[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || The Prolamin Proteins of Maize, Sorghum and Coix

7. The Prolamin Proteins of Maize, Sorghum and Coix

CRAIG E. COLEMAN, JOANNE M. DANNENHOFFER andBRIAN A. LARKINSDepartment ofPlant Sciences. University ofArizona. Tucson. AZ 85721. USA

ABSTRACT. The prolamin proteins of maize and its close relatives, sorghum and Coix , sharecommon structural features. These proteins are classified as 00- , (3-, , - and 8-types, based onstructural homology and solubility characteristics. Each of these proteins has a distinct spatialdistribution within the protein bodies, which form as accretions within the lumen of the roughendopl asmic reticulum. There is increasing evidence that several of these proteins, notablythe (3- and -y-zeins, play important roles in initiating and organizing the protein body. Theseprolamins are devoid of lysine and tryptophan, two essential amino acids for monogastricanimals. Since prolamins account for approximately 70% of the seed protein , these grains areof inferior quality for human and livestock nutrition. Interest in improving the protein quality ofthese cereal s led to the ident ification of several maize mutants that alter the pattern of prolaminsynthesis. One of these , opaque2, corresponds to a defective transcription factor that regulateso-zein gene expression , while another,floury2, is a mutant o -zein protein. Both mutations cancause pleiotropic effects that increase the lysine content of the seed, and hence the proteinquality of the grain . Another mutant , opaqu el S, alters the synthesis of the -y-zein protein.This reduce s the number of protein bodies that form, which appears to have a major effect ofthe texture of the grain. By over-producing -y-zein in an opaque2 mutant, plant breeders haveproduced a new type of corn, "Quality Protein Maize", which has excellent protein quality.

1. Introduction

Prolamin is a type of cereal seed protein that is soluble in alcohol and whosefunction is storage of nitrogen and amino acids for the germinating seedling(Osborne, 1908; Osborne and Mendel, 1914). Maize prolamin, the zein frac-tion , is similar to that of sorghum and Coix (Job 's Tears), which are known askafirin and coixin, respectively. In these species , prolamin aggregates formprotein bodies within the lumen of the endoplasmic reticulum of endospermcells. These proteins are unlike the prolamins in the more distantly relatedcereals, wheat, barley, oats and rice. Of the three panacoid species, maize isthe most well-characterized. As maize grain is the most abundantly produced,it is not surprising that zein has been the focus of much research. Consequent-ly, in this chapter we concentrate on what is known about zein, but we broadenthe discussion to include kafirin and coixin when appropriate.Zein proteins are primarily synthesized in the kernel endosperm, a special-

ized tissue devoted to the accumulation of protein and starch. Hydrolysis ofthese reserves upon seed germination provides a rich source of metabolitesfor the emerging seedling. The triploid endosperm arises from the union of asingle sperm nucleus with two polar nuclei of the central cell of the embryo

B.A. Larkins and IX. Vasil (eds.s.Cellular and Molecular Biology of Plant Seed Development. 257- 288.@ 1997 Kluwer Academic Publishers,

258 Craig E. Coleman et al.

kDa29.0

18.4

14.3

MW Zein

'Ya

Kafirin

Fig. 1. SOS-polyacrylamide gel separation of zein and kafirin. Proteins were extracted frommaize and sorghum endosperm and stained with Coomassie Brilliant Blue (Wallace et aI.,1990). The molecular weight of size markers (lane MW) is shown on the left. Greek lettersidentify the respective zein and kafirin protein classes.

sac. Zein and starch synthesis begins 10-14 days after pollination (DAP),coincident with several other developmental event s that may be triggeredby a rapid decrease in the cytokinin to auxin ratio (Lur and Setter, 1993).Mitotic activity ceases in the central region of the endosperm, but continuesin the subaleurone layer, near the periphery of the tissue. The central cellsbegin to enlarge, coincident with amplification of the entire nuclear genome.This process of DNA endoreduplication results in ploidy levels that averagefrom 90e to woe (Kowles and Phillips, 1988).Although the entire genome isamplified, the function of DNA endoreduplication may be to provide multiplecopies of genes involved in the synthesis of protein and carbohydrate.Starch accumulation is greatest toward the center of the endosperm, while

the largest quantity of zein is found within the more peripheral cell layers.The starchy cells in the interior of the endo sperm form a soft, chalky tissueat maturity, while the proteinacious outer endosperm is hard and translucent.The outermost endosperm cell layer, the aleurone, remains viable throughmaturation and dessication , and it produces hydrol ytic enzymes that digestthe protein and carbohydrate reserves upon germination. At maturity, 90% (byweight) of the maize seed is endosperm, with the remainder consisting of theembryo , testa and pericarp. For further details on endosperm development,see review by Lopes and Larkins (1993).

The Prolamin Proteins ofMaize, Sorghum and 'Coix' 259

2. Characterization of Zein

2.1. Extrac tion ofZein

Procedures for isolating zein take advantage of its solubility in alcoholic solu-tions. The first publi shed procedure was developed by Osborne and Mendel(1914) and is based on sequential extraction of milled flour in various solvents.Thu s, albumin, globulin, prolamin and glutelin are obtained by consecutiveextraction with water, saline, alcohol and alkali or acid, respectively. Thismethod has drawbacks, because some proteins form insoluble aggregates andothers are soluble in more than one solvent. Landry and Moreaux (1970)modified the Osborne-Mendel procedure by incorporating the reducing agent,8-mercaptoethanol in the alcohol and alkali solvents, which generated twonew fractions. Although this method has been employed by researchers formany years, it also has limitation s, particularly in analysis and quantificationof zein. A simpler procedure for zein extraction was developed by Wallace etal. (1990). With this method total endosperm protein is solubilized in a high-pH borate buffer containing SDS and ,8-mercaptoethanol and subsequentlydivided into alcohol-inso luble (non-zein) and alcohol -soluble (zein) fractionsby addition of ethanol to a final concentration of 70%. The alcohol-insolublefraction consists of a mixture of albumin, globulin and glutelin proteins. Thisprocedure results in a more quantitative prolamin exraction, and it has theadvantage of yielding a single fraction containing all of the zein proteins.Separation of zein by SDS-PAGE yields polypeptides with apparent molec-ular masses of 27-,22-, 19- , 16-, 15- and lO-kDa (Figure 1). Proteins withsimilar masses are isolated from sorghum (Figure I) and Coix.

2.2 . Nomenclature

The variety of methods for extracting zein from maize endosperm has led to acomplex nomenclature for these proteins. Several attempts have been made todevelop a systematic class ification of the zein polypeptides based on solubilityproperties and mobility through gels (Hartings et al., 1984; Wilson, 1985,1986). Esen (1986 ) described a zein fractionation procedure using differenti alsolubility that separated zeins into three classes, and he later proposed thatthey be called a -zein, ,8-zein, and -y-zein (Esen, 1987). Nucleotide sequencingof zein genes further helped to refine this nomenclature system, so that theseclasses now identify polypeptides that are related structurally rather than onlyby solubility (Thompson and Larkin s, 1989). This nomenclature system iswidely used by many laboratories, and it has also been used to classify therelated prolamins from sorghum (Shull et aI., 1991) andCoix (Ottoboni et aI.,1993).


A c

Fig . 2. Potential arrangements of a-helices in the 22-kOa a-zein protein. The cylinders in(A) and (C) represent the 10 a -helices that comprise the molecule ; the Q's above and belowthe cylinders correspond to glutamine residues at the ends of the helices . (B) and (0) showcross-sectional views of the molecule and illustrate the antiparallel orientation of the helices.The circles represent each of the 10 a-helices and are numbered according to their position inthe molecule (i.e., helix I is closest to the N-terminus) . 'Up' and 'down ' refer to the orientationof the helix from the N- to the C-terminus relative to the plane of the page. The arrows denotethe direction of the helical turn. (A) and (B) were adapted from Argos et al., 1982; (C) and (0)were adapted from Garratt et al. (1993).

2.3. a-Zein

The most abundant class of prolamin in maize is a-zein, constituting about70% of the total fraction. On SDS-polyacrylamide gels (Figure 1) a-zeinappears as two broad bands with approximate molecular masses of 19- and22-kDa. These bands are composed of a complex mixture of polypeptidesthat are the products of a multigene family. In addition to the size differences,there is a significant amount of charge heterogeneity that allows the a-zeinpolypeptides to be separated by isoelectric focusing (Righetti et aI., 1977).As many as 40 a-zein polypeptides have been resolved by two-dimensionalgel electrophoresis (Wall et aI., 1984). This microheterogeneity between a-zeins has been exploited for genotypic identification of inbreds and hybrids(Hartings et aI., 1984; Wilson, 1985; Smith and Smith, 1988) . Polypeptidesstructurally related to a-zein are found in sorghum and Coix, where they arealso the most abundant class of prolamin (DeRose et aI., 1989; Ottoboni etaI., 1990, 1993).Several genes encoding a-zein, o-kafirin and o-coixin have been cloned

and sequenced, revealing some features of the protein structure. These pro-lamins are extremely rich in glutamine and proline, as well as the hydrophobic


TABLE I

Amino acid composition, deduced from cDNA clones,of a -prolamins (mol%)

19-kDa 22-kDa 22-kDa 25-kDao-zein! o-zeirr' o-kafirirr' o -coixin"

Asn 4.5 5.3 6.0 5.3Asp 0.5 0.0 0.4 0.4Thr 3.2 2.8 4.0 1.6Ser 7.3 6.9 6.0 6.2Gin 19.5 20.7 24.6 21.8Glu 0.5 0.8 0.4 0.8Pro 10.0 8.9 7.7 8.6Gly 0.9 0.8 1.6 1.6Ala 13.6 13.8 14.9 16.5Cys 0.9 0.4 0.4 0.4Val 2.7 6.9 4.4 5.8Met 0.5 2.0 0.8 0.8lie 4.5 4.5 5.6 4.5Leu 19.5 17.1 15.3 16.0Tyr 3.6 2.8 2.8 2.9Phe 5.5 3.3 2.4 3.3His 0.5 1.2 1.2 1.6Lys 0.0 0.0 0.0 0.0Arg 1.4 1.6 0.8 1.2Trp 0.0 0.0 0.4 0.4

I ClonecZl9Cl; Mark setal. , 19852 Clone pZ22.3; Mark s and Larkins, 19823 Clone pGK.l ; DeRose et aI., 19894 Clone pBCX25.1O; Ottoboni et aI., 1993.

residues alanine and leucine, but they completely lack lysine and tryptophan(Table 1). The core of the a -zein protein is a series of peptide repeats approx-imately 20 amino acids in length . Each of the repeats is flanked by clustersof glutamine residues (Pedersen et al., 1982). There are 9 such repeats in the19-kDa molecule and 10 in the 22-kDa molecule (Garratt et al., 1993). Circu-lar dichroism measurements and a solution conformational analysis suggestthat these repeats form antiparallel a-helices (Figure 2) (Argos et al., 1982;Tatham et al., 1993). The position of polar amino acids in these helices is con-served, and these residues may be important for establishing the conformationof the molecule, or in intermolecular interactions that facilitate aggregationof the polypeptides (Garrat et al., 1993). The predicted molecular weight of

262 Craig E . Coleman et al .

o -zeins is 4 to 5 kDa larger than the apparent molecular weight observed onSDS-polyacrylamide gels. This difference may be due to the unusual struc-tural features of the polypeptides, which could alter their mobility in thepolyacrylamide matrix.It is common to divide the o -zeins into two groups based upon their

apparent molecular weight: namely, 19-kDa o-zeins and 22-kDa o-zeins. Infact, there is about 90% homology between proteins within each size-class ,but only about 60% homology when comparing proteins between the twogroups. Still, the molecular weight of a particular a-zein polypeptide maynot accurately reflect the degree of homology with other o -zeins, Severalcases have been reported in which a 19-kDa a-zein appeared to be morehomologous to 22-kDa o-zeins or in which a 22-kDa a -zein appeared tobe more similar to 19-kDa o-zeins (Spena et al., 1982; Marks et al., 1985;Esen et al., 1987). These observations demonstrate that grouping of thesepolypeptides by molecular weight does not accurately reflect the evolutionaryand genetic relationships underlying the structure of the multigene familyencoding the proteins. Nevertheless, this system is the most practical, and inmany cases it is legitimate.

2.4 . ,,(-Zein

The second-most abundant class of zeins are the cysteine-rich -y-zeins. Thereare two bands, 16- and 27-kDa, identified as "(-zeins on SDS-polyacrylamidegels, each corresponding to one or two polypeptides. These proteins aresoluble in alcoholic and aqueous solutions in the presence of reducing agent.Because of the unusual solubility properties of the 27-kDa -y-zein, it hasbeen variously referred to as alcohol-soluble glutelin (Paulis and Wall, 1971),reduced-soluble protein (Wilson et al., 1981; Vitale et al., 1982), and glutelin -2 (Prat et al., 1985). In contrast to the o-zeins, the predicted molecular weightof these polypeptides, based on cDNA sequences, is much smaller than theapparent molecular weight observed on SDS-polyacrylamide gels (Prat eta1. , 1985, 1987). This size discrepancy can be explained by the high prolinecontent, which could generate an unusual secondary structure and retardmovement of the molecules through the polyacrylamide matrix. The ,,(-kafirinand -y-coixin proteins are structurally similar to -y-zein, although "(-coixin issomewhat smaller than the other two proteins (Taylor et al., 1989; de Barroset al., 1991; Leite et al., 1991). Like the o-zeins, these proteins lack lysineand tryptophan (Table 2).Because -y-zein is rich in cysteine and requires reducing agent for sol-

ubilization, it has been difficult to obtain information about the secondarystructure of the molecule; however, it has some interesting primary structuralfeatures. The N-terminal half of the 27-kDa -y-zeincontains 8 tandem repeatsof the hexapeptide PPPVHL, fol1owedby the so-called Pro-X linker region, astretch of 22 amino acids in which nearly every other residue is proline. The


TABLE 2

Amino acid composition, deduced from cDNA clones,of -y-prolamins (mol%)

27-kDa 16-kDa , -kafirin2 , -coixin3

, -zein' -y-ze in '

Asn 0.0 0.6 0.0 0.0Asp 0.0 0.0 0.0 0.0Thr 4.4 3.7 4.7 4.4Ser 3.9 5.5 5.2 3.3GIn 14.7 18.9 I \.9 18.9Glu \.0 \.8 \.0 1.1Pro 25.0 15.2 23.3 20.0Gly 6.4 9.1 8.8 6.7Ala 4.9 7.9 5.7 6.7Cys 7.4 7.3 7.8 7.8Val 7.4 4.9 6.2 5.0Met 0.5 \.8 \.0 1.7lie 2.0 0.6 2.6 3.9Leu 9.3 8.5 8.3 7.2Tyr 2.0 4.9 2.1 2.2Phe \.0 4.3 \.6 2.2His 7.8 2.4 7.8 5.6Lys 0.0 0.0 0.0 0.0Arg 2.5 \. 8 2.1 3.3Trp 0.0 0.6 0.0 0.0

) Prat et al., 19872 de Barros et al., 19913 Leite et al., 1991

16-kDa 'Y-zein has only two degenerate copies of the PPPVHL hexapeptide,whi le the 'Y-kafirin and -y-coixin proteins have 4 and 3 cop ies, respectively.The C-terrninal half of the protein is rich in glutamine and cysteine, andcontains two short sections that have some homology to ,8-zein. There arealso some motifs that are homolgous to sequences in the 0 /,8- and -y-gliadinsfrom wheat and ,8-hordein from barley, suggesting a phylogenetic relationshipbetween these proteins (Prat et aI., 1985).

2.5. ,8- and 6-Zeins

The two sma llest maize pro lamins are rich in methionine, and are knownas ,8- and 6-zein. These proteins have apparent molecular weights of about


TABLE 3

Amino acid composition of ,B-prolamins(mol%). Based on deduced amino acidsequences from cDNA of ,B-zein and,B-coixin, and direct determination of,B-kafirin

,B-zein l

,B-kafirin2

,B-coixin3

Asx 2.5 3.3 1.7Thr 2.5 4.6 4.6

Ser 5.0 4.6 5.2

Glx 18.1 17.7 15.6

Pro 8.8 9.7 7.5Gly 8.8 6.8 8.7

Ala 13.8 13.4 16.2

Cys 4.4 4.9 5.2Val 1.9 5.2 4.0

Met 11.3 5.7 11.6

lie 0.6 2.3 0.6Leu 10.0 12.0 9.8

Tyr 8.8 3.0 5.8Phe 0.0 1.9 0.6

His 0.0 0.9 0.0Lys 0.0 0.5 0.0Arg 3.1 2.7 2.3

I Pedersen et aI., 19862 Shull et al., 19923 Leite et al., 1992

14 and 10 kDa, respectively (Figure I). The relatively high level of cys teineand the fact that reducing agent is requi red for its effic ient so lubilization,suggests that j3-zein is involved in intra- and intermolecul ar chemical cross -linkages. Circular dichroism measurements and computer modeling sugges tthat the secondary structure of j3-zein is mostly j3-strand and tum (Pedersenet aI., 1986). An equivalent polypeptide from sorghum, j3-kafirin, cross-reactswith j3-zein antiserum (Shull et aI., 1992), and a gene encoding a j3-coixinhas been cloned and sequenced (Le ite et aI., 1992). Besides having higherlevels of cysteine and methionine than the o -zeins, j3-zein has less glutamine,

The Prolamin Proteins of Maize, Sorghum and 'Coix' 265

Fig . 3. Low magnification of a developing (18 dap) maize endosperm cell. CW: cell wall;ER: endoplasmic reticulum; N: nucleus; PB: protein body; ST: starch grain. Bar: 1.0pm.

Fig.4. Electron micrographs of protein bodies in peripheral cell layers of sorghum endosperm.Bar: 0.5 tun . (A) Low magnification field of protein bodies. (B) Polyribosomes (arrow) onsurface of protein bodies. (C) Concentric pattern of dark-staining proteinaceous material. (D)Concentric pattern and darkly stained core . (E) Darkly stained peripheral layer and core. Takenfrom Shull et al. (1992).

leucine and proline (Table 3). Surprisingly, (3-kafirin contain s about half themethionine of (3-ze in and (3-coixin.The o-ze in protein is struc tura lly unrelated to any of the other zeins,

although its am ino acid compos ition is similar to (3-zein (Kirihara et aI.,


1988a, b). Some structural similarities, based on computer modeling, havebeen noted between the 10-kDa J-zein and the Brazil nut 2S storage pro-tein, which is also very methionine-rich. The inbred BSSS-53 shows a 30%increase in methionine over normal inbred s due to high levels of this pro-tein (Phillips and McClure, 1985). Recently, a gene has been isolated frommaize that encodes a second methionine-rich polypeptide that is 50% largerand is 76% homologous to the 10 kDa J-zein gene (Chui and Falco, 1995).Deduced amino acid sequences for both polypeptides predict a central corethat is about 50% methionine, flanked by N-terminal and C-terminal regionsthat have lower methionine contents. Equivalent polypeptides have not yetbeen identified in sorghum or Coix.

3. Protein Body Formation

Ultrastructural analyses of maize, sorghum and Coix endosperm demonstratea high level of similarity in the structure of protein bodies (Figures 3 and4) (Lending et al., 1988; Taylor et al., 1985; Krishnan et aI., 1989; Shull etal., 1992; Targon et al., 1992). Fully developed protein bodies are spheri-cal with a diameter of I-211m. Both zeins and kafirins are synthesized onmembrane-bound polysomes and have signal peptides which target them tothe lumen of the ER. The mechanism whereby these protein s remain in theER is not known, since they lack canonical HDEL/KDEL retention signals;however, some clues have been obtained from studies on the 27-kDa -y-zein(Torrent et aI., 1994; Geli et aI., 1994). Removal of the PPPVHL repeatsfrom the 27-kDa -y-zein permits the truncated protein to move freely throughthe secretory pathway in transgenic Arabidopsis leaves. Full length proteinand truncated versions missing either the C-terminus or the Pro-X regionremained associated with membrane-bound vesicles from transformed Ara-bidopsis leaves and frog oocytes injected with mRNA. Proline-rich regionshave been implicated in protein-protein interactions (Williamson, 1994), andso the hexapeptide-repeat region of the -y-zein protein may be responsiblefor interactions that prevent normal movement of the polypeptide through thesecretory pathway. It does not appear that retention of -y-zein within the ERis the result of binding to the membrane surface, since the protein diffusesfreely through the lumen of the ER of frog oocytes (Lee et al., 1995).Protein interactions that allow -y-zein to be retained in the ER could also

be responsible for the initiation of protein bodies since, along with tJ-zein,')'-zein is the first of the zein polypeptides to appear during developmentof the protein body. In the most immature endosperm cells, protein bodiesare small and spherical, and they stain darkly, as viewed with an electronmicroscope (Figure 5) (Lending and Larkins, 1989). Immunocytochemicalanalyses demonstrate that this dark-staining material is composed entirely of13- and -v-zein. Later, as the protein body matures, lighter-staining material


.~

.; ~.

;

,..

Fig . 5. Developmental pattern of protein body formation in maize endosperm. The mostimmature protein body is on the left and development is from left to right. Greek letterdesignations in the lower figure indicate the location of the corresponding zein classes asdeterm ined by immunolocalization. Adapted from Lending and Larkins (1989).

appears as locules within the matrix of {3- and -y-zein. This lighter-stainingsubstance is mostl y a -zein, but J -zein has also been localized to this region.Growth of the protein body occurs as additional a -zein accumul ates withinthe interior. Eventually, the locul es of light-staining material fuse, until atmaturity the protein body consists of a large central core of a -zein surroundedby a thinner outer shell of {3- and -y-zein.Several attempts have been made to synthesize a -zein in foreign hosts,

such as Xenopus oocytes (Hurkman et aI., 1981; Wallace et aI., 1988), yeast(Coraggio et al., 1986, 1988),Acetabularia (Langridge et aI., 1985), tobacco(Schemthaner et aI., 1988; Ohtani et aI., 1991) and petunia (Williamson etaI., 1988). In the case of the oocytes, membrane-bound zein aggregates weredetected with density similar to maize protein bodies, but it is not knownif these structures are organized comparable to maize protein bodies. Fur-thermore, there were differences in the density of the aggregates, dependingon whether a -zein mRNA alone or total zein mRNA was injected into theoocytes.Zein genes have been expressed in a variety of tissues in transgenic tobacco

and petun ia, depending on the spec ificity of the promoter. Nevertheless, inno case have significant levels of a -zein protein been detected in any of thetissues examined.Th is is depite the fact that the amount of transcript producedappeared to be sufficient to synthesize detectable quantities of protein. Thisimpli es that either a -zein mRNA is poorly translated in these organisms, orthe protein is unstable. In transgenic petunia seeds, a small amount of a-zein


protein was located as an amorphous inclusion appressed against the cell wall(Wallace et aI., 1989). This is similar to the observation with yeast, wherea-zein accumulated to 5% of the total protein, but remained as an unorganizedmass within the ER (Coraggio et aI., 1988).Undoubtedly, there are interactions between the various zeins that lead

to formation of a protein body. Studies with heterologous systems clearlydemostrate that a-zein alone is incapable of organizing into a protein body.This is consistent with the fact that a-zein does not appear until after 13- and-y-zein coalesce and initiate protein body development. These observationssuggest that the stability of a-zein and its inclusion in protein bodies isdependent upon the presence of 13- and -y-zein. In this light, it is interesting tonote that f3-zein alone is able to form protein-body-like structures in transgenictobacco leaves and seeds (Bagga et aI., 1995). These structures appear as acluster of lobes, rather than a single sphere, which suggests 'Y-zeinmay alsobe required to generate distinct protein bodies. The nature of the interactionsbetween the zeins that cause protein body formation, and the protein domainsinvolved are yet to be determined.In addition to interactions between the zeins, formation of a protein body

appears to require the action of molecular chaperones. The ER-resident b-70 protein , which has been shown to be a homolog of an immunoglobulinbinding protein (BiP) (Fontes et aI., 1991; Marocco et aI., 1991), appears toplaya role in zein folding and assembly (Boston et aI., 1991). BiP is over-expressed in several endosperm mutants that are characterized by abnormalprotein body morphology, and it has been localized to the periphery of theprotein body (Zhang and Boston, 1992). BiP also co-localizes with -y-zeinduring its accumulation in transgenic Arabidopsis leaves (Geli et aI., 1994).A role for BiP in the association of rice prolamins in the ER lumen has beendemonstrated (Li et aI., 1993).

4. Zein Gene Structure and Regulation

4.1. Genomic Organization and Gene Structure

Genes encoding a-zein proteins occur in a complex multigene family con-taining between 70 and 100 members (Hagen and Rubenstein, 1981; Wilsonand Larkins, 1984). Since this estimate exceeds the number of polypeptidesidentified by various electrophoretic techniques, it is possible that many of themembers of the gene family are pseudogenes, or that there are many dupli-cated sequences. In-frame stop codons have been observed in several a-zeingenomic clones, implying that they are pseudogenes (Spena et aI., 1983; Kridlet aI., 1984; Wandelt and Feix, 1989; Liu and Rubenstein, I 992a, b; Thomp-son et aI., 1992). The o-kafirin gene family may be somewhat less complexthan its counterpart in maize, as there appear to be only about 20 copies


dzrl

0 12

013

1

010

fll /0408

2 3 4

fl2

0 1

5

p-zein

014

6

02

e*B30&.zein

05

y-zein

015

7 8

06f13

07

9 10

Fig. 6. Schematic diagram of the 10 maize chromosomes showing the location of zein genes,zein regulatory genes, and mutant loci that generate an opaque phenotype. The locations ofa -zein gene clusters are shown with boxes.

in the genome (DeRose et aI., 1989). DNA from related species, includingTripsacum dactyloides and teosint e species, cross -hybridizes to a -zein cDNAprobes, indicating that zein homologues are present. As with sorghum, themultigene family in these spec ies appears to be substantially less complexthan in maize (Wilson and Larkins, 1984). Thu s, it appears that the a -zeingene family was less complex prior to the divergence of maize and sorghum,and that substantial gene dupli cation occured followin g the separation.Alpha-ze in genes were mapped to the long and short arms of chromosome 4, theshort arm of chromosome 7, the long arm of chromosome 10, and near thecentromere of chromosome I (Figure 6) (Hartin gs et aI., 1984; Shen et aI.,1994).A single locus near the centromere on the long arm of chromosome 7 may

contain either one or two copies of the gene encoding the 27-kDa , -zeinprotein (Das and Messing, 1987). When there are two functional copies ofthe gene they are contained in 12-kb tandem repeats separated by a 2-kbspacer region. These two genes occur in the same transcriptional orientation;by convention, the upstream gene is designated A and the downstream geneis referred to as B. In the inbred line W22 there is very high homology


between the two genes, especially in the 5' flanking region where the onlydifference is a dinucleotide insertion in the B gene. There is progressively lesshomology between the two genes toward the 3' end, with the most significantdifference being a 1.8-kb insertion in the B gene several hundred base pairsdownstream from the termination codon. In those cases where there is only asingle ')'-zein gene, it appears to have arisen from a two-step recombinationevent. The recombined gene, called Ra, is generated from a cross-over eventin the 3' flanking region, and a second recombination in the 5' region that maybe a deletion, insertion, or inversion. The new gene consists of the entire 5'flanking and coding sequences of the A gene, and the 3' flanking sequencesof the B gene beginning about 230 bp downstream of the termination codon(Das et aI., 1990).Despite the extensive homology between the A and B genes there is a

disproportionate accumulation of transcripts. In the inbred lines A188, W22andW23 there is 2.5-times more B than A RNA. The opposite is true in otherlines, such as Tuxpefio,where there is twice as much A than B gene transcript(Or et aI., 1993). In lines containing the Ra gene, the amount of A genetranscript is comparable to lines with both genes. There is strong evidencethe difference in the level of transcripts is not due to variation in transcriptionrates, but rather to stability of the mRNA (Or et aI., 1993). This suggests apost-transcriptional mechanism for the control of gene expression. Most ofthe differences between the two genes are in the 3' end, but it is unclear whichof these alterations are responsible for the disparity in mRNA stability. Notonly is this an interesting system for the study of post-translational controlof gene expression, but the information has potential agronomic value sincethe level of ')'-zein in the endosperm is closely correlated to kernel hardness(Dannenhoffer et al., 1995; Lopes et al., 1995).The genes encoding (3- and 8-zein are single copy and are mapped to the

short arm of chromosome 6 and the short arm of chromosome 7, respectively.

4.2. Control a/Zein Gene Expression

There is good evidence that expression of zein genes is under strict spatialand temporal control (Faccio Dolfini et al., 1992); however, very little isknown about the mechanisms responsible for this regulation. Nearly all cerealprolamin genes have a common promoter element, called the endosperm orprolamin box, located about 300 bp upstream of the translation start codon(Kreis et al., 1985). Usually, this element is bipartite, consisting of a so-called'endosperm motif' (TGTAAAG) that resembles the SV40 enhancer coresequence and a motif that is similar to the GCN-4 binding sequences in yeastand the mammalian Jun and API transcription factor binding sites (Mulleret aI., 1995). Panacoid prolamin gene promoters have a conserved lS-bpelement that contains the 7-bp endosperm motif, but not the GCN-4 motif. In-y-zein, ')'-kafirin and -y-coixin gene promoters, GCN-4-like motifs have been

The Prolamin Proteins ofMaize, Sorghum and 'Coix ' 27 1

identified, but not in such close proximit y to the endosperm motif (de Freitaset al., 1994). There is no evidence that the GCN-4 motifs in these promotersinteract with the endos perm motif as they do in non-panacoid prolamin genepromoters. The endos perm motif in 22-kDa a -zein gene promoters may act asa tissue-specific enhancer (Quayle and Feix, 1992). It is protected by proteinsin endosperm nuclear extrac ts in footprinting assays, but a specific proteinfactor has not been isolated or characterized (Maier et al., 1987).Thu s far, the only transcriptional activator of zein genes charac terized to

any appreciable extent is the product of theOpaque2 (02) gene. This gene hasbeen cloned (Schmidt et al., 1987; Motto et al., 1988), and the deduced aminoacid sequence shows the protein contains a domain with strong homolog y toa group of transcription factors known as bZIP proteins (Hartings et al., 1989;Schm idt et al., 1990). The bipartite bZIP domain consists of a basic motif,rich in positively-charged amino acids, flanked by a ' leucine zipper' motifconsisting of evenly spaced leucine residues embedded within tandem heptadrepeats. The basic motif is responsible for binding the protein to DNA, whilethe ' leucine zipper' is believed to be involved in dimerization of the protein(Aukerman et al. , 1991; Schmidt et al., 1992). The 02 protein specificallyregulates the transcription of 22-kDa a-zein genes (Kodrzycki et al., 1989),and a ribosome-inactivating protein known as b-32 (Lohmer et al., 1991; Basset al., 1992). A second bZIP-type protein, OHP-l , has been identified in maizeendosperm, and it has the ability to form heterod imers with 0 2 (Pysh et al.,1993). The OHP-l prot ein does not have a transactivation domain, and soits function remains to be determ ined. A homolog of the maize 02 gene hasbeen cloned from sorghum and it presumably has similar functions (Pirovanoet al., 1994).The 0 2 protein binds one or more sites within the promoter of 22-kDa

a-zein genes (Schmidt et al., 1992; MUller et al., 1995). Comparison of thesesites yields a consensus sequence of TYCACGTRR. This motif is locatedin close proximity to the endos perm box. It has been sugges ted that the 0 2motif may play a role in the response of zein gene expression to nitrogenlevels, although it is unlikely that the 02 protein itself is involved (MUller etal., 1995; see Chapter X). The OHP- l protein also binds to these sites, but theabsence of a transcriptional activator in this protei n sugges ts it may act as anegative regulator of zein gene express ion (Pysh et al., 1993). The 02 proteinbind s to a different type of site in the b-32 gene, with a consensus sequenceof GATGAPyPuTGPu (Lohmer et al., 1991). A single binding site for the 02protein was identified in the promoter of an o -coixin gene (Yunes et al., 1994).Interestingly, the sequence of this site, GACATGTC , is more closely relatedto the b-32 con sensus sequence than to the 22-kDa a -zein sequence. Thissuggests that the 0 2 protein may bind a wide range of cis-acting sequences.The majority of a -zein gene transcripts initiate approximately 40-65 bp

upstream from the translat ional start codon, yielding mRNAs with an approxi-mate length of900 nucl eotides (Kriz et al., 1987).The presence of much larger


transcipts on northern blots led to the speculation that precursor mRNAs aresynthesized from a promoter (P I) located about I kb further upstream fromthe major promoter (P2) (Langridge et aI., 1982; Langridge and Feix, 1983).Sequence compari son showed there are consensus promoter elements in thePI region, and a variety of experiments were performed to test the relativestrengths of the two promoters in vitro and in vivo, with conflicting results(Langridge and Feix, 1983; Langridge et aI., 1984; Boston et aI., 1986). Asplice site in the 5' flanking region between the two promoters was identified,and it was suggested that it might playa role in processing the larger a -zeintranscript s (Brown and Feix, 1990). Nevertheless, it has not been shown thatthis splicing occurs in vivo, and con sequently, the functional significance ofthe the P I promoter, if any, remains to be determined.Overexpression of o-zein in the naturally-ocurring variant BSSS53 has

been linked to a single genetic locu s, dzrl , that maps to chromosome 4Swithin a cluster of 22-kDa a-zein genes (Benner et aI., 1989; Chaudhuriand Mes sing, 1995). There is co-dominance between the dzrl +BSSS53 andwild-type alleles, since dosage of the former allele is correlated to the levelof o-zein protein in the endosperm (Chaudhuri and Messing , 1994). It is alsobelieved that the product of this alle le exerts its effect on o-zein througha post-transcript ional mechani sm, since transcriptional rates in BSSS53 arecomparable to rates in the low-e-zein lines W22 , W23 and Mo 17, yet thesteady-state level of transcripts is significantly higher (Cruz-Alvarez et al.,1991; Schickleret aI., 1993). Exactly how posttransciptional control is exertedhas yet to be determined. Interestingly, parental imprinting has been observedfor some of the dzrl alleles (Chaudhuri and Messing, 1994) , although thephysiological significance of this observation is not clear.

4.3. Genes Affec ting Zein Synthesis and Processing

Several genetic mutations that affect the accumulation or processing of zeinsin maize endosperm have been identified, and are shown on the chromosomalmap (Figure 6). These mutations are easy to identify due to the distinctivephenotype which they condition: maize kernel s that are defective in the syn-thesis or processing of zeins have a soft, starchy endosperm that is opaqueto transmitted light, rather than the hard , translucent endosperm of wild-typekernels. The recessive mutations opaque2 (02) and opaque7 (07) result ina specific decrease in accumulation of 22-kDa a -zein and 19-kDa a -zein,respectively (Motto et aI., 1989), while the opaque15 (015) mutation exertsits effect primarily on the 27-kDa -y-zein (Dannenhoffer et aI., 1995). Therecessive mutation opaque6 (06), and the dominant or semi-domiant muta-tions floury2 (jl2), floury3 (jl3), Defective endosperm-Bst) (De* B30) , andMucuronate (Mc) cause a more general reduction in accumulation of all zeinclasses (Motto et aI., 1989).

The Prolamin Proteins ofMaize. Sorghum and 'Coix' 273

The mechanism that causes kernel softness in these mutants is poorlyunderstood, but it is clear that there are multiple pathways that can lead tothe aberrant phenotype. Generally, models have been proposed suggestingthese mutations affect genes encoding zein regulatory proteins (Soave etaI., 1981; Soave and Salamini, 1983; Motto et aI., 1989; Schmidt, 1992);nevertheless, only the 02 mutation has been shown to affect a regulatory gene .Some mutations originally thought to affect regulatory genes have since beenshown to affect other types of genes. For instance, 06 was found to be allelic topro] (Manzocchi et aI., 1986), a mutation that require s supplementation withcertain amino acids for normal growth (Rizzi et ai, 1992), and there is strongevidence that the fl2 mutation affects processing of an a -zein protein (Lopes etaI., 1994; Coleman et aI., 1995). The existence of additional mechanisms thatgenerate an opaque phenotype is further supported by the fact that a numberof opaque mutants, including opaquel, opaque5,flouryl , and hornyl, haveno observable alteration in their pattern of zein accumulation. Several of themore well-characterized opaque endosperm mutants are described below.

4.3.1. Opaque2Two well-studied opaque mutants in maize , 02 andfl2, were originally report-ed by Emerson et al. (1935 ). Both mutants gained prominence when it wasdiscovered that the level of lysine and tryptophan in the endosperm is greatlyenhanced compared to the wild type (Mertz et aI., 1964; Nelson et aI., 1965).Since maize grain is normally deficient in these two essential amino acids, thisfinding generated a great deal of excitement about the potential exploitation ofthe mutants to enhance nutritional quality. Unfortunately, it was soon discov-ered that the se mutants mani fested several undesirable characteristics, suchas reduced yield, increased levels of damage during harvesting and handling,and a greater susceptibility to attack by pathogens.Because the 02 gene encodes a transcriptional activator that regulates

expression of 22-kDa a -zein genes, it is not surprising that mutant 02 alle-les result in lowered amounts of these proteins in the endo sperm; however,reduced accumulation of other cla sses of zeins is also observed. This phe-nomenon is most likely a pleiotropic effect, since there is no evidence thatthe 02 protein binds to the promoter of other zein genes (Ueda et ai, 1992).Lower zein content in 02 endosperm results in protein bodie s that are aboutone-fifth to one-tenth the normal size. The smaller protein bodies presumablyalter the packing of starch grains during seed dessication and give the kernelthe characteristic soft texture. There is usually a coordinate increase in theamount of non-zein protein in the endosperm of 02 mutants. It is temptingto speculate that this is simply the result of diverting nitrogen resources fromone protein type to another, but there is evidence that the level of certain non-zein proteins is more elevated than others (Habben et aI., 1993). On the otherhand, there are some non -zein proteins, such as b-70 and lysine -ketoglutaratereductase , that are reduced in 02 endosperm compared to wild type (Marocco


"Y-zein

o 1 2 3

Fig . 7. lmmunodetection of a-zein proteins in mature endosperm flour of W64A ,W64Ao2,W64Ajf2, W64Ao2jf2, and their reciprocal FI crosses . Total zein extracts were separated witha 7.5-18% SDS-polyacrylamide gradient gel. Proteins were electro blotted onto a nitrocellulosefilter and treated with a-zein polycolonal antibodies. The large arrow marks the 24-kDa a-zeinassociated with the jf2 phenotype, and the small arrow marks a 45-kDa dimer of the 24 kDaa-zein. Genotype designations arc shown above each lane.

et aI., 1991; Brochetto-Braga et aI., 1992). Clearly, there are other cellularprocesses that are disrupted by the 02 mutation, besides the formation ofprotein bodies .

4.3.2. Quality protein maizeThe soft, starchy phenotype of the 02 mutant can be converted to wild typethrough the activity of genetic modifiers. Breeding programs at the Inter-national Maize and Wheat Improvement Center (CIMMYT; Villegas et ai,1992) and Pietermaritzburg, South Africa (Geevers and Lake, 1992) madeuse of these modifiers to develop 02 varieties that have a hard, translucentendosperm. These varieties, known as Quality Protein Maize (QPM), haveenhanced nutritional quality due to the 02 effect of raising the level of lysineand tryptophan. QPM varieties are being developed in many parts of the worldfor both human and livestock consumption (Graham et aI., 1990; Bressani,1992; Knabe et aI., 1992; Graham, 1993), although currently their popularityis greater in Third World countries than in developed nations.The mechanism by which the modifier genes convert the starchy endosperm

of 02 to a normal phenotype is poorly understood, but some clues have beenuncovered by observation of biochemical changes in modified 02 endosperm.QPM varieties have levels of a-zein comparable to unmodified 02 lines, butthe level of -y-zein is increased 2-3 fold (Wallace et aI., 1990). The modifiergenes behave semidominantly and a correlation exists between dosage of

The Prolamin Proteins ofMaize , Sorghum and 'Coix ' 275

modifier genes, the level of -y-zein and the degree of modification. (Figure 7)(Geetha et al., 1991; Lopes and Larkins, 1991). There is a slight negativecorrelation between endosperm modification and accumulation of lysine-richnon-zein proteins (Lopes and Larkins, 1995), consistent with earlier reportsthat some decrease in lysine and tryptophan are associated with modificationof 02 endosperm (Ortega and Bates, 1983; Bjamason and Vasal, 1992). Theincreased -y-zein in QPM is the result of elevated steady-state levels of -y-zeinmRNA (Geetha et aI., 1991). Furthermore, the 02 modifiers act in a polarfashion on the -y-zein locus , since a much greater effect is observed on thelevel of A gene than B gene transcript. Because the level of -y-zein genetranscription is similar between 02 and modified 02 mutants, it appears thatthe modifiers increase the steady-state level of -y-zein transcripts through apost-transcriptional mechanism (Or et aI., 1993).The genes responsible for endospermmodification remain uncharacterized,

but progress has been made in identifying the number and location of geneticloci that are responsible for phenotypic conversion. Segregation ofF2 progenygenerated from a cross between 02 and modified 02 suggest that modificationis controlled by two independent loci (Lopes and Larkins, 1995) . Since -y-zeinprotein accumulation is consitently correlated with endosperm modification,it is not surpri sing that RFLP-mapping data point to the -y-zeinlocus itself as astrong candidate for an effector of the vitreous phenotype (Lopes et aI., 1995).Specifically, the tandem arrangement of A and B -y-zein genes is required,although not sufficient for endosperm modification. A second modifier locuswas mapped near the telomere of chromosome 7L, and presumably encodesa trans-acting factor that affects the stable accumulation of -y-zein.

4.3 .3.0paqueI5The 015 mutation, which negatively affects -y-zein accumulation, has beenmapped to the same location on chromosome 7L as an 02 modifier gene(Dannenhoffer et aI., 1995). This mutation results in a 2-3 fold reductionin , -zein synthesis, coincident with a reduction in the number, but not thesize of protein bodies. Interestingly, 015 preferentially reduces the level ofthe -y-zein A gene mRNA. This polar effect implies a post-transcriptionalmechanism since 5' promoter sequences are virtually indentical between theA and B genes. The 015 gene has not yet been isolated ; however, it is temptingto speculate that it is a mutant allele of an 02 modifier, since both map tothe same location and both control -y-zein synthe sis. This is encouraging fordeveloping gene-tagging strategies to identify modifier genes , because it ismuch easier to screen for tagged opaque mutants than to screen for mutationsthat fail to modify 02 endosperm.

4.3.4. Floury2The fl2 mutation results in a general decrease in zein synthesis and increasedaccumulation of non-zein proteins, similar to 02. There is also a measurable


Fig. 8. The degree of modification of 02 maize endosperm is correlated with the amount of-y-zein. An SDS-polyacrylamide gel of total zein protein s from W64A02(0), Pool 34 QPM(3) and their reciprocal crosses ( I, 2) is shown on the left ; the position of 27-kDa -y-zein ismarked and the number of doses of modi fier genes (0-3) indicated at the top of each lane.Cross-sections of these kernels are shown on the right, with the number corresponding to thedosage of modifier genes in the endosperm,

g, + g, ~ ~~~ + " .... .. .. .. .. .. ..

~~+ + ~ ~ + + g, g, ~ ~ g, g,1~ ~I1 2 3 4 5 II 7 8 9 10 11 12 13 1415 16

kD

45

Fig. 9. Transmission electron micrograph of prote in bodies in W64A ji2 maize endosperm.M: mitochondrion; bar: 0.5 J1.m .

reduction in polyribosomes in fl2 endosperm (Jone s 1978). Not only areft2 protein bodies smaller than normal, but they are also severly deformed(Figure 8). The pattern of zeins for the o2ft2 double mutant is more similar

The Prolamin Proteins ofMaize , Sorghum and 'Coix' 277

to the 02 parent, indicating that 02 is epistatic over fl2 (Figure 9). This isparticularly evident by observing the unusual 24-kDa a -zein that is typicallyfound in fl2 endosperm (indicated with a large arrow in Figure 9). Thisprotein is only present when there is at least one functional 02 gene. Also,the intensity of this protein band increases with dosage of the fl2 allele,reflecting the semi-dominant property of the mutation.The 24-kDa protein band can be resolved into at least four polypeptides

(Lopes et al., 1994) . Three of the polypeptides are the product of a gene, orgenes, loosely linked to the fl2 locus . It is not clear why these polypeptideshave a slower mobility in SDS-polyacrylamide gels than normal o -zeins, butthe fact that they are sometimes observed in normal endosperm indicates theyare not responsible for the fl2 phenotype. The fourth polypeptide has alwaysbeen observed infl2 endosperm, even in the absence of the other three proteinsdescribed above. The deduced amino acid sequence from the gene encodingthis polypeptide shows it is a typical 22-kDa a -zein except for an alanineto valine substitution at the C-terminus of the signal peptide, and a histidineinsertion in the seventh a-helical repeat (Coleman et al., 1995). N-terminalsequencing of the purified protein shows the signal peptide remains attached,suggesting the valine substitution is sufficient to block cleavage by signalpeptidase. The uncleaved signal peptide may serve as a membrane anchorand thus interfere with the normal process of protein translocation .The alterations in the primary structure of this a -zein protein are consistent

with the observed phenotypic characteristics of the ji2 mutant. It is not hardto envision overexpression of BiP in response to the disruption in proteintranslocation. Furthermore, a gain-of-function mutation such as fl2, wouldbe expected to behave dominantly or semi-dominantly, and additional alleleswould be difficult to obtain, as is the case withfl2. The inability of the mutanta-zein to move into the center of the protein body could create mutliple,hydrophobic foci on the surface of protein bodies , and thereby disrupt thenormal organization ofproteins in these structures. Twoother opaque mutants ,De *B30 and Me have phenotypes very similar to fl2; however, both of them aredominant (Salamini et al., 1979, 1983). These mutants exhibit reduced levelsof zein synthesis and increased levels of non-zein proteins very similar to fl2 .The De*B30 mutation has a more pronounced effect on the 22-kDa o-zeins,while Me appears to exert its effect in a more general sense . In both mutants ,protein body symmetry is lost , as in fi2, and there is a dosage-dependentincrease in the level of BiP (Boston et al., 1991; Marocco et al., 1991; Zhangand Boston, 1992). Interestingly, De*B30 maps to a cluster of 19-kDa a-zeingene s on chromosome 4L , suggesting the intriguing possibility that, like fi2,it also affects translocation or folding of an a-zein protein.

4.3 .5. Opaque7The maize 07 mutant exhibits a preferential decrease in accumulation of the19-kDa o-zeins, but a somewhat smaller reduction in all other zein classes

278 Craig E. Coleman et al .

is also observed (Di Fonzo et al., 1979). A synergistic relationship has beenproposed between 02 and 07 since double mutant kernels exhibit reducedlevels of both 19- and 22-kDa o-zeins (Di Fonzo et aI., 1980). This invitesthe possibility that 07 affects a regulatory locus encoding a transcriptionalactivator that interacts with 19-kDa a-zein genes, the same way in which02 interacts with 22-kDa a-zein genes. Unfortunately, there is no molecularevidence to support this notion, and the 07 locus remains uncharacterized.

4.3.6. Sorghum high lysine mutantsCertain mutants of sorghum with elevated levels of endosperm lysine havebeen identified and are referred to as high lysine (hi) (Singh and Axtell, 1973).The increased amount of lysine in these lines is presumably due to geneticmutation, since the trait is heritable, but, unfortunately, there are no reportsof the identification and characteri zation of the gene or genes responsible forthe phenotypic effects. These mutants exhibit reduced yield, similar to high-lysine mutants in maize; however, they differ from the maize mutants in thatthe elevation in lysine is accompanied by an increase in total protein (Ejeta andAxtell, 1987). The molecular phenomenon that underlies the overproductionof lysine in mutant sorghum lines is unknown, but it may be related to thesame mechanism as in maize (Habben et aI., 1995).

5. The Effect of Zein on Endosperm Traits

5.1. Lysine Content

Interest in maize opaque mutants is due to the fact that several of them arecharacterized by higher levels of lysine than normal. In this regard , the devel-opment of QPM is a significant achievement, as it remedies the phenotypicdefects of standard 02 mutants, while maintaining protein quality. Neverthe-less, the level of lysine in QPM is still below the standard set by the Foodand Agriculture Organization (FAO) of the United Nations. Further efforts toincrease the lysine content of maize and other cereals would be enhanced byknowledge of the physiological and biochemical basis for the enhanced levelof lysine-containing proteins in 02.As already discussed, the 02 mutation reduces synthesis of a subset of

a -zein polypeptides and indirectly decreases the level of all other zeins.Coinciden tally, there is an increase in the level of certain non-zein proteinsthat are rich in lysine (Habben et al. 1993). Among these are proteins typicallyoverexpressed in response to stress, such as chymotrypsin inhibitor, trypsininhibitor, and catalase. This is consistent with previous findings that levelsof RNase and trypsin inhibitor are also increased in 02 endosperm (Halim etaI., 1973; Wilson, 1975). This pattern of gene expression suggests that the 02mutation may induce a stress response, perhaps as the result of altered nitrogen


metabolism conditioned by the reduction in zein synthesis . Confirmation ofthis hypothesis awaits further characterization of the subset of proteins thatis elevated in high-lysine maize.Another protein that was found to be elevated in 02 endosperm is elongation

factor-l a (EF-I a) (Habben et al., 1995). In fact, a very high correlation wasobserved between the levels of lysine and EF-l a in 02 endosperm. Despitethe fact that 10% of the amino acid residues in EF-I a are lysine, it alonecannot account for the increase in lysine observed in 02 endosperm. Thisprotein was originally described as the factor responsible for the binding ofaminoacyl-tRNAs to the ribosome (Merrick, 1992), but it is unlikely that itselevation in 02 endosperm is related to this function . This is because otherprotein synthesis factors, such as elongation factor-2, that would be expectedto rise in parallel with EF-l a, do not exhibit the same relationship to lysinecontent. EF-I a is also known to be associated with the cytoskeleton (Merrick,1992; Durso and Cyr, 1994; Shiina et al., 1994), and this role provides a moreattractive explanation for the elevated levels of the protein in 02 endosperm .There is some evidence that the cytoskeleton is involved in the synthesisof zeins through an interaction with the rough endoplasmic reticulum thatsurrounds the protein bodies (Stankovic et al., 1993). A significant amount ofEF-l a cosediments with protein bodies in sucrose density gradients (Habbenet al., 1993) . Perhaps changes in the quantity or quality of zeins affects thenumber, morphology or surface area of protein bodies in such a way that itincreases the development of the cytoskeleton, indirectly affecting the levelof EF-la . Efforts to identify additional components that interact with thecytoskeleton and that are coordinately elevated in 02 endosperm are criticalto testing this intriguing possibility.

5.2. Kernel Hardness

Grain hardness is important to preserving maize seed during harvesting, andit also determines the quality of the milled product (Pratt et al., 1995). Whenthe amount of zein in the endosperm is reduced, such as in the 02, 07,015 and ji2 mutants, there is an accompanying loss of kernel density andhardness. Environmental effects that diminish zein synthesis, such as reducednitrogen, also result in grain that is soft and chalky, similar to the opaquekernel mutants (Tsai et al., 1978). The interior starchy endosperm of maizeand sorghum grain contains much less zein or kafirin, respectively, than theperipheral hard endosperm (Dombrink-Kurtzman and Bietz, 1993; Mazharand Chandrashekar, 1995). This observation led to the conclusion that theamount and type of prolamin in the endosperm is a strong determinant ofkernel hardness.The restoration of a normal phenotype in QPM varieties implies a correla-

tion between the amount of -y-zein in the endosperm and the hardness of thekernel (Wallace et al., 1990). Loss of seed density in 015 mutants provides


further evidence for the correlation between kernel density and -y-zeincontent(Dannenhoffer et al., 1995). Introduction of 02 modifier genes into normalgenotypes led to increased accumulation of -y-zein; however, the degree ofkernel hardness was impacted to only a certain level and it was not verywell correlated to the -y-zein content (Moro et al., 1995). In fact, the traitmost highly correlated to kernel hardness was the level of cysteine (Moro etal., 1995). This is interesting, since it is easy to envision that intermoleculardisulfide linkages might playa role in the interaction between protein bodies,and thus be important in determining kernel hardness. Clearly, there may beother cysteine-rich proteins, besides -y-zein, that provide disulfide linkages,although none have been identified.Perhaps zein composition influences kernel hardness only insofar as it alters

the size , shape, number and distribution of protein bodies. In 02, 07, andfl2endosperm,protein bodies are considerably smaller than normal, and, as in thecase of fl2 , severely malformed. In 015 endosperm the protein bodies have anormal shape and size, but there are fewer of them than in normal endosperm.The soft, starchy endosperm core also contains fewer protein bodies comparedto the vitreous outer endosperm. Differences in the shape and distribution ofstarch granules can also be observed between hard and soft endosperm. Hardendosperm consists of compact, polygonal-shaped starch grains that appearto be directly associated with protein bodies and the protein matrix , whilesoft endosperm is composed of spherical, loosely-aggregated starch grains(Robutti et al. 1974). Given the close association between protein bodies andstarch grains in mature endosperm, it is easy to see how alterations in the size,shape and distribution of protein bodies may ultimately affect the texture ofthe endosperm by changing the shape and distribution of starch granules.

6. Conclusion

There have been great advancements in our knowledge of maize, sorghumand Coix prolamins , but there is still much to be learned. We know how zeinsare spatially arranged within the protein body, but little is known about themolecular interactions that direct the formation of the protein body. Clearlythere are additional proteins involved in trafficking zeins to their site ofdeposition, but these remain to be identified and characterized. It is alsoimportant to learn how the quantity and quality of zein influences the structureof the protein body, and how its organization ultimately affects the texture ofthe kernel. There are many unresolved questions about the developmental andspatial control of zein gene expression.Another interesting aspect that remainsto be explored is the role of the cytoskeleton in protein body formation. Manyendosperm mutants with an opaque phenotype have been identified, but mostof them remain to be characterized and their effect upon zeins and proteinbodies determined. Additional opaque mutants may yet be found. It is clear

The Prolamin Proteins of Maize. Sorghum and 'Coix' 281

that the characteri zat ion of these mutants will help answer many questionsabout zein synthesis and protein body formation, and the development of thevitreous endosperm phenotype.

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