[20, 21]. (4) Examples of associations between metabolites and
biotic or abiotic stress tolerance have been uncovered which hopefully will pave the way toward the discipline of functional metabolomics [5] which will likely be founded using the model plant
Arabidopsis thaliana.
2 Materials
2.1 Plant material
1. A large collection of Arabidopsis thaliana natural accessions
such as the 314 natural accessions commonly referred to as
the Arabidopsis HapMap collection ([22]; see Note 1). Alternatively, segregating populations resulting from biparental
crosses such as introgression lines or recombinant inbred
lines [23] or from multi-parental crosses following Magic like
crossing schemes ([24]; see Note 2).
2. Suitable greenhouse or growth chamber conditions that are
large enough to accommodate the populations in a manner
that facilitates rapid harvest of samples from individual plants.
3. For validation experiments, knockout lines can be ordered
from stock centers and genotyped as described in [25].
2.2 GC-MS
2.2.1 Sampling and
Extraction
1. Argon.
2. Centrifuge (capable of 3700 g), (e.g., Allegra
® x-15R, Beckman Coulter).
3. Methanol gradient grade for liquid chromatography.
4. MilliQ water approx. 0.055 μS/cm.
5. Oscillating ball mill MM200 (e.g., Retsch GmbH and Co.KG,
Haan, Germany) or alternatively a pestle and mortar.
6. Ribitol, purity !99.0%; 0.2 mg/mL in dH 2 O.
7. Speed vacuum concentrator (e.g., SPD111V-230, ThermoElectron Corporation, Langenselbold, Germany).
8. Schott glass AR-GLASS
® culture tubes (soda-lime)
9. Thermoblock (capable of heating to up to 70
C).
10. Liquid nitrogen supply.
11. Vortex.
12. Scalpel blades, aluminum foil, 6-well plates, spatula, balance,
microfuge tubes (2 mL).
2.2.2 Derivatization
1. Methoxyamine hydrochloride, purity 98%. Store at room temperature under dry atmosphere.
2. N-methyl-N-trimethylsilyltrifluor(o)acetamide
(MSTFA).
MSTFA should be stored in opaque glass bottles under
Metabolomic Analysis of Natural Variation in Arabidopsis
395
biotic or abiotic stress tolerance have been uncovered which hopefully will pave the way toward the discipline of functional metabolomics [5] which will likely be founded using the model plant
Arabidopsis thaliana.
2 Materials
2.1 Plant material
1. A large collection of Arabidopsis thaliana natural accessions
such as the 314 natural accessions commonly referred to as
the Arabidopsis HapMap collection ([22]; see Note 1). Alternatively, segregating populations resulting from biparental
crosses such as introgression lines or recombinant inbred
lines [23] or from multi-parental crosses following Magic like
crossing schemes ([24]; see Note 2).
2. Suitable greenhouse or growth chamber conditions that are
large enough to accommodate the populations in a manner
that facilitates rapid harvest of samples from individual plants.
3. For validation experiments, knockout lines can be ordered
from stock centers and genotyped as described in [25].
2.2 GC-MS
2.2.1 Sampling and
Extraction
1. Argon.
2. Centrifuge (capable of 3700 g), (e.g., Allegra
® x-15R, Beckman Coulter).
3. Methanol gradient grade for liquid chromatography.
4. MilliQ water approx. 0.055 μS/cm.
5. Oscillating ball mill MM200 (e.g., Retsch GmbH and Co.KG,
Haan, Germany) or alternatively a pestle and mortar.
6. Ribitol, purity !99.0%; 0.2 mg/mL in dH 2 O.
7. Speed vacuum concentrator (e.g., SPD111V-230, ThermoElectron Corporation, Langenselbold, Germany).
8. Schott glass AR-GLASS
® culture tubes (soda-lime)
9. Thermoblock (capable of heating to up to 70
C).
10. Liquid nitrogen supply.
11. Vortex.
12. Scalpel blades, aluminum foil, 6-well plates, spatula, balance,
microfuge tubes (2 mL).
2.2.2 Derivatization
1. Methoxyamine hydrochloride, purity 98%. Store at room temperature under dry atmosphere.
2. N-methyl-N-trimethylsilyltrifluor(o)acetamide
(MSTFA).
MSTFA should be stored in opaque glass bottles under
Metabolomic Analysis of Natural Variation in Arabidopsis
395