Data for A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Haas, B.E., Saif, F., Bolger, E., Doran, L., Rosales, E., Kim, A., Burgess, S.J., Long, S.P. March 21, 2026. Data for: “A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants.” FigShare. DOI: 10.6084/m9.figshare.31828477.

Software posted on 2026-03-21, 11:01 Authored by Steven Burgess, Benjamin Haas

Scripts for the manuscript “A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants” by Haas et al.

Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome-wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild-type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost-efficient alternative to investigate transcript leaders by co-expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO₂ assimilation during sun–shade transitions.

FigShare: qPCR and plant data, R scripts

Illinois Data Bank: Gene fragments/backbones, vectors, primers, RT-qPCR primer sequences/performance parameters, mean fluorescence, R packages