AJPS  Vol.5 No.18 , August 2014
Tissue Specific Expression of a Terpene Synthase in Nicotiana benthamiana Leaves
Abstract: To study transient tissue specific linalool emission and to examine its fate, plastid targeted dual linalool/nerolidol synthase (FaNES1) was introduced into Nicotiana benthamiana leaves under LTP1, SUC2, RbcS and CaMV35 promoters. Tissue specificity of the promoters was tested with β-glucuronidase (GUS) reporter gene. Promoter::GUS/FaNES1 fusion was confirmed with colony PCR and agro-infiltrated into six weeks old N. benthamiana leaves. Eight days post inoculation (dpi), promoter::GUS constructs were examined with GUS histochemical staining at 1.5 h, 3.5 h, 5.5 h and 16 h incubation times. After 4/8 days, FaNES1 construct agro-inoculated leaves were assessed for linalool emissions and its conjugates using a dynamic headspace system and LC-MS, respectively. There was high affinity of promoters to their respective cell-types although it was not as specific as in stable transformation. This is possibly due to activations of many copies of transiently introduced promotes by few transcription factors of the respective promoter that naturally exist in untargeted cell-types. GUS staining intensity was stronger in leaf veins and injured sites compared to other plant tissues under all heterologous promoters, and it was gradually increased with increase in incubation times that could be explained by promoters wounding responses and/ or GUS leakage to unstained sites. Linalool emission was obtained in the same pattern under all promoters; it was higher at day 4 than day 8. Its concentration declined 44, 12, 4.5 and 4 folds at 8 dpi under LTP1, SUC2, RbcS and CaMV35S promoters, respectively. Conversely, linalool conjugates were significantly increased at day 8. These might be due to T-DNA degradations and/or protein modifications 4 dpi. LTP1 promoter was the least efficient to drive both GUS and FaNES1 possibly due to immature plastids in epidermal cells and/or its weak performance. Hence, to study FaNES1 activity in transient assay it is suggested to use relatively shorter duration and longer inoculation times for linalool and its conjugates, respectively.
Cite this paper: Juneidi, S. , Ting, H. and der Krol, A. (2014) Tissue Specific Expression of a Terpene Synthase in Nicotiana benthamiana Leaves. American Journal of Plant Sciences, 5, 2799-2810. doi: 10.4236/ajps.2014.518296.

[1]   Dorothea, T. (2006) Terpene Synthases and the Regulation, Diversity and Biological Roles of Terpene Metabolism. Current Opinion in Plant Biology, 9, 297-304.

[2]   Mahmoud, S.S. and Croteau, R.B. (2002) Strategies for Transgenic Manipulation of Monoterpene Biosynthesis in Plants. Trends in Plant Science, 7, 366-373.

[3]   Jonathan Gershenzon, M.E. (2000) Regulation of Monoterpene Accumulation in Leaves of Peppermint. Plant Physiology, 122, 205-214.

[4]   Lücker, J., Bouwmeester, H.J., Schwab, W., Blaas, J., Van Der Plas, L.H.W. and Verhoeven, H.A. (2001) Expression of Clarkia S-Linalool Synthase in Transgenic Petunia Plants Results in the Accumulation of S-Linalyl-β-d-glucopyranoside. The Plant Journal, 27, 315-324.

[5]   Aharoni, A., Giri, A.P., Deuerlein, S., Griepink, F., De Kogel, W.J., Verstappen, F.W.A., Verhoeven, H.A., Jongsma, M.A., Schwab, W. and Bouwmeester, H.J. (2003) Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants. Plant Cell, 15, 2866-2884.

[6]   Aharoni, A., Jongsma, M., Kim, T.-Y., Ri, M.-B., Giri, A., Verstappen, F., Schwab, W. and Bouwmeester, H. (2006) Metabolic Engineering of Terpenoid Biosynthesis in Plants. Phytochemistry Reviews, 5, 49-58.

[7]   Davidovich-Rikanati, R., Sitrit, Y., Tadmor, Y., Iijima, Y., Bilenko, N., Bar, E., Carmona, B., Fallik, E., Dudai, N., Simon, J.E., Pichersky, E. and Lewinsohn, E. (2007) Enrichment of Tomato Flavor by Diversion of the Early Plastidial Terpenoid Pathway. Nature Biotechnology, 25, 899-901.

[8]   Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991) A DNA Transformation-Competent Arabidopsis Genomic Library in Agrobacterium. Nature Biotechnology, 9, 963-967.

[9]   Sanger, F.A., Nicklin, S. and Coulson, A.R. (1977) DNA Sequencing with Chain Termination Inhibitors. Proceedings of the National Academy of Sciences of USA, 74, 5463-5467.

[10]   McCormac, A., Elliott, M. and Chen, D. (1998) A Simple Method for the Production of Highly Competent Cells of Agrobacterium for Transformation via Electroporation. Molecular Biotechnology, 9, 155-159.

[11]   Main, D.G., Reynold, S. and Jill, S.G. (1995) Methods in Molecular Biology, Electroporation for Agrobacterium. Humana Press Inc, Totowa, 405-512.

[12]   Yang, Y., Li, R. and Qi, M. (2000) In Vivo Analysis of Plant Promoters and Transcription Factors by Agroinfiltration of Tobacco Leaves. The Plant Journal, 22, 543-551.

[13]   Zottini, M., Barizza, E., Costa, A., Formentin, E., Ruberti, C., Carimi, F. and Lo Schiavo, F. (2008) Agroinfiltration of Grapevine Leaves for Fast Transient Assays of Gene Expression and for Long-Term Production of Stable Transformed Cells. Plant Cell Reports, 27, 845-853.

[14]   Yang, T., Stoopen, G., Yalpani, N., Vervoort, J., de Vos, R., Voster, A., Verstappen, F.W.A., Bouwmeester, H.J. and Jongsma, M.A. (2011) Metabolic Engineering of Geranic Acid in Maize to Achieve Fungal Resistance Is Compromised by Novel Glycosylation Pattern. Metabolic Engineering, 13, 414-425.

[15]   De Vos, R.C.H., Moco, S., Lommen, A., Keurentjes, J.J.B., Bino, R.J. and Hall, R.D. (2007) Untargeted Large-Scale Plant Metabolomics Using Liquid Chromatography Coupled to Mass Spectrometry. Nature Protocols, 2, 778-791.

[16]   Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS Fusions: Beta-Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants. EMBO Journal, 6, 3901-3907.

[17]   Ranjan, A., Ansari, S.A., Srivastava, R., Mantri, S., Asif, M.H., Sawant, S.V. and Tuli, R. (2009) A T9G Mutation in the Prototype TATA-Box TCACTATATATAG Determines Nucleosome Formation & Synergy with Upstream Activator Sequences in Plant Promoters. Plant Physiology, 151, 2174-2186.

[18]   Truernit, E. and Sauer, N. (1995) The Promoter of the Arabidopsis thaliana SUC2 Sucrose-H+ Symporter Gene Directs Expression of β-Glucuronidase to the Phloem: Evidence for Phloem Loading and Unloading by SUC2. Planta, 196, 564-570.

[19]   Torbert, K.A., Gopalraj, M., Medberry, S.L., Olszewski, N.E. and Somers, D.A. (1998) Expression of the Commelina Yellow Mottle Virus Promoter in Transgenic Oat. Plant Cell Reports, 17, 284-287.

[20]   Bara ski, R. and Puddephat, I. (2004) Tissue Specific Expression of β-Glucuronidase Gene Driven by Heterologous Promoters in Transgenic Cauliflower Plants. Acta Physiologiae Plantarum, 26, 307-315.

[21]   Lojda, Z. (1970) Indigogenic Methods for Glycosidases. Histochemistry & Cell Biology, 23, 266-288.

[22]   Mascarenhas, J.P. and Hamilton, D.A. (1992) Artifacts in the Localization of GUS Activity in Anthers of Petunia Transformed with a CaMV35S-GUS Construct. Plant Journal, 2, 405-408.

[23]   Martin, V.J.J., Piteral, D.J., Withers, S.T., Newman, J.D. and Keasling, J.D. (2003) Engineering a Mevalonate Pathway in Escherichia coli for Production of Terpenoids. Nature Biotechnology, 21, 796-802.

[24]   Nap, J.P., Spanje, M., Dirkse, W.G., Baarda, G., Mlynarova, L., Loonen, A., Grondhuis, P. and Stiekema, W.J. (1993) Activity of the Promoter of the Lhca3.St.1 Gene, Encoding the Potato Apoprotein 2 of the Light-Harvesting Complex of Photosystem I, in Transgenic Potato and Tobacco Plants. Plant Molecular Biology, 23, 605-612.

[25]   Maghuly, F., Khan, M.A., Fernandez, E.B., Druart, P., Watillon, B. and Laimer, M. (2008) Stress Regulated Expression of the GUS-Marker Gene (uidA) under the Control of Plant Calmodulin and Viral 35S Promoters in a Model Fruit Tree Rootstock: Prunus incisa × serrula. Journal of Biotechnology, 135, 105-116.

[26]   Outchkourov, N.S., Peters, J., de Jong, J., Rademakers, W. and Jongsma, M.A. (2003) The Promoter-Terminator of Chrysanthemum SMALL Rbcs1directs High Expression Levels in Plants. Planta, 216, 1003-1012.

[27]   Baroux, C., Blanvillain, R., Moore, R. and Gallois, P. (2001) Transactivation of BARNASE under the AtLTP1 Promoter Affects the Basal Pole of the Embryo and Shoot Development of the Adult Plant in Arabidopsis. The Plant Journal, 28, 503-515.

[28]   Chen, X., Yauk, Y.K., Nieuwenhuizen, N.J., Matich, A.J., Wang, M.Y., Perez, R.L., Atkinson, R.G. and Beuning, L.L. (2010) Characterisation of an (S)-linalool Synthase from Kiwifruit (Actinidia arguta) that Catalyses the First Committed Step in the Production of Floral Lilac Compounds. Functional Plant Biology, 37, 232-243.

[29]   Lavy, M., Zuker, A., Lewinsohn, E., Larkov, O., Ravid, U., Vainstein, A. and Weiss, D. (2002) Linalool and Linalool Oxide Production in Transgenic Carnation Flowers Expressing in Clarkia breweri Linalool Synthase Gene. Molecular Breeding, 9, 103-111.

[30]   Lindbo, J.A. (2007) TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector. Plant Physiology, 145, 1232-1240.

[31]   Miyamoto, T., Nakamura, T., Nagao, I. and Obokata, J. (2000) Quantitative Analysis of Transiently Expressed mRNA in Particle-Bombarded Tobacco Seedlings. Plant Molecular Biology Reporter, 18, 101-107.

[32]   Raguso, R.A. and Pichersky, E. (1995) Floral Volatiles from Clarkia breweri: Recent Evolution of Floral Scent and Moth Pollination. Plant Systematics and Evolution, 194, 55-67.

[33]   Fujiwara, M., Kazama, Y., Abe, T. and Itoh, R. (2010) Plastid Replication in Leaf Epidermis: Insights from the atminE1 Mutant of Arabidopsis. 21st International Conference on Arabidopsis Research, Yokohama, 6-10 June 2010, 203-204.