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Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

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Abstract

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Δ9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibiotic-insensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the Δ9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the “binding-type” vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

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References

  • Bock R (2007) Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming. Curr Opin Biotechnol 18:100–106

    Article  PubMed  CAS  Google Scholar 

  • Bock R, Khan MS (2004) Taming plastids for a green future. Trends Biotechnol 22:311–318

    Article  PubMed  CAS  Google Scholar 

  • Buhot L, Horvàth E, Medgyesy P, Lerbs-Mache S (2006) Hybrid transcription system for controlled plastid transgene expression. Plant J 46:700–707

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  PubMed  CAS  Google Scholar 

  • Craig W, Lenzi P, De Palma M, Saggese P, Dix PJ, Curran NM, Medgyesy P, Carbone V, Scotti N, Grillo S and Cardi T (2004) Plastid transformation for the production of unsaturated fatty acids: molecular and biochemical characterization of tobacco transplastomic plants expressing Δ9 desaturase genes. Proceedings of the XLVIII Italian Society of Agricultural Genetics—SIFV-SIGA Joint Meeting Lecce, Italy, September 15–18, 2004

  • Crawford RV, Hilditch TP (1950) The component fatty acids of tobacco-seed oils. J Sci Food Agric 1:230–234

    Article  CAS  Google Scholar 

  • Cséplő A, Maliga P (1984) large scale isolation of maternally inherited lincomycinresistance mutations, in diploid Nicotiana plumbaginifolia protoplast cultures. Mol Gen Genet 196:407–412

    Article  Google Scholar 

  • Daniell H, Kumar S, Dufourmantel N (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends Biotechnol 23:238–245

    Article  PubMed  CAS  Google Scholar 

  • Darmstadt GL, Mao-Qiang M, Chi E, Saha SK, Ziboh VA, Black RE, Santosham M, Elias PM (2002) Impact of topical oils on the skin barrier: possible implications for neonatal health in developing countries. Acta Paediatr 91:546–554

    Article  PubMed  CAS  Google Scholar 

  • De Palma M, Grillo S, Massarelli I, Costa A, Balogh G, Vigh L, Leone A (2008) Regulation of desaturase gene expression, changes in membrane lipid composition and freezing tolerance in potato plants. Mol Breed 21:15–26

    Article  CAS  Google Scholar 

  • Dix PJ, Kavanagh TA (1995) Transforming the plastome: genetic markers and DNA delivery systems. Euphytica 85:29–34

    Article  CAS  Google Scholar 

  • Dix PJ, McKinley CP, Mc Cabe PF (1990) Antibiotic resistant mutants of Solanum nigrum. In: Nijkamp HJJ, Van Der Plas LHW, Van Aartijk J (eds) Progress in plant cellular and molecular biology. Kluwer Academic Publishers, Dordrecht, Boston, London, pp 169–174

    Google Scholar 

  • Flint HL, Boyce BK, Beattie DJ (1967) Index of injury—a useful expression of freezing injury to plant tissues as determined by the electrolytic method. Can J Plant Sci 47:229–230

    Article  Google Scholar 

  • Giannelos PN, Zannikos F, Stournas S, Lois E, Anastopoulos G (2002) Tobacco seed oil as an alternative diesel fuel: physical and chemical properties. Ind Crop Prod 16:1–9

    Article  CAS  Google Scholar 

  • Golds T, Maliga P, Koop HU (1993) Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Biotechnology 11:95–97

    Article  CAS  Google Scholar 

  • Graham IA, Larson T, Napier JA (2007) Rational metabolic engineering of transgenic plants for biosynthesis of omega-3 polyunsaturates. Curr Opin Biotechnol 18:1–6

    Article  CAS  Google Scholar 

  • Grevich JJ, Daniell H (2005) Chloroplast genetic engineering: recent advances and future perspective. Crit Rev Plant Sci 24:83–107

    Article  CAS  Google Scholar 

  • Hornung E, Saalbach I and Feussner I (2004) Production of conjugated fatty acids in plants. Proceedings of the 16th International Plant Lipid Symposium. Budapest, Hungary, June 1–4, 2004

  • Horváth EM, Peter SO, Joët T, Rumeau D, Cournac L, Horváth GV, Kavanagh TA, Schäfer C, Peltier G, Medgyesy P (2000) Targeted inactivation of the plastid ndhB gene in tobacco results in enhanced sensitivity of photosynthesis to moderate stomatal closure. Plant Physiol 123:1337–1349

    Article  PubMed  Google Scholar 

  • Ishizaki-Nishizawa O, Fujii T, Azuma M, Sekiguchi K, Murata N, Ohtani T, Toguri T (1996) Low-temperature resistance of higher plants is significantly enhanced by a nonspecific cyanobacterial desaturase. Nat Biotechnol 14:1003–1006

    Article  PubMed  CAS  Google Scholar 

  • Kachroo P, Shanklin J, Shah J, Whittle EJ, Klessig DF (2001) A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Proc Natl Acad Sci USA 98:9448–9453

    Article  PubMed  CAS  Google Scholar 

  • Kachroo A, Shanklin J, Whittle E, Lapchyk L, Hildebrand D, Kachroo P (2007) The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis. Plant Mol Biol 63:257–271

    Article  PubMed  CAS  Google Scholar 

  • Kavanagh TA, O’Driscoll KM, McCabe PF, Dix PJ (1994) Mutations conferring lincomycin, spectinomycin, and streptomicyn resistance in Solanum nigrum are located in three different chloroplast genes. Mol Gen Genet 242:675–680

    Article  PubMed  CAS  Google Scholar 

  • Kavanagh TA, Thanh ND, Lao NT, McGrath N, Peter SO, Horvath EM, Dix PJ, Medgyesy P (1999) Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events. Genetics 152:1111–1122

    PubMed  CAS  Google Scholar 

  • Knutzon DS, Thompson GA, Radke SE, Johnson WB, Knauf VC, Kridle JC (1992) Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene. Proc Natl Acad Sci USA 89:2624–2628

    Article  PubMed  CAS  Google Scholar 

  • Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K (1994) Genetic enhancement of cold tolerance by expression of a gene for chloroplast ω-3 fatty acid desaturase in transgenic tobacco. Plant Physiol 105:601–605

    PubMed  CAS  Google Scholar 

  • Letawe C, Boone M, Piérard GE (1998) Digital image analysis of the effect of topically applied linoleic acid on acne microcomedones. Clin Exp Dermatol 23:56–58

    Article  PubMed  CAS  Google Scholar 

  • Lightner J, Wu J, Browse J (1994a) A mutant of Arabidopsis with increased levels of stearic acid. Plant Physiol 106:1443–1451

    PubMed  CAS  Google Scholar 

  • Lightner J, James DW, Dooner HK, Browse J (1994b) Altered body morphology is caused by increased stearate levels in a mutant of Arabidopsis. Plant J 6:401–412

    Article  CAS  Google Scholar 

  • Lutz KA, Maliga P (2007) Construction of marker-free transplastomic plants. Curr Opin Biotechnol 18:107–114

    Article  PubMed  CAS  Google Scholar 

  • Madoka Y, Tomizawa K-I, Mizoi J, Nishida I, Nagano Y, Sasaki Y (2002) Chloroplast transformation with modified accD operon increases acetyl-CoA carboxylase and causes extension of leaf longevity and increase in seed yield in tobacco. Plant Cell Physiol 43:1518–1525

    Article  PubMed  CAS  Google Scholar 

  • Maliga P (2002) Engineering the plastid genome of higher plants. Curr Opin Plant Biol 5:164–172

    Article  PubMed  CAS  Google Scholar 

  • Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313

    Article  PubMed  CAS  Google Scholar 

  • Mc Cabe PF, Timmons PJ, Dix PJ (1989) A simple procedure for the isolation of streptomycin resistant plants in Solanaceae. Mol Gen Genet 216:132–137

    Article  CAS  Google Scholar 

  • McGrath Curran N, Shiel K, Nugent J, Kavanagh T and Dix P (2003) Chloroplast transformation of tobacco using non-bacterial selectable marker genes. Proceedings of the 7th International Congress of Plant Molecular Biology, Barcelona, Spain, June 23–28, 2003

  • McKeon TA, Stumpf PK (1982) Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J Biol Chem 257:12141–12147

    PubMed  CAS  Google Scholar 

  • Merlo AO, Cowen N, Delate T, Edington B, Folkerts O, Hopkins N, Lemeiux C, Skokut T, Smith K, Woosley A, Yang Y, Young S, Zwick M (1998) Ribozymes targeted to stearoyl-ACP Δ9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves. Plant Cell 10:1603–1622

    Article  PubMed  CAS  Google Scholar 

  • Moon BY, Higashi SI, Gombos Z, Murata N (1995) Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants. Proc Natl Acad Sci USA 92:6219–6223

    Article  PubMed  CAS  Google Scholar 

  • Moon H, Hazebroek J, Hildebrand DF (2000) Changes in fatty acid composition in plant tissues expressing a mammalian Δ9 desaturase. Lipids 35:471–479

    Article  PubMed  CAS  Google Scholar 

  • Murata N, Wada H (1995) Acyl-lipid desaturases and thier importance in the tolerance and acclimatization to cold of cyanobacteria. Biochem J 308:1–8

    PubMed  CAS  Google Scholar 

  • Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzephysiol 78:453–455

    Google Scholar 

  • Nandi A, Krothapalli K, Buseman CM, Li M, Welti R, Enyedi A, Shah J (2003) Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase. Plant Cell 15:2383–2398

    Article  PubMed  CAS  Google Scholar 

  • Nugent GD, ten Have M, van der Gulik A, Dix PJ, Uijtewaal BA, Mordhorst AP (2005) Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep 24:341–349

    Article  PubMed  CAS  Google Scholar 

  • O’Neill C, Horvath GV, Horvath E, Dix PJ, Medgyesy P (1993) Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. Plant J 3:729–738

    Article  PubMed  CAS  Google Scholar 

  • Rumeau D, Bécuwe-Linka N, Beyly A, P. C, Cuiné S, Genty B, Medgyesy P, Horvath E, Peltier G (2004) Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit. Plant Biotechnol J 2:389–399

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Scotti N, Monti L, Cardi T (2003) Organelle DNA variation in parental Solanum spp. genotypes and nuclear-cytoplasmic interactions in Solanum tuberosum (+) S. commersonii somatic hybrid-backcross progeny. Theor Appl Genet 108:87–94

    Article  PubMed  CAS  Google Scholar 

  • Somerville C, Browse J, Jaworski JG, Ohlrogge JB (2000) Lipids. In: Buchanan BGW, Jones R (eds) Biochemistry & molecular biology of plants. American Society of Plant Physiologists, Rockville, USA

    Google Scholar 

  • Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530

    Article  PubMed  CAS  Google Scholar 

  • Tetlow IJ, Rawsthorne S, Raines C, Emes MJ (2005) Plastid metabolic pathways. In: Moller SG (ed) Plastids. Blackwell Publishing, Oxford, pp 60–125

    Google Scholar 

  • Wakasugi T, Sugita M, Tsudzuki T, Sugiura M (1998) Update gene map of tobacco chloroplast DNA. Plant Mol Biol Rep 16:231–241

    Article  CAS  Google Scholar 

  • Wilkinson L, Hill MA, Vang E (1992) SYSTAT: statistics. Version 5.2 edn. SYSTAT Inc., Evanston

    Google Scholar 

  • Zaborowska Z, Starzycki M, Femiak I, Swiderski M, Legocki AB (2002) Yellow lupine gene encoding stearoyl-ACP desaturase-organization, expression and potential application. Acta Biochim Pol 49:29–42

    PubMed  CAS  Google Scholar 

  • Zhou MX, Glennie Holmes M, Robards K, Helliwell S (1998) Fatty acid composition of lipids of australian oats. J Cereal Sci 28:311–319

    Article  CAS  Google Scholar 

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Acknowledgements

The work was partially funded by the European Union 5th Framework Programme, project “The Plastid Factory” (QLK3-CT-1999-00692). We thank Dr. T. Toguri, Kirin Brewery Co., Yokohama, Japan for generously providing the desaturase cDNA from A. nidulans, and Ms. A. Morgese, Mr. P. Palmieri, Mr. A. Piccolo and Ms. L. Sannino for their excellent technical assistance.

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Authors and Affiliations

  1. CNR-IGV, Institute of Plant Genetics-Portici, Portici, NA, Italy

    Wendy Craig, Paolo Lenzi, Nunzia Scotti, Monica De Palma, Stefania Grillo & Teodoro Cardi

  2. CNR-ISA, Institute of Food Science, Avellino, AV, Italy

    Paola Saggese & Virginia Carbone

  3. Plant Cell Biology Laboratory, Biology Department, NUI-Maynooth, Maynooth, Ireland

    Noreen McGrath Curran, Peter Medgyesy & Philip J. Dix

  4. Plant Molecular Biology Laboratory, Smurfit Institute of Genetics, Trinity College, Lincoln Place Gate, Dublin 2, Ireland

    Alan M. Magee & Tony A. Kavanagh

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  1. Wendy Craig
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  2. Paolo Lenzi
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  3. Nunzia Scotti
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  4. Monica De Palma
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  11. Philip J. Dix
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  12. Stefania Grillo
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  13. Teodoro Cardi
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Correspondence to Teodoro Cardi.

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Contribution no. 104 from CNR-IGV, Portici.

Wendy Craig and Paolo Lenzi contributed equally to the work.

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Craig, W., Lenzi, P., Scotti, N. et al. Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance. Transgenic Res 17, 769–782 (2008). https://doi.org/10.1007/s11248-008-9164-9

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