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     

Pea legumin overexpressed in wheat endosperm assembles into an ordered paracrystalline matrix

Legumin, a major component of pea seed storage vacuoles, is synthesized by a number of paralogous genes. The polypeptides are cleaved posttranslationally and can form mixed hexamers. This heterogeneity hampers structural studies, based on the production of hexamer crystals in vitro. To study a single type of homogenous legumin we produced pea legumin A in transgenic wheat (Triticum aestivum) endosperm where prolamins are predominant and only small amounts of globulins accumulate in separate inclusions. We demonstrated that the legumin precursor was cleaved posttranslationally and we confirmed assembly into 11S hexamers. Legumin was deposited within specific regions of the inclusion bodies. Angular legumin crystals extended from the inclusion bodies into the vacuole, correlating with the high legumin content. This suggests that the high-level production of a single type of legumin polypeptide resulted in the spontaneous formation of crystals in vivo. The use of a heterologous cereal system such as wheat endosperm to produce, isolate, and recrystallize homogenous 11S legume globulins offers exciting possibilities for structural analysis and characterization of these important seed storage proteins.     

Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.

Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.

When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA.

     

Inheritance of legumin and albumin contents in a cross between round and wrinkled peas

Summary Legumin and albumin are the fractions of pea seed proteins preferred to vicilin because of their high sulfur amino acid contents. The joint inheritance of legumin and albumin contents was studied in a cross between to contrasting lines of peas — one with high legumin and low albumin, and the other with low legumin and medium to high albumin. Single seed determinations were made in the parental, F₁; F₂ and backcross generations using rocket immunoelectrophoresis. In the non-segregating generations (P₁, P₂ and F₁), legumin and albumin contents were negatively correlated (r=–0.50). The estimates of correlation coefficients in the segregating generations (F₂, BC₁ and BC₂) were also about –0.5. However, the two estimates based on the round and on the wrinkled seeds separately in the F₂ generation were not significantly different from zero. At least four individual round F₂ seeds showed the desired recombination of high legumin with high albumin indicating that the unfavorable correlation can be broken. In this cross legumin content showed predominantly additive genetic variation whereas the dominance variance was the largest component for albumin content. A combined relative sulfur index, proposed as a convenient measure for selection, showed a narrow sense heritability of 47%. In general these results support the view that sulfur amino acid content of peas can be improved by breeding, but that the required selection regime must take both legumin and albumin content into account.     

Legumin of Vicia faba major: accumulation in developing cotyledons,purification, mRNA characterization and chromosomal location of coding genes

Summary Experiments were carried out on Vicia faba major involving (1) determination of the pattern of legumin accumulation during seed development, (2) protein purification from mature cotyledons, (3) the characterization of legumin mRNA, and (4) the chromosomal localization of the genes coding for legumins. In developing cotyledons the synthesis of legumin begins 28 days after petal desiccation (DAPD), and 4 days after initiation of vicilin synthesis. The two subunits (_A and _A) of legumin A appear 2 days earlier than those (_B and _B) of legumin B. While the accumulation of vicilin peaks on the 30th DAPD, that of legumin continues during further seed development, and the synthesis of legumin mRNA peaks on the 37th DAPD. Northern blot hybridizations using two DNA plasmids containing cDNA inserts with sequence homology to the A- and B-type legumin genes, respectively, indicated that legumin mRNAs extracted from cotyledons 36 DAPD band below the 18S RNA band. In addition, a faint band below that of the 25S RNA band can be observed in legumin mRNAs extracted from cotyledons at an earlier developmental stage (30 DAPD). By means of polyacrylamide gel electrophoresis in the presence or absence of SDS and 2-mercaptoethanol, two fractions could be eluted after zonal isoelectric precipitation of the globulins from mature seeds: one fraction contains mainly vicilin, the other, legumin. In situ hybridization showed that legumin genes are arranged in two clusters: the genes coding for legumin A are located in the longer arm of the one between the two shortest subtelocentric chromosome pairs whose centromere is in a less terminal position; those coding for legumin B are located in the non-satellited arm of the longer submetacentric pair.     

Correlated in situ hybridisation and immunochemical studies of legumin storage protein deposition in pea (Pisum sativum L.)

Legumin and vicilin are the major globulin seed storage proteins of pea. They are synthesised predominantly in the cotyledons where they are sequestered within membrane-bounded vacuolar protein bodies. In situ hybridisation histochemistry, with both biotinylated and ³⁵S-labelled cDNA probes, has been used to visualise the temporal and spatial patterns of distribution of legumin and vicilin mRNAs during seed development. These patterns have been compared with those of storage protein deposition which have been determined by immunocytochemistry. Results indicate that within the cotyledons high levels of legumin and civilin mRNAs are restricted to the storage parenchyma tissues, whilst the epidermal cells and vascular parenchyma do not show such accumulation. The tissues of the embryo axis also show differential levels of expression, although where present the levels of mRNAs appear much lower than in the cotyledons. Throughout the embryo the patterns shown by in situ hybridisation are similar to those shown by immunocytochemistry, although the transient endosperm of early seed development does not show such a correlation.     

The characterisation of vicilin during seed development in Vicia faba (L.)

Summary Vicilin and legumin were extracted from developing seeds at different stages using the classical method of repeated isoelectric precipitations. The subunits of these two protein fractions were separated by SDS gel electrophoresis, and it was shown that the sub-unit structure of vicilin changed during development whereas that of legumin did not. Thus vicilin is not a single protein.Vicilin was formed prior to legumin during seed development although the rate of synthesis of the latter was faster, so that in the mature seed the ratio of legumin to vicilin was about 4:1 by weight.     

Isoelectric-focusing properties and carbohydrate content of pea (Pisum sativum) legumin

Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an `acidic' subunit (mol.wt. about 40000) and a `basic' subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the `basic' subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0–5.3 for the `acidic' subunits, and 8.3–8.7 for the `basic' subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The `basic' and `acidic' subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.     

Influence of Sulfur Nutrition on Developmental Patterns of Some Major Pea Seed Proteins and Their mRNAs

In addition to the marked reduction in legumin synthesis and legumin mRNA levels reported earlier (Chandler, Higgins, Randall, Spencer 1983 Plant Physiol 71: 47-54), pulse labeling of S-deficient Pisum sativum L. seeds showed that a high relative level of total vicilin (vicilin plus convicilin) synthesis was maintained throughout the entire phase of protein accumulation, whereas in nondeficient seeds vicilin synthesis is largely confined to the first half of this phase. Fractionation of pulse-labeled proteins on Na-dodecylsulfate-polyacrylamide gels showed that the synthesis of the M_r 50,000 family of vicilin polypeptides was increased and greatly extended in S-deficient seeds whereas that of convicilin was slightly reduced. Other changes apparent from pulse-labeling experiments include a depression, to different degrees, in the synthesis of three major albumin polypeptides.

The level of the mRNAs for seven major seed proteins was followed throughout development of control and sulfur-deficient seeds. In all cases, the changes in each mRNA closely reflected the pattern of synthesis of its corresponding polypeptide seen by pulse labeling. S-deficient seeds showed an elevated level of M_r 50,000 vicilin mRNA which remained high throughout seed formation, whereas legumin mRNA levels were greatly reduced at all stages of development.

When S-deficient plants were given an adequate supply of sulfate midway through seed development, there was a shift toward the protein synthesis profile characteristic of healthy plants. The synthesis of legumin and two albumins rapidly increased and the synthesis of M_r 50,000 vicilin declined more slowly. Similar responses were seen in detached, S-deficient seeds supplied directly with adequate sulfate.

     

Proteins arising during the late stages of embryogenesis in Pisum sativum L.

Two abscisic acid (ABA)-responsive seed proteins, ABR17 and ABR18 (ABA-responsive 17000-M_r and 18000-M_r, respectively), previously found to be induced in cultured embryos of pea (Pisum sativum L.) are major components synthesised during normal seed desiccation. The ABR17 and ABR18 proteins showed different patterns of accumulation. The ABR18 protein was abundant in the testa during early seed development but in desiccating seed it was synthesised in the embryo, indicating spacial as well as temporal regulation of expression. The ABR18 protein was undetectable soon after germination but reappeared after adding ABA. The ABR17 protein was not detected in the testa but appeared in the embryo just prior to maximum fresh weight. The ABR17 protein continued to be synthesised during germination and was also present in non-stressed leaves. A high level of endogenous ABA or added ABA increased levels of translatable ABR17 mRNA. The ABR17 and ABR18 proteins were further characterised so as to help determine their structure and function. Neither protein appeared to contain a signal peptide but both proteins appeared to be glycosylated. The proteins had similar amino-acid compositions and limited Nterminal analysis showed 56% sequence identity. Neither protein had any significant N-terminal sequence homology to any of the late embryogenesis-abundant (LEA) proteins or dehydrins. Both proteins, however, show striking homology with a pea disease-resistance-response protein and the major birch pollen allergen, indicating that the ABR17 and ABR18 proteins may be members of a distinct group of stress-induced proteins.Abbreviations ABA (±) cis,trans-abscisic acid - ABR17 M_r-17200 ABA-responsive protein - ABR18 M_r-18 100 ABA-responsive protein - FW fresh weight - I_gG immunoglobulin G - LEA late embryogenesis-abundant - M_r apparent molecularmass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station.     

Cyclin-dependent kinase-like proteins in pea nuclei: their presence and role in cell proliferation

Summary Although many putative cdk (cyclin-dependent kinase) homologue genes have been identified in higher plants, their function and involvement in cell proliferation are still unclear. In this work we investigated the presence and distribution of cdk-like proteins in root tip meristem nuclei at different germination times (before, during, and after the onset of cell proliferation) and in nuclei of differentiated leaves. Nuclear cdk-like proteins were found in the root meristem throughout seed germination with a higher amount in actively proliferating cells, but were not detected in differentiated leaf. Characterization of the detected pea cdk-like proteins by immunoblotting led to the identification of two specific principal proteins of 33.2 and 34 kDa with the cdk conserved motif PSTAIRE. The p33.2 protein was also recognized by the anti-human p33^cdk2 antibody, suggesting that the p33.2 and p34 proteins could be pea homologues of human p33^cdk2 and p34^cdk1, involved in the G₁-S and G₂-M transitions, respectively. Additional analysis of pea cdk protein localization has shown partial localization of these proteins at DNA replication sites during the G₁ to S transition. These microscopical and biochemical data support the hypothesis that, in pea nuclei as in mammals, many PSTAIRE-cdks are present with different functions related to cell proliferation, one of which is probably involved in the control of the G₁-S transition.Abbreviations Cdk cyclin-dependent kinase - HU hydroxyurea - BrdU bromodeoxyuridine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101 - PI propidium iodide     

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein contentof pea seeds (Pisum sativum L.) as compared to soya bean seeds(Glycine max L. Merrill) might be due to faster degradationof the pea storage proteins during development of the seed.Pea and soya bean cotyledons were subjected to a ‘pulse-chase’experiment using [³H]glycine in in-vitro cultures. In peas,legumin had a half-life of 146 days, while vicilin had a half-lifeof 39 days. There was no measureable degradation of soya beanstorage proteins. Even with the pea storage proteins, the half-liveswere so much longer than the maturation time of seeds that degradationof storage proteins could not account for the lower proteincontent of peas as compared to soya beans. The validity of theseresults was indicated by the finding that non-storage proteinshad much shorter half-lives and that omission of a carbon ora nitrogen source greatly accelerated degradation. Labelledglycine was found to be a good probe for protein turnover studiesbecause it was very rapidly metabolized. Glycine max L. Merrill, soya bean, Pisum sativum, L. pea, protein turnover, storage proteins, legumin, vicilin     

Variation in amino acid composition of legumin from Pisum

Pisum; Leguminosae: pea: amino acid composition: seed storage proteins: legumin.     

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean α‐amylase inhibitor gene

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for α‐amylase inhibitor‐1 (αAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean αAI1 protein and the corresponding αAI1‐free segregating lines and non‐GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for αAI1. Proteomic analysis showed that in addition to the presence of αAI1, 33 other proteins were differentially accumulated in the αAI1‐expressing GM lines compared with their non‐GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of αAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium‐mediated transformation events. Sixteen proteins were identified after MALDI‐TOF‐TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin‐containing protein. Two proteins were uniquely expressed in the αAI1‐expressing GM lines and one new protein was present in both the αAI1‐expressing GM lines and their αAI1‐free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.