Reaction #1876809

Reaction equation

o-anisic acid

→

Salicylic Acid

Solvents

methanol

Conditions

Workup

1. 1
   ExtractionSalicylic acid was extracted
2. 2
   Otherorontii-infected leaves (7 dpi)
3. 3
   Temperaturewas frozen in glass tubes with liquid nitrogen
4. 4
   Otherstored at −80° C
5. 5
   Othersonicated for 20 minutes
6. 6
   OtherThe supernatant was transferred to a new tube
7. 7
   Extractionthe pellet was re-extracted with 2 ml of 90% methanol
8. 8
   Otherdried
9. 9
   workup.ADDITIONFor total SA, 500 μl β-glucosidase (80 U/ml in 100 mM sodium acetate, pH 5.2; Sigma, St. Louis, Mo.) was added to each sample
10. 10
    OtherThe samples were sonicated for 5 minutes
11. 11
    workup.WAITincubated for 90 minutes at 37° C
12. 12
    workup.ADDITIONFor both total and free SA samples, 2.5 ml 5% trichloroacetic acid was added
13. 13
    Othersonicated for 5 minutes
14. 14
    workup.WAITcentrifuged at 3000 rpm for 15 minutes
15. 15
    ExtractionThe supernatant was extracted twice with 2.5 ml of a 1:1 (v/v) mixture of ethyl acetate
16. 16
    Othervacuum dried
17. 17
    Temperaturefrozen at −80° C
18. 18
    Othersonicated for 5 minutes
19. 19
    Filtrationfiltered through a 0.22 μm nylon
20. 20
    Filtrationfilter

Procedure

Salicylic acid was extracted and analyzed by HPLC using a modification of the methods described in Meuwly et al., 1993. For salicylic acid analysis, 0.3 to 0.5 gFW leaf tissue from E. orontii-infected leaves (7 dpi) was frozen in glass tubes with liquid nitrogen and either used directly or stored at −80° C. The frozen tissue was ground in liquid nitrogen to a fine powder using a chilled glass rod. Three ml of 90% methanol and 250 ng o-anisic acid (internal standard) were added to each sample. Samples were vortexed, sonicated for 20 minutes, and centrifuged for 20 minutes at 3000 rpm in a table-top centrifuge. The supernatant was transferred to a new tube, and the pellet was re-extracted with 2 ml of 90% methanol. The two supernatants were combined, divided into two portions of equal volumes (for total SA and free SA measurements), vacuum dried, and frozen at −80° C. For total SA, 500 μl β-glucosidase (80 U/ml in 100 mM sodium acetate, pH 5.2; Sigma, St. Louis, Mo.) was added to each sample. The samples were sonicated for 5 minutes, vortexed, covered with foil, and incubated for 90 minutes at 37° C. For both total and free SA samples, 2.5 ml 5% trichloroacetic acid was added, and the samples were vortexed, sonicated for 5 minutes, and centrifuged at 3000 rpm for 15 minutes. The supernatant was extracted twice with 2.5 ml of a 1:1 (v/v) mixture of ethyl acetate:cyclopentane. The organic phases were combined, vacuum dried, and frozen at −80° C. Just prior to loading samples on the HPLC, each was resuspended in 250 μl of 20% methanol, vortexed, sonicated for 5 minutes, and filtered through a 0.22 μm nylon filter.

Source