HIGH-THROUGHPUT, QUANTITATIVE BIOASSAY FOR STRIGOLACTONE ACTIVITY IN PLANTA

(1)

HIGH-THROUGHPUT, QUANTITATIVE BIOASSAY FOR

STRIGOLACTONE ACTIVITY IN PLANTA

Wajeeha Saeeda,b_{, Ivan Visentin}a_{, Emma Artuso}c_{, Chiara Lombardi}c_{, Cristina Prandi}c_{, Elena}

Sanchezd_{, Pilar Cubas}d_{, Zahid Ali}b_{, Andrea Schubert}a_{, Francesca Cardinale}a

a_{University of Turin, DISAFA, Largo P. Braccini 2, Grugliasco (TO) – ITALY;}b_COMSATS

Institute of Information Technology,Department of Biosciences, Park Road, Islamabad

-PAKISTAN; b_{University of Turin, Department of Chemistry, via P. Giuria 7, Turin – ITALY;}

d_{National Center of Biotechnology, Department of Plant Molecular Genetics, Calle Darwin nº 3,}

Madrid - SPAIN

e-mail: [email protected]

Available bioassays for Strigolactone (SL) activity in planta are hardly quantitative, rather labour-intensive and time-consuming; conversely, bioassays on parasitic plants and arbuscular mycorrhizal fungi may not reflect perfectly hormonal activity in planta. A truly quantitative, high-throughput bioassay for SL activity is therefore missing, yet needed to generate a homogeneous structure-activity relationship (SAR) dataset for SL-related molecules. As other plant hormones, SL act as interfacial molecules, promoting and stabilizing protein-protein interactions; in this case, between the ligand-binding moiety of the SL receptor complex (the α/β hydrolase D14) and the co-receptor moiety (the F-box MAX2). Such interaction promotes further binding between MAX2 and its target(s), leading to ubiquitination and degradation of the latter by the proteasome machinery. D14 itself is a target for proteasome-dependent destruction, which explains why fluorescence of D14::GFP fusion proteins quenches upon SL treatment in transgenic Arabidopsis1_.

We exploited this molecular network to implement a quantitative activity assay, inversely correlating luminescence to perception of SL-related molecules in transgenic Arabidopsis expressing D14::LUC under the control of D14 endogenous promoter. The reporter lines have been tested in a concentration range of various functional and/or fluorescent derivatives of SL. Our initial results show that though indirect, the bioassay has an acceptable dynamic range, is relatively simple to execute, up-scalable and robust enough to be exploitable for SAR studies. In addition, being centered on the SL-D14 interaction event, it could lead to a more detailed understanding of the SL perception/signal transduction model.

1_{F. Chevalier, K. Nieminen, J.C. Sánchez-Ferrero, M. L. Rodríguez, M. Chagoyen, C.S. Hardtke,}

P. Cubas, Plant Cell 2014, 26, 1134–1150