Vicilin Protein Research Articles

Transient expression of storage-protein genes during early embryogenesis of Vicia faba: synthesis and metabolization of vicilin and legumin in the embryo, suspensor and endosperm

Seed storage proteins are thought to be accumulated exclusively in the cell-expansion phase of embryogenesis and metabolized during germination and seedling growth. Here we show by a sensitive immunohistological technique that the two Vicia faba L. storage proteins vicilin and legumin are accumulated in substantial amounts in the suspensor and coenocytic endosperm and to a lesser extent in the mid-globular embryo. Both proteins appear and disappear at precise stages specific for each tissue. In the endosperm the accumulation starts around 12 d after pollination (DAP). After a maximum attained at 14–15 DAP, storage proteins are degraded within about 4 d. Accumulation is restricted to that part of the endosperm which covers the embryo and displays the highest levels of endoploidy (maximum 96n). In all other parts of the endosperm, storage proteins do not appear to accumulate, although storage-protein-specific mRNA synthesis takes place. In the suspensor, storage proteins are already observed at 6 DAP and disappear very quickly at approximately 10 DAP. Low amounts of legumin and vicilin are also detectable in the mid-globular embryo, but disappear completely as the embryo enters the heart stage. We conclude that storage proteins of Vicia faba accumulated transiently during early seed development are used as nutritive reserves for the growing embryo.

The 5[prime] Flanking Regions of Vicilin and Napin Storage Protein Genes Are Down-Regulated by Desiccation in Transgenic Tobacco.

Drying of seeds, when imposed prematurely, elicits a switch in metabolism; events unique to development, such as synthesis of storage protein, are terminated, whereas syntheses associated with germination and growth are initiated. To determine the role of desiccation in down-regulating the expression of genes for storage proteins, the desiccation responsiveness of the 5[prime] and 3[prime] regulatory regions of the genes encoding the pea storage protein vicilin and the Brassica napus storage protein napin was tested in transgenic tobacco seed. Chimeric genes were introduced into tobacco; these genes consisted of the coding region of the reporter gene for [beta]-glucuronidase (GUS) and 5[prime] and/or 3[prime] regions from the vicilin or napin genes or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation insensitive. In transgenic seed expressing the gene constructs containing the vicilin or napin promoters, GUS activities declined during late seed development, and more dramatically, after imbibition of mature dry seed or prematurely dried seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral promoter replaced the storage-protein gene 5[prime] region. Transient expression assays support the hypothesis that premature drying down-regulates the expression of the storage-protein gene promoter. Following desiccation, this region may become insensitive to positive controlling factors; alternatively, changes to trans-acting factors may occur.

Protein storage vacuoles form de novo during pea cotyledon development.

We have investigated the formation of protein storage vacuoles in peas (Pisum sativum L.) in order to determine whether this organelle arises de novo during cotyledon development. A comparison of different stages in cotyledon development indicates that soluble protease activities decline and the amounts of storage proteins and the integral membrane protein of the protein body, alpha-TIP, increase during seed maturation. On linear sucrose density gradients we have been able to distinguish between two separate vesicle populations: one enriched in alpha-TIP, and one in TIP-Ma 27, a membrane protein characteristic of vegetative vacuoles. Both vesicle populations possess, however, PPase and V-ATPase activities. Conventionally fixed cotyledonary tissue at an intermediate stage in cotyledon development reveals the presence of a complex tubular-cisternal membrane system that seems to surround the pre-existing vacuoles. The latter gradually become compressed as a result of dilation of the former membrane system. This was confirmed immunocytochemically with the TIP-Ma 27 antiserum. Deposits of the storage proteins vicilin and legumin in the lumen, and the presence of alpha-TIP in the membranes of the expanding membrane system provide evidence of its identity as a precursor to the protein storage vacuole.

Role of desiccation in the termination of expression of genes for storage proteins

AbstractThe final stage of development of orthodox seeds is maturation drying, a process often accompanied by a dramatic decline in storage-protein synthesis. When desiccation is imposed prematurely at certain stages prior to the completion of development, a switch in synthetic events is elicited. events unique to development, such as synthesis of storage protein, are terminated, while syntheses associated with germination and growth are initiated. We investigated whether desiccation plays a key role in effecting a decline in expression of genes for storage proteins by acting directly upon the regulatory regions of these developmental genes. The desiccation responsiveness of the 5′ and 3′ regulatory regions of the gene for the pea storage protein vicilin was tested in transgenic tobacco seed. Chimaeric genes were introduced into tobacco, these genes consisted of the coding region of the reporter gene for β-glucuronidase (GUS) and 5′ and 3′ regions from the vicilin gene, or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation-insensitive – those from the cauliflower mosaic virus (35S) and nopaline synthase genes. In transgenic seed expressing the gene constructs containing the vicilin 5′ upstream region, GUS activities declined dramatically after imbibition in mature seed and after rehydration of prematurely dried tobacco seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral (35S) promoter replaced the vicilin 5′ upstream region. The 3′ downstream region of the gene construct did not significantly affect the patterns of changes in GUS activities after seed rehydration. In studies of transient gene expression in castor bean cotyledons, premature desiccation led to termination ofGUSgene expression only when the gene construct contained the vicilin 5′ upstream region. This region may respond directly to desiccation; alternatively, changes totrans-acting factors important for expression of genes for storage-proteins may occur as a result of drying.

Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants.

Gene constructs were designed to test the effect of the endoplasmic reticulum (ER)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35S promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDEL-coding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steady-state levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. Immunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin. It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

The expression of a chimeric cauliflower mosaic virus (CaMV-35S) — pea vicilin gene in tobacco

A gene encoding the 7S pea seed protein vicilin was modified for general expression in transgenic tobacco by fusion of the cauliflower mosaic virus 35S enhancer to the promoter region of the gene. The 7S seed protein was accumulated in leaves, stems, roots, seeds and callus but the level of expression varied widely in the different organs. Vicilin per unit of total soluble protein was higher in younger (upper) leaves (0.01%) than in older (lower) leaves (0.001%) indicating that vicilin was less stable than the average leaf protein. The vicilin in tobacco leaves was cleaved preferentially at or near one of the two post-translational cleavage sites used in pea seeds and in a manner very similar to that which occurs in tobacco seeds with either the unmodified vicilin gene or the 35S-vicilin construct. Leaf vicilin was also assembled into a normal 7S oligomer. These results indicate that a seed protein gene can be modified for expression in most organs of a transgenic host and that expression proceeds with a high level of fidelity. Minor variations in expression were related to selective cleavage of the polypeptide chain and to the production of two mRNA size classes instead of one. The fact that the seed protein was moderately stable in the heterologous environment augurs well for the engineered expression of other seed protein genes in transgenic hosts.

An analysis of seed development inPisum sativum L.

Indirect immunofluorescence has been used to examine the temporal and spatial distribution of cells containing the storage protein vicilin, in immature pea embryos grown in vivo and in culture. The occurrence of the storage protein was investigated in both separated cells and cryosectioned embryos. In culture, a rapid cessation of cell division in embryos was observed. The proportion of cells containing vicilin was increased up to three-fold in culture depending on the initial fresh weight of the embryo. The DNA levels of the separated cotyledon cells from embryos grown in vitro were compared to those from ones grown in vivo. The maximum DNA content of the cell population was increased though the minimum DNA level of cells containing storage protein was unaltered at 5 C. In addition, no alteration was seen in the spatial localization of vicilin-containing cells within the parenchyma region of the cotyledons. Some cells of the axes of in vitro grown embryos, however, were seen to contain the storage protein which was absent from the same weight embryos taken from the plant. Cells of the epidermal and provascular regions did not contain vicilin in any of the embryos investigated here. These data have been related to the cellular development of the pea embryo and provide evidence for a hypothesis that there is a relationship between cell expansion and deposition of storage protein.

The problem of refolding and reassembling recombinant vicilin subunits

The reassociation of a storage protein (pea (Pisum sativum L) vicilin) expressed in yeast (Saccharomyces cerevisiae MC16 (ade 2–1, leu 2–3, lys 2–1, his 4–712FS, SUF 2)) was investigated. The corresponding protein in French, bean (Phaseolus vulgaris L), termed phaseolin, was used as a model to produce a methodology for the refolding of the recombinant protein. Phaseolin was successfully renatured from its dissociated state in 5 M guanidinium chloride. Application of this rationale to yeast-derived vicilin proved unsuccessful. The nature of the yeast vicilin and its failure to refold and reassemble correctly are discussed together with the further experimental approaches required.

Structure of the globular protein vicilin revealed by scanning tunnelling microscopy

Scanning tunnelling microscopy (s.t.m.) has been used to study the structure of the non-crystalline globular protein vicilin. Molecules were deposited on amorphous carbon substrates and imaged both in air and in vacuo without additional sample preparation. Current-voltage plots of an individual protein molecule are also reported. The s.t.m. images are compared with conventional transmission electron micrographs and with a model of vicilin based on small-angle synchrotron X-ray scattering data.

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5' and 3' regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M r∼50000 together with a range of smaller polypeptides.

Amino acid composition of storage proteins of a promising chickpea (Cicer arietinum L) cultivar

AbstractThe amino acid compositions of flours made from the cotyledons and from the whole seeds of a disease‐resistant, stable and high‐yielding cultivar of chickpea (Cicer arietinum L) cv H75–35, known locally as Gaurav, have been analysed. These, together with similar analyses of the albumin, globulin, legumin and vicilin protein fractions, have been compared with those of other legumes. Seed protein content was 19·8 % with globulin constituting 62·4 % of the total seed protein. The ratios of albumin to globulin and legumin to vicilin were 1:4 and 6:1, respectively. The proportion of basic amino acids in the albumin was low whereas the reverse was true in the globulin. The legumin fraction seems to be superior in terms of total essential amino acids to those from other sources. Sulphur amino acids were the most limiting, followed by tryptophan or threonine depending on the fraction. However, the ratio of methionine to cystine was high (2·76:1).The extent of the sulphur amino acid deficiency was assessed, and possible approaches for its improvement are outlined.

Use of intrinsic fluorescence to follow the denaturation of vicilin, a storage protein from Vicia faba

Excitation of native vicilin protein at 274 nm resulted in a single emission peak at 304 nm attributed to tyrosine fluorescence. Changes in the quantum yield of the tyrosine fluorescence with addition of both urea and guanidine hydrochloride (GdnHCl) were only significant at intermediate denaturant concentrations, and not suitable for assessment of conformational change. Emission spectra for the tryptophan residues were obtained by excitation of the vicilin at 295 nm. The wavelength of maximum tryptophan emission shifted from 347 to 353 nm with the addition of denaturants (2.0 M GdnHCl or 3.0 M urea). At higher concentrations of denaturant, the quantum yield values for tryptophan also increased, reflecting a gradual change in the conformation of vicilin. The degree of polarization for both tyrosine and tryptophan fluorescence decreased significantly at denaturant concentrations slightly above those at which the protein showed the first signs of unfolding. This was further indication of a variety of conformational states for vicilin with increasing levels of denaturant.

Convicilin mRNA from pea (Pisum sativum L.) has sequence homology with other legume 7S storage protein mRNA species.

Nucleotide-sequence analysis of a complementary-DNA clone for convicilin, one of the storage proteins from pea (Pisum sativum L.) seeds, shows it to be homologous with the 7S legume seed storage proteins vicilin, conglycinin and phaseolin. Convicilin is more similar to vicilin than to phaseolin or to conglycinin. Significant areas of sequence difference are discussed.

LR white resin and improved on-grid immunogold detection of vicilin, a pea seed storage protein

LR White resin combined with post-embedding immunogold labelling was used to localize the storage protein vicilin within developing pea seed cotyledons by electron microscopy. Fine structural preservation is comparable to that obtained with Spurr's resin, and antibody labelling is improved. More gold binds to protein bodies, to rough endoplasmic reticulum and to Golgi vesicles, and in addition, vicilin was detected within Golgi cisternae, a site not previously observed.

Periodate-acid treatment of sections permits on-grid immunogold localization of pea seed vicilin in ER and Golgi

The legume seed reserve protein vicilin has been localized in developing pea cotyledons by immunogold labelling on sections of glutaraldehyde-osmium-fixed, resin-embedded tissue. By treating sections with periodate and acid before antibody labelling, a 20-fold increase in specific antibody binding was observed. The densest label occurred over vacuolar contents, previously identified as the site of protein storage, and over electron-dense cisternae occasionally seen to be continuous with the vacuolar contents. Vicilin was also associated with the rough endoplasmic reticulum, the implied site of synthesis, and in electron-dense Golgi vesicles which, we suggest, are a vehicle by which newly synthesized protein is relocated into the vacuole.

Cloning of pea storage protein genes

Vicilin and legumin are the major storage proteins of Pisum sativum . Complementary DNAs (cDNAs) have been produced from poly(A) + mRNA isolated from developing seeds and specific storage protein cDNAs cloned into pBR322. The amino acid sequences predicted from the cDNA sequences have been compared with the actual amino acid sequences derived from the purified protein subunits. These comparisons have confirmed that the legumin α and β subunits as initially synthesized are covalently joined together and that a small peptide is subsequently removed by endoproteolysis to give the disulphide linked subunits of the mature seed legumins. Similar comparisons between the predicted amino acid sequence of vicilin cDNA clones and the amino acid sequence determined on the isolated subunits has shown that some of the 50000 M r type subunits are subsequently cleaved to give three subunits as products, i.e. polypeptides of 19000 M r (α), 13500 M r (β) and 12500 M r or 16000 M r if glycosylated (γ). In addition to these three subunits, cleavage at one or other of the two potential cleavage sites, results in a 33000 M r polypeptide (α + β) and a 31000 M r polypeptide tentatively identified as β + γ. The presence of the sequence Lys-Glu-Asn leads to cleavage on the carboxy side of Asn at the β :γ cleavage site whereas the sequence Gly-Leu-Arg does not lead to cleavage. Comparable sequence data for the α: β processing site do not exist. Comparisons of the cDNA and amino acid sequence disclose the presence of a 15 or 16 amino acid residue vicilin leader sequence as well as a 12 amino acid residue C-terminal peptide which is also removed. The codon usage of the messenger RNAs for the storage proteins are similar to those of other plant proteins and differ somewhat from animal messenger RNAs. Complementary DNAs for specific storage proteins when used to probe different restriction enzyme digests of pea genomic DNA reveal the presence of a small number of legumin and vicilin coding sequences (two to five for legumin and three to seven for vicilin) that occur as single copies except for one vicilin sequence present in two to three copies. Genetic mapping experiments using whole plants locate both the main legumin and the vicilin genes on chromosome 7. The main legumin subunits are coded by genes located at a single Mendelian locus Lg-1 located on the short arm of chromosome 7 very close to the rub locus and the vicilin gene is located 16 map units away close to the r locus. Gene libraries prepared with size fractionated partial restriction enzymic digests of pea genomic DNA ligated into both phage λ L47 and phage λ gt wes have led to the isolation of at least three similar but different legumin genomic sequences. Comparison of the λ and cDNA legumin clones suggests the presence of at least one intron in the former. Legumes in general contain two major seed storage protein types, vicilin and legumin (Derbyshire et al. 1976). Seeds of Pisum sativum (L) have significant amounts of both proteins and since a considerable body of knowledge exists about pea physiology and genetics, this species is a good choice for the study of storage protein genes. Peas are also one of the world’s major legume crops. Since the storage proteins are found only in the tissues of the developing seed (Millerd 1975) and then only in significant amounts during the middle and late stages of development, it was suspected from the onset that the genes responsible for the storage protein would belong to the class of developmentally regulated genes, i.e. those that are only switched on in specific tissues over restricted periods of time.

Role of the endoplasmic reticulum in the synthesis of reserve proteins and the kinetics of their transport to protein bodies in developing pea cotyledons.

Developing pea (Pisum sativum L.) cotyledons were labeled with radioactive amino acids, glucosamine, and mannose in pulse an pulse-chase experiments to study the synthesis, glycosylation, and transport of the reserve proteins vicilin and legumin to the protein bodies. Tissue extracts were fractionated on sucrose gradients to isolate either the endoplasmic reticulum (ER) or the protein bodies. Immunoaffinity gels were used to determine radioactivity in the reserve proteins (legumin and vicilin). After pulse-labeling for 45 min with amino acids, about half the total incorporated radioactivity coincided closely with the position of the ER marker enzyme NADH-cytochrome c reductase at a density of 1.13 g . cm-3 on the sucrose gradient. Both radioactivity and enzyme activity shifted to a density of 1.18 g . cm-3 in the presence of 3 mM MgCl2 indicating that the radioactive proteins were associated with the rough ER. Approximately half of the incorporated radioactivity associated with the rough ER was in newly synthesized reserve protein and this accounted for 80% of the reserve protein synthesized in 45 min. Trypsin digestion experiments indicated that these proteins were sequestered within the ER. In pulse-chase experiments, the reserve proteins in the ER became radioactive without appreciable lag and radioactivity chased out of the ER with a half-life of 90 min. Radioactive reserve proteins became associated with a protein body-rich fraction 20-30 min after their synthesis and sequestration by the ER. Pulse-chase experiments with radioactive glucosamine and mannose in the presence and absence of tunicamycin indicated that glycosylation of vicilin occurs in the ER. However, glycosylation is not a prerequisite for transport of vicilin from ER to protein bodies. Examination of the reserve protein polypeptides by SDS PAGE followed by fluorography showed that isolated ER contained legumin precursors (Mr 60,000-65,000) but not the polypeptides present in mature legumin (Mr 40,000 and 19,000) as well as the higher molecular weight polypeptides of vicilin (Mr 75,000, 70,000, 50,000, and 49,000). The smaller polypeptides of vicilin present in vicilin extracted from protein bodies (Mr 12,000-34,000) were absent from the ER. The results show that newly synthesized reserve proteins are preferentially and transiently sequestered within the ER before they move to the protein bodies, and that the ER is the site of storage protein glycosylation.

An Acid Protease Produced by Monilinia fructigena in vitro and in Infected Apple Fruits, and its Possible Role in Pathogenesis

SUMMARY: The fungus Monilinia fructigena grown in a liquid medium containing protein secreted a protease with many similarities to one extractable from apple fruits infected with this pathogen but absent from healthy fruit. Enzymes from both sources degraded several protein substrates optimally at pH 3·4. At this pH, haemoglobin was degraded optimally at 47 °C, while at 37 °C both enzymes were most stable at pH 5. The pI values for the enzymes were close to 4·0, and their action was inhibited by pepstatin and diazoacetyl-norleucine methyl ester plus copper ions but unaffected by p-hydroxymercuribenzoate, EDTA or trypsin inhibitors. Mercaptoethanol was essential for the recovery of enzyme activity from acrylamide gels in which the in vitro and in vivo enzymes were electrophoretically homogeneous. The in vitro enzyme gave a positive reaction for glycoproteins after purification with a Sepharose-haemoglobin affinity medium. Both enzymes were single units with a molecular weight of 37000–40000 on sodium dodecyl sulphate (SDS)-poryacrylamide gels; gel filtration indicated a molecular weight of 34000–38000. The purified in vitro enzyme had no aminopeptidase or carboxypeptidase activity; the products of haemoglobin digestion were peptides with molecular weights on SDS--polyacrylamide gels of 2000-12000. Purified in vitro protease did not damage apple fruit tissue or cultured apple cells, lyse plant protoplasts or release proteins from isolated fruit cell walls. The protease had no effect on active fungal or host enzymes that might have a role in host/pathogen interactions, and did not induce phaseollin formation in bean cotyledons. The plant proteins vicilin, legumin and heat-denatured peroxidase were hydrolysed at pH 3·5 and spinach nitrate reductase was inactivated. Nontural protease inhibitors were detected in healthy or infected apples. We suggest that in vivo the protease might be involved in the nutrition of the pathogen.

Structural Aspects of Protein Accumulation in Developing Pea Cotyledons. Iv. Effects of Preparative Procedures on Ultrastructural Integrity.

The pea storage proteins legumin and vicilin have previously been shown to be deposited in the vacuoles of cells of young developing cotyledons. By electron microscopy, the appearance of vacuole contents, and in particular of the peripheral protein deposits, are known to vary even in controlled and reproducible conditions of plant growth. This observation suggests an influence of specimen preparation on appearance. Different fixative solutions used on developing pea-seed cotyledons affected the appearance of the cytoplasm and its contents only slightly. The appearance of vacuole contents was greatly modified by the type of buffer used as a fixative vehicle and by osmotic conditions induced by added sucrose, and less by the primary fixative used. From a comparison with living protoplasts from cotyledon cells, it was concluded that the non-membrane-bound peripheral protein deposits are not artefacts of preparation. It is suggested that sucrose may play a role in the transition of vacuoles to protein bodies.

Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.).

Investigations of the vicilin fraction of the storage proteins of pea (Pisum sativum L.) have shown that its major components are a number of protein species of Mr 170 000. Convicilin (Mr 280 000, composed of 71 000-Mr subunits) is a separable component of this fraction. The vicilin proteins are composed principally of approximately equal to 50 000-Mr polypeptides, but also contain a number of smaller polypeptides. The sub-unit polypeptide composition of vicilin changes during seed development quantitatively and qualitatively. Vicilin sub-units have been shown to be synthesised as polypeptides of Mr approximately equal to 50 000 by means of pulse-labelling experiments in vivo, and synthesis of vicilin in vitro directed by mRNA, polysomes and microsomes extracted from pea cotyledons in cell-free translation systems. Polypeptides then undergo two distinct types of proteolytic modification: (a) co-translational removal of a small polypeptide (Mr less than 1000); (b) 'nicking' of polypeptide chains in assembled vicilin molecules, which occurs more than 4 h after their initial synthesis. The basic structure of the vicilin molecule is thus a multimer, possibly a trimer, of approximately equal to 50 000-Mr subunits. The heterogeneity of the initially synthesised 50 000-Mr subunits accounts not only for the several different 50 000-Mr polypeptides found in vicilin, but also for the range of minor polypeptides, since the 'nicking' points will differ among subunits. It also accounts for the observed partial separation of vicilin into different molecular species, since different subunit combinations will give rise to molecules with different properties. Vicilin is also glycosylated and this is a source of further variation.