Abstract
Purpose of work: The purpose of this study was to determine if Arabidopsis protoplast transfection could be scaled up, from the commonly used cell-based studies, to be used in triterpenoid production assays as an in planta alternative/complement to other expression systems.
Enzyme activities are often identified using heterologous expression systems such as yeast cells. These systems, however, may be incompatible for expressing enzymes involved in specialized (secondary) metabolism. Previous reports with long-term in planta expression systems show that the activity of the triterpenoid pathway can be enhanced by expressing enzymes catalyzing initial steps in the pathway. Here we show that triterpenoid production can also be enhanced in Arabidopsis mesophyll protoplasts after transfection. This system is designed to quantify changes in productivity of a plant metabolic pathway within 48 h and, as proof of concept, we show a significantly increased production of a triterpenoid by transiently expressing squalene synthase 1 (SQS1) from 0.5 pg/protoplast in mock-transfected protoplasts to 2.7 pg/protoplast in constitutively expressing SQS1 protoplasts.
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References
Buschhaus C, Jetter R (2012) Composition and physiological function of the wax layers coating Arabidopsis leaves: β-amyrin negatively affects the intracuticular water barrier. Plant Physiol 160:1120–1129
Busquets A, Keim V, Closa M et al (2008) Arabidopsis thaliana contains a single gene encoding squalene synthase. Plant Mol Biol 67:25–36
Greer S, Wen M, Bird D et al (2007) The cytochrome P450 enzyme CYP96A15 is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis. Plant Physiol 145:653–667
Kribii R, Arró M, Arco A et al (1997) Cloning and characterization of the Arabidopsis thaliana SQS1 gene encoding squalene synthase. Eur J Biochem 249:61–69
Lee MH, Jeong JH, Seo JW et al (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45:976–984
Marks MD, Betancur L, Gilding E et al (2008) A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses. Plant J 56:483–492
Mirjalili M, Moyano E, Bonfill M et al (2011) Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures. Biol Plantarum 55:2373–2393
Nuringtyas TR, Choi YH, Verpoorte R et al (2012) Differential tissue distribution of metabolites in Jacobaea vulgaris, Jacobaea aquatica and their crosses. Phytochemistry 78:89–97
Rasbery JM, Shan H, LeClair RJ et al (2007) Arabidopsis thaliana squalene epoxidase 1 is essential for root and seed development. J Biol Chem 282:17002–17013
Rontein D, Onillon S, Herbette G et al (2008) CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4 (5), 11 (12)-diene into the cyclic ether 5 (12)-oxa-3 (11)-cyclotaxane. J Biol Chem 283:6067–6075
Schilmiller AL, Last RL, Pichersky E (2008) Harnessing plant trichome biochemistry for the production of useful compounds. Plant J 54:702–711
Seo JW, Jeong JH, Shin CG et al (2005) Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation. Phytochemistry 66:869–877
Tiwari S, Wang S, Hagen G, Guilfoyle TJ (2006) Transfection assays with protoplasts containing integrated reporter genes. Methods Mol Biol 323:237
Wang E, Gan S, Wagner GJ (2002) Isolation and characterization of the CYP71D16 trichome-specific promoter from Nicotiana tabacum L. J Exp Bot 53:1891–1897
Wang S, Chang Y, Guo J, Chen JG (2007) Arabidopsis ovate family protein 1 is a transcriptional repressor that suppresses cell elongation. Plant J 50:858–872
Yerger EH, Grazzini RA, Hesk D et al (1992) A rapid method for isolating glandular trichomes. Plant Physiol 99:1–7
Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572
Acknowledgments
This work was supported by NSERC Discovery Grants (nos. 298264-09 and 262461-08), the Canada Research Chairs program, and Canada Foundation for Innovation Leaders Opportunity grants to G.O.W and R. J. We thank Christopher Buschhaus for providing 35Spro::LUP4 seeds and Luke Busta for technical assistance with GC and GC–MS analyses.
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Johnson, E.E., Jetter, R. & Wasteneys, G. Rapid induction of the triterpenoid pathway in Arabidopsis thaliana mesophyll protoplasts. Biotechnol Lett 36, 855–858 (2014). https://doi.org/10.1007/s10529-013-1427-8
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DOI: https://doi.org/10.1007/s10529-013-1427-8