Plant Physiol. (1983) 72, 161-1650032-0889/83/72/0161/05/$00.50/0

Oat Seed GlobulinSUBUNIT CHARACTERIZATION AND DEMONSTRATION OF ITS SYNTHESIS AS A PRECURSOR'

Received for publication October 12, 1982 and in revised form January 5, 1983

GREGORY WALBURG AND BRIAN A. LARKINSDepartment ofBotany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907

ABSTRACT

Tlhe predominant storage protein of oat (Avena sativa L.) seeds is asaline-soluble globulin with a mol wt of 320,000 which is composed of sixlarge (Mr = 35,000 to 40,000) and six smali (Mr = 20,000 to 25,000)subunits. Experiments were conducted to hurther describe the subunitpolypeptides and to identify the initial translation products of globulinmRNAs. Approximately 20 large subunits and 10 small subunits wereresolved by two-dimensional gel analysis. The large and small subunits hadacidic and basic isoelectric points, respectively. Disulfide-linked complexesof one large and one small subunit were isolated by extraction in bufferlacking a reducing agent. The NH2-terminal sequence of the small subunitswas homologous to a small subunit of soybean glycinin. Immunoprecipita-tion of in vitro translation products of poly(A)+ RNA with anti-oat globulinsera yielded Mr = 60,000 to 68,000 polypeptides. In vivo labeling ofspikelets with radioactive amino acids resulted in high amounts of incor-poration into polypeptides with Mr - 65,000 to 68,000 which were immu-noprecipitated with anti-globulin sera. These two results suggest oatglobuln is synthesized as a higher mol wt precursor which is subsequentdyprocessed to yield the large and small subunit polypeptides.

The major storage proteins in the seeds of most cereals are thealcohol-soluble prolamines. In contrast, up to 50%o of the proteinmass in oat (Avena sativa) seeds consists of a saline-solubleglobulin; the prolamine, avenin, constitutes only about 10o of theprotein. Earlier experiments (19) showed oat seed globulin was amultimeric protein containing subunits with mol wt of 20,000 to25,000 and 35,000 to 40,000. The holoprotein, with a mol wt of320,000 and sedimentation coefficient of 12S, was found to containsix of the large and six of the small subunits (19). These charac-teristics of oat globulin correspond to those described for the 11Sseed globulins which are present in many dicots and in somemonocots as well (5, 16).We conducted experiments to describe further the oat globulin

protein and to extend the comparison with other llS globulins.Inasmuch as synthesis in vitro of oat globulin was not definitelydemonstrated (15), we translated oat mRNAs in a cell-free systemand immunoprecipitated products with antisera induced againstoat globulin to identify globulin polypeptides. Results ofthis studyindicate that oat globulin resembles 11S seed proteins in a numberof features, including synthesis of globulin subunits as higher molwt precursors. A preliminary report of this research was published(21).

' Joumal Paper No. 9231 of the Purdue University Agricultural Exper-iment Station.

MATERIALS AND METHODS

Protein Preparation. Mature dry oat (Avena sativa L.) seedswere dehulled and ground to a fine powder. Globulin was ex-tracted by stirring powdered seeds in 10 volumes of buffer A (1 MNaCl, 10 mm 2-mercaptoethanol, 0.2 mm phenylmethylsulfonylfluoride in 0.1 M Tris, pH 8.5) at 25°C for 1.5 h. The resultingsuspension was filtered and then centrifuged at 10,000g for 10 minto remove cellular debris. The supernatant was dialyzed againstdistilled water for 48 h at 4°C. Precipitated material was collectedby centrifugation at 25,000g for 20 min and lyophilized or redis-solved in buffer A and applied to a Sepharose 6B column (2.5 x90 cm) equilibrated in the same buffer.

Total globulin was separated into the two subunit classes bychromatography on DEAE-Sephadex columns (2.5 x 40 cm)equilibrated in buffer B (6 M urea, 10 mm 2-mercaptoethanol, 0.1M Tris, pH 8.5) at 25°C. Prior to chromatography, lyophilizedprotein was dissolved in 6 M guanidine HCI, 0.1 mm EDTA, 0.2M Tris-HCl, pH 7.6, and reduced by incubation with 150 mg DTTfor 4 h under N2. After reduction, S-alkylation was by incubationin the dark for 2 h with 300 ILI of redistilled 4-vinylpyridine. Thesample was desalted by chromatography on a Sephadex G-25column (2.5 x 30 cm) equilibrated in 9% HCOOH. Peak fractionswere pooled and lyophilized. About 300 mg of alkylated proteinwas dissolved in buffer B and applied to the DEAE column. Thecolumn was washed with buffer B until all nonadsorbed polypep-tides were eluted. Retained polypeptides were released by elutionwith buffer B containing 0.4 M NaCl.

Gel Electrophoresis. Protein samples were analyzed by electro-phoresis in SDS-polyacrylamide slab gels (10), consisting of a 12.5or 15% acrylamide resolving gel and a 5% acrylamide stacking gel(75:1, acrylamide:bisacrylamide). Gels were stained with Coomas-sie brilliant blue to visualize polypeptides. Gels containing radio-active polypeptides were dried and fluorographed (13). Two-di-mensional electrophoresis was as described by O'Farrell et al. (18),with the first dimension nonequilibrium electrofocusing gels con-taining pH 2 to 11 mixed ampholytes. Approximately 200 jig ofglobulin chromatographed on Sepharose 6B was loaded onto thefirst dimension gel. The protein was focused at 400 v for 4 h. Thegels were then removed from the tubes, equilibrated in SDS bufferfor 2 h, and transferred to slab gels, and proteins were electropho-resed as above.

Sequence Analysis. NH2-terminal sequence analysis was per-formed on a Beckman 890C Sequencer as described by Hermod-son et al. (9). Amino acid phenylthiohydantoins were identifiedby GC and HPLC. No identification was made unless the peak tobackground ratio was greater than three and the quantity of theamino acid was consistent with those of the preceding cycles.

Preparation of Antisera. Antibody synthesis was induced by theinjection of total oat globulin protein or purified large subunitsinto New Zealand White rabbits. The initial injection was 1 mg ofprotein in Freund's complete adjuvant followed by weekly boosterinjections of 1 mg protein in Freund's incomplete adjuvant for 3

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WALBURG AND LARKINS

weeks. Beginning 2 weeks after the last injection, the rabbits werebled from an ear vein. The blood was allowed to clot at roomtemperature for 2 h and then was kept at 40C overnight tofacilitate collection of sera. The blood was centrifuged for 30 minat 3000g at 40C, after which the clear supernatant was removedand stored at -20'C.

Polyribosome and RNA Isolation. Developing oat seeds wereharvested approximately 20 d after anthesis, frozen in liquid N2,and stored at -80'C. Polyribosomes were isolated as previouslydescribed (11). The pelleted polyribosomes were resuspended inH20, frozen dropwise in liquid N2, and stored in liquid N2.Poly(A)+ RNA was isolated from polyribosomal preparations bythree cycles of affinity chromatography on oligo(dT)-cellulose in30-ml Corex tubes (12). Alternatively, total RNA was extractedfrom homogenized frozen seeds with hot borate buffer (8).Poly(A)+ RNA was again isolated by oligo(dT)-cellulose chro-matography. RNA preparations were analyzed by electrophoresisin agarose gels containing methylmercury hydroxide (1).

In Vitro Protein Synthesis and Immunopreciptation. Polyribo-somes and poly(A)+ RNA preparations were translated in a cell-free system prepared from wheat germ as previously described(1 1). Polypeptides were labeled with either 0.5 uCi of [35S]methi-onine or 1.0 IuCi of [3H]leucine. Typically, 1 to 2 ,ug of RNA or 1to 2 Am.0 units of polyribosomes were added per 50-1d reaction.Translation reactions were incubated for 60 min at 27°C andterminated by cooling on ice with the addition of 2 volumesacetone. Samples for electrophoresis were dissolved in SDS buffer(1.0%1o SDS, 1% 2-mercaptoethanol, 50 mm Tris-HCl, pH 7.5) andboiled for 2 min prior to electrophoresis. For immunoprecipita-tion, samples were dissolved in NET buffer (150 mm NaCl, 10 mmNa2-EDTA, 10 mm Tris, pH 8.3, 1% Triton-X-100), reacted with4 p1 of antisera induced against either total oat globulin or largesubunits, and precipitated with Staphylococcus aureus ghosts asdescribed by Lingappa et al. (14).

In Vivo Labeling of Polypeptides. Spikelets with 1 cm of stemtissue remaining were excised from greenhouse-grown oat plantsapproximately 10 d after anthesis. After excision, spikelets wereimmediately placed in 200-tl Microfuge caps (Eppendorf) con-taining 30 p1 of labeling solution (5 ,uCi [TMS]methionine in 76 mmP04, pH 7.2) and allowed to take up the solution for 1 h at 30°C.The spikelets were frozen in liquid nitrogen after the incubationperiod. The frozen seeds were then dehulled and homogenized in500 pl of buffer D (10 mm Tris, pH 7.3, 150 mm NaCl, 1 mMphenylmethylsulfonyl fluoride, and 0.5% Triton X-100). Afterhomogenization, the suspensions were centrifuged for 5 min in anEppendorfmicrofuge to remove insoluble material. The supernantwas then analyzed on SDS-polyacrylamide gels or immunoprecip-itated as described (14) with the addition of 10 pl of antisera.

RESULTS

Subunit Characterization. Globulin extracted from matureseeds was separated by electrophoresis in SDS-polyacrylamidegels to resolve subunit polypeptides. Figure 1 shows the patternproduced under reductive conditions; total SDS-soluble seed pro-tein is shown for comparison. The two size classes of globulinsubunits (Mr = 20,000-25,000 and Mr = 35,000-40,000) eachcontain several polypeptides, as indicated by the multiple bandsin the gel.

Analysis of globulin by two-dimensional electrophoresis (Fig.2) reveals further the heterogeneity present within each group ofsubunits. Some 20 to 30 polypeptides are present in the cluster oflarge subunits, and approximately 5 to 15 polypeptides are presentwithin the group of small subunits. Both the large and smallsubunits are heterogeneous with respect to mol wt and isoelectricpoint; the subunit heterogeneity is greatest in the isoelpctric focus-ing dimension.

Figure 2 also illustrates the difference in isoelectric points

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FIG. 1. Pattern of globulin (lane 1) and total SDS-soluble protein (lane2) isolated from mature oat seeds and separated by electrophoresis in a12.5% SDS-polyacrylamide gel. Proteins v ere visualized by Coomassieblue staining. Mol wt marker proteins are in lane 3, with numbersindicating Mr x l0-3.

between the large and small subunits. The large subunits arefocused in the acidic region of the gel (pH 4-5), while the smallsubunits are focused in the basic region of the gel (pH 7-8). Theseresults agree with the isoelectric points found for the subunits ofa number of other I1S globulins; generally, the large subunitshave isoelectric points ranging from pH 4 to 6 and the smallsubunits have isoelectric points ranging from pH 7.5 to 9 (2, 6).

Electrophoresis of globulin extracted in buffer lacking reducingagent produced a broad band of polypeptides which have mobil-ities corresponding to mol wt of 50,000 to 65,000 (Fig. 3). Whenthese polypeptides were exposed to reducing agent and separatedby electrophoresis, the Mr = 50,000 to 65,000 bands disappearedand polypeptides which co-migrated with the large and smallglobulin subunits appeared. These data are consistent with thehypothesis that extraction of seeds without reducing agent yieldsdisulfide-linked complexes of one large and one small subunit.These complexes are then broken to produce free subunits whenexposed to a disulfide bond reductant.To characterize further the globulin subunits, isolated acidic

and basic polypeptides were subjected to NH2-terminal aminoacid sequence determinations. Although the sample of basic sub-units examined contained several different polypeptides as shownby gel electrophoresis, only one sequence was apparent in the first18 amino acids (Fig. 4). No phenylthiohydantoin amino acidswere released when the acidic subunits were subjected to Edman

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OAT SEED GLOBULIN

pH 4.0 75

Mr

-40,000

-20,000

FIG. 2. Two-dimensional gel pattern of oat globulin. The first dimension was nonequilibrium isoelectric focusing, run from left (acidic) to right(basic). The second dimension was SDS-polyacrylamide gel electrophoresis. Polypeptides were visualized by staining with Coomassie blue. The numberson the right indicate the approximate mol wt of the two subunit groups.

1 5 10 15AVENA SATIVA G L E E N F C D L E A (R) E N I E N P

GLYCINE MAX G®( E E N I C T L K L H E N I A R P

VICIA FABA G L E E T V/ C T V/AK L R L/E N I A/G () P

PISUM SATIVUM G L E E T V/l C T A K L R L/E N I A/G P S

FIG. 4. The NH2-terminal amino acid sequences of the basic subunitsfrom A. sativa globulin and the II S globulins from G. max, V. faba, andP. sativum. The legume sequences are from Reference 3. Regions ofhomology are boxed to highlight them. Data in parentheses indicateuncertainty.

FIG. 3. Electrophoretic pattern of oat glol

ducing agent (lane 1) and the same protein afteof reductant (lane 2). Mol wt marker proteinsindicating Mr X 10-3.

degradation. This suggested that the NH2-terminal amino acid(s)of these polypeptides were blocked.Comparison of the basic polypeptide sequence determined with

X43 sequences from the basic subunits of IIS globulins from Viciafaba, Pisum sativum, and Glycine max (Fig. 4) shows considerablehomology. The oat subunits share 10 amino acids with the Glycinesequence, 10 amino acids with the Vicia sequence, and 9 aminoacids with the Pisum sequence. The three legume species share

30 from 12 to 16 amino acids within this region of sequence.Synthesis. To examine the synthesis of globulin polypeptides,

polyribosomes and poly(A)+ RNA isolated from immature oatseeds were translated in the wheat germ protein-synthesizingsystem. When the translation products from either polysomes orpoly(A)+ RNA were immunoprecipitated with antisera raised

21 against oat globulin, a set of polypeptides with mol wt of 60,000to 68,000 was isolated (Fig. 5). The same result was obtained whenthe products were precipitated with antisera raised against total

_PA oat globulin or with antisera raised against acidic subunits. No14 translation products were immunoprecipitated which coincidedwith the mature acidic and basic subunits. These results suggestedthat the Mr = 60,000 to 68,000 polypeptides were precursors tothe oat globulin subunits, as has been shown for soybean, pea,

buhin extracted without re- and broadbean 11S globulins (4, 20).,r incubation in the presence To substantiate this hypothesis, short term labeling of newlyare in lane 3, with numbers synthesized polypeptides in spikelets was performed. After a 1-h

incubation period in the presence of radioactive amino acids, a

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WALBURG AND LARKINS

i

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FIG. 5. Total in vitro translation products (lanes 1 and 3) and globulinprecursors (lanes 2 and 4) immunoprecipitated from total translationproducts with anti-oat globulin serum (lane 2) and with anti-large subunitserum (lane 4) and separated by electrophoresis in SDS-polyacrylamidegels. Translation products were labeled by the addition of 1.0 MCi of[35Slmethionine. The gel was dried after electrophoresis and the radioactivepolypeptides were visualized by fluorography. Mobilities of mol wtmarkers in a lane of the same gel were determined by the position ofstained bands, with numbers indicating M, X l0-3.

polypeptide which migrated in gels with mobility similar to the invitro globulin translation products appeared prominently on fluo-rographs (Fig. 6, lane 1). These polypeptides were immunoprecip-itated with anti-globulin sera (Fig. 6, lane 2).

DISCUSSION

The results oftwo-dimensional electrophoresis demonstrate thatboth the large and small subunits of oat globulin consist ofcomplex mixtures of polypeptides. The 20 to 30 different largesubunits and 5 to 15 small subunits represent a substantially largernumber of subunit polypeptides than has been reported for other11S proteins (5). In comparison, six large and five small subunitswere isolated from soybean glycinin (17), although heterogeneityin amino acid sequences of these proteins indicates that multipleforms are present (N. C. Nielsen, personal communication).Inasmuch as the number of oat globulin polypeptides present

presumably reflects the number of genes encoding globulins, agene family is likely to encode these proteins. There is also apotential for a number of allelic forms of the globulin genes, dueto the hexaploid nature of the oat genome. Post-translationalmodification of the gene products might also partially account forthe multiplicity of globulin polypeptides observed.The sequence homology for oat globulin basic polypeptides and

the Vicia, Glycine, and Pisum sequences is the most fundamentalsimilarity between these proteins described. Homology at this levelsuggests that all 11S globulins may have derived from a commonancestral gene. If they have descended from a common gene,strong selective pressure was present to maintain this sequence,because monocots and dicots diverged millions of years ago.Indeed, most of the amino acid replacements which are found inthe sequence could have resulted from single nucleotide changes

18

FIG. 6. Fluorograph of polypeptides synthesized in vivo, labeled with[ISlmethionine, and separated by electrophoresis in a 15% SDS-polyacryl-amide gel. Lane I shows total SDS-soluble products and lane 2 thepolypeptides immunoprecipitated with anti-globulin sera as described in"Materials and Methods." The numbers on the right indicate Mr X 10-3of marker proteins run in a lane of the same gel and visualized byCoomassie staining.

in the triplet codon.Taken together, results of the in vitro and in vivo protein

synthesis experiments strongly argue that the initial translationproducts of globulin mRNAs are higher mol wt precursors whichare subsequently processed to yield the Mr = 40,000 and 20,000subunits. The immunoprecipitated precursor polypeptides are suf-ficiently large to include both the large and small subunits, as wasshown for pea, soybean, and broadbean 1 IS globulins (4, 20). Ifthe structure of the oat precursors resembles that of the other 11Sprecursors, the arrangement of the subunits in the precursorpolypeptide would be NHracidic-basic-COOH (4).The complexes of disulfide-linked M, = 20,000 and 40,000

polypeptides isolated by extraction in the absence of reducingagent are the fundamental subunit of the globulin protein. Datafrom the glycinin molecule showed that only specific large andsmall subunits were linked in these complexes, and the specificpairing were shown to be the pairs which occurred in the precursormolecule (N. C. Nielsen, personal communication). Thus, thedisulfide linkage occurs before the proteolytic cleavage of the twopolypeptides takes place in order for the specific linkages to bemaintained.An mRNA molecule of approximately 2,000 base pairs is

necessary to encode the Mr = 68,000 precursor polypeptides; thesoybean glycinin mRNAs were shown to be 2,100 base pairs inlength and to migrate slightly faster than the 18S ribosomal RNA

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OAT SEED GLOBULIN

in gels (7, 20). Analysis of a poly(A)+ RNA preparation indenaturing gels revealed a prominent band slightly smaller thanthe 18S ribosomal RNA. Because the message for oat globulinwould be expected to occur in high concentrations, we believe thisprominent RNA band is likely to contain oat globulin mRNAs.We have synthesized cDNAs from this RNA, cloned them intobacteria, and are currently screening those clones for globulinsequences. These cloned DNAs will be used for further analysesof oat globulin synthesis.

Acknowledgments-We thanl Dr. Paul Staswick for amino acid sequence deter-minations, Dr. Donald Foard for assistance with antisera preparation and immuno-precipitations, and Dr. Niels Nielson for helpful suggestions.

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3. CASEY R, JIF MARCH, E SANGER 1981 N-terminal amino acid sequence of a-subunits of legumin from PisLom sativum. Phytochemistry 20: 161-163

4. CROY RRD, GW LYcm, JA GATEHousE, JN YARWOOD, D BOULTER 1982Cloning and analysis of cDNAs encoding plant storage protein precursors.Nature 295: 76-79

5. DERBYSHIRE E, DJ WRIGHT, D BouLTR 1976 Legumin and vicilin, storageproteins of legume seeds. Phytochemistry 15: 3-24

6. GATEHOUSE JA, RRD CROY, D BoULTER 1980 Isoelectric-focusing propertiesand carbohydrate content of pea (Pisum sativwm) legumin. Biochem J 185:497-503

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9. HERMODSON M, G ScHmsR, K KuRAcHI 1977 Isolation, crystallization, andprimary amino acid sequence of human platelet factor 4. J Biol Chem 252:6276-6279

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11. LARKNs BA, WJ HuRKaN 1978 Synthesis and deposition of zein in proteinbodies of maize endosperm. Plant Physiol 62: 256-263

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14. LINGAPPA UR, JR LINGAPPA, R PRAsAD, KE EBNER, G BLOBEL 1978 Coupledcell free synthesis, segregation, and core-glycosylation of a secretory protein.Proc Natl Acad Sci USA 25: 2338-2342

15. LuTHE DS, DM PETERSON 1977 Cell-free synthesis ofglobulin by developing oat(Avena sativa L.) seeds. Plant Physiol 59: 836-841

16. Mu±zDD A 1975 Biochemistry of legume seed proteins. Annu Rev Plant Physiol26: 53-72

17. MoREIA MA, MA HERMODSON, BA LARuNs, NC NIELSEN 1979 Partial char-acterization of the acidic and basic polypeptides of glycinin. J Biol Chem 254:9921-9926

18. O'FAaRELL PZ, HM GOODMN, PH O'FaRRELL 1977 High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12: 1133-1142

19. PETERSON DM 1978 Subunit structure and composition of oat seed globulin.Plant Physiol 62: 506-509

20. Turn NE, VH THANH, NC NELSEN 1981 Purification and characterization ofmRNA from soybean seeds. J Biol Chem 256: 8756-8760

21. WALBURG G, B LARKINs 1982 The structure and synthesis of I IS globulin fromoat seeds. Plant Physiol 69: S-123

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