Skip to main content
Plant Physiology logoLink to Plant Physiology
. 1994 Nov;106(3):929–939. doi: 10.1104/pp.106.3.929

The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

D R Gallie 1, T E Young 1
PMCID: PMC159615  PMID: 7824660

Abstract

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Buchman A. R., Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988 Oct;8(10):4395–4405. doi: 10.1128/mcb.8.10.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Callis J., Fromm M., Walbot V. Introns increase gene expression in cultured maize cells. Genes Dev. 1987 Dec;1(10):1183–1200. doi: 10.1101/gad.1.10.1183. [DOI] [PubMed] [Google Scholar]
  4. Chandler P. M., Huiet L. Primer extension studies on alpha-amylase mRNAs in barley aleurone. I. Characterization and quantification of the transcripts. Plant Mol Biol. 1991 Apr;16(4):627–635. doi: 10.1007/BF00023427. [DOI] [PubMed] [Google Scholar]
  5. Dickey L. F., Gallo-Meagher M., Thompson W. F. Light regulatory sequences are located within the 5' portion of the Fed-1 message sequence. EMBO J. 1992 Jun;11(6):2311–2317. doi: 10.1002/j.1460-2075.1992.tb05290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gallie D. R., Feder J. N., Schimke R. T., Walbot V. Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression. Mol Gen Genet. 1991 Aug;228(1-2):258–264. doi: 10.1007/BF00282474. [DOI] [PubMed] [Google Scholar]
  7. Gallie D. R., Lucas W. J., Walbot V. Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell. 1989 Mar;1(3):301–311. doi: 10.1105/tpc.1.3.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gallie D. R., Sleat D. E., Watts J. W., Turner P. C., Wilson T. M. The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucleic Acids Res. 1987 Apr 24;15(8):3257–3273. doi: 10.1093/nar/15.8.3257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gallie D. R., Walbot V. Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation. Nucleic Acids Res. 1992 Sep 11;20(17):4631–4638. doi: 10.1093/nar/20.17.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gopalakrishnan B., Sonthayanon B., Rahmatullah R., Muthukrishnan S. Barley aleurone layer cell protoplasts as a transient expression system. Plant Mol Biol. 1991 Mar;16(3):463–467. doi: 10.1007/BF00023996. [DOI] [PubMed] [Google Scholar]
  11. Hillmer S., Bush D. S., Robinson D. G., Zingen-Sell I., Jones R. L. Barley aleurone protoplasts are structurally and functionally similar to the walled cells of aleurone layers. Eur J Cell Biol. 1990 Jun;52(1):169–173. [PubMed] [Google Scholar]
  12. Huang M. T., Gorman C. M. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 1990 Feb 25;18(4):937–947. doi: 10.1093/nar/18.4.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klein T. M., Roth B. A., Fromm M. E. Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6681–6685. doi: 10.1073/pnas.86.17.6681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Luehrsen K. R., de Wet J. R., Walbot V. Transient expression analysis in plants using firefly luciferase reporter gene. Methods Enzymol. 1992;216:397–414. doi: 10.1016/0076-6879(92)16037-k. [DOI] [PubMed] [Google Scholar]
  15. Matzke A. J., Stöger E. M., Schernthaner J. P., Matzke M. A. Deletion analysis of a zein gene promoter in transgenic tobacco plants. Plant Mol Biol. 1990 Mar;14(3):323–332. doi: 10.1007/BF00028769. [DOI] [PubMed] [Google Scholar]
  16. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mozer T. J. Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid. Cell. 1980 Jun;20(2):479–485. doi: 10.1016/0092-8674(80)90634-0. [DOI] [PubMed] [Google Scholar]
  18. O'Neill S. D., Kumagai M. H., Majumdar A., Huang N., Sutliff T. D., Rodriguez R. L. The alpha-amylase genes in Oryza sativa: characterization of cDNA clones and mRNA expression during seed germination. Mol Gen Genet. 1990 Apr;221(2):235–244. doi: 10.1007/BF00261726. [DOI] [PubMed] [Google Scholar]
  19. Radicella J. P., Brown D., Tolar L. A., Chandler V. L. Allelic diversity of the maize B regulatory gene: different leader and promoter sequences of two B alleles determine distinct tissue specificities of anthocyanin production. Genes Dev. 1992 Nov;6(11):2152–2164. doi: 10.1101/gad.6.11.2152. [DOI] [PubMed] [Google Scholar]
  20. Roth B. A., Goff S. A., Klein T. M., Fromm M. E. C1- and R-dependent expression of the maize Bz1 gene requires sequences with homology to mammalian myb and myc binding sites. Plant Cell. 1991 Mar;3(3):317–325. doi: 10.1105/tpc.3.3.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shimamoto K., Ackermann M., Dierks-Ventling C. Expression of zein in long term endosperm cultures of maize. Plant Physiol. 1983 Dec;73(4):915–920. doi: 10.1104/pp.73.4.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Skriver K., Olsen F. L., Rogers J. C., Mundy J. cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7266–7270. doi: 10.1073/pnas.88.16.7266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Unger E., Parsons R. L., Schmidt R. J., Bowen B., Roth B. A. Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells. Plant Cell. 1993 Aug;5(8):831–841. doi: 10.1105/tpc.5.8.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Walker J. C., Howard E. A., Dennis E. S., Peacock W. J. DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase 1 gene. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6624–6628. doi: 10.1073/pnas.84.19.6624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Young T. E., DeMason D. A., Close T. J. Cloning of an alpha-amylase cDNA from aleurone tissue of germinating maize seed. Plant Physiol. 1994 Jun;105(2):759–760. doi: 10.1104/pp.105.2.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zhang S., Mehdy M. C. Binding of a 50-kD Protein to a U-Rich Sequence in an mRNA Encoding a Proline-Rich Protein That Is Destabilized by Fungal Elicitor. Plant Cell. 1994 Jan;6(1):135–145. doi: 10.1105/tpc.6.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES