The legumin gene family: structure of a B type gene of Vicia faba and a possible legumin gene specific regulatory element.

The field bean, Vicia faba L. var. minor, possesses two sub-families of 11 S legumin genes named A and B. We isolated from a genomic library a B-type gene (LeB4) and determined its primary DNA sequence. Gene LeB4 codes for a 484 amino acid residue prepropolypeptide, encompassing a signal peptide of 22 amino acid residues, an acidic, very hydrophilic alpha-chain of 281 residues and a basic, somewhat hydrophobic beta-chain of 181 residues. The latter two coding regions are immediately contiguous, but each is interrupted by a short intron. Type A legumin genes from soybean and pea are known to have introns in the same two positions, in addition to an extra intron (within the alpha-coding sequence). Sequence comparisons of legumin genes from these three plants revealed a highly conserved sequence element of at least 28 bp, centered at approximately 100 bp upstream of each cap site. The element is absent from the equivalent position of all non-legumin and other plant and fungal genes examined. We tentatively name this element "legumin box" and suggest that it may have a function in the regulation of legumin gene expression.     

Use of synthetic oligodeoxyribonucleotides and primed cDNA as probes for pea (Pisum sativum L.) RNA and genomic DNA sequences

Two oligonucleotide sequences were synthesised by a solid-phase phosphotriester method. One of these sequences, A was a copy of part of a characterised cDNA clone encoding the basic subunit of legumin, a seed storage protein of Pisum sativum L. (garden pea); the other sequence B was predicted to be complementary to the 5 region of legumin mRNA on the basis of the amino acid sequence of legumin acidic subunits and most likely codon usage. Sequence A was shown to hybridise specifically to a legumin cDNA clone and to legumin mRNA. Sequence B did not hybridise specifically to legumin mRNA and was concluded not to be correctly complementary to legumin mRNA. Sequence A was used as a primer for cDNA synthesis using pea seed mRNA as a template. The cDNA so produced hybridised specifically to a legumin cDNA clone, to legumin mRNA, and to sequences encoding legumin in a restriction digest of pea genomic DNA. It is suggested that such oligonucleotide primed cDNAs may be of general value in probing eukaryotic genomic DNA.     

Characterization and evolutionary relationship of methionine-rich legumin-like protein from buckwheat.

We have isolated and characterized a full-length cDNA for legumin-like storage polypeptide from buckwheat seed (Fagopyrum esculentum Moench) and compared its deduced amino acid sequence with those from different representatives of dicots, monocots and gymnosperms. The cDNA sequence was reconstructed from two overlapping clones isolated from a cDNA library made on mRNA of buckwheat seed at the mid-maturation stage of development. Analysis of the deduced amino acid sequence revealed that this specific buckwheat storage polypeptide should be classified in the methionine-rich legumin subfamily present in the lower angiosperm clades, a representative of which was first characterized in Magnolia salicifolia (clone B 14). The fact that a methionine-rich legumin coexists together with methionine-poor legumins in buckwheat should be an important element regarding the evolutionary position of buckwheat. This may also be supporting evidence that the B14 ortholog was not lost in evolution but was protected under pressure of an increased need for sulfur. Using primers designed from characterized cDNA, we also isolated its corresponding gene from buckwheat genomic DNA and analyzed the characteristic exon/intron structure. The firstly identified two-intron structure of buckwheat legumin gene is an important contribution to study of methionine-rich legumins in lower angiosperms.     

Cloning and structural analysis of DNA encoding an A2B1a subunit of glycinin

The partial DNA sequence of a glycinin gene in a genomic clone and a homologous cDNA clone were determined. They have nearly identical nucleotide sequences and encode the basic polypeptide and part of the acidic polypeptide for an A2B1a glycinin subunit. The protein primary structure deduced from the DNA sequence is in close agreement with the amino acid sequence of the subunit determined chemically and confirms assignment of part of the amino acid sequence in the basic component where we were able to establish an overlap using conventional approaches. The coding part of the basic subunit is interrupted by a 625-base pair A + T-rich intron whose boundaries correlate with the established consensus sequences for the exon-intron junctions. Comparison of the nucleotide sequence of the basic subunit of pea legumin gene with that of the gene for A2B1a subunit reveals 70% homology in coding regions, although there is considerably less in the 3'-flanking regions.     

Isolation and characterization of a minor legumin and its constituent polypeptides from Pisum sativum (pea).

The purification and characterization of a minor legumin species from Pisum sativum is described. Electrophoretic data indicate that it corresponds to a legumin subunit pair previously designated L1. The beta-polypeptides of the minor legumin have a phenylalanine N-terminus. This is the first time that an amino acid other than glycine has been reported as the N-terminus of the basic polypeptides from legumin-like proteins from any plant species. Sequence analyses of the isolated alpha- and beta-polypeptides of the minor legumin show that it does not correspond to any of the three legumin gene families that have previously been defined on the basis of DNA hybridizations and genetic analyses.     

Organization and mapping of legumin genes in Pisum

Summary The organization of three classes of legumin gene has been studied by exploiting restriction fragment length polymorphisms. Hybridization of cDNA probes, representing different sub-families of legumin gene, to DNA from F₂ and F₆ plants from selected crosses showed that there was a tight linkage of individual genes within each of two sub-families. One of these sub-families has been mapped to a locus on chromosome 7 close to r, while the other maps to a locus near a on chromosome 1. The results from one of the crosses analyzed indicated that a third class of legumin gene is also linked to a.     

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.     

Two genes encoding ''minor'' legumin polypeptides in pea (Pisum sativum L.). Characterization and complete sequence of the LegJ gene.

A genomic clone from pea (Pisum sativum L.) contains all of one gene encoding a 'minor' (B-type) legumin polypeptide, and most of a second very similar gene. The two genes, designated LegJ and LegK, are arranged in tandem, separated by approx. 6 kb. A complete sequence of gene LegJ and its flanking sequences is given, with as much of the sequence of gene LegK as is present on the genomic clone. Hybridization of 3' flanking sequence probes to seed mRNA, and sequence comparisons with cDNA species, suggested that gene LegJ, and probably gene LegK, was expressed. The partial amino acid sequences of 'minor' legumin alpha- and beta-polypeptides were used to confirm the identity of these genes. The transciption start in gene LegJ was mapped. The 5' flanking sequence of gene LegJ contains a sequence conserved in legumin genes from pea and other species, which is likely to have functional significance in control of gene expression. Sequence comparisons with legumin genes and cDNA species from Vicia faba and soya bean show that separation of legumin genes into A- and B-type subfamilies occurred before separation of the Viciae and Glycinae tribes.     

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.     

Polymorphism of legumin subunits from field bean (Vicia faba L. var. minor) and its relation to the corresponding multigene family

Legumin, which amounts to approximately 55% of the seed protein in field beans (Vicia faba L. var. minor), is a representative of the 12S storage globulin family. The 12S storage globulins are hexameric holoprotein molecules composed of different types of polymorphic subunits encoded by a multigene family. Type-A legumin subunits contain methionine whereas type-B are methionine-free subunits. Sequencing of two different type A-specific cDNAs, as well as an FPLC/HPLC-based improvement of subunit fractionation and peptide mapping with subsequent partial amino-acid sequencing, permit the assignment of some of the polymorphic legumin subunits to members of the multigene family. Two different type A subunits (A1 and A2) correspond to the two different cDNA clones pVfLa129 (A2) and 165 (A1), but microheterogeneity in the amino-acid sequences indicates that polymorphic variants of both representatives of this type may exist. Two groups of published type B-specific gene sequences (LeB7, and LeB2, LeB4, LeB6, respectively) are represented by two polymorphic subunit fractions (B3I, B3II, and B4I, B4II). A seventh clone, LeB3, encodes one of the large legumin subunits that is only a minor component of the legumin seed protein complex.     

Anchoring of genetic linkage maps to the chromosome complement of Vicia faba L.

Using amplification of marker sequences with DNA from a set of distinct microdissected Vicia faba L. chromosomes covering the entire genome, we could unambiguously show that the linkage group I.B, which includes the pseudogene of legumin B4 (ψ1) and was previously ascribed to the metacentric chromosome I, actually belongs to chromosome IV. By considering the breakpoints of the translocated BKH chromosomes III and IV, even the subchromosomal position of loci LG085 and CNGC4 could be inferred. Anchoring all linkage groups to distinct faba bean chromosomes will facilitate quantitative trait locus fine mapping and gene identification using synteny, and will boost the development of efficient markers for selection in breeding programs.     

Different legumin protein domains act as vacuolar targeting signals.

Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons. We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles. To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively. In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole. Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole. A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles. With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain. We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences.     

Detection of legumin gene DNA sequences in pea by in situ hybridization

In order to detect low copy number sequences in pea using biotin-labelled probes we optimised some aspects of the in situ hybridization technique. We found protoplast preparations to be superior to standard squashes in terms of their signal: noise ratio. Heat and alkali denaturation of chromosomal DNA were both more effective than acid denaturation. A comparison of antibody-fluorochrome and streptavidin-enzyme conjugates showed the streptavidin-alkaline phosphatase conjugate to be the most sensitive detection system. Using the optimised method, we were able to detect a single site for a 13.5 kb legumin gene clone.     

Legumin Synthesis in Developing Cotyledons of Vicia faba L

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.     

The legumin gene family: structure and evolutionary implications of Vicia faba B-type genes and pseudogenes

We have characterized several Vicia faba genes encoding methionine residue-free group B subunits of the 11S or legumin storage proteins. The respective gene subfamily consists of 10 to 15 members, six of them having been studied by DNA sequence analysis. Four functional genes (LeB2, LeB4, LeB6, LeB7) are highly homologous in their coding region and 0.3 kb of their 3 flanking sequences. On the other hand, two pseudogenes (LeB1, LeB5) have accumulated a large number of mutations including an identical 0.7 kb internal deletion; they are both flanked by a repetitive element. Analysis of sequence changes show that transitions are nearly double as frequent as transversions. CpG is the most infrequent dinucleotide whereas TpA is significantly underrepresented in exon sequences. End points of deletions are correlated with short direct repeats and preferentially found in the two introns. Our studies indicate that the Vicia faba legumin B gene subfamily contains a group of expressed, highly homologous genes as well as more diverged pseudogenes.     

Legumin heterogeneity inPisum

Analyses of heterogeneity in legumin subunit patterns, legumin precursor polypeptides, and restriction fragments containing legumin genes have shown thatPisum (pea) genotypes vary in the extent of gene and polypeptide divergence but not in the degree of gene reiteration. Genotypes containing single and multiple ^M subunits had the same numbers of legumin genes. The potential value of this heterogeneity in genetical analyses is outlined.This work was supported by the Agricultural and Food Research Council via a grant-in-aid to the John Innes Institute. We acknowledge financial support from Agrigenetics Corporation, Boulder, Colorado, and from the CEC Biomolecular Engineering Programme, Contract GBI-4-113-UK.