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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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
- Heterologous expression
- Squalene epoxidase
- Squalene synthase