Imaging of multiple fluorescent proteins in canopies enables synthetic biology in plants

Abstract

Summary Reverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields. Genetically encoded fluorescent proteins (FPs) have revolutionized plant biology paradigms in gene expression, protein trafficking and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 25 years ago, modern phenomics has largely ignored fluorescence as a transgene expression device despite the burgeoning FP colour palette available to plant biologists. Here, we show a new platform for stand‐off imaging of plant canopies expressing a wide variety of FP genes. The platform—the fluorescence‐inducing laser projector (FILP)—uses an ultra‐low‐noise camera to image a scene illuminated by compact diode lasers of various colours, coupled with emission filters to resolve individual FPs, to phenotype transgenic plants expressing FP genes. Each of the 20 FPs screened in plants were imaged at >3 m using FILP in a laboratory‐based laser range. We also show that pairs of co‐expressed fluorescence proteins can be imaged in canopies. The FILP system enabled a rapid synthetic promoter screen: starting from 2000 synthetic promoters transfected into protoplasts to FILP‐imaged agroinfiltrated Nicotiana benthamiana plants in a matter of weeks, which was useful to characterize a water stress‐inducible synthetic promoter. FILP canopy imaging was also accomplished for stably transformed GFP potato and in a split‐GFP assay, which illustrates the flexibility of the instrument for analysing fluorescence signals in plant canopies.

Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

“…7H ). Furthermore, the eYGFPuv-based biosensor has the potential of enabling advanced plant biotechnology and synthetic biology by combining fluorescent reporters and plant imaging 34 . However, as a new reporter gene, some characteristics of eYGFPuv still remain unclear, e.g., the stability of eGFPuv mRNA and protein in the transgenic plants, such as multiple generations of annual plants or multiple years of perennial plants.…”

Benchmarking organic electrochemical transistors for plant electrophysiology

“…This leads to a very relevant conundrum in the study of plant wound response: not being able to identify cellular origins without invasive techniques; but also changing the wound response by using said techniques. This problem is now starting to be surmounted by the use of voltage-sensitive dyes and genetic mutants (Zhao et al, 2015;Farmer et al, 2020;Rigoulot et al, 2021). Nonetheless, a better integration of molecular-based approaches and electrophysiological techniques still needs to be developed.…”

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology

“…5f,g). This is of particular interest in a context of modern agriculture, where phenomics has the prospect to bring together high-dimensional phenotypic and molecular data together with their interaction with environmental stimuli 43,59 .…”

“…Plant fluorescence patterns were characterized using the Fluorescence-Inducing Laser Projector (FILP), a custom-built instrument for imaging fluorescence in plants. FILP imaging was performed on 3-week-old transplastomic lines in pots 43 . GFP fluorescence in plants was acquired at 150 ms exposure time using 465 nm excitation and 525 nm emission notch (50 nm) filter.…”