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Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings

Plant Cell Reports volume 33, pages 89–97 (2014)Cite this article

Abstract

Key message

An Agro-mediated transformation method has been adapted in Catharanthus roseus seedlings for transient overexpression. Our results suggest that Agro-mediated methods may induce defense-related genes, which should be considered in its application.

Abstract

The Fast Agro-mediated Seedling Transformation (FAST) method, which involves the co-cultivation and transient transformation of young seedlings with Agrobacterium, was adapted and optimized in Catharanthus roseus. We investigated the optimal conditions for Gus expression by varying the Agrobacterium density (OD600 = 0.29 and 0.50), A. rhizogenes strain (15834 and R1000), and co-cultivation time in liquid (2, 12, or 24 h) followed by incubation time on solid media (1 or 2 days). Transformation efficiency was assessed quantitatively in terms of average GUS intensity per cotyledon surface area and percentage of cotyledons transformed. GUS staining was observed in 100 % of cotyledons co-cultivated with A. rhizogenes (OD600 = 0.50) co-transformed with the Mas promoter-driven Gus and pSoup helper plasmids, in the presence of 0.01 % v/v Silwet L-77 for 24 h in liquid followed by 2-days on solid media. In addition, we observed that co-cultivation with Agrobacterium strongly induced Zct1 and Orca3, two transcription factors known to regulate defense-related alkaloid biosynthesis in C. roseus. Homologous transcription factors regulate defense responses in many plant species. Therefore, possible induction of defense-related genes by Agro-mediated transformation should be a consideration in experimental design.

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References

  • Aerts RJ, Verpoorte R (1992) The influence of tryptophan and tryptamine feeding and light on alkaloid biosynthesis in Cinchona seedlings. Planta Med 58(2):150–152. doi:10.1055/s-2006-961417

    CAS  PubMed  Article  Google Scholar 

  • Aerts RJ, Gisi D, De Carolis E, De Luca V, Baumann TW (1994) Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings. Plant J 5(5):635–643

    CAS  Article  Google Scholar 

  • Chen X, Equi R, Baxter H, Berk K, Han J, Agarwal S, Zale J (2010) A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings. Biotechnol Biofuels 3(1):9. doi:10.1186/1754-6834-3-9

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Collu G, Unver N, Peltenburg-Looman AM, van der Heijden R, Verpoorte R, Memelink J (2001) Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett 508(2):215–220. doi:S0014-5793(01)03045-9

    CAS  PubMed  Article  Google Scholar 

  • Crane C, Wright E, Dixon R, Wang Z-Y (2006) Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens-transformed roots and Agrobacterium rhizogenes transformed hairy roots. Planta 223(6):1344–1354. doi:10.1007/s00425-006-0268-2

    CAS  PubMed  Article  Google Scholar 

  • De Boer K, Tilleman S, Pauwels L, Vanden Bossche R, De Sutter V, Vanderhaeghen R, Hilson P, Hamill J, Goossens A (2011) APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis. Plant J 66:1053–1065

    Google Scholar 

  • De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inze D, Goossens A, Hilson P (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J 44(6):1065–1076. doi:10.1111/j.1365-313X.2005.02586.x

    PubMed  Article  Google Scholar 

  • Di Fiore S, Hoppmann V, Fischer R, Schillberg S (2004) Transient gene expression of recombinant terpenoid indole alkaloid enzymes in Catharanthus roseus leaves. Plant Mol Biol Rep 22(1):15–22. doi:10.1007/bf02773344

    Article  Google Scholar 

  • Farrell LB, Beachy RN (1990) Manipulation of β-glucuronidase for use as a reporter in vacuolar targeting studies. Plant Mol Biol 15(6):821–825

    CAS  PubMed  Article  Google Scholar 

  • Goklany S, Loring RH, Glick J, Lee-Parsons CWT (2009) Assessing the limitations to terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy root cultures through gene expression profiling and precursor feeding. Biotechnol Prog 25(5):1289–1296

    PubMed  Article  Google Scholar 

  • Guevara-Garcia A, Mosqueda-Cano G, Arguello-Astorga G, Simpson J, Herrera-Estrella L (1993) Tissue-specific and wound-inducible pattern of expression of the mannopine synthase promoter is determined by the interaction between the positive and negative cis-regulatory elements. Plant J 4(3):495–505

    CAS  PubMed  Article  Google Scholar 

  • Jefferson RA (1989) The GUS reporter gene system. Nature 342(6251):837–838

    CAS  PubMed  Article  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907

    CAS  PubMed  Google Scholar 

  • Kazan K (2006) Negative regulation of defence and stress genes by EAR-motif-containing repressors. Trends Plant Sci 11(3):109–112. doi:10.1016/j.tplants.2006.01.004

    CAS  PubMed  Article  Google Scholar 

  • Langridge W, Fitzgerald K, Koncz C, Schell J, Szalay A (1989) Dual promoter of Agrobacterium tumefaciens mannopine synthase genes is regulated by plant growth hormones. Dev Biol 86:3219–3223

    CAS  Google Scholar 

  • Lee-Parsons CWT, Erturk S, Tengtrakool J (2004) Enhancement of ajmalicine production in Catharanthus roseus cell cultures with methyl jasmonate is dependent on timing and dosage of elicitation. Biotechnol Lett 26(20):1595–1599

    CAS  PubMed  Article  Google Scholar 

  • Li J-F, Park E, von Arnim A, Nebenfuhr A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5(1):6

    PubMed Central  PubMed  Article  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. doi:10.1006/meth 2001.1262

    CAS  PubMed  Article  Google Scholar 

  • Lorenzo O, Piqueras R, Sanchez-Serrano J, Solano R (2002) ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell Online 15(1):165–178. doi:10.1105/tpc.007468

    Article  Google Scholar 

  • McGrath KC, Dombrecht B, Manners JM, Schenk PM, Edgar CI, Maclean DJ, Scheible W-R, Udvardi MK, Kazan K (2005) Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression. Plant Physiol 139(2):949–959

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Memelink J, Gantet P (2007) Transcription factors involved in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Phytochem Rev 6(2–3):353–362

    CAS  Article  Google Scholar 

  • Memelink J, Verpoorte R, Kijne JW (2001) ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism. Trends Plant Sci 6:212–219

    CAS  PubMed  Article  Google Scholar 

  • Menke FLH, Champion A, Kijne JW, Memelink J (1999a) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J 18(16):4455–4463

    CAS  PubMed  Article  Google Scholar 

  • Menke FLH, Parchmann S, Mueller MJ, Kijne JW, Memelink J (1999b) Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in Catharanthus roseus. Plant Physiol 119:1289–1296

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Pauw B, Hilliou FAO, Martin VS, Chatel G, de Wolf CJF, Champion A, Pre M, van Duijn B, Kijne JW, van der Fits L, Memelink J (2004) Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus. J Biol Chem 279(51):52940–52948

    CAS  PubMed  Article  Google Scholar 

  • Pre M, Atallah M, Champion A, De Vos M, Pieterse CM, Memelink J (2008) The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense. Plant Physiol 147(3):1347–1357. doi:10.1104/pp.108.117523

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Rico A, Bennett MH, Forcat S, Huang WE, Preston GM (2010) Agroinfiltration reduces ABA levels and suppresses Pseudomonas syringae-elicited salicylic acid production in Nicotiana tabacum. PLoS ONE 5(1):e8977. doi:10.1371/journal.pone.0008977

    PubMed Central  PubMed  Article  Google Scholar 

  • Slater A, Scott NW, Fowler MR (2008) Plant Biotechnology: the genetic manipulation of plants, 2nd edn. Oxford University Press, Oxford

  • van der Fits L, Memelink J (2000) ORCA3, a Jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289(5477):295–297

    PubMed  Article  Google Scholar 

  • van der Fits L, Memelink J (2001) The jasmonate-inducible AP2/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element. Plant J 25(1):43–53

    PubMed  Article  Google Scholar 

  • van der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R (2004) The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr Med Chem 11(5):607–628

    Article  Google Scholar 

  • van der Meer IM (2006) Agrobacterium-mediated transformation of petunia leaf discs. Methods in Molecular Biology. In: Loyola-Vargas V, Vazquez-Flota F (eds) Plant Cell Culture Protocols vol 318, 2nd edn. Humana Press Inc., Totowa

  • Zhang H, Memelink J (2009) Regulation of secondary metabolism by jasmonate hormones. In: Lanzotti AEOAV (ed) Plant-derived natural products. Springer Science + Business Media, LLC. doi:10.1007/978-0-387-85498-4_8

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Acknowledgments

This work was supported by the National Science Foundation (NSF-CAREER, Grant No. BES-0134511; NSF-CBET Grant No. 1033889). We would like to thank Dr. Jian-Feng Li and Dr. Andreas Nebenführ (University of Tennessee, Knoxville, Tennessee) for providing the pVKH-NLS-YFP-GUS plasmid and plasmid map and Dr. Hans-Joerg Jacobsen (Leibnitz University, Hannover, Germany) for providing the pGII0229TR-GUS-CP-LUC and pSoup plasmids.

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Authors and Affiliations

  1. Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA

    Jessica Weaver & Erin J. Cram

  2. Department of Chemical Engineering, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA

    Sheba Goklany, Noreen Rizvi & Carolyn W. T. Lee-Parsons

  3. Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA

    Carolyn W. T. Lee-Parsons

Authors
  1. Jessica Weaver
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  2. Sheba Goklany
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  3. Noreen Rizvi
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  4. Erin J. Cram
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  5. Carolyn W. T. Lee-Parsons
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Corresponding author

Correspondence to Carolyn W. T. Lee-Parsons.

Additional information

Communicated by K. Kamo.

J. Weaver and S. Goklany contributed equally.

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Weaver, J., Goklany, S., Rizvi, N. et al. Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings. Plant Cell Rep 33, 89–97 (2014). https://doi.org/10.1007/s00299-013-1514-2

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  • DOI: https://doi.org/10.1007/s00299-013-1514-2

Keywords

  • Catharanthus roseus
  • Agrobacterium-mediated transient transformation
  • Orca APETALA2/ETHYLENE Response Factor (AP2/ERF) family
  • Zct zinc finger protein family
  • Transcription factor
  • Plant defense response