Bean lectins

Summary Seeds of forty bean cultivars having different lectin types based on two-dimensional isoelectric focusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE) were analyzed for quantities of lectin, phaseolin and total protein. Significant differences were found among groups of cultivars with different lectin types for the quantity of lectin and phaseolin. Cultivars with more complex lectin types based on IEF-SDS/PAGE tended to have higher quantities of lectin and lower quantities of phaseolin than cultivars with simple lectin types. An association between lectin type and the quantity of lectin and phaseolin was found also in the seeds of F₂ plants that segregated in a Mendelian fashion for two lectin types. Seeds from plants with the complex lectin type had more lectin and less phaseolin than seeds from plants with the simple lectin type. Therefore, the genes controlling qualitative lectin variation also may influence the quantitative variation of lectin and phaseolin. The results of this study are related to other studies on the quantitative variation for seed proteins and to the possible molecular basis for variation in the quantity of lectins in beans.     

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from -carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an -helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core -barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isolecuine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressedin vivo.     

Bean (Phaseolus vulgaris L.) protoplasts as a model system to study the expression and stability of recombinant seed proteins

A suitable protocol for the transient expression of seed protein genes in protoplasts derived from cell suspension cultures of common bean has been established. Preliminary analyses of cultures to verify the synthesis of phaseolin – actively accumulated by the starting tissue, the developing cotyledon – showed that the protein was no longer synthesised after 5 days of culture. Transient expression of a phaseolin sequence, driven by a constitutive promoter, resulted in the accumulation of the correctly glycosylated and assembled protein. This system, when compared to tobacco protoplasts, largely avoids phaseolin fragmentation and the presence of contaminant polypeptides in the immunoprecipitates. Therefore, bean protoplasts are a good system to study the expression of wild-type as well as in-vitro-modified bean seed proteins. Received: 26 November 1996 / Revision received: 10 February 1997 / Accepted: 15 February 1997     

Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope himet (Hoffman et al., 1988, Plant Mol. Biol. 11, 717–729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent. However, the protein was sufficiently destabilized to be degraded by vacuolar proteases.Abbreviations ER endoplasmic reticulum - BiP binding protein - IgG immunoglobulin G - M_r relative molecular mass The mention of vendor or product does not imply that they are endorsed or recommended by the US Department of Agriculture over vendors of similar products not mentionedThis work was supported by a grant from the National Science Foundation (Cell Biology) to M.J. Chrispeels and a fellowship from the Ministry of Education and Science, Spain-Fullbright Program to J.J. Pueyo. We thank H. Pelham for a gift of the constructs containing c-myc-SEKDEL and cmyc-FEHDEL and for a gift of anti-HDEL monoclonal antibodies. The original HiMet phaseolin construct was made by L. Hoffman and the phaseolin-HDEL or KDEL and HiMet-HDEL or KDEL constructs were made by D. Hunt as part of his doctoral research.     

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Summary Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Zeolin is a recombinant storage protein that can be used to produce value-added proteins in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

The specific features of plants make them particularly suitable for the production of recombinant proteins and alfalfa is one of the recommended plant production systems. We have transformed alfalfa with a gene coding for a chimaeric protein made previously by fusing phaseolin to the N-terminal region of γ-zein and have analyzed the accumulation of this fusion protein, named zeolin. Zeolin was expressed both in T0 Regen SY alfalfa plants and in the progeny resulting from the sexual cross between Regen SY transformants and alfalfa cv. Adriana plants. In some alfalfa plants a 95 kDa zeolin glycosylated polypeptide is the most abundant polypeptide detected by Western-blot analysis, whereas in tobacco the most abundant zeolin polypeptide has a molecular mass around 60 kDa, expected for intact zeolin. Zeolin has been stably accumulated in alfalfa leaves because it forms endoplasmic reticulum-located protein bodies in the cell. As regards zeolin quantisation, in the progeny alfalfa plants a value of about 0.22–0.28 mg of zeolin / g of fresh leaf weight has been estimated. Michele Bellucci and Francesca De Marchis contributed equally to this work.     

Bean arcelin

Summary SDS-PAGE of seed proteins from the seeds of a nondomesticated bean of Mexican origin (Phaseolus vulgaris L., PI 325690) revealed the presence of a novel 38 kd protein which appeared to be neither an altered phaseolin nor a lectin fraction. The protein was named arcelin, after Arcelia, the town in the state of Guerrero near which PI 325690 had been collected. The pure line, UW 325, was derived by self fertilization of the plant from a single arcelin-containing seed of PI 325690. Despite a low percentage seed phaseolin (14.6%), seed phenotype, seed germination, plant growth, pollen fertility, and percentage seed protein of UW 325 were normal. Analyses of F₂ and F₃ seeds from a single F₁ plant of the cross SanilacXPI 325690-3 revealed that arcelin expression was inherited as a single gene and that presence was dominant to absence of arcelin. The mean percentage phaseolin in the seeds of homozygous dominant Arc/Arc F₃ families (14.0%) was significantly lower than that of the homozygous recessive arc/arc seeds (44.7%). The distribution of percentage phaseolin values for seeds within segregating families was bimodal and nonoverlapping. Without exception, seeds containing arcelin (Arc+phenotype) contained a lower percentage phaseolin than seeds lacking arcelin (Arc-phenotype). Although arcelin presence was associated with low percentage phaseolin, the Arc/Arc and Arc/arc genotypes were similar for seed weight and percentage total seed protein.     

Immunocytochemical localization of phaseolin and phytohemagglutinin in the endoplasmic reticulum and Golgi complex of developing bean cotyledons

Development of legume seeds is accompanied by the synthesis of storage proteins and lectins, and the deposition of these proteins in protein-storage vacuoles (protein bodies). We examined the subcellular distribution, in developing seeds of the common bean, Phaseolus vulgaris L., of the major storage protein (phaseolin) and the major lectin (phytohemagglutinin, PHA). The proteins were localized using an indirect immunocytochemical method in which ultrathin frozen sections were immunolabeled with rabbit antibodies specific for either PHA or phaseolin. Bound antibodies were then localized using goat-anti-rabbit immunoglobulin G adsorbed onto 4- to 5-nm colloidal gold particles. The sections were post-fixed with OsO₄, dehydrated, and embedded in plastic on the grids. Both PHA and phaseolin exhibited a similar distribution in the storage-parenchyma cells, being found primarily in the developing protein bodies. Endoplasmic reticulum and Golgi complexes (cisternal stacks and associated vesicles) also were specifically labeled for both proteins, whereas the cytosol and other organelles, such as mitochondria, were not. We interpret these observations as supporting the hypothesis that the transport of storage proteins and lectins from their site of synthesis, the rough endoplasmic reticulum, to their site of deposition, the protein bodies, is mediated by the Golgi complex.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - PBS phosphate-buffered saline - PHA phytohemagglutinin