No abstract

No abstract

MS performed the experiments, wrote the manuscript, and processed and analyzed data, AY wrote the manuscript and carried out metabolic analysis, CB carried out genotyping experiments, YC analyzed data, VS analyzed data, SH carried out genotyping and metabolic analysis, EK performed GLS measurements, CK peformed initial gtr1/2 experiment, AL verified analytical methods and assisted with statistical aid, H N-E provided gtr1/2 mutants and initial analysis, DK provided all the GLS mutants and GLS related measurements from the population, RA conceived the experimental design, supervised the work, provided funding, and wrote the manuscript. All authors have reviewed the final version of the manuscript and approved it and therefore are equally responsible for the integrity and accuracy of its content.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate–protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.

The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and distribution of the specialized plant metabolite glucosinolates. Due to representing both antinutritional and health-promoting compounds, there is increasing interest in characterizing GTRs from various plant species. We generated seven artificial glucosinolates (either aliphatic or benzenic) bearing different fluorophores (Fluorescein, BODIPY, Rhodamine, Dansylamide, and NBD) and investigated the ability of GTR1/2/3 from Arabidopsis thaliana to import the fluorescent glucosinolates (F-GSLs) into oocytes from Xenopus laevis. Five out of the seven F-GSLs synthesized were imported by at least one of the GTRs. GTR1 and GTR2 were able to import three F-GSLs actively above external concentration, while GTR3 imported only one actively. Competition assays indicate that the F-GSLs are transported by the same mechanism as non-tagged natural glucosinolates. The GTR-mediated F-GSL uptake is detected via a rapid and sensitive assay only requiring simple fluorescence measurements on a standard plate reader. This is highly useful in investigations of glucosinolate transport function and provides a critical prerequisite for elucidating the relationship between structure and function through high-throughput screening of GTR mutant libraries. The F-GSL themselves may also be suitable for future studies on glucosinolate transport in vivo.