MTBLS2: Comparative LC/MS-based profiling of silver nitrate-treated Arabidopsis thaliana leaves of wild-type and cyp79B2 cyp79B3 double knockout plants    

Christoph Böttcher , Steffen Neumann

Study Submission Date: 22-May-2012   Study public release date: 22-May-2012

Indole-3-acetaldoxime (IAOx) represents an early intermediate of the biosynthesis of a variety of indolic secondary metabolites including the phytoanticipin indol-3-ylmethyl glucosinolate and the phytoalexin camalexin (3-thiazol-2′-yl-indole). Arabidopsis thaliana cyp79B2 cyp79B3 double knockout plants are completely impaired in the conversion of tryptophan to indole-3-acetaldoxime and do not accumulate IAOx-derived metabolites any longer. Consequently, comparative analysis of wild-type and cyp79B2 cyp79B3 plant lines has the potential to explore the complete range of IAOx-derived indolic secondary metabolites.

Organism(s):
Arabidopsis thaliana (thale cress)

Study Design Description:
  • Plant Metabolomics
  • Comparative LC/MS-based profiling
  • Pathway Elucidation

Publications
The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) converts cysteine-indole-3-acetonitrile to camalexin in the indole-3-acetonitrile metabolic network of Arabidopsis thaliana.
Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data

Experimental Factors
  • genotype: [ cyp79 , Col-0 ]
  • replicate: [ Exp2 , Exp1 ]
Protocol Description
Sample collection Arabidopsis thaliana Col-0 and cyp79B2 cyp79B3 lines were grown for about six weeks after cold stratification (3 days, 4°C) in parallel on a soil/vermiculite mixture (3/2) in a growth cabinet with 8 h light (~150 µE m^-2 s^-1) at 22°C and 16 h dark at 20°C to developmental stage 3.5. Plants were sprayed with an aqueous solution of silver nitrate (5 mM) in order to stimulate accumulation of indolic secondary metabolites and incubated for 48 h in a growth cabinet under the same conditions as described above. Rosette leaves of six individual plants per genotype were harvested, immediately frozen in liquid nitrogen, pooled and stored at –80°C until analysis. Two independent experiments (biological replicates) were performed on independent sets of plants grown at different times.
Extraction Plant material was homogenized in liquid nitrogen using a pestle and mortar and aliquots of 100 +/- 5 mg were weighed into pre-cooled 2-ml, round bottom tubes. After addition of 200 µL methanol/water, 80/20 (v/v) pre-cooled at –40°C the samples were immediately vortexed for 15 s, sonicated for 15 min at 20°C and centrifuged for 10 min at 19000 x g. The supernatants were transferred to new 2-ml tubes and the remaining pellets subjected to a second extraction using 200 µL methanol/water, 80/20 (v/v). The combined extracts were evaporated to dryness in a vacuum centrifuge at 30°C, thoroughly reconstituted in 200 µL methanol/water, 30/70 (v/v) and filtered using 0.2-µm PTFE syringe filters. Four extracts (technical replicates) were prepared for each of the four leaf pools.
Chromatography Chromatographic separations were performed on an Acquity UPLC system (Waters) equipped with a HSS T3 column (100 x 1.0 mm, particle size 1.8 µm, Waters) applying the following binary gradient at a flow rate of 150 µL/min: 0-1 min, isocratic 95% A (water/formic acid, 99.9/0.1 (v/v)), 5% B (acetonitrile/formic acid, 99.9/0.1 (v/v)); 1-16 min, linear from 5 to 45% B; 16-18 min, isocratic 95% B; 18-20 min, isocratic 5% B. The injection volume was 3.0 µL (full loop injection).
Mass spectrometry Eluting compounds were detected from m/z 100-1000 using a micrOTOF-Q II hybrid quadrupole time-of-flight mass spectrometer (Bruker Daltonics) equipped with an Apollo II electrospray ion source in positive and negative ion mode using following instrument settings: nebulizer gas, nitrogen, 1.4 bar; dry gas, nitrogen, 6 L/min, 190°C; capillary, –5000 V; end plate offset, -500 V; funnel 1 RF, 200 V; funnel 2 RF, 200 V; in-source CID energy, 0 V; hexapole RF, 100 V; quadrupole ion energy, 5 eV; collision gas, nitrogen; collision energy, 7 eV; collision RF 150/350 V (timing 50/50); transfer time, 70 µs; pre pulse storage, 5 µs; pulser frequency, 10 kHz; spectra rate, 3 Hz. Mass spectra were acquired in centroid mode. Mass calibration of individual raw data files was performed on lithium formate cluster ions obtained by automatic infusion of 20 µL 10 mM lithium hydroxide in isopropanol/water/formic acid, 49.9/49.9/0.2 (v/v/v) at a gradient time of 18 min using a diverter valve.
Data transformation Raw data files were converted to mzData format using the vendor-specific CompassXport (http://www.bdal.de/) and processed using the XCMS package (http://bioconductor.org/packages/release/bioc/html/xcms.html). XCMS settings for processing LC/MS data with findPeaks.centWave() were prefilter=(3,200); snthr=5; ppm=25; peakwidth=(5,12). For alignment group.density() function with parameters minfrac=0.75 and bw=2 was used.
Metabolite identification Differential features detected in narrow retention time windows displaying a high chromatogram correlation were grouped. In combination with the raw data, individual compound mass spectra were reconstructed and features annotated as cluster ion, quasimolecular ion, or in-source fragment ion. Annotated lists of all differential feature sets are provided in Supplemental DataSet S5 of DOI: 10.1007/s11306-012-0401-0 online. Afterwards, targeted CID-MS/MS experiments of quasimolecular ions (MS2) were performed using QTOF-MS. Based on accurate mass measurements, putative elemental compositions were calculated, filtered by isotope abundance and the number of double bond equivalents and electron parity (Kind and Fiehn, 2006), and checked for consistency in relation to elemental compositions calculated for fragment ions and observed neutral losses (Konishi et al., 2007). Mass spectral characterization of CID mass spectra can be found in Supplemental Data Set S6 (DOI: 10.1007/s11306-012-0401-0) online. Identification was based on reference spectra in MassBank (MSI Level 2 and 3), and in some cases on authentic standards (MSI Level 1).

Technology - Platform: mass spectrometry - metabolite profiling - micrOTOF-Q II ESI TOF (Bruker)
Source replicate genotype
Ex2-cyp79-48h-Ag-2_1-B,4_01_9830 Exp2 cyp79
Ex2-Col0-48h-Ag-4_1-A,2_01_9833 Exp2 Col-0
Ex1-cyp79-48h-Ag-4_1-B,2_01_9825 Exp1 cyp79
Ex2-cyp79-48h-Ag-4_1-B,4_01_9834 Exp2 cyp79
Ex2-cyp79-48h-Ag-1_1-B,3_01_9828 Exp2 cyp79
Ex2-cyp79-48h-Ag-3_1-B,3_01_9832 Exp2 cyp79
Ex1-Col0-48h-Ag-2_1-A,1_01_9820 Exp1 Col-0
Ex2-Col0-48h-Ag-3_1-A,4_01_9831 Exp2 Col-0
Ex1-Col0-48h-Ag-1_1-A,1_01_9818 Exp1 Col-0
Ex2-Col0-48h-Ag-2_1-A,3_01_9829 Exp2 Col-0
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Hexosyl indole-3-carboxylate C15H17NO7 118.06505 299.419500 7.664658e+03 7.554282e+03 8.201915e+03 8.010598e+03 4.340455e+02 4.483271e+02 4.152530e+02 3.610310e+02 7.643618e+03 8.957460e+03 8.446990e+03 8.189397e+03 3.944030e+02 3.563160e+02 4.698117e+02 6.586958e+02
Description Formula m/z Retention time Smiles Inchi Ex1-Col0-48h-Ag-1 Ex1-Col0-48h-Ag-2 Ex1-Col0-48h-Ag-3 Ex1-Col0-48h-Ag-4 Ex1-cyp79-48h-Ag-1 Ex1-cyp79-48h-Ag-2 Ex1-cyp79-48h-Ag-3 Ex1-cyp79-48h-Ag-4 Ex2-Col0-48h-Ag-1 Ex2-Col0-48h-Ag-2 Ex2-Col0-48h-Ag-3 Ex2-Col0-48h-Ag-4 Ex2-cyp79-48h-Ag-1 Ex2-cyp79-48h-Ag-2 Ex2-cyp79-48h-Ag-3 Ex2-cyp79-48h-Ag-4
Indol-3-ylmethylamine (CID472107) C9H10N2 130.06545 121.309200 1.249611e+05 1.138784e+05 1.217397e+05 1.322581e+05 1.738169e+04 1.479092e+04 1.866853e+04 1.693713e+04 1.545710e+05 1.563897e+05 1.587602e+05 1.608485e+05 1.706545e+04 1.829369e+04 1.734161e+04 2.014217e+04
4-Methoxy-indol-3-ylmethylglucosinolate (CID9576738) C17H22N2O10S2 161.07827 270.794400 7.629423e+03 8.677716e+03 1.004428e+04 1.018451e+04 8.083200e+02 7.788642e+02 1.179137e+03 9.012768e+02 9.600794e+03 1.334583e+04 1.121301e+04 1.120058e+04 7.942137e+02 1.117613e+03 9.232602e+02 9.249443e+02
Methyl indole-3-carboxylate (CID589098) C10H9NO2 176.07003 630.681000 9.250563e+03 9.820634e+03 9.736468e+03 9.857022e+03 2.973238e+02 3.017280e+02 5.687201e+02 3.086554e+02 1.703326e+04 1.725702e+04 1.675971e+04 1.494328e+04 2.331763e+02 1.585951e+02 2.181946e+02 3.155066e+02
Methyl indole-3-carboxylate (CID589098) C10H9NO2 198.05311 630.654000 5.110339e+03 4.146565e+03 4.660061e+03 4.403397e+03 1.253720e+03 9.428730e+02 1.072886e+03 9.412162e+02 7.474541e+03 6.952984e+03 8.047602e+03 6.723343e+03 9.051840e+02 8.300888e+02 9.597375e+02 9.543495e+02
Camalexin (CID636970) C11H8N2S 216.03539 625.431000 7.464447e+03 7.806548e+03 7.214133e+03 6.910273e+03 3.017011e+02 3.800909e+02 4.239305e+02 2.932831e+02 7.879613e+03 7.440832e+03 7.771898e+03 6.724082e+03 3.747694e+02 3.562498e+02 3.410974e+02 3.555763e+02
Camalexin (CID636970) C11H8N2S 217.04316 625.566000 5.220521e+03 5.285962e+03 5.022604e+03 4.866686e+03 6.076903e+02 6.026374e+02 5.142811e+02 5.176469e+02 6.042066e+03 5.268268e+03 5.558949e+03 5.272832e+03 4.520154e+02 4.425914e+02 5.049489e+02 3.709017e+02
Camalexin (CID636970) C11H8N2S 233.03800 625.266000 1.007807e+04 1.081607e+04 1.043308e+04 1.119202e+04 4.537466e+02 5.948897e+02 8.047517e+02 5.062963e+02 1.123108e+04 1.113297e+04 1.098395e+04 1.195447e+04 5.527826e+02 7.074000e+02 5.329056e+02 4.615374e+02
3-Hydroxy-3-(thiazol-2-yl)indolin-2-one C11H8N2O2S 233.03842 334.275000 1.202915e+04 1.133048e+04 1.257899e+04 1.160849e+04 7.840299e+02 4.634129e+02 1.026850e+03 6.022139e+02 1.424490e+04 1.364508e+04 1.426954e+04 1.344697e+04 2.606697e+02 5.452510e+02 4.738531e+02 6.587468e+02
2-Formamidophenyl-2'-thiazolylketone C11H8N2O2S 233.03911 540.714600 1.347337e+04 1.270898e+04 1.258257e+04 1.372369e+04 2.785400e+02 2.585797e+02 2.236403e+02 3.078211e+02 1.628563e+04 1.831195e+04 1.616030e+04 1.809257e+04 3.148720e+02 2.980952e+02 7.114308e+02 2.714298e+02
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