Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A ) Bayesian inference (MrBayes) tree (s.d.< 0.01) of the A. thaliana NPF family with reduced phylogenies of NPF1.x, NPF3.x, NPF4.x, NPF5.x, NPF6.x, NPF7.x, NPF8.x and NPF2.1–7 clades (x denotes the subfamily number) as previously annotated . Numbers in brackets indicate the number of genes in reduced phylogeny. Green circles at nodes represent a posterior probability of 1 (maximum is 1). Values at nodes separated by a backslash represent MrBayes values below 1 in red, followed by RAxML generated bootstrap values in black (only reported when Mrbayes value is below 1). GTR1, GTR2, GTR3 and 3 other homologs tested in B ) are highlighted with red branches. For non-reduced phylogeny, see . ( B ) The chemical structure of 4-methylthiobutyl glucosinolate (4MTB) and indol-3-ylmethyl glucosinolate (I3M). ( C ) GTR1, GTR2, GTR3, NPF2.14, NPF2.13 and NPF2.8 were expressed individually in 15 X. laevis oocytes and transport activity was measured in the presence of 0.2 mM 4MTB (black bars) or 0.2 mM I3M (green bars). 4MTB or I3M accumulated within oocytes were quantified by LC-MS analyses in 3 × 5 oocytes for each gene. Error bars represent ± s.d. n = 3, experiment repeated two times; nd=none detected.

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Generated, Activity Assay, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A – B ) Normalized IV (Current-Voltage) curve of 4MTB ( A )- and I3M ( B )-induced currents for GTR1 (black circles)- and GTR3 (green circles)-expressing oocytes exposed to 100 µM substrate at pH5. Both GTR1 and GTR3 currents were normalized to GTR1 currents elicited at saturating 4MTB concentrations and at a membrane potential of −60 mV (Error bars represent ± s.e., n = 6, experiment repeated two times). ( C – D ) Time-dependent accumulation of I3M ( C ) and 4MTB ( D ), respectively, relative to assay media concentration in GTR1- and GTR3-expressing oocytes. Accumulated 4MTB or I3M were quantified by LC-MS in 3 × 5 oocytes for each gene after 3, 4 and 5 hr of incubation in a standard pH5 Kulori buffer containing 0.2 mM I3M or 0.2 mM 4MTB (error bars represent ± s.d. n = 3). Dotted line represents media concentration. ( E – F ) Normalized I3M-induced currents for GTR3 ( E ) or GTR1 ( F ) measured at a membrane potential of −60 mV and pH 5 plotted against increasing I3M concentrations. The saturation curve was fitted with a Michaelis-Menten equation represented by a solid line. Each oocyte dataset was normalized to currents elicited at 0.8 mM I3M concentration at −60 mV. The insert shows the apparent K m as a function of membrane potential. Error bars represent ± s.e. of mean, n = 6, experiment repeated two times.

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Expressing, Membrane, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A – B ) Time-dependent I3M ( A ) and 4MTB ( B ) uptake by GTR1- and GTR3-expressing oocytes. This data was shown in as up-concentration relative to assay media concentration. Here substrate amounts are shown in pmol per oocyte. Accumulated 4MTB or I3M was quantified by LC-MS in 3 × 5 oocytes for each gene after 3, 4 and 5 hr of incubation in a standard pH5 kulori buffer containing 0.2M I3M or 0.2 µM 4MTB. Error bars represent ± s.d. of mean for data, n = 3.

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Expressing, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: Normalized I3M-induced currents of GTR2-expressing oocytes measured at a membrane potential of −60 mV and pH 5 were plotted against increasing I3M concentrations. The saturation curve was fitted with a Michaelis-Menten equation – represented by a solid line. Insert shows apparent K m as a function of clamped membrane potential. Each oocyte dataset was normalized to I3M-induced currents elicited at 0.8 mM I3M concentration at −60 mV. The insert shows the apparent K m as a function of membrane potential. Error bars represent ± s.e. of mean for data obtained from six different oocytes per experiment, experiment repeated two times.

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Expressing, Membrane, Concentration Assay

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A – D ) Competition for uptake of I3M and 4MTB into oocytes expressing GTR1 or GTR3. ( A ) Quantification of 4MTB uptake when oocytes were exposed to high 4MTB concentration (2 mM) alone or in combination with low concentration of I3M (0.2 mM). ( B ) Quantification of I3M uptake when low I3M concentration (0.2 mM) was competed with high concentration of 4MTB (2 mM). ( C ) Quantification of I3M when oocytes were exposed to high I3M concentration (2 mM) alone or in combination with low concentration of 4MTB (0.2 mM). ( D ) Quantification of 4MTB uptake when oocytes were exposed to low I3M concentration (0.2 mM) alone or in combination with high concentration of 4MTB (2 mM). Accumulated 4MTB ( A and D ) or I3M ( B and C ) was quantified by LC-MS in 3 × 5 oocytes for each gene. Two tailed T-test, **p<0.001 vs non-competed, *p<0.05 vs non-competed. NS= not significantly different (Error bars represent ± s.d. of mean for data obtained from three times five different oocytes per experiment). ( E – G ) Quantification of nitrate and glucosinolate competition assays. ( E ) Quantification of I3M uptake in GTR3-expressing oocytes when saturating I3M concentration (0.1 mM) is competed with high concentration of NO 3 - (10 mM) or saturating concentration of 4MTB (0.1 mM). ( F ) Quantification of 4MTB uptake in GTR3-expressing oocytes when saturating 4MTB concentration (0.1 mM) is competed with high concentration of NO 3 - (10 mM). ( G ) Quantification of NO 3 - uptake in GTR3-expressing oocytes when high concentration of NO 3 - (10 mM) is competed by 0.1 mM 4MTB or saturating concentration of I3M (0.1 mM). Accumulated I3M ( E ) or 4MTB ( F ) was quantified by LC-MS in 3 × 5 oocytes for each gene. Accumulated NO 3 - ( G ) was quantified by ICP-MS in three oocytes for each gene. Error bars represent ± s.d. of mean, n = 3. Groups in subfigures are determined by one-way ANOVA followed by Holm-Sidak post-hoc analysis (p<0.05).

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Expressing, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A – B ) Indole glucosinolate content in ( A ) root and ( B ) rosette of non-grafted hydroponically grown wildtype, gtr3 , gtr1 gtr2 dko and gtr1 gtr2 gtr3 tko plants. The box is determined by the 25th and 75th percentiles. The whiskers are determined by the 5th and 95th percentiles. Median and mean are shown as line and square. Groups in subfigures are determined by one-way ANOVA followed by Tukey HSD Calculator multiple comparison post-hoc analysis (p<0.05). Data presented is one of two individual experiments, each containing 8–12 repeats (n) (see – for individual glucosinolate data points and individual n; error bars and parentheses are s.d. ( C ) Indole glucosinolate concentrations of micro-grafted 3-week-old plate-grown Arabidopsis wildtype (Col-0) and mutants. Rosettes and roots from wild type (wt), the glucosinolate biosynthesis null mutant myb28 myb29 cyp79b2 cyp79b3 (qko) and the gtr1 gtr2 gtr3 mutant (tko) were reciprocally grafted using 4-day-old seedlings. Glucosinolate content in the rosette and roots was quantified by LC-MS in 3-week-old plants. Data presented is one of two individual experiments, each containing 8–16 repeats (n) (see – for individual glucosinolate data points and individual n; error bars and parentheses are s.d. Groups in subfigures are determined by one-way ANOVA (p<0.05). n.d. none detected. 10.7554/eLife.19466.013 Figure 4—source data 1. Glucosinolate content in rosettes of hydroponically grown wildtype, gtr3 , gtr1 gtr2 dko and gtr1 gtr2 gtr3 tko plants . Glucosinolate content in rosettes of three-week-old hydroponically grown plants determined by LC-MS. Data presented is one of two individual experiments. Data are given as means and standard deviation (SD) for individual glucosinolates (nmoles/mg FW), total short-chained aliphatic glucosinolates (SC), total long-chained aliphatic glucosinolates (LC), total aliphatic glucosinolates (AG) and total indole glucosinolates (IG). Differences were tested by ANOVA followed by Post-hoc Tukey HSD Calculator multiple comparison (3mtp, 3-methylthiobutylglucosinolate; 3msp, 3-methylsulfinylpropylglucosinolate; 4mtb, 4-methylthiobutylglucosinolate; 4msb, 4-methylsulfinylbutylglucosinolate;5msp,5-methylsulfinylpentylglucosinolate;7mth,7-(methylthio)heptylglucosinolate,7msh,7-ethylsulfinylheptylglucosinolate;8mso, 8-methylsulfinyloctylglucosinolate; I3M, indol-3-ylmethylglucosinolate; 4MOI3M, 4-methoxy-indol-3-ylmethylglucosinolate; NMOI3M, n,-methoxyindol-3-ylmethylglucosinolate). 10.7554/eLife.19466.014 Figure 4—source data 2. Glucosinolate content in roots of hydroponically grown wildtype, gtr3 , gtr1 gtr2 dko and gtr1 gtr2 gtr3 tko plants . Glucosinolate content in roots of three-week-old hydroponically grown plants determined by LC-MS. Data presented is one of two individual experiments. Data are given as means and standard deviation (SD) for individual glucosinolates (nmoles/mg FW), total short-chained aliphatic glucosinolates (SC), total long-chained aliphatic glucosinolates (LC), total aliphatic glucosinolates (AG) and total indole glucosinolates (IG). Differences were tested by ANOVA followed by Post-hoc Tukey HSD Calculator multiple comparison (3mtp, 3-methylthiobutylglucosinolate; 3msp, 3-methylsulfinylpropylglucosinolate; 4mtb, 4-methylthiobutylglucosinolate; 4msb, 4-methylsulfinylbutylglucosinolate;5msp,5-methylsulfinylpentylglucosinolate;7mth,7-(methylthio)heptylglucosinolate,7msh,7-ethylsulfinylheptylglucosinolate;8mso, 8-methylsulfinyloctylglucosinolate; I3M, indol-3-ylmethylglucosinolate; 4MOI3M, 4-methoxy-indol-3-ylmethylglucosinolate; NMOI3M, n,-methoxyindol-3-ylmethylglucosinolate). 10.7554/eLife.19466.015 Figure 4—source data 3. Glucosinolate content in rosettes of micro-grafted plants. Glucosinolate content in rosettes of three-week-old micro-grafted plants determined by LC-MS. Data presented is one of two individual experiments. Data are given as means and standard deviation (SD) for individual glucosinolates (nmoles/mg FW), total short-chained aliphatic glucosinolates (SC), total long-chained aliphatic glucosinolates (LC), total aliphatic glucosinolates (AG) and total indole glucosinolates (IG). Differences were tested by ANOVA followed by Post-hoc Tukey HSD Calculator multiple comparison (3mtp, 3-methylthiobutylglucosinolate; 3msp, 3-methylsulfinylpropylglucosinolate; 4mtb, 4-methylthiobutylglucosinolate; 4msb, 4-methylsulfinylbutylglucosinolate;5msp,5-methylsulfinylpentylglucosinolate;7mth,7-(methylthio)heptylglucosinolate,7msh,7-ethylsulfinylheptylglucosinolate;8mso, 8-methylsulfinyloctylglucosinolate; I3M, indol-3-ylmethylglucosinolate; 4MOI3M, 4-methoxy-indol-3-ylmethylglucosinolate; NMOI3M, n,-methoxyindol-3-ylmethylglucosinolate). 10.7554/eLife.19466.016 Figure 4—source data 4. Glucosinolate content in roots of micro-grafted plants. Glucosinolate content in roots of three-week-old micro-grafted plants determined by LC-MS. Data presented is one of two individual experiments. Data are given as means and standard deviation (SD) for individual glucosinolates (nmoles/mg FW). total short-chained aliphatic glucosinolates (SC). Total long-chained aliphatic glucosinolates (LC). total aliphatic glucosinolates (AG) and total indole glucosinolates (IG). Differences were tested by ANOVA followed by Post-hoc Tukey HSD Calculator multiple comparison (3mtp. 3-methylthiobutylglucosinolate; 3msp. 3-methylsulfinylpropylglucosinolate; 4mtb. 4-methylthiobutylglucosinolate; 4msb. 4-methylsulfinylbutylglucosinolate;5msp.5-methylsulfinylpentylglucosinolate;7mth.7-(methylthio)heptylglucosinolate.7msh.7-ethylsulfinylheptylglucosinolate;8mso. 8-methylsulfinyloctylglucosinolate; I3M. indol-3-ylmethylglucosinolate; 4MOI3M. 4-methoxy-indol-3-ylmethylglucosinolate; NMOI3M. n.-methoxyindol-3-ylmethylglucosinolate).

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Comparison, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Standard Deviation

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A ) Selected part of Bayesian inference (MrBayes) tree (s.d. < 0.01) of GTR homologs from selected species (Full phylogenetic tree of NPFs from selected species is found as ). Green circles at nodes represent a posterior probability of 1 (maximum is 1). Values at nodes separated by a backslash represent MrBayes values below 1 in red, followed by RAxML generated bootstrap values in black (only reported when Mrbayes value is below 1). Asterix indicates that Cp17.188 lacks the highly conserved EXXE[R/K] motif involved in proton-coupling . Subclades with green, purple and pink background denote the GTR1 subclade, GTR3 subclade and GTR-like subclade (genes that cluster with GTR homologs from C.papaya) , respectively. Genes in bold were tested for glucosinolate transport in B ). ( B ) Uptake of 4MTB and I3M by X. laevis oocytes expressing selected GTR homologs (bold) from A. thaliana , B. rapa , C. papaya , T. cacao and M. esculenta from the colored subclades and Me15G176100, which clusters outside the GTR-like subclade). Genes were expressed individually in X. laevis oocytes and transport activity was measured in the presence of 0.2 mM 4MTB (black bars) or 0.2 mM I3M (green bars) at external pH 5. Dotted line represents substrate concentration in external media. Accumulated 4MTB or I3M was quantified by LC-MS in 5 × 1 oocytes for each gene (Error bars represent ± s.d. of mean, n = 5, experiment repeated two times). ( C ) 4MTB (black circles)- and I3M (green circles)-induced currents in oocytes expressing GTR homologs that showed glucosinolate uptake in B ). Expressing oocytes were exposed to 0.2 mM 4MTB or I3M and induced currents were measured at membrane potentials clamped between 0 mV and −180 mV in 20 mV increments at pH 5 (Error bars represent ± s.d. of mean, n = 4, experiment repeated two times). ( D ) Normalized 4MTB-induced currents of CpGTRL2 (Cp17.190) measured at a membrane potential of −60 mV at pH 5 plotted against increasing 4MTB concentrations. The saturation curve was fitted with a Michaelis-Menten equation represented by a solid line (Error bars represent ± s.d. of mean for data obtained from four different oocytes per experiment). Each oocyte dataset was normalized to currents elicited at 1 mM 4MTB concentration at −60 mV. The insert shows the apparent K m as a function of membrane potential.

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Generated, Expressing, Activity Assay, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Membrane

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A – D ) 4MTB (black circles)- and I3M (green circles)-induced currents in oocytes expressing GTR homologs that showed glucosinolate uptake in . Expressing and non-expressing oocytes were exposed to 0.2 mM 4MTB or I3M and induced currents were measured at membrane potentials between 0 mV and −180 mV in 20 mV increments at pH5 (Error bars represent ± s.d. of mean for data obtained from four different oocytes per experiment, experiment repeated two times).

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Expressing, Membrane

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: ( A ) Uptake of prunasin and linamarin in X. laevis oocytes expressing GTR homologs from A. thaliana , B. rapa , C. papaya , T. cacao and M.esculenta . Genes were expressed individually in X. laevis oocytes and transport activity was measured in the presence of 0.2 mM prunasin (red bars) or linamarin (blue bars). Accumulated prunasin or linamarin were quantified by LC-MS in 5 × 1 oocytes for each gene (Error bars represent ±s.d. of mean for data obtained from five different oocytes per experiment, experiment repeated two times). Dotted line represents media substrate concentration. None of the genes accumulated linamarin to detectable levels. ( B ) Bayesian inference tree (MrBayes tree) showing selected part of M. esculenta NPF phylogenetic tree (closest homologs of Me14G074000). Green circles at nodes represent a posterior probability of 1 (maximum is 1). Values at nodes separated by a backslash represent MrBayes values below 1 in red, followed by RAxML generated bootstrap values in black (only reported when Mrbayes value is below 1). Scale bar indicates number of substitutions per site. Subclades coloured green, pink and purple mark genes that cluster with GTRs, NPF2.12/13 and NPF2.8, respectively, in . Full phylogenetic tree of M.esculenta NPFs is found as . Genes in bold were assayed for prunasin, linamarin, 4MTB and I3M uptake. ( C ) Accumulation of prunasin and linamarin in X.laevis oocytes expressing closest homologs of Me14G074000 from M. esculenta . Genes were expressed individually in X. laevis oocytes and transport activity was measured in the presence of 0.2 mM prunasin (red bars) or 0.2 mM linamarin (blue bars). Accumulated prunasin or linamarin was quantified by LC-MS in 5 × 1 oocytes for each gene (Error bars represent ± s.d. of mean for data obtained from five different oocytes per experiment, experiment repeated two times). Only MeCGTR1 accumulated linamarin to detectable levels. Dotted line represents substrate concentration in external media. ( D ) Prunasin (red circles)- and linamarin (blue circles)-induced currents in oocytes expressing Me14G74000, MeCGTR1, Me15G176100 and non-expressing oocytes, respectively. Expressing and non-expressing oocytes were exposed to 0.2 mM prunasin or 0.2 mM linamarin and induced currents were measured at membrane potentials between 0 mV and −180 mV in 20 mV increments at pH5. ( E – F ) Normalized prunasin ( E ) or linamarin ( F ) induced currents elicited in MeCGTR1-expressing oocytes measured at a membrane potential of −60 mV and pH 5 plotted against increasing prunasin ( E ) or linamarin ( F ) concentrations. The saturation curve was fitted with a Michaelis-Menten equation represented by a solid line (error bars are s.d.; n = 4). Each oocyte dataset was normalized to currents elicited at 0.8 mM prunasin ( E ) or 1 mM linamarin ( F ) concentration at −60 mV. The insert shows the apparent K m as a function of membrane potential (error bars are s.d.; n = 3–4 oocytes).

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Expressing, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Generated, Membrane

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet: Genes were expressed individually in X. laevis oocytes and transport activity was measured in the presence of 0.2 mM 4MTB (black bars) or 0.2 mM I3M (green bars). Accumulated 4MTB or I3M was quantified by LC-MS in 5 × 1 oocytes for each gene (Error bars represent ± s.d. of mean for data obtained from five different oocytes per experiment, experiment repeated two times). Dotted line represents media substrate concentration.

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: eLife

Article Title: Origin and evolution of transporter substrate specificity within the NPF family

doi: 10.7554/eLife.19466

Figure Lengend Snippet:

Article Snippet: 4-methylsulfinylbutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) were obtained from C 2 Bioengineering ( http://www.glucosinolates.com/ ) and CFM Oskar Tropitzsch GmbH, Marktredwitz ( http://www.cfmot.de/ ), respectively.

Techniques:

Journal: bioRxiv

Article Title: Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory

doi: 10.1101/2021.02.03.429630

Figure Lengend Snippet: A , Levels of I3M, 1MO-I3M, and 4MO-I3M in Col-0 and cyp79b2 cyp79b3 ( cyp79b2b3 ) leaves supplemented with IGs. (water/-mite), untreated leaves immediately frozen after being cut from intact plant; (+mite), leaves challenged with mites for 48h. Values were log2 transformed for statistical analysis. B , Mite fitness upon feeding on Col-0 and cyp79b2 cyp79b3 ( cyp79b2b3 ) leaves supplemented with I3M, 1MO-I3M or 4MO-I3M. The total numbers of deposited feces (top panels) and eggs (bottom panels) were recorded 48 h after the addition of 10 mites per leaf. Experiments were performed in five biological replicates/trial and in four independent trials. Data represent the mean ± SE of four trials. Significant differences (p ≤ 0.05) are indicated by different letters.

Article Snippet: The 3-indolylmethyl glucosinolate or glucobrassicin (I3M), the neoglucobrassicin (1MO-I3M), and the 4-methoxyglucobrassicin (4MO-I3M) potassium salts were purchased from Extrasynthese (France), with the catalog numbers 2525, 2519 S, and 2522 S, respectively.

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory

doi: 10.1101/2021.02.03.429630

Figure Lengend Snippet: A , Mite fecundity upon direct application of 0.23, 2.3 or 4.6 mM I3M to mites. Mites were treated for 19 h with I3M solutions and were subsequently transferred to bean leaves. Mite fecundity was determined at 24 and 48 h after treatment. B , The total number of deposited feces and eggs over 24 h of feeding on bean leaf disk treated with 2.4 mM of I3M, 1MO-I3M, or 4MO-I3M. Experiments were performed in three (in A) and five (in B) biological replicates/trial and in three independent trials. Data represent the mean ± SE of three trials. Significant differences (p ≤ 0.05) are indicated by different letters.

Article Snippet: The 3-indolylmethyl glucosinolate or glucobrassicin (I3M), the neoglucobrassicin (1MO-I3M), and the 4-methoxyglucobrassicin (4MO-I3M) potassium salts were purchased from Extrasynthese (France), with the catalog numbers 2525, 2519 S, and 2522 S, respectively.

Techniques:

Journal: bioRxiv

Article Title: Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory

doi: 10.1101/2021.02.03.429630

Figure Lengend Snippet: A , Fecundity of 10 mites feeding for 48 h on Col-0, myc2 myc3 myc4 ( myc234 ) and cyp79b2 cyp79b3 ( cyp79b2b3 ) leaves. B , Levels of I3M, 1MO-I3M and 4MO-I3M in Col-0 and myc2 myc3 myc4 ( myc234 ) leaves supplemented with 2.4 mM I3M for 6 h and kept in water for 16 h before mite addition. (-I3M/-mite), untreated leaves were immediately frozen after being cut from intact plant; (+ mite) leaves challenged with mites for 48h. C , Fecundity of 10 mites upon feeding for 48 h on Col-0, cyp79b2 cyp79b3 ( cyp79b2b3 ) and myc2 myc3 myc4 ( myc234 ) leaves supplemented with 4.8 mM I3M or 1MO-I3M for 24 h before mite addition. Experiments were performed in five biological replicates/trial and in three independent trials. Data represent the mean ± SE of three trials. Significant differences (p ≤ 0.05) are indicated by different letters.

Article Snippet: The 3-indolylmethyl glucosinolate or glucobrassicin (I3M), the neoglucobrassicin (1MO-I3M), and the 4-methoxyglucobrassicin (4MO-I3M) potassium salts were purchased from Extrasynthese (France), with the catalog numbers 2525, 2519 S, and 2522 S, respectively.

Techniques:

Journal: Molecular Plant Pathology

Article Title: Differential roles of glucosinolates and camalexin at different stages of Agrobacterium ‐mediated transformation

doi: 10.1111/mpp.12672

Figure Lengend Snippet: The effects of indole glucosinolate (iGS) and aliphatic glucosinolate‐isothiocyanates (aGS‐ITCs) on Agrobacterium transformation efficiency. Seven‐day‐old Col‐0 seedlings were treated with indol‐3‐ylmethylglucosinolate (I3M) (a, b) and aGS‐ITCs (c, d) during Agrobacterium infection, followed by analysis for β‐glucuronidase (GUS) staining (a, c) and GUS activity assay (b, d). The results are presented as the mean ± standard error of the mean (SEM) from three independent experiments ( n ≥ 20), and the GUS activity in (d) is presented as a log 2 value. Significant differences from the control groups methanol (MeOH) and dimethylsulfoxide (DMSO) are indicated [one‐way analysis of variance (ANOVA) with Dunnett's test, * P < 0.05, ** P < 0.01, *** P < 0.001]. Scale bar, 1 cm.

Article Snippet: The GSs were quantified with the given references, including I3M for iGSs, 4MTB for methylthioalkyl GSs and 4MSOB for methylsulfinylalkyl GSs, purchased from AppliChem (Darmstadt, Germany).

Techniques: Transformation Assay, Infection, Staining, Activity Assay, Control

Journal: bioRxiv

Article Title: A versatile method to expand and compare transporter substrate spectra

doi: 10.1101/2025.07.11.664331

Figure Lengend Snippet: Presence of indole-glucosinolates glucobrassicin (I3M), 4-methoxyglucobrassicin (4MOI3M), and neoglucobrassicin (NMOI3M) in oocytes exposed to 1:100 seedling media. NPF2.10 in brown, NPF2.11 in red, and NPF2.9 in pink. Oocytes were assayed at pH 5 for 1 hour; replicates consisted of five oocytes each, and media samples consisted of 5µl each. Control oocytes in blue and seedling media in green. Combined extracted ion chromatogram for I3M (447.0537 +/-0.005 m/z) and 4NMOI3M and NMOI3M (477.0643 +/-0.005 m/z).

Article Snippet: Neoglucobrassicin (NMOI3M), glucobrassicin (I3M), 4-methoxy-3-indolylmethyl glucosinolate (4MOI3M) sinalbin (pOHB), glucoraphanin (4msb) afzelin (K3R), isoquercetin (Q3G), quercitrin (Q3R), and astragalin (K3G) where purchased from Extrasynthese.

Techniques: Control

Journal: bioRxiv

Article Title: A versatile method to expand and compare transporter substrate spectra

doi: 10.1101/2025.07.11.664331

Figure Lengend Snippet: The metabolic differences of the extracts and transporter activity drive the chemical profile of the oocytes . Unsupervised bicluster analysis of oocytes assayed with empty buffer (pH5), and control (mock) and GTR-expressing oocyte samples assayed with either seed (Seed) or seedling (Sling) derived media. The relative intensities of metabolic features are represented on a color scale. Columns represent samples, and rows represent metabolic features. Oocytes were assayed at pH 5 for 1 hour with either 1:100 dilutions of seed or seedling extract; replicates consisted of five oocytes each. pH5 oocytes were assayed with an empty pH 5 buffer for 1 hour. Metabolic features identified with chemical standards as indole glucosinolates in black font: glucobrassicin (I3M), neoglucobrassicin (NMOI3M), and 4-methoxyglucobrassicin (4MOI3M). Annotated glucosinolates in blue font: 3-sinapoyloxypropylglucosinolate (3sin), 4-sinapoyloxybutylglucosinolate (4sin), glucoraphasatin (dH4mtb), 6′-O-benzoyloxy-glucoerucin (6’bz4mtb). Annotated flavonoid Quercitrin (Q3R) in purple. Ward’s biclustering of the 100 most significant features (ANOVA). Sig. P-value <0.05. Features displaying a Relative Standard deviation of >30% in the QC were filtered. Features are logarithmically transformed (Log10) and Pareto scaled. Identification of indole glucosinolate-derived features was performed with co-analyzed chemical standards.

Article Snippet: Neoglucobrassicin (NMOI3M), glucobrassicin (I3M), 4-methoxy-3-indolylmethyl glucosinolate (4MOI3M) sinalbin (pOHB), glucoraphanin (4msb) afzelin (K3R), isoquercetin (Q3G), quercitrin (Q3R), and astragalin (K3G) where purchased from Extrasynthese.

Techniques: Activity Assay, Control, Expressing, Derivative Assay, Standard Deviation, Transformation Assay

Journal: bioRxiv

Article Title: A versatile method to expand and compare transporter substrate spectra

doi: 10.1101/2025.07.11.664331

Figure Lengend Snippet: Glucosinolate import generates most of the clusters separating selected NPF substrate spectra, while phenylpropanoid import contributes to the separation of some additional samples . Unsupervised bicluster analysis of control (mock), and selected NPF-expressing oocyte samples assayed with 1:20 dilution of seedling-derived media. The relative intensities of metabolic features are represented on a color scale. Columns represent samples and rows metabolic features. Metabolic features identified with chemical standards are in black: glucobrassicin (I3M), neoglucobrassicin (NMOI3M), 4-methoxyglucobrassicin (4MOI3M), and Kaempferol-3-O-rhamnoside (K3R). Annotated glucosinolates in blue font: 3-sinapoyloxypropylglucosinolate (3sin), 4-sinapoyloxybutylglucosinolate (4sin), 4-Hydroxyglucobrassicin (4OHI3M), and 6′-O-benzoyloxy-4-sinapoyloxybutylglucosinolate (6’bz4sin). Features deriving from annotated phenylpropanoids in purple: Two isomers of disinapoyl hexose (DSH’’ and DSH’’’). Ward’s biclustering of the 100 most significant features (ANOVA). Sig. P-value <0.05. Oocytes were assayed at pH 5 for 1 hour with 1:20 dilutions of seedling extract; replicates consisted of five oocytes each. Features displaying a Relative Standard deviation of >30% in the QC were filtered. Features are logarithmically transformed (Log10) and Pareto scaled.

Article Snippet: Neoglucobrassicin (NMOI3M), glucobrassicin (I3M), 4-methoxy-3-indolylmethyl glucosinolate (4MOI3M) sinalbin (pOHB), glucoraphanin (4msb) afzelin (K3R), isoquercetin (Q3G), quercitrin (Q3R), and astragalin (K3G) where purchased from Extrasynthese.

Techniques: Control, Expressing, Derivative Assay, Standard Deviation, Transformation Assay