Metabolism of trypsin-inhibitory proteins in the germinating seeds of kidney bean (Phaseolus vulgaris)

Summary A number of proteins with trypsin-inhibitory activity was separated by isoelectric focusing and their amounts measured in the extracts of the seeds of kidney bean at various stages of germination up to 16 days.The total trypsin inhibitor content of the dormant seed, 2.2 mg per g bean rose to about 3.6 mg by the seventh day and declined slowly after the tenth day of germination. The individual trypsin inhibitors however, appeared to change independently of each other and some components disappeared almost completely with the progress of germination. The emergence of an inhibitor not found in the dormant seed was also observed. Some of the inhibitor proteins attained a maximum concentration by the 7–8th day of germination. This coincided with a similar maximum in the general protein and proteolytic enzyme content of the germinating bean seeds. The results obtained suggested that the main function during germination of these protein components might not be related to their trypsin-inhibitory activity.     

The toxicity of Jack bean (Canavalia ensiformis) cotyledon and seed coat proteins to the cowpea weevil (Callosobruchus maculatus)

The seeds of the Jack bean, Canavalia ensiformis (L) DC are known to contain several toxic substances that prevent their utilisation as food for humans and animals. The lectin concanavalin A and the enzyme urease are the best known of these proteins. We have found that many proteins present in the seeds of the Jack bean, like trypsin inhibitors and canatoxin, are detrimental to the development of the bruchid insect Callosobruchus maculatus (F) (Coleoptera: Bruchidae). Among these proteins, canavalin (vicilin, 7S globulin) was found to be expressed in the seed coat. We suggest that seed coat canavalin, in addition to other detrimental proteins expressed in this tissue, may have been of importance in the evolutionary discrimination of the seeds of this legume by non-pest bruchids.     

Amino Acid Sequence of Mung Bean Trypsin Inhibitor and Its Modified Forms Appearing during Germination

The amino acid sequence of the major trypsin inhibitor, F, of ungerminated mung beans (Vigna radiata [L.] Wilczek) was determined by a combination of automatic solid phase and manual sequencing techniques. F is a typical Bowman-Birk-type proteinase inhibitor with 80 amino acid residues and exhibits a high degree of identity with the other sequenced members of the Bowman-Birk family of inhibitors. Thin layer peptide maps of mung bean inhibitors E and C (which appear during germination) indicate that both are derived from inhibitor F by limited specific proteolysis. Loss of the carboxyl-terminal residues 77 to 80 from F produces inhibitor E, while the loss of an additional two carboxyl-terminal residues, the loss of the amino-terminal residues 1 to 8, and an internal cleavage at Ala³⁵-Asp³⁶ produces inhibitor C from E. Another inhibitor species, E′, was isolated from ungerminated seeds. It differs from F in the loss of residues 1 to 6. The majority of the proteolytic cleavages noted in the F-E-C-E′ system are at peptide bonds involving aspartyl residues.     

Isolation and Partial Characterization of a Subtilisin Inhibitor from the Mung Bean (Vigna radiata)

The subtilisin inhibitor (MBSI-A) from the mung bean (Vigna radiata (L.) Wilczek) seed has been purified to homogeneity. MBSI-A consists of a single polypeptide chain of 119 residues, with a high content of glutamic acid/glutamine, aspartic acid/asparagine, valine, threonine, and proline (19, 12, 10, 9, and 8 residue percent, respectively). MBSI-A is a potent inhibitor of subtilisin Carlsberg, but is inactive toward bovine trypsin and α-chymotrypsin and the plant cysteinyl proteinase papain. The MBSI is located exclusively in the cytosol of the seed cotyledon cell, unlike the mung bean trypsin inhibitor (MBTI), which is located primarily in the protein bodies. Both MBSI and MBTI accumulate in the seed during the most active period of reserve protein accumulation, 12 to 18 days after flowering. During germination MBSI, like MBTI, is broken down beginning 2 to 3 days after seed imbibition. The disappearance of MBSI-A is accompanied by the transient appearance of a new inhibitor species, MBSI-D. The amino acid composition of MBSI-D suggests that it may be produced by the loss of approximately 20 amino acid residues from MBSI-A.     

Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase

The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either α-mannosidase or N-acetyl-β-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.     

Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.     

Nature of proteinase inhibitors released from soybeans during imbibition and germination

Proteinase inhibitors are released to a smaller extent from soybeans which have been pre-equilibrated to an atmosphere of high relative humidity (r.h.) compared to those equilibrated to low r.h. Seeds pre-equilibrated to high r.h. also exhibited better germination. Regardless of the states of seed hydration, both Kunitz and Bowman-Birk soybean trypsin inhibitors are released in parallel with respect to each other and to other proteins at germination times up to 100 hr. After 48 hr of germination, new forms of trypsin inhibitor appear in the leachate which react with anti-Bowman-Birk trypsin inhibitor antibodies. No new forms of Kunitz trypsin inhibitor were observed immunochemically during the first 100 hr of germination.     

Endoplasmic Reticulum of Mung Bean Cotyledons: ACCUMULATION DURING SEED MATURATION AND CATABOLISM DURING SEEDLING GROWTH

Homogenates of mung bean cotyledons were subjected to equilibrium density centrifugation on linear sucrose gradients and the positions of the various organelles determined by assay of marker enzymes. Measurement of phospholipid distribution on such gradients showed that the major peak of phospholipid at a density of 1.11 to 1.13 grams per cubic centimeter coincided with the position of the endoplasmic reticulum (ER), confirming ultrastructural evidence that storage parenchyma cells are rich in ER. Germination and seedling growth were accompanied by a rapid decline in ER-associated phospholipid but a marked increase in the ER marker enzyme NADH cytochrome c reductase. Similar experiments with developing seeds indicated that the amount of ER-associated phospholipid increases during cotyledon expansion reaching a maximum during seed maturation. There was no subsequent decline during seed desiccation, instead ER-associated phospholipid levels were maintained in the dry seed until germination when catabolism was initiated 12 to 24 hours after the start of imbibition. This timing indicates that the observed ER breakdown is not an expression of the overall senescence of the cotyledons, but may represent the dismantling of the extensive rough ER used for reserve protein synthesis during cotyledon development.     

Mobilization of seed protein reserves

The mobilization of seed storage proteins upon seed imbibition and germination is a crucial process in the establishment of the seedling. Storage proteins fold compactly, presenting only a few vulnerable regions for initial proteolytic digestion. Evolutionarily related storage proteins have similar three-dimensional structure, and thus tend to be initially cleaved at similar sites. The initial cleavage makes possible subsequent rapid and extensive breakdown catalyzed by endo- and exopeptidases. The proteolytic enzymes that degrade the storage proteins during mobilization identified so far are mostly cysteine proteases, but also include serine, aspartic and metalloproteases. Plants often ensure early initiation of storage protein mobilization by depositing active proteases during seed maturation, in the very compartments where storage proteins are sequestered. Various means are used in such cases to prevent proteolytic attack until after imbibition of the seed with water. This constraint, however, is not always enforced as the dry seeds of some plant species contain proteolytic intermediates as a result of limited proteolysis of some storage proteins. Besides addressing fundamental questions in plant protein metabolism, studies of the mobilization of storage proteins will point out proteolytic events to avoid in large-scale production of cloned products in seeds. Conversely, proteolytic enzymes may be applied toward reduction of food allergens, many of which are seed storage proteins.     

A Bacterial Indole-3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean (Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery

Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver a bacterial IAA-Asp hydrolase into cells of mung bean (Vigna radiata) seeds and measured its effects on mung bean seed germination. IAA-Asp hydrolase inhibited seed germination about 12 h after the enzyme was delivered into cells of mung bean seeds both covalently and noncovalently. Mung bean seed germination was delayed by 36 h when the enzyme protein was noncovalently attached to the AID peptide and longer than 60 h when the enzyme protein was covalently attached to the AID peptide. Root elongation of mung bean plants was inhibited as much as 90% or 80%, respectively, when the IAA-Asp hydrolase was delivered with the AID peptide by covalent or noncovalent association. Further thin-layer chromatography analysis of plant extracts indicated that the levels of IAA increased about 12 h after treatment and reached their peak at 24 h. This result suggests that IAA-Asp hydrolase may increase IAA levels and inhibit seed germination of mung bean plants and that the AID peptide is a new, rapid, and efficient experimental tool to study the in vivo activity of enzymes of interest in plant cells.     

Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

Thiamine binding and metabolism in germinating seeds of selected cereals and legumes.

The basic characteristics of thiamine metabolism in germinating seeds of maize (Zea mays), oat (Avena sativa), faba bean (Vicia faba) and garden pea (Pisum sativum) are presented with a special emphasis of a possible thiamine storage function of seed thiamine-binding proteins (TBPs). Seeds were germinated for 6 d in the dark. Thiamine-binding activity in seeds decreased during germination by 50% in cereals and by 30% in legumes. The degradation of TBPs was also detected by polyacrylamide gel electrophoresis. The total thiamine content decreased rapidly to 20-40% of the initial value in cereal seeds during first 3 d of germination while in legume seeds thiamine content started changing from the fourth day and dropped by 50% at the sixth day. A composite pattern was found for the changes in thiamine pyrophosphate (TPP) contribution to total thiamine during seed germination. A peak of the coenzyme percentage was usually detected at the second day of germination. Another gain of TPP was often seen toward the sixth day of germination. The activity of thiamine pyrophosphokinase (EC 2.7.6.2) was high in resting legume seeds and did not significantly change during germination. In contrast, the low activity of this thiamine-activating enzyme in cereal seeds progressively increased during germination. Thiamine phosphate synthase (EC 2.5.1.3) was also detected in seeds and was shown to contribute significantly to the balance of thiamine compounds during seed germination.     

萌发绿豆子叶衰老期间蛋白质代谢的变化

萌发绿豆子叶自然衰老过程中可溶性蛋白质含量一直下降;从衰老开始到衰老前期,总游离氨基酸含量明显上升;但游离氨基酸各组分在子叶衰老期间的变化趋势并不相同。~3H-亮氨酸掺入蛋白质试验和多聚核糖体的相对量及其与总核糖体的比值(P/T)测定都证明在子叶衰老前期有蛋白质的新合成。子叶衰老期间。氨肽酶活性明显降低;而以酪蛋白为底物的蛋白水解酶活性却急剧上升,承担着催化蛋白质降解的主要功能。     

Isolation and function of a low molecular weight protein of mung bean embryonic axes

A low molecular weight protein from dry mung bean (Vigna radiata) embryonic axes has been purified to near homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. It shows a molecular weight of about 12,000 in sodium dodecyl sulfate-polyacrylamide gels and a sedimentation coefficient of about 2 S in sucrose gradients. This protein occurs in greater amounts in dry axes than in dry cotyledons, and it dramatically disappears during early germination of the seed. Affinity chromatography tests do not indicate it as a trypsin inhibitor or as a glycoprotein. It is a water-soluble cytoplasmic protein exhibiting an amino acid composition characteristic of storage proteins with a high content of glutamic acid/glutamine. We suggest that it is a low molecular weight storage albumin.Abbreviations Asx aspartic acid/asparagine - BSA bovine serum albumin - Con A concanavalin A - EB extraction buffer - Glx glutamic acid/glutamine - HA hydroxylapatite - PB phosphate buffer - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation

We utilized a proteomic approach to investigate seed development in Medicago truncatula, cv Jemalong, line J5 at specific stages of seed filling corresponding to the acquisition of germination capacity and protein deposition. One hundred twenty proteins differing in kinetics of appearance were subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry. These analyses provided peptide mass fingerprint data that identified 84 of them. Some of these proteins had previously been shown to accumulate during seed development in legumes (e.g. legumins, vicilins, convicilins, and lipoxygenases), confirming the validity of M. truncatula as a model for analysis of legume seed filling. The study also revealed proteins presumably involved in cell division during embryogenesis (beta-tubulin and annexin). Their abundance decreased before the accumulation of the major storage protein families, which itself occurs in a specific temporal order: vicilins (14 d after pollination [DAP]), legumins (16 DAP), and convicilins (18 DAP). Furthermore, the study showed an accumulation of enzymes of carbon metabolism (e.g. sucrose synthase, starch synthase) and of proteins involved in embryonic photosynthesis (e.g. chlorophyll a/b binding), which may play a role in providing cofactors for protein/lipid synthesis or for CO2 refixation during seed filling. Correlated with the reserve deposition phase was the accumulation of proteins associated with cell expansion (actin 7 and reversibly glycosylated polypeptide) and of components of the precursor accumulating vesicles, which give rise to a trypsin inhibitor on maturation. Finally, we revealed a differential accumulation of enzymes involved in methionine metabolism (S-adenosyl-methionine synthetase and S-adenosylhomo-cysteine hydrolase) and propose a role for these enzymes in the transition from a highly active to a quiescent state during seed development.     

滇重楼种子水浸液对三种植物种子萌发和幼苗生长的影响

利用滇重楼(Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz.)种子外种皮和胚乳的水浸液对白菜(Brassica pekinensis(Lour.)Rupr.)、绿豆(Vigna radiata(Linn.)Wilczak)、小麦(Triticum aestivum L.)种子进行处理,研究滇重楼种子水浸液对3种植物种子萌发、幼苗生长和保护酶活性的影响,并利用GC-MS方法对滇重楼种子内源抑制物的成分进行分析。结果显示,不同浓度滇重楼外种皮、胚乳水浸液对上述3种受体植物的发芽率、苗高、根长及鲜重均产生影响,其作用强度和水浸液的浓度有关,总体上表现出低促高抑的双重浓度效应。滇重楼种子水浸液对白菜的影响作用最强,对绿豆的影响作用最弱,且胚乳水浸液的影响较外种皮强。不同浓度滇重楼种子外种皮和胚乳水浸液均能影响3种植物幼苗体内保护酶的活性,随着水浸液浓度的升高,叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)活性总体增加,与对照相比差异显著。白菜、小麦过氧化氢酶(CAT)活性减少,与对照相比差异显著;绿豆过氧化氢酶(CAT)活性增加,但与对照相比无显著差异。利用GC-MS方法从胚乳和外种皮水浸液中分别检出8种和2种物质。研究结果表明滇重楼种子中存在内源抑制物质,可能是导致种子休眠的原因;种子水浸液可能通过影响植物幼苗保护酶的活性进而影响其正常生长;有机酸类物质可能是滇重楼种子内源抑制物之一。     

Immunochemical identification of mung bean actin like protein and its cellular involvement during germination

Actin like protein, extracted and purified fromVigna radiata (mung bean) seedling, has been found to give positive enzyme-linked immunosorbent assay with mouse monoclonal antiactin antibody. In vivo studies show that cytochalasin B at sublethal dose inhibits the chromosomal movement at metaphase stage during germination. Fromin vitro studies it is found that the actin like protein isolated from mung bean seedling has a cytochalasin B binding property with a Kd value 1.2 × 10⁻⁵ M. From these two specific observations it appears probable that the biological function of mung bean actin like protein is to take part in cell division process directly or indirectly during the time of seedling development.     

Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata)

The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl₂. It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4°C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.     

Isolation and cDNA cloning of an Em-like protein from mung bean (Vigna radiata) axes

Until now no 'early-methionine-labelled' (Em) proteins have been reported in the Fabaceae. To check whether a previously isolated low-molecular mass albumin from dry mung bean embryonic axes possibly corresponded to an Em-like protein, the protein was purified, sequenced and its cDNA clone isolated and characterized. N-terminal sequencing of cyanogen bromide cleavage products of the protein revealed homology with previously described Em-like proteins from other species. Analysis of cDNA clones encoding the mung bean Em protein revealed the presence of two classes of Em proteins and confirmed their homology to the previously characterized Em-like proteins. In vivo labelling and northern blot analysis further demonstrated that the mung bean protein is synthesized during early germination of the axes and that abscisic acid (ABA) extends its synthesis. It appears, therefore, that legumes also contain maturation-specific, ABA-responsive Em-like proteins.