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Int J Mol Sci
2023 Jan 04;242:. doi: 10.3390/ijms24020920.
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Artificial Fluorescent Glucosinolates (F-GSLs) Are Transported by the Glucosinolate Transporters GTR1/2/3.
Kanstrup C
,
Jimidar CC
,
Tomas J
,
Cutolo G
,
Crocoll C
,
Schuler M
,
Klahn P
,
Tatibouët A
,
Nour-Eldin HH
.
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The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and distribution of the specialized plant metabolite glucosinolates. Due to representing both antinutritional and health-promoting compounds, there is increasing interest in characterizing GTRs from various plant species. We generated seven artificial glucosinolates (either aliphatic or benzenic) bearing different fluorophores (Fluorescein, BODIPY, Rhodamine, Dansylamide, and NBD) and investigated the ability of GTR1/2/3 from Arabidopsis thaliana to import the fluorescent glucosinolates (F-GSLs) into oocytes from Xenopus laevis. Five out of the seven F-GSLs synthesized were imported by at least one of the GTRs. GTR1 and GTR2 were able to import three F-GSLs actively above external concentration, while GTR3 imported only one actively. Competition assays indicate that the F-GSLs are transported by the same mechanism as non-tagged natural glucosinolates. The GTR-mediated F-GSL uptake is detected via a rapid and sensitive assay only requiring simple fluorescence measurements on a standard plate reader. This is highly useful in investigations of glucosinolate transport function and provides a critical prerequisite for elucidating the relationship between structure and function through high-throughput screening of GTR mutant libraries. The F-GSL themselves may also be suitable for future studies on glucosinolate transport in vivo.
Figure 2. Uptake of the 7 fluorescent glucosinolates, analyzed by LCMS. Xenopus laevis oocytes expressing GTR1, GTR2, GTR3, or water-injected Mock were used in import assay. Numbers above X-axis refer to n number of single oocytes analyzed over 3 batches; the points show the signal from individual samples, and different shapes and colors represent different oocyte batches. Assay conditions: 1 h assay, pH 5, 50 μM intended concentration of the F-GSL (Dotted horizontal lines represent the average measured concentration of F-GSL in the external media in the 3 assays, color correlates to different batches). GSL-B-Fluorescein lacks the assay concentration line since the concentration is 6–10 times higher than the uptake (i.e., not visible in the plot). Statistics: Letters indicate statistical significance comparing the transporters (one-way ANOVA (model: GSL~ID), Tukey post hoc test, p < 0.05). ANOVA table in Supplementary.
Figure 3. Uptake of the 5 fluorescent glucosinolates imported in Figure 2, analyzed on plate reader. Xenopus laevis oocytes expressing GTR1, GTR2, GTR3, or water-injected Mock were used. Numbers above X-axis refer to n number of single oocytes analyzed over 3 batches; the points show the signal from individual samples, and different shapes and colors represent different oocyte batches. Assay conditions: 1 h assay, pH 5, 50 μM intended external concentration of the glucosinolates (Dotted horizontal lines represent the average measured concentration of F-GSL in the external media in the 3 assays, color correlates to different batches). GSL-B-Fluorescein lacks the media line since the media are 6–10 times higher than the uptake (i.e., not visible in the plot). Statistics: Letters indicate statistical significance comparing the transporters (one-way ANOVA (model: GSL~ID), Tukey post hoc test, p < 0.05). ANOVA table in Supplementary.
Figure 4. Competition assays. Xenopus laevis oocytes expressing GTR1, GTR2, GTR3, or water-injected Mock were used. Numbers above X-axis refer to n number of single oocytes analyzed over 1–2 batches. Upper: 4MTB uptake analyzed by LCMS; 1 h assay, pH 5, 50 μM intended concentration of the 4MTB alone or in combination with one of the F-GSLs also at 50 μM. (Dotted horizontal lines represent the average measured concentration of 4MTB in the external media averaged over each assay. In order of assay: 100 pmol/μL, 161 pmol/μL, 86 pmol/μL, and 87 pmol/μL). Lower: F-GSL uptake analyzed on plate reader; 1 h assay, pH 5, 50 μM intended concentration of the F-GSL alone or in combination with 4MTB at 50 μM. (Dotted horizontal lines represent the average measured concentration of GSL in the external media averaged over each assay.) Statistics: one-way ANOVA within gene groups (model: GSL~Assay), using Dunnett post hoc test (single assay as reference, against the equimolar assays), p-value: *** < 0.001, ** < 0.01, and * <0.05.
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