A quantitative ratiometric sensor for time-resolved analysis of auxin dynamics

Abstract

Time-resolved quantitative analysis of auxin-mediated processes in plant cells is as of yet limited. By applying a synergistic mammalian and plant synthetic biology approach, we have developed a novel ratiometric luminescent biosensor with wide applicability in the study of auxin metabolism, transport, and signalling. The sensitivity and kinetic properties of our genetically encoded biosensor open new perspectives for the analysis of highly complex auxin dynamics in plant growth and development.

StrigoQuant: A genetically encoded biosensor for quantifying strigolactone activity and specificity

“…Furthermore, StrigoQuant also comprises a renilla luciferase (REN), incorporated as a normalization element. The synthetic construct encodes REN and the SM-FF fusion protein separated by a self-processing 2A peptide, allowing for cotranslational cleavage and resulting in stoichiometric expression of the sensor elements from a single transcript ( 27 , 28 ). By monitoring the activity of both luciferases of StrigoQuant, it becomes possible to observe the initial steps of SL signaling (namely, perception) over the SMXL6 SM and to translate substrate specificity, activity, and concentration to SM-FF degradation, with the unaffected REN activity for normalization (Fig.…”

Synthetic Botany

“…Examples include a light-inducible gene expression system activated by red light and switched off by far-red light (Müller et al 2014) as well as a range of in vivo biosensors to monitor levels of cytokinin (Müller and Sheen 2008;Zurcher et al 2013), auxin (Wend et al 2013), plant pathogens (Liu et al 2011;Fethe et al 2014), and elicitors (Koschmann et al 2012). These are examples of synthetic circuits that have been constructed in plants using methods for DNA manipulation, transgene regulation, transformation, and analysis to yield basic systems for chassis engineering and genetic network design in plant synthetic biology.…”

“…Traits of agricultural importance successfully introduced to plants using recombinant DNA technology include herbicide resistance (Comai et al 1985), drought resistance (Kumar et al 2014), pest resistance (Bates et al 2005), pathogen resistance (Brunner et al 2011;Horvath et al 2012;Jones et al 2014), abiotic stress resistance (Jaglo-Ottosen et al 1998), enhanced photosynthetic capacity (Ku et al 2001), im-proved nitrogen use efficiency (Yanagisawa et al 2004), and added nutritional value (Ye et al 2000). However, although plants hold a unique promise for bioproduction at the gigatonne scale, efforts in genetic engineering of plants are lagging compared with microbial systems (Antunes et al 2009(Antunes et al , 2011Liu et al 2011Liu et al , 2013Koschmann et al 2012;Wend et al 2013;Zurcher et al 2013;Fethe et al 2014;Mül-ler et al 2014).…”

Advanced imaging techniques for the study of plant growth and development

“…To measure temporal dynamics of such agents in vivo , readouts from highly specific molecular sensors are required. Tools based on changes in probe intensity, color, localization and energy transfer [17, 18] together with a wide array of sensors used to assay a variety of key molecules such as sugars, amino acids, ions such as ammonium and hormones are now available [18–21]. Among these, a new sensor has been engineered, that enables dynamic changes in endogenous auxin levelsto be detected.…”