Degradation Of Storage Proteins Research Articles

Water Stress and Storage-protein Degradation during Germination of Impatiens Seed

Water uptake by impatiens (Impatiens wallerana Hook. f. cv. Super Elfin Coral) seeds was measured as an increase in fresh weight every 24 hours during 144 hours of germination. Seeds absorbed most of the water required for germination within 3 hours of imbibition and germinated at 60% to 67% moisture on a dry-weight basis. Germination started at 48 hours and was complete by 96 hours at 25C. Water stress of -0.1, -0.2, -0.4, and -0.6 MPa, induced by polyethylene glycol 8000, reduced germination by 13%, 49%, 91%, and 100%, respectively, at 96 hours. Under the same water-stress conditions, increases in fresh weight were inhibited by 53%, 89%, 107%, and 106%, respectively. Three distinct groups of storage proteins were present in dry seed; their estimated molecular weights were 1) 35, 33, and 31 kDa; 2) 26, 23, and 21 kDa; and 3) two bands <14 kDa. Major depletion of storage proteins coincided with the completion of germination. Water potentials that inhibited germination also inhibited degradation of storage proteins. During germination under optimum conditions, the soluble protein fraction increased, coinciding with a decrease in the insoluble fraction.

Purification and Characterization of Zein-Degrading Proteases from Endosperm of Germinating Maize Seeds

We have purified a group of four proteases (molecular mass 26-33 kilodalton) from germinating seeds of maize (Zea mays L. var W64A) using ammonium sulfate and isoelectric precipitations, anion exchange chromatography, and electroelution from preparative nondenaturing polyacrylamide gels. Their appearance in the endosperm of germinating seeds coincides with the onset of zein degradation. We have shown that these proteases degrade zeins dissolved in alcoholic solution as well as aggregated in protein bodies from developing maize kernels. The apparent molecular weights and net negative charges of each of these proteases are very similar. Additionally, they are inhibited by thiol-blocking agents and activated by reducing compounds. These characteristics suggest that they are a group of cysteine proteases involved in the first steps of storage protein degradation.

Purification and some properties of an aminopeptidase from endosperms of ×Haynaldoticum sardoum Meletti et Onnis.

Aminopeptidase activity was detected in dry and germinating seeds of x Haynaldoticum sardoum. The enzyme was partly purified from germinating seeds and preliminary characterization was carried out. The aminopeptidase showed a pH optimum of 8.0, a molecular weight estimated at 89 000 by Sephadex G-100 gel filtration and an isoelectric point of 4.5. The enzyme hydrolysed p-nitroanilides, but was unable to break down exogenous native proteins. Activity was severely inhibited by p-chloromercuribenzoate, p-hydroxy-mercuribenzoate and bestatin, slightly inhibited by iodoacetamide and ethylenediaminetetraacetic acid and not influenced by 2-mercaptoethanol, phenylmethanesulphonyl fluoride and polyamines. A possible physiological role for the enzyme in the final stages of storage protein degradation is suggested.

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 (Mr) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a Mr 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.

Morphometric analysis of reserve substances and ultrastructural changes during caulogenic determination and loss of competence of Eastern White pine (Pinus strobus) cotyledons in vitro

Lipid, starch, and protein reserves were monitored cytochemically and, along with certain ultrastructural changes, were quantified morphometrically during adventitious shoot determination or loss of competence in cotyledons from cultured Pinus strobus zygotic embryos. Initial expiants were rich in lipid, which declined during culture. There was more lipid retention in explants on cytokinin medium by day 5 than on basal medium. However, by day 7, five- and six-celled clusters showed greater lipid utilization than most other cells on cytokinin, which may have indicated a shift towards shoot determination in these cells. Except for an initially greater retention on cytokinin, a similar pattern for storage protein degradation was observed in both treatments. Starch levels increased during culture, but did not differ between treatments during the first 7 days. Vacuolation increased during culture and was greater on basal medium than on cytokinin. Relative nuclear size also increased, but was greater on cytokinin. Relative mitochondrial area increased during culture, except in noncluster cells on basal medium. While few differences were noted between cell types within each treatment, distinct differences in nuclear size, vacuolation, and lipid content existed between cells on cytokinin and basal medium. These coincided with the timing of caulogenic determination in cytokinin-treated expiants. The loss of lipid reserves, as well as ultrastructural changes associated with the maturation of cells on basal medium, was associated with the loss of competence.

Properties, synthesis, and accumulation of storage proteins in Pieris rapae L.

AbstractTwo kinds of storage proteins (SP‐1, SP‐2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP‐1 and SP‐2 dropped after pupation as the proteins were being deposited in fat bodies. SP‐2 is present in a larger amount than SP‐1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body.SP‐1 has a molecular weight of 500,000 and consists of one type of subunit (Mr 77,000), while SP‐2 has a molecular weight of 460,000 and is composed of two types of subunits (Mr 80,000 and 69,000). The pl values of SP‐1 and SP‐2 were determined to be 6.97 and 7.06, respectively.Fat body cells from 1‐day‐old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage.Morphological changes in the fat body tissues were observed during the larval‐pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.

Formation and Degradation of Storage Proteins in the Embryo of Sinapis alba

The synthesis and accumulation of storage proteins of Sinapis alba was shown in a continuous line of SDS gels during the development of embryos up to maturation. The synthesis for the large storage protein (13 s) starts at day 14 after pollination and stops 34 days after pollination. The small protein (1.7s) is synthesized up to the full maturation of the seeds. The phase of storage protein synthesis is not coordinated with the change in water status in the developing seeds or embryos. Freshly isolated embryos can germinate but are able to continue storage protein synthesis for at least 10 hours after isolation when they are younger than 35 days. This synthesis can be stimulated slightly by treatment with 1μM ABA. Embryos older than 40 days do not synthesize the large storage protein, and the synthesis can not be induced with 1μM ABA under the conditions of germination. This external ABA concentration, however, delays the degradation of storage proteins during germination by about 2 days in mature and 4 days in immature embryos. The appearance of the pattern of the newly formed «germination proteins» is retarded by the same time.

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7-10 d following seed germination. We assayed cotyledon extracts for protease activity by using [(3)H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [(3)H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M r=30 000). Optimum hydrolysis of [(3)H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [(3)H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.

Degradation of storage proteins in germinating seeds

The criteria for the participation of proteases in the mobilization of storage proteins during seed germination are formulated. The proteases that satisfy these criteria, namely the acid cysteine endopeptidases, serine carboxypeptidases and neutral aminopeptidases, are reviewed. The possibility of other seed proteases participating in storage protein degradation is discussed. The course of 11S and 7S storage protein degradation through the action of endogenous and exogenous proteases is described. The 11S and 7S proteins are modified during the early stages of proteolysis and the effects of these modifications on the regulation of breakdown are examined. A scheme for 11S protein degradation in germinating seeds is presented.

AXIAL CONTROL OF AMYLASE SYNTHESIS AND SECRETION IN COTYLEDONS OF AGROSTEMMA GITHAGO

SUMMARY In Agrostemma githago (Caryophyllaceae) starch reserves are located outside the embryo in the perisperm. One of the main degradative enzymes, α-amylase, is synthesized in and secreted by the cotyledons. The synthesis and secretion of amylase were shown to be under axial control. Benzylaminopurine (BAP)1 or gibberellic acid (GA3) replaced axis requirement. BAP probably acted by a general enhancement of metabolism, whereas GA3 had, like the axis, a specific effect on the cotyledons. The degradation of storage proteins in the cotyledonary tissue was controlled in a similar way. Two arguments raised in the literature against hormonal regulation of reserve breakdown in dicots are 1) that the axis withdraws otherwise inhibitory hydrolysis products from the cotyledons and 2) that reduced activity of hydrolytic enzymes in detached cotyledons is an artefact due to lack of oxygen. These arguments were shown to be most likely invalid.

Hormonal Regulation of the Development of Protease and Carboxypeptidase Activities in Barley Aleurone Layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA(3)) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA(3). In contrast, GA(3) is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA(3) is similar to that observed for alpha-amylase. Treating aleurone layers with both GA(3) and abscisic acid prevents all the GA(3) effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA(3) response, it is concluded that the proteases represent the GA(3)-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.

Precociously germinating rapeseed embryos retain characteristics of embryogeny

We compared the germination of Brassica napus L. embryos at three stages of development-mid-cotyledon, maturation and mature dry-to determine at which stage they acquired the capacity for normal germination and seedling development. Embryos were removed from the seed and cultured on hormone-free medium, allowing them to germinate. The transition from embryogeny to germination was monitored both morphologically and biochemically, using synthesis of 12 S storage protein as a marker of embryogeny. The mature embryos (dry seeds) set the standard for normal seedling development: radicle emergence, hypocotyl extension and cotyledon expansion occurred within 2 d and true leaves were formed within a week of germination. Rocket immunoelectrophoresis indicated that the storage proteins in seedlings from mature dry embryos were completely degraded within a week. In contrast, the midcotyledon-stage embryos appeared to germinate abnormally, retaining many embryonic characteristics. Although the roots emerged, the hypocotyls did not elongate and secondary cotyledons instead of leaves were formed at the shoot apex. Also, the seedlings continued to synthesize and accumulate storage proteins. The maturation-stage embryos did develop into normal-looking seedlings, but complete degradation of storage proteins required several weeks, presumably reflecting continued synthesis and turnover. We conclude that embryogenic and germination-specific processes can occur concurrently and that the capacity to develop as normal seedlings is acquired gradually during the maturation process.

Biosynthesis and degradation of storage protein in spores of the fungus Botryodiplodia theobromae.

Muiridin, a spore-specific protein of the fungus Botryodiplodia theobromae, comprises about 25% of the mature pycnidiospore protein. It has an apparent molecular weight of 16,000 to 17,000 and is rich in glutamine, asparagine, and arginine. Muiridin is synthesized in developing spores via a precursor with an apparent molecular weight of 24,000. Two other polypeptides present in young developing spores with apparent molecular weights of 18,000 and 15,000 are immunologically related to muiridin. We propose a pathway for muiridin synthesis. Muiridin is actively degraded during the germination of spores from 30-day-old cultures. This degradation is independent of exogenous amino acids in the germination medium. In contrast, glutamine and, to a lesser extent, asparagine partially inhibit the degradation of muiridin during germination of spores from 7-day-old cultures.

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein content of pea seeds ( Pisum sativum L.) as compared to soya bean seeds {Glycine max L. Merrill) might be due to faster degradation of 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-life of 39 days. There was no measureable degradation of soya bean storage proteins. Even with the pea storage proteins, the half-lives were so much longer than the maturation time of seeds that degradation of storage proteins could not account for the lower protein content of peas as compared to soya beans. The validity of these results was indicated by the finding that non-storage proteins had much shorter half-lives and that omission of a carbon or a nitrogen source greatly accelerated degradation. Labelled glycine was found to be a good probe for protein turnover studies because it was very rapidly metabolized.

Phytochrome-mediated accumulation of free amino acids in embryonic axes of New York lettuce seeds.

Red light given to New York lettuce seeds incubated on a 0.3 m mannitol solution increased the amount of free amino acids in the embryonic axes, far-red light reversed this effect. Germination in response to red light given at various times during imbibition was closely related to the amount of free amino acids accumulated in the axes in response to the red light. The analyses of the free amino acids that accumulated in the axes of seeds incubated on 0.3 m mannitol indicated that the free amino acids that accumulated on red-light irradiation were produced by the degradation of storage protein. This is evidence that red-light-increased germination may be caused by the accumulation of free amino acids in the embryonic axes of New York lettuce seeds.

Protein Synthesis and Degradation in Anabaena During Nitrogen Starvation

SUMMARY: Nitrogen starvation and heterocyst development were induced in the cyanobacterium Anabaena 7120 by growth in nitrogen-free medium or by treatment with the amino acid analogue methionine sulphoximine. During the first 6 h of nitrogen deprivation, amino acid levels and rates of protein synthesis, as measured by the incorporation of [3H]leucine, decreased to 50 to 70% of those in ammonia-grown organisms; after this time there was no difference between the rates of protein synthesis in ammonia-grown and nitrogen-starved cultures. The period 4 to 12 h after the onset of starvation was marked by the release of [3H]leucine from previously labelled proteins at a rate 6 to 7·5 times that of ammonia-grown organisms. These results are consistent with the idea that nitrogen starvation in cyanobacteria causes a reduction in protein synthesis and leads to the rapid degradation of storage proteins. In rapidly growing Anabaena 7120, the doubling time for total cell protein was estimated to be 14·9 ± 1·0 h and the half-life was 139 ± 88 h.

Protein Degradation and Proteolytic Activities in Germinating Field Beans (Vicia faba L., var. minor)

Protein degradation in germinating seeds of field beans ( Vicia faba L., var. minor) was investigated biochemically and by electron microscopy. Rapid nitrogen transfer from cotyledons into the germ starts at the 7th day of germination simultaneously with the beginning of rapid protein degradation. Decrease of nitrogen in the cotyledons and increase in the germ, respectively, accompany corresponding changes in the dry weights of these organs. Legumin (11 S storage protein) degradation commences between 7th and 8th day previous to the breakdown of vicilin (7 S storage protein). On acid polyacrylamide gels no changes in amount and mobility of vicilin and legumin were observed before the 9th and 7th days respectively, whereas under denaturing conditions degradation products were already visible at earlier germination stages. It indicates hidden endoproteolytic cleavages. In immunological as well as in basic electrophoresis mobility changes were observed previous to the rapid degradation of the respective proteins. Amido-N of the proteins does not change parallel to the mobility shifts. Two peaks of acid endopeptidase activity were measured during germination, 1 at the 3th and 2 at the 9th day, the latter being much more pronounced than the former. BAPAase 1) activity starts to increase at the 7th day. Changes in the electron density of protein bodies were already visible previous to the onset of rapid protein degradation in the cotyledons, but the translucent areas appearing inside the protein bodies from the 4th day on may also be interpreted in terms of preparation artefacts. The results are in accordance with a two-step hypothesis of storage protein breakdown in field bean cotyledons.

Density labeling of proteins with 13C-labeled amino acids : No accumulation of an inactive α-amylase precursor in barley aleurone cells

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus 13C-labeled amino acids and [ 3H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [ 3H]leucine was negligible in comparison to that of 13C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of 13C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with 13C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with 13C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.

Proteolytic enzyme activity and storage protein degradation in cotyledons of germinating peanut (Arachis hypogaea L.) seeds.

Proteolytic enzyme activity and storage protein degradation in cotyledons of germinating peanut (Arachis hypogaea L.) seeds.

Histochemical and Biochemical Observations on Storage Protein Metabolism and Protein Body Autolysis in Cotyledons of Germinating Mung Beans

Storage protein hydrolysis in the cotyledons of germinating mung beans (Phaseolus aureus Roxb.) was examined by histochemical techniques, and the autolytic capacity of isolated protein bodies was studied with biochemical methods. The localization of endopeptidase activity within the cotyledons was studied using an India ink-gelatin film technique. After 24 hours of imbibition, a low level of endopeptidase activity was found throughout the storage tissues of the cotyledons. A marked increase in activity was noted in cells farthest from the vascular bundles 48 to 60 hours after the start of imbibition. The decrease in storage protein followed the same spatial distribution starting in the cells farthest from the bundles. The cotyledons contain a population of cells in various stages of endopeptidase activity enhancement and storage protein degradation. A wave of endopeptidase activity moves progressively through the cotyledons towards the vascular bundles leaving behind areas devoid of stored reserves and low in endopeptidase activity. Observations on the morphology of protein bodies during germination indicate that the membrane surrounding them remains intact, while the reserves disappear. This result suggests that the protein bodies may be undergoing autolysis. To determine whether this may indeed be the case, protein bodies were isolated from the meal of mung bean seeds using an aqueous medium containing 80% glycerol. The protein body preparations and the cytoplasm were assayed for the presence of a number of enzymes which may be involved in the breakdown of the storage proteins. The protein bodies contained all, or nearly all, of the carboxypeptidase, alpha-mannosidase, N-acetyl-beta-glucosaminidase, and caseolytic activity. The cytoplasm contained all, or most, of the leucine aminopeptidase and the trypsin-like activity (benzoyl arginine-p-nitroanalide as substrate). Incubation of the isolated protein bodies resulted in the release of amino acids. An analysis of the products of hydrolysis indicated that very little, if any, storage protein was being hydrolyzed during the incubation. Hydrolysis of the storage proteins present in the protein bodies was greatly accelerated by the addition of extracts from the cotyledons of 4-day-old seedlings. The results suggest that new enzymic activities not present in the protein bodies isolated from dry seeds must either be activated or synthesized and possibly added to the protein bodies before storage protein breakdown can begin.