The zein proteins of maize endosperm

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Reviews The zein proteins of maize endosperm Brian A. Larkins, Karl Pedersen, M. David Marks and Deborah R. Wilson The storage protein in maize seed is composed of a group of alcohol-soluble polypeptides called zeins. These proteins are synthesized and deposited in the endosperm during seed development and are metabolized during seed germination. The proteins in seeds have been of interest to biochemists for many years and were among the first proteins to be isolated and characterized. In cereals, such as maize, proteins comprise approximately 10% of the seed's dry weight, and most of this is made up of a fraction called 'storage protein'. There is no clear definition of what constitutes a 'storage protein', but there is general agreement that it: (1) has no enzymatic activity, (2) provides nitrogen for the germinating seedling, (3) usually accumulates in aggregates called protein bodies, (4) is often composed of a related group of polypeptides. In maize and other cereals (including wheat and barley) the storage proteins are unusual in that they are soluble in alcoholic solutions. This was first demonstrated by T . B . Osborn I who also showed that these proteins have a high content of proline and glutamine. For this reason he called them 'prolamins'. Subsequently, the prolamin from maize (Zea mays L.) was given the trivial name 'zein'; the prolamin from barley (Hordeum vulgare L.) was given the trivial name 'hordein' etc.

tion with alcohol and a reducing agent removes zein-2 which contains Mr 22 030 and Mr 19 000 polypeptides as well as some Mr 15 000 and Mr 10 000 polypeptides. Another component of the zein-2 fraction is a larger protein of around Mr 27 000 which is called 'water soluble alcohol-soluble glutelin' or 'reduced soluble (RS) protein '2. It can readily be separated from the other proteins because of its solubility in water or dilute salt solution. The Mr 22 000 and Mr 19 000 zeins have similar amino acid compositions. Glutamine (20%), leucine (20%), alanine (14%), proline (9%) and serine (7%) are the most prevalent amino acids and there is little or no lysine or tryptophan 2. The amino acid composition of the Mr 15 000 and Mr 10 000 proteins is similar; however, these have a higher content of methionine, cysteine, tyrosine and glyA

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cine. The RS protein differs from the others by having a higher proline content (25%); it is also high in histidine, cysteine, glycine and valine, and lower in glutamine and leucine. The absence of iysine and tryptophan as well as the high leucine/isoleucine ratio in zein is responsible for the poor nutritional quality of these proteins 3. Analysis of the Mr 22 000 and Mr 19 000 zeins by isoelectric focusing reveals a number of differently charged species; however, the Mr 15 000, Mr 10 000, and the RS-protein contain only one or two forms (Fig. 1B). The complexity of the Mr 22 000 and M r 19 000 proteins is not caused by deamidation. It has been shown to be genotype specific and to be inherited in a single Mendelian fashion4. Zein synthesis is initiated in the endosperm of the seed approximately 12 days after pollination, and continues until maturity which is around 50 days after pollination. Messenger RNAs that direct zein synthesis are localized on membrane bound polyribosomes. The primary translation products have 'signal peptides' which are cleaved as the proteins enter the lumen of the rough endoplasmic reticulum (RER) 5'6. Once inside the RER, the polypeptides associate to form dense insoluble masses called protein bodies. This process appears to be protein dependent as it can also occur in Xenopus laevis oocytes injected with zein mRNAs 7.

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Characterization and synthesis of zein proteins 27 000Zein proteins are typically extracted 22 000~ t from maize seeds with 70% ethanol or 1900091[D,~lmllb 55% isopropanol in the presence or absence of a reducing agent such as 2mercaptoethanol. The protein fraction 15 0 0 0 extracted by alcohol alone is called zein10 0001 (Ref. 2), and it consists of a mixture of polypeptides with apparent Mr of 22 0130 and 19 000 (Fig. 1A). Sequential extrac- Fig. 1. Analysis of maize zein proteins by SDS polyacrylamide gel electrophoresis. Panel A shows a oneBrian A. Larkins, Karl Pedersen, M. David Marks and Deborah R. Wilson are at the Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA.

dimensional separation of proteins extracted from mature endosperm with 70% ethanol (lane 1) or 70% ethanol plus 1% 2-mercaptoethanol (lane 2). Apparent tool. wts are indicated on the left. Panel B shows a two-dimensional separation of zein proteins from protein bodies of developing endosperm; proteins extracted with 70% ethanol containing 1% 2-mercaptoethanol. The non-equilibrium IEF gradient between p H 9.6 and 4.0 is le]~ to right; apparent tool. wts correspond to values lef~ of panel A.

1984, Elsevier Science Publishers B V., Anmterdara 0376 - 5067/84/$02.01)

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Zein proteins appear to have a highly ordered structure The insolubility of zein proteins makes them difficult to characterize, and until recently little was known except their apparent size, amino acid composition, and NH2-terminal sequence. However, analyses of full-length cDNA clones of zein mRNAs have revealed their primary amino acid sequence as well as some of their structural features 8"9. Zein preproteins have a 'signal peptide' of 21 amino acids. The size of the mature proteins is nearly Mr 5 000 larger than expected, based on their mobility on SDS polyacrylamide gels. Although the reason for this is unclear, it may be related to their structure. Both the Mr 22 000 and Mr 19 000 zeins contain a conserved peptide of 20 amino acids that is tandemly repeated nine times in the central part of the protein. These repeats vary slightly in their amino acid sequence, but they can be averaged to obtain a consensus repeated sequence. Analysis of this sequence on an a-helical wheel shows that polar amino acids are distributed in three symmetrical sites (Fig. 2A) 1°. If the repeated peptides are folded back on one another in an antiparallel fashion, two of the three polar groups can form hydrogen bonds with adjacent helices (Fig. 2B). The nine repeats could interact to form a roughly cylindrical molecule which would be somewhat collapsed to accommodate the non-polar side chains of the amino acids. Aggregation of the polypeptides within the RER would be facilitated by the third polar group which is exposed on the surface of the peptide. The clustering of glutamine residues at the ends of the repeats would also contribute to packing interactions within the protein body (Fig. 2C). Although this is a hypothetical model for the structure of these proteins, circular dichroism measurements of mixtures of zein polypeptides indicate an a-helical content consistent with these structural predictions. Furthermore, electron microscopy of zein proteins indicate they are rod-shaped molecules, which is also consistent with these structural predictions. A sequence analysis of a genomic clone for the Mr 15 000 zein has recently been completed. Although the amino acid composition of this protein is similar to that of the Mr 22 000 and Mr 19 000 zeins, it does not contain the same repeated peptide. The protein has some a-helical structure, but its conformation is uncertain. A clone corresponding to the RS-protein has not yet been identi-

POLAR

LEU 8

pRO4

TYR16

ALAli

LEU 3

~4_A~0

LEU~7

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C

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Fig. 2. Structural model for the Mr 22 000 and M, 19 000 maize zein proteins. (A) Analysis of consensus repeat on an a-helical wheel. (B) Organization of nine repeated a-helices in a cross-section view of a protein molecule. The numbers 1, 2 and 3 on each repeat indicate the positions of the three polar regions. 'Up' and "Down' indicate the antiparallel orientation of the repeated peptides. (C) Model for arrangement of zein proteins within the protein body. "Q' indicates the positions of glutamine residues at the ends of the repeated peptides. (From Argos et al. (1982).)

fled; however, the protein is structurally distinct from the other alcohol-sohible polypeptides. NHz-terminal sequence analysis of the RS protein showed a tandemly repeated hexapeptide (ProPro-Pro-Val-His-Leu) which occurs at least eight times following the first 11 amino acids 11. Thus, the Mr 15 000 and Mr 27 000 proteins appear to be structurally distinct from the Mr 22 000 and Mr 19 000 zeins. Zein gene structure, organization, and expression Zein cDNA clones hybridize to multiple restriction enzyme fragments of maize nuclear DNA, indicating that the genes occur in multigene families12"13. There are discrepancies regarding the total number of zein genes in the maize

genome which result in part from the methods used for analysis and differences in hybridization criteria 13. We determined that the Mr 22 000 zeins constitute a highly homologous family (approximately 90% sequence homology) with approximately 25 members. There are at least two subfamilies of Mr 19 000 sequences with a combined total of 55 members. We detected only one or two genes for the Mr 15 000 protein. Although the hybridization pattern differs somewhat in DNA restriction enzyme digests of the related maize grasses Tripsacum dacteloides and Teosinte sp., zein genes also occur in multigene families in these species 13. Their seeds contain alcohol-soluble proteins that migrate on SDS polyacrylamide gels similar to proteins from modem inbreds; however, clones of zein

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genes have not been isolated, so we do 19 000 zeins. The opaque-2 and opaque- accumulates in significant amounts in not know their structural similarities. 7 mutations appear to act through differ- the normal genotype, but its function Analyses of a number of different zein ent mechanisms, since in double mutant and subceUular localization are unknown. combination their effects are additive 19. genomic clones by R-looping and D N A Much remains to be learned about sequencing have shown that the genes Interestingly, the opaque-6 mutation is how these mutations alter the synthesis do not contain intervening sequences 9'14. lethal in the homozygous state 2°. of zein proteins. Additional research The flanking regions contain typical The floury-2 mutation appears to act may eventually help to explain how the through a different mechanism than the expression of this large multigene family ' C C A T ' ' T A T A ' and ' A A T A A A ' sequences which are presumably in- opaque mutations. Floury-2, which is on is regulated. volved in transcription initiation and the short a n n of chromosome 4, affects polyadenylation. Feix and co-workersl 5,16 zein synthesis in a dosage dependent Acknowledgement Our research is supported by grants found evidence for the transcription of manner, i.e. zein synthesis is progreszein m R N A precursors. In Northern sively reduced as the number of floury-2 from the National Science Foundation. hybridization blots of total endosperm genes is increased. Other mutants, such References R N A they detected RNAs of 900, 1 800, as floury-1 and floury-3, show a similar 10sborn, T. B. (1908) Science 28, 417-427 2 800, and 3 800 nucleotides. The R N A dosage effect; seeds homozygous for 2 Wilson, C. M. (1983) in Seed Proteins: Bioof 900 bases corresponds to the m a t u r e floury-3 will not germinate z°. In the chemistry, Genetics, NutritiveValue(Gottschalk, W. and Muller, H. P., eds), pp. 271-307, mRNA. The larger RNAs, which account homozygous condition floury-2 causes Martinus Nijhoff/Junk for a significant portion of the total about a 40-45% reduction in zein syn3 Nelson, O. E. (1969) Advances in Agronomy transcripts, are presumed to be pre- thesis. In contrast to opaque-2 and 21,171-194 cursors. Based on R-loop analysis 15 and opaque-7, all zein polypeptides are re4 Righetti, P. G., Gianazza, E., Viotti, A. and in vitro transcription ~6 these precursors duced to a similar extent in the floury-2 Soave, C. (1977) Planta 136, 115--123 arise from promotor sites that are 1 000 mutant. Double mutant combinations 5 Larkins, B. A. and Hurkman, W. J. (1978) nucleotides or more beyond the 5' end with either opaque-2 or opaque-7 are Plant Physiol. 62, 256-263 6 Burr, A., Burr, F. A., Rubenstein, I. and of the gene. A n interesting feature of epistatic to floury-2 (Ref. 19). Simon, M. N. (1978) Proc. Natl Acad. Sci. these transcripts is that they vary in size The defective endosperm-B 30 (De-B USA 75, 696-700 during endosperm development 17. 30) mutation is dominant and causes a 7 Hurkman, W. J., Smith, L. D., Richter, J. and 30% reduction in zein synthesis. De-B Larkins, B. A. (1981)J. CellBiol. 89, 292-299 30 is closely linked with the opaque-2 Mutations differentially affect the 8 Geraghty, D., Peifer, M. A., Rubenstein, I. expression of zein gene families locus, and like opaque-2 it causes a and Messing, J. (1981) Nucleic Acids Res. 9, 5163-5173 Many different mutations affect zein significant reduction in the synthesis of 9 Pedersen, K., Devereux, J., Wilson, D. R., synthesis. Two of these, opaque-2 and the Mr 22 000 zeins. Sheldon, E. and Larkins, B. A. (1982) Cell29, From genetic linkage analyses Carlo floury-2, were identified during a search 1015-1026 to find more nutritional genotypes 3. Soave and Francesco Salamini and their 10 Argos, P., Pedersen, K., Marks, M. D. and Since zein accounts for 50% of the total colleagues have shown that zein genes Larkins, B. A. (1982) J. Biol. Chem. 257, endosperm protein and contains little or occur primarily on chromosomes 4 and 9984-9990 no lysine and tryptophan, these amino 7 (Ref. 18). Genes encoding the M r 11 Esen, A., Bietz, J. A., Paulis, J. W, and Wall, J. S. (1982) Nature 296, 678--679 acids are limiting when maize is used as 19 000 zein are mainly on chromosome the principal source of protein in the 7 and are linked with the opaque-2 locus 12 Hagen, G. and Rubenstein, I. (1981) Gene 13, 239-249 diets of monogastric animals. and the De-B 30 locus. Genes encoding 13 Wilson, D. R. and Larkins, B. A. Z MoL The mutations that affect zein syn- the M r 22 000 zeins are on chromosome Evol. (in press) thesis are either recessive, semidominant 4 and are linked with the floury-2 locus. 14 Wienand, U., Langridge, P. and Feix, G. or dominant is. The opaque-2 mutation, Chromosome 4 also has three tightly (1981) Mol. Gen. Genet. 182, 440--444 which is on the short arm of chromosome linked Mr 19 000 zein genes that are 15 Langridge,P., Pintor-Toro, J. A. and Feix, G. (1982) Mol. Gen. Genet. 187, 432-438 7, is a simple Mendelian recessive. In close to the floury-2 locus. The mechanisms by which these muta- 16 Langridge, P. and Feix, G. (1983) Cell 34, this mutant zein synthesis begins several 1015-1022 tions affect zein synthesis are unknown. days later than in the normal genotype. 17 Langridge, P., Pintor-Toro, J. A., Feix, G. It proceeds at a slightly slower rate, and Some of the mutant endosperms are (1982) Planta 156, 166-170 ceases midway through endosperm known to have a reduced level of mem- 18 Salamini,F. and Soave, C. (1982)in Maizefor development. As a result, opaque-2 brane-bound polyribosomes and a correBiological Research (Sheridan, W. F., ed.), pp. 155-160. Plant Molec. Biol. Assn. seeds have about 50% less zein than the sponding reduction in zein mRNAs. In addition, the synthesis of certain non- 19 DiFonzo, N., Fornasari, E., Salamini, F., normal genotype. Reggiani, R. and Soave, C. (1980)J. Heredity Other recessive mutations such as zein proteins is affected. A soluble pro71,379--402 opaque-6 and opaque-7 also cause a tein of Mr 32 000 is missing in opaque-2 20 Ma, Y. and Nelson, O. (1974) Maize Genet. significant reduction in zein synthesis. In mutants 21. However, this protein appears Coop. News. 48, 16--19 contrast with opaque-2, opaque-7 causes to be the product of the opaque-6 rather 21 Soave, C., Tardoni, L., DiFonzo, N. and a reduction in the synthesis of the Mr than the opaque-2 locus. This protein Salamini, F. (1981) Cell 27, 403-410