TY - JOUR
T1 - Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates
AU - Chung, Yi Chia
AU - Cheng, Hao Yu
AU - Wang, Wei Tung
AU - Chang, Yen Jui
AU - Lin, Shih Ming
N1 - Funding Information:
We would like to thank Dr. David Drew at Stockholm University, Sweden, for kindly providing the plasmids and yeast strains used to complete this study. The authors gratefully acknowledge the use of EM000900 of MOST 110-2731-M-006-001 belonging to the Core Facility Center of National Cheng Kung University. The authors would like to acknowledge funding from the Ministry of Science and Technology (MOST) of Taiwan (MOST 109-2636-B-006-012; MOST 110-2636-B-006-012) to S.M.L.
Funding Information:
We would like to thank Dr. David Drew at Stockholm University, Sweden, for kindly providing the plasmids and yeast strains used to complete this study. The authors gratefully acknowledge the use of EM000900 of MOST 110-2731-M-006-001 belonging to the Core Facility Center of National Cheng Kung University. The authors would like to acknowledge funding from the Ministry of Science and Technology (MOST) of Taiwan (MOST 109-2636-B-006-012; MOST 110-2636-B-006-012) to S.M.L.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=85127061081&partnerID=8YFLogxK
U2 - 10.1038/s41598-022-09115-x
DO - 10.1038/s41598-022-09115-x
M3 - Article
C2 - 35332238
AN - SCOPUS:85127061081
SN - 2045-2322
VL - 12
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 5097
ER -