Storage-protein hydrolysis and protein-body breakdown in germinatedZea mays L. seeds

Torrent, Margarita; Geli, Maribel; Ludevid, M. Dolors

doi:10.1007/bf02411414

Cited by 23 publications

(14 citation statements)

References 26 publications

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“…In this case, however, there was no differential deg-radation of the 27-kD zein or the a-zeins, probably because the two types of protein were equally accessible to the proteases. This reinforces the idea that in cereals the hydrolysis of storage proteins occurs sequentially from the surface of the protein bodies (21).…”

Section: Resultssupporting

confidence: 84%

“…Using immunolocalization techniques, Torrent et al. (21) demonstrated that the 27 kD y-zein disappeared before the 19 and 22 kD a-zeins from protein bodies isolated from germinating seeds. This result is consistent with the observation that the 27 kD y-zein occupies a peripheral position in protein bodies (14)(15)(16), while the a-zeins make up the core of the deposit (14,15).…”

Section: Resultsmentioning

confidence: 99%

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

Barros¹,

Larkins²

1990

Plant Physiol.

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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.Storage proteins provide nitrogen, sulfur, and carbon skeletons for the developing embryo during seed germination. Degradation of these proteins in germinating seeds appears to follow a common pattern in many plant species (20). Hydrolysis of the proteins is initiated by the action of cysteine proteinases that cleave internal peptide bonds without causing extensive modifications. The first cysteine proteinase to act, designated proteinase A, is synthesized de novo. The limited hydrolysis by proteinase A generates sites for the action of proteinase B, another type of cysteine proteinase, which is inactive against the native substrate. The carboxyl-ends exposed by the concerted action of proteinases A and B are attacked by carboxypeptidases, and these are normally serine proteases. Finally, tri-and dipeptides are degraded into amino acids by the action of amino-and dipeptidases.Evidence in support of this model comes from angiosperm as well as gymnosperm species (20). Vetch ( Vicia sativa L.) was one of the first species in which the components of this model were identified, isolated, and characterized (19). In mung bean (Vigna radiata L.), the proteolytic enzymes in germinating seeds were also extensively characterized (4), and most of the components of this model are recognized. In cereals, the pathway of storage protein degradation is not as well defined as in dicotyledons. However, the participation of proteinase A followed by the action of carboxypeptidases has been observed in several cereal species (20). 297In maize (Zea mays L.), the storage proteins comprise a group of alcohol-soluble polypeptides called zeins (6). Zeins accumulate in the endosperm during seed development and make up about 50% of total endosperm proteins in mature seeds. They can be divided into subclasses based on solubility and apparent molecular weights: a-zeins, molecular masses 19 and 22 kD, are readily soluble in 70% ethanol; y-, molecular masses 16 and 27 kD, (-, molecular mass 14 kD, and 6-zeins, molecular mass 10 kD, require reducing agents to be solubilized in ethanol (13).Zein degradation begins around the second DAG3, and by 8 DAG most of the protein in the endosp...

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

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

Barros¹,

Larkins²

1990

Plant Physiol.

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“…As a rule, TN and PN quantities in the endosperm decrease and that decreasing is more strongly expressed at the interval 48-72 h. Calcium ions influence in this interval is also effective and lead to significant decreasing of TN and PN and to increasing of NPN compared with control seedlings. Undoubtedly this is due to the proteases activity which is elevated strongly on the second day from the beginning of germination (9,13). During gennination the NPN steadily increase and this increasing is very well expressed especially from 48 to 72 h. These results are in good agreement with the conclusions of some investigators assuming that these changes occur at the early stages of germination and then further progress (8,11).…”

Section: Resultssupporting

confidence: 81%

“…Hydrolysis of the proteins is initiated by the action of cysteine proteinases that cleave internal peptide bonds without causing extensive modifications (1). The storage proteins hydrolysis has been studied for a long time in germinating seeds isolated at different germination times, the starting points being the establishment of protein degradation and the detection of proteolytic activity in seeds (2,8,13). However some aspects of storage proteins degradation remains unclear up to now and it is difficult to provide a generalized scheme for these processes in germinating seeds.…”

Section: Introductionmentioning

confidence: 99%

Effect of Calcium Ions on Protein Composition in Germinating Maize Seeds

Stefanov

1998

Biotechnology & Biotechnological Equipment

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“…However, they have been detected by others in b-32 preparations from both normal kernels and pfoline-7 mutants and may reflect cleavage by endogenous proteases during purification Di Fonzo et al, 1988). Further support for a proteolytic activation of b-32 was found in the demonstration of increases in RIP activity ( Figure 6) coincident with the onset of protease synthesis and storage protein degradation during germination (Moureaux, 1979;Torrent et al, 1989;de Barros and Larkins, 1990;Hay et al, 1991).…”

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confidence: 70%

A maize ribosome-inactivating protein is controlled by the transcriptional activator Opaque-2.

et al. 1992

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Although synthesis of the cytosolic maize albumin b-32 had been shown to be controlled by the Opaque-2 regulatory locus, its function was unknown. We show here that b-32 is a member of the large and widely distributed class of toxic plant proteins with ribosome-inactivating activity. These ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that remove a single base from a conserved 28s rRNA loop required for elongation factor l u binding. Cell-free in vitro translation extracts were used to show that both maize and wheat ribosomes were resistant to molar excesses of b-32 but not to the dicotyledonous RIP gelonin. We extracted RIP activity from kernels during seed maturation and germination. The amount of RIP activity increased during germination, although the amount of b-32 protein remained fairly constant. Expression of a maize RIP gene under the control of an endosperm-specific transcriptional regulator may be an important clue prompting investigation of the biological basis for RIP expression in seeds of other plants. INTRODUCTIONRibosome-inactivating proteins (RIPs) are a widely distributed group of toxic plant proteins that catalytically inactivate eukaryotic ribosomes (for review, see Stirpe and Barbieri, 1986). RlPs function as N-glycosidases to remove a specific adenine in a conserved loop of the large rRNA . This irreversible modification renders the ribosome unable to bind elongation factor l a , thereby blocking translation. Because this translational inhibitory activity is toxic, RlPs have been tested extensively for use as immunotoxins and antiviral agents and more recently for their effects on protozoa, insects, and fungi (Barbieri and Stirpe, 1982;Cenini et al., 1988;Gatehouse et al., 1990;Leah et al., 1991).RIP activities have been found in the seed, root, leaf, or sap of more than 50 different plant species (Gasperi-Campani et al., 1985). Two forms of RlPs have been described (Stirpe and Barbieri, 1986). Type 1 RlPs such as pokeweed antiviral protein, trichosanthin, the barley translation inhibitor, and gelonin are monomeric enzymes, each with an approximate M, of 30,000 (Irvin, 1975;Stirpe et al., 1980;Asano et al., 1984;Maraganore et al., 1987;Yeung et al., 1988). Type 2 RlPs such as ricin, abrin, and modeccin are highly toxic heterodimeric proteins, each with an approximate M, of 60,000 in which one polypeptide with RIP activity (A-chain) is linked by a disulfide bridge to a galactose-binding lectin (B-chain; Pihl, 1973, 1982;Stirpe et al., 1978).Type 1 RlPs and the A-chain of type 2 RlPs have basic isoelectric points, and many have signal peptides that are not To whom correspondence should be addressed. present in the mature protein (Stirpe and Barbieri, 1986). Although RlPs share biological activity, they typically exhibit similarities of <50%, and antibodies raised against RlPs seldom cross-react with RlPs from distantly related species (Ready et al., 1988). The maize b-32 protein has homology with severa1 previously characterized RIPs, yet it is a singlechain acidic protein tha...

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Storage-protein hydrolysis and protein-body breakdown in germinatedZea mays L. seeds

Cited by 23 publications

References 26 publications

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

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

Effect of Calcium Ions on Protein Composition in Germinating Maize Seeds

A maize ribosome-inactivating protein is controlled by the transcriptional activator Opaque-2.

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