Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

SUMMARY: Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation‐based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)‐based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co‐expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA‐INSENSITIVE, GAI; REPRESSOR‐of‐ga1‐3, RGA; RGA‐like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA‐oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta. Significance Statement: Using the degradation‐based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)‐based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. [ABSTRACT FROM AUTHOR]