Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan, Taiwan.
Institute of Tropical Plant Sciences and Microbiology, National Cheng Kung University, Tainan, Taiwan.
Sci Rep. 2022 Mar 24;12(1):5097. doi: 10.1038/s41598-022-09115-x.
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.
硫代葡萄糖苷（GLS）是一类参与抵御食草动物的次生代谢物。在拟南芥中，葡萄糖苷转运蛋白 1（AtGTR1）通过质子梯度驱动的过程，以高亲和力转运 GLS。除了转运 GLS 外，AtGTR1 还转运植物激素茉莉酸异亮氨酸（JA-Ile）和赤霉素（GA）。然而，对于 AtGTR1 广泛的底物特异性的机制知之甚少。在这里，我们通过酵母摄取实验来表征 AtGTR1 的底物偏好，结果表明 GLS 转运速率与底物的疏水性呈负相关。有趣的是，AtGTR1 对疏水性较高的 GLS 表现出更高的底物亲和力，表明存在一个疏水性的底物结合口袋。此外，竞争实验表明，JA、水杨酸（SA）和吲哚-3-乙酸（IAA）在酵母中与 GLS 竞争运输，表明 AtGTR1 与这些植物激素可能存在相互作用。为了进一步表征 AtGTR1 的功能特性，突变实验证实保守的 EXXEK 基序和 Arg166 对于 GLS 转运功能至关重要。此外，纯化的 AtGTR1 采用同源二聚体构象，可能受到 Thr105 磷酸化的调节。磷酸模拟突变 T105D 降低了其蛋白表达并完全阻断了其 GLS 转运功能，表明磷酸化在 AtGTR1 中起着重要作用。综上所述，本研究调查了与 GLS 转运相关的各种因素，并增加了我们对 AtGTR1 底物偏好的了解。这些发现有助于理解植物中防御性 GLS 分布是如何被调控的，并可用于提高农业中作物的品质。
