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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_9, © Springer Science+Business Media, LLC 2013

Chapter 9

Pro fi ling of Jasmonic Acid-Related Metabolites and Hormones in Wounded Leaves

Yusuke Jikumaru , Mitsunori Seo , Hideyuki Matsuura , and Yuji Kamiya

Abstract

The endogenous concentration of N -jasmonoyl- L -isoleucine (JA-Ile) is regulated by the balance between biosynthesis and deactivation and controls plant developmental processes and stress responses. Therefore, pro fi ling of its precursors and metabolites is required to understand the mechanism by which the JA-Ile concentration is regulated. Also, other hormones, such as indole-3-acetic acid, abscisic acid, salicylic acid, and ethylene, have been suggested to interact with JA-Ile signaling. Pro fi ling of these hormones and their metabolites should give us insights into their interaction mode. Liquid chromatography-electrospray ionization-tandem mass spectrometry has enabled us to develop a highly sensitive and high-throughput comprehensive quanti fi cation analysis of phytohormones.

Key words Plant hormone , Quanti fi cation , Liquid chromatography-electrospray ionization-tandem mass spectrometry , Hormone interaction , Stable isotope-labeled internal standard

N -jasmonoyl- L -isoleucine (JA-Ile) is involved in the responses of plants to environmental stimuli, such as wounding and pathogen infections, through the regulation of gene expression. Early steps of the JA-Ile biosynthesis occur in the plastid membrane where linolenic acid is converted to 12-oxo-phytodienoic acid (OPDA) by the consecutive actions of lipoxygenase, allene oxide synthase, and allene oxide cyclase. OPDA is then transported to the peroxi-some and converted into jasmonic acid (JA) by successive reactions catalyzed by OPDA reductase and β -oxidation enzymes. In turn, JA is converted into JA-Ile by the enzyme jasmonate-resistant1 to function as a signaling molecule. Among the jasmonate-related compounds tested (JA-amino acid conjugates, JA, OPDA, and methyl-JA), only JA-Ile has been shown to promote the interaction between CORONATINE INSENSITIVE1 and the JA ZIM-domain (JAZ) repressor proteins to regulate the downstream gene

1 Introduction

114 Yusuke Jikumaru et al.

expression [ 1– 3 ] . Recently, a cytochrome P450 monooxygenase CYP94B3 that converts JA-Ile into 12-hydroxy-JA-Ile (12-OH-JA-Ile) had been identi fi ed [ 4, 5 ] . The loss-of-function mutant cyp94b3 had enhanced sensitivity to exogenous JA-Ile, whereas overexpres-sion of CYP94B3 resulted in insensitivity to applied JA, indicating that this enzyme plays a crucial role in regulating endogenous JA-Ile levels and, hence, the JA-Ile-mediated responses. As physi-ological responses triggered by JA-Ile depend on its endogenous concentration, its precise determination is important. The endog-enous levels of JA-Ile are regulated by the balance between biosyn-thesis and deactivation. Thus, to understand the detailed regulatory mechanisms that control JA-Ile concentration, it is necessary to analyze its metabolites (JA-Ile and its related compounds, OPDA, JA, and 12-OH-JA-Ile) as well (hereafter, referred to as jasmonates) ( see Fig. 1 ).

Responses to environmental stimuli are not merely regulated by JA-Ile alone, but rather by the combination or the interaction with other hormones, such as indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), and ethylene (ET). IAA suppresses jasmonate signals through the induction of JAZ1/TIFY10 [ 6 ] ; the ABA receptor PYR/PYL/RCAR proteins mediate jasmonate sig-naling [ 7 ] ; SA suppresses jasmonate-responsive gene expression downstream of jasmonate biosynthesis [ 8 ] ; and the synergistic effects by ET and jasmonates are regulated by the ET-stabilized

O

COOH

O

a b c

d e

f g h

COOH

O

OCHN

HC COOH

O

OCHN

HC COOH

OH

O

OHCOOH

COOH

OH H2N COOH

NH

COOH

Fig. 1 Structures of target compounds. ( a ) OPDA. ( b ) JA. ( c ) JA-Ile. ( d ) 12-OH-JA-Ile. ( e ) IAA. ( f ) ABA. ( g ) SA. ( h ) ACC

115JA Hormone Profi ling

transcription factors ETHYLENE INSENSITIVE3/ETHYLENE INSENSITIVE-LIKE1 [ 9 ] . Therefore, to obtain more insight into these interactions, analysis of the concentrations of multiple hor-mones is required. In comparison to gas chromatography-electron impact ionization-mass spectrometry, liquid chromatography-elec-trospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is more suitable to analyze simultaneously plant hormones and their related compounds. ESI can produce protonated/deproto-nated molecules to obtain further speci fi c product ions in collision-induced dissociation, allowing a highly speci fi c determination of target compounds in impurity-rich plant extracts [ 10 ] .

1. Methanol (MeOH). 2. Acetonitrile (MeCN). 3. Distilled water. 4. 80 % (v/v) MeCN. 5. 80 % (v/v) MeCN containing 1 % (v/v) acetic acid. 6. Water containing 1 % (v/v) acetic acid. 7. Water containing 1 % (w/v) ammonia. 8. Oasis HLB (30 mg, 1 cc) (Waters, Milford, MA, USA). 9. Oasis MCX (30 mg, 1 cc) (Waters). 10. Oasis WAX (30 mg, 1 cc) (Waters). 11. Internal standards: D 6 -ABA (Icon Isotopes, Summit, NJ,

USA); d 5 -OPDA, d 4 -ACC (Olchemim Ltd, Olomouc, The Czech Republic); d 2 -IAA, d 6 -SA (Sigma-Aldrich, St. Louis, MO, USA); d 2 -JA (Tokyo Kasei, Tokyo, Japan). 13 C 6 -JAIle and 13 C 6 -12-OH-JA-Ile were prepared as described [ 5, 11 ] .

1. Liquid chromatographer 1200 (Agilent, Santa Clara, CA, USA). 2. Triple Quad LC/MS 6410 (Agilent). 3. Column ZORBAX Eclipse XDB-C18 (2.1 mm × 50 mm,

1.8 μ m) (Agilent). 4. Column ZORBAX AQUA (2.1 mm × 50 mm, 1.8 μ m)

(Agilent). 5. Spectrometry software MassHunter™ version B.01.03 (Agilent). 6. MeCN containing 0.05 % acetic acid. 7. Water containing 0.01 % acetic acid. 8. MeCN containing 0.1 % formic acid. 9. Water containing 0.1 % formic acid.

2 Materials

2.1 Sample Extraction and Puri fi cation

2.2 LC-ESI-MS/MS Analysis

116 Yusuke Jikumaru et al.

Here we describe a fundamental procedure to quantify jasmonates, IAA, ABA, SA, and the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) ( see Fig. 1 ). Because of its volatility and small molecular weight, LC-ESI-MS/MS is not suitable for the analysis of ET itself, but, instead, the precursor ACC can be ana-lyzed to determine the ET biosynthesis rate ( see Note 1 ). Extraction and partial puri fi cation are based on the procedure as described [ 12 ] ( see Fig. 2 ). A set of results obtained from Arabidopsis thali-ana leaves collected at 0 or 1 h after wounding is shown in Table 1 . The endogenous concentrations of jasmonates increased as reported previously [ 13 ] , whereas those of other hormones did not change after 1 h of wounding. For the analysis of cytokinins ( trans -zeatin, isopentenyladenine, and dihydrozeatin), gibberel-lins (GA 12 , GA 15 , GA 24 , GA 9 , GA 4 , GA 51 , GA 34 , GA 53 , GA 44 , GA 19 , GA 20 , GA 1 , GA 29 , and GA 8 ), and brassinosteroids (castasterone and brassinolide) from vegetative tissues, large amounts of sam-ples and additional puri fi cation steps are required ( see Note 2 ). It is important to avoid cross-contaminations ( see Note 3 ).

1. Prepare plant material as required (approximately 5 mg of freeze-dried samples were used for the analysis presented in Table 1 ) ( see Note 4 ).

3 Methods

3.1 Sample Extraction and Puri fi cation

80% MeCN1% acetic acid

Extracts

Water1% acetic acid

OPDA, JA, JA-Ile, 12-OH-JA-Ile,IAA, ABA, SA

ACC

Water1% Acetic acid

ACC

Water1% ammonia

80% MeCN1% acetic acid

HL

B

HL

B

MC

X

MC

X

MC

X

80% MeCNWater1% Acetic acid

80% MeOH1% Acetic acid

OPDA, JA, JA-Ile, 12-OH-JA-Ile,IAA, ABA

WA

X

WA

X

WA

X

LC-ESI-MS/MSLC-ESI-MS/MS

SA LC-ESI-MS/MS

Fig. 2 Overview of the puri fi cation by cartridge columns

117JA Hormone Profi ling

2. Freeze the materials immediately in liquid nitrogen and store at −80 °C until use. If required, material dry weights can be measured after lyophilization ( see Note 5 ).

3. Grind the plant material into powder and add 1 mL of 80 % MeCN containing 1 % acetic acid as an extraction solvent, to, at least, 100 times the volume of the dry weight material.

4. Add internal standards so that their ratio to the endogenous compounds will be approximately 1 (Table 1 ) ( see Note 6 ) and extract for 1 h at room temperature.

5. Centrifuge the extracts at 14,000 × g for 10 min at room tem-perature and collect the supernatant.

6. Extract the pellets with the same volume of extraction solvent again for 10 min and collect the supernatants after centrifugation.

7. Evaporate MeCN in the combined supernatants to obtain extracts in acetic acid-containing water ( see Note 7 ).

8. To obtain ACC-containing fractions, apply the extracts after step 3 to an Oasis HLB column cartridge ( see Note 8 and Subheading 3.2 for equilibration) and collect together the fl ow-through and 1 mL of water containing 1 % acetic acid.

9. Elute the other target compounds (OPDA, JA, JA-Ile, 12-OH-JA-Ile, IAA, ABA, and SA) with 2 mL of 80 % MeCN containing 1 % acetic acid. Keep 100 μ L of this eluate for SA analysis.

10. Apply the fraction containing ACC to an Oasis MCX column cartridge ( see Subheading 3.2 , for equilibration and regeneration).

Table 1 Endogenous hormone concentrations in wounded leaves of Arabidopsis and concentrations of internal standards (in ng/g dry weight)

Hormones

Control Wounded (1 h)

Endogenous Internal standard Endogenous Internal standard

OPDA 12.3 × 10 3 10.0 × 10 3 11.4 × 10 6 10.0 × 10 6

JA 8.13 10.0 9.23 × 10 3 10.0 × 10 3

JA-Ile 1.09 1.00 1.00 × 10 3 1.00 × 10 3

12-OH-JA-Ile 4.34 5.00 15.5 × 10 3 15.0 × 10 3

IAA 58.9 50.0 56.1 50.0

ABA 33.7 50.0 50.9 50.0

SA 1.66 × 10 3 2.00 × 10 3 1.58 × 10 3 2.00 × 10 3

ACC 2.39 × 10 3 2.00 × 10 3 2.43 × 10 3 2.00 × 10 3

118 Yusuke Jikumaru et al.

11. Wash the column with 1 mL of water containing 1 % acetic acid and then with 80 % MeCN containing 1 % acetic acid.

12. Elute ACC with 2 mL of water containing 1 % ammonia. 13. Evaporate MeCN in 1.9 mL of eluate after step 4 to obtain

extracts in water containing acetic acid. 14. Apply the extracts after step 7 to the Oasis WAX column car-

tridge ( see Subheading 3.2 , for equilibration and regeneration). 15. Wash the column with 1 mL of water containing 1 % acetic

acid and then with 2 mL of 80 % MeCN. 16. Elute the remainder of the target compounds (OPDA, JA,

JA-Ile, 12-OH-JA-Ile, IAA, and ABA) with 2 mL of 80 % MeCN containing 1 % acetic acid.

17. Dry the fractions containing the target compounds after step 4 (SA), step 6 (ACC), and step 9 (OPDA, JA, JA-Ile, 12-OH-JA-Ile, IAA, and ABA).

18. Dissolve the residue in 100 μ L of MeOH ( see Note 9 ) and transfer to the vials for LC-ESI-MS/MS.

19. Evaporate MeOH in the vials and dissolve the extracts in 100 μ L (for ACC) or 30 μ L (for SA, and for OPDA, JA, JA-Ile, 12-OH-JA-Ile, IAA, and ABA) of water containing 1 % acetic acid.

20. Inject 1 μ L (for ACC) or up to 15 μ L (for SA, and for OPDA, JA, JA-Ile, 12-OH-JA-Ile, IAA, and ABA) to LC-ESI-MS/MS for the analysis ( see Note 10 ).

1. For the Oasis HLB column, wash with 1 mL of MeCN and then with MeOH.

2. Equilibrate with 1 mL of initial solvent (water containing 1 % acetic acid).

3. For the Oasis MCX column, wash with 1 mL of MeCN and then with MeOH. After regeneration with 0.5 mL of 0.1 M HCl, equilibrate with 1 mL of initial solvent (water containing 1 % acetic acid).

4. For the Oasis WAX column, wash with 1 mL of MeCN and then with MeOH. After regeneration with 0.5 mL of 0.1 M NaOH, equilibrate with 1 mL of initial solvent (water containing 1 % acetic acid).

1. Set the LC conditions as follows: fl ow rate, 200 μ L/min. Composition and gradients of solvents are listed in Table 2 .

2. Set the MS/MS conditions: Capillary, 4,000 V; desolvation temperature, 300 °C; gas fl ow, 9 L/min; and nebulizer, 30 psi. Other parameters for analysis are listed in Table 3 .

3. Determine the areas of peaks from each compound with the spectrometer software (MassHunter™ version B.01.03) and

3.2 Column Cartridge Equilibration

3.3 LC-ESI-MS/MS Analysis

119JA Hormone Profi ling

calculate the endogenous concentrations of the target compounds.

Typical MS/MS chromatograms obtained in Arabidopsis leaves 1 h after wounding are presented in Fig. 3 .

Table 2 LC conditions

Method no. Column Solvent A Solvent B Composition of solvent B

1 XDB-C18 Water containing 0.01 % acetic acid

MeCN containing 0.05 % acetic acid

3–70 % B over 30 min

2 AQUA Water containing 0.1 % formic acid

MeCN containing 0.1 % formic acid

3–98 % B over 10 min

3 XDB-C18 Water containing 0.1 % formic acid

MeCN containing 0.1 % formic acid

0–3 % B over 5 min

Table 3 Parameters for LC-ESI-MS/MS analysis

Hormone LC method no. Retention time (min) a Charge MS/MS ( m / z )

Collision energy Fragmentor

OPDA 1 24.7 − 291/165 12 160

D 5 -OPDA 1 24.7 − 296/170 12 160

JA 1 15.6 − 209/59 10 150

D 2 -JA 1 15.6 − 211/59 10 150

JA-Ile 1 18.6 − 322/130 14 160

13 C 6 -JA-Ile 1 18.6 − 328/136 14 160

12-OH-JA-Ile 1 12.2 − 338/130 20 150

13 C 6 -12-OH-JA-Ile 1 12.2 − 344/136 20 150

IAA 1 11.8 − 174/130 18 110

D 2 -IAA 1 11.8 − 176/132 18 110

ABA 1 13.4 − 263/153 8 140

D 6 -ABA 1 13.3 − 269/159 8 140

SA 3 6.3 − 137/93 16 100

D 6 -SA 3 6.3 − 141/97 16 100

ACC 2 0.8 + 102/56 12 50

D 4 -ACC 2 0.7 + 106/60 12 50

a Retention time of deuterium-labeled internal standards is slightly shorter than that of nonlabeled endogenous com-pounds ( see Note 11 )

120 Yusuke Jikumaru et al.

1. ACC is generated by the reaction of ACC synthase and then converted to ET by ACC oxidase. ACC synthase has been shown to be a key regulatory enzyme in the ET biosynthesis [ 14 ] .

2. Cation exchange cartridge puri fi cation is required to analyze basic compounds, such as cytokinins as described [ 10 ] . Because of their low concentrations, large amounts of plant material and additional puri fi cations are required for analysis of gibberellins [ 15 ] and brassinosteroids [ 16 ] .

3. To avoid cross-contamination, all cartridges, glassware, and plasticwares must not be reused.

4. The amount of plant material needed for the analysis depends on the endogenous concentrations of the target compounds and on the type of material (species, tissue, physiological state,

4 Notes

a b c

d e

f g h

Time (min)

22 23 24 25 26Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

100

15 16Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

100

18 19 20 21Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

100

11 12

Rel

ativ

e ab

unda

nce

(%)

0

100

11 12Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

100

1 2 3 4Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

100

5 6 7 8Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

100

13 14Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

100

Fig. 3 MS chromatograms of target compounds. ( a ) OPDA. ( b ) JA. ( c ) JA-Ile. ( d ) 12-OH-JA-Ile. ( e ) IAA. ( f ) ABA. ( g ) SA. ( h ) ACC. JA-Ile gives two peaks ( see Note 12 )

121JA Hormone Profi ling

and treatment). A preliminary analysis is carried out to esti-mate the amount of samples needed.

5. Endogenous concentrations of jasmonates have been reported to increase rapidly within a few minutes after wounding [ 17 ] . For accurate determination of the concentration of jasmonates, it is recommended to fl ash-freeze plant materials just after sam-pling without measuring their fresh weight. Lyophilized samples are stable at room temperature under dry conditions and hormone concentrations per dry weight can be calculated.

6. The amount of internal standards added here depended on the dry weight of the samples. The following amounts of internal standards were added to approximately 5 mg of dry weight. For control leaves, 50 ng of d 5 -OPDA, 50 pg of d 2 -JA, 5 pg of 13 C 6 -JA-Ile, 25 pg of 1 3C 6 -12-OH-JA-Ile, 250 pg of d 2 -IAA, 250 pg of d 6 -ABA, 10 ng of d 6 -SA, and 10 ng of d 4 -ACC were added to the extracts, whereas for wounded leaves, 50 μ g of d 5 -OPDA, 50 ng of d 2 -JA, 5 ng of 13 C 6 -JA-Ile, 75 ng of 13 C 6 -12-OH-JA-Ile, 250 pg of d 2 -IAA, 250 pg of d 6 -ABA, 10 ng of d 6 -SA, and 10 ng of d 4 -ACC were supplemented. The accumula-tion of wound-induced impurities must not be ignored. When endogenous concentrations of JA increase, accumulation of unknown impurities that have a 211/59 of MS/MS transition can also be observed. This peak is overlapping and cannot be discriminated from d 2 -JA that is added as the internal standard. To minimize the effect of this overlap, d 2 -JA has to be added a 1,000 times higher than the amounts of the control sample. Other internal standards for jasmonates have to be added in the same manner.

7. Because of the volatility of JA, the eluate should not be kept for a long time under a negative pressure after it is completely dry.

8. The amount of HLB resin must be adapted to the dry weight of the starting material. Generally, the dry weight of the sample is less than fi ve times that of the resin.

9. Sonicate the extracts for a short time in MeOH to help dissolu-tion. Do not transfer debris to the vial for LC-ESI-MS/MS analysis. If necessary, the MeOH solution is fi ltrated.

10. Because of its high polar nature, the amount of ACC retained on the column used here is limited. The injection volume must not be increased, because nonretained ACC will give a broad peak that is not identical. If necessary, dilute the extract.

11. Due to the isotope effect, the retention time of the deuterium-labeled internal standard is slightly shorter than that of the corresponding nonlabeled compounds (e.g., d 6 -ABA is 4 s shorter than ABA and d 2 -JA is 2 s shorter than JA). This infor-mation is important to be sure of the identity of the compounds in impurity-rich mass chromatograms.

122 Yusuke Jikumaru et al.

12. JA-Ile gives two peaks of diastereomers: (−)-JA-Ile at a short retention time and (+)-7-iso-JA-Ile at a long retention time. (+)-7-iso-JA-Ile is converted to (−)-JA-Ile during extraction and puri fi cation in protic solvent. Both peaks are used for the quanti fi cation of endogenous JA-Ile.

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