Proteolysis of the main storage protein of buckwheat seeds at the early stage of germination

The changes in the main storage protein of seeds of buckwheat ( Fagopyrum esculentum Moench cv. Shatilovskaya 5), 13S globulin, were studied during seed germination. During the first three days of germination the 13S globulin is subjected to a limited proteolysis, which consists in the splitting of some of its subunits into large polypeptide fragments. Insignificant changes in the sedimentation coefficient of the 13S globulin during the first days of germination as well as immunochemical data, indicate that the limited proteolysis of the 13S globulin does not cause any major changes in its structure.
Dormant buckwheat seeds contain a proteolytic enzyme (a metalloproteinase), which can cause limited proteolysis of 13S globulin. The proteinase hydrolyzed some subunits of the 13S globulin to high molecular weight fragments. In the presence of sodium dodecylsulphate the electrophoretic pattern of 13S globulin, isolated from 3-day-old buckwheat seedlings, was almost identical to that of 13S globulin from dormant seeds hydrolyzed with metalloproteinase. It is suggested that the proteolysis of 13S globulin observed in vitro may also take place in vivo in the course of seed germination.     

Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds.

A homogeneous preparation of metalloproteinase, purified 1000-fold, was obtained from buckwheat (Fagopyrum esculentum) seeds. The Mr of the enzyme, determined by SDS/PAGE, was 34,000 (it was 39,000 by gel chromatography). Its pH optimum was 8.0-8.2 with 13 S globulin, from buckwheat seeds, as substrate. Atomic-absorption spectroscopy revealed the presence of one Zn2+ ion per enzyme molecule. The enzyme was completely inhibited by EDTA (1 mM), zincone (1 mM) and 1, 10-phenanthroline (1 mM). The metalloproteinase performed limited proteolysis of the following seed storage proteins: 13 S globulin from buckwheat seeds and 11 S globulin from soybean (Glycine max) seeds. It hydrolysed three peptide bonds formed by the amino groups of Leu15, Tyr16 and Phe25 in the oxidized B-chain of insulin. In its main properties the enzyme is similar to metalloproteinases of animal and bacterial origin.     

The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds

Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH₄)₂SO₄ fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (M_r) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a M_r of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - M_r apparent molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Protein Bodies from Buckwheat Seed Cotyledons: Isolation and Characteristics

Protein bodies were isolated from cotyledons of dry buckwheatseeds by homogenization in acetone with subsequent purificationin a 1.26 g cm^–3 to 1.53 g cm^–3 linear density gradientof a mixture of acetone with CCI₄. The purified fraction ofprotein bodies with globoids (PB I) had a buoyant density of1.48–1.51 g cm^–3 and was intact according to microscopicdata. Localization of hydrolytic enzymes and proteinase inhibitorsin the PB I fraction and in the fraction of the cytoplasm andmembrane material (CMM) was studied. It was shown that acidhydrolytic enzymes, such as aspartic proteinase, carboxypeptidase,acid phosphatase, -D-mannosidase and N-acetyl-ß-glucosaminidase,as well as chymotrypsin and trypsin inhibitors were predominantlylocalized in the PB I. BAPAase and SH-activated caseinase activitieswere equally distributed between the PB I and CMM fraction.The activities of leucine aminopeptidase and SH-independentcaseinase were noticeably predominant in the CMM fraction. Key words: Buckwheat, subcellular fractionation, protein bodies, hydrolases     

Effects of the embryonic axis and phytohormones on proteolysis of the storage protein in buckwheat seed

The role of the embryonic axis in regulation of proteolysis of the main storage protein was studied in buckwheat ( Fagopyrum esculentum ) seed. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that removal of the embryonic axis had no effect on the first stage of hydrolysis, that is proteolytic modification, of 13S globulin. This modification took place in the growing seedlings also in the presence of cycloheximide, i.e. it was due to an enzyme present in dry seed. However, in isolated cotyledons the 13S globulin was not degraded completely. Incubation of isolated cotyledons with cytokinins, gibberellic acid and indoleacetic acid could not replace the excised embryonic axis. At the same time, proteolysis of the 13S globulin in the growing seedlings was strongly inhibited by casein hydrolyzate. It is suggested that a complete proteolysis of the modified storage protein is regulated by the concentration of hydrolysis products at the site of hydrolysis. The embryonic axis serves, most probably, as a site of efflux of the products of protein hydrolysis in the cotyledons during seedling growth and thus regulates the course of proteolysis.
Abscisic acid (10–100 μ M ) was without effect on modification of the 13S globulin, but suppressed the complete proteolysis of the protein by inhibiting, apparently, the synthesis of the cysteine proteinase in the growing seedlings.     

荞麦属种间13S球蛋白基因序列比较研究

荞麦13S球蛋白是荞麦种子中的一类主要贮藏蛋白。本研究选用荞麦属植物甜荞栽培种及其野生类型、苦荞栽培种及其野生类型、毛野荞、左贡野荞、细柄野荞和硬枝万年荞6个种共44份材料,进行PCR特异性扩增、测序得到荞麦13S球蛋白基因的保守片段序列。对序列进行差异分析,结果发现44份供试材料13S球蛋白基因片段的285个排列位点中不变位点为24个,多态性位点S为261个(含简约信息位点数198个和单型可变位点63个),序列总突变位点Eta为503个。野生甜荞种内13S球蛋白基因序列差异明显高于栽培甜荞,但野生苦荞种内13S球蛋白基因序列差异仅稍高于栽培苦荞。推测其一方面可能与繁殖方式有关,另一方面可能与荞麦驯化过程中通常只有少数野生型群体被驯化有关。系统聚类分析发现栽培甜荞与野甜荞亲缘关系近,与左贡野荞亲缘关系次之;栽培苦荞与野苦荞亲缘关系近,与毛野荞亲缘关系次之;细柄野荞和硬枝万年荞的13S蛋白基因片段序列差异较小,说明其亲缘关系较近。上述结果为荞麦属种间遗传多样性与进化关系研究提供了理论依据。     

Ultrastructural localization of glycinin and beta-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

beta-Conglycinin (7 S globulin) and glycinin (11 S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7 S globulin could not be detected.     

Ultrastructural localization of glycinin and β-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

Summary -Conglycinin (7S globulin) and glycinin (11S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7S globulin could not be detected.     

黄瓜种子萌发过程中Ca~(2 )分布的变化

采用细胞化学方法 ,研究了黄瓜种子中贮藏Ca2 的分布特点及其在萌发过程中的变化动态。干种子的子叶细胞中贮藏有大量的蛋白体、油脂体 ,Ca2 沉淀颗粒大量分布于胞质、胞间隙以及细胞质膜上。大多数蛋白体中有 1至数个圆球形或椭圆体形含Ca2 的球状晶体。相比之下 ,胚芽和胚根细胞中Ca2 较少。种子萌发早期 ,子叶中的贮藏钙及晶体溶解释放出的Ca2 部分转运到生长发育中的胚芽和胚根中。随着萌发的继续 ,胚根和胚芽细胞中的Ca2 不会持续增多 ,反而下降     

Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination. Received: 14 February 2000 / Accepted: 16 August 2000     

甜荞不同品种不同海拔地区种子蛋白组分含量研究

该研究通过分析甜荞10个品种在4个不同海拔栽培的种子蛋白质组分(清蛋白、球蛋白、醇溶蛋白和谷蛋白)的含量变异,以揭示不同荞麦品种之间以及不同栽培地点甜荞种子蛋白组分的变异规律。结果表明:在所有甜荞品种种子蛋白组分含量中清蛋白谷蛋白球蛋白醇溶蛋白。其中,种植于海拔最低的内蒙古通辽的甜荞种子平均球蛋白含量最高(1.081%),而种植于海拔1 450 m的河北甜荞谷蛋白平均含量最高(2.805%);海拔2 620 m的青海甜荞清蛋白平均含量为4.750%,而在海拔最高的西藏日喀则收获的甜荞种子的醇溶蛋白最高(平均为0.393%)。另外,蒙0530在4个地区的平均种子清蛋白和谷蛋白含量都最高,而球蛋白含量最高的品种是赤甜荞1号,定甜荞2号的种子醇溶蛋白含量最高。双因素方差分析表明,种子清蛋白含量品种间变异达极显著水平,不同地点间的种子醇溶蛋白含量达极显著水平,而地点和品种两个因素对种子球蛋白含量和谷蛋白含量的变异都有极显著影响。相关性分析表明,赤甜荞1号的醇溶蛋白含量与海拔呈显著正相关,蒙0530的球蛋白含量与海拔呈显著负相关,其他品种蛋白组分与海拔的相关性不显著。该研究结果对于甜荞优质品种培育和栽培以及推广都有一定的指导意义。     

Seed storage proteins in developing somatic embryos of alfalfa: defects in accumulation compared to zygotic embryos

Somatic embryos of alfalfa (Medicago sativa L.) synthesizedall of the major storage proteins of zygotic embryos; an 11Sglobulin (medicagin), a 7S globulin (alfin), and a 2S albumin(LMW). In zygotic embryos (cotyledons and/or axis) these storageproteins accounted for 30%, 10%, and 20%, respectively, of thetotal extractable protein. In somatic embryos the 7S proteinwas predominant while the 11S (particularly subfamily I) and2S proteins were present in lower amounts. Analysis of cultivarsand selfed seed of the embryogenic clone (RL34) demonstratedthat these differences were predominantly physiologically, ratherthan genetically, based. The accumulated 7S and 11S storageproteins of somatic embryos were processed normally, aggregatedas oligomers, and were deposited in protein bodies. This wasnot the case for the 2S storage protein. In somatic embryosthat protein was localized in the cytoplasm rather than in proteinbodies, the site of deposition in zygotic embryos. Key words: Medicago (alfalfa), zygotic/somatic embryos (seeds), storage proteins, immunolocalization     

Subcellular localization of the 2S globulin narbonin in seeds of Vicia narbonensis

Narbonin is a 2S protein from the globulin fraction of narbon bean (Vicia narbonensis L.) cotyledons. Its amino acid composition and the pattern of its regulated accumulation in developing seeds led to the suggestion that narbonin could be a storage protein. Therefore, it was expected to be present in protein bodies of the storage tissue cells. Comparison of the cDNA-derived amino acid sequence with a directly determined partial N-terminal sequence revealed that the primary translation product of narbonin mRNA lacks a transient N-terminal signal peptide (V.H. Nong et al., 1995, Plant Mol Biol 28: 61–72). Narbonin polypeptides that had been synthesized in a cell-free translation system supplemented with dog pancreas microsomes were not protected against degradation by posttranslationally added proteases (protease protection assay). In accordance with the lack of a signal peptide this indicates that the polypeptide was not cotranslationally sequestered into the microsomes. The protein-body fraction that had been isolated from mature narbon bean cotyledons by a non-aqueous gradient centrifugation procedure was free of narbonin; this was found in the soluble cell fraction. In electron micrographs, narbonin could be localized in the cytoplasm using the immuno gold-labelling technique. Previously, it had already been shown that narbonin is too slowly degraded during narbon bean germination to act as a storage protein. From all these results it has to be concluded that narbonin is a cytoplasmic protein which does not belong to the storage proteins in the restricted sense. Other possible functions are discussed. Received: 18 November 1996 / Accepted: 28 February 1997     

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.Abbreviations DAA days after anthesis - IgG immunoglobulin G - M_r apparent molecular mass - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis     

Polypeptide components of oligomeric legumin-like thiamin-binding protein from buckwheat seeds characterized by partial amino acid sequencing and photoaffinity labeling

Among thiamin-binding proteins that ubiquitously occur in plant seeds, that of common buckwheat became a model of extensive studies of the chemical mechanism of ligand-protein interaction. In this work, the polypeptide components of buckwheat seed thiamin-binding protein (BSTBP) are identified and characterized. We suggest that BSTBP is probably a fraction of major storage 13 S globulin (legumin), has an average molecular mass of 235 kDa and comprises hexamers of 57-kDa and 38-kDa subunits in variable combinations. Each subunit is a pair of disulfide-linked polypeptide chains, 36 kDa plus 24 kDa and two-times 22 kDa, respectively. The N-terminal sequences of 22-kDa and 24-kDa components show strict homology with those reported for basic subunits of buckwheat legumin. By photoaffinity labeling of BSTBP with 4-azido-2-nitrobenzoylthiamine, it is shown that the 36-kDa chain plays the major role in thiamin binding, but the other chains may also be variably involved. Putative thiamin-binding fragments are identified and sequenced.     

Primary structure and antiplatelet mechanism of a snake venom metalloproteinase, acurhagin, from Agkistrodon acutus venom

Acurhagin has been characterized as a P-III hemorrhagic metalloproteinase. We herein report the complete sequence of acurhagin by molecular cloning. Analysis of the cDNA-predicted amino acid sequence encoding acurhagin precursor revealed that this mosaic Asn-linked glycoprotein possesses a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domains (189/205/102/114 residues), with an overall 87% identity to that of jararhagin, an integrin alpha2beta1-cleaving metalloproteinase. Acurhagin has a Ser-Glu-Cys-Asp sequence in the disintegrin-like domain instead of the typical Arg-Gly-Asp motif. In contrast to inhibiting fibrinogen-integrin alphaIIbbeta3 interaction by disintegrins, acurhagin selectively showed a dose-dependent inhibition on platelet aggregation induced by collagen, and suppression on tyrosine phosphorylation of several signaling proteins in convulxin-stimulated platelets. Although the immobilized acurhagin was shown to bind platelet GPVI and collagen in a primary structure- and steric conformation-dependent manner, respectively, the mechanism of acurhagin under short incubation is mainly through its binding to GPVI and collagen, instead of binding to alpha2beta1, or cleaving platelet membrane glycoproteins. Moreover, the molecular conformation maintained by divalent cations is required for the proteolytic activity of acurhagin toward extracellular matrix fibronectin. Taken together, these results suggest that all the three domains in mature acurhagin may cooperatively contribute to its biological function.     

Mature Amaranthus hypochondriacus seeds contain non-processed 11S precursors

Amaranth is a dicotyledonous plant whose major seed storage proteins are globulins and glutelins. An unique feature of amaranth seeds is the presence of a fraction named albumin-2, that is extractable with water only after an exhaustive extraction of globulins and albumin-1. In this work, we tested the hypothesis that albumin-2 fraction could be constituted by a non-processed 11S globulin (proglobulin). To this end, the gene encoding the amaranth 11S subunit was cloned and expressed in Escherichia coli. Subsequently, the recombinant proglobulin and albumin-2 purified from seeds were treated with a sunflower vacuolar processing enzyme (VPE). A 55 kDa component of albumin-2 was specifically cleaved into 38 and 17-15 kDa polypeptides, as a consequence of this endoproteolytic cleavage a change of the oligomeric state from trimeric to hexameric was observed. Amaranth 11S globulin fraction was not modified under these proteolysis conditions. Using VPE-specific antibodies, it was shown that amaranth expresses a 57 kDa VPE, and that both developing and mature amaranth seeds have VPE activity, although the increase of this activity during amaranth seed development is higher than that observed for sunflower seeds. These results confirm the presence of unprocessed 11S precursors in mature amaranth seeds; this phenomenon cannot, however, be attributed to low VPE activity during developing of amaranth seeds.     

Etude de la globuline des graines de Tournesol (Helianthus annuus L.) accumulation au cours de la maturation et localisation intracellulaire

Protein synthesis and accumulation in growing sunflower (Helianthus annuus L.cv.Airelle) seeds are studied. The salt soluble fraction, globulin, is the main soluble protein component. The earlier stages of seed development (10 days after flowering) are characterized by high M_r polypeptides (74, 58 and 44 kDa). Later stages mainly show nature globulin polypeptides. Thus, protein synthesis in seed occurs at a specific period of seed development which follows a period of fast cell divisions (0–14 days after flowering). Protein bodies are isolated and their protein composition analyzed. Globulin subunits are the main polypeptides of protein bodies soluble fraction. Mature globulin is only stored in protein bodies.      

MAIGO2 is involved in exit of seed storage proteins from the endoplasmic reticulum in Arabidopsis thaliana

Seed storage proteins are synthesized on the endoplasmic reticulum (ER) as precursors and then transported to protein storage vacuoles, where they are processed into mature forms. Here, we isolated an Arabidopsis thaliana mutant, maigo2 (mag2), that accumulated the precursors of two major storage proteins, 2S albumin and 12S globulin, in dry seeds. mag2 seed cells contained many novel structures, with an electron-dense core that was composed of the precursor forms of 2S albumin. 12S globulins were segregated from 2S albumin and were localized in the matrix region of the structures together with the ER chaperones lumenal binding protein and protein disulfide isomerase, which were more abundant in mag2 seeds. The MAG2 gene was identified as At3g47700, and the MAG2 protein had a RINT-1/TIP20 domain in the C-terminal region. We found that some MAG2 molecules were peripherally associated with the ER membrane. MAG2 had an ability to bind to two ER-localized t-SNAREs (for target-soluble NSF [N-ethylmaleimide-sensitive fusion protein] attachment protein receptor; At Sec20 and At Ufe1). Our findings suggest that MAG2 functions in the transport of storage protein precursors between the ER and Golgi complex in plants.     

Biochemical and immunocytochemical localization of a BAPAase in developing and mature lupin cotyledons

Summary Activity measurements and specific antibodies were used to detect and localize in developing and mature cotyledons ofLupinus albus seeds an endopeptidase, active on BAPA, previously isolated from the same seeds. Total activity and enzyme amount were highest at full seed maturation and then declined during germination. Protein bodies were isolated from mature dry cotyledons under anhydrous conditions with a yield of intact organelles of about 80% as assessed by dot blotting with antibodies to lupin legumin-like storage globulin. Activity assays on the isolated protein bodies indicated that 72% of BAPAase activity was associated with these organelles. Quantitative immunocytolocalization with antibodies to the enzyme on thin sections of mature lupin cotyledons confirmed that 75% of the enzyme was located inside the protein bodies. The possible involvement of the BAPAase in the proteolytic processing of the storage proteins during seed ontogeny is discussed.Abbreviations BAPA N-benzoyl-L-arginine-4-p-nitroanilide - DAF days after flowering - EM electron microscopy - NaPi sodium phosphate buffer - LRW London resin white - SDS sodium dodecylsulphate - PAGE polyacrylamide gel electrophoresis