2.3 Metabolite
Extraction
1. Methanol with 0.01% butylated hydroxytoluene (BHT).
2. Metabolite standards: 10 μL 0.1 nmol/μL lidocaine and
0.1 nmol/μL camphosulfonic acid.
3. Chloroform.
4. 0.1% Formic acid in LC grade water.
5. SpeedVac lyophilizer.
2.4 Mass
Spectrometry Using
Acquire X
1. High-resolution Orbitrap Fusion Tribrid mass spectrometer.
2. Vanquish™ UHPLC liquid chromatography.
3. Accucore C18 (100 Â 2.1) column.
4. Acquire X MS
n data acquisition program.
5. Solvent A: 0.1% Formic acid in water.
6. Solvent B: 0.1% Formic acid in acetonitrile.
2.5 Data Analysis
1. Compound Discover™ 3.0 Software for data analyses.
3 Methods
3.1 Bacterial
Treatment of Plants
(See Note 1)
1. Grow Arabidopsis plants for 5 weeks in short-day conditions.
2. Inoculate one leaf on each plant with either Pst DC3000
(OD600 ¼ 0.02) (treatment) or 10 mM MgCl 2 (solution for
resuspension of Pst) (control) using a needless syringe into a
fully expanded rosette leaf.
3.2 Harvesting of
Guard Cell Samples
(See Note 2)
1. To obtain 100 mg of freeze-dried guard cells for each sample,
harvest systemic, uninfected leaves of Columbia (Col-0) wildtype Arabidopsis at 72 h after the inoculation. Fully expanded
leaves should be collected from 5-week-old plants. Roughly
300 leaves from 108 plants are required to obtain 100 mg of
freeze-dried guard cell sample.
2. To obtain guard cell samples, 300 systemic or uninfected leaves
(after removing the mid-vein of each leaf) were put in 500 mL
ice water and blend using a high-speed blender for 1 min.
3. Filter material through a nylon mesh to remove broken mesophyll cells and other types of cells. Then the material retained
was put in 500 mL ice water, followed by blending again for
1 min.
4. Repeat step 3 again to obtain enriched stomatal guard cells.
5. Transfer guard cell samples from the nylon mesh to labeled
15 mL centrifuge tubes and immediately freeze in liquid nitrogen, then transfer to a À80
C freezer.
Untargeted Metabolomics of Arabidopsis Stomatal Immunity
417
Extraction
1. Methanol with 0.01% butylated hydroxytoluene (BHT).
2. Metabolite standards: 10 μL 0.1 nmol/μL lidocaine and
0.1 nmol/μL camphosulfonic acid.
3. Chloroform.
4. 0.1% Formic acid in LC grade water.
5. SpeedVac lyophilizer.
2.4 Mass
Spectrometry Using
Acquire X
1. High-resolution Orbitrap Fusion Tribrid mass spectrometer.
2. Vanquish™ UHPLC liquid chromatography.
3. Accucore C18 (100 Â 2.1) column.
4. Acquire X MS
n data acquisition program.
5. Solvent A: 0.1% Formic acid in water.
6. Solvent B: 0.1% Formic acid in acetonitrile.
2.5 Data Analysis
1. Compound Discover™ 3.0 Software for data analyses.
3 Methods
3.1 Bacterial
Treatment of Plants
(See Note 1)
1. Grow Arabidopsis plants for 5 weeks in short-day conditions.
2. Inoculate one leaf on each plant with either Pst DC3000
(OD600 ¼ 0.02) (treatment) or 10 mM MgCl 2 (solution for
resuspension of Pst) (control) using a needless syringe into a
fully expanded rosette leaf.
3.2 Harvesting of
Guard Cell Samples
(See Note 2)
1. To obtain 100 mg of freeze-dried guard cells for each sample,
harvest systemic, uninfected leaves of Columbia (Col-0) wildtype Arabidopsis at 72 h after the inoculation. Fully expanded
leaves should be collected from 5-week-old plants. Roughly
300 leaves from 108 plants are required to obtain 100 mg of
freeze-dried guard cell sample.
2. To obtain guard cell samples, 300 systemic or uninfected leaves
(after removing the mid-vein of each leaf) were put in 500 mL
ice water and blend using a high-speed blender for 1 min.
3. Filter material through a nylon mesh to remove broken mesophyll cells and other types of cells. Then the material retained
was put in 500 mL ice water, followed by blending again for
1 min.
4. Repeat step 3 again to obtain enriched stomatal guard cells.
5. Transfer guard cell samples from the nylon mesh to labeled
15 mL centrifuge tubes and immediately freeze in liquid nitrogen, then transfer to a À80
C freezer.
Untargeted Metabolomics of Arabidopsis Stomatal Immunity
417