Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates

Abstract

Glucosinolates (GLSs) are a group of secondary metabolites that are involved in the defense of herbivores. In Arabidopsis thaliana, Glucosinolate Transporter 1 (AtGTR1) transports GLSs with high affinity via a proton gradient-driven process. In addition to transporting GLSs, AtGTR1 also transports phytohormones, jasmonic acid-isoleucine (JA-Ile), and gibberellin (GA). However, little is known about the mechanisms underlying the broad substrate specificity of AtGTR1. Here, we characterized the substrate preference of AtGTR1 by using a yeast uptake assay, and the results revealed that GLS transport rates are negatively correlated with the hydrophobicity of substrates. Interestingly, the AtGTR1 showed a higher substrate affinity for GLSs with higher hydrophobicity, suggesting a hydrophobic substrate binding pocket. In addition, competition assays revealed that JA, salicylic acid (SA), and indole-3-acetic acid (IAA) competed with GLS for transport in yeast, suggesting a potential interaction of AtGTR1 with these phytohormones. To further characterize the functional properties of AtGTR1, mutagenesis experiments confirmed that the conserved EXXEK motif and Arg166 are essential for the GLS transport function. In addition, the purified AtGTR1 adopts a homodimeric conformation, which is possibly regulated by phosphorylation on Thr105. The phosphomimetic mutation, T105D, reduced its protein expression and completely abrogated its GLS transport function, indicating the essential role of phosphorylation on AtGTR1. In summary, this study investigated various factors associated with the GLS transport and increased our knowledge on the substrate preferences of AtGTR1. These findings contribute to understanding how the distribution of defense GLSs is regulated in plants and could be used to improve crop quality in agriculture.

Artificial Fluorescent Glucosinolates (F-GSLs) Are Transported by the Glucosinolate Transporters GTR1/2/3

“…Although GTRs are promiscuous towards the side chain [ 7 , 25 ], it is not given that they would accept artificial GSLs to which an entire fluorophore molecule had been added to the side chain. To investigate whether any of the seven F-GSL are transported by the GTRs, we performed transport assays, wherein GTR1, GTR2, and GTR3 expressed in Xenopus laevis oocytes were exposed to 50 μM external concentration of each individual F-GSL at pH 5 for 1 h. To make sure the F-GSL are stable and remain intact during the assays, we first conducted three independent experiments where oocyte homogenates were analyzed by LCMS ( Figure 2 ).…”

“…Most studies have used Xenopus oocytes as expression hosts and have characterized transport activity directly by measuring imported glucosinolate levels via LCMS or indirectly by measuring the currents elicited by TEVC electrophysiology. LCMS has also been used to measure GTR-mediated glucosinolate import in other heterologous hosts, including yeast [ 25 ], cotton cells [ 26 ], and insect cells (unpublished data) [ 27 ]. In one example, glucosinolate import was detected by monitoring the influx of radiolabeled glucosinolates that were generated by feeding radiolabeled tyrosine to CYP79A1 overexpressing Arabidopsis plants [ 7 , 28 ].…”

“…So far, GTR1 and GTR2 have been shown to transport glucosinolates with near similar preference irrespective of the structure of the glucosinolate side chain [ 7 , 25 ]. In comparison, GTR3 displays a strong preference for tryptophan-derived glucosinolates [ 8 ].…”

“…Accordingly, the F-GSL provides the prerequisites for conducting high-throughput screening of mutant libraries in studies aimed at unraveling the structural basis underlying substrate specificity in the GTRs. In this context, it is noteworthy that GTR1 and GTR2 have so far been shown to have almost completely overlapping substrate preference [ 7 , 8 , 25 ]. Here, the similar uptake of GSL-B-DNS and GSL-B-NBD but varying uptake of GSL-A-Dansylamide by GTR1 and GTR2, respectively, indicates that they harbor distinct substrate preferences.…”

“…Based on GSL-N 3 precursors, different small fluorophores can be attached to the sidechain of different GSL-core structures [ 23 , 24 ]. Notably, the characterization of GTRs has revealed that they are notoriously promiscuous towards the glucosinolates’ amino side chain [ 7 , 25 ]. This prompted us to explore whether artificial fluorescent glucosinolates are accepted as substrates by the GTRs.…”