A Protoplast-Based Bioassay to Quantify Strigolactone Activity in Arabidopsis Using StrigoQuant

Abstract

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors maybe also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, a genetically encoded, ratiometric sensor, StrigoQuant, was developed and allowed the quantification of the activity of a wide concentration range of SLs.StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL GR24. We then show the recording of the luminescence signal and how obtained data are processed and used to assess/determine SL perception.

Engineering and implementation of synthetic molecular tools in the basidiomycete fungusUstilago maydis

“…Dual-reporters are widely used in synthetic biology applications and consist of one reporter for monitoring the expression of a gene of interest, and a second one that serves as internal control thereby yielding a direct comparison. Such an approach increases robustness and enables in situ normalization, rendering the quantification of gene expression relatively independent of biological sample variation, including reporter absolute expression levels, cell health, and metabolic load, among other factors [17,33]. We first characterized the expression of two strains comprising a single reporter each: RLuc under the constitutive promoter P O2tef (Fig.S2A), and FLuc expressed under the inducible promoter P CRG , which is activated upon a change of carbon source from glucose to arabinose (Fig.S2B).…”

Engineering and Implementation of Synthetic Molecular Tools in the Basidiomycete Fungus Ustilago maydis

“…Dual reporters are widely used in synthetic biology applications and consist of one reporter for monitoring the expression of a gene of interest, and a second one that serves as an internal control, thereby yielding a direct comparison. Such an approach increases robustness and enables in situ normalization, rendering the quantification of gene expression relatively independent of biological sample variation, including reporter absolute expression levels, cell health, and metabolic load, among other factors [17,33]. We first characterized the expression of two strains comprising a single reporter each: RLuc under the constitutive promoter P O2tef (Figure S2A) and FLuc expressed under the inducible promoter P CRG , which is activated upon a change of carbon source from glucose to arabinose (Figure S2B).…”

Strigo-D2 – a bio-sensor for monitoring the spatio-temporal pattern of strigolactone signaling in intact plants

“…The sensors responded as expected to various natural or synthetic SLs with high specificity, sensitivity and quantitative resolution demonstrating the suitablity of using proteolytic targets of the SL signaling pathway to estimate respective signaling levels. However, described sensors use partly bioluminescence as a readout hampering cellular imaging and requiring continuous supply of luciferin, the substrate of the luciferase reporter [35,47,48]. Moreover, ratiometric SL sensors have only been tested successfully in transient expression systems so far [35,[47][48][49] leaving open the question towards their performance in stable transgenic plant lines and in the context of an SL-controlled process.…”

“…However, described sensors use partly bioluminescence as a readout hampering cellular imaging and requiring continuous supply of luciferin, the substrate of the luciferase reporter [35,47,48]. Moreover, ratiometric SL sensors have only been tested successfully in transient expression systems so far [35,[47][48][49] leaving open the question towards their performance in stable transgenic plant lines and in the context of an SL-controlled process. In comparison, a sensor stably integrated in plants expressing the luminescently labeled D2 domain of SMAX1 under the control of the ubiquitously active UBQ10 promoter was shown recently to faithfully report KAR signaling (pRATIO2251-SMAX1D2 [35]).…”

“…With the unexcelled advantage of revealing relative molecule levels with high spatio-temporal resolution and within a physiological range, sensors have been developed for measuring small molecules like calcium, sugars and various plant hormones [42][43][44][45][46]. For SLs, ratiometric biosensors exploiting the levels of the SMXL6 or SMXL7 proteins as indication for the activity of the SL signaling pathway were characterized previously [35,[47][48][49]. The sensors responded as expected to various natural or synthetic SLs with high specificity, sensitivity and quantitative resolution demonstrating the suitablity of using proteolytic targets of the SL signaling pathway to estimate respective signaling levels.…”