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 acidisoleucine (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, a competition assay revealed that the presence of JA, salicylic acid (SA), and indole-3-acetic acid (IAA) inhibits the transport of GLSs in yeast, suggesting a potential regulatory mechanism of AtGTR1. 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.

Ablation of glucosinolate accumulation in the oil crop Camelina sativa by targeted mutagenesis of genes encoding the transporters GTR1 and GTR2 and regulators of biosynthesis MYB28 and MYB29

“…The truncated CsGTR1 and CsGTR2 proteins still contain the ETFEK motif which is essential for proton coupling and active glucosinolate transport (Jørgensen et al ., 2015 ). However, further amino acids required for transport and protein stability are eliminated and transmembrane domains abolished (Chung et al ., 2021 ). Therefore, the mutant loci presumably represent null alleles.…”

Mechanistic insight into substrate specificity of plant glucosinolate transporters

“…The dockings indicate strong interactions between the selectivity filter and the side chains for glucosinolates being transported and show that the core glucosinolate structure interacts with two conserved residues that are assumed to be involved in general substrate recognition in the POT family. Whereas interactions with the two conserved residues have previously been observed in dockings of I3M, 4MTB, 4MSB and benzyl in NPF2.10 using inward and outward facing conformations (Chung et al 2022, Peña-Varas et al 2022), but the selectivity filter has not been described before.…”

Glucosinolate catabolism maintains glucosinolate profiles and transport in sulfur-starvedArabidopsis

“…So, if the low expression of these HSPs in bglu28/30 under −S led to negatively affected cell membrane integrity, GSL stored in the RLA cells of bglu28/30 could be leaked and finally loaded to phloem, which caused the observation that GSL transport was enhanced in these mutants. Besides, the reduced expression of HPSs is likely to indicate the change of intracellular pH, which may increase the proton gradient-driven activity of the GTRs (Triandafillou et al, 2020; Chung et al, 2022). To clarify these possibilities, the contribution of HSPs to the phenotypes of bglu28/30 under −S needs to be investigated.…”