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Cited by 14Drug Transport and Resistance MechanismsPlant Molecular Biology ResearchPlant nutrient uptake and metabolism

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Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates

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Chung, Yi-Chia, Cheng, Hao-Yu, Wang, Wei-Tung, Chang, Yen-Jui, Lin, Shih-Ming·2022·Scientific Reports
DOI 10.1038/s41598-022-09115-xPMC PMC8948214

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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.

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Figures

Figure 1 Yeast-expressed AtGTR1 localized to the cell membrane and showed the GLS transport activities. (a) Microscopic observation of AtGTR1-GFP-expressing yeast cells. The scale bar represents 5 μm. (b–d) The yeast membrane fractions isolated from AtGTR1-GFP-expressing and AtGTR1-expressing yeasts were analyzed by (b) SDS-PAGE, (c) western blotting, and (d) in-gel fluorescence. The primary antibody is an anti-His-tag antibody. The filled triangle and filled circle indicate the predicted positions of the AtGTR1-GFP and AtGTR1 bands, respectively. The open triangle and open circle indicate the predicted positions of dimeric AtGTR1-GFP and AtGTR1 bands, respectively. (e) Time-dependent GLS accumulation via AtGTR1. Yeast cells expressing AtGTR1 were incubated with 100 μM GTR for different lengths of time, and then GLS contents of the yeasts were quantified by RP-HPLC. (f) pH profile of the GLS transport of AtGTR1. Yeast cells were incubated in the assay medium containing 100 μM GTR with various pH for 1 h and then lysed to quantify the GLS contents by RP-HPLC. All data points with error bars represented the mean ± SD of three independent repeats.
Figure 1 Yeast-expressed AtGTR1 localized to the cell membrane and showed the GLS transport activities. (a) Microscopic observation of AtGTR1-GFP-expressing yeast cells. The scale bar represents 5 μm. (b–d) The yeast membrane fractions isolated from AtGTR1-GFP-expressing and AtGTR1-expressing yeasts were analyzed by (b) SDS-PAGE, (c) western blotting, and (d) in-gel fluorescence. The primary antibody is an anti-His-tag antibody. The filled triangle and filled circle indicate the predicted positions of the AtGTR1-GFP and AtGTR1 bands, respectively. The open triangle and open circle indicate the predicted positions of dimeric AtGTR1-GFP and AtGTR1 bands, respectively. (e) Time-dependent GLS accumulation via AtGTR1. Yeast cells expressing AtGTR1 were incubated with 100 μM GTR for different lengths of time, and then GLS contents of the yeasts were quantified by RP-HPLC. (f) pH profile of the GLS transport of AtGTR1. Yeast cells were incubated in the assay medium containing 100 μM GTR with various pH for 1 h and then lysed to quantify the GLS contents by RP-HPLC. All data points with error bars represented the mean ± SD of three independent repeats.CC BY · PubMed Central
Figure 2 Substrate preference of AtGTR1 depends on the hydrophobicity of transported GLSs. (a) Molecular structures of the seven GLSs used in this study. The corresponding type is marked at the top. (b) Transport activity of AtGTR1 for the seven tested GLSs as measured by yeast uptake assay. The GLS transported in yeast cells were quantified by RP-HPLC. Letters above bars indicate statistically significant differences between groups (one-way ANOVA, p < 0.05). Bar values are the means of three independent replicates. Error bars indicate the SD. (c) RP-HPLC chromatogram of each desulfo-GLS standard. The compound name and retention time are marked at the top of the peaks. (d) The transport activity of each GLS was plotted against its retention time. The linear correlation function is shown, and the dashed line shows the confidence interval of 95%. Each data point represents mean ± SD (n = 3). The pink color represents aliphatic GLSs, the green color represents benzenic GLSs, and the blue color represents indolic GLSs.
Figure 2 Substrate preference of AtGTR1 depends on the hydrophobicity of transported GLSs. (a) Molecular structures of the seven GLSs used in this study. The corresponding type is marked at the top. (b) Transport activity of AtGTR1 for the seven tested GLSs as measured by yeast uptake assay. The GLS transported in yeast cells were quantified by RP-HPLC. Letters above bars indicate statistically significant differences between groups (one-way ANOVA, p < 0.05). Bar values are the means of three independent replicates. Error bars indicate the SD. (c) RP-HPLC chromatogram of each desulfo-GLS standard. The compound name and retention time are marked at the top of the peaks. (d) The transport activity of each GLS was plotted against its retention time. The linear correlation function is shown, and the dashed line shows the confidence interval of 95%. Each data point represents mean ± SD (n = 3). The pink color represents aliphatic GLSs, the green color represents benzenic GLSs, and the blue color represents indolic GLSs.CC BY · PubMed Central
Figure 3 AtGTR1 showed high affinity and low efficiency for transporting GTR and GBS. The kinetic curve of AtGTR1 transporting (a) GRA, (b) GTR, and (c) GBS was measured by yeast uptake assay in the substrate concentration range of 2.5–320 μM. Michaelis–Menten kinetic curves were fitted using nonlinear regression, and the apparent Km for GRA, GTR, and GBS are 249.2, 13.6, and 8.2 μM, respectively. Each data point represents the mean of three independent replicates (mean ± SD).
Figure 3 AtGTR1 showed high affinity and low efficiency for transporting GTR and GBS. The kinetic curve of AtGTR1 transporting (a) GRA, (b) GTR, and (c) GBS was measured by yeast uptake assay in the substrate concentration range of 2.5–320 μM. Michaelis–Menten kinetic curves were fitted using nonlinear regression, and the apparent Km for GRA, GTR, and GBS are 249.2, 13.6, and 8.2 μM, respectively. Each data point represents the mean of three independent replicates (mean ± SD).CC BY · PubMed Central
Figure 4 The addition of JA, IAA, and SA inhibited the transport of GLS via AtGTR1. (a) Yeast GLS uptake activity was evaluated in the presence of various acidic phytohormones. In each experiment, yeast cells expressing AtGTR1 were incubated with 25 μM GLS and 500 μM phytohormones for 1 h. GLS contents in the yeast cells were quantified by RP-HPLC and were normalized to the average value of the control group. Ctl control group, without adding phytohormones, JA jasmonic acid, GA gibberellic acid, SA salicylic acid, IAA indole-3-acetic acid, and ABA abscisic acid. Bar values are the means of three independent replicates. Error bars indicate SD. Significant differences were assessed by unpaired two-tailed Student t-test and are indicated by **p < 0.01. (b–d) Concentration–response curves for the inhibition of GLS uptake by addition of (b) JA, (c) IAA, and (d) SA. Various concentrations of the phytohormones were treated to inhibit GLS transport in yeast uptake assay. The GLS contents in yeast cells were quantified and normalized to the mean of the control group. Concentration–response curves were fitted using nonlinear regression. Corresponding IC50 values for JA, IAA, and SA are 937.8 μM, 762.1 μM and 630.0 μM, respectively. Each data point represents mean ± SD (n = 3).
Figure 4 The addition of JA, IAA, and SA inhibited the transport of GLS via AtGTR1. (a) Yeast GLS uptake activity was evaluated in the presence of various acidic phytohormones. In each experiment, yeast cells expressing AtGTR1 were incubated with 25 μM GLS and 500 μM phytohormones for 1 h. GLS contents in the yeast cells were quantified by RP-HPLC and were normalized to the average value of the control group. Ctl control group, without adding phytohormones, JA jasmonic acid, GA gibberellic acid, SA salicylic acid, IAA indole-3-acetic acid, and ABA abscisic acid. Bar values are the means of three independent replicates. Error bars indicate SD. Significant differences were assessed by unpaired two-tailed Student t-test and are indicated by **p < 0.01. (b–d) Concentration–response curves for the inhibition of GLS uptake by addition of (b) JA, (c) IAA, and (d) SA. Various concentrations of the phytohormones were treated to inhibit GLS transport in yeast uptake assay. The GLS contents in yeast cells were quantified and normalized to the mean of the control group. Concentration–response curves were fitted using nonlinear regression. Corresponding IC50 values for JA, IAA, and SA are 937.8 μM, 762.1 μM and 630.0 μM, respectively. Each data point represents mean ± SD (n = 3).CC BY · PubMed Central
Figure 5 EXXEK and Arg166 are involved in the transport function of AtGTR1. The GLS transport activity of AtGTR1 mutants. The GLS transport activity was determined by yeast uptake assay, and the protein expression of these mutants was confirmed by western blot. The primary antibody is an anti-His-tag antibody. Bar values with error bar represent mean ± S.D. (n = 3); **p < 0.01; N.D. indicates nondetectable.
Figure 5 EXXEK and Arg166 are involved in the transport function of AtGTR1. The GLS transport activity of AtGTR1 mutants. The GLS transport activity was determined by yeast uptake assay, and the protein expression of these mutants was confirmed by western blot. The primary antibody is an anti-His-tag antibody. Bar values with error bar represent mean ± S.D. (n = 3); **p < 0.01; N.D. indicates nondetectable.CC BY · PubMed Central
Figure 6 Purified AtGTR1 primarily adopts a homodimeric conformation. (a) SEC chromatogram of AtGTR1 showed that the major eluted peak corresponded to molecular size near 158 kDa. Points indicate the retention volume of each standard protein marker with labeled molecular sizes (kDa). (b,c) The AtGTR1 purified by IMAC and SEC were respectively analyzed by (b) SDS-PAGE and (c) western blot with anti-His tag antibody. The filled triangle indicates the AtGTR1 monomer, whereas the open triangle indicates the predicted dimer.
Figure 6 Purified AtGTR1 primarily adopts a homodimeric conformation. (a) SEC chromatogram of AtGTR1 showed that the major eluted peak corresponded to molecular size near 158 kDa. Points indicate the retention volume of each standard protein marker with labeled molecular sizes (kDa). (b,c) The AtGTR1 purified by IMAC and SEC were respectively analyzed by (b) SDS-PAGE and (c) western blot with anti-His tag antibody. The filled triangle indicates the AtGTR1 monomer, whereas the open triangle indicates the predicted dimer.CC BY · PubMed Central
Figure 7 Phosphomimetic mutant of AtGTR1 lost GLS transport function and protein expression in yeast. (a) GLS transport activity of T105 mutants was determined by yeast uptake assay. The GLS uptake was quantified by RP-HPLC and showed as vertical bars with errors (mean ± SD, n = 3); N.D. indicates nondetectable. (b) Western blot of yeast cells expressing AtGTR1 and its T105 mutants. An anti-His-tag antibody was used as the primary antibody. The filled triangle indicates the AtGTR1 monomer, whereas the open triangle indicates degraded AtGTR1.
Figure 7 Phosphomimetic mutant of AtGTR1 lost GLS transport function and protein expression in yeast. (a) GLS transport activity of T105 mutants was determined by yeast uptake assay. The GLS uptake was quantified by RP-HPLC and showed as vertical bars with errors (mean ± SD, n = 3); N.D. indicates nondetectable. (b) Western blot of yeast cells expressing AtGTR1 and its T105 mutants. An anti-His-tag antibody was used as the primary antibody. The filled triangle indicates the AtGTR1 monomer, whereas the open triangle indicates degraded AtGTR1.CC BY · PubMed Central

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Cites

Show all 51 references

Research works printed but not linked: 3

  1. Prabhala, B. K., Rahman, M., Nour-eldin, H. H., Jørgensen, F. S. & Mirza, O. PTR2/POT/NPF Transporters: What Makes Them Tick? Advances in Protein Chemistry and Structural Biology Vol. 123 (Elsevier Ltd, 2021).
  2. Longo, A., Miles, N. W. & Dickstein, R. Genome mining of plant NPFs reveals varying conservation of signature motifs associated with the mechanism of transport. Front. Plant Sci. 871, 1–17 (2018).
  3. Jørgensen, M. E. et al. Origin and evolution of transporter substrate specificity within the NPF family. Elife 6, e19466 (2017). · DOI

Metadata

Authors
Chung, Y.-C., Cheng, H.-Y., Wang, W.-T., Chang, Y.-J., & Lin, S.-M.
Affiliations
National Cheng Kung University (TW)
Corresponding author
Lin, Shih-Ming
ORCID
Lin, Shih-Ming
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2022-03-24
Volume
12
Issue
1
Pages
5097-5097
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-022-09115-x
PMC
PMC8948214
OpenAlex
W4226267209
Cited by
14
References
51
Citations by year
2026: 3 · 2025: 4 · 2024: 2 · 2023: 3 · 2022: 2
FWCI
1.15
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
Ministry of Science and Technology (MOST) of Taiwan (MOST 109-2636-B-006 -012)
Topics
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant nutrient uptake and metabolism
Keywords
substrate specificity, substrate hydrophobicity, substrate binding pocket, gibberellin, salicylic acid, indole-3-acetic acid, phosphorylation, homodimer, Arabidopsis thaliana
MeSH terms
Glucosinolates, Plant Growth Regulators, Saccharomyces cerevisiae, Arabidopsis, Gene Expression Regulation, Plant, Membrane Transport Proteins, Arabidopsis Proteins, Hydrophobic and Hydrophilic Interactions

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Chung, Y.-C., Cheng, H.-Y., Wang, W.-T., Chang, Y.-J., & Lin, S.-M. (2022). Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates. Scientific Reports. https://doi.org/10.1038/s41598-022-09115-x

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