Plant Molecular Biology 5: 165-173, 1985 © 1985 Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Developmental expression of sunflower I1S storage protein genes Randy D. Allen, Craig L. Nessler & Terry L. Thomas Department of Biology, Texas A & M University, College Station, TX 77843, U.S.A.

Keywords: cDNA, ~. gtl l, expression vector, seed development, storage protein, sunflower (Helianthus annuus L.)

Summary Helianthinin is the major globulin storage protein of sunflower seeds. Antiserum prepared against purified helianthinin was used to study storage protein accumulation in developing sunflower embryos. Synthesis of helianthinin begins within 7 days after anthesis (DAA) and it accumulates rapidly until 19 DAA. Storage protein synthesis then slows until seed maturation at 30 DAA. Anti-helianthinin was also used to select specific cDNA recombinants from a bacteriophage~, gt 11 cDNA expression library prepared from immature sunflower embryo polyadenylated RNA. One of these recombinants, ~. gt 11H3, contained a 300 bp cDNA insert which was used as a hybridization probe for RNA gel blot and RNA dot blot analyses. This probe hybridizes with an approximately 1.9 kb sunflower embryo transcript. Helianthinin mRNA is present in embryos 7 DAA and accumulates to maximum prevalence at 12 DAA which corresponds to the period of maximum helianthinin accumulation. Soon after 12 DAA helianthinin RNA begins to decrease. Full length helianthinin mRNA is not detectable in mature seeds or in germinated seedlings. These results indicate that synthesis of the 11S sunflower seed storage protein occurs during a specific stage of embryogenesis and is regulated by the accumulation of helianthinin mRNA.

Introduction Accumulation of seed storage proteins is a closely regulated biosynthetic process of great agronomic and economic importance. Storage proteins of most dicotyledonous species are synthesized exclusively in developing embryos (reviewed in 15). Therefore, seed storage protein synthesis represents an excellent system for investigation of tissue specific gene expression. Sunflower seeds accumulate large amounts of a few major protein species during Abbreviations: bp: base pair DMSO: dimethylsulfoxide kb: kilobase kd: kilodalton mw: molecular weight PAGE: polyacrylamide gel electrophoresis SDS: sodium dodecyl sulfate

embryo development. These proteins can be divided into two classes: the water soluble 2S albumins and the salt soluble l lS globulins (29). The I IS protein of sunflower, which has been named helianthinin (22), resembles legumin-like seed storage proteins of other plant species (reviewed in 24). Helianthinin has a molecular weight of about 305 kd and exists as a hexamer of non-identical subunits (22). Each subunit consists of one of two groups of larger acidic polypeptides (mw. 30-40 kd) and a smaller basic polypeptide (mw. 27-23 kd) linked by disulfide bonds (6). Polypeptides which compose the subunits of all 11S seed proteins so far investigated are derived by proteolytic processing from larger precursor molecules (15). Globulin seed storage proteins of most dicotyledonous species are encoded by families of genes (2, 27). The existence of various forms of helianthinin subunits suggests that this protein is also a

166 product of multiple genes (6). Synthesis and accumulation of storage proteins and associated synthesis of corresponding mRNAs have been studied intensively in members of several agronomically important agiosperm families including the Fabaceae (3, 13, 25), Malvaceae (8, l 1), Brassicaceae (4, 5), and Poaceae (14, 17). Although the overall patterns of expression of storage protein genes are similar, each system has unique characteristics that facilitate specific experimental approaches. In this report we describe the isolation of a bacteriophage h recombinant cDNA that encodes helianthinin, the major globulin storage protein of sunflower seeds. This cDNA recombinant and polyclonal antibodies directed against sunflower 11S protein allowed us to examine the ontogenic expression of this storage protein gene at the mRNA and protein levels. This is the first description of storage protein gene expression in the highly specialized Asteraceae family.

Materials and methods

Plant materials Sunflower seeds (Helianthus annuus L. cv. Giant grey stripe, Northrup King Seed Co. Minneapolis, MI) were obtained locally. Plants were field grown and embryos were dissected from achenes 5, 7, 9, 12, 15, 19, and 30 days after anthesis (DAA). Isolated embryos were immediately frozen in liquid nitrogen and stored at -70°C. Purification o f 11 S storage protein and preparation o f antisera Dry sunflower seed embryos were ground in hexane and extracted with l M NaC1. The extract was dialyzed for 15 h against distilled water and the precipitated globulin fraction was pelleted by centrifugation. Helianthinin was purified from the raw globulin fraction by gel filtration chromatography. The raw globulin pellet was resuspended in running buffer (0.05 M phosphate buffer pH 8, 1 M NaC1, 1 mM phenylmethylsulfonyl fluoride) and applied to a 60 cm × 0.5 cm column of Biogel A-1.5. Fractions were monitored by total protein determination and the purity of helianthinin was checked by SDS polyacrylamide gel electrophoresis (SDS-

PAGE). Approximately 80 ~g of protein of purified helianthinin was emulsified in Freund's complete adjuvent and injected subcutaneously at two sites on the back of a female New Zealand white rabbit. Booster injections in incomplete adjuvent were given 2 and 3 weeks after initial injection and the rabbit was bled 2 weeks later. Serum was prepared by centrifugation and IgGs were purified by affinity chromatography over a Staphylococcus aureus protein ASepharose column and precipitation with 50% saturated (NH4)2SO 4. The pellet was resuspended in borate buffered saline and dialyzed against additional borate buffered saline. Dialyzed anti-helianthinin IgG (2 mg protein/ml) was stored at -20°C. Protein blot analysis Protein extracts of developmentally staged embryos were subjected to S D S - P A G E on 12.5% polyacrylamide gels and the proteins were electrophoretically transferred to nitrocellulose (28). Dot blots were prepared using a dot blot manifold. Nitrocelulose was washed with phosphate buffered saline (PBS), protein samples in 100 #l of PBS, were applied and filtered under vacuum. After an additional wash with PBS the filter was removed from the manifold and air dried. All immunochemical procedures were carried out at 4°C. Identical methods were used for protein gel blots, protein dot blots and bacteriophage screens. Filters were washed 3 times for 30 min in a blocking solution of 0.1% NP-40, 1% bovine serum albumin, and 0.02% sodium azide in PBS (NBA-PBS). Blots were incubated in antibody diluted l:100 with NBA-PBS for 12 h, washed 3 times with NBA-PBS and incubated for 3 h with 2.5 #Ci of [125I] Staphylococcus aureus protein A in 50 ml NBA-PBS. Blots were washed 6 times for 15 min in NBA-PBS, rinsed with PBS and air dried. Autoradiographs were exposed for 2 h at -70°C with an intensifying screen. R N A isolation RNA from 5, 7, 9, 12, 15, and 19 DAA embryos, mature seed (30 DAA) and germinated seedlings (2 days after imbibition) was prepared by the methods of Galau et al. (11). This procedure involves extraction of homogenized embryos with phenol/CHC13 : isoamyl alcohol (24: I) and successive precipitation

167 of RNA with 2 M LiC1, 3.75 M KOAC and ethanol. Poly A RNAs were purified by oligo(dT) cellulose chromatography (1).

Construction and screening of gtl 1 cDNA library AMV reverse transcriptase was used to synthesize oligo(dT) primed first strand cDNA from poly A RNA of 9 15 DAA sunflower embryos. Second strand synthesis was performed using DNA polymerase I Klenow fragment followed by S1 nuclease digestion. Eco RI sites were protected with Eco RI methylase, and uneven ends were repaired with additional Poll Klenow fragment. T4 DNA ligase was used for blunt-end ligation of phosphorylated synthetic Eco RI linkers to the cDNA. Double stranded cDNA, with linkers, was digested with Eco RI and size fractionated on a Biogel A-50 column. Double stranded cDNA was ligated into the Eco RI site of the bacteriophage expression vector X gt 11 (30) and the ligated mixture packaged in vitro. Packaged phage were plated on the host strain KM392 [LE392 Alacu169 supF hsdR hsdM+]. The library was amplified on the host strain KM392pMC9 (KM392 laciq+). Detailed methods of cDNA library construction are available from the authors and will be published elsewhere. Anti-helianthinin antibodies were used to screen the sunflower embryo h g t l l cDNA library for helianthinin specific sequences. Recombinant phage were plated on the host strain KM392. Nitrocellulose filters were applied to plates after 6 h growth at 37°C, plates were incubated for an additional 12 h then cooled to 4°C and the filters removed. Filters were immunologically treated as described above and autoradiographed for 12 h at -70°C with an intensifying screen. To eliminate antibody reaction to E. coli or ,k gt 11 determinants, anti-helianthinin antibodies were preadsorbed for 12 h to filters prepared from plates containing nonrecombinant h gtl 1 plated on KM392. Six bacteriophage plaques that reacted with anti-helianthinin antibodies were selected and purified by additional rounds of screening. One of these bacteriophage recombinants, designated X gt I 1H3, was used in all subsequent analyses.

RNA hybridization X gtl 1H3 was digested with Eco RI and the cohe-

sive termini filled in with [32p] dATP and d T T P (New England Nuclear, 3000 Ci/mMole) with DNA polymerase I large fragment. The labeled Eco RI fragment was purified by electrophoresis on a 1.5%agarose gel. Total RNAs from developmentally staged embryos was either dotted onto nitrocellulose using a dot blot manifold or was separated by electrophoresis after denaturation with glyoxal and D M S O on a 1% agarose gel and transferred to nitrocellulose by blotting. The labeled cDNA probe was denatured by boiling and hybridized to filters for 16 h at 42°C in 50% formamide, 25 mM phosphate buffer pH 6.8, 5 X SET(1 X S E T = 150 mM NaCI, 20 mM tris pH 7.8, 1 mM EDTA), 0.1% SDS, 10% dextran sulfate, 5 X Denhardt's solution (1 X Denhardt's = 0.02% Ficoll, 0.02% BSA, 0.02% polyvinylpyrrolidone), 100 #g/ml denatured calf thymus DNA, 50 #g/ml poly A and 10/~g/ml poly C. After hybridization the filters were washed for 1 h in 4 X SET wash (4 X SET + 0.025 M phosphate buffer, 0.2% SDS), 1 h in 2 X SET wash, and 2 h in 1 X SET wash all at 60°C. Filters were dried and exposed for autoradiography at -70°C with an intensifying screen.

Hybrid selection The cDNA insert of h gt I 1H3 was subcloned into the Eco RI site of pUCI9. This plasmid (pUCH3) was used to hybrid select helianthinin mRNA from 12 DAA embryo total RNA as described by Parnes (19). Hybrid selected RNA and 12 DAA embryo total RNA were translated in vitro using a rabbit reticulocyte cell free system. In vitro translation products of total RNA were immunoprecipitated with anti-helianthinin antibodies. Translation products were separated by S D S - P A G E and the gel dried and exposed for autoradiography for 72 h.

Results

Accumulation of l l S storage protein in sunflower embryos The ontogenic accumulation of sunflower storage protein was initially analyzed by SDS-PAGE. Helianthinin polypeptide bands are easily distinguished in gels of 7, 9, 12, 15 and 19 DAA sunflower embryo protein extracts stained with Coomassie

168

Fig. 1. A. SDS-PAGE of developmentally staged sunflower embryos. Five micrograms of total protein was loaded in each lane. After electrophoresis the gel was stained with coomassie brilliant blue. a, t~',and ~ helianthinin polypeptides are marked. Embryo age is indicated as days after anthesis (DAA). Standard molecular weight values, in kd × 103, indicated at the left of the figure are for: bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme.

blue (Fig. 1). Large acidic polypeptides (or a n d a', 38-42 kd a n d 30-32 kd respectively) a n d smaller basic polypeptides (/3, 23-27 kd) are present by day 7 D A A a n d the relative intensity of these b a n d s increases d r a m a t i c a l l y t h r o u g h 19 D A A . Polypeptides f r o m a gel as in Fig. 1 were transferred to nitrocellulose a n d reacted with antibodies raised against purified h e l i a n t h i n i n . ~ a n d a ' polypeptides are easily detected in these blots (Fig. 2A) but reaction of a n t i - h e l i a n t h i n i n with/3 polypeptides is less intense. This result could indicate t h a t / 3 polypeptides are less a n t i g e n i c t h a n e~ polypeptides b u t a d d i t i o n a l b l o t t i n g experiments have s h o w n that/3 polypeptides are also transferred less efficiently to the nitrocellulose. With the e x c e p t i o n of a g r o u p of b a n d s at a b o u t 50 kd, little if a n y non-specific ant i b o d y reaction with other sunflower seed proteins was observed. We believe that these 50 kd b a n d s may represent h e l i a n t h i n i n precursor molecules. The relative increase of h e l i a n t h i n i n in e m b r y o protein extracts can be easily seen with i m m u n o - d o t blots (Fig. 2B). M a x i m a l h e l i a n t h i n i n a c c u m u l a tion occurs between 9 a n d 15 D A A . After this time, h e l i a n t h i n i n storage p r o t e i n synthesis appears to be a t t e n u a t e d . These d a t a are graphically s u m m a r i z e d in Fig. 6.

Fig. 2. A: Immuno-gel blot analysis of total sunflower embryo protein extracts from developing embryos 9 and 15 DAA are shown in lanes B and C. Lane A shows 15 DAA embryo proteins stained with coomassie blue. Protein was electrobloned from gels onto nitrocellulose and reacted with anti-helianthinin polyclonal antibodies followed by reaction with 1251protein A. Bands were visualized by autoradiography for I h. Helianthinin polypeptides a, a' and/~ are indicated. B: lmmuno-dot blot analysis of developmentallystaged sunflower embryos. One microgram (top row) or 0.5 #g (bottom row) of total protein was loaded on each dot and the filter reacted as in Fig. 2A.

169

300 bp Fig. 3. Restriction endonuclease cleavage site map of h gtllH3 sunflower l IS seed storage protein (helianthinin) eDNA clone. Restriction enzyme sites: H, Hpa II; L, Sal I; M, Mnl 1; R, Eco RI; S, SSt I; T, Taq 1. Enzymes which do not digest Xgt I 1H3 eDNA insert include Eco RI, Barn HI, HinD I11, Hae il, Pvu I and Pst I. Shaded regions indicate the h gtl 1 lac Z gene.

Construction and screening o f sunflower expression o f eDNA library

Fig.4. A: Developmentallystaged embryo RNA dot blot hybridized with helianthinin eDNA probe. One microgram (top row) or 0.5/.Lg(bottom row) of total RNA was loaded on each dot and the filter was hybridized with a 32p labeled 300 bp Eco RI fragment of X gt I 1H3 and autoradiographed for-12 h. Ribosomal 18S and 28S RNA markers are indicated. B: RNA gel blot analysis of staged embryo RNA hybridized with helianthinin eDNA probe. Two micrograms of total RNA from 5, 7, 9, 12, 15, 19 DAA, mature (M) and germinated (G) sunflower embryo was denatured with glyoxal and DMSO and run on a 1% agarose gel and transferred to nitrocellulose. The filter was hybridized and autoradiographed as described in figure legend 4A and Materials and methods.

A eDNA library from sunflower embryo poly A R N A was c o n s t r u c t e d to facilitate isolation of helia n t h i n i n a n d o t h e r sunflower storage p r o t e i n genes. Size selected d o u b l e s t r a n d e d e D N A was cloned into a u n i q u e Eco RI site of the e x p r e s s i o n v e c t o r b a c t e r i o p h a g e h g t l I to generate a l i b r a r y of 1 X 105 r e c o m b i n a n t s . The Eco RI site in h g t l 1 is located 53 bp f r o m the 3' t e r m i n u s of the lac Z gene (30). The inserted e D N A sequence, if present in the a p p r o p r i a t e r e a d i n g frame, can be expressed as a fusion p o l y p e p t i d e . This fusion p o l y p e p t i d e is c o m p o s e d p r i m a r i l y o f the f l - g a l a c t o s i d a s e p r o t e i n with the p r o d u c t o f the e D N A fused to its c a r b o x y terminus. These fusion p o l y p e p t i d e s can be detected in h g t l I e x p r e s s i o n libraries by screening with antibodies directed a g a i n s t proteins o f interest. The s u n f l o w e r e D N A l i b r a r y was p l a t e d on KM392, a strain with a deletion in the lac region of the E. coli c h r o m o s o m e which includes the gene that e n c o d e s the lac repressor. Thus the k gt 11 lac Z gene is constitutively expressed when p l a t e d on K M 3 9 2 a n d fusion p r o t e i n s a c c u m u l a t e as the p h a g e u n d e r g o their lytic cycle. Plates were replicated o n t o nitrocellulose filters a n d the filters were screened with the a n t i - h e l i a n t h i n i n a n t i b o d y used in the e x p e r i m e n t s of F i g u r e s 2 A a n d 2B. S e v e r a l r e c o m b i n a n t b a c t e r i o p h a g e plaques were detected by this m e t h o d . One o f these plaques, d e s i g n a t e d k g t l I H3 was isolated a n d the p h a g e were r e p l a t e d a n d rescreened. D N A o f these p h a g e was purified a n d the 300 bp e D N A insert was c h a r a c t e r i z e d by restriction enzyme m a p p i n g (Fig. 3).

Representation o f helianthinin m R N A in sunflower embryos We used the e D N A insert o f h gt I 1 H3 to investigate the a c c u m u l a t i o n o f h e l i a n t h i n i n m R N A d u r ing s u n f l o w e r seed o n t o g e n y . The 300 bp e D N A

170 insert of ~ gt 1 1H3 was excised with Eco RI and the cohesive Eco RI termini labeled with 32p using DNA polymerase I Klenow fragment. This probe was hybridized with RNA of developmentally staged sunflower embryos in RNA dot blot (Fig. 4A) and RNA gel blot (Fig. 4B) experiments. The H3 eDNA probe hybridized specifically with a 1.9 kb transcript on embryo RNA gel blots (Fig. 4B). Hybridization of H3 eDNA was first detectable with 7 DAA embryo RNA, under the conditions used, and increased greatly in 9 and 12 DAA embryo RNA. Equivalent amounts of RNA were loaded on each lane so the increase in helianthinin transcripts represents a dramatic accumulation of these mRNAs. The fraction of helianthinin RNA in embryo RNA decreased slightly by 15 DAA and a more pronounced decrease was seen by 19 DAA. The developmental accumulation of helianthinin transcripts is more evident in RNA dot blot experiments (Fig. 4A). These data are presented graphically in Fig. 6. Titration experiments using endfilled H3 as probe and RNA dot blots containing increasing amounts of RNA were used to quantitate the prevalence of helianthinin mRNA in era-

Fig. 5. Autoradiographof in vitro translation products from 12

DAA embryo RNA. Lane A represents products of total mRNA. The bands indicated specifically immunoprecipitate with anti-helianthinin antibodies (lane B) and represent helianthinin precursor proteins of approximately 50, 52 and 58 kd. Translation of helianthinin mRNA purified by hybrid selection givesa single 52 kd polypeptideband (lane C).

bryo RNA. We estimate that helianthinin mRNA constitutes 0.045% of the total RNA in 7 DAA embryos. This fraction increases to 0.22% in 12DAA embryos then decreases to 0.15% in 19 DAA embryos. Long autoradiographic exposures of dot blots of mature seed RNA showed faint hybridization with the H3 probe (data not shown). However, RNA gel blot analysis showed no detectable hybridization. No hybridization of the H3 probe to RNA of germinated seeds was ever detected. We estimate that our RNA dot blot procedure is sufficiently sensitive to detect helianthinin mRNA that is approximately 20-30 fold less prevalent than in 7 DAA embryo RNA. The eDNA insert o f h gtl 1H3 was subcloned into the plasmid vector pUC19. This recombinant plasmid (pUCH3) was used for hybrid selection of helianthinin mRNA. A series of polypeptides ranging in molecular weight from 50 to 58 kd, are the predominate in vitro translation products of 12 DAA embryo RNA (Fig. 5 lane A). These polypeptides are the expected size for helianthinin precursor molecules and at least three bands in this region immunoprecipitate with anti-helianthinin antibodies (Fig. 5 lane B). I n v i t r o translation of mRNA purified by hybrid selection with pUCH3 DNA yields a single, 52 kd polypeptide band (Fig. 5, lane C). The presence of 3 differently sized polypeptides which immunoprecipitate with anti-helianthinin may represent helianthinin precursors encoded by mRNAs transcribed from different genes. However, under hybrid selection conditions, the eDNA insert of pUCH3 recognizes mRNAs which encode helianthinin polypeptides of a single size. Data from immuno-dot blots (Fig. 2A) and RNA dot blots (Fig. 2B) were quantitated and are graphically displayed in Fig. 6. Storage protein mRNA begins to accumulate rapidly during the interval between 7 and 9 DAA and reaches maximum prevalence around 12 DAA. Helianthinin transcripts then undergo an apparent stochastic decay. By 19 DAA the prevalence of helianthinin mRNA is reduced by 50% and helianthinin transcripts are undetectable in mature seeds. Helianthinin polypeptides, on the other hand, accumulate much more slowly. At 12 DAA, the period of maximum mRNA prevalence, only about 50% of the final amount of helianthinin has been produced. However, the most rapid rate of accumulation occurs during the interval between 9 and 15 DAA which

171

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Fig. 6. Relative accumulation of helianthinin mRNA and protein. Protein and RNA dot blots as show in Figs 2B and 4A were excised and the hybridized radioactivity of individual spots was determined by gamma or scintillation counting. The mean bound cpm value minus background of four replicate protein and RNA dot blots was determined for each embryo developmental stage. Maximum standard deviation for any point is ± 60 cpm.

corresponds exactly with the period of maximal mRNA concentration. As the level of helianthinin mRNA decreases between 15 and 19 DAA, the accumulation of storage protein is also attenuated. These results strongly suggest that biosynthesis of the 11S storage protein in sunflower embryos is regulated by the accumulation of helianthinin mRNA.

Discussion

Seed storage protein synthesis in many species begins during the cell enlargement phase of embryo development and may continue, closely followed by dry weight increase, until the seeds begin to desiccate (7). Sunflower embryos at 7 DAA have fully formed cotyledons and embryonic axes and are about 2 mm in length. Embryos reach their full size of about 1 cm in length by 15 DAA and dry weight accumulation continues slowly until seed dessication begins around 20 DAA. Sunflower seeds are mature at 30 DAA (21). We have found that the most rapid deposition of helianthinin in sunflower embryos occurs between 9 and 15 DAA which corresponds with the cell enlargement phase of embryo development. Seed storage proteins are largely protected from

proteolytic degradation after deposition and prior to germination (18). Therefore, the rate of storage protein accumulation is primarily determined by the rate of synthesis. We have shown that helianthinin accumulation in sunflower embryos correlates with the level of helianthinin mRNA. The rapid and specific build up of globulin storage protein during the cell enlargement stage of sunflower embryo development is due to the accumulation of helianthinin mRNA in these tissues. As the level of helianthinin mRNA begins to drop in mid maturation embryos, the rate of helianthinin protein accumulation concomitantly decreases• Helianthinin mRNA could not be detected in RNA of mature seed embryos or germinated seedlings. Storage protein mRNA levels have been measured in embryos of a variety of legumes by in vitro translation (9, 16, 25) or by nucleic acid hybridization (3, 12, 13). In all these cases, storage protein synthesis appears to be regulated by the prevalence of mRNAs encoding these polypeptides. Although the role of posttranscriptional processing events in the accumulation of seed protein mRNAs cannot be excluded, it is more probable that the primary control parameter is the rate at which transcripts are initiated from storage protein genes. Understanding the mechanisms of ontogenically regulated gene expression is a basic problem of developmental biology. The signals that cause specific sets of genes to be expressed in a precisely timed developmental program remain a enigma and the genomic sequences that respond to these signals have not been characterized. The phytohormone abscisic acid can promote storage protein synthesis in isolated embryos of some species (4, 26) but this effect is not universal (8). The action of other embryonic factors, which may act in concert with phytohormones on gene expression during differentiation a n d development has not been fully investigated. Although it is premature to formulate general paradigms of gene regulation in plant development, a pattern does seem to be emerging. Expression of genes involved in development does not appear to be dependent on their genomic position nor are they regulated as groups but as individual units• Fischer and Goldberg (10) found that soybean glycinin genes are not closely linked to each other or to other genes expressed during embryogenesis. Likewise, mRNAs for various globulin, lectin, and albumin storage proteins in pea em-

172 bryos accumulate and disappear independently (15). In addition, Goldberg and colleages have recently found that several developmentally controlled genes which are present on an approximately 20 kb fragment of soybean genomic DNA are expressed in the appropriate tissues when they are introduced into tobacco plants via a Ti plasmid (R. Goldberg, pers. comm.). These and other gene transfer experiments imply that the cis regulatory elements for many genes of interest are located sufficiently near the gene to be transferred on relatively small (a few kb) DNA segments, and these cis elements must respond to an array of t r a n s acting factors. Accumulation of seed storage proteins and their associated mRNAs represents an excellent example of the ontogenic expression of multiple genes. Use of the sunflower storage protein system may provide a distinct advantage in studies that involve Ti plasmid transformations because, unlike most legumes, sunflower plants can be regenerated from tissue culture (20). This will allow analysis of modified genes and their flanking sequences in a homologous genomic environment which may facilitate identification of cis and t r a n s factors involved in the ontogenic control of sunflower storage protein gene expression.

Acknowledgement This work was supported in part by USDA grants 5332R6070 to C.L.N. and 84CRCR11391 to T.L.T.

References 1. Aviv H, Leder P: Purification of biologically active globin mRNA by chromatography on oligothymadilic acid-cellulose. Proc Natl Acad Sci USA 69:1408 1412, 1977. 2. Brown JWS, Ma Y, Bliss FA, Hall TC: Genetic variation in the subunits of globuiin-I storage protein of French bean. Theor Appl Genet 59:83-88, 1981. 3. Chandler PM, Higgins TJV, Randall PJ, Spencer D: Regulation of legumin levels in developing pea seeds under conditions of sulfur deficiency: rates of legumin synthesis and levels of legumin mRNA. Plant Physiol 71:47-54, 1983. 4. Crouch ML, Sussex 1M: Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro. Planta 153:64 74, 1981. 5. Crouch ML, Tenbarge KM, Simon AE, Ferl AE: cDNA clones for Brassica napus seed storage proteins: evidence

from nucleotide sequence analysis that both subunits of napin are cleaved from a single precursor polypeptide. J Mol Appl Genet 2:273 284, 1983. 6. Dalgalarrondo M, Raymond J, Azanza JL: Sunflower seed proteins: Characterization and subunit composition of the globulin fraction. J Exper Bot 35:1618 1628, 1984. 7. Dure LS III: Seed formation. Ann Rev Plant Physiol 26:259 278, 1975. 8. Dure LS 111, Galau GA: Developmental biochemistry of cottonseed embryogenesis and germination. Xlll. Regulation of biosynthesis of principal storage proteins. Plant Physiol 68:187-194, 1981. 9. Dure LS, Galau GA, Greenway S: Changing protein patterns during cotton cotyledon embryogenesis as shown by in vivo and in vitro synthesis, lsr J Bot 29:293 306, 1981. 10. Fischer RL, Goldberg RB: Structure and flanking regions of soybean seed protein genes. Cell 29:651-660, 1982. 11. Galau GA, Legocki AB, Greenway SC, Dure LS: Cotton messenger RNA sequences exist in both polyadenylated and nonpolyadenylated forms. J Biol Chem 266:2551-2560, 1981. 12. Gatehouse JA, Evans I M, Brown D, Croy RRD, Boulter D: Control of storage-protein synthesis during seed development in pea (Pisum sativum L.). Biochem J 208:119 127, 1982. 13. Goldberg RB, Hoschek G, Ditta GS, Breidenbach RW: Developmental regulation of cloned superabundant embryo mRNAs in soybean. Dev Biol 83:218-231, 1981. 14. Greene FC: Expression of storage protein genes in developing wheat ( Triticum aestivum) seeds. Plant Physiol 71:40-46, 1983. 15. Higgins TJV: Synthesis and regulation of major proteins in seeds. Ann Rev Plant Physiol 35:191 221, 1984. 16. Higgins TJV, Spencer D: Precursor forms of pea vicilin subunits: modification by microsomal membranes during cell-free translation. Plant Physiol 65:205 211, 1981. 17. Larkins BA, Hurkman WJ: Synthesis and deposition ofzein in protein bodies of maize endosperm. Plant Physiol 62:256-263, 1978. 18. Madison JT, Thompson JF, Meunster AE: Turnover of storage protein in seeds of soya bean and pea. Ann Bot 47:65 73, 1981. 19. Parnes JR, Velan B, Felsenfeld A, Ramanathan L, Ferrini U, Appella E, Sidman JG: Mouse /3 2 microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs. Proc Natl Acad Sci USA 78:2253-2257, 1981. 20. Paterson KE, Everett NP: Regeneration of Helianthus annuus plants from callus. Amer J Bot 71:42, 1984. 21. Robinson RG: Sunflower phenology year, variety, and date of planting effects on day and growing degree-day summations. Crop Science 11:635 638, 1971. 22. Schwenke KD, Pahtz W, Linow KJ, Schultz M: On oil seed proteins Part 11. Purification, chemical composition, and some physico-chemical properties of the I Is globulin (Helianthinin) in sunflower seed. Die Nahrung 23:241-254, 1979. 23. Schuler MA, Schmidt ES, Beachy RN: Closely related families of genes code for the a, and a' subunits of the 7S storage protein complex. Nucleic acids Res 10:8255 8244, 1982.

173 24. Spencer D, Higgins TJV: Seed maturation and deposition of storage proteins. In: Smith H, Grierson D (eds) The molecular biology of plant development. University of California Press, Berkeley, 1982. 25. Sun SM, Mtitschler MA, Bliss FA, Hall TC: Protein synthesis and accumulation in bean cotyledons during growth. Plant Physiol 61:918-923, 1978. 26. Sussex IM, Dale RMK: Hormonal control of storage protein synthesis in Phaseolus vulgaris. In: Rubenstein I, Phillips RL, Green CE, Gegenbach BG (eds) The plant seed: development, preservation and germination. Academic Press, New York, 1979, pp 129-141. 27. Thomson JA, Schroeder HE: Cotyledonary storage proteins of Pisum sativum II. Hereditary variation in components of the legumin and vicilin fractions. Aust J Plant Physiol

5:281-294, 1978. 28. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4353, 1979. 29. Youle, RJ, Huang AHC: Occurrence of low molecular weight and high cysteine containing albumin storage proteins in oilseeds of diverse species. Amer J Bot 68:44 48, 1981. 30. Young RA, Davis RW: Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci (USA) 80:1194-1198, 1983. Received 26 February 1985; in revised form 19 June 1985; accepted 24 June 1985.