[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || The Storage Proteins of Rice and Oat

8. The Storage Proteins of Rice and Oat

DOUGLAS G. MUENCH and THOMAS W. OKITAInstitute ofBiological Chemistry. Washington State University. Pullman, WA 99164-6340.USA

ABSTRACT. Rice and oat are unique among the cultivated plants in that their seed reservetissues contain significant amounts of globulins and prolamins, the two classes of storageproteins that are present in plants . Unlike other cereals which accumulate prolamins as theirprimary nitrogen reserve, the major class of storage proteins in rice and oat are the gluteiins andl2S globulins, respectively. These proteins are highly homologous at the primary sequencelevel to the leguminous l lS globulins . This similarity in protein structure extends to themolecular level with regard to the organization and complexity of the multigene families thatcode for these proteins, and the biochemical and cellular events that lead to the synthesis,transport, packaging and assembly of these proteins into protein bodies. In contrast , the riceand oat prolamins share little sequence homology and only some of the cellular events that leadto their deposition into protein bodies . Rice packages prolamin and glutelin in separate proteinbodies, a process that may be initiated by the active sorting of their mRNAs to specific domainsof the endoplasmic reticulum, whereas the avenins and globulins of oat are deposited withinthe same protein inclusion body, although as separate protein aggregates. The expression of therice and oat storage proteins is regulated at the transcriptional and post-transcriptional levels.This is conspicuously evident in oat where globulin mRNAs are translated at a much higherrate than avenin mRNAs . In this review, we discuss the latest developments on the organizationand expresssion of the rice and oat storage protein genes and the cellular pathways that resultin the assembly and deposition of coded proteins into protein bodies, as well as recent geneticefforts to modify the protein quality of these cereals .

1. Introduction

The storage proteins function as a reserve of nitrogen, carbon, and sulfurand are synthesized during seed development and then utilized during ger-mination. These proteins, which lack any enzymatic or structural role, varyconsiderably in molecular weight and net charge from species to species.Despite these differences, the storage proteins share many properties : theyare a dominant class of proteins that are accumulated in seed tissue, they areassembled and packaged into membrane delimited protein bodies, and theyare rich in the amidated amino acids, glutamine and asparagine (Shotwelland Larkins, 1989). Based on solubility properties, the storage proteins wereinitially grouped into three classes: the salt-soluble globulins, the alcohol-soluble prolamins, and the insoluble glutelins. More recent studies on the

B.A. Larkins and IX. Vasil ieds.t. Cellular and Molecular Biology (~f Plan Sad D C\'e!0I'n!t'lII . 289- 330.© 1997 Kluwcr Academi c Publishers .

290 Douglas G.Muench and Thomas W. Okita

primary sequences of the storage proteins from many plant spec ies indicatethat these protein s fall into two broad clas ses , globulins and prolamins, withthe insoluble gluteiins sharing homology with either the prol amin s or globu-lins, depending on the plant spec ies (Shotwell and Larkins, 1989). The majorglobulins consist of two different species that sediment at 7S and I IS. Eventhough the 7S and liS globulins display very different molecular we ightsand possess different quaternary structures , their polypeptides share signifi-cant structural homology, indicating that the 7S and II S genes evolved froma common ancester (Argos et aI., 1985). The alcohol-soluble prolamins, themajor storage proteins of cereals (Kries et aI., 1985), comprise a much morediverse group of protein s at the primary sequence level (Bietz, 1982). Nev-ertheless , the prolamins can be classified as being either sulfur-rich (solublein alcohol solutions with reducing agent) or sulfur-poor (soluble in alcoholsolutions alone).Most plant species accumulate either globulins or prolamins in their seed

storage tissues. Rice and oat are atypical in that they accumulate both types ofstorage proteins in the endosperm (Juliano, 1972). In contrast to other cereals,the major storage protein in these plants is not a prolamin but a globulin whichcompri ses about 60-85% of the total protein on a weight basis (Colyer andLuthe , 1984; Li and Okita, 1993; Robert et al., 1985). Prol amins constitute arelatively small proportion of the total seed prot ein , 18 to 20% in rice (Li andOkita, 1993; Ogawa et aI., 1987) and 2.5% to 15% in oat (Boyer et al. , 1992;Colyer and Luthe, 1984; Peterson and Smith, 1976).Rice and oat , as well as the other major cereal spec ies, provide an impo rtant

source of protein for humans and livestock (Shotwell and Larkin s, 1989). Theoverall quantity and quality of the seed protein is dict ated by the contentand amino acid composition of the storage proteins. The globulins are low inmethionine and cysteine, while the prolamins are low in lysine, threonine andtryptophan. Despite being the major food for many people in Asia, Africa andSouth America , rice contains the lowest protein content (ca. 5%) of the majorcereals. Moreover, a considerable percentage of this pro tein is not easilydigested by monogastric animals (Resurreccion and Juliano, 1982; Tanakaet aI., 1975b). On the other hand , oat seeds contain not only a high proteincontent (ca. 15%) but also have a much better balance of amino acid s than theother major cereals, due to their high globulin, low prolamin ratios (Boyeret aI., 1992). In view of their importance as food , efforts have been aimedat improving the qualit y and quantity of the oat and rice storage proteinsthrough plant breeding methods. Although much of this earlier work has beenunsuccessful, the implementation of new screening procedures to identifygenetic traits that influence protein quality and quantity, together with theemployment of modem genetic enginee ring approaches, have improved theprospects of modifying the protein quality of these cerea ls. Such approacheshave also been employed to identify rice lines that are deficient in protein,a trait desired by the Japanese sake indu stry. In this chapter, we discuss

The Storage Proteins ofRice and Oat 291

the structure, organization and regulation of the oat and rice storage proteingenes, the structures of the coded proteins, the biochemical and cellularprocesses responsible for their packaging within protein bodies, and effortsto genetically manipulate their levels.

2. The Storage Proteins of Rice

2.1. Structural Chara cteristics ofRice Storage Proteins

Glutelins . The insoluble glutelins comprise about 75% of the total seed pro-tein in rice (Li and Okita, 1993). Despite their general insolubility, the riceglutelins display a subunit composition, heterogeneity, and charge distribu-tion similar to that of the 12S globulins of oat and 11 S globulins of legumes,suggesting that these proteins are homologous (Robert et aI., 1985; Wen andLuthe, 1985). When analyzed by SDS polyacrylamide gel electrophoresis ,a single major polypeptide band of 55-57 kD is evident under nonreducingconditions while two major polypeptide bands at 20-23 kD (basic subunits)and 32-35 kD (acidic subunits) are observed under reducing conditions. Thispolypeptide pattern is similar to that displayed by the lIS globulins (Julianoand Boulter, 1976; Robert et al., 1985; Wen and Luthe, 1985; Yamagata etal., 1982). This relationship to the l lS globulins is also evident as seen inresults obtained by pulse-chase studies where the 55-57 kD polypeptide bandis post-translationally processed to yield the acidic and basic subunits (Luthe,1983; Yamagata et al., 1982). Additionally, in vitro translation of endospermmembrane-bound polysomes yielded only the 55-57 kD glutelin, suggestingthat the acidic and basic subunits are proteolytic products of this precursor(Furuta et aI., 1986; Yamagata et al., 1982).A structural relationship between the rice glutelin and 11S globulins was

established by the near identical N-terminal amino acid sequences obtainedby Edman degradation of their basic subunits (Zhao et al., 1983) and byimmunological cross -reactivity studies (Krishnan and Okita, 1986; Robert etaI., 1985). The extent of homology between the rice glutelin and lIS globulinswas determined by the comparison of the primary sequences of these proteinsas derived from cDNA sequences (Higuchi and Fukazawa, 1987; Masumuraet al., 1989a; Okita et al., 1989; Takaiwa et al., 1986; 1987a; 1989; Wang etal., 1987). Rice glutelins share about 30-35% amino acid sequence identityto the lIS storage proteins (Higuchi and Fukazawa, 1986; Takaiwa et aI.,1986; 1987a). Such structural similarity is evident not only in the primarysequence of the mature polypeptide but also in the signal peptide . Moreover,the positions of cysteine residues involved in linking the two subunits of thesoybean glycinin (Staswick et al., 1984) are also conserved in the rice glutelin,suggesting that these residues playa similar role in maintaining the secondarystructure of the rice glutelin (Higuchi and Fukazawa, 1987; Takaiwa et aI.,1986).


Although the rice glutelin and 11S globulins share extensive sequencehomology, this similarity is not distributed uniformly throughout the primarysequences of these proteins. The basic subunits from these proteins are moreconserved than the acidic subunit, and within each subunit the first half ismore conserved than the second half (Higuchi and Fukazawa, 1987) . Themost divergent region is near the C-terminus of the acidic subunit. Thisregion, labeled the hypervariable region by Argos et aI. (1985), appears totolerate large insertions of variable size and charge. This is quite evident inthe hypervariable region of the rice glutelin, which is highly hydrophobicand may account for its reduced solubility as compared to the negativelycharged hypervariable region of the lIS globulins (Higuchi and Fukazawa,1987). In addition to the hypervariable region, the acidic subunit containstwo additional but smaller regions that display sequence variabil ity amongthe glutelins and lIS globulins (Okita et aI., 1989; Takaiwa et aI., 1991a).The capacity of the hypervariable region, as well as the two smaller variableregions, to tolerate mutations suggests that they may be potential targets forgenetic manipulation (Argos et aI., 1985).

Prolamins . The alcohol-soluble prolamins comprise about 18 to 20% ofthe total endosperm protein in rice (Li and Okita, 1993; Ogawa et aI., 1987).This value is much higher than originally estimated (Juliano, 1972) , sinceprolamins are more efficiently solubilized in 50% n-propanol than in otheralcohol solutions (Sugimoto et aI., 1986) . The prolamins exhibit considerablepolymorphism with regard to their sizes. In the rice japonica variety Kinmaze,the major prolamin is a 13 kD species with two minor species at 16 kD and10 kD (Kumamaru et aI., 1988) . In M201 , an early California japonica line,only a single prolamin band at 14 kD is observed (Krishnan and Ok ita, 1986),whereas Tainung 67 possesses two major prolamin specie s at 15.5 kD and14.2 kD (Shyur et aI., 1994). Based on the analysis of cDNA sequences,the 13-16 kD prolamins are structurally related, while the 10 kD speciescomprises a distinct class . The 13-16 kD prolamins can be divided into twoclasses based on their content of sulfur-containing amino acids.The polymorphism evident between different rice lines is also evident with-

in a line itself. Analysis of the prolamins from Kinmaze (Hibino et aI., 1989)and Tainung 67 (Shyur et aI., 1994) by two dimensional polyacrylamide gelelectrophoresis revealed nine and 10 species, respectively, having isoelectricpoints between pH 6.0 to 8.1. This polymorphism at the protein level of the13-16 kD prolamins is reflected by the complexity of cDNA sequences iso-lated. Kim and Okita (1988a, b) identified two classes of prolamins basedon restriction enzyme physical maps, extent of cross-hybridization, and thededuced amino acid sequence from cDNA clones. Class I prolamins containhigh levels of glutamine and hydrophobic amino acids and are low in lysine,histidine and sulfur-containing amino acids (Kim and Okita, 1988a, b; Shyurand Chen, 1990; Shyur et aI., 1992). Class II prolamins differ from class I pro-lamins in that their coded proteins contain a high content of sulfur-containing


amino acid s (3% methionine and 5.9% cysteine).The amino acid sequences ofclass I and II are 54-63% identical, with major differences due to two blocksof amino acid insertions/deletions and clusters of amino acid replacements(Kim and Okita, 1988b). A third cla ss of prolamin was isolated by Masumuraet al. (1990) and Shyur and Chen (1993) . The class III prolamins containabout the same methionine and cysteine content as class II, but share only71% identity, differences being attributable to amino acid delet ions ratherthan substitutions (Masamura et al., 1990; Shyer and Chen , 1993). A verysulfur-rich cDNA belonging to the less abundant 10 kD class of prolamins hasbeen isolated (Masumura et al. , 1989b ) and shows little homology to the otherprolamin classes. Aside from molecular weight, this protein is distingui shedby its high levels of methionine (20%), cysteine (10%), glutamine (16%) andproline (6%) residues.The primary sequences of the rice prolamins are distinct from those in

maize and the small grains cereals of the Triticeae, e.g wheat and barley(Bietz, 1982) . Prol amin s of the major cereal s conspicuously possess tan-dem repeats of a con served proline-rich peptide whereas, the rice prolaminslack this feature. Despite this major difference in overall structure, the riceprolamins share two peptide motifs with other cereal prolamins. The signalpeptide is almo st identi cal to the maize zein leader sequence (Masumura etal. , 1990) and displays moderate homology to the signal peptides of other pro-lamins. A common octapeptide consensus sequence (QQQCCQQL) which isconserved in the sulfur-containing prolamins is also found in the prolamin sfrom maize, wheat, rye and barley (Masumura et al., 1990). The rice pro-lamin s are immunologically distinct from the prolamins of wheat and oat, butantibodies against them are weakly reactive with prolamin polypeptides ofmaize, sorghum and barle y (Okita et al., 1988; Shyer et al., 1994).

Globulin s. The salt-soluble globulins, representing only 2 to 8% of thetotal endosperm protein , comprise a minor component of the storage protein sin rice. The globulin fraction is composed of two polypeptides of 23-27 kDand 16 kD (Krishnan et al., 1992; Komatsu and Hirano, 1992; Luthe, 1992;Perdon and Juliano, 1978; Tanaka et al., 1980; Yamagata et al. , 1982). Thelarger 26 kD species, called a -globulin, appears to run anomalously on SDSpolyacrylamide gels (Pan and Reeck, 1988). Molecular mass estimations bygel filtration (Perdon and Juliano, 1978) and (more definitively) sedimentationequilibrium (Pan and Reeck, 1988), however, indicate a much smaller sizeof 16.7-18 kD. Both the 26 kD a -globulin and 16 kD globulins are initiallysynthes ized as preproteins about 2,000 daltons larger than the mature form,sugges ting the presence of a signal peptide (Krishnan et al., 1992). This viewis con sistent with the transport and deposition of these polypeptides in theglutelin-containing PB-II protein bodi es (Krishnan et al., 1992; Tanaka et al.,1980).Recombinant cDNA clones have been isolated for the a -globulins by

screening a Xgt expression library using anti-globulin . The cDNA sequence

294 Douglas G.Muench and Thomas W Okita

contains a single open reading frame that codes for a 19 kD polypeptidepossessing a 22 amino acid signal peptide. The amino acid compositionof the derived protein is similar to the measured compositions of purifieda -globulins, being rich in glutamine/glutamate and arginine residues anddeficient in lysines (Krishnan and Pueppke, 1993; Pan and Reeck, 1988;Shorrosh et aI., 1992). Several peptide stretches of the a -globulin share sig-nificant homology to the wheat glutenins. Thi s structural relationship wasalso evident immunologically, as antibodies raised against the a-globulinscross-react with the wheat high molecular weight glutenins (Krishnan andPueppke, 1993).Komatsu and Hiramu (1992) have characterized an a -globulin from the rice

variety Norin, 29 which has biochemical properties distinct from those iso-lated in other varietie s. Although Norin exhibits a similar apparent molecularmass on SDS polyacrylamide gels and shares short stretches of homology withthe wheat glutenins, as well as other known a -globulins, its amino acid com-position is distinct. In addition to being rich in glutamine/glutamate, it containsvery high levels of glycine (20.6 mole%) and significant amounts of lysine(5.2 mole%). The high mole percentage of glycine and glutamine/glutamateresidue s is also a feature of the wheat glutenins and supports the close struc-tural relationship of these proteins. Because of the significant levels of lysine,the Norin a -globulin may be an ideal candidate for genetic manipulationefforts to increase protein quality of this cereal.

2.2. Mutants Affecting Protein Quality in Rice Seeds

The nutrition al value of rice glutelin is superior to prolamin due to its greaterdigestability by human s and higher lysine content (Ogawa et al. 1987; Tanakaet al. 1975a; 1975b). Researche rs have used mutagene sis as a tool to modifyprotein concentrations in an effort to increase the protein quality and quantityof rice seeds. A number of mutants have been isolated which show variationsin the normal profile patterns of storage proteins. These were obtained byscreening approximately 3000 rice lines mutagenized by treatment of fertil-ized egg cells with N-methyl-N-nitrosourea for variations in protein profilesby SOS-PAGE (Kumamaru et aI., 1988) . Seventeen mutants were isolatedwhich had different protein profiles as compared to the parent plant varietyKinmaze. These mutant s were categorized into four cla sses represented bymutant lines ern 21, em 1675, em 1787 and em 1834 (Figure I). Relative tothe Kinmaze profile, em 21 contains lower amounts of the 13b kD prolaminpolypeptides while em 1675 contains lower levels of IO kD , 13a kD and 16kO prolamin polypeptides. em 1834 possesses high level s of the 10 kD and16 kO polypeptides, but low amounts of the 13b kO polypeptide band. Theern 1787 mutant is much different than the previou s three, in that it containshigh amounts of the 57 kO proglutelin polypeptide, and low amounts of theglutelin acidic and basic subunits. In addition to having different prol amin


01. wt.r, 103

)

57

37 -39 (

2622-23 (

161310

1 2 3 4 5

Fig. I . 50 S-PAGE analysis of storage proteins extracted from the endosperm of the ricevariety Kinmaze (lane 1), and mutants ern 21 (lane 2), em 1675 (lane 3), em 1787 (lane 4) andlane 5 (eM 1834). From Kumamaru et al., ( 1988) with permission of the author and publisher.

compositions, em 21 and em 1834 have increased glutelin levels, and althoughthey have reduced amounts of the 13 kD prolamins, their overall prolamincontent is higher than Kinmaze (Ogawa et al., 1989).The prolamin protein bodi es of the 13 kD prolamin deficient mutants,

em 21 and cm 1834, display a distinct morphology from those observed inKinmaze (Ogawa et al., 1989). The protein bodie s from these mutants lackthe typical concentric ring s, and they are more uniformily electron dense .Thi s change is consistent with the view that the sulfur-rich prolamins (classesII-IV) are assembled in the central core of the protein body while the sulfur-poor prolamins (class I) are located at the periphery (Hibino et aI., 1989;Masamura et al., 1989b; Resurreccion et al., 1993). The lower levels of thesulfur-poor 13b kD prolamin s would therefore result in a protein body ofuniform electron density.Clvl 1787 , which has 1O-fold higher amounts of the 57 kD proglutelin and

corresponding reduced level s of the acidic and basic subunits, also accumu-lates protein bodies which differ in size, shape and electron density fromthose observed in wild -type endosperm cells (Ogawa et aI., unpubl.). Insteadof the usual concentric ringed PB-I and irregularly shaped PB-lI, all of the


protein bodies are of small, uniform size (0.5 /-Lm in diameter) and of uniformelectron density. Based on biochemical analysis of protein body fractionspartially purified by sucrose density gradient centrifugation and immunocy-tochemical analysis of thin sections of developing endosperm, Ogawa et al.(unpubl.) concluded that em 1787 is defective in transporting the 57 kD prog-lutelin between the ER to the vacuole due to a reduction in Golgi function.This not only results in a high proportion of proglutelin not being processedinto subunits but also their retention in ER-delimited vesicles (Ogawa et aI.,unpubI.).Inheritance patterns of several of these mutant lines show that they are

the result of single gene mutations (Kumamaru et aI., 1987; 1990). em 21and em 1787 are inherited as single gene, recessive traits. Interestingly, em1675 and em 1834 show maternal expression of nuclear encoded genes, asthe endosperm of seeds produced from the cross of em 1675 x Kinmaze andem 1834 x Kinmaze always shows the protein profile of the maternal parent.Analysis of segregating F2 plants indicated that em 1675 is controlled by asingle recessive gene, whereas em 1834 is controlled by a single dominantgene. The basis of the maternal expression is not known. These single genemutants appear to have pleiotropic effects since the expression of severalpolypeptides is modified in each mutant.Iida et al. ( 1993) have isolated a mutant, nm 67, showing a reduced amount

of glutelin and a high content of prolamin. nm 67 has reduced band intensityof all of the polypeptides in the acidic and basic fractions of glutelin, includingthe complete loss of one of the acidic polypeptides. The 13 kD prolamin bandis increased in intensity in this mutant. This trait is controlled by a singledominant gene. Since prolamin containing PB-I is not digestable by pepsin(Ogawa et al. 1987), a mutant such as this may be important for individualsrequiring a low protein diet, e.g. those with kidney disease (lida et aI., 1993).Such a low protein mutant may also be useful for the sake industry wherethe use of high protein rice results in the formation of fermented wine withseveral undesirable traits.Screening of storage protein mutants will likely be an important approach

to improve seed protein quality. Initial nutritional studies of the four emmutants in the diet of rats demonstrated that em 1787 has the highest proteinquality based, on amino acid content and digestability (Eggurn et al., 1994).The enhanced protein quality of em 1787 warrants further effort in mutationalbreeding efforts.

2.3. Organization and Structure ofRice Storage Protein Genes

Glutelins - The genes encoding both the glutelin and prolamin polypeptidesare organized in complex multigene families. Glutelins are composed of twolarge subfamilies, A and B, which share 60 to 65% identity at the amino acidlevel (Takaiwa et aI., 1991a), with each subfamily containing several classes


of genes. The divergence is readily apparent in the signal peptide, wherethe leader sequence from the A subfamily is homologous to that of the 11Sglobulins from pea and soybean, while the 5' leader from subfamily B showsalmost no similarity other than the sequence CXXLLCXGS. Other regions ofdivergence between these subfamilies are the hypervariable region and twosmaller variable regions in the acidic subunit and the C-terminal region of thebasic subunit (Takaiwa et al ., 1991a).A number of genes have been characterized in each subfamily (Okita et

aI., 1989 ; Takaiwa et al. 1987b; 1991a; Takaiwa and Oono, 1991). The Asubfamily is composed of at least three classes, AI , A2, and A3 (Takaiwa andOono, 1991; Takaiwa et al ., 1991a). Comparable genes were isolated by Okitaet al. (1989) and called Gt2, Gtl, and Gt3, respectively. Each class containsabout five to eight gene copies. Gtl (A2) and Gt2 (Al ) have coding regionswhich are more similar to each other at the nucleotide levels (95%) than theyare to Gt3 (81%). Specific portions of all three glutelin clones, however, arequite similar. The 5' regions from -183 bp to the translation start sites of thesegenes show a high degree of sequence similarity, and S I nuclease mappingdemonstrated that transcription start site s are conserved between all membersof the A subfamily. Other conserved regions include the TATA and CAATboxes and polyadenylation signal sequences. 5' flanking sequences upstreamof -183 are conserved between Gtl and Gt2, but not between Gtl/Gt2 andGt3 . This divergence in the presumed promoter regions between Gtl/Gt2 andGt3 is consistent with the observed steady state patterns of their mRNAsduring seed development. Gtl/Gt2 transcripts steadily increase throughoutseed development, whereas Gt3 transcripts attain a max imum level at about10 days post-anthesis and then decline at subsequent stages (Ok ita et al.,1989).Takaiwa et al. (1991 a) isolated three members of the B subfamily. Each

of these classes, denoted as B I, B2 , and B3, were estimated to contain fivecopies per haploid genome. Although one of these genes is a pseudogene,all members contain three introns within the coding regions located at thesame sites as those in subfamily A. The exons are 80% to 88% homologouswithin this subfamily and 60% homologous to subfamily A members. Strongsequence conservation is evident at the site of post -translational cleavage,which forms the basic and acidic glutelin subunits, as found in the lISlegume storage protein genes (Takaiwa et aI., 1991a).The nucleotide similarity between the coding regions of the l lS legume

storage protein genes and the rice glutelins does not extend to the regulatoryregions. Differences between their promoters are expected as these two genesare active in different tissue types, the glutelins in the endosperm and the lISstorage proteins in the embryo (Okita et aI., 1989). In addition to the codingsequence conservation between the glutei ins and 1IS globulins, the positionof the three introns within the coding sequences are similar. The relatednessof their coding regions indicates that, despite the lack of enzymatic activity


and cellular structural role, these proteins remain evolutionarily conserved.This suggests that the primary amino acid sequence may be important in thesynthesis, transport, and/or assembly of these proteins in the protein body(Okita et al., 1989).

Prolamins - At least ten prolamin cDNAs and three genomic DNAs havebeen cloned so far from rice (Feng et al., 1990; Kim and Okita, 1988a, b;Masumura et al., 1989b; 1990; Shyer and Chen, 1990; 1993; Wen et al.,1993; Yamagata et al., 1992). Alignment of the cDNAs to their respectivegenomic sequences indicates that the coding sequences and 3' untranslatedregions are devoid of introns. Therefore, the rice prolamin genes appear to besimilar to other cereal prolamin genes in lacking introns . Close examinationof the divergence of the 5' untranslated sequences in the 13-16 kD prolamincDNAs, however,may suggest the presence of such a structural unit. Despitethe relative high abundance of prolamin mRNAs (about 5% of total poly (A)+RNA), full-length cDNA sequences for the 13-16 kD have not been isolated.As estimated by Northern blot analysis (Kim and Okita , 1988b; Masumuraet al., 1990), the prolamin transcript is about 900 nucleotides in length. Thecoding sequence and 3' untranslated region comprise about 445 nucleotidesand 107 nucleotides, respectively. Assuming a poly (A)+ tail of about 100nucleotides, the 5' untranslated region is about 250 nucleotides in length. ThecDNA clones containing the longestmRNA sequences were identified by Kimand Okita (1988b). The 5' untranslated region of pProl 7 is 200 nucleotides inlength; the closely related pProl 14 contains only 164 nucleotides, while theclass II prolamin, pProl 17, has only 165 nucleotides in the 5' untranslatedregion. Inspection of these cDNAs revealed a bipartite arrangement of DNAsequences in their 5' untranslated regions. Sequences upstream of -60 fromthe translation initiation codon are G-C-rich, whereas sequences downstreamfrom this site are A-T-rich. The proximal domain of the 5' untranslated regionof Prol 7 and Prol 14 shows very close identity to the corresponding region ofthe genomic clone, whereas the distal domain diverges from the genomic DNAsequences (Kim and Okita, 1988b). Likewise, the pProl 17 sequence divergesfrom its corresponding genomic DNA sequences at almost the identical site(Okita , unpubl.). The divergence at -60 bp between the class I and II prolamincDNAs and their corresponding genomic clones may be due to the presenceof an intron. Although the typical 3' splice site consensus sequences are notobserved, the sequences at -60 resemble those from the unique 3' splicesite of intron 2 of the rice endosperm ADP-glucose pyrophosphorylase gene(Anderson et al., 1989).The prolamin genes of rice are in much higher copy numbers than the

glutelins. Similar to the prolamin genes of maize (Shotwell and Larkins,1989) and wheat (Reeves and Okita, 1988), the prolamin gene family in therice cultivar M201 consists of 80 to 100 copies per haploid genome (Kimand Okita, 1988b). Somewhat lower copy numbers were estimated by otherlaboratories, which may reflect the use of different rice lines and/or different


A

B

a

• • • ••b

••••• •c d

• • • • •

510152025 5 10 15 2025 5 10 15 20 25OAF

510 20 25

Fig . 2. Temporal accumulation of glutelin and prolamin mRNAs in developing rice seeds.A) Dot blot analysis of total RNA (2 Ji g, upper row; 0.2 Jig, lower row) B) Northern blot oftotal seed RNA. Blots were probed with Gt l (panel a), Gt3 (panel b), Prol 14 (panel c), Pro117 (panel d). RNA was isolated from seeds at various days after flowering (DAF). From Kimet al., (1993) with permission of the author and publisher.

experimental procedures employed in hybridiz ation . Masumura et al. (1990)showed that under stringe nt hybridization and washing conditions there wereabout 7 copies of the class II prolamin s. This value was supported by Shyerand Chen (199 3), who estimated that there were 30 to 50 genes encoding theclass I, II and III prolamin s. At least some of these genes are tightly clusteredin the rice genome . A lambda phage genomic clone isolated by Kim andOkita (l 988b) contained four tandemly repeated 2.5 kb EcoRI fragment s,each containing class I prolamin gene sequences (Kim and Okita, 1988b).

2.4 . Expression of Rice Glutelin and Prolamin Genes

Analysis of the steady state levels of glutelin and prolamin transcripts andresults obtained by nuclear run-on transcription studie s demonstrate that thesegenes are not coordinately expressed during seed development. In 5 day-old developing seeds, there are equimolar amounts of glutelin and prolamintranscripts (Kim et aI., 1993). Thi s glutelin/prolamin mRNA ratio, however,subsequently declines such that by 15 days, there is a 40% molar excessof prolamin transcripts. The non-equivalent expression of storage proteingenes is also reflected within a gene family. The mRNAs of the Gtl and Gt2classes are present in 5 day old developing seeds and their levels continu eto increase throughout seed development. In contrast, the Gt3 class mRNAsatta in maximum levels at 10 days after flowering, which later steadily declineduring seed development (Kim et aI., 1993; Figure 2). Transcript s of the Bsubfamily increase from 6 to 14 days after flowering, and then decrease in a


A0 ·25 0 ·5 2 4n9

Globin

B

c 50

20

a: 10

o

Gt 1 Gt 3 AoI14 Pro.tr Bluesctipt

~~r-. ~

~ ~~ ~ I-~ ~~ ~~ ~~ ~~ ~

~ ~~ ~~ ~- u rL

15

DAF

5 OAf

15 OAf

• Gil[J GI3

• Pro! ,.

Pro! 17

0 B SC~

Fig. 3. Nuclear run-on transcription analysis of rice prolamin and glutelin gene expression.A) Dot blot hybridization to obtain an arbitrary standard curve used to estimate the extent ofstorage protein mRNA hybridization. B) Nucle i were isolated from seeds harvested 5 and 15days after flowering (OAF) and radioact ive run-on tran scripti on was performed. The resultingRNA was hybridized to DNA dot blots each containing cDNA plasmid of Gt l, Gt3, Prol 14,Prol 17 or bluescript plasmid . C) Densitometric scans of dot blot analysis from panels A and Bwere obtained and the relative transcriptional activities plotted. From Kim et al. , (1993) withperm ission of the author and publisher.

manner similar to that observed for Gt3 mRNA (Takaiwa et al., 1991a). Theinduction of the different prolamin mRNA classes also does not appear to becoordinate (Figure 2). During the early stages of seed development , the steadystate mRNA levels of class II (Prol 17) genes are higher than those of theclass I (Prol 14) genes. At later stages (25 days post-anthesis), the tran scriptlevels of the two gene classes are equal (Figure 2; Kim and Okita, 1988b;Kim et.al., 1993; Shyur et al., 1992).


TABLE I

Seed storage protein levels (nmol/seed)"

Days after flowering

Protein 10 15 20 25

Prog lutelin

a -glutelin

,B-glutelin

a -glutelin/,B-glutelin

Total glu telin"

Prolamin

Glutelin/pro lamin

% prolamin of totalstorage protein"

0040504

5.2

1.04

5.7

3.31.7 (6 .2)C

13.6

0.9

20.2

22.00.92

22.0

14.7

1.5 (5.4)

15.4

1.4

29.0

30.0

0.97

30.9

25.3

1.2 (4.4)

17.4

2.849 .0

49 .0

1.0

51.8

43.31.2 (4.3)

18.6

a Average from three indepe ndent experiments.b Repre sents the total levels of prog lutelin assuming no proteolytic processing.C Numbers in parenthesis repre sent the ratio of glutelins to prolamins on aweight basis .d On a weight basis.

Transcriptional activ ities of glutelin and prolamin genes as measured bynuclear run -on transcription assays are not consistent with the estimatedsteady-state levels of their mRNAs. The run-on transcript ional activity of theclass I prolam in genes is quit e high compared to class II in 5 day old seeds(Figure 3) , where as the class I steady-state mRNA levels are low as comparedto class II (Figure 2). Converse ly, the Gt I/Gt2 run-on transcription rate is aboutone quarter of that observed for Gt3 in 5 day-old seeds (Figure 3), even thoughthe steady state levels of these transcripts are about equal (Figure 2; Kim et aI.,1993). These data indicate that glute lin and prolamin mRNA concentrationsare controlled at both the transcriptional and post-transcriptional levels.Quant ification of prolamin and glutelin polypept ide levels were estimated

by immunoblot analysis of storage protein from developing seed (Li andOkit a, 1993). At 10 days post-anthesis glutelin synthesis predominates suchthat there is a 58% molar excess of glutelin polypeptides over prolamins(Table 1). In older develop ing seeds, prolamin synthesis exceeds that forglutelin, which results in only a 17% difference in the molar levels of thesepro teins at 25 days post-anthesis. The greater abundance of glutelin thanpro lamin on a weight basis is due mainly to the large differences in themolecul ar mass of these proteins (52 kD vs 14 kD; Table 1). Pulse-chaselabell ing studies of developing seeds are also consi stent with the observationthat prolam in synthesis increases during the latter half of seed development. In15 day-old seed, there is a 39% molar excess of 14C-leucine incorporation into


glutelin over prolamins . In 20 day-old seeds, however, the incorporation ratesinto these storage proteins are the same, suggesting that they are synthesizedat the same rate (Li and Okita, 1993). Therefore, the higher levels of prolamintranscripts over glutelin transcripts during mid to late seed development isconsistent with the increase in prolamin synthesis during this period. Theabundance of total glutelin polypeptides over total prolamin polypeptidesthroughout development, however, suggests that the synthesis of prolamin andglutelin is controlled at the translational level as well. Kim et al. (1993) haveshown that glutelin mRNAs are associated with membrane bound poly somesat a higher frequency than prolamin mRNAs, as more prolamin transcriptsare found free in the cytoplasm. The increased recruitment of glutelin mRNAto the membrane may account for the difference in translation efficiencybetween the glutelins and prolamin s.

Promoter Analysis - A number of potential regulatory motifs that maycontrol the expression of the prolamin and glutelin genes have been identifiedby DNA sequence analysis, gel retardation assays, DNase I footprinting andpromoter deletion analysis in transgenic plants. One regulatory motif thatplays a major a role in overall quantitative expression of several monocotstorage protein genes is the 'endosperm box ' or '-300 element' (Colot et aI.,1987; Kries et aI., 1985; Matske et al., 1990; Takaiwa et aI., 1991b; Thomasand Flawell, 1990). Elements similar to the '- 300 element' are present at sixto seven copies in Gtl and Gt2, but only once in Gt3. These differences inthe promoter regions of Gtl/Gt2 and Gt3 suggest that these genes are notsubject to identical regulatory processes, a view supported by the differentaccumulation patterns exhibited by their mRNAs during seed development.Therefore, even closely related genes of the same subfamily are not controlledidentically.Several glutelin genes of the A subfamily have been the focus of study to

identify the role of regulatory elements responsible for endosperm-specificexpression. Leisy et al. (1989) showed that 980 bp of the Gt3 promoter 5' tothe transcription start site was able to direct expression of a chloramphenicolacetyl transferase (CAT) reporter gene construct in developing seeds of trans-genic tobacco. Some non-seed expression was observed in some but not allplants, suggesting some leakiness in the control of the transgene . MaximumCAT activity occurred 10 days prior to the peak level of endogenous proteinaccumulation, indicating that the temporal expre ssion ofthe transgene was notcoordinate with tobacco storage protein production. This temporal pattern ofpromoter activity is consistent with the fact that development of the endospermprecedes development of the embryo, the site of storage protein accumula-tion in tobacco seeds. In a follow-up study using a f3-g1ucuronidase (GUS)reporter system, the same Gt3 promoter was expressed solely in developingendosperm of transgenic tobacco seeds (Zhao et aI., 1994). This 5' promoterdeletion analysis of the Gt3 promoter sequence identified two domains, -346to -263 (domain I) and -945 to -726 (domain II), which were required for


maximum expres sion of a GUS reporter gene in transgenic tobacco. Deletionof domain II resulted in a decrease in GUS activity, as well as a shift in tem-poral expression from 16-20 days after flowering to about 24 days. Deletionthrough domain I completely eliminated expression .Therefore, domain I con-tains elements important for quantitative and endosperm-specific expression,whereas domain II is required for temporal and quantitative control (Zhao etaI., 1994).A series of 3' deletions of the Gt3 promoter (-945 bp to +7 bp from

the transcription initiation site) linked to a 90 bp constitutive cauliflowermosaic virus (CaMV) core promoter sequence was also analyzed in transgenictobacco (Croissant-Sych and Okita , 1996.). Other than the expression in roottissue directed by the -90 CaMV 35S core promoter sequences (Benfeyet a!., 1989), reporter gene activity with the intact Gt3-CaMV 35S corepromoter hybrid was only observed in developing endosperm. Moreover, thetemporal GUS expression pattern during seed development was identical tothat observed for the Gt3 promoter alone. These observations indicate that theGt3 regulatory motifs responsible for spatial and temporal expression functionin combination with the core promoter elements of CaMV35S. Successive 3'deletions showed that two regions of the Gt3 promoter, +7 bp to -181 bp and-278 bp to -482 bp, were essential for maximal expression. GUS activity wasreduced about 50% upon deletion from +7 to -181. This reduction was likelydue to the removal of three possible regulatory elements (the AACA motif,and Boxes I and II, see below) present in all A subfamily genes. Successivedeletions from -278 to -482 resulted in a gradual decrease in promoteractivity, a result consistent with those obtained from the 5' promoter deletionanalysis . The region between -320 and -482 bp also contains a negativeregulatory element, as its deletion resulted in the appearance of significantGUS expression in leaf tissue (Croissant-Sych and Okita, 1996).The identification of regulatory regions of the Gt3 promoter by in vivo

promoter deletion studies are supported by in vitro protein-DNA interactionexperiments. Three seed-specific DNA binding activities were located withindomain I and domain II as determined by gel retardation and DNase foot-printing experiments (Zhao and Okita, 1994). The DNA binding activities ofthese nuclear factors coincide with the steady state levels of the Gt3 mRNAsduring seed development. One of the binding activities (BP-l) interacts withthe domain I containing sequence CAACACA. This sequence motif, whichis conserved in the 5' flanking regions of a number of other monocot anddicot genes (Goldberg, 1986; Kries et aI., 1985; Maier et a!., 1988; Mullerand Knudson, 1993; So and Larkins , 1991), is similar to the opaque-2 bind-ing motif (Schmidt et a!., 1992). Domain II contains two DNA binding siteslocated between -861 bp to -838 bp and -837 bp to -788 bp. Both segmentsare rich in A-T nucleotides. Although the exact binding sequences could notbe discerned , both segments contain known sequence binding motifs. The


distal sequence contains a modified octomer binding site while the proximalsequence contains two modified '-300-Iike elements' (Zhao and Okita, 1994).Promoter analysis of an A2 (Gt 1) gene demonstrated that only 44 I nucleo-

tides of the 5' regulatory region were required for proper spatial, temporaland quantitative expression of a GUS reporter gene in tobacco (Takaiwa etaI., I991b). Significant GUS activity was observed in endosperm tissue ofdeveloping seeds while only background levels were found in the developingembryo, stem or leaves. Further deletion from --441 bp to -237 bp resultedin a complete abolishment of GUS activity in developing seeds. Inspectionof the promoter region downstream of --441 revealed the presence of threedirect repeats each containing homology to the '-300 bp endosperm box'.Gel retardation and DNase I footprinting analysis of the 5' regulatory region

of a glutelin Gt2 gene identified five boxes that interact with nuclear proteins(Kim and Wu, 1990).These putative regulatory elements are located at - 103 to-86 (Box I), -164 to -146 (Box ll), -122 to -1 08 (Box Ill), -206 to -189 (BoxIV) and -595 to -575 (Box V). Boxes I and II are common to all of the genesof the A subfamily and may be responsible for the partial reduction in reportergene activity when removed from the Gt3 promoter (Crissant-Sych and Okita,unpub!.) . Gel retardation and methylation interference footprinting of the A2(Gtl) promoter demonstrated binding of a seed-specific protein to a regioncorresponding to Box II of the Gt2 promoter (Takaiwa and Oono, 1990) . BoxesIII, IV and V are only evident in the Gt2 (AI) and Gtl (A2) genes, implyingthat these elements may be important in the differential expression of mRNAseen between the Gtl/Gt2 and Gt3 genes. Elements within Boxes I and IIIshowed similarities to the '-300 element' of cereal promoters and containpotential Jun/GCN4-like and octomer-like transcription factor binding sites(Kim and Wu, 1990).Zheng et al. (1993) analyzed the 5' promoter region of the Gt 1 gene in

transgenic rice, and demonstrated that a major regulatory element is locatedfar upstream from the transcriptional start site. Deletion of a promoter segmentfrom -5.1 kb to -1 .8 kb resulted in a 20-fold decrease in GUS expression.Deletion from -1.8 kb to -507 or-399 bp did not lower expression significant-ly, however, each reduced the frequency of transformants showing high GUSactivity. Deletion to -214 completely eliminated GUS expression. Simulta-neous substitution mutations corresponding to the protein binding boxes I toIV in the A2 class promoter described by Kim and Wu (1990), had a drasticeffect on transcriptional activity of a promoter construct containing the -1.8kb of the 5' upstream region, suggesting that these elements interact synergi s-tically. Of these elements , box II appears critical as its removal reduced the-1.8 kb promoter activity 35-fold. The se authors also showed that the TATAbox and a conserved 13 base pair element conserved in all glutelin genes (theAACA motif; Takaiwa et aI., 1991b) are critical cis-elements, as mutationsin these regions in the -1.8 kb promoter construct lowered the expression inthis promoter region 10-fold or greater.

The Storage Proteins of Rice and Oat 305

Few prolamin genomic clones have been isolated from rice (Feng et al.,1990; Kim and Okita, 1988b; Wen et al., 1993), and only a few promoterdissection studies have been performed on these genes. The promoter region-680 to -18 of a class I prolamin gene could direct endosperm-specificexpression of a GUS reporter gene in transgenic tobacco (Zhou and Fan,1992; 1993). Two putative upstream regions (named prolamin box I andprolamin box II) were identified, each of which contained a -300 box-likesequence. Successive 5' deletions downstream to -470, to -430 and to -290retained the ability to direct transcription in an endosperm-specific manner intransgenic plants. Quantitative GUS assays showed that plants transformedwith promoter fragment -470 had eight times the expression of the -430promoter fragment. The region between -470 and -430 contains prolaminbox II, suggesting that this sequence is important in quantitative expression(Zhou and Fan, 1995).

2.5. Synthesis and Deposition ofRice Storage Proteins

Since rice seeds produce both major types of storage protein, the modes bywhich these proteins are synthesized and packaged into protein bodies arelikely different from those observed for other cereals, which utilize prolaminas their major storage protein. Analysis of developing endosperm cells byelectron microscopy revealed the presence of two morphologically distinctprotein bodies, spherical electron-lucent protein bodies (PB I) and irregularlyshaped electron-dense protein bodies (PB II; Bechtel and Juliano, 1980;Harrisand Juliano, 1977; Ivanova, 1974; Krishnan et al., 1986;Ogawa et al., 1987;Tanaka et aI., 1980). Early electron microscopic studies of these two types ofprotein bodies suggested that they were formed by different cellular processes.The spherical PB-I was initially described as being cytoplasmic and boundedby a double membrane, whereas PB-II is irregular and secreted into thevacuole (Ivanova, 1974). The spherical PB-I has been categorized as eitherlarge or small. The large ones were shown to form within the cisternae ofrough endoplasmic reticulum (RER), had a lamellar-like structure, and wereenclosed by a single, ribosome studded membrane (Bechtel and Juliano,1980). The smaller spherical PB-I were thought to represent a distinct part ofthe population that formed later in development, around 14DAA (Bechtel andJuliano, 1980; Bechtel and Pomeranz, 1978). They formed within the ER likethe large smooth protein bodies, but they differed slightly morphologically,lacking dense centers and concentric rings (Bechtel and Juliano, 1980). Theirregularly-shaped PB-II, often called crystalline protein bodies, are depositedinto the vacuole and are often associated with the Golgi apparatus, suggestingthat this structure is involved in their synthesis (Bechtel and Juliano, 1980).Small spherical Golgi vesicles are thought to discharge into larger vesicles,which in tum discharge into the crystalline protein body vacuole (Oparka andHarris, 1982).


..

a

•

Fig .4. Protein-A gold immunocytochemistry showing the localization of glutelin and prolaminin rice endosperm cells . a) Section showing the specificity of prolamin antibody to the sphericalprolamin protein body (S). Arrowhead indicates the attachment of the RER to these proteinbodies . GL, glutelin protein body. b) Electron-dense vesicles are labelled with glutelin antibodyaround the Golgi apparatus (G). Smaller electron-lucent vesicles labelled with glutelin antibody(arrowheads) are located proximal to the Golgi apparatus. Bar equals I mm . From Krishnanet al., (1986) with permission of the author and publisher.


Tanaka et aI. (1980) obtained relatively pure preparations ofPB-I and PB-II.Biochemical analysis revealed that the PB-I are highly enriched for prolaminsand PB-II contains glutelins and globulins. Immunocytochemical analysis ofthin sections of developing endosperm using antibodies specific for prolaminand glutelin confirmed this observation (Krishnan et aI., 1986). Glutelinswere only detected in the irregularly shaped electron dense protein bodies,while the prolamins were evident in both the large and small spherical proteinbodies. In addition, many of the anti-prolamin antibodies reactive to sphericalprotein bodies also showed a direct attachment to the rough endoplasmicreticulum. This observation provides clear evidence that the spherical PB-I areproduced by the direct deposition of prolamins into the ER lumen (Figure 4a;Krishnan et al., 1986), a process similar to the formation of maize proteinbodies containing zein (Shotwell and Larkins, 1989). In contrast, vesiclescontaining proteinaceous deposits reactive to glutelin antibodies are eitherroutinely observed in close proximity to the Golgi complex or appear directlyassociated with this organelle (Figure 4b; Krishnan et aI., 1986). In the sub-aleurone layer, i.e, the outermost 2-3 cell layers of endosperm, anti-glutelinwas observed to react with proteinaceous deposits in the vacuole, suggestingthat these proteins are packaged in the vacuole via the Golgi complex in thebulky endosperm.When viewed at the electron microscopic level, the irregularly shaped PB-

II appear to have a complex internal organization (Krishnan and Pueppke,1993). The single protein inclusion body appears to be composed of distinctprotein aggregates exhibiting differential staining. This view is supported bythe location of globulin polypeptides within PB-II as determined by immuno-cytochemistry. The a-globulin and 16 kD species are co-localized in thedarkly staining areas of the PB-II which are usually located at the peripheryof the inclusion body. Vesicles associated with the Golgi complex were alsolabeled with anti -globulin, indicating a role for this organelle in the transportof globulins to PB-II (Krisknan and Pueppke, 1993).

The Segregation ofStorage Protein mRNAs on Rough ER - The assembly ofprolamin polypeptides within the ER lumen to form an intraci sternal proteingranule raises a que stion regarding protein trafficking. Although glutei insand globulins are transported to a separate subcellular compartment fromthe prolamins, they nevertheless are initially located in the ER lumen, fromwhere they are transported to the Golgi complex. In view of their commonlocalization within the ER lumen, rice endosperm cells must utilize very effi-cient sorting processes so that prolamin depo sition in the ER lumen does notimpede the transport of glutelins and globulins to the vacuolar compartment.Efforts to understand how prolamin and glutelin are packaged into two dif-

ferent compartments led to the novel discovery that rice prolamin and glutelinmRNAs are not randomly distributed on the ER membranes, but instead aresegregated on distinct ER types. Some indirect, yet very interesting, data sup-port the hypothesis for the segregation of the glutelin and prolamin mRNAs


to specific ER membranes. Yamagata et al. (1986) showed that polyadenylat-ed mRNA isolated from purified PB-I directed the in vitro synthesis of onlyprolamins. These results suggest that prolamin mRNAs are highly enrichedon the ER membranes that delimit these protein bodies. Kim et al. (1993)demonstrated that glutelin mRNAs were present at more than two-fold excessover prolamin transcripts in membrane-bound polysome fractions isolatedfrom crude microsomal fractions. As these microsomal fractions are highlyenriched in rough ER membrane vesicles, the biased distribution of glutelinmRNAs to the microsomal fraction suggests that these transcripts are enrichedon ER membranes.Li et al. (1993a) conducted a comprehensive biochemical and in situ

hybridization study to assess the intracellular location of the rice storageprotein mRNAs. As viewed by electron microscopy, developing endospermcells possess two morphologically distinct ER membranes: the cisternal ER(C-ER) consisting of single lamellar membranes distributed throughout thecell, and the ER that delimits the prolamin protein bodies (PB-ER; Li et aI.,1993a). Subcellular fractions enriched for both types of ER membranes wereobtained and analyzed for their mRNA content. The results revealed thatglutelins are present at a 2-fold molar excess relative to prolamin transcriptsin the C-ER enriched fraction, whereas prolamin transcripts are present at ala-fold molar excess over the glutelin transcripts in the PB-ER fraction (Liet aI., 1993a). High resolution in vitro and in situ hybridization techniqueswere employed to confirm the distribution of the mRNAs to their respec-tive ER membranes (Li et aI., 1993a). Hybridization of prolamin and glutelinRNA probes to a chemically fixed protein body fraction demonstrated that theprolamin mRNAs are associated specifically with the surface of the prolamin-containing spherical protein bodies (PB-I). Consistent with the relative levelsof mRNAs estimated by blot hybridization, there is greater than Ifl-fold moreprolamin mRNA than glutelin mRNA associated with prolamin protein bod-ies. Very little hybridization of either probe occurred with the glutelin proteinbodies. Immunogold in situ hybridization of ultrathin rice endosperm sec-tions was performed to confirm that the segregation of mRNA molecules invivo (Li et aI., 1993a). Significant hybridization of antisense glutelin probeto C-ER membranes was visible, whereas antisense prolamin probes gavegreatly reduced hybridization signals to those membranes. A ratio of 2.3:1glutelin :prolamin was obtained, again similar to the blot hybridization datadescribed earlier.Double in situ hybridization using digoxigenin and biotin to label antisense

prolamin and glutelin probes allowed similtaneous quantification of eachprobe (Figure 5; Li et aI., 1993a). The results from these experiments agreedwell with those obtained by in vitro hybridization and blot hybridization.Therefore, irrespective of the technique employed, the same conclusion wasattained. Prolamin mRNAs are specifically localized to the PB-ER, whereasglutelin mRNAs are enriched on the C-ER. The initial targeting process of


C· ERo

(0 30 "T-"------------,

Fig. 5. Double-label in situ hybridization of rice prolamin and glutelin mRNAs in endospermsections. Antisense prolamin RNA probe was labelled with digoxigenin and antisense glutelinRNA was labelled with biotin . A) C-ER membranes showing preferential binding of glutelintranscripts (5 nm gold particles, barely visible) over prolamin transcripts (15 nm gold particles) .B) Protein body (P) showing the abundance of prolamin transcripts associated with the PB-ER.C) Gold particle densities from C-ER and PB-ER. Bar equals 0.25 J-tm. From Li et al., (1993a)with permission of the author and publisher.

310 Douglas G. Muench and Thomas W Okita

the glutelins and prolamins to their distinct protein bodies is facilitated by thesegregation of their transcripts on the C-ER and PB-ER, respectively.The targeting of a number of mRNAs to specific regions of the cytoplasm

has been observed in many animal cells (summarized by Okita et al., 1994;St. Johnson, 1995). The basis for such intracellular localization is obvious formany maternal or zygotic mRNAs of eggs and oocytes, and in specializedcell types with pronounced polar structures, such as neuronal cells, fibroblastsand epithelial cells. The most widely accepted mechanism which accountsfor the non-random localization of mRNAs is a stepwise localization pathway(Yisraeli et aI., 1990). This involves cellular factors that recognize specificsignals on the mRNA and facilitate its transport to a specific intracellularlocalization. Upon reaching its destination, the mRNA is then anchored,thereby ensuring translation at the destination site. Both mRNA transportand anchoring processes appear to rely on components of the cytoskeleton.Yisraeli et al. (1990) have shown using cytoskeletal inhibitors that micro-tubules are involved in the transport of Vg I to the vegetal half of the oocyte,while microfilaments are important for anchoring of the RNA to the cortex.Likewise, localization of bicoid to the anterior pole of the Drosophila eggis dependent on microtubules (Pokrywka and Stephenson, 1991), whereasmicrofilaments are suspected in the transport of actin mRNAs (Sundell andSinger, 1991).Recent studies are beginning to decipher the signals and mechanisms

responsible for mRNA localization. The localization of (Y- and fJ-actin(Kislauskis et al., 1993), Vgl (Mowry and Melton, 1992), bicoid (Macdonaldand Struhl, 1988), nanos (Gavis and Lehman, 1992), oskar (Kim-Ha et aI.,1991) and pair rule transcripts (Davis and Ish-Horowicz, 1991) are dependenton their 3'-untranslated regions . The targeting signal of bicoid (Macdonaldand Struhl, 1988) and Vg1 (Mowry and Melton, 1991) spans several hun-dred nucleotides, suggesting multiple cis-elements for protein binding thatare responsible for the various steps in RNA movement and anchoring tospecific regions of the cell. This has been demonstrated for oskar, where the3'-untranslated region contains separate elements which mediate differentsteps in mRNA movement and localization (Kim-Ha et al., 1991). Very littleinformation is known about the proteins that recognize these signal elements,although genetic and biochemical studies (St. Johnson et al., 1992; Schwartzet al., 1992) have identified candidates that may be involved in the transportprocess . Protein factors involved in RNA anchoring have yet to be identified.Preliminary evidence suggests that prolamin mRNAs are attached to the pro-tein body surface by an RNA binding activity, in addition to the ribosomereceptor of the protein translocation complex. The association of mRNA tothis second RNA receptor is detergent resistant but salt sensitive (Okita etaI., unpubl.). This stepwise localization pathway is one possible mechanismwhereby prolamin and glutelin transcripts are segregated to different domainsof the ER (Figure 6b).


PB·ER

(c)

Glutelin po lysome

C·ER

Secre tory pathway

(b) mRNA withouttopoqernc Signal(e.g . glutelin?)

5' - ~~ 3'

/Chain irutranon and dOCKing

Secretory pathway

Prolamin e polysom e

(d)

Glutelin mRNA

ProlaminemRNA

Rough ER f J' I! BI~O-'"

~~'l~• ~B"

/ ~~,~p

C·ER

Fig. 6. Possible mechanisms for sorting of mRNAs to different parts of the ER. a) Differentialtrafficking of mRNA in the embryonic epithelial cells of Drosophila . The nucleus separates theapical and basal parts of the cell, and asymetrical export ofmRNA from the nucleus could allowfor segregation of mRNAs to apical and basal ERs for translation . b) Pretranslational sortingof prolamin mRNA to the protein bodies (PB) by association of the transcript with a specificreceptor located near or at the protein body surface. c) Cotranslational sorting suggests thedifferential recognition of signal peptides of nascent prolamin and glutelin to a heterogenouspopulation of SRPs which associate to different domains of the ER. d) Post-translationalsorting of prolamin mRNA on PB-ER by enrichment due to the association of prolamin withthe lumenal chaperonin, BiP. From Okita et al., (1994) with permission of the author andpublisher.

312 Douglas G.Muench and Thoma s W Okita

In addition to the stepwise localization pathway, there are three otherpossible mechani sms that could account for the nonrandom localization ofstorage protein RNAs on the ER. In embryonic cells of Dro sophila, severalRNA species are localized speci fically to the apical and basal regions ofthe cell (Figure 6a; Banerjee et aI., 1987). As the nucleus appears to serveas a physical barrier between the two sub-compartments, these RNA s arelocalized to apical or basal regions by asymmetric export from the nucleus.This mechanism, however, does not appear to be applicable in the rice system,as confocal microscopic analysis indicates that the C-ER/PB-ER membranecomplex and nucleus are not polarly arranged. Instead, the ER complexis located symmetrically around the nucleus near the periphery of the cell(Franceschi, unpubl. ).Two other possible mechanisms of mRNA locali zation are dependent on

translation. One possibility is that there are multiple pathways leading tothe translat ion of mRNAs on the ER. The signal peptides of prolamins andglutelins may recognize distinct SRPs which, in tum, are recognized by spe-cific SRP receptors located on the C-ER and PB-ER, respectively (Figure 6c;Li et aI., 1993a; Masumura et al. , 1990 ; Okita et aI., 1994). Consistent withthis mechanism of co-translational sorting via specialized signal peptides andSRPs is that the rice storage protein s have very distinct signal peptides. Asfirst recognized by Masumura et al. (1990), the prolamin signal peptide dis-plays significant homology to the maize zein leader sequence. As the maturecoding sequences of these proteins are totally unrelat ed other than a commonoctomer (QQQCCQQL) motif, the similarity of their signal pept ides may beresponsible for the common intracistern al site of protein deposition (Li et aI.,1993a). The glutelin signal peptide bears no resemblance to the rice and maizeprolamin signal peptide s. The hydrophobic region of the signal peptide con-tains a unique sequence (CXXLLCXGS) which is the only conse rved motifin the leader sequence between the glutelin A and B subfamilies (Takaiwa etaI., 1991a). Such a motif may be recognized by a distinct SRP species andtransported directly to the C-ER.Alternatively, enrichment of prolamin RNAs on the PB-ER may be the

end-result of the assembly process of the mature polypeptide. Bec ause ofthe high ionic strength of the ER lumen and the hydrophobic nature of theprolamins, it was simply assumed that prolamins assembled into an intracis-ternal protein granule by a spontaneous, self-directed process. This view wassupported by the translation of zein transcipts in Xenopus oocytes , where thenewly synthesized zein polypeptides assembled into struc tures possessing asedimentation on density gradients similar to native protein bodi es (Wallaceet aI., 1988). If this assembly process occurred during the translocation of thenascent polypeptide across the ER into the lumen, then it may stabilize theER-association of the translational complex.Despite the attractiveness of this self-assembly model for intracistemal

protein body formation, this mechanism is not applicable, as proper folding


and assembly of prolamin polypeptides is dependent on BiP, the lumenalchaperone (Li et aI., 1993b). Results from immunocytochemical studies andimmunoblot analysis of enriched ER and protein body fraction s revealed thatBiP is primarily distributed within the PB-ER, and accounts for about 4%of the total protein in the protein body fraction (Li et aI., 1993b). Detergentwashed protein bodies retain about 70% of BiP, indicating that BiP asso-ciates with the membrane-free components of this organelle. BiP is presentas a complex with mature prolamin polypeptides, as demonstrated by the co-sedimentation of BiP and prol amin as a large molecular weight complex andby the ability of anti-prolamin to co-immunoprecipitate BiP and mature pro-lamin polypeptides (Li et aI., 1993b). This BiP/prolamin complex is sensitiveto ATP. BiP also forms an ATP-insensitive complex with nascent polypeptidechains attached to polysomes. The presence of nascent chain/BiP complex,as well as the salt-stable but ATP-sensitive complex observed with both theintact prolamin polypeptides and the protein aggregate, suggests that BiPserves to retain prolamin polypeptides within the ER in addition to its role inprotein folding and assembly. Moreover, the existence of the two complexessuggests that the assembly of newly synthesized prolamin onto the proteinaggregate is not strictly a co-translational proces s, but that the translocationof prolamin and assembly onto the protein aggregate may be sequentiallyindependent events. The restricted localization of high levels of BiP to theperipheral regions of the PBs and the presence of distinct populations of BiP-prolamin complexes are con sistent with a model where prolamin folding andassembly to form protein bodies is mediated by BiP. This model proposes thatBiP initially bind s to the nascent prolamin polypeptide in an ATP-insensitivemanner as it moves through the ER membrane,maintaining the polypeptide ina competent state for deposition into the protein body. After protein synthesisis completed, BiP remains complexed to the prolamin polypeptide to facilitatefolding in the ER lumen. Upon interaction of this complex with the proteinbody surface, it dissociates at the expense of ATP hydroly sis, and BiP is thenrecycled. Thi s dissociation and recycling explains the presence of BiP at theperiphery of the protein body. Initial aggregation of prolamin into a proteinbody structure would likely occur when a critical level of BiP/prolamin com-plex is reached within the ER lumen, resulting in the aggregation of prolaminpolypeptides (Li et aI., 1993b).Although the involvement ofBiP in prolamin PB formation appears distinct

from the localization ofprolamin RNAs to the PB-ER , there may be a possiblerelationship between the two processes. The formation of a stable complexbetween BiP and the nascent prolamin chain could potentially stabilize theassociation of the translation complex onto the PB-ER. The net effect wouldbe an increase in the residence time of prolamin transcripts on the ER and,in tum, result in preferential enrichment of these transcripts on the PB-ER(Figure 6d). This mechanism may be responsible for the several-fold increaseof prolamin mRNA over glutelin mRNA on the PB-ER (Okita et aI., 1994).


3. The Storage Proteins of Oat

3.1. Structure of the Oat Globulins and Avenins

12S Globulins - Like rice, oat accumulates both globulins and prolamins(avenins). The mature oat seed contains about 15 to 20% protein, of whichthe relative amounts of avenin and globulin protein were initially estimatedat about 10-15% and 70-85%, respectively (Colyer and Luthe, 1984; Frey,1951; Peterson and Smith, 1976). More recent protein analysis by Boyeret al. (1992) have demonstrated the globulins to be 26-fold more abundantthan avenins on a weight basis. These authors noted that the differences inthe relative amounts of these storage proteins were due to variable extrac-tion methods employed by different investigators or to the different cultivarsexamined. Early studies demonstrated that the complete solubilization of oatseed globulins required much higher salt concentrations (I M NaCl) than thenormal 0.4 m solution used to extract the 11 S globulins from legumes (Peter-son, 1978).Other than this higher salt requirement, however, the oat globulinsshare many properties with the liS globulins . The native globulins sedimentat a value of 12S corresponding to a globular molecular mass between 327and 369 kD (Brinegar and Peterson, 1982a; Peterson, 1978). Electrophoresisof reduced and unreduced globulins indicated that globulins are composedof acidic and basic subunits of 32.5-37.5 kD and 22-24 kD, respectively,linked by a disulfide bond to yield a 53-58 kD heterodimer (Brinegar andPeterson, 1982a; Peterson, 1978; Walburg and Larkins, 1983). Based on themolecular mass estimations , the structure of the native globulin is deduced tobe a hexamer of 53-58 kD heterodimers, a quaternary structure identical tothe 11 S globulins (Shotwell and Larkins, 1989). In addition, the globulins aresynthesized as preproproteins, as cleavage of the signal peptide and process-ing into the acidic and basic subunits occurs (Adeli et aI., 1984; Brinegar andPeterson, 1982b; Walburg and Larkins, 1983). Amino-terminal amino acidsequencing of the basic polypeptide reveals a direct relationship to the basicsubunits of the lIS globulins of Glycine, Vicia and Pisum species (Walburgand Larkins, 1983).The isolation and sequencing of oat globulin cDNAs confirmed the relat-

edness of the oat globulins to the II S globulins of legumes and rice glutelins(Shotwell et al., 1988; Walburg and Larkins , 1986). The deduced primarysequence of a full-length globulin cDNA, pOG2, shows 31% amino acididentity with soybean glycinin, 38% identity with pea legumin, and 70%identity with rice glutelin. The corresponding protein has a signal peptideof 24 amino acids followed by an acidic polypeptide of 293 amino acidsand a basic polypeptide of 20 I amino acids. The deduced protein sequenceis rich in the amidated amino acids, glutamine and aspargine, which com-prise about 20% of the total amino acid content. It is also characteristicallydeficient in sulfur-containing amino acids, a feature seen in other globu-


lins (Shotwell et a!., 1988). Each subunit contains only a single conservedcysteine residue, indicating that only a single disulfide bond links the twosubunits. One unique characteristic of the oat globulins is the presence ofan eight amino acid, glutamine-rich peptide sequence repeated four or fivetimes in the hypervariable region of the acidic polypeptide (Shotwell et a!.,1988). As the hypervariable region is believed to reside on the surface of theoligomeric protein in contact with the solvent (Plietz and Damaschun, 1986),these repeats confer to this region of the protein a very hydrophobic charac-ter, which may account for the requirement of higher salt concentrations forsolubilization of the protein.The close identity of the oat 12S globulin with the rice glutelin reflects the

direct phylogenetic relationship between oat and rice. Although highly homol-ogous at the primary sequence level , the oat 12S globulin and rice glutelindisplay three major structural differences. In addition to the glutamine-richoctomer peptide repeat in the hypervariable region , the oat polypeptide con-tains several additional amino acid residues at the carboxy terminus of thebasic polypeptide, and lacks a highly charged peptide (RREVEER) that islocated in the middle of the acidic subunit of the rice glutelin (Shotwell et a!.,1988).

Avenins - Early studies showed that the oat prolamin, avenin, is mostefficiently extracted from endosperm tissue with 45% ethanol (Kim et aI.,1978). Eight bands of avenin protein were visualized by starch gel elec-trophoresis and were divided into three classes designated as a, (3, and ry(Kim et a!., 1978; Robert et a!., 1983). More sensitive two-dimensional gelelectrophoresis resolved 12 avenin polypeptides with sizes ranging between22 and 37 kD (Pernollet et al., 1987). Three of these polypeptides, one aand two ry , were sequenced by Edman degradation. The three proteins havevery similar amino termini and display a consensus repeat of a seven aminoacid peptide PFVQQQQ. Other than a common (3-tum secondary structurein the N-terminal region, these proteins show little homology with the aminotermini of prolamins from other cereal s. Pernollet et aI. (1987) suggestedthe (3-turn structure may be important for the compact deposition patterns ofthese proteins within the protein body.A more complete amino acid sequence of the avenins was deduced from

cDNA sequences (Chesnut et a!., 1989). The derived primary sequences ofthree avenin cDNA clones share 50-72% identity to each other, and are richin glutamine and proline residues. Consistent with the strong immunologicalrelationship with the low molecular weight hordeins of barley and with the ry-secalins of rye (Festenstein et aI., 1987), the avenin primary sequences possessa close sequence (>68%) and structural relationship to the wheat a-, (3- and-y-gliadins and the related barley B-hordeins (Chesnut et a!., 1989). Likewheat and barley prolamins, the avenins can be divided into seven regions: a19 amino acid signal peptide, amino and carboxyl terminal regions containingunique sequences, two regions of tandem repeats separated by a 63 amino acid


conserved region, and a short polyglutamine tract. Both tandem repeats areglutamine-rich, the first having a repeat length of six to eight amino acids andthe second having eight to eleven amino acids. Three to five of these repeatsare present in each protein. These clones represent two subfamilies of aveninsand appear to correspond to the (Y- and -y-type avenin polypeptides. A cDNAisolated by Fabijanski et al. (1988) was found to be very sulfur-rich and hadhigh homology with oat globulin . This clone , however, did not resemble anyof the other recombinant avenin sequences, and likely represents a differentavenin class or an artifact.

3.2. Organization and Structure ofOat Storage Protein Genes

The complex pattern of 12S globulin and avenin polypeptides observed bypolyacrylamide gel electrophoresis (Chesnut et aI., 1989; Fabijanski et aI.,1985) suggests that, like seed proteins from other plants, these proteins arecoded by complex multigene families. This view is substantiated by thediverse sequence polymorphism displayed by cDNA and genomic clones (seebelow) and by Southern blot hybridization studies. Reconstructive Southernblot analysis of genomic DNA ofAvena sativa indicated that there are approx-imately 25 avenin genes and 50 globulin genes per haploid genome (Chesnutet aI., 1989). The main cause for the multiplicity of storage protein genesis due to the hexaploid nature of Avena sativa, which is composed of A, Cand D genomes. Although the progenitor of the D genome is not known, itis clear that the A and C homoeologous chromosomes do not donate equiva-lent gene copies, especially for the globulins. Potential progenitors of the Agenome, e.g. A. hirtula and A. longiglumis, contain about 20 globulin copiesper haploid genome, whereas A . clauda and A . pilosa, potential progenitorsof the C genome, possess only 3-6 copies (Chesnut et aI., 1989; Potier, 1994).At least some of the hybridizable restriction fragments on Southern blots areprobably due to pseudogene sequences, some of which have been cloned forboth globulin and avenin genes (Potier, 1994; Shotwell et aI., 1990).Based on cross hybridization of isolated globulin cDNAs, four classes of

globulins were identified (Walburg and Larkins, 1986). Four oat globulingenomic clones have been characterized and appear to represent two of thefour gene classes. Clones OG I-El (Shotwell et aI., 1990) and asglof (Schu-bert et aI., 1990) have 99% DNA sequence identity to one another, but differfrom two other genomic clones, Glav-l and Glav-3 (Tanchak et aI., 1995). TheGlav clones are highly homologous in the coding and non-coding regions.Although the deduced amino acid sequences of Glav-l and Glav-3 are approx-imately 85% identical to OG I-El and asglofi, their 5' and 3' non-coding DNAsequences differ substantially. The differences in the 5' upstream region sug-gest that these genes may be subject to different regulation at transcription(Tanchak et aI., 1995).


The transcriptional initiation site ofOG l-E1resides 38 base pairs upstreamof the ATG initiation codon, and putative TATAand CAATboxes are presentas well (Shotwe ll et aI., 1990 ; Tanch ak et aI., 1995). The sequence context atthe translation init iation site of the globulin genes is typical for plant genes,containing seven of nine nucleotides in the plant consensus AACAATG-Gc. Polyadenylat ion sites are located at approximately 30 and 80 base pairsdownstream of the termination codon in all four genomic clones . In general,the globulin genes contain three introns, located at similar positions in the11S globulins (Shotwe ll et aI., 1990 ; Tanchak et aI., 1995). An exceptionis observed in the Glav-l genomic clone (Tanchak et a!., 1995). This genecontains an additiona l intron (intron 2') which contains the standard splicesite consensus sequences, and a new exon (exon 2' ). Exon 2 ' is unique as it iscomposed of two tandem heptamer repeats followed by an 11 residue repeti -tion of the residues at the C-terminal end of exon 1. It is not known whetherthe Glav-I gene is functional , as a portion of its 5' flanking sequence wasunable to direct signifi cant express ion of a GUS reporter gene in transgenictobacco (Potier, 1994).Few avenin genomic DNA clones have been isolated. A single genomic

clon e from a lambda library contained four avenin gene copies (Shotwell etaI., 1990). Sequence analysis showed that one of clustered genes belonged tothe -y-avenin subfamily, whereas a second belonged to the a-avenin subfami-ly. The isolation of this genomic DNA fragment demonstrates that the aveningenes within and between subfamilies are clustered in the genome (Shotwellet aI., 1990). The transcriptional start site of one of the avenin genomic clones(AY45-X 1) resides 73 bp upstream of the ATG start codon, and potentialTATA and CAAT sequences are present at - 38 and - 68. Polyadenylationsequences are located at 80 bases and 139 bases downstream of the termin a-tion codon (Shotwell et aI., 1990). An interesting characteristic of the aveninsequence is the presence of inverted sequence repeats in the amino acid repeatreg ions which could potentially form secondary structures in the correspond-ing mRNAs. As described later, these secondary structures may playa role intranslational regul ation of the avenins (Shotwell et aI., 1990).

3.3. Expression of Oat Globulin and Avenin Genes

The globulins and avenin s are first visible by Coomassie blue staining orimmunoblot analysis of seed extracts six days after anthesis, and both continu eto increase in amounts up to 16 days after anthesis (Chesnut et aI., 1989;Luthe, 1987). Consistent with this pattern of storage protein accumulation,the steady state levels of globulin and avenin mRNAs increase in parallelbeginning at 4 DAA and atta in a maximum level at 8 DAA (Chesnut et aI.,1989). Th is coordinate accumulation of the storage protein mRNAs is not,however, reflected in the 5' flanking regions of these genes, which do not


appear to share any common potential regulatory sequences (Potier, 1994;Shotwell et aI., 1990; Tanchak et aI., 1995).Quantitative estimations of globulin and avenin mRNA levels indicated that

these storage proteins are under both transcriptional and post-transcriptionalcontrols. Although there are twice as many globulin genes as avenin genes,the avenin mRNA levels are equal to or greater than those for globulin (Boyeret aI., 1992; Chestnut et al., 1989). Assuming that the number of pseudogenesequences is the same for the avenins and globulins, the avenin genes are eithertranscribed at higher rates than the globulin genes or the globulin mRNAs arenot as stable as the avenin transcripts (Chestnut et aI., 1989).The most conspicuous level of gene regulation affecting the expression

of the oat storage protein genes is at translation. This was first suggestedby Fabijanski and Altosaar (1985) who showed that globulins represented agreater proportion of the in vitro translated products when synthesized frompolysomes as compared to poly(A)+ RNA prepared from these polysomes.Addition of nuclease-treated oat poly somes to the in vitro translation systemcontaining poly(A)+ RNA resulted in an increase in globulin synthesis. As thenuclease treated polysomes were incapable of protein synthesis alone, theseauthors proposed the existence of factors that influenced the translationalefficiency of globulin mRNAs .Translational control of storage protein synthesis is readily apparent when

the relative molar ratios of transcripts and protein are compared. Using anenzyme-linked immunosorbent assay to quantify the relative amounts of thestorage proteins in developing and mature seed, Boyer et al. (1992) estimatedthat there are approximately 10- to l l-fold greater amounts of globulin thanavenin, on a molar level. This estimate was also consistent with the results ofpulse-chase experiments where the rate of 35S-incorporation into globulinswas about nine-times higher than that observed for avenins. The synthesisrate of these proteins, however, is inconsistent with the relative levels of theirtranscripts. As discussed above, globulin and avenin mRNAs are present atequivalent concentrations (Boyer et aI.,1992; Chestnut et aI., 1989).Several possibilities were evaluated to determine the underlying basis for

translational control of oat storage protein synthesis. To determine if the dif-ference in globulin and avenin synthesis is due to differential loading of theirmRNAs on the rough endoplasmic reticulum (RER), Boyer et al. (1992) iso-lated membrane-bound polysomes and characterized the associated mRNA.The majority of the avenin and globulin mRNA was associated with the mem-brane bound polysomes and not free polysomes, suggesting that the efficiencyof translation initiation and recruitment of translation complexes to the RERis the same for the two mRNA species. The efficiency of translation of thesetwo transcripts was also shown to be similar, as sucrose density gradients ofpolysomes showed similar loading of ribosomes (Boyer et aI., 1992). AveninmRNAs possess inverted repeat sequences at the 5' end which have the poten-tial to form hairpin structures known to cause ribosome stalling and, in tum,


97.469.0

30.0

M 1 2 3

...... Globulin

~ Avenin

Fig. 7. Co-translation of equimolar amounts of globulin and avenin mRNAs in vitro. RNAswere translated alone (avenin, lane I; globulin, lane 3) or together (lane 2) in a wheat germcell-free system, and analyzed by SDS-PAGE and ftuorography.m, Molecular weight standards.Arrowheads point to full-length avenin and globulin polypeptides. From Boyer et al. (1992)with permission of the author and publisher.

decreased intitiation (Shotwell et aI., 1990). Since in vitro translation ratesusing equimolar amounts of synthetic avenin and globulin mRNA producedequal amounts of avenin and globulin (Figure 7), the potential hairpin struc-tures observed in avenin mRNAs do not appear to affect initiation or cause adecrease in translatability (Boyer et aI., 1992).Based on the evaluation of several possible mechanisms, Boyer et al. (1992)

concluded that elongation or termination of translation were the most like-ly explanations for the preferential translation of globulin mRNAs . Theseauthors noted that codon usage differs substantially for avenin and globulingenes. This is readily evident by the usc of G + C at the third position ofthe codon, where globulins mRNAs contain 43% G + C whereas avenin scontain 72% G + C. These authors speculated that the translation rates mayvary between the avenin and globulin mRNA because the distribution of iso-accepting tRNAs may be better coordinated for the translation of globulinmRNA versus avenin mRNA. Moreover, codon usage may also affect theenergetic interaction between codons and anticodons, and that a higher per-


centage of favorable interactions may enhance translation. A final hypothesisinvolves the differences in translation termination codons. Avenin mRNAsterminate translation with the less favored UAA codon, whereas globulin uti-lizes the favored stop codon UGA (Angenon et a!., 1990). The less favorableUAA codon may cause stalling of the avenin mRNA-translation complexprior to their release, which would then reduce the rate of translation (Boyeret a!., 1992).

Promoter Analysis - The 5' regulatory region of the OG1-E1 globulingenomic clone does not contain any of the regulatory elements found fre-quently in the promoters of other seed storage proteins. The only sequencewhich resembles any previously identified element is at position -162, whichis similar to a CACA element identified in soybean storage protein genes(Shotwell et aI., 1990). Glav-I and Glav-3 contain several putative regulato-ry elements. At position -104 a ]un/GCN4 sequence motif, TGAGTCA, isfound. This element is located in regions previously shown to bind putativetranscription factors in other cereal genes (Tanchak et a!., 1995). An A/T-rich direct repeat sequence located in the -250 region of Glav-l contains asequence similar to the ' prolamin box' core element found in the promotersof several cereal protein genes. The Glav-I core element deviates from theconsensus by the insertion of two purines. Further analysis of the OG l-EIgenomic clone also showed the presence of this element in its promoter, indi-cating that this element may be important in the tissue-specific expressionof oat protein as well. In addition to the prolamin box, Glav-l and Glav-3also appear to have a large duplicated sequence of approximately 70 basepairs within the region -1068 to -882. The significance of this duplicationis not known, as no previously known elements have been identified with it(Tanchak et aI., 1995).Potier (1994) has shown that the Glav-3 promoter, but not Glav-l , is able to

direct the endosperm specific expression of a GUS reporter gene in tobacco.Glav-3 has a single endosperm box at -250. To determine the functionalsignificance of the -250 region, Potier (1994) deleted the Glav-3 promoterat three different positions in the vicinity of -250. The -259 deletion left theprolamin box intact, the -247 deletion removed half of the box, while the-237 deletion removed the entire endosperm box . GUS assays in these plantsrevealed only very low levels of expression for all three constructs, indicatingthat this promoter segment alone is not directly responsible for quantitativeglobulin expression (Potier, 1994).The oat asglof gene promoter was analyzed in transgenic tobacco using

a GUS reporter system (Schubert et aI., 1994). The asglof gene , which isexpressed only in endosperm and aleurone cells of oat , directed the transcrip-tion of the transgene not only in endosperm tissue, but also in the provasculartissue, the presumptive root tip, and the shoot apical meristem of the embryoof transgenic tobacco seed. A similar pattern of expression was also evi-dent for the intact asglof gene in transgenic tobacco as determined by in situ


hybridization. When the same plants were analyzed by immunostaining, how-ever, oat globulin protein was only detected in endosperm tissue (Manteuffeland Panitz, 1993; Schubert et aI., 1994). These results indicated that, althoughaberrant spatial transcription of the oat asglof transgene occurs during seeddevelopment in transgenic tobacco, exten sive post-transcriptional regulatoryprocesses influence the expression at the protein level.Two avenin genes possess similar 5' upstream DNA sequences (Potier,

1994; Shotwell et aI., 1990) . These promoters contain a prolamin box locatednear the -300 region upstream of the translation start site. Putative CAATand TATAboxes are also observed in these promoters (Potier, 1994; Shotwellet aI., 1990). An avenin gene promoter containing 406 base pairs of the 5'flanking sequence is capable of directing endosperm-specific GUS activity(Potier, 1994). A high degree of nucleotide identity was shown between theoat, wheat, barley and rye prolamin promoters, emphasizing the importanceof these elements in prolamin gene expression (Potier, 1994).Transformation studies using avenin and globulin promoters showed that

the transgenes used different transcription start sites than those observed inoat (Potier, 1994). Possible TATA and CAAT boxes were identified whichcould be utilized in the synthesis of the globulin mRNA in tobacco, but noTATA box could be identified in the avenin mRNA which would playa rolein the different transcription start site used in tobacco (Potier, 1994). Thissuggests that transcriptional mechanisms in dicots are not identical to thosein monocots, although temporal and spatial expression appears similar.

3.4. Synthesis and Deposition ofGlobulins and Avenins

Like other seed storage proteins, the avenins and globulins are synthesizedon the RER and transported into its lumen. The synthesis of oat globulin as ahigh molecular weight (58 to 62 kD) precursor protein has been shown by invitro and in vivo protein synthesis studies (Adeli and Altosaar, 1983; Brine-gar and Peterson, 1982; Matlashewski et al., 1982; Rossi and Luthe, 1983;Walburg and Larkins, 1983). Initial reports that described oat globulin mRNAassociation with membrane-bound polysomes were presented by Luthe andPeterson (1977). Using a cell-free system, they showed that membrane boundpolysomes synthesized about two-thirds more of a globulin-like protein thanfree polysomes. Adeli and Altosaar (1983) isolated RER from pulse labeledoat seed and immunologically identified proglobulin polypeptides within it.Poly somes bound to the RER were removed and translated in vitro, and wereshown to direct the synthesis of the proglutelin polypeptide, whereas freepolysomes did not.The localization of oat globulin and avenin within the protein body was

demonstrated by the characterization ofpurified protein bodies (Donhowe andPeterson, 1983). Initial microscopic evidence showed that storage proteinaccumulation was initiated within the ER, but protein body accumulation


Fig.8. Double label in sit u hybridization showing the deposition of globulin and avenin withinthe same protein body of an endosperm cell from an 8 day old seed. A, avenin; G, globulin.Bar equals 0.5 utu. From Lending et al. (1989) with permission of the author and publi sher.

was within the vacuole (Saigo et aI., 1983). The lack of dictyosomes andthe presence of direct continuities of the ER and the vacuole suggested thatthe newly synthesized protein was deposited into the vacuole directly fromthe ER. Further pulse-chase experiments involved the fractionation of tissueextracts by sucrose gradients (Adeli et aI., 1984). After a one hour pulse,radioactivity was associated with the ER, however, after a 20 hour chase, muchof the radioactivity shifted to the protein bodies, indicating the transport of thenewly synthesized polypeptides to the protein body. Protein bodies isolatedafter various chase period s showed that the globulin proprotein which wasdetected after 2 hours of chase gradually disappeared, and the acidic and basicsubunits appeared and assembled into the 12S oligomer (Adeli et aI., 1984).Double-label immunogold staining of oat endosperm cell s demonstrated

that the globulins and the avenin s were deposited within the same protein body(Lending et aI., 1989, Figure 8). The globulins, which con stituted the bulk ofthe protein body, stained darkly with uranyl acetate and lead citrate, whereasthe avenins were a minor component and stained lightly. The proportionsof avenins and globulins support earlier estimations of the protein ratiosin oat seeds. Immunostaining was also evident within the RER , and it was


demonstrated that the aggregation of the two protein types is spatially distinct.Avenin polypeptides appear to aggregate within the ER and then migrate inan undetermined fashion to the vacuole, since avenin aggregates within theRER and the vacuole are similar in morphology. Conversely, the globulinsaggregate within the vacuole rather than in the ER. These authors suggest thationic strength, pH changes or post-translational modification are responsiblefor the aggregation process, which would involve the assembly of globulinhexamers and deposition of the protein. The deposition of globulins withinthe vacuole would occur around the preexisting deposits of avenin (Lendingeta\.,1990).

4. Conclusions

Unlike many other cultivated plants which normally accumulate eitherprolamin-type or globulin-type proteins, rice and oat synthesize and accumu-late both classes of storage proteins. These plants are able to do so becausethey utilize novel biochemical and cellular processes that are not readily evi-dent in other plant systems. In rice, prolamins and glutelins are packagedinto separate protein bodies . To circumvent potential problems of proteintrafficking, rice segregates the glutelin and prolamin mRNAs to different ERmembranes. Oat appears to use a simpler process and localizes both storageproteins within the same protein body. These storage proteins, however, arenot randomly distributed within the protein body but instead exist as discreteprotein aggregates. Aggregation of the oat avenins appears to occur in the ER,and thereby the folding and assembly processes responsible for the formationof this initial protein aggregate may be identical to that observed for the riceprolamins. These cellular processes may also be responsible, together withother translational control mechanisms, for the reduction in avenin synthesiscompared to globulin synthesis.The molecular cloning of rice and oat storage protein genes has provided

valuable information on the structure of these proteins and their capacity totolerate mutations. Although oat seeds are high in protein content, they aredeficient in methionine and cysteine. Specific regions of the 12S oat globulinprimary sequences have been identified which may tolerate the incorporationof methionine and cysteine and thereby potentially increase the level of theseessential amino acids in this grain. In contrast to oat, rice seeds are low inprotein content and their prolamin protein bodies are not digestable by mono-gastric animals. As our understanding of the molecular mechanisms of genetranscription, post-transcriptional regulation, and processing becomes moreadvanced, we can attempt to over-express engineered storage protein genesin these plants. In addition to molecular approaches, modification of glutelinand prolamin ratios in rice seeds using chemical mutagenesis demonstratesthe potential of this technique in the improvement of seed quality. Although


mutagenesis often results in deleterious pleiotropic effects in addition to thedesired modification, this technique still offers promise, as demonstrated bythe high protein quality of the em 1787 mutant (Eggum et al., 1994).

Acknowledgements

The authors' research on the rice storage proteins is supported by USDANRICGP grant No. 94-37304-1174, by the Rockefeller Foundation Programin Rice Biotechnology and by Project 0590, Washington State UniversityCollege of Agriculture and Home Economics. The authors express their gra-ditude to Drs. Illimar Altosaar, Ching-San Chen, Yun-Liu Fan, Brian Larkins,Hikaru Satoh , Fumio Takaiwa and Kunisuke Tanaka for providing reprints,preprints and figures.

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[Advances in Cellular and Molecular Biology of Plants] Cellular and Molecular Biology of Plant Seed Development Volume 4 || The Storage Proteins of Rice and Oat