Simple, Rapid, and Simultaneous Assay of ... - Semantic Scholar

ANALYTICAL SCIENCES NOVEMBER 2012, VOL. 28

1081

2012 © The Japan Society for Analytical Chemistry

Simple, Rapid, and Simultaneous Assay of Multiple Carboxyl Containing Phytohormones in Wounded Tomatoes by UPLC-MS/MS Using Single SPE Purification and Isotope Dilution Jihong FU,*,** Jinfang CHU,** Xiaohong SUN,** Jide WANG,* and Cunyu YAN**† *College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi 830046, P. R. China **National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, P. R. China

An efficient simplified isotope dilution method was developed to determine four carboxyl containing phytohormones simultaneously in 200 mg of fresh tomato tissues using ultra high performance liquid chromatography–triple quadrupole mass spectrometry (UPLC-MS/MS) with negative electrospray ionization. The four phytohormones are indole-3-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA). Only one purification step of Oasis MAX solid phase extraction (SPE) was employed to enrich target phytohormones after crude extraction. In addition, two endogenous isomers of JA, (–)-JA and (+)-7-iso-JA, were separated directly. The validated method has been applied to monitor changes of JA, SA, IAA, and ABA in both local and systemic leaves of wild-type and transgenic 35S::prosystemin (35S::PS) tomato lines. Meanwhile, the JA burst amplified by the overexpressed prosystemin in 35S::PS was verified. Furthermore, the spatial and temporal changes of JA, SA, ABA, and IAA were analyzed. (Received July 4, 2012; Accepted September 25, 2012; Published November 10, 2012)

Introduction In the new OMICS era, to the systematic study of plant functional genes and their molecular mechanisms is becoming increasingly important for the biologist. Phytohormones, which are important endogenous active small molecules, play a central role in regulating physiological processes such as cell division, enlargement and differentiation, seed germination and dormancy, bud formation, flowering, apical dominance, and senescence.1,2 Phytohormone crosstalk is a fine-tuned network for the plants to adapt to various external stress.3–5 For wound signaling, some of the involved components interact with each other in the plants defense responses, suggesting that crosstalk events may regulate temporal and spatial activation of different defenses. Research on the function and metabolism of phytohormones depends upon the availability of highly sensitive and specific methods for the quantitation of phytohormones. Numerous analytical techniques have been developed in recent years. Immunoassay based techniques for semi-quantitation, such as radio immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), have had limitations owing to the cross-reactivity Gas chromatography–mass spectrometry of antibodies.6,7 (GC-MS) for phytohormone quantitation and profiling is being used less and less because its disadvantages, which include laborious and time-consuming sample pretreatment.8–21 Among the different analytical techniques available for phytohormones analysis, liquid chromatography–mass spectrometry (LC-MS) is a very promising technique for improving detectability. Targeted To whom correspondence should be addressed. E-mail: [email protected]

†

analysis of phytohormones can be carried out by LC-MS with multiple reaction monitoring (MRM) in a tandem mass spectrometer. Current LC-MS methods have mainly focused on detection of specific classes of phytohormones, such as auxin,22,23 abscisic acid (ABA),24–26 jasmonates,27,28 salicylic acid (SA) and cytokinins.29–31 Simultaneous determination of trace multiple phytohormones by LC-triple quadrupole mass spectrometry (MS/MS), which is a method that had been attracting increasing attention and which could provide detailed information for the understanding of molecular mechanisms of crosstalk, is still characterized by the time-consuming pretreatment and separation processes as well as co-eluted interferences of matrix. For example, three kinds of phytohormones in 1.5 g of plant tissues had been determined by LC-MS in 20 min.32,33 Furthermore, six kinds of phytohormones including IAA, ABA, SA, jasmonic acid (JA), cytokinins, and gibberellins have been analyzed simultaneously by LC-MS/MS with co-elution of indole-3-acetic acid (IAA) and SA.34 Kojima developed a multiple phytohormone profiling method using a derivatization technique called MS-probe modification, which can improve detection sensitivity significantly, but the process of derivatization is a little difficult to handle in a biological laboratory.35 Although the ultrasound-assisted extraction and dispersive solid-phase extraction allow for the detection of more phytohormones, the wet handling and concentration process using a rotatory evaporator are time-consuming steps in Fan’s work.36 In this work, an efficient and sensitive quantitation method based on isotope dilution strategy by ultra high performance liquid chromatography (UPLC)-MS/MS with negative electrospray ionization (ESI) and MRM was developed for the silmultaneous determination of multiple carboxyl-containing

1082

ANALYTICAL SCIENCES

phytohormones, including IAA, ABA, JA, and SA. Structures of all the phytohormones as well as isotope labeled internal standards characterized by the carboxyl group are illustrated in Fig. 1. In our work, only one purification step of anion ion exchange solid-phase extraction (SPE) was employed, corresponding to a simple sample pretreatment. The utilization of four isotope labeled internal standards that were commercially available gave much more reliable quantitative results by eliminating the matrix effect considering the co-elution. Four target phytohormones in a relatively small quantity of fresh plant tissue, 200 mg were separated effectively within 5 min and with 2 min equilibration. The method was then applied to monitor levels of endogenous phytohormones in the mechanically wounded tomatoes. The spatial and temporal changes of phytohormones in tomato leaves were determined, providing information for research on phytohormone crosstalk.

from CDN Isotopes Inc. (Pointe-Claire, Quebec, Canada). HPLC-grade methanol and acetonitrile were obtained from Fisher Scientific (Fair Lawn, NJ). Analytical-grade formic acid, acetic acid, and sodium diethyldithiocarbamate trihydrate were purchased from Fluka Sigma. Ammonium hydroxide of analytical grade was obtained from Beijing Chemical Works (Beijing, China). Oasis MAX columns (150 mg/6 cc) were obtained from Waters (Milford, MA). Each 10 pmol/μL stock solution of IAA, ABA, JA, and SA was prepared in methanol. Aliquots were then diluted with methanol when needed. Distilled water was supplied by a PURELAB ultra purification system (ELGA, High Wycombe, UK).

Experimental Chemicals and reagents Certified analytical standards of SA, IAA, ABA and JA were purchased from Sigma (St. Louis, MO). Deuterated ABA (2H6-ABA) was obtained from OlChemIm Ltd. (Olomouc, Czech Republic). 2H5-JA, 2H4-SA and 2H2-IAA were purchased

Fig. 1 Chemical structures of four phytohormones (SA, IAA, ABA and JA) and their isotope-labeled internal standards (2H4-SA, 2H2-IAA, 2H -ABA, and 2H -JA). 6 5

NOVEMBER 2012, VOL. 28

The overall layout of the developed method The overall method layout for the quantitative analysis of multiple carboxyl-containing phytohormones is illustrated in Fig. 2. The key rate-limiting step of frozen plant tissue grinding could be replaced by multi-parallel microscale ball-mill, and purification could be performed on multi-parallel high-throughput anion exchange extraction plates for higher throughput. The sample treatment was simplified to a single SPE column purification for effectively removing contaminants, such as lipids and chlorophyll. Sample extraction and cleanup procedures Frozen tomato samples were ground in liquid nitrogen with mortar and pestle. The internal standards (2H2-IAA 100 pmol, 2H -ABA 45 pmol, 2H -SA 140 pmol, and 2H -JA 95 pmol, see 6 4 5 Fig. 1 for the structures) were added to 200 mg of ground powder. The powder was extracted with 2 mL methanol and kept overnight at –20° C, then centrifuged at 4° C for 15 min at 18000 rpm. The supernatant was collected, dried under nitrogen, then dissolved in 1 mL ammonia solution (5%). The crude extracts were further purified by Oasis MAX SPE column, which had been sequentially preconditioned with 4 mL methanol, 4 mL water, and 4 mL ammonia solution (5%). After the samples were loaded, SPE columns were sequentially washed with 4 mL ammonia solution (5%), 4 mL water, and 4 mL methanol. SA, IAA, ABA, and JA were eluted with 4 mL methanol contain 10 or 5% formic acid. The eluent was dried under nitrogen gas and finally dissolved in 200 μL water/methanol (20:80, v/v) for further analysis.

Fig. 2 The overall layout of the developed method with two ion selection channels: MS1, monitoring precursor ion; CID, collision cell; MS2, monitoring product ion; IS, internal standards; P, phytohormones.


1083

Table 1 Optimized mass spectrometry parameters for the quantitation of target phytohormones Compound

Function/min

Retention time/min

Parent ion (m/z)

Daughter ion (m/z)

Cone voltage/V

Collision energy/eV

SA H4-SA IAA 2 H2-IAA ABA 2 H6-ABA JA 2H -JA 5

1.0 – 2.5 1.0 – 2.5 2.5 – 3.0 2.5 – 3.0 3.0 – 3.55 3.0 – 3.55 4.0 – 5.0 4.0 – 5.0

1.93 1.92 2.69 2.68 3.33 3.30 4.28 4.27

137.0 141.1 174.1 176.2 263.1 269.3 209.1 214.25

92.9 96.8 130.1 132.0 153.0 159.0 58.9 61.78

24 24 19 19 27 27 27 27

15 15 10 10 10 10 15 15

2

Plant material and wounding model design Tomato plants, both WT (Lycopersicon esculentum cv. Castlemart) and transgenic 35S::prosystemin (35S::PS), which accumulates high levels of wound response proteins in the absence of wounding due to overexpressing of the PROSYSTEMIN gene,37 were grown in a greenhouse under 16 h light/8 h dark at 30° C/24° C daylight cycle with light at 200 μE m–2 S–1 and relative humidity of 60 ± 10%. Wounding experiments were performed on two-week old seedlings with two fully expanded leaves by nipping the midvein with a hemostat as shown in Fig. 1S (Supporting Information). The lower wounded leaves (local) were used for analysis of the local wounding response and the upper undamaged leaves (systemic) were used for analysis of the systemic wounding response. The undamaged and damaged leaves were collected at different times such as 5, 15, 30, 60, 90, 120, and 180 min after wounding, frozen in liquid nitrogen, and stored at –80° C for further use. Liquid chromatography and mass spectrometry LC system of Waters ACQUITY UPLC (Waters, Milford, MA) was used with a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm i.d., 1.7 μm). Four phytohormones were separated with a mobile phase consisting of acetonitrile and water, both of which contained 0.05% acetic acid (v/v). The gradient run was at a flow rate of 0.5 mL/min with initial 15% acetonitrile, which was then increased to 40% in 5 min and further increased to 80% in the next 0.5 min. The injection volume for all samples was 5 μL and the column temperature was 35° C. The UPLC system was coupled online with Waters Quattro Premier XE mass spectrometer (Micromass, Manchester, UK) equipped with an ESI source. The eletrospray capillary voltage was operated at 2.80 kV in the negative ion mode. The optimized parameters with ESI source obtained by infusion of each standard solution of 10 pmol/μL in acetonitrile/water (50:50, v/v) at 10 μL/min were the following: source temperature, 110° C; desolvation temperature, 350° C; desolvation gas flow, 600 L/h; cone gas flow, 60 L/h; multiplier, 650 V. Quantitative analysis was performed in MRM mode with four timesegmented scannings as shown in Table 1.

Results and Discussion UPLC-MS/MS optimization and MRM transition selection For the phytohormone quantitation, the mass spectrometer parameters were optimized. Table 1 summarizes the optimum parameters for targets and internal standards. For the MRM with negative ESI, the deprotonated molecule ions [M–H]– were chosen as precursor ions. All of these acidic phytohormones produced the characteristic fragment ions of [M–H–CO2]–, which were the only fragment ions of IAA (m/z 130.1) and SA

(m/z 92.9), respectively, and was thus chosen as the MRM channels for IAA (Fig. 3a) and SA (Fig. 3d). The most intensive product ions of ABA corresponding to a negatively charged cyclohexenone ring fragment resulted from the cleavage of the side chain, as showed in Fig. 3b.38 The dominant product ion m/z 58.9, corresponding to [CH3COO]–, was chosen for JA (Fig. 3c). The UPLC gradient needed to be optimized to enhance sensitivity and to lower ionization suppression. Methanol and acetonitrile with variable pH values were tested for these acidic phytohormones. Although the carboxyl group could be ionized in the negative mode in either neutral or basic environments, higher pH values resulted in undesirable peak shape, after optimization, acetonitrile and water, both containing 0.05% acetic acid, could maintain a balance between the retention and the negative ionization. Four unlabeled phytohormone standards were separated effectively within 5 min and with 2 min of equilibration, which was superior to the HPLC-MS analyses taking 30 min. Four time-segmented function channels of MS were chosen and sensitivity was improved by increasing the dwell time. The total ion chromatograms of the standards with MRM mode are shown in Fig. S2 (Supporting Information). After a series of optimizations, the quantitation of four phytohormones based on isotope dilution strategy by UPLC-MS/MS was established. Equations of linear regression related to the concentration ratios to area ratios are listed in Table 2, together with correlation coefficients higher than 0.99. The limit of detection (LOD) and the limit of quantification (LOQ) for these phytohormones were estimated to be in the range of 4 × 10–5 – 0.35 and 1 × 10–4 – 0.85 pmol, respectively. The average relative standard deviations (RSDs) of the areas and the retention times were 6.61 and 0.43%, respectively (n = 6), illustrating that the stability of sample solutions and the method’s repeatability were all acceptable. Epimerization separation of endogenous (–)-JA Among these four phytohormones, endogenous (–)-JA can be biosynthesized into its final bioactive form of (+)-7-iso-jasmonoyl-L-isoleucine ((+)-7-iso-JA-Ile), which can further trigger the plant immunity response.39,40 The synthesized JA consists of two pairs of enantiomers, (–)-JA and (+)-JA, as well as (+)-7-iso-JA and (–)-7-iso-JA, each of which could not be separated on C18 column due to chiral centers of C-3 and C-7 shown in Fig. S3 (Supporting Information). The former trans pair is thermally more stable than the corresponding cis one. In plants, due to the keto-enol tautomerism at C-7 as well as sterical hindrance, the epimerization of endogenous (–)-JA and (+)-7-iso-JA reaches a dynamic equilibrium with an approximate ratio of 9:1.41–43 The synthesized JA has another pair of diasteromers, (+)-JA and (–)-7-iso-JA, due to the keto-enol tautomerism at C-7. Remarkably, most of the JA quantitation

1084

ANALYTICAL SCIENCES


Fig. 3 Fragmentation patterns for phytohormone standards analyzed in the negative-ion mode. (a) Precursor (m/z 174.1) and product (m/z 130.1) ions of IAA, (b) precursor (m/z 263.1) and product (m/z 153.0) ions of ABA, (c) precursor (m/z 209.1) and product (m/z 58.9) ions of JA, (d) precursor (m/z 137.0) and product (m/z 92.9) ions of SA. Table 2 Quantitation results of four phytohormones under optimized UPLC-MS/MS conditions Phytohormone standard

Regression equation

Correlation coefficient

Concentration range/pmol

LOD/pmol

LOQ/pmol

SA IAA ABA JA

y = 0.4496x – 4.3284 y = 0.3373x + 0.1617 y = 1.1223x + 0.0465 y = 1.9399x + 0.4971

0.9996 0.9999 0.9997 0.9999

15 – 230 1 – 50 0.06 – 2.50 0.1 – 60

0.35 0.3 4 × 10–5 0.02

0.7 0.85 1 × 10–4 0.04

y, peak area ratio of the standard and the internal standard; x, concentration ratio of the standard (pmol); tm, retention time; LOD, limits of detection; LOQ, limits of quantification.

distribution as shown in Fig. 4.44,45 Four stereoisomers resulted in two peaks under optimized UPLC-MS/MS conditions, the larger of which consisted of a racemic mixture of deuterium labeled (–)-JA and (+)-JA, and the smaller of which consisted of deuterium labeled (–)-7-iso-JA and (+)-7-iso-JA. For the same retention time and ionization behavior of enantiomers on the achiral reverse phase chromatographic column, the synthesized deuterium labeled JA used as the internal standard for isotope dilution quantitation of endogenous JA was appropriate. The relative concentration of endogenous (–)-JA was obtained in the experiment due to the racemic internal standard. Furthermore, this method allowed for the determination of bioactive (+)-7-iso-JA and (+)-7-iso-JA-Ile as well. Fig. 4 Separation of 2H5-JA by the optimized UPLC-MS/MS method. 2H5-JA was separated into two peaks, each of which consisted of a pair of enantiomers. The higher one contains 2H5-(–)-JA and 2H -(+)-JA; the lower one contains 2H5-(+)-7-iso-JA and 5 2H -(–)-7-iso-JA. The ratio of peak areas resulted from different steric 5 hindrance.

methods have neglected the separation of these four stereoisomers. But the optimized UPLC-MS/MS method provided baseline separation of these two pairs of diasteromers with the peak area ratio corresponding to their natural

Optimization of plant extract pretreatment and recoveries of target phytohormones Interferences such as the lipids, chlorophyll as well as other neutral and basic substances in plant extracts may seriously affect the quantitative results of acidic phytohormones. Reverse phase SPE columns, such as C18 or HLB type, could concentrate acidic phytohormones.46 However, a large volume of plant tissue used in such methods means reverse phase SPE column can not eliminate interferences effectively. MAX SPE column used in Dobrev’s work allowed for the use of a smaller amount of plant tissue for IAA and ABA determination, which implies the method could offer a better practical application for acidc


1085

Fig. 5 UPLC-MS/MS MRM chromatograms of IAA, ABA, JA, and SA in the undamaged wild-type leaves (a, b, c, and d). MRM transitions: (a) IAA, 174.1 > 130.1; 2H2-IAA, 176.2 > 132.0, (b) ABA, 263.1 > 153.0; 2H6-ABA, 269.3 > 159.0, (c) JA 209.1 > 58.9; 2H5-JA, 214.3 > 61.8, (d) SA, 137.0 > 92.9; 2H4-SA, 141.1 > 96.8.

phytohormones.47 The dual-step SPE strategy also employed by Ge providing ideal GA and CTK quantization results. These multi-step SPE methods mean lower target compound recoveries and lower sample throughput.48 In the present study, the main objectives were to simplify the procedures of extraction and purification and to develop a rapid and sensitive detection method for the determination of the four acidic phytohormones. Quantitative information for multiple phytohormones, fewer SPE steps, quick LC program and higher recovery should be considered in the new phytohormone determination method. Considering the same physiochemical properties of the carboxyl group, an Oasis MAX SPE column was used for purification. The column was filled with divinylbenzene and N-vinylpyrrolidone based copolymer sorbent and possesses mixed-mode functionalities of reverse phase and anion exchange properties. The sample was loaded on the preconditioned Oasis MAX columns in alkaline solution, which could increase the retention of the weak hydrophobic and acidic phytohormones. The wash of SPE columns with 4 mL ammonia solution (5%) was to remove the salts and block the ionized analytes on the ion exchange sorbent. Water and methanol were applied to remove neutral and basic interference. Finally, the targeted phytohormones were eluted with 4 mL methanol containing 10% formic acid. The recoveries of SA, IAA, ABA and JA were 92, 81, 92 and 87% on Oasis MAX columns, respectively. Herein, the recovery of SA eluted with 4 mL methanol containing 5% formic acid was only 64%, the stronger acidic character of SA (pKa 2.97) compared with other phytohormones enabled higher recovery of SA when the formic acid content was increased to 10%. Besides, this sample handling procedure also provided the potential for direct separation of SA from other carboxyl-containing phytohormones on the MAX SPE column.

Determination of target phytohormones in wounded tomato leaves In order to verify the feasibility of the new method, wild-type (WT) tomato leaves were used first to determine SA, IAA, ABA, and JA simultaneously. The MRM chromatograms of the target phytohormones in WT tomato leaves are shown in Fig. 5. The content of these four phytohormones was about 65.68 ± 6.39, 31.22 ± 1.72, 672.88 ± 58.55, and 6.09 ± 0.43 pmol/g fresh weight (FW) (n = 4), respectively. Then a wounding model of tomatoes was constructed for the determination of multiple carboxyl-containing phytohormones and to monitor the systemic phytohormones response to injury. The WT and 35S::PS tomato lines were chosen to determine multiple acidic phytohormone spatial and temporal distribution. The 35S::PS tomato accumulates high levels of wound response proteins in the absence of wounding because of overexpression of the prosystemin.49 WT and 35S::PS tomatoes were mechanically wounded with a hemostat across the midvein of fully expanded leaves. Samples were collected from wounded (local) and unwounded (systemic) leaves at the designed time intervals as described in the Exeperimental section (Fig. S1). A rapid burst of JA, the key molecule responding to tissue wounding, was verified in both WT and 35S::PS lines. JA was found to accumulate rapidly in local wounded leaves, with concentration increasing from initial 6.09 ± 0.43 and 11.52 ± 0.88 pmol/g FW to 2059.41 ± 185.85 and 2678.44 ± 133.57 pmol/g FW for WT at 15 min and for 35S::PS at 30 min, respectively, corresponding to approximately 338-fold (P < 0.01) and 232-fold (P < 0.01) increments (Fig. 6, JA). In the systemic unwounded leaves, the JA concentrations of WT at 15 min and 35S::PS at 30 min were 156-fold and 50-fold higher than initial values, respectively. The systemic JA level was typically lower than that of the local damaged leaves for both, WT and 35S::PS, with concentrations reaching only about 46 and 21% of the maximum of JA burst, respectively. The local as well as systemic JA burst of both WT and 35S::PS gradually declined to

1086

ANALYTICAL SCIENCES


Fig. 6 Concentration changes of phytohormones in wounding model in wild-type and 35S::PS tomato leaves. The damaged leaves (local) and undamaged leaves (systemic) were collected at indicated times after wounding. Values are expressed as means ± SD (n = 4). ■, WT-local; ▲, 35S::PSlocal; ●, WT-systemic; ▼, 35S::PS-systemic.

almost initial levels in 24 h, which is consistent with earlier literatures.50,51 The 35S::PS line showed an amplified JA burst in the damaged tomato leaves (Fig. 6, JA, green line) and the JA level in the systemic leaves remained higher related to those of WT (Fig. 6, JA, blue line). Usually, resistance induced by SA, another vital phytohormone related to tissue necrosis, is effectively against a diverse range of pathogens, including bacteria, viruses, fungi and oomycetes with different defense signaling pathway with JA.52,53 Herein, the concentration of SA distributes in a relatively narrow range from 665.68 ± 6.39 to 1314.80 ± 75.73 pmol/g FW (Fig. 6, SA). The damaged leaves withered after handling with hemostat within 15 min. The spatial accumulation of ABA in damaged leaves was relatively greater than for undamaged leaves in both WT and 35S::PS (Fig. 6, systemic line and local line). Slight increases of ABA levels from 573.70 ± 27.62 and 642.74 ± 55.04 at 5 min to 1896.25 ± 236.79 and 1902.97 ± 246.82 pmol/g FW at 120 min were observed in the WT and 35S::PS damaged leaves, respectively (Fig. 6, ABA, green line and black line), which suggests that the dehydration of damaged leaves resulting from mechanical wounding was directly related to ABA content change.54 Although exogenous jasmonate can promote local auxin accumulation in the basal meristem of WT roots,55 the endogenous JA burst had no significant effect on IAA biosynthesis in damaged and undamaged leaves of WT and 35S::PS (Fig. 6, IAA). Further experiments are being conducted to verify the crosstalk effect between JA and IAA in the wounding model.

Acknowledgements The authors thank Prof. Jiayang Li for his warmhearted help on the lab startup. We also sincerely thank Prof. Chuanyou Li and Dr. Jiuhai Zhao for providing wild-type and transgenic tomato

lines as well as for their help with plant cultivation. This work was supported by the National Natural Science Foundation of China (Grant Nos. 90917017, 90817008 and 91117016) and the Scientific Research Starting Foundation for Returned Overseas Chinese Scholars, Ministry of Education, P. R. China 2009 (Grant No. 1341).

Supporting Information This material is available free of charge on the Web at http:// www.jsac.or.jp/analsci/.

References 1. P. J. Davies, “Plant Hormones: Biosynthesis, Signal Transduction, Action!”, 2004, Springer, Dordrecht, The Netherlands. 2. A. Gomez-Cadenas, J. Mehouachi, F. R. Tadeo, E. Primo-Millo, and M. Talon, Planta, 2000, 210, 636. 3. J. Liu, S. Mehdi, J. Topping, P. Tarkowski, and K. Lindsey, Mol. Syst. Biol., 2010, 6, 373. 4. E. Loreti, G. Povero, G. Novi, C. Solfanelli, A. Alpi, and P. Perata, New Phytol., 2008, 179, 1004. 5. M. Fujita, Y. Fujita, Y. Noutoshi, F. Takahashi, Y. Narusaka, K. Yamaguchi-Shinozaki, and K. Shinozaki, Curr. Opin. Plant Biol., 2006, 9, 436. 6. A. Grayling and D. E. Hanke, Phytochemistry, 1992, 31, 1863. 7. Z. Y. Xin, X. Zhou, and N. G. Zhang, J. Nanjing Agric. Univ., 1998, 21, 19. 8. S. J. Neill, R. Horgan, and J. K. Heald, Planta, 1983, 157, 371. 9. X. Li, C. La Motte, C. Stewart, N. Cloud, S. Wear-Bagnall,


1087

and C.-Z. Jiang, J. Plant Growth Regul., 1992, 11, 55. 10. J. Ueda, K. Miyamoto, and S. Kamisaka, J. Chromatogr., A, 1994, 658, 129. 11. D. M. Ribnicky, T. J. Cooke, and J. D. Cohen, Planta, 1997, 204, 1. 12. J. Engelberth, T. Koch, G. Schuler, N. Bachmann, J. Rechtenbach, and W. Boland, Plant Physiol., 2001, 125, 369. 13. P. H. Duffield and A. G. Netting, Anal. Biochem., 2001, 289, 251. 14. A. Muller, P. Duchting, and E. W. Weiler, Planta, 2002, 216, 44. 15. C. Birkemeyer, A. Kolasa, and J. Kopka, J. Chromatogr., A, 2003, 993, 89. 16. E. A. Schmelz, J. Engelberth, J. H. Tumlinson, A. Block, and H. T. Alborn, Plant J., 2004, 39, 790. 17. A. J. Koo, H. S. Chung, Y. Kobayashi, and G. A. Howe, J. Biol. Chem., 2006, 281, 33511. 18. L. S. Barkawi, Y. Y. Tam, J. A. Tillman, B. Pederson, J. Calio, H. Al-Amier, M. Emerick, J. Normanly, and J. D. Cohen, Anal. Biochem., 2008, 372, 177. 19. F. J. Zhang, Y. J. Jin, X. Y. Xu, R. C. Lu, and H. J. Chen, Phytochem. Anal., 2008, 19, 560. 20. L. S. Barkawi, Y. Y. Tam, J. A. Tillman, J. Normanly, and J. D. Cohen, Nat. Protoc., 2010, 5, 1609. 21. P. S. Blake, J. M. Taylor, and W. E. Finch-Savage, Plant Growth Regul., 2002, 37, 119. 22. F. Matsuda, H. Miyazawa, K. Wakasa, and H. Miyagawa, Biosci., Biotechnol., Biochem., 2005, 69, 778. 23. J. Chamarro, A. Ostin, and G. Sandberg, Phytochemistry, 2001, 57, 179. 24. A. R. Ross, S. J. Ambrose, A. J. Cutler, J. A. Feurtado, A. R. Kermode, K. Nelson, R. Zhou, and S. R. Abrams, Anal. Biochem., 2004, 329, 324. 25. R. Zhou, T. M. Squires, S. J. Ambrose, S. R. Abrams, A. R. Ross, and A. J. Cutler, J. Chromatogr., A, 2003, 1010, 75. 26. A. Gomez-Cadenas, O. J. Pozo, P. Garcia-Augustin, and J. V. Sancho, Phytochem. Anal., 2002, 13, 228. 27. S. Tamogami and O. Kodama, J. Chromatogr., A, 1998, 822, 310. 28. X. Liu, Y. Yang, W. Lin, J. Tong, Z. Huang, and L. Xiao, Chin. Sci. Bull., 2010, 55, 2231. 29. G. Segarra, O. Jauregui, E. Casanova, and I. Trillas, Phytochemistry, 2006, 67, 395. 30. K. Hoyerova, A. Gaudinova, J. Malbeck, P. I. Dobrev, T. Kocabek, B. Solcova, A. Travnickova, and M. Kaminek, Phytochemistry, 2006, 67, 1151. 31. O. Novak, E. Hauserova, P. Amakorova, K. Dolezal, and M. Strnad, Phytochemistry, 2008, 69, 2214. 32. A. Durgbanshi, V. Arbona, O. Pozo, O. Miersch, J. V. Sancho, and A. Gomez-Cadenas, J. Agric. Food Chem.,

2005, 53, 8437. 33. S. Giannarelli, B. Muscatello, P. Bogani, M. M. Spiriti, M. Buiatti, and R. Fuoco, Anal. Biochem., 2010, 398, 60. 34. X. Pan, R. Welti, and X. Wang, Phytochemistry, 2008, 69, 1773. 35. M. Kojima, T. Kamada-Nobusada, H. Komatsu, K. Takei, T. Kuroha, M. Mizutani, M. Ashikari, M. Ueguchi-Tanaka, M. Matsuoka, K. Suzuki, and H. Sakakibara, Plant Cell Physiol., 2009, 50, 1201. 36. S. Fan, X. Wang, P. Li, Q. Zhang, and W. Zhang, J. Sep. Sci., 2011, 34, 640. 37. C. Li, M. M. Williams, Y. T. Loh, G. I. Lee, and G. A. Howe, Plant Physiol., 2002, 130, 494. 38. S. Kitamura, N. Jinno, S. Ohta, H. Kuroki, and N. Fujimoto, Biochem. Biophys. Res. Commun., 2002, 293, 554. 39. W. P. Suza, M. L. Rowe, M. Hamberg, and P. E. Staswick, Planta, 2010, 231, 717. 40. S. Fonseca, A. Chini, M. Hamberg, B. Adie, A. Porzel, R. Kramell, O. Miersch, C. Wasternack, and R. Solano, Nat. Chem. Biol., 2009, 5, 344. 41. C. Wasternack, Ann. Bot., 2007, 100, 681. 42. R. A. Creelman and J. E. Mullet, Annu. Rev. Plant Physiol. Plant Mol. Biol., 1997, 48, 355. 43. M. J. Mueller and W. Brodschelm, Anal. Biochem., 1994, 218, 425. 44. L. Holbrook, P. Tung, K. Ward, D. M. Reid, S. Abrams, N. Lamb, J. W. Quail, and M. M. Moloney, Plant Physiol., 1997, 114, 419. 45. R. Kramell, J. Schmidt, G. Schneider, G. Sembdner, and K. Schreiber, Tetrahedron, 1988, 44, 5791. 46. S. Hou, J. Zhu, M. Ding, and G. Lv, Talanta, 2008, 76, 798. 47. P. I. Dobrev, L. Havlicek, M. Vagner, J. Malbeck, and M. Kaminek, J. Chromatogr., A, 2005, 1075, 159. 48. L. Ge, J. W. Yong, S. N. Tan, X. H. Yang, and E. S. Ong, J. Chromatogr., A, 2004, 1048, 119. 49. B. McGurl, M. Orozco-Cardenas, G. Pearce, and C. A. Ryan, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 9799. 50. G. Glauser, E. Grata, L. Dubugnon, S. Rudaz, E. E. Farmer, and J. L. Wolfender, J. Biol. Chem., 2008, 283, 16400. 51. A. J. Koo, X. Gao, A. D. Jones, and G. A. Howe, Plant J., 2009, 59, 974. 52. J. A. Ryals, U. H. Neuenschwander, M. G. Willits, A. Molina, H. Y. Steiner, and M. D. Hunt, Plant Cell, 1996, 8, 1809. 53. M. E. Alvarez, Plant Mol. Biol., 2000, 44, 429. 54. G. F. Birkenmeier and C. A. Ryan, Plant Physiol., 1998, 117, 687. 55. J. Sun, Y. Xu, S. Ye, H. Jiang, Q. Chen, F. Liu, W. Zhou, R. Chen, X. Li, O. Tietz, X. Wu, J. D. Cohen, K. Palme, and C. Li, Plant Cell, 2009, 21, 1495.