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1.
Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pKI of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F2 seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F2 seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
Bean arcelin     
Summary Crude proteins from seeds of wild bean accessions of Mexican origin were analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE). Several accessions had electrophoretic patterns showing unique protein bands. When analyzed by two-dimensional isoelectric focusing (IEF)-SDS/PAGE, four protein variants which had electrophoretic mobilities similar to each other but different from the other major seed proteins, phaseolin and lectin, were observed. All four variants, which have not been described in cultivated beans, were tentatively named arcelin proteins and designated as arcelin 1, 2, 3 and 4. Arcelins 3 and 4 had polypeptides that comigrated on two-dimensional gels and these variants occurred in accessions that were collected in the same location. Analysis of single F2 seeds from crosses among arcelin-containing lines and from crosses between cultivated beans lines without arcelin and arcelin-containing lines revealed that differences in arcelin polypeptide expression were inherited monogenically. The alleles for different arcelin variants were codominant to each other and dominant to the absence of arcelin. The gene(s) controlling arcelin proteins were unlinked to those controlling phaseolin expression and tightly linked to genes controlling the presence of lectin proteins (< 0.30% recombination). The possible origins of arcelin genes and their potential role in bruchid resistance are discussed.  相似文献   

5.
Summary The relationship between available methionine concentration and the levels of phaseolin — the major seed storage proteins of the common bean — was studied using three groups of genetic materials: First, the F2 progenies of interspecific crosses between P. vulgaris cultivars and aP. coccineus subsp. coccineus line (cv. Mexican Red Runner) having no detectable phaseolin; second, the F2 progenies and segregating F3 families of crosses between cultivated P. vulgaris lines and a Mexican wild bean accession (PI 325690-3) carrying a gene producing a reduction in phaseolin content; third, two inbred backcross populations: SanilacxBush Blue Lake 240 (population 2) and Sanilacx15R 148 (population 6). Total seed N levels were determined by micro-Kjeldahl, phaseolin levels by rocket immunoelectrophoresis and available methionine levels by the Streptococcus zymogenes bioassay. Our results indicate that in all the genetic materials studied, with the exception of population 6, higher phaseolin levels lead to increased available methionine concentration. Although phaseolin has a low methionine concentration, it is actually a major source of available methionine in common bean seeds, because it represents a large part of total seed nitrogen and because limited differences exist between the methionine concentrations of the different protein fractions. This contrasts with the situation in cereals such as maize, barley and sorghum, where increased levels of the major limiting amino acid (lysine) can be achieved through a decrease in the amounts of the main seed storage protein fraction (prolamines). In population 6, no relationship was observed between available methionine and phaseolin content. Other factors, such as additional methionine-rich polypeptides or the presence of tannins, might obscure the positive relationship between phaseolin and available methionine content in population 6.  相似文献   

6.
Phaseolin is the most abundant storage protein of bean seeds. To modify its amino-acidic composition by protein engineering, for the improvement of its nutritional value, regions which could be modified without detrimental effects on structural features of the protein must be identified. Data presented here, on the characterisation of the major storage protein of lima bean (Phaseolus lunatus L.) seeds, a phaseolin-like glycoprotein, provide good indications on one of such region. Phaseolus lunatus phaseolin consists of four major oligomers containing two subunit classes. Polypeptides of one class show a molecular mass ranging from 38.5 kDa to 32 kDa, while the molecular mass of polypeptides belonging to the other class ranges from 27 kDa to 21 kDa. The subunits originate from the cleavage of precursor forms, with molecular masses of 58 kDa and 54 kDa, which are still present — in residual amounts — in the mature protein. Comparison of their N-terminal sequences with those of the subunits demonstrate that cleavage occurs in a region of the molecule that instead remains uncleaved in phaseolins of the other species. Since this region can accommodate such a drastic modification, we suggest it could be a good candidate for in vitro manipulation.  相似文献   

7.
Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride–pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation.  相似文献   

8.
Bean lectins     
Summary Single seeds of over 100 bean cultivars were analyzed by two-dimensional electrophoresis. The cultivars could be classified into eight groups by virtue of their G2/albumin electrophoretic patterns: TG2, SG2, VG2, PrG2, BG2, MG2, PG2, and PiG2, The polypeptide compositions of these types were largely inter-related having particular polypeptides in common. It was possible to correlate the G2/albumin patterns with agglutinating activity of cow and rabbit blood cells as measured by the agglutination ratio (minimum concentration of extract required to agglutinate cow blood cells: minimum concentration of extract required to agglutinate rabbit blood cells). The active lectin polypeptides were identified by extracting lectins from agglutinated erythrocytes and by comparing the qualitative similarities and differences of the G2/albumin patterns and their agglutination activities. A reference catalogue of over 100 bean cultivars giving their phaseolin and G2/albumin electrophoretic patterns, and agglutination ratios is presented.  相似文献   

9.
In this report, we describe the efficient expression and glycosylation, in insect cells, of -phaseolin polypeptides (M r 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (M r 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/106 cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.  相似文献   

10.
We have designed protein molecules based on an -helical coiled-coil structure. These proteins can be tailored to complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean -conglycinin promoters. Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds. Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression. Plants transformed with the soybean -conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations. However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable. This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.  相似文献   

11.
An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wti–) and mutant phaseolin glycoforms (dgly 1, dgly 2 and dgly 1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly 1 and dgly 2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly 1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton  相似文献   

12.
Selection and random genetic drift are the two main forces affecting allele frequencies in common bean breeding programs. Therefore, knowledge on allele frequency changes attributable to these forces is of fundamental importance for breeders. The changes in frequencies of alleles of biochemical markers were examined in F2 to F7 populations derived from crosses between cultivated Mesoamerican and Andean common bean accessions (Phaseolus vulgaris L.). Biochemical markers included the seed proteins phaseolin, lectin and other seed polypeptides, and six isozymes. The Schaffer’s test detected a high significant linear trend of the 63% of the polymorphic loci studied, meaning that directional selection was acting on those loci. Associations between seed size traits, phaseolin seed-storage protein and isozyme markers were detected based on the comparisons of the progeny genotypic means. In the interracial populations the intermediate form PhaH/T, b6, and Rbcs 98 alleles had a positive effect on seed size. In the inter-gene pool populations, a higher transmission of Mesoamerican alleles in all loci was showed, although the Andean alleles PhaT, Skdh 100 , Rbcs 98 , and Diap 100 showed positive effects on seed weight. Our results suggest that phaseolin and other seed proteins markers are linked to loci affecting seed size. These markers have good potential for improving the results of the selection and should be considered as a strategy for germplasm enhancement and to avoid the reduced performance of the inter-gene pool populations.  相似文献   

13.
14.
Phaseolin seed protein variability in a group of 8 wild and 77 cultivated common bean (Phaseolus vulgaris) accessions was determined using 1-dimensional SDS/ PAGE and 2-dimensional IEF-SDS/PAGE. Wild common bean accessions exhibited the 'CH' and 'B' patterns, previously undescribed among either wild or cultivated common beans. The cultivated genotypes showed (in decreasing frequency) the previously described 'S,' T,' and 'C phaseolin patterns as well as the new 'B' pattern similar to the pattern identified in a Colombian wild common bean accession. In the northeastern part of the Colombian bean-growing region, the cultivars exhibited almost exclusively an 'S' phaseolin type, while in the south-western part, the 'T' and 'C phaseolin cultivars were more frequent. Seed size analysis indicated that 'T' and 'C' phaseolin cultivars had larger seeds than 'S' and 'B' phaseolin cultivars. Our results suggest that Colombia is a meeting place for Andean and Middle American common bean germplasms, as well as a domestication center for the common bean.  相似文献   

15.
Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of Mr=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (Mr=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - Mr relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis  相似文献   

16.
PHA quantity and activity dynamics during early germination of bean seed were investigated. Electrophoretic characteristics, subunits composition and carbohydrate-binding specificity of lectin extracted from white kidney bean cv. Bilozerna were studied. It was shown that investigated lectin consisted of 2 subunits E and L with molecular weight 34 and 36 kDa, respectively, analogously to purified PHA ("Serva", Germany), and specifically bound N-acetyl-D-galactosamin and galactose. During germination both quantity and activity of PHA were dramatically decreasing in embryonic axes and in cotyledons, possibly, as a result of the lectin release from seeds to the environment. It is very likely that one of the defence mechanisms of germinating seeds is related with the releasing of lectins that are able to bind components of the bacterial cell wall and to inhibit their growth.  相似文献   

17.
Phaseolin and lectin-related polypeptides, the abundant oligomeric glycoproteins of bean seeds, are synthesized on the endoplasmic reticulum (ER) and then transported to the storage vacuole via the Golgi apparatus. Glycosylation and folding are among the major modifications these proteins undergo in the ER. Although a recurrent role of N-glycosylation is on protein folding, in previous studies on common bean (Phaseolus vulgaris) seeds we demonstrated that the oligosaccharide side-chains are not required for folding, intracellular transport and activity of storage glycoproteins. We show here that in lima bean (Phaseolus lunatus), incubation of the developing cotyledon with tunicamycin to prevent glycosylation has a dramatic effect on the intracellular transport of the storage glycoproteins. When lacking their glycans, phaseolin and lectin-related polypeptides misfold and are retained in the ER as mixed aggregates to which the chaperone BiP irreversibly associates. The lumen of the ER becomes enlarged to accommodate the aggregated polypeptides. Intracellular transport of legumin, a naturally unglycosylated storage protein, is mostly unaffected by the inhibitor, indicating that the observed phenomenon specifically occurs on glycoproteins. Furthermore, recombinant lima bean phaseolin synthesized in tobacco protoplasts is also correctly folded and matured in the presence of tunicamycin. To our knowledge, this is the first report that describes in detail the block of intracellular transport of vacuolar glycoproteins in plant cells due to aggregation following glycosylation inhibition.  相似文献   

18.
Many of the proteins that are translocated into the endoplasmic reticulum are glycosylated with the addition of a 14-saccharide core unit (Glc3Man9GlcNAc2) to specific asparagine residues of the nascent polypeptide. Glucose residues are then removed by endoplasmic reticulum-located glucosidases, with diglucosylated and monoglucosylated intermediates being formed. In this study, we used a cell-free system constituted of wheat germ extract and bean microsomes to examine the role of glucose trimming in the structural maturation of phaseolin, a trimeric glycoprotein that accumulates in the protein storage vacuoles of bean seeds. Removal of glucose residues from the N-linked chains of phaseolin was blocked by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin. If glucose trimming was not allowed to occur, the assembly of phaseolin was accelerated. Conversely, polypeptides bearing partially trimmed glycans were unable to form trimers. The effect of castanospermine on the rate of assembly was much more pronounced for phaseolin polypeptides that have two glycans but was also evident when a single glycan chain was present, indicating that glycan clustering can modulate the effect of glucose trimming on the rate of trimer formation. Therefore, the position of glycan chains and their accessibility to the action of glucosidases can be fundamental elements in the control of the structural maturation of plant glycoproteins.  相似文献   

19.
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  相似文献   

20.
The storage protein phaseolin accumulates during seed development in protein bodies in cotyledons of the common bean Phaseolus vulgaris. Hall et al. (In L Van Vloten-Doting, TC Hall, eds, Molecular Form and Function of the Plant Genome, 1985 Plenum Press, In press) recently reported the expression of a gene coding for phaseolin and the accumulation of phaseolin protein in developing seeds of tobacco plants regenerated from transformed callus cells. The protein did not accumulate in other organs of the plants. Mature seeds from normal and transformed tobacco plants were obtained and the subcellular distribution of phaseolin in the seeds was examined using both light and electron microscopic immunocytochemical methods. Phaseolin was found in six of seven transformed tobacco embryos examined, but was present in only one endosperm of five. When present, phaseolin was located exclusively in the protein bodies of the embryonic and endospermic cells. Furthermore, phaseolin was restricted solely to the amorphous matrix of the protein bodies and was excluded from the globoid and proteinaceous crystalloid components of these organelles. The subcellular location of phaseolin in seeds from transformed tobacco plants is similar to that seen in mature seeds of the common bean indicating that in the transformed cells the protein is targeted to the right subcellular compartment.  相似文献   

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