Synthetic Promoter Screening Using Poplar Mesophyll Protoplast Transformation
Yongil Yang,
Yuanhua Shao,
Timothy Chaffin,
Amir Ahkami,
Eduardo Blumwald,
C. Neal Stewart Jr.
Affiliations
Yongil Yang
Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USADepartment of Plant Sciences, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USA
Yuanhua Shao
Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USADepartment of Plant Sciences, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USA
Timothy Chaffin
Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USADepartment of Plant Sciences, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USA
Amir Ahkami
Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, WA, USA
Eduardo Blumwald
Department of Plant Sciences, University of California, Davis, CA, USA
C. Neal Stewart Jr.
Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USADepartment of Plant Sciences, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USA
Plant protoplasts are useful to study both transcriptional regulation and protein subcellular localization in rapid screens. Protoplast transformation can be used in automated platforms for design-build-test cycles of plant promoters, including synthetic promoters. A notable application of protoplasts comes from recent successes in dissecting synthetic promoter activity with poplar mesophyll protoplasts. For this purpose, we constructed plasmids with TurboGFP driven by a synthetic promoter together with TurboRFP constitutively controlled by a 35S promoter, to monitor transformation efficiency, allowing versatile screening of high numbers of cells by monitoring green fluorescent protein expression in transformed protoplasts. Herein, we introduce a protocol for poplar mesophyll protoplast isolation followed by protoplast transformation and image analysis for the selection of valuable synthetic promoters.Graphical overview
