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.

     

Another class of vicilin gene in Pisum

We describe a novel class of vicilin gene in Pisum corresponding to a precursor polypeptide of M_r 68000. The mRNA corresponding to this polypeptide accumulates during seed development in a pattern similar to that of convicilin. Hybridization and sequence analyses show that this vicilin gene class is less homologous to other Pisum vicilin gene classes than these last are to each other. Gene-copy-number estimates and decreased stringency hybridizations of the gene class described here show that the Pisum vicilin gene family is bigger and more complex than hitherto reported.Abbreviations cDNA complementary DNA - M_r relative molecular mass - SDS sodium dodecyl sulphate - SSC 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0 This work was supported by the Agricultural and Food Research Council via a grant-in-aid to the John Innes Institute.     

Pulse-labeling Studies on Protein Synthesis in Developing Pea Seeds and Evidence of a Precursor Form of Legumin Small Subunit

Intact cotyledons were taken from pea seeds at various stages during seed development and pulse-labeled with ¹⁴C-amino acids. Salt-soluble proteins then were extracted and fractionated on Na dodecyl sulfate-polyacrylamide gels. Storage proteins in these extracts were identified by their binding to immunoaffinity columns. The labeling studies showed that the synthesis of storage protein polypeptides accounts for a major part of total protein synthesis of developing cotyledons between 10 and 22 days after flowering. The distribution of the incorporated radioactivity between individual storage protein polypeptides varied with stage of development. For example, the synthesis of the 50 kilodalton complex of vicilin subunits dominated the early stages of protein accumulation but was a negligible proportion of the total incorporation in the later stages. On the other hand, the 75 kilodalton vicilin subunit was synthesized throughout this entire period. The major small subunit of legumin (20 kilodaltons) was not detected by either Coomassie blue staining or by 2-hour labeling during this period. It was found to arise during the desiccation phase of seed maturation from a long-lived precursor with a relative electrophoretic mobility equivalent to 19 kilodaltons.     

The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.).

A third storage protein, distinct from legumin and vicilin, has been purified from the seeds of pea (Pisum sativum L.). This protein has been named 'convicilin' and is present in protein bodies isolated from pea seeds. Convicilin has a subunit mol.wt. of 71 000 and a mol.wt. in its native form of 290 000. Convicilin is antigenically dissimilar to legumin, but gives a reaction of identity with vicilin when tested against antibodies raised against both proteins. However, convicilin contains no vicilin subunits and may be clearly separated from vicilin by non-dissociating techniques. Unlike vicilin, convicilin does not interact with concanavalin A, and contains insignificant amounts of carbohydrates. Limited heterogeneity, as shown by isoelectric focusing, N-terminal analysis, and CNBr cleavage, is present in convicilin isolated from a single pea variety; genetic variation of the protein between pea lines has also been observed.     

Measurement of gene number for seed storage proteins in Pisum.

We have measured the numbers of genes coding for the three seed storage proteins, vicilin, convicilin and legumin, in a number of Pisum genotypes of variant protein composition. No difference in gene number existed among P. sativum genotypes for any of the proteins. There were differences in the number of genes coding for individual proteins with approximately 11 genes (per haploid genome) for vicilin, 8 genes for legumin and 1 gene for convicilin.     

Studies on Changing Protein Levels in Developing and Germinating Seeds of Lathyrus sativus L.

SDS-polyacrylamide gel electrophoresis of seed protein extracts showed synthesis of major legumin and vicilin polypeptides from 30 OAF onwards though very faint polypeptide bands could be seen at early stage of 10 OAF. Globulin synthesis enhanced and dominated in the second half of seed development. In germinating seeds, legumin polypeptides (mol wt 75–66, 45–41 kD) and vlcllin polypeptides (mol wt 54 kD) were degraded Increasingly from 2nd to 6th day of germination. Globulins and glutellns decreased and albumins Increased from 2nd to 6th day of seed germination.     

Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons

Cotyledons of developing pea seeds (pisum sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies, The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulse-labeled (1-2 h), newly synthesized vicilin was present as a series of polypeptides with M(r) 60,000-65,000, and newly synthesized vicilin was present as series of polypeptides with M(r) 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J Cell Biol., 93:5- 14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (M(r) 40,000 and 19,000) and smaller vicilin polypeptides (M(r) 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm). Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 8S and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (M(r) less than 49,000). These results indicate that the smaller vicilin polypeptides (M(r) less than 49,000) arise delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (M(r) more than 49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of M(r) 40,000 and 19,000.     

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.     

The role of glycosylation in storage-protein synthesis in developing pea seeds

Intact pea (Pisum sativum L.) cotyledons were incubated with [¹⁴C]glucosamine at several stages of seed development and the resultant radioactive proteins were analysed by gel electrophoresis combined with immunoaffinity chromatography and sucrose gradient fractionation. Glucosamine was incorporated into at least five vicilin polypeptides (approx. molecular weight 70,000; 50,000, two components; 14,000, two components). No incorporation was detected into the subunits of legumin. Tunicamycin at 50 g/ml largely inhibited glucosamine incorporation but had little effect on the incorporation of ¹⁴C-labelled amino acids into cotyledon proteins, including vicilin. The assembly of vicilin polypeptides into full-sized protein oligomers (7–9 S) was also unaffected by tunicamycin. Chromatography on concanavalin A confirmed that glycosylation of cotyledon proteins was inhibited by tunicamycin. It is concluded that glycosylation of most cotyledonary proteins involves lipid-linked sugar intermediates, but that glycosylation itself is not an essential step in the synthesis of vicilin polypeptides nor in their assembly into oligomers.Abbreviations IgG immunoglobulin G - M W_t approximate molecular weight based on electrophoretic mobility relative to that of protein standards - SDS-PAGE Na-dodecyl sulfate-polyacrylamide gel electrophoresis     

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.     

Legumin and vicilin,storage proteins of legume seeds

The structure, location in the seed and distribution of the storage protein of legume seeds are described. Methods which have been employed for the extraction, purification and characterisation of seed globulins are reviewed in relation to modern biochemical practice. The physical, chemical and immunological characteristics of the classical legumin and vicilin preparations from Pisum sativum are summarised and the distributions of proteins with sedimentation coefficients and/or immunological determinants similar to those of legumin and vicilin, are tabulated. The structure and composition of various purified legumin and vicilin-type proteins from a variety of legumes, are compared.     

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 _r50000 together with a range of smaller polypeptides.     

Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than M_r～50 000 (i.e., M_r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M_r～50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than M_r～50 000 arose by endoproteolytic cleavage of parent molecules within the M_r～50 000 group. Cleavage in different M_r 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than M_r～50 000, with the possible exception of the M_r34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the M_r～50 000 group.     

Leishmania tropica: Surface antigens of intracellular and flagellate forms

Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (M_r 67,000, M_r 50,000, M_r 68,000, and M_r 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (M_r 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (M_r 94,000 and M_r 43,000).     

Sequestration of pea reserve proteins by rough microsomes

Free polysomes, polysomes released from membranes, and rough microsomal vesicles isolated from developing cotyledons of Pisum sativum L. cv. Burpeeana were used to direct cell-free protein synthesis in a wheat germ system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the polypeptide products had molecular weights ranging from 12,000 to 74,000. Some of the polypeptides migrated during electrophoresis with the same mobility as polypeptides present in legumin and vicilin preparations. By the use of rabbit antibodies raised against pea reserve proteins it was established that polysomes released from membranes and rough microsomes directed the synthesis of polypeptides that were related to reserve proteins whereas free polysomes did not.     

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

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