2010 32 5 1056-1060 http / /xuebao. jxau. edu. cn Acta Agriculturae Universitatis Jiangxiensis E - mail ndxb7775@ sina. com * / 510642 JA SA SA JA GC JA SA / 50 mmol /L = 7 /3 JA SA - HS - SPME FID 60 1 min 250 15 /min 250 3 min MeJA MeSA SA JA - Q948 A 1000-2286 2010 05-1056 - 05 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 510642 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 2010-07 - 20 30870390 1982 * 1965 1985 6 1988 9 Annual Revlew of Entomdogy Journal of Chemical Ecology Allelopathy Journal 30 7 70 SCI 26 Springer E-mail rszeng@ scau. edu. cn
5 1057 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 1 1. 1 Lycopersicon esculentum Mill. cv. Jin Bao 10% H 2 O 2 8 min 10 d 24 cm 18 cm 12 cm 24 h 3 1 1 5 mm 2 h Fig. 1 Trapping of methylated plant signaling compounds on Tenax filters 2 mm 170 ~ 180 2 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 3 2 1 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 3 1. 2 45 d - 80 1. 3 SA JA 7 1. 3. 1 SA JA Engelberth and Engelberth Engelberth et al 10 0. 05 ~ 0. 15 g 2 ml
1058 32 1 ml /50 mmol /L = 7 /3 FastPrep 4. 5 m /s 30 s 1 ml 4. 5 m/s 15 s 4 ml N 2 1. 3. 2 100 μl / = 9 /1 4 μl 2 mmol /L 30 min 30 4 μl 2 mol /L 30 min Tenax 50 mg 6 mm10 cm 1 80 3 min 300 μl - 20 GC 1. 3. 3 300 μl 300 μl 2 N 2 1. 3. 4 MeJA MeSA HPLC 1 000 20 μg /ml MeJA Sigma - Aldrich 1 000 400 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 1. 3. 5 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 1. 3. 6 GC HP - 5 30 m 320 μm 0. 25 μm 60 1 min15 /min 250 3 min 280 300 N 2 1 ml /min FID 1 μl 1. 3. 7 JA SA 0 100 200 400 ng JA SA GC 2 2. 1 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. 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 7. 532 11. 250 Area = 30. 120 012 1 Amt - 25. 753 772 Area = 37. 343 753 1 Amt - 5. 068 504 5 0. 5 ~ 40. 0 0. 999 21 0. 100 030 0. 225 ~ 18. 000 0. 999 86 0. 080 680 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 4 3 1 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 12-13 ELISA 14 LC LC - MS /MS JA SA 15
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