145

Alain Goossens and Laurens Pauwels (eds.), Jasmonate Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1011, DOI 10.1007/978-1-62703-414-2_12, © Springer Science+Business Media, LLC 2013

Chapter 12

Jasmonic Acid–Amino Acid Conjugation Enzyme Assays

Martha L. Rowe and Paul E. Staswick

Abstract

Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione- S -transferase:JAR1 fusion protein produced in Escherichia coli , including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas chromatography/mass spectrometry.

Key words Adenylation , Conjugation , Jasmonic acid , Hormone , GH3 protein , Enzyme

Jasmonic acid (JA) is an important signaling compound involved in both plant development and defense responses. The active form of JA in signaling reactions is an amino acid conjugate, primarily JA-isoleucine (JA-Ile). The major enzyme responsible for the JA-Ile formation in Arabidopsis thaliana is JASMONATE RESISTANT1 (JAR1), also designated GH3.11 [ 1, 2 ] . JAR1 is a member of the fi re fl y luciferase superfamily that forms an adeny-lated JA intermediate prior to condensation to the amino acid (Fig. 1 ).

The GH3 family is widely distributed in plants and other enzymes of the GH3 family conjugate amino acids to other plant hormones, especially indole-3-acetic acid [ 3 ] , or small carboxylic acids [ 4 ] . The methods presented here for the JAR1 assay would be applicable to the related enzymes with minor modi fi cations. In addition to a rapid qualitative assessment of conjugate synthesis by thin-layer chromatography (TLC) [ 2 ] , we have used two procedures to quan-tify the conjugation activity: one measures the production of the

1 Introduction

146 Martha L. Rowe and Paul E. Staswick

conjugation product by gas chromatography/mass spectrometry (GC/MS) [ 5 ] and the other the production of adenosine mono-phosphate (AMP) by high-performance liquid chromatography (HPLC) [ 6 ] . We also included an adenosine triphosphate (ATP)-pyrophosphate (PPi) isotope exchange protocol that detects the enzyme-adenylating activity by carboxylic acid-dependent exchange of 32 P-PPi into ATP (substrate + Mg-ATP ↔ substrate-AMP + PPi) [ 1 ] . This assay can be used to test the adenylating activity of an enzyme on carboxylic acids without requiring the knowledge of which amino acid the enzyme prefers [ 1 ] . An enzyme-coupled assay for adenylation has been described elsewhere [ 4 ] .

The glutathione- S -transferase (GST): JAR1 construct made from JAR1 cDNA makes production and puri fi cation of proteins expressed in Escherichia coli simple and fast. Alternate protein expression systems could be used as well. The fusion protein is isolated by binding with glutathione-agarose beads. The target enzyme activity can be assessed qualitatively with the fusion pro-tein still bound to the glutathione-agarose beads or eluted proteins of known concentrations can be used for kinetic studies.

It should be noted that the JAR1 enzyme of both Arabidopsis and tomato ( Solanum lycopersicum ) have a strong preference for (3R,7S)-JA, also called (+)-7-iso-jasmonic acid, which is the isomer synthesized in plants [ 5 ] . However, this cis -isomer is unstable and constitutes only a small fraction of commercially available prepara-tions of (±)-JA. Therefore, the kinetic activity will probably be underestimated by using the isomeric mixture as a substrate. Although (3R,7S)-JA is not generally commercially available, it can be prepared through a laborious process [ 5 ] .

In our laboratory, a pGEX-4T expression vector in E. coli BL21 cells is used according to the general procedures outlined by the supplier (GE Healthcare, Little Chalfont, UK). Prepare solutions in ultrapure water and use HPLC-grade reagents for HPLC and GC/MS. Dispose of all hazardous materials appropriately.

2 Materials

E + ATP + JA [ E-JA-AMP E-JA-Ile ] E + JA-Ile

Ile

PPi AMP

Fig. 1 Mechanism of JAR1 enzyme activity. The enzyme performs two distinct steps, adenylation of the carboxyl group of JA followed by condensation of the amino acid to the activated carboxyl of JA. JA jasmonic acid, E enzyme, Ile isoleucine

147JA Conjugation Assays

1. 1× phosphate-buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ): 8.2 g NaCl, 0.2 g KCl, 2.68 g Na 2 HPO 4 ⋅7H 2 O, and 0.24 g KH 2 PO 4 added to 1 L of water. The pH should approximately be 7.3 without adjustment.

2. Glutathione agarose (Sigma-Aldrich, St. Louis, MO, USA): For preparation, see Subheading 3.1 .

1. LB medium: 25 g/L LB Miller (EMD Millipore, Billerica, MA, USA). In 1-L fl asks, place 250 mL LB medium and autoclave.

2. Ampicillin: 50 mg/mL stock, fi lter sterilized before use and aliquoted into sterile microfuge tubes for storage at −20 °C. Add 250 μ L of 50 mg/mL ampicillin to 250 mL LB medium for a fi nal concentration of 50 μ g/mL.

3. 1 M isopropyl β - D -1-thiogalactopyranoside (IPTG): Add 2.38 g of IPTG to 10 mL of water and vortex until dissolved. Filter sterilize and store at −20 °C.

4. Sonicator: 130-W high-intensity ultrasonic processor with 6-mm diameter probe (Autotune Model GE130; Sonics and Materials, Newtown, CT, USA) ( see Subheading 3.3 for use).

1. 20 % (v/v) Triton X-100: 4 mL of Triton X-100 dissolved in 16 mL water ( see Note 1 ).

2. 10 mM reduced glutathione in 50 mM Tris–HCl (pH 8): Prepare 50 mM Tris–HCl (pH 8) from 1 M stock solution or dissolve 0.12 g Tris base in 15 mL water, adjust pH to 8.0, and bring to fi nal volume of 20 mL. Add 0.06 g reduced glutathi-one (Sigma-Aldrich) to 20 mL of 50 mM Tris–HCl (pH 8), dissolve, and fi lter sterilize before storing at 4 °C.

1. 1 M Tris–HCl (pH 8.0): Dissolve 6.06 g Tris base in 40 mL water, adjust pH to 8.0, and bring to fi nal volume of 50 mL.

2. 25 mM ATP: Dissolve 0.552 g ATP in 10 mL water for a 100-mM stock solution and dilute with water to obtain 25 mM ATP. Divide into small aliquots in microfuge tubes and store at −20 °C ( see Note 2 ).

3. 25 mM MgCl 2 : Dissolve 0.092 g MgCl 2 in 10 mL water for a 100-mM stock solution and dilute with water to obtain 25 mM.

4. 5 mM JA: Dissolve 21 mg (±)-JA (Sigma-Aldrich) in 1 mL of 100 % ethanol to make a 100-mM stock solution and dilute this stock solution with ethanol.

5. 100 mM dithiothreitol (DTT): Dissolve 0.55 g DTT in 10 mL water.

6. 10 mM pyrophosphate (PP i ): Dissolve 0.045 g NaP 2 O 7 . 10H 2 O

(Sigma-Aldrich) in 10 mL water.

2.1 Glutathione-Agarose

2.2 E. coli Growth and Sonication

2.3 Protein Puri fi cation and Elution

2.4 Isotope Exchange Assays

148 Martha L. Rowe and Paul E. Staswick

7. 32 P-PP i : Dilute 32 P-tetrasodium pyrophosphate (1 mCi at 10 μ Ci/ μ L) to 10× with water for a fi nal activity of 1 μ Ci/ μ L.

8. PEI-F cellulose TLC Sheets: 20 × 20 cm sheets can be pur-chased (J.T. Baker Baker- fl ex Pre-Coated Flexible TLC Sheets, PEI-F Cellulose; Avantor Performance Materials, Center Valley, PA, USA) and cut into strips (10 cm long) for TLC. Spots can also be cut out of developed chromatograms for quantitative scintillation counting.

9. X-ray fi lm: Kodak Bio-Max MS fi lm or equivalent. 10. Chromatogram developer (1.9 M formic acid and 1 M LiCl):

Dissolve 4.24 g LiCl in 80 mL water, add 8 mL of 88 % formic acid, and bring to a fi nal volume of 100 mL.

1. 50 mM Tris–HCl (pH 8.5): Dissolve 0.30 g Tris in 40 mL water. Adjust pH to 8.5 and bring to a fi nal volume of 50 mL.

2. 25 mM ATP: See Subheading 2.4 . 3. 25 mM MgCl 2 : See Subheading 2.4 . 4. 25 mM JA: See Subheading 2.4 . 5. 2 mM amino acids for most amino acid solutions: Dissolve

0.05 mmol amino acid in 1 mL water to obtain a 50-mM stock solution. For a few amino acids that are not readily soluble in water (aspartic acid, glutamic acid, and tyrosine), dissolve them in a minimum amount of 1 N NaOH, and dilute with water to a fi nal volume of 1 mL for the stock solution. Cysteine should optimally always be made fresh because it converts to cystine in solution. Make 2 mM solutions by adding 40 μ L of the 50 mM stock to 960 μ L water.

6. 100 μ M AMP: Dissolve 0.0035 g AMP in 1 mL of water to make a 10-mM stock and dilute with water to obtain 100 μ M.

7. Stop buffer: Add 0.34 g KH 2 PO 4 to 50 mL water to make a 50-mM solution.

1. Silica gel plates: TLC Silica Gel 60F 254 glass plates, 20 × 20 cm (EMD Millipore).

2. Developing solution: Ethyl acetate (55 %), chloroform (35 %), formic acid (10 %) [v/v].

3. Vanillin reagent: Dissolve 3 g vanillin in 50 mL ethanol and add 500 μ L H 2 SO 4 .

4. We use a tube-type chromatography sprayer (Sigma-Aldrich) to wet the TLC plate.

All solutions should be fi ltered prior to use (Whatman nylon membrane fi lters, 0.45 μ m, 47 mm diameter) (GE-Healthcare).

2.5 JA–Amino Acid-Conjugating Enzyme Assays

2.6 Thin-Layer Chromatography

2.7 HPLC Detection of AMP

149JA Conjugation Assays

1. Ultra IBD reverse-phase column, length, 150 mm; diameter 4.6 mm dia; particle size 5.0 μ m (Restek, Bellefonte, PA, USA).

2. 100- μ L limited-volume polypropylene HPLC vials (Restek) or regular vials with microvolume insert.

3. Solution A: 50 mM potassium phosphate (pH 5.5) with 0.01 % sodium azide to impede microbial contamination.

4. Solution B: 50 % methanol.

1. 0.5 M Tris–HCl (pH 8.8): Dissolve 3.02 g Tris base in 40 mL water. Adjust pH to 8.8 and bring fi nal volume to 50 mL.

2. 10 mM JA ( see Subheading 2.4 ). 3. 100 mM ATP, 100 mM MgCl 2 , and 100 mM DTT ( see

Subheading 2.4 ). 4. 5 mM amino acid ( see Subheading 2.4 ). 5. Internal standards: Will vary with the speci fi c amino acid to be

assayed. We use a stable isotope of Ile to synthesize [ 13 C 6 ]-JA-Ile, or amino acid conjugates can be synthesized with dihydro-jasmonic acid with a mixed anhydride reaction [ 2 ] .

6. 2 M (trimethylsilyl)diazomethane (Sigma-Aldrich). 7. Isooctane (2,2,4-trimethylpentane; EMD Millipore).

1. Add a small amount (approximately 0.5 mL) of dry glutathi-one-agarose to a clean 15-mL polypropylene conical centrifuge tube and add approximately 5 mL cold 1× PBS buffer.

2. Thoroughly suspend the glutathione-agarose beads in the buffer and allow to hydrate overnight at 4 °C to obtain a fi nal volume of 1–2 mL. Centrifuge (250 × g ) the hydrated beads brie fl y in a benchtop centrifuge and discard the PBS supernatant.

3. Add fresh PBS (5–10 mL), resuspend beads, and centrifuge again.

4. Repeat washing of the glutathione-agarose beads three to four times.

5. After fi nal wash, leave enough PBS buffer so that the bead bed volume is approximately equal to the free buffer volume for a 50 % suspension that can be kept refrigerated for at least 2 weeks.

1. Inoculate 5 mL of LB broth supplemented with 50 μ g/mL ampicillin to the pGEX expression vector-containing E. coli .

2. Grow overnight at 37 °C with shaking.

2.8 Assay and Sample Preparation for GC/MS

3 Methods

3.1 Preparation of Glutathione-Agarose

3.2 Growth and Harvest of Cells

150 Martha L. Rowe and Paul E. Staswick

3. In the morning, add the 5 mL of overnight culture to 250 mL of the 25- μ g/mL ampicillin-containing LB medium in a 1-L fl ask.

4. Grow at 37 °C with shaking for approximately 2 h, until OD 600 = 0.6–0.8.

5. Induce production of the fusion protein by adding IPTG to a fi nal concentration of 0.1 mM and by shaking at room tem-perature for 5–6 h.

6. Pour culture into a 250-mL polypropylene centrifuge bottle and centrifuge for 10 min at 5,000 × g .

7. Discard the supernatant back into the culture fl ask for autoclaving.

8. Drain the bottle upside down brie fl y on paper towels. 9. Freeze bottles with bacterial pellets for later use or transfer

them to an ice bucket for immediate use.

1. Add 10 mL of cold PBS to each pellet generated in Subheading 3.2 .

2. Keep cells on ice and resuspend pellets gently by agitating the ice bucket on a rotary shaker at low speed or by using a pipettor to break up the pellet.

3. Transfer the cell suspensions to cold clean 50-mL open-mouth polypropylene centrifuge tubes and keep in the ice bucket.

4. Prepare a 250-mL beaker fi lled with ice and a little water. 5. To disrupt the bacterial cells and release the produced proteins

without damage, sonicate with settings as follows: amplitude, 40 %; pulse, 3 s; and time, 3 min ( see Note 3 ).

6. Put centrifuge tube with suspended cells in beaker of ice/water. 7. Rinse probe with water. 8. Lower probe into the tube so that it does not quite touch the

bottom ( see Note 3 ) . 9. Push START button on the machine with pulse ON for 3 s,

and OFF for 1 s continually for 3 min ( see Note 4 ). 10. Repeat procedure with the remaining samples. 11. Rinse probe with 70 % ethanol. 12. Turn dials OFF, and then power OFF. 13. Add 500 μ L of 20 % Triton X-100 to each tube (1 % fi nal

concentration).

1. Spin sonicated cells in a refrigerated high-speed centrifuge at 12,000 × g for 10 min at 4 °C.

2. With a 200- μ L pipettor with trimmed tip, transfer 100–200 μ L of washed glutathione-agarose (50 % suspension) into a 15-mL conical polypropylene centrifuge tube with cap.

3.3 Cell Sonication

3.4 Binding Fusion Protein to Glutathione-Agarose

151JA Conjugation Assays

3. Pour the supernatant carefully into the conical tube with the glutathione-agarose suspension.

4. Close tube tightly and place on rotator for 30 min ( see Note 5 ). 5. Spin tube brie fl y in a benchtop centrifuge at approximately

250 × g , just long enough to pull down the glutathione-agarose beads.

6. Pour off supernatant into the used empty centrifuge bottle. 7. Add 1 mL of cold 1× PBS to the tube and transfer the entire

volume into a 1.5-mL microfuge tube for further washings. 8. Centrifuge the microfuge tube brie fl y and discard the

supernatant. 9. Add 1 mL of cold 1× PBS to the tube, resuspend the beads,

and centrifuge brie fl y (250 × g ). 10. Discard the supernatant and repeat washings with 1× PBS

three times. Beads can be stored at 4 °C for at least 2 weeks, as long as microbial growth does not occur.

11. Check the binding of the protein to the beads on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels ( see Notes 6 and 7 ).

1. After a fi nal 1× PBS wash of the fusion protein-bound glutathi-one-agarose beads, remove as much buffer as possible from the beads ( see Note 8 ).

2. Add a volume of reduced glutathione (10 mM in 50 mM Tris, pH 8 buffer) approximately equal to the bed volume of the beads.

3. Put on rotator for 10 min. 4. Open lid of microfuge tube and hold it over another clean

microfuge tube ( see Note 9 ). 5. Very carefully puncture a very small hole at the end of the

bead-containing tube with the sharp end of a jeweler’s forceps or syringe needle.

6. Set this tube into the clean tube and tape them together. 7. In the benchtop centrifuge with whatever adaptor that will

hold them, spin at approximately 500 × g for approximately 30 s, until the agarose beads in the top tube are white and look dry ( see Note 10 ) .

8. Use the eluted fusion protein for enzyme assays. Puri fi ed enzymes can be stored at 4 °C for a few days or inde fi nitely in a freezer, although solubility of individual proteins after freezing should be tested fi rst.

1. Prepare the reaction buffer minus the 32 P-PP i as follows: 75.4 μ L of water, 10.0 μ L of 1 M Tris–HCl (pH 8.0), 5.6 μ L of 25 mM MgCl 2 , 5.6 μ L of 25 mM ATP, 2.0 μ L of 100 mM DTT, and 4 μ L of 10 mM PP i .

3.5 Elution of Bound Proteins with Reduced Glutathione

3.6 Isotope Exchange Assay

152 Martha L. Rowe and Paul E. Staswick

2. Add 5 μ L of 5 mM JA to the reaction tube. 3. In the isotope work area, add 1–2 μ L of 32 P-PP i (1 μ Ci/ μ L)

for every 100 μ L of reaction buffer. 4. Add 10 μ L of reaction buffer with the isotope to each reaction

tube. 5. Add 5 μ L of glutathione-agarose-fusion protein or eluted

fusion protein to each reaction tube. 6. Let the reaction proceed for 10 min. If the bead-bound enzyme

is used, gently fl ick tubes occasionally during the reaction to resuspend the agarose beads that have settled to the bottom.

7. Mark PEI-F Cellulose strips with a pencil by placing dots where the reaction mixes will be spotted, approximately 1.5 cm from the bottom edge; place another dot on the right side of the strip for orienting the thin-layer strip on the X-ray fi lm.

8. At the end of the reaction time, spot 1.5 μ L of the reaction mixtures on the dots at the bottom of PEI-F Cellulose strip.

9. Use a small amount of the last reaction mixture to spot the orienting dot on the right side of the strip.

10. Let the thin-layer strip dry. 11. Develop the strip in 1.9 M formic acid and 1 M LiCl. 12. Let solvent rise to within approximately 2 cm of the strip top

in approximately 20 min. 13. Remove the strip and let dry on paper towel. 14. Wrap the thin-layer strip in a plastic wrap and expose to X-ray

fi lm for approximately 3 h. 15. Develop fi lm (Fig. 2 ) which is suf fi cient for qualitative activity

assessment.

PPi

ATP

none JA MeJA

Fig. 2 Autoradiogram of a thin-layer chromatograph from the isotope exchange assay. Origin is at the bottom and the ATP and PPi positions are indicated by arrows . The reactions contained JA, the methyl ester of JA (MeJA), or no car-boxylic acid as substrate, as indicated above. MeJA is unreactive because it lacks a free carboxyl group

153JA Conjugation Assays

16. For quantitative activity measurements, use the developed X-ray fi lm as a guide to mark the location of the radioactive spots that can be cut out and counted in a scintillation counter.

This procedure is based on the use of eluted fusion proteins for the enzyme assay. Products can be detected by TLC ( see Subheading 3.8 ) or, because AMP is a product of the con-jugation reaction, the released AMP can be quanti fi ed by HPLC ( see Subheading 3.9 ). For TLC product detection, reactions can also be done with enzymes bound to the agarose beads ( see Note 11 ). For HPLC, AMP control reactions should be run with each set of conjugation assays to develop a standard curve that compensates for possible AMP metabolism during the reactions.

To assess the conjugating enzyme activity with different amino acids, the following reaction is set up, but the procedure may be modi fi ed to measure the activity at different amino acid or hor-mone concentrations.

1. Immediately before use, prepare a stock solution for four reactions by mixing together 30 μ L of water, 25 μ L of 100 mM Tris–HCl (pH 8.5), 4 μ L of 5 mM MgCl 2 , 4 μ L of 5 mM ATP, 2 μ L of 25 mM JA, and 10 μ L of enzyme preparation. For each reaction, 18 μ L of the stock solution plus 6 μ L of 2 mM amino acid will be needed.

2. For control reactions, substitute water for the amino acid; for the AMP measurement by HPLC, substitute the amino acid with dilutions of the 100 μ M AMP preparation to construct a standard curve.

3. Place 6 μ L of the variable substrate (amino acid or AMP standard) in a microfuge tube.

4. Add 18 μ L of the reaction mix at time 0 ( see Note 12 ). 5. At the end of the desired reaction time, add 96 μ L of stop buffer

(50 mM KH 2 PO 4 ) and vortex both to dilute the reaction and lower the pH to a point where the reaction will essentially stop ( see Note 13 ).

6. For analysis by TLC, spot the reaction directly to the plate without addition of stop buffer to maintain the product concentration.

1. Use a pencil to mark spot locations on a silica gel plate approxi-mately 1.5 cm from the bottom edge.

2. Apply 2 μ L of the reaction mixture to a spot and let dry. 3. Reapply 2 μ L twice more for a total of 6 μ L ( see Notes 14

and 15 ). 4. When the plate is dry, develop in a mixture of ethyl

acetate:chloroform:formic acid (55:35:10 %).

3.7 Enzyme Conjugation Reactions

3.8 Thin-Layer Chromatography

154 Martha L. Rowe and Paul E. Staswick

5. Remove the plate from the developing tank and allow to dry approximately for 5 min.

6. Spray with vanillin reagent, blot lightly, and bake at 100 °C for 10–15 min ( see Note 16 and Fig. 3 ).

1. Place the reaction samples (120 μ L) in microvolume HPLC vials, making sure that no air bubbles are caught at the vial bottom.

2. Set injection volume of HPLC at 50 μ L, detector wavelength at 260 nm, and pump at 1 mL per minute. Use the following HPLC cycle to separate AMP from ATP and other possible products: 90 % solution A and 10 % solution B isocratic for 5 min, 100 % solution B for 5 min, and 90 % solution A and 10 % solution B for 10 min ( see Note 17 ).

3. Wash the column and equilibrate for the next run. 4. Use the area under the AMP peak with a standard AMP curve

to calculate the enzyme activity or the fraction (AMP/AMP + ATP) ( see Note 18 ).

1. For each reaction, prepare 130 μ L of 2× reaction mix: 80 μ L of water, 20 μ L of 0.5 M Tris–HCl (pH 8.8), 20 μ L of 10 mM (−)JA, 4 μ L of 100 mM MgCl 2 , 4 μ L of 100 mM ATP, and 2 μ L of 100 mM DTT.

2. Prepare the stop solution: 540 μ L of water, 90 μ L of glacial acetic acid, and 45 μ L of the appropriate internal standard at the desired concentration.

3.9 Reaction Quanti fi cation of AMP Production by HPLC Detection

3.10 Reaction Quanti fi cation of Conjugates by GC/MS Detection

Origin

JA

Con

juga

tes

I K L M N P Q R S T V W Y

Fig. 3 Thin-layer chromatogram of JAR1 enzyme reactions with JA and indicated amino acids. The origin and migration point for JA are indicated with arrows , where JA conjugates migrate to positions below JA. Individual amino acids included in each enzyme reaction are indicated with standard single-letter abbreviations

155JA Conjugation Assays

3. Use a known quantity of internal standard to account for any sample loss during the subsequent processing.

4. Add 50 μ L of 5 mM amino acid to 130 μ L of 2× reaction mix. 5. Start reaction by adding 20 μ L of the enzyme; mix well, and

note the time. 6. At each desired time point, withdraw 45 μ L of the assay mixture

into the tube containing 150 μ L of stop solution and vortex. 7. Add 200 μ L of chloroform to each sample, vortex, centrifuge

(250 × g ) brie fl y, and pipette chloroform (lower phase) into a 6-mL disposable glass tube (100 × 13 mm).

8. Repeat the chloroform extraction of the assay mixture and combine with the fi rst.

9. Dry sample under N 2 in fume hood ( see Note 19 ). 10. Add 100 μ L of 100 % methanol. 11. Vortex well and roll tube slightly to make sure that the sample

is fully dissolved from the tube walls. 12. In a fume hood, add 5 μ L of 2 M (trimethylsilyl)diazomethane

(derivatizing agent); cover the sample with para fi lm and leave for 30 min.

13. Add 1 μ L of glacial acetic acid to stop the reaction and inactivate the remaining derivatizing agent.

14. Dry the sample again under N 2 and resuspend it in 100 μ L isooctane.

15. Vortex and roll tubes slightly. 16. Transfer the sample to a glass vial with a vial insert appropriate

for the GC instrument used. 17. For the quanti fi cation by GC/MS, follow the procedure

depending on the available instruments. The Finnigan Trace GC with a DB-5ht column (15 m, 0.25 mm, 0.1 L) coupled to a Finnigan DSQ mass spectrometer was operated in electron ionization mode; the injector port was at 280 °C and the col-umn gradient was 60–300 °C in 25 min. The amount of JA-Ile was obtained from the molecular ion-integrated peak areas with the isotope dilution method described previously [ 7 ] .

1. First, a gel-like product will be formed, but heating and gentle agitation will eventually bring it into solution.

2. ATP can break down after multiple freeze–thaw cycles. It is best to prepare a large amount and to subdivide it into small portions to be frozen and used as needed.

4 Notes

156 Martha L. Rowe and Paul E. Staswick

3. Use ear protection when operating the sonicator . 4. At the end of the sonication, the suspension in tubes should be

slightly paler because the cells have burst open. 5. Fusion proteins with GST moiety will bind to the glutathione

bound to the agarose beads. 6. To check the protein availability and approximate amounts,

use a trimmed pipette tip to transfer 10 μ L of the fusion pro-tein-bound beads to a microfuge tube. Add an equal amount of 2× SDS-PAGE loading buffer and boil for 5 min. Place on ice until ready to load on an SDS-PAGE gel. The fusion pro-tein will now be free of the beads and only the liquid need to be loaded.

7. A rough measure of the enzyme activity can also be determined while the protein is still bound to the glutathione-agarose beads ( see Subheading 2.7 ), but quanti fi cation of the protein is dif fi cult. For speci fi c activities, the protein must fi rst be eluted from the beads.

8. The volume of the beads should be approximately half the volume of the suspension originally used.

9. Be sure to open the microfuge tube before puncturing. Positive pressure from warming may have built up in the tube and the enzyme-containing liquid will leak out as the hole is punctured.

10. If the beads have come into the catch tube, the hole was too large.

11. A more rapid qualitative endpoint assessment of the enzyme activity or substrate preferences can be done with the GST fusion protein still attached to the glutathione-agarose beads. After Subheading 3.4 , step 5 , and without the fi nal centrifuga-tion, allow beads to settle for a few minutes, and remove most of the PBS so that only beads loosely remain in the solution. Add 5 μ L of the suspended beads to the 20- μ L reactions in a 1.5-mL conical centrifuge tube. The end of the micropipette tips may need to be cut to produce a hole large enough for beads to enter the tips. Place the tube on a slowly rotating tube mixer so that the long axis of the microfuge tube is parallel with the rotation axis. For this purpose, pieces of foam with holes for tubes have been attached to the rotator. The small sample volume will stay at the “bottom” of the tube, while the beads will tumble into the solution, ensuring that the enzyme is mixed with the solution over an extended time, as desired. For endpoint assays, we often incubate the reaction for several hours or overnight.

12. Addition of the reaction mix to the subsequent tubes is spaced at fi xed intervals of approximately 15–30 s.

157JA Conjugation Assays

13. The reaction time should be determined empirically when doing kinetics so that product formation is proportional to enzyme concentration.

14. If too much assay mixture is applied to the TLC plate at once, the spot becomes too large and diffuse; hence 2 μ L is applied at a time after drying between applications.

15. JA and the desired conjugates can be used as standards on the plate.

16. JA will move near the solvent front and will appear as a dark brown or a purple spot. Conjugates will have lower retention values, depending on the amino acid (Fig. 3 ).

17. Exact parameters may need to be determined for other instru-ments and columns.

18. A typical chromatogram will show ATP coming off the column early, followed by AMP.

19. Placing the tubes into a 50 °C water bath or dry bath will speed the solvent evaporation.

Acknowledgments

This research has been supported in part by funds from the Hatch Act and additionally by the National Science Foundation (Award IOS-0744758).

References

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2. Staswick PE, Tiryaki I (2004) The oxylipin sig-nal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. Plant Cell 16:2117–2127

3. Staswick PE, Serban B, Rowe M, Tiryaki I, Maldonado MT, Maldonado MC, Suza W (2005) Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell 17:616–627

4. Okrent RA, Brooks MD, Wildermuth MC (2009) Arabidopsis GH3.12 (PBS3) conjugates

amino acids to 4-substituted benzoates and is inhibited by salicylate. J Biol Chem 284:9742–9754

5. Suza WP, Rowe ML, Hamberg M, Staswick PE (2010) A tomato enzyme synthesizes (+)-7- iso -jasmonoyl- L -isoleucine in wounded leaves. Planta 231:717–728

6. Guranowski A, Miersch O, Staswick PE, Suza W, Wasternack C (2007) Substrate speci fi city and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1). FEBS Lett 581:815–820

7. Cohen JD, Baldi BG, Slovin JP (1986) 13 C 6 -[benzene ring]-indole-3-acetic acid. A new inter-nal standard for quantitative mass spectral analysis of indole-3-acetic acid in plants. Plant Physiol 80:14–19