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.     

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.

     

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.     

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.     

Biosynthesis and processing of legumin-like storage proteins in Lupinus angustifolius (lupin).

Synthesis, secretion and post-translational proteolysis of the storage proteins in cotyledons of Lupinus angustifolius L. (lupin) have been examined in vivo and in vitro by using a combination of pulse-chase experiments with [3H]- or [35S]-labelled amino acids, subcellular fractionation and cell-free translation from poly(A)+ (polyadenylylated) RNA or membrane-bound polyribosomes. Related polypeptides were identified by immunoprecipitation, separation on sodium dodecyl sulphate/polyacrylamide gels and fluorography. The synthesis and processing of two proteins were compared. Conglutin alpha, the 11 S protein, was found as a family of precursor polypeptides of Mr 68000-88000 when translated from poly(A)+ RNA under conditions where signal segments were not cleaved, and Mr 64000-85000 both when sequestered into the endoplasmic reticulum and when accumulated in the protein bodies. Pulse-chase labelling showed that cotyledons from early stages of development were completely incapable of further proteolysis of these precursors. Nevertheless, in the same juvenile cotyledons, the precursors of the minor storage protein conglutin gamma, two polypeptides with Mr 50000-51000, were proteolytically cleaved to mature subunits of Mr 32000 and 17000 within 2 h. Further cleavage of the precursors of conglutin alpha into families of mature subunits of Mr 21000-24000 and 42000-62000 was detected in more mature cotyledons. A model is proposed which suggests that the mature subunits are produced by a single proteolytic cleavage of each of the three major precursors of conglutin alpha and also suggests that a close similarity exists between these subunits and those of other legumin-like proteins. The enzyme responsible for this cleavage, which appears at a specific stage in the middle of cotyledonary development, seems to be an integral part of the programmed developmental sequence in these pods.     

The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons

Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm³ (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.     

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     

Poly(A)-containing RNA in early embryogenesis of sea urchins

The content, biosynthesis and template activity of poly(A)+ RNA in the early stages of sea urchin development have been studied. The amount of poly(A)+ RNA reaches a maximum at the middle blastula stage in polyribosomes and at the 8-blastomere stage in the cytoplasm. Poly(A)+ RNA synthesis becomes noticeable at the 64-blastomere stage and the spectrum of newly synthesized molecules is different from that at the middle blastula stage. The products of translation in vitro of poly(A)+ RNA at all the stages studied show insignificant differences and contain a major group of polypeptides of molecular mass 10-20 kDa.     

Precursor Forms of Pea Vicilin Subunits: MODIFICATION BY MICROSOMAL MEMBRANES DURING CELL-FREE TRANSLATION

Polyribosomal RNA isolated from pea cotyledons at various developmental stages programmed the cell-free synthesis of polypeptides which were recognized by antibodies specific for pea storage proteins. There were quantitative and qualitative changes in the template activity during seed maturation. Most of the polysomal RNA was associated with the membrane fraction, and all of the template for storage protein occurred in this fraction. Using RNA from a stage of seed maturation at which the synthesis of the high-molecular weight vicilin polypeptides predominate, it was found that the major translation products, although antigenically recognizable as storage protein, did not coincide with the authentic vicillin polypeptides on denaturing polyacrylamide gels. The addition during translation of microsomal membranes from dog pancreas or pea cotyledons resulted in the appearance of new polypeptides which did coincide with some of the authentic vicilin polypeptides (in the apparent molecular weight regions of 75,000 and 50,000) and were antigenically recognizable as storage protein. Other translation products related to storage protein were not visibly altered in their electrophoretic mobility by the addition of membranes. Microsomal membranes treated with Triton X-100 were not effective in modifying the cell-free products. The modified vicilin polypeptides and at least two other translation products were protected from proteolytic degradation, suggesting that they were sequestered within microsomal vesicles. Thus, these storage protein components may be synthesized by a mechanism analogous to that described for membrane and secretory proteins (Blobel G, B Dobberstein 1975 J Cell Biol 67: 835-851).     

Poly(A) length,cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B. Clegg, 1982, Dev. Biol.89, 362–378). To shed light on the significance of these changes, we analyzed the size distribution of total poly(A) from embryos at different stages of development by gel electrophoresis and hybridization with [³H]poly(U). The number-average size of poly(A) tracts varies only slightly, from 61 to 77 nucleotides, indicating that the changes in poly(A) content are due primarily to changes in the number of poly(A) sequences, i.e., the number of poly(A)+ mRNA. From these data, the number of poly(A)+ mRNA can be estimated as follows: ovulated egg, 1.7 × 10⁷; one-cell embryo, 2.4 × 10⁷; late two-cell, 0.7 × 10⁷; late eight-cell, 1.3 × 10⁷; and early blastocyst, 3.4 × 10⁷. These results suggest the elimination of the bulk of maternal poly(A)+ mRNA at the two-cell stage, to be replaced by newly synthesized mRNA derived from the embryonic genome. To study the synthesis of poly(A)+ mRNA, we cultured mouse embryos in vitro with [³H]adenosine and analyzed the labeled poly(A)+ RNA as to molecular size, length of the poly(A) tail, and relative distribution of label in poly(A) vs internal locations. We observed an active incorporation of label into large-molecular-weight (average size about 2 kb) poly(A)+ RNA at all stages from the one-cell to the blastocyst. However, in the one-cell embryo, about 70% of the label was localized in the poly(A) tail, suggesting cytoplasmic polyadenylation, and only about 30% was localized in the remainder of the molecule, suggesting the complete new synthesis of a small amount of poly(A)+ RNA. Differences in the size distribution of the labeled poly(A) as compared with the total poly(A) in the one-cell embryo indicate that the labeling is not due to a general turnover of poly(A) tails, but rather to the polyadenylation of previously nonpolyadenylated, stored RNA. Significant new synthesis of poly(A)+ RNA is evident from the two-cell stage onward and most likely accounts for the sharp rise in the number of poly(A)+ RNA molecules by the early blastocyst stage.     

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.     

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.     

Legumin Synthesis in Developing Cotyledons of Vicia faba L

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.     

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.     

Sequence complexity of messenger RNA in cotyledons of developing pea (Pisum sativum) seeds

The hybridization kinetics of poly(A)⁺-RNA preparations from the cotyledons of developing pea (Pisum sativum seeds to complementary DNAs have shown that the number of distinct sequences in poly(A)⁺ -RNA decreases from ca 20 000 at the early stage of cotyledon development to ca 200 at a late stage of cotyledon development. The decrease in sequences is accounted for entirely by the disappearance of ‘rare’ poly(A)⁺ -RNAs (< 10³ copies/cell) as seed development proceeds. There is an increase (1–6) in very abundant poly(A)⁺-RNA sequences (? 5 × 10⁵ copies/cell) from early- to mid-developmental stages, concomitantly with the increase in the synthesis of seed-specific storage protein polypeptides. In agreement with the continuing synthesis of most of these polypeptides to the end of seed development, the number of very abundant poly(A)⁺-RNAs is maintained to the late cotyledon development stage. Abundant poly(A)⁺-RNA sequences (ca 10⁴ sequences/cell) increase from 80 to 180 during development, possibly corresponding to the polypeptides which are not storage proteins but are known to be accumulated in pea seeds. Hybridization of single-copy pea genomic DNA sequences to poly(A)⁺-RNA from developing seeds showed that ca 5 % of the single-copy sequences were present in mRNA from mid-development cotyledons. In addition, hybridization of cDNA prepared against poly(A)⁺-RNA from nuclei of early development cotyledons to the corresponding cytoplasmic polysomal poly(A)⁺-RNA showed that the cytoplasmic poly(A)⁺-RNA contained ca 50 % of the sequences present in the nuclei. These results are discussed and interpreted in the light of existing results from similar systems.     

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.