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Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter

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Abstract

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protein (BCCP), and carboxytransferase (α-CT, β-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

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References

  1. An G, Binary Ti vectors for plant transformation and promoter analysis. Meth Enzymol 153: 292–305 (1987).

    Google Scholar 

  2. Ausubel FM: Current Protocols in Molecular Biology. Green Publishing Associates/Wiley-Interscience, New York (1990).

    Google Scholar 

  3. Bearson SR, Lamppa GK: Developmental regulation of an acyl carrier protein gene in vegetative and reproductive tissues. Plant Mol Biol 22: 255–267 (1993).

    Google Scholar 

  4. Bearson SR, Vander Heiden MG, Lamppa GK: Identification of domains in an Arabidopsis acyl carrier protein gene promoter required formaximal organ-specific expression. Plant Mol Biol 26: 1947–1959 (1994).

    Google Scholar 

  5. Baldet P, Alban S, Axiotis S, Douce R: Localization of free and bound biotin in cells from green pea leaves. Arch Biochem Biophys 303: 67–73 (1993).

    Google Scholar 

  6. Caddle MS, Lussier RH, Heintz NH: Intramolecular DNA triplexes, bent DNA and DNA unwinding elements in initiation region of an amplified dihydrofolate reductase repicon. J Mol Biol 211: 19–33 (1990).

    Google Scholar 

  7. Choi JK, Ke J, Sun J, Yu F, Wurtele ES, Nikolau BJ: Molecular biology of the plastidic acetyl-CoA carboxylase of Arabidopsis thaliana. Presented at the 12th International Symposium on Plant Lipids, Toronto, Canada (1996).

  8. de Boer GJ, Fawcett T, Slabas A, Nijkamp J, Stuije A: Analysis of theArabidopsis enoyl-ACP reductase promoter in transgenic tobacco. Presented at the 12th International Symposium on Plant Lipids, Toronto, Canada (1996).

  9. de Silva J, Loader NM, Jarman C, Windust JHC, Hughes SG, Safford R: The isolation and sequence analysis of two seed expressed acyl carrier protein genes from Brassica napus. Plant Mol Biol 14: 537–548 (1990).

    Google Scholar 

  10. de Silva J, Robinson SJ, Safford R: The isolation and functional characterization of a B. napus acyl carrier protein 50-flanking region involved in the regulation of seed storage lipid synthesis. Plant Mol Biol 18: 1163–1172 (1992).

    Google Scholar 

  11. Doyle JJ, Schuler MA, Godette WD, Zenger V, Beachy RN, Slightom JL: The glycosylated seed storage proteins ofGlycine max and Phaseolus vulgaris. J Biol Chem 261: 9228–9238 (1986).

    Google Scholar 

  12. Elborough KM, Winz R, Markham JE, White AJ, Slabas AR: Biotin carboxyl carrier protein of the multisubunit form of acetyl CoA carboxylase from oil seed rape, Brassica napus: cloning and comparative analysis of expression during oilseed rape embryogenesis of the two forms of ACCase. Presented at the 12th International Symposium on Plant Lipids, Toronto, Canada (1996).

  13. Evans DE, Taylor PE, Singh MB, Knox RB: The interrelationship between the accumulation of lipids, protein and the level of acyl carrier protein during the development of Brassica napus L pollen. Planta 186: 343–354 (1992).

    Google Scholar 

  14. Enjuto M, Lubreras V, Marin C, Boronate A: Expression of the Arabidopsis HMG2 gene, encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, is restricted to meristematic and floral tissues. Plant Cell 7: 517–527 (1995).

    Google Scholar 

  15. Gavel Y, von Heijne G, A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett 261: 455–458 (1990).

    Google Scholar 

  16. Gilmour DS, Thomas GH, Elgin SCR: Drosophilla nuclear proteins bind to regions of alternating C and T residues in gene promoters. Science 245: 1487–1490 (1989).

    Google Scholar 

  17. Gornicki P, Haselkorn R: Wheat acetyl-CoA carboxylase. Plant Mol Biol 22: 547–552 (1993).

    Google Scholar 

  18. Gornicki P, Podkowinski J, Scappino LA, DiMaio J, Ward E, Haselkorn R: Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure. Proc Natl Acad Sci USA 91: 6860–6864 (1994).

    Google Scholar 

  19. Harwood JL: Fatty acid metabolism. Annu Rev Plant Physiol Plant Mol Biol 39: 101–138 (1988).

    Google Scholar 

  20. Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 378–405 (1987).

    Google Scholar 

  21. Knutzon DS, Lardizabal KD, Nelsen JS, Bleibaum JL, Davies HM, Metz JG: Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrate. Plant Physiol 109: 999–1006 (1995).

    Google Scholar 

  22. Konishi T, Sasaki Y: Compartmentalization of two form of acetyl-CoA carboxylase in plants and the origin of their toler ance towards herbicides. Proc Natl Acad Sci USA 91: 3598–3601 (1994).

    Google Scholar 

  23. Laliberte JF, Nicolas O, Durand S, Morosoli P: The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants. Plant Mol Biol 18: 447–451 (1992).

    Google Scholar 

  24. Lamppa G, Jacks C: Analysis of two linked genes coding for the acyl carrier protein (ACP) from Arabidopsis thaliana (Columbia). Plant Mol Biol 16: 469–474 (1991).

    Google Scholar 

  25. Lessard PA, Allen RD, Bernier F, Crispino JD, Fujiwara T, Beachy RN: Multiple nuclear factors interact with upstream sequences of differentially regulated β-conglycinin genes. Plant Mol Biol 16: 397–413 (1991).

    Google Scholar 

  26. Li S, Cronan JE: Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalizes the first committed step of lipid biosynthesis. J Bact 175: 332–340 (1993).

    Google Scholar 

  27. Lu Q, Wallrath LL, Granok H, Elgin SCR: (CT)n.(GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcription activation of the Drosophilla hsp26 gene. Mol Cell Biol 13: 2802–2841 (1993).

    Google Scholar 

  28. JB, Browse J, Somerville CR: The genetics of plant lipids. Biochim Biophys Acta 1082: 1–26 (1991).

    Google Scholar 

  29. Ohlrogge JB, Jaworski JG: Regulation of fatty acid synthesis. Annu Rev Plant Physiol Plant Mol Biol 48: 109–136 (1997).

    Google Scholar 

  30. Paszkowski J, Peterhans A, Bilang R, Filipowicz W: Expression in trasgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes. Plant Mol Biol 19: 825–836 (1992).

    Google Scholar 

  31. Post-Beittenmiller D, Jaworski JG, Ohlrogge JB: In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis. J Biol Chem 266: 1858–1865 (1991).

    Google Scholar 

  32. Post-Beittenmiller D, Roughan PG, Ohlrogge JB: Regulation of plant fatty acid biosynthesis: analysis of acyl-CoA and acyl-ACP substrate pools in spinach and pea chloroplasts. Plant Physiol 100: 923–930 (1992).

    Google Scholar 

  33. Post-Beittenmiller MA, Hlousek-Radojcic A, Ohlrogge JB: DNA sequences of a genomic clone encoding an Arabidopsis acyl carrier protein (ACP). Nucl Acids Res. 17: 1777 (1989).

    Google Scholar 

  34. Quigley F, Martin WF, Cerff R: Intron conservation across the prokaryote-eukaryote boundary: structure of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize. Proc Natl Acad Sci USA 85: 2672–2676 (1988).

    Google Scholar 

  35. Rempel HC, Nelson LM: Analysis of conditions for agrobacterium-mediated transformation of tobacco cells in suspension. Transgen Res 4: 199–207 (1991).

    Google Scholar 

  36. Roesler KR, Shorrosh BS, Ohlrogge JB: Structure and expression of an Arabidopsis acetyl-coenzyme A carboxylase gene. Plant Physiol 105: 611–617 (1994).

    Google Scholar 

  37. Roesler KR, Savage LJ, Shintani DK, Shorrosh BS, Ohlrogge JB: Co-purification, co-immunoprecipitation, and coordinate expression of acetyl-coenzyme A carboxylase activity, bioyin carboxylase, and biotin carboxyl carrier protein of higher plants. Planta 198: 517–525 (1996).

    Google Scholar 

  38. Roessler PG, Ohlrogge JB: Cloning and chaterization of the gene that encodes acetyl-coenzyme A carboxylase in the alga Cyclotella cryptica. J Biol Chem 268: 19254–19259 (1993).

    Google Scholar 

  39. Sambrook J, Fritch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  40. Vagelos RP: Regulation of fatty acid biosynthesis. Curr Top Cell Regul 4: 119–166 (1971).

    Google Scholar 

  41. Sasaki Y, Konishi T, Nagano Y: The compartmentation of acetyl-coenzyme A carboxylase in plants. Plant Physiol 108: 445–449 (1995).

    Google Scholar 

  42. Shapiro MB, Senapathy P: RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications of gene expression. Nucl Acids Res 15: 7155–7174 (1987).

    Google Scholar 

  43. Shintani DK, Ohlrogge JB: Feedback inhibition of fatty acid synthesis in tobacco suspension cells. Plant J 7: 577–587 (1995).

    Google Scholar 

  44. Shorrosh BS, Dixon RA, Ohlrogge JB: Molecular cloning, characterization and elicitation of acetyl-CoA carboxylase from alfalfa. Proc Natl Acad Sci USA 91: 4323–4327 (1994).

    Google Scholar 

  45. Shorrosh BS, Roesler KR, Shintani DK, van de Loo FJ, Ohlrogge JB: Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-Coenzyme A carboxylase from tobacco. Plant Physiol 108: 805–812 (1995).

    Google Scholar 

  46. Slocombe SP, Piffanelli P, Fairbairn D, Bowra S, Hatzopoulos P, Tsiantis M, Murphy DJ: Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene. Plant Physiol 104: 1167–1176 (1994).

    Google Scholar 

  47. van Hoof A, Green PJ: Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a position-dependent manner. Plant J 10: 415–424 (1996).

    Google Scholar 

  48. Vancanneyt G, Schmid R, O'Connor-Sanchez A, Willmitzer L, Rocha-Sosa M: Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220: 245–250 (1990).

    Google Scholar 

  49. Wakil SJ, Stoop JK, Joshi VC: Fatty acid synthesis and its regulation. Annu Rev Biochem 52: 537–579 (1983).

    Google Scholar 

  50. Weinreb A, Collier DA, Birshtein BK, Wells RD: Left-handed Z-DNA and intramolecular triplex formation at the site of an unequal sister chromatid exchange. J Biol Chem 265: 1352–1359 (1990).

    Google Scholar 

  51. Wells RD, Collier DA, Hanvey JC, Shimizu M, Wohlrab F: The chemistry and biology of unusual DNA structures adopted by oligopurine oligopyrimidine sequence. FASEB J 2: 2939–2949 (1988).

    Google Scholar 

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Authors and Affiliations

  1. Department of Botany and Pathology, Michigan State University, East Lansing, MI, 48824, USA

    Xiaoming Bao, Basil S. Shorrosh & John B. Ohlrogge

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  1. Xiaoming Bao
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  2. Basil S. Shorrosh
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  3. John B. Ohlrogge
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Bao, X., Shorrosh, B.S. & Ohlrogge, J.B. Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter. Plant Mol Biol 35, 539–550 (1997). https://doi.org/10.1023/A:1005881006620

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