Abstract
Chimeric genes composed of the β-phaseolin promoter, an α-zein coding sequence and its modified versions containing lysine codons, and a β-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. α-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa α-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an α-zein content that was approximately 0.003% of the total seed protein. The amount of α-zein in other transgenic plants varied between 1 × 10−4% and 1 × 10−5% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the α-zein protein. Polysomes translating α-zein mRNA isolated from tobacco seeds contained fewer ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized α-zein was degraded in tobacco seeds with a half-life of less than 1 hour.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
AnG: Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthetase promoter in transformed tobacco cells. Plant Physiol 81: 86–91 (1986).
ArgosP, PedersenK, MarksMD, LarkinsBA: A structural model for maize zein proteins. J Biol Chem 257: 9984–9990 (1982).
BevanM: Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12: 8711–8721 (1984).
BoronatA, MartinezMC, ReinaM, PuigdomenechP, PalauJ: Isolation and sequencing of a 28 kD glutelin-2 gene from maize. Common elements in the 5′ flanking regions among zein and glutelin genes. Plant Sci 47: 95–102 (1986).
BradfordMM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).
BurrB, BurrFA, RubensteinI, SimonMN: Purification and translation of zein mRNA from maize endosperm protein bodies. Proc Natl Acad Sci USA 75: 696–700 (1978).
BustosMM, GuiltinanMJ, JordanoJ, BegumD, KalkanFA, HallTC: Regulation of β-glucuronidase expression in transgenic tobacco by an A/T-rich, cis-acting sequence found upstream of a French bean β-phaseolin gene. Plant Cell 1: 839–853 (1989).
ChenZ-L, PanP-S, BeachyRN: A DNA sequence element that confers seed-specific enhancement to a constitutive promoter. EMBO J 7: 297–302 (1988).
DellaportaSL, WoodJ, HicksJB: Maize DNA miniprep. In: MalmbergR, MessingJ, SussexI (eds) Molecular Biology of Plants, pp. 36–37. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1985).
EsenA: Separation of alcohol-soluble proteins (zeins) from maize into three fractions by differential solubility. Plant Physiol 80: 623–627 (1986).
FeinbergAP, VogelsteinB: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
GaliliG, KawataEE, CuellarRE, SmithLD, LarkinsBA: Synthetic oligonucleotide tails inhibit in vitro and in vivo translation of SP6 transcripts of maize zein cDNA clones. Nucleic Acids Res 14: 1511–1524 (1986).
GreenwoodJS, ChrispeelsMJ: Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco. Plant Physiol 79: 65–71 (1985).
HagenG, RubensteinI: Complex organization of zein genes in maize. Gene 13: 239–249 (1981).
HoffmanLM, DonaldsonDD, BooklandR, RashkaK, HermanEM: Synthesis and protein body deposition of maize 15 kD zein in transgenic tobacco seeds. EMBO J 6: 3213–3221 (1987).
HoffmanLM, DonaldsonDD, HermanEM: A modified storage protein is synthesized, processed, and degraded in the seeds of transgenic plants. Plant Mol Biol 11: 717–729 (1988).
HorschRB, FryJE, HoffmanNL, EichholtzD, RogersSG, FraleyRT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
KiriharaJA, HunspergerJP, MahoneyWC, MessingJW: Differential expression of a gene for methionine-rich storage potein in maize. Mol Gen Genet 211: 477–484 (1988).
KodrzyckiR, BostonRS, LarkinsBA: The opaque 2 mutation of maize differentially reduces zein gene transcription. Plant Cell 1: 105–114 (1989).
LaemmliUK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
LarkinsBA, HurkmanWJ: Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol 62: 256–263 (1978).
LarkinsBA, PedersenK, MarksMD, WilsonDR: The zein proteins of maize endosperm. Trends Biochem Sci 9: 306–308 (1984).
LaskeyRA, MillsAD: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56: 335–341 (1975).
LendingCR, LarkinsBA: Changes in the zein composition of protein bodies during maize endosperm development. Plant Cell 1: 1011–1023 (1989).
ManiatisT, FritschEF, SambrookJ: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
MarksMD, LarkinsBA: Analysis of sequence microheterogeneity among zein messenger RNAs. J Biol Chem 257: 9976–9983 (1982).
MarksMD, LindellJS, LarkinsBA: Nucleotide sequence analysis of zein mRNAs from maize endosperm. J Biol Chem 260: 16451–16459 (1985).
MeltonDA, KriegPA, RebagliatiMR, ManiatisT, ZinnK, GreenMR: Efficient in vitro syntheses of biological active RNA and RNA hybridization probes from plasmids containing bacteriophage SP6 promoter. Nucleic Acids Res 12: 7035–7056 (1984).
MessingJ, CarlsonJ, HagenG, RubensteinI, OlesonA: Cloning and sequencing of the ribosomal RNA genes in maize: The 17S region. DNA 3: 31–40 (1984).
NelsonOE: Genetic modification of protein quality in plants. Adv Agron 21: 171–194 (1969).
MurashigeT, SkoogR: A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15: 473–497 (1962).
PedersenK, ArgosP, NaravanaSVL, LarkinsBA: Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15000. J Biol Chem 261: 6279–6284 (1986).
PetersonGL: Simplification of the protein assay method of Lowry et al. which is more generally applicable. Ann Biochem 83: 346–356 (1977).
SchernthanerJP, MatzkeMA, MatzkeAJM: Endosperm-specific activity of a zein gene promoter in transgenic tobacco plants. EMBO J 7: 1249–1255 (1988).
Sengupta-GopalanC, ReichertNA, BarkerRF, HallTC, KempJD: Developmentally regulated expression of the bean β-phaseolin gene in tobacco seed. Proc Natl Acad Sci USA 82: 3320–3324 (1985).
TowbinH, StaehelinT, GordonJ: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 67: 4350–4354 (1979).
UengP, GaliliG, SapanaraV, GoldsbroughPB, DubeP, BeachyRN, LarkinsBA: Expression of a maize storage protein gene in petunia plants is not restricted to seeds. Plant Physiol 86: 1281–1285 (1988).
WallaceJC, GaliliG, KawataEE, CuellarRE, ShotwellMA, LarkinsBA: Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes. Science 240: 6623–6624 (1988).
Wallace JC, Ohtani T, Lending CR, Lopes M, Williamson JD, Shaw KL, Gelvin SB, Larkins BA: Factors affecting physical and structural properties of maize protein bodies. In: Lamb C, Beachy RN (eds) Plant Gene Transfer, UCLA Symposium on Molecular and Cellular Biology New Series vol 129. Alan R. Liss, New York, in press.
WilliamsonJD, GaliliG, LarkinsBA, GelvinSB: The synthesis of a 19 kilodalton zein protein in transgenic Petunia plants. Plant Physiol 88: 1002–1007 (1988).
WangSZ, EsenA: Primary structure of a proline-rich zein and its cDNA. Plant Physiol 81: 70–74 (1986).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ohtani, T., Galili, G., Wallace, J.C. et al. Normal and lysine-containing zeins are unstable in transgenic tobacco seeds. Plant Mol Biol 16, 117–128 (1991). https://doi.org/10.1007/BF00017922
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00017922