Materials and Reagents

BD^TM Tuberculin syringe, 1 ml (Becton Dickinson, catalog number: 309659 )
Kimwipes, 1-Ply (Fisher, catalog number: 06-666 )
Microfuge tubes, 1.5 ml (Sarstedt, catalog number: 72.690.300 )
Disposable test tubes, 13 x 100 mm (VWR, catalog number: 47729-572 )
GC vials, 2 ml (Thermo Scientific, catalog number: C4000-1 )
Screw caps (Agilent; catalog number: 5182-0717 )
Pasteur pipets (Fisher, catalog numbers: 13-678-20A and 13-678-20C )
Micro capillary pipets (Kimble, catalog number: 71900-20 )
Pseudomonas syringae expressing avrRpt2 (a gift from Dr. Barbara Kunkel, Washington University in St. Louis)
Arabidopsis thaliana ecotype Col-0 and mutant ald1 plants
Liquid nitrogen (American Welding & Gas, 22 Psi)
Magnesium chloride hexahydrate (Fisher, catalog number: BP214-500 )
Acetonitrile (ACROS, catalog number: 26827-0040 )
Hydrochloric acid (36.5-38% w/w, approximately 12 N) (Fisher, catalog number: A144-212 )
! CAUTION: The reagent is corrosive and volatile; wear protective gloves and handle it in a fume hood.
L-Norvaline (ACROS, catalog number: 6600-40-4 )
L-Pipecolic acid (TCI, catalog number: P1404 )
Sodium hydroxide (Fisher, catalog number: BP359-500 )
Methanol (Fisher, catalog number: A452-4 )
Pyridine (EMD Millipore, PX2020-6 )
Propyl chloroformate (Beantown Chemical, catalog number: 133200-100G )
CAUTION: This reagent is extremely toxic, volatile, flammable and corrosive and should be handled in a fume hood. Store at 4 °C in a closed jar containing some calcium sulfate desiccant (DRIERITE).
Anhydrous calcium sulfate desiccant (W. A. Hammond DRIERITE Company LTD, catalog number: 13005 ), which is used for storage of propyl chloroformate
Chloroform (EMD Millipore, catalog number: CX1058-1 )
CAUTION: The reagent is toxic and volatile and should be handled in a fume hood.
Sodium bicarbonate (Fisher, catalog number: BP328-1 )
Anhydrous sodium sulfate (Fisher, catalog number: S-421 )
10 mM Magnesium chloride (see Recipes)
Extraction buffer (see Recipes)
Internal standard (see Recipes)
1 M Sodium hydroxide (see Recipes)
50 mM Sodium bicarbonate (see Recipes)

Equipment

Plastic pestle (Axygen Scientific, catalog number: PES15BSI )
Milli-Q Advantage A10 Water purification system (EMD Millipore, model: Z00Q0V0WW )
Autoclave (Consolidated Stills & Sterilizers, model: SSR3APB )
Scissors (Harbor Freight Tools, catalog number: 62458 )
Balance (METTLER TOLEDO, model: AT261 )
Benchtop Dewar Flask (Thermo Scientific, catalog number: 10-194-100B )
Vortexer with a Styroform platform (Labnet, model: S0100 )
Table top centrifuge (Eppendorf, model: 5417C ), which is used for centrifuging microfuge tubes
Parker Source TriGas Generator (Parker Hannifin, model: LCMS-5000NA , compressor model: SF120872PH)
Table-top swing-arm centrifuge (Clay Adams, model: Dynac 0101 ), used for centrifuging disposable test tubes
Latex bulbs, 2 ml (VWR, catalog number: 82024-554 )
Gas chromatograph-mass spectrometer (GC-MS) (Agilent, model: 7890A-5977 )
GC column, HP-5MS (Agilent, catalog number: 19091J-413 )
Computer for GC-MS (Dell, model: 3430 )
Syringe for manual GC injection, 5 µl (Hamilton, catalog number: 87993 )

Software

MassHunter Workstation for qualitative and quantitative analysis (Agilent, software version: 10.0)
Enhanced MassHunter Workstation for data acquisition (Agilent, software version: 10.0)
MS spectral library (NIST)

Procedure

Equipment setup
1. Gas chromatography (GC) set up
  Chromatography: perform chromatography with a 30-m capillary HP-5MS column. Use helium as the carrier gas at a constant pressure of 14 psi. Set the injection port temperature to 240 °C and the transfer line temperature to 280 °C. Samples are run in splitless mode. Use the following temperature program for chromatography as shown in Table 1.
  
  Table 1. GC temperature used for profiling norvaline and pipecolic acid
2. Mass spectrometer (MS) set up
  Automatically tune the instrument according to the manufacturer’s instructions.

Sample preparation and analysis
1. For pathogen assays, the largest three leaves of ~4 weeks old plant are infiltrated with either 10 mM MgCl₂ or the pathogen Pseudomonas syringae avrRpt2 using a needleless syringe from the abaxial side of the leaves. After leaf infiltration, remove excess droplets of 10 mM MgCl₂ or pathogen suspension with Kimwipes. Figure 2 shows a pot of Arabidopsis thaliana ecotype Columbia-0 plants just after pathogen infiltration (Figure 2).
  
  Figure 2. A picture of Arabidopsis thaliana ecotype Col-0 plants after infiltration with Pseudomonos syringae expressing avrRpt2. Infiltrated leaves were marked with a white marker.
2. About 24 h later, collect ~100 mg infiltrated leaf tissue. The entire leaf was sampled using a pair of scissors.
3. Weigh the tissue with a balance that is accurate to the 1 mg digit. Record the weight of each sample. ∆CRITICAL STEP
4. Place the tissue in a microfuge tube, cap the tube, and drop it into a bucket of liquid nitrogen. Frozen tissues can be stored at -80 °C indefinitely. •PAUSE POINT
5. Grind the tissue with a plastic pestle.
6. Continue to the next step before the tissue is thawed. CRITICAL STEP
7. Add 400 µl of extraction buffer.
8. Add 20 µl of internal standard.
9. Vigorously shake for 15 min on a vortexer at room temperature.
10. Centrifuge for 5 min at 18,000 x g.
11. Supernatant is transferred to a 13 x 100 mm glass test tube. Extract can be stored at -80 °C indefinitely. •PAUSE POINT
12. Dry the supernatant with a stream of nitrogen gas. Dried extract can be stored at -80 °C indefinitely. •PAUSE POINT
13. Add 200 µl of 1 M sodium hydroxide.
14. Add 167 µl of methanol and 34 µl of pyridine.
15. Vortex vigorously and break up any pellets with a Pasteur pipet.
16. Add 20 µl of propyl chloroformate and vigorously vortex for 30 s. Use a glass micro capillary pipette instead of a plastic pipette tip to avoid contamination of plasticizers. CRITICAL STEP
17. Add another 20 µl of propyl chloroformate and vigorously vortex for 30 s.
18. Add 400 µl of chloroform and vortex vigorously for 10 s.
19. Add 400 µl of µl sodium bicarbonate (50 mM) and vortex vigorously for 10 s.
20. Centrifuge for 2 min with a table-top swing-arm centrifuge at about 80% speed (approximately 1,250 x g).
21. The lower layer is transferred to a new 13 x 100 mm glass test tube with a Pasteur pipet. The lower phase obtained after the transfer is approximately 0.375 ml.
22. Dry the chloroform extract by adding approximately 40 µl of anhydrous sodium sulfate and vortex until clear.
23. Centrifuge for 1 min with a table-top swing-arm centrifuge at about 80% speed (approximately 1,250 x g).
24. Transfer chloroform extract to a GC vial with a Pasteur pipet.
25. Inject 0.5-1 µl to GC/MS for analysis using selective ion monitoring mode (SIM) using quantifier ions 158 and 170 and qualifier ions 72 and 128.

GC-MS data from standard compound analysis
1. Figure 3 shows the total ion chromatogram (Figure 3A) and corresponding MS of norvaline (Figure 3B left panel) and Pip (Figure 3B right panel) standards run in scan mode.
  
  Figure 3. GC-MS analysis of norvaline (NV) and pipecolic acid (Pip) using scan mode. (A) GC-MS total ion chromatogram of equal weight (2 µg each) mixture of L-norvaline (NV) and L-pipecolic acid (Pip) derivatized with propyl chloroformate as described in B using scan mode. L-norvaline, N-propoxycarbonyl, propyl ester (at 8.2 min) and L-pipecolic acid, N-propoxycarbonyl, propyl ester (at 9.9 min) are the two major peaks. The bottom panel (B) shows mass spectra of NV- and Pip-N-propoxycarbonyl propyl esters. The loss of propoxycarbonyl group produces major ions 158 from NV and 170 from Pip derivatives.
2. Figure 4 shows the GC-MS data from standard compound analysis using selective ion monitoring (SIM) mode. Based on the mass spectra shown in Figure 1, two major ions for each compound were used to monitor NV (158 and 72) and Pip (170, 128) in SIM mode.
  
  Figure 4. GC-MS analysis of norvaline (NV) and pipecolic acid (Pip) using selective ion monitoring (SIM) mode. (A) GC-MS total ion chromatogram of equal weight (2 µg each) mixture of NV and Pip derivatized with propyl chloroformate as described in B using SIM mode. NV- (at 8.2 min) and Pip-N-propoxycarbonyl propyl ester (at 9.9 min) are the two major peaks. The bottom panel (B) show mass spectra of NV- and Pip-N-propoxycarbonyl propyl esters in SIM mode selected for the major ions 158.2 and 72.2 (NV) and 172.2 and 128.0 (Pip). The dwell time for each ion was set at 25 ms. The SIM mode was set to monitor ions 158 and 72 in the time interval 7.4-9.1 min and ions 170 and 128 from 9.1-10.5 min. The relative abundance of major and qualifier ions monitored in SIM and scan modes was similar.

Pip levels in Arabidopsis leaf tissue
1. GC-MS data for pipecolic acid analysis from Arabidopsis thaliana leaf tissue samples using scan (Figure 5) and SIM (Figure 6) modes.
  
  Figure 5. GC-MS analysis of extracts prepared from Col-0 and ald1 plants analyzed using scan mode. (A) Total ion chromatograms of mock (10 mM MgCl₂)- and pathogen (Pseudomonas syringae expressing avrRpt2) inoculated wild-type Col-0 and mutant ald1 plants. The leaf tissues were sampled at 24 h post treatment and NV (2 µg) was used as an internal standard as described in Procedure B. The ald1 plants did not accumulate Pip after pathogen infection. The bottom panels (B) show MS spectra of NV- and Pip-N-propoxycarbonyl propyl esters.
  
  Figure 6. GC-MS analysis of extracts prepared from Col-0 and ald1 plants analyzed using SIM mode. (A) Chromatograms of mock (10 mM MgCl₂)- and pathogen (Pseudomonas syringae expressing avrRpt2) inoculated wild-type Col-0 and mutant ald1 plants. The leaf tissues were sampled at 24 h post treatment and NV (2 µg) was used as an internal standard as described in Procedure B. The ald1 plants did not accumulate Pip after pathogen infection. The bottom panels (B) show MS spectra of NV- and Pip-N-propoxycarbonyl propyl esters in SIM mode selected for the major ions 158.2 and 72.2 (NV) and 172.2 and 128.0 (Pip).
2. Relative Pip levels in Col-0 and ald1 plants.
  Figure 7 shows the relative Pip levels in Col-0 and ald1 plants inoculated with mock or pathogen. The pathogen inoculated wild-type plants accumulated ~18-fold higher Pip levels compared with the mock inoculated plants. In contrast to wild-type plants, the ald1 accumulate reduced basal levels of Pip and did not show any increase in Pip levels after pathogen infection.
  
  Figure 7. Pip levels in Arabidopsis wild-type Col-0 and Pip deficient ald1 plants post 24 h of inoculation with mock (10 mM MgCl₂) or pathogen (P. syringae expressing avrRpt2). Plants were inoculated with 10⁵ CFU ml^-1in 10 mM MgCl₂. Error bars indicate standard deviations (n = 3) and astetisks indicate statistical significane (P > 0.001).

Data analysis

MassHunter Workstation 10.0 was used for qualitative and quantitative data analysis. The ion ratios between quantitative and qualifier ions were used as an additional measure to access proper identification of the analytes using SIM mode. Pipecolic acid levels were calculated based on the standard curves generated using varying concentration of NV and Pip standards.
Statistical analysis was performed using Student t-test. For pathogen assays, ~16 plants/ genotype/treatment were analyzed in a single experiment. At least 4 technical replicates/genotype/treatment were plated. For metabolite quantification, ~12 plants/genotype/treatment were analyzed in each experiment. Experiments were repeated at least two-three times with a different set of plants. Unless otherwise mentioned error bars indicate SD.

Notes

The protocol has shown excellent reproducibility and been used to analyze numerous extractions reported in our recent studies (Wang et al., 2018; Lim et al., 2020).

Recipes

10 mM Magnesium chloride
1. Weigh 20.33 g of magnesium chloride hexahydrate and dissolve it in Milli-Q water to a final volume of 100 ml to obtain 1 M MgCl₂
2. Autoclave 1 M MgCl₂along with 1 liter of Milli-Q water at 121 °C for 30 min using a liquid cycle
3. After the solutions have cooled down to room temperature, take 1 ml of the 1 M MgCl₂ and add it to 99 ml of the autoclaved Milli-Q water, mix well to obtain 10 mM MgCl₂
Extraction buffer
1. Add 8.33 ml of concentrated HCl to 91.67 ml of Milli-Q water and mix well to obtain 1 N HCl
2. Take 1 ml of the 1 N HCl, add it to 99 ml of Milli-Q water, mix well to obtain 0.01 N HCl
3. Mix 25 ml of acetonitrile with 75 ml of the 0.01 N HCl and use this solution as extraction buffer
Internal standard
Prepare 100 ng/µl norvaline (wt/vol) in Milli-Q water
1 N Sodium hydroxide
Weigh 40 g of NaOH pellets and dissolve it in Milli-Q water to a final volume of 100 ml to obtain 10 N NaOH, then dilute 10-fold with Milli-Q water
50 mM Sodium bicarbonate
Weigh 420 mg NaHCO₃ and dissolve it in Milli-Q water to a final volume of 100 ml

Acknowledgments

This work was supported by grants from National Science Foundation (MCB#0421914 and IOS#051909), Kentucky Science and Engineering Foundation (#1244), and Kentucky Soybean Board (3084113467). We thank John Johnson for technical help. This protocol was adapted from previous work (Villas-boas et al., 2003; Kugler et al., 2006).

Competing interests

The authors declare no conflict of interest.

References

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