak-3 protein binds selectively to concanavalin A.     

Measurement of gene number for seed storage proteins in Pisum.

We have measured the numbers of genes coding for the three seed storage proteins, vicilin, convicilin and legumin, in a number of Pisum genotypes of variant protein composition. No difference in gene number existed among P. sativum genotypes for any of the proteins. There were differences in the number of genes coding for individual proteins with approximately 11 genes (per haploid genome) for vicilin, 8 genes for legumin and 1 gene for convicilin.     

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     

The comparison of seed proteins of several representatives of the genusPisum with respect to their relationships An immunological comparison

Using immunochemical methods a comparison was made of a complex of water soluble (albumins) and salt soluble (globulins) seed proteins, especially vicilin and legumin, in selected species of the genusPisum, to determine the degree of their taxonomic relationship. Within the genusPisum the interspecific differences betweenP. abyssinicum, P. cinereum,P. elatius, P. fulvum, P. sativum, andP. syriacum are much smaller, and thus the taxonomic distances are shorter than is the casee.g. in the genusPhaseolus. In spite of this fact one may state thatPisum sativum, P. elatius andP. syriacum constitute a definite group, whereasP. abys-sinicum, P. cinereum and P.fulvum have longer taxonomic distances.     

Glycoprotein Metabolism in the Cotyledons of Pisum sativum during Development and Germination

The glycoprotein nature of legumin and vicilin, the reserve globulins in the cotyledons of Pisum sativum was studied. Legumin from mature seed was found to contain 1% neutral sugars (mannose and glucose) and 0.1% amino sugar (glucosamine), whereas vicilin contained 0.3% neutral sugar (mannose) and 0.2% amino sugar (glucosamine). On the basis of the incorporation of ¹⁴C-labeled glucosamine, it appeared that not all of the component subunits of the reserve proteins are glycosylated to the same extent. In addition, it has been established that glycosylation occurs after peptide synthesis. During seed development there was a change in neutral sugars and amino sugar ratio in vicilin. During germination, the neutral sugars and the amino sugar content of the glycoproteins declined. These findings are discussed in relation to the synthesis and degradation of the glycosyl component of the glycoproteins.     

Phaseolus coccineus L. Storage Proteins. Extraction and Characterization

Phaseolus coccineus storage globulins were extracted from mature cotyledons, purified and characterized. Three major proteins were separated. A component showing erythroagglutinating activity was thoroughly purified by thyroglobulin-Sepharose chromatography. The relative molecular masses of the three fractions are Mr = 330, 178, and 500 kDa as determined by polyacrylamide gel electrophoresis (PAGE). They correspond to the proteins found in other systems and classified as phytohaemagglutinin (PHA), vicilin and legumin, respectively. Electrophoretic analyses under denaturating conditions (SDS-PAGE) evidenced the major subunits for the three proteins. Isoelectrofocusing of the isolated proteins indicated a large heterogeneity for vicilin. Part I.     

The characterisation of vicilin during seed development in Vicia faba (L.)

Summary Vicilin and legumin were extracted from developing seeds at different stages using the classical method of repeated isoelectric precipitations. The subunits of these two protein fractions were separated by SDS gel electrophoresis, and it was shown that the sub-unit structure of vicilin changed during development whereas that of legumin did not. Thus vicilin is not a single protein.Vicilin was formed prior to legumin during seed development although the rate of synthesis of the latter was faster, so that in the mature seed the ratio of legumin to vicilin was about 4:1 by weight.     

What is pea legumin — Is it glycosylated?

Since there is some question as to whether or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of Pisum sativum L. supplied with [¹⁴C]-labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387–400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509–520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40–70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263–279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used.     

Coffee seeds contain 11S storage proteins

A legumin-like seed protein was purified from the endosperm of coffee ( Coffea arabica L. cv. Colombia). In contrast to legumes, where efficient storage globulin extraction requires buffered saline solutions well above the acidic pK_I of the globulins, coffee legumin is readily extracted with acidic aqueous buffers. The coffee legumin migrates like other 11S storage globulins in sucrose gradients. Subunits of coffee legumin have an apparent molecular mass of about 55 kDa after one-dimensional SDS-polyacrylamide gel electrophoresis in the absence of a reducing agent. In the presence of 2-mercaptoethanol, two polypeptides appear that have apparent molecular masses of 33 and 24 kDa. Two full-length cDNAs were generated from mRNA of developing seeds that were more than 98% homologous. They had open reading frames of 1 458 and 1 467 bp. Each encoded legumin precursors of 486 and 489 amino acids, respectively (M_r=54 136 and 54 818). Examination of a 5' promoter region from a coffee legumin gene revealed a putative legumin-box. Genomic DNA from C . arabica was digested with six different restriction endonucleases. After separation of the fragments by electrophoresis, single discrete fragments on DNA blots hybridized strongly to a cDNA probe for the acidic chain. Other fragments that hybridized weakly with this probe were visible after hybridization at very low stringency. DNA from other species and commercially important cultivars that comprise the genus Coffea produced similar results. Immunocytochemical studies revealed that some legumin was detected in the cytoplasm in mature coffee seeds, but that the majority of it was in large storage vacuoles that accounted for most of the cell volume.     

Electron microscopy of seed-storage globulins

The quaternary structures of a range of seed globulins, including examples of both the so-called 7 S and 11 S types, have been examined by electron microscopy. The legume 7 S proteins, phaseolin (bean), beta-conglycinin (soybean), and vicilin (pea), appear as flat discs of diameter ca. 8.5 nm and thickness ca. 3.5 nm formed by association of three subunit domains. Phaseolin converts to an 18 S tetramer at acid pH, and images recorded under these conditions suggest that four of the 7 S protomer discs associate to form the faces of a regular tetrahedron. The classical 11 S seed globulins, cucurbitin (pumpkin) and legumin (pea), are approximately spherical molecules of diameter ca. 8.8 nm composed of six subunits. In contrast, the hexameric 10 S storage protein from lupin seed, conglutin gamma, appears toroidal in shape with outer diameter ca. 10.3 nm and thickness ca. 2.2 nm. These results indicate that constraints imposed on seed proteins by their role in sustaining the germinating plant may have allowed a variety of different globulin structures to accumulate in the protein-storage bodies of seeds.     

Protein patterns associated with <Emphasis Type="Italic">Pisum sativum</Emphasis> somatic embryogenesis

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.