Simultaneous Quantification of Jasmonic and Salicylic Acids in Tomato Plants by Gas Chromatography

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Ευλάλιος Λύκος
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1 http / /xuebao. jxau. edu. cn Acta Agriculturae Universitatis Jiangxiensis E - mail ndxb7775@ sina. com * / JA SA SA JA GC JA SA / 50 mmol /L = 7 /3 JA SA - HS - SPME FID 60 1 min /min min MeJA MeSA SA JA - Q948 A N 2 Simultaneous Quantification of Jasmonic and Salicylic Acids in Tomato Plants by Gas Chromatography SONG Yuan-yuan XU Jian-feng LIANG Xiao-ting SU Yi-juan XIE Li-jun ZENG Ren-sen * Key Laboratory of Ecological Agriculture /Ministry of Agriculture Institute of Tropical and Subtropical E- cology South China Agricultural University Guangzhou China Abstract Jasmonic and salicylic acids are important phytohormones and plant defensive signaling compounds. Here a new method was developed for the simultaneous quantification of the two signaling compounds in tomato leaves by using headspace - solid phase microextraction HS - SPME and gas chromatography with hydrogen ion flame detector FID. Leaf samples were extracted by mixture of acetone and citric acid 50 mmol /L V /V = 7 /3 and ethyl acetate. Then the supernatant was dried by N 2 and subsequently methylated with trimethylsilyldiazomethane. The volatilized compounds were collected by using headspace - solid * Annual Revlew of Entomdogy Journal of Chemical Ecology Allelopathy Journal SCI 26 Springer rszeng@ scau. edu. cn

2 phase microextraction on Tenax adsorbents and eluted with n - hexane. Eluted samples were analyzed by u- sing GC with hydrogen ion flame detector FID. The temperature gradient was increased from 60 1 min to 250 in a rate of 15 /min and held on 3 min at 250. The final chromatographic peaks of JA and SA in the samples were identical to the authentic compounds. This method will be useful for simultaneous quantification of JA and SA in plants. Key words gas chromatograph signal compounds jasmonic acid salicylic acid methylation headspace - solid phase microextraction 1 Salicylic acid SA Jasmonic acid JA 2-3 SA systemic acquired resistance SAR JA MeJA 4 SA JA SA JA ELISA 5 - HPLC - MS /MS 6 - GC - MS 7-9 GC SA JA Lycopersicon esculentum Mill. cv. Jin Bao 10% H 2 O 2 8 min 10 d 24 cm 18 cm 12 cm 24 h mm 2 h Fig. 1 Trapping of methylated plant signaling compounds on Tenax filters 2 mm 170 ~ h 25 ± 1 16 h 150 μe m 2 s 8 h 60% 7 d 50 ml 5 ml 1 mol /L KNO 3 5 ml 1 mol /L Ca NO ml 1 mol /L MgSO 4 2 ml 1 mol /L KH 2 PO 4 1 ml H 3 BO 3 1 ml MnCl 2 1 ml Zn- SO 4 1 ml CuSO 4 1 ml Fe - EDTA 1 L d SA JA SA JA Engelberth and Engelberth Engelberth et al ~ g 2 ml

3 ml /50 mmol /L = 7 /3 FastPrep 4. 5 m /s 30 s 1 ml 4. 5 m/s 15 s 4 ml N μl / = 9 /1 4 μl 2 mmol /L 30 min 30 4 μl 2 mol /L 30 min Tenax 50 mg 6 mm10 cm min 300 μl - 20 GC μl 300 μl 2 N MeJA MeSA HPLC μg /ml MeJA Sigma - Aldrich μg /ml MeSA Sigma - Aldrich 20 μg /ml MeJA 400 μg /ml MeSA MeJA /MeSA = 9 /1 18 μg /ml MeJA 40 μg /ml MeSA - 20 GC JA SA 25 μl 80 μg /ml JA 125 μl 160 μg /ml SA 100 μl 20 μg /ml MeJA 200 μg /ml MeSA - 20 GC GC HP m 320 μm μm 60 1 min15 /min min N 2 1 ml /min FID 1 μl JA SA ng JA SA GC JA SA MeJA MeSA MeJA MeSA 2 JA SA MeJA MeSA Fig. 2 2 MeJA MeSA JA SA Profiles of GC chromatography of authentic MeJA MeSA and JA derived MeJA and SA derived MeSA in tomato leaves

5 1059 2. 2 GC - FID JA SA 0. 225 ~ 18 ng /μl 0. 5 ~ 40 ng /μl r 0. 999 2 0. 999 8 JA LOD 0. 08 ng /μlsa LOD 0. 1 ng /μl 1 Kallenbach et al 11 Tab.

4 GC - FID JA SA ~ 18 ng /μl 0. 5 ~ 40 ng /μl r JA LOD ng /μlsa LOD 0. 1 ng /μl 1 Kallenbach et al 11 Tab. 1 1 GC - FID JA SA Retention time linear regression equation and limit of detection of JA and SA detected by GC - FID /min /Compound Retention time Linear regression / ng μl - 1 Coefficient / ng / μl - 1 a Area = Amt Area = Amt ~ ~ JA SA 65% 90% 3 Schmelz et al 8 GC - MS JA SA 80% 96% SA JA Fig. 3 3 JA SA Recovery rates of jasmonic and salicylic acids in the GC analysis 2. 3 MeJA MeSA MeJA MeSA 95% MeJA MeSA 300 μl 600 μl 3 JA SA JA SA 4 MeJA MeSA μg ~ ng /g Fig. 4 Effects of adsorbent reuse times on the adsorbability JA of the adsorbent on MeJA and MeSA 7 JA SA GC - MS ELISA 14 LC LC - MS /MS JA SA 15

5 JA SA 3 JA SA ELISA 5 HPLC - MS JA SA 6 GC - FID GC JA SA GC JA SA Engelberth et al 10 / 50 mmol /L = 7 / 3 JA SA 50 mmol /L / = 3 /7 9 Engelberth / / = 2 /1 / JA SA Wu et al 6 JA SA / 50 mmol /L = 7 /3 JA SA GC SA JA GC JA SA r JA SA GC - FID JA SA ng /μl 0. 1 ng /μl JA SA 1 Van Dam N M. How plants cope with biotic interactions J. Plant Biol Kazan K Manners J M. Jasmonate signaling toward an integrated view J. Plant Physiol Loake G Grant M. Salicylic acid in plant defence - the players and protagonists J. Curr Opin Plant Biol Hummel G M Schurr U Baldwin I T et al. Herbivore - induced jasmonic acid bursts in leaves of Nicotiana attenuata mediate short - term reductions in root growth J. Plant Cell Environ Xin Z Y Zhou X Zhang N G. The development of an indirect ELISA for jasmonic acid J. J Nanjing Agric Univ Wu J Q Hettenhausen C Medlau S et al. Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata J. Plant Cell Engelberth M J Engelberth J. Monitoring plant hormones during stress responses J. J Vis Exp Schmelz E A Engelberth J Alborn H T et al. Simultaneous analysis of phytohormones phytotoxins and volatile organic compounds in plants J. Proc Natl Acad Sci USA Engelberth J Schmelz E A Alborn H T et al. Simultaneous quantifi cation of jasmonic acid and salicylic acid in plants by vapor - phase extraction and gas chromatography - chemical ionization - mass spectrometry J. Anal Biochem Engelberth J Koch T Schuler G et al. Ion channel - forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling. Cross talk between jasmonate and salicylate signaling in lima bean J. Plant Physiol Kallenbach M Baldwin I T Bonaventure G. A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC - MS /MS J. Plant Method Weber H Vick B A Farmer E E. Dinor - oxo - phytodienoic acid A new hexadecanoid signal in the jasmonate family J. Proc Natl Acad Sc USA Mueller M J Brodschelm W. Quantification of jasmonic acid by capillary gas chromatography - negative chemical ionization - mass spectrometry J. Anal Biochem Albrechet T Kehlen A Stahl K et al. Quantification of rapid transient increases in jasmonic acid in wounded plants u- sing a monoclonal antibody J. Planta Tamogami S Kodama O. Quantifi cation of amino acid conjugates of jasmonic acid in rice leaves by high - performance liquid chromatography turboionspray tandem mass spectrometry J. J Chromatogr A Lan Y P Han Z H Xu X F. Accumulation of jasmonic acid in apple seedlings under water stress J. Acta Horti Sin Liu Y Pan Q H Zhan J C et al. Response of endogenous salicylic acid and jasmonates to mechanical injury in pea leaves J. Sci Agr Sin

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