Characteristics of two types of legumin genes in the field bean (Vicia faba L.var. minor) genome as revealed by cDNA analysis

Nucleotide sequence analysis of cDNA clones coding for field bean legumin precursor polypeptides revealed two different types, called A and B. Although homologous, both types differ in several sequence characteristics. Comparison with similar data from soybean and recent findings from pea leads to the following conclusions: (i) the two types of legumin genes described represent two subfamilies, A and B, which are probably of widespread occurrence; (ii) legumin genes or subunits can best be placed in either subfamily A or B by sequence homology, in addition B-type subunits contain generally fewer (or none at all in V. faba) Met residues as compared to A-type subunits; (iii) members of one subfamily from different species are more homologous than members of either subfamily within a species, therefore the two subfamilies must have arisen long before speciation of the genera Glycine, Pisum and Vicia; (iv) during speciation members of the B-subfamily diverged significantly more than members of the A-subfamily.     

Specific subunit pairs of legumin from Vicia faba

Four pairs of disulphide-linked acidic (α) and basic (β) subunits were isolated from legumin of Vicia faba. Pairing between α- and β-subunits is nonrandom, supporting the view that each subunit pair arises from a common precursor polypeptide, already containing intramolecular disulphide bonds, when cleavage to the subunit pair takes place. The subunit pairs belong to two structural types: type A contains Met, whereas type B lacks Met. In addition to these four subunit pairs, at least two more pairs are present in legumin in minor amounts.     

Electrophoretic seed albumin patterns and species relationships inVicia sect.Faba (Fabaceae)

Electrophoretic analysis of seed albumins (PAGE) covered 173 accessions representing nine species ofVicia sect.Faba. The number of albumin bands recorded in particular species varied from three inV. eristaloides to 23 inV. faba; in total, 38 bands were distinguished in the investigated material. The examined species, exceptV. eristalioides, showed intraspecific variation with respect to the number and relative staining intensity of albumin bands; individual variation was especially marked inV. faba and inV. narbonensis. Hierarchical clustering of the investigated taxa was based onBhattacharyya distances calculated from the electrophoretic data. The taxa grouped in three main clusters.Vicia faba and the rather remotely relatedV. kalakhensis formed one cluster. The second cluster was composed ofV. narbonensis distantly related toV. hyaeniscyamus. The third cluster comprised three subgroups: 1.V. johannis, V. galilaea andV. serratifolia, 2.V. eristalioides, and 3.V. bithynica. The obtained results are discussed with reference to taxonomic relationships inVicia sect.Faba.     

TheTy1-copia group retrotransposons inVicia species: copy number,sequence heterogeneity and chromosomal localisation

We present an in-depth study of theTy1-copia group of retrotransposons within the plant genusVicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploidVicia species chosen to represent large (V. faba, 1C=13.3 pg), medium (V. melanops, 1C=11.5 pg) and small (V. sativa, 1C=2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, withV. faba containing approximately 10⁶ copies,V. melanops about 1000 copies andV. sativa 5000 copies. The degree of sequence heterogeneity ofTy1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons inV. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.     

Taxonomy, evolution and domestication of Vicia in relation to aphid resistance

Thirty Vicia species and 28 V. faba cultivars were evaluated for Aphis fabae resistance by measuring rates of aphid population increase (r_m) on the host plants. A subset of these species (22) and cultivars (five) were evaluated for resistance to Acyrthosiphon pisum and Megoura viciae. For the same subset of 22 Vicia species and all three aphid species, positive correlations were observed between the concentration and numbers of leaf and stem non-protein amino acids and the level of aphid resistance. Correlations were also observed between trichome density, on some organs of the host, and both nymphal survival and development time. Aphid resistance, together with these possible physical and chemical resistance mechanisms, showed a pattern which was described in relation to the taxonomy of the genus Vicia and the degree of domestication of the host. The least advanced Vicia species were most resistant whilst the more specialised species and those most closely related to V. faba and V. faba itself were most susceptible. Within each taxonomic group (subgenus or section), the most domesticated species were least resistant. The merits of utilising the resistance found in some V. faba cultivars and in other wild Vicia species were considered.     

Isoenzyme variation in the wild relatives ofVicia faba (Fabaceae)

Electrophoretic analysis of five enzyme systems, LAP, PGI, SKDH, SOD and 6-PGDH, among 102Vicia accessions representingV. bithynica and seven species of theV. narbonensis complex, namelyV. eristalioides, V. kalakhensis, V. johannis, V. galilaea, V. serratifolia, V. narbonensis andV. hyaeniscyamus, has been performed. The recorded variation was tentatively assigned to 41 allelic genes at eight loci; intraspecific variation was observed in all species except forV. eristalioides. The results obtained were compared with the corresponding data reported earlier forV. faba. Hierarchical grouping of the investigated taxa, includingV. faba, was based onNei's genetic identities calculated from the allozyme frequency data.Vicia faba andV. bithynica were shown to be most distantly related to one another and to the remaining species investigated.Vicia serratifolia appeared to be a peripheral member of theV. narbonensis complex. The results are discussed with reference to genetic diversity and taxonomic relationships of the species under study.     

Immunochemical behaviour of legumin and vicilin from Vicia faba: a survey of related Proteins in the leguminosae subfamily faboideae

An antiserum specific for the legumin and vicilin of Vicia faba was used to examine extracts of seeds of taxa of the Fabeae and Trifolieae for the presence of related storage proteins, Proteins related to legumin were found to be widely distributed, indicating considerable conservation of the genetic information for this protein. Only Pisum sativum contained a protein immunochemically identical with the vicilin of V. faba; the equivalent proteins of all other genera tested here were immunochemically different from vicilin.     

The relationship between ribosomal repeat length and genome size in Vicia

The organization of the ribosomal RNA genes was examined in several species of Vicia in an attempt to determine whether a relationship exists between genome size and ribosomal repeat length. Species within this genus exhibit a sevenfold variation in haploid DNA content. Our data suggest that species with an intermediate genome size maintain one predominant Eco RI class of ribosomal repeat of about 9 kilobases (kb). In contrast, the smallest and largest genomes of Vicia possess one major and several minor classes. The possible relationship between repeat classes among species is discussed. We examined the species with the smallest (V. villosa) and largest (V. faba) genomes in closer detail by R-loop analysis of a satellite DNA from Hoechst 33258 dye-CsCl gradients. Heterogeneity was found in the length of the ribosomal repeat for both species, but no appreciable difference was observed in the distribution of these lengths, which averaged 11–12 kb. This heterogeneity is associated with the nontranscribed spacer region. Intervening sequences were not found in either the 25S or 18S coding regions of the ribosomal repeat of either of these two plants. A putative ribosomal RNA precursor of 7 kb was identified for both species.     

The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium–Vicia symbiosis.     

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     

TheTy1-copia group retrotransposons inVicia species: copy number, sequence heterogeneity and chromosomal localisation

We present an in-depth study of theTy1-copia group of retrotransposons within the plant genusVicia, which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploidVicia species chosen to represent large (V. faba, 1C=13.3 pg), medium (V. melanops, 1C=11.5 pg) and small (V. sativa, 1C=2.3 pg) genomes within the genus. The copy numbers of the retrotransposons are all high but vary greatly, withV. faba containing approximately 10⁶ copies,V. melanops about 1000 copies andV. sativa 5000 copies. The degree of sequence heterogeneity ofTy1-copia group elements correlates with their copy number within each genome, but neither heterogeneity nor copy number are related to the genome size of the host. In situ hybridization to metaphase chromosomes shows that the retrotransposons inV. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotransposon evolution.     

Phylogenetic relationships betweenVicia faba (Fabaceae) and related species inferred from chloroplasttrnL sequences

The chloroplasttrnL intron from 46 differentVicia accessions, representing five of the nine sections of the genusVicia subg.Vicia sensuMaxted (1991a) were amplified by the Polymerase Chain Reaction (PCR) using oligonucleotide primers homologous to conserved regions intrnL. The products fell into two distinct groups; those of approximately 250 nt and those of around 450 nt in length. Of these, products from 17 differentVicia species were cloned and their nucleotide sequences determined. Multiple alignments were assembled and phylogenetic trees constructed by the weighted least-squares distance method. ALathyrus latifolius trnL intron sequence was used as an outgroup. The resulting trees clearly group and separate the sectt.Narbonensis, Bithynica andFaba species but were less able to distinguish species from sectt.Hypechusa andPeregrinae. Based on these sequence data,V. faba appears to be more distant from sect.Narbonensis than sectt.Hypechusa andPeregrinae. The results are in general agreement with a recent treatment ofVicia subg.Vicia (Maxted 1993) and lend further support to placingV. faba in the monospecific sect.Faba.     

Ribosomal DNA repeat unit polymorphism in 49 Vicia species

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of Vicia ribosomal DNA (rDNA), more particularly among V. faba and its close relatives and the taxa within three (Narbonensis, Villosa, Sativa) species' complexes. Total genomic DNA of 90 accessions representing 49 Vicia species was restricted with 11 enzymes, and the restriction fragments were probed with three ribosomal clones. Twenty-eight repeat unit length classes were identified. The number of length classes (1–2) per accession did not correspond to the number of nucleolar organizing regions (NORs). The number of rRNA genes was independent of the 2C nuclear DNA amount present in the taxon. Each of the 90 accessions had 2 (rarely 1)-4 DraI sites. Those taxa with the same number of DraI sites generally could be distinguished from each other by different configurations. Probing of the DNA samples digested with tetranucleotide recognition restriction endonucleases emphasized differences between divergent spacer regions and enabled relative homologies between the coding regions to be established. Overall, rDNA restriction site variation among the species showed a good correlation with taxonomic classification. The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex. rDNA diversity within two other species complexes (Villosa, Sativa), on the other hand, was more extensive than expected. With few exceptions, data on the two complexes give evidence of taxon-specific divergences not seen with other approaches. The restriction site variability and repeat length heterogeneity in the rDNA repeat exhibited startling differences between V.faba and its close wild relatives included in the Narbonensis species complex. This analysis provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.     

The ra locus and legumin synthesis in Pisum sativum

Cellulose acetate electrophoresis has been used to resolve the storage proteins of peas into their constituent groups. Comparisons of 171 randomly chosen genotypes representing primitive forms, subspecies, and cultivars of peas, of seven near-isogenic lines for round and wrinkled and of two F₂ populations have shown that wrinkled seed has a lower proportion of legumin than round seed. The extent of the reduction varies with the background genotype; some of the wrinkled forms had less than one-third as much legumin as their isogenic round forms. This effect of the r_a locus on storage protein composition provides the first example in peas of a mutant analogous to the op 2 and fl 2 mutants in maize. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to discriminate the 40 kdalton (α subunits) of legumin. On the basis of the data obtained from F₂ populations derived from genotypes with distinct α subunit patterns, it was shown that the structural genes for the α subunit polypeptides of legumin are on chromosome 7, and closely linked to the r_a locus.     

Chloroplast DNA diversity in Vicia faba and its close wild relatives: implications for reassessment

To obtain new information on phylogenetic relationships between wild and cultivated broad bean, restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNAs from Vicia faba and eight subspecies/species of its close wild relatives grouped together in the Narbonensis complex was carried out using 14 restriction endonucleases. The molecular sizes of the cpDNAs obtained were similar (122.6–123.4 kbp), indicating that they had all lost one of inverted repeats. Among the more than 300 sites surveyed, the three subspecies within V. narbonensis, which exhibit just as many types of karyotypes, were shown to have identical cp fragment patterns. Genetic distances between all of the pairs of species were calculated from RFLP data. The cpDNA diversity within the Narbonensis complex was found to be more extensive than expected, except for the genetic relationship between V. hyaeniscyamus and V. johannis in which a total of three mutations were detected among the 300 sites sampled, thereby showing their close relatedness. The cpDNA of V. faba vis-a-vis its wild relatives also exhibited startling differences, indicating a clear division of Vicia species into two distinct lineages. This analysis unambiguously provides new evidence that the wild species grouped in the complex did not contribute their plastomes to the evolution of V. faba, and hence none of the species can be considered to be putative allies of broad bean. The present study also demonstrates profound cpDNA diversity among closely related species that have lost one of inverted repeats.     

The Structure of Legumin of Vicia faba L.--a Reappraisal

Two-dimensional electrophoretic studies have shown a wide rangeof heterogeneity in the subunits of legumin isolated from seedsof Vicia faba L. As many as 10 disulphide-linked subunit pairsin the mol. wt. range 37 000–79 000 have been observed.Each subunit pair separated on reduction by 2-mercaptoethanolinto a large acidic and a small basic subunit, each of whichwas shown to be heterogeneous in charge by isoelectric focusing.More heterogeneity was found in the large subunits (mol. wt.range 23 000–58 000; pl range 4.6–6.1) than in thesmall subunits (mol. wt. range 21 000–23 000; pl range8.2–8.5). Most legumin molecules seemed to be formed byrandom association of subunit pairs, although one subunit pairassociated only with itself to give a molecular type separableunder non-dissociating conditions.     

The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.     

Genetic diversity and relationships in some <Emphasis Type="Italic">Vicia</Emphasis> species as determined by SDS-PAGE of seed proteins

To evaluate the genetic diversity of some Vicia species, seed proteins of 160 accessions (30 of Vicia faba, 15 of V. narbonensis, 82 of V. sativa and 25 of V. ervilia and 8 accessions of other Vicia species) were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The dendrogram showed that the two outcrossing species V. faba and V. villosa were the most distant among all species (average percent disagreement value PDV 0.47 and 0.45, respectively). The tree was divided into small clusters of two species each. V. narbonensis fell in one cluster with V. michausai (at PDV = 0.35) and V. lutea (var. hirta) fell in one cluster with V. serococorpes (at PDV = 0.32) whereas, V. ervilia fell in one cluster with V. sativa (at PDV = 0.27). The V. sativa subspecies, however, were closely related (PDV < 0.1). In general, this study did not prove any relationship between the studied storage proteins and the geographical distribution or ecological needs of the studied accessions.     

Characterization of Pisum sativum and Vicia faba microsymbionts in Morocco and definition of symbiovar viciae in Rhizobium acidisoli

In this work, we analyzed the diversity of seventy-six bacteria isolated from Pea and faba bean nodules in two regions of Morocco. The molecular diversity was realized using the analysis of the sequences of 16S rRNA and six housekeeping genes (recA, glnII, atpD, dnaK, rpoB and gyrB) and two symbiotic genes (nodA and nodC).The phylogeny of the 16S rRNA gene sequences revealed that all strains belong to the genus Rhizobium, being related to the type strains of R. leguminosarum, R. laguerreae, R. indigoferae, R. anhuiense and R. acidisoli. The housekeeping genes phylogenies showed that some strains formed a subclade distinct from the rhizobial species that usually nodulate Vicia faba and Pisum sativum which are closely related to R. acidisoli FH23 with sequence similarity of 98.3%.Analysis of the PGPR activities of the different isolates showed that the strains related to R. laguerreae were able to solubilize phosphates and to produce siderophores and auxin phytohormone. However, R. acidisoli strain F40D2 was unable to solubilize phosphates although they produce siderophores and IAA.The phylogenetic analysis of the nodA and nodC sequences showed that all isolated strains were closely related with the strains of symbiovar viciae. The nodulation tests confirmed the ability to nodulate V. faba and P. sativum but not Cicer arietinum or Phaseolus vulgaris. Hence, in Morocco P. sativum is nodulated by R. laguerreae; whereas V. faba is nodulated by R. laguerreae and the symbiovar viciae of R. acidisoli which has been not previously described in this species.     

Cytological characterization of Vicia oroboides Wulfen in Jacq

Vicia oroboides, a rare taxon belonging to section Atossa of subgenus Vicia, was recovered and analysed by means of cytological and karyological methods with the aim of both characterising this species and integrating our knowledge on phylogeny of subgenus Vicia. Automated karyotype analysis and nuclear DNA content have been determined after Feulgen’s reaction; chromosome banding was performed by fluorochrome staining to evidence heterochromatic blocks along the chromosome complement. The chromosome number is in line with the values of the species of section Atossa; the GC- and AT-rich sites were identified by CMA and DAPI staining. Karyomorphological parameters, based on symmetry indices, provide information about the phylogenetic position of this species inside the subgenus Vicia. DNA content is reported for the first time.