Synthetic Promoter Screening Using Poplar Mesophyll Protoplast Transformation

Yongil Yang; Yuanhua Shao; Timothy A Chaffin; Amir H Ahkami; Eduardo Blumwald; C Neal Stewart Jr

doi:10.21769/BioProtoc.4660

Synthetic Promoter Screening Using Poplar Mesophyll Protoplast Transformation

Bio Protoc. 2023 Apr 20;13(8):e4660. doi: 10.21769/BioProtoc.4660.

Authors

Yongil Yang^{1

2}, Yuanhua Shao^{1

2}, Timothy A Chaffin^{1

2}, Amir H Ahkami³, Eduardo Blumwald⁴, C Neal Stewart Jr^{1

2}

Affiliations

¹ Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USA.
² Department of Plant Sciences, University of Tennessee Institute of Agriculture, Knoxville, Tennessee, USA.
³ Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, WA, USA.
⁴ Department of Plant Sciences, University of California, Davis, CA, USA.

Abstract

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.

Keywords: Fluorescence; Image analysis; Mesophyll protoplasts; Poplar; Synthetic promoter.