Ortho-Anisic Acid as Internal Standard for the Simultaneous Quantitation of Salicylic Acid and Its Putative Biosynthetic Precursors in Cucumber Leaves

Abstract

Salicylic acid (SA) acts as an endogenous signal for the induction of systemic acquired resistance (SAR) in plants infected by pathogens. In order to study SA biosynthesis in plants, a quantitative method using an internal standard was developed for the simultaneous measurement of SA and three of its putative bioprecursors, namely trans-cinnamic acid (CA), trans-ortho-hydroxycinnamic acid (oHCA), and benzoic acid (BA). ortho-Anisic acid (oANI) was found to be a suitable internal standard for all four compounds. It was not present as endogenous compound in cucumber leaves, no substance interfered with its detection, and it followed identical losses as SA, CA, oHCA, and BA during the extraction procedure. Baseline separation of these four compounds and of eight other plant phenolics was achieved in 35 min using deactivated reversed-phase HPLC. On-line detection was performed fluorimetrically for SA, oANI, and oHCA (with the excitation and emission wavelengths optimized for each compound) and with uv detection for CA and BA. Correlation equations for SA, CA, oHCA, and BA using oANI as internal standard were linear for a broad range of concentrations. The limits of detection (in ng/g fresh weight, FW) obtained during routine analyses were as follows: SA, 4; oHCA, 100; CA, 150; and BA, 500. The method was used to quantify levels of free and acid-hydrolyzed bound SA in cucumber plants either 5 days postinoculation with Pseudomonas lachrymans or after mock-inoculation with water on their first leaf. Free SA increased 33-fold locally (to 382.0 ng/g FW) in the infected leaf and 4.2-fold systemically (to 21.3 ng/g FW) in the second leaf compared to controls. Bound SA reached 4399 and 287.3 ng/g FW in the first and second leaves of infected plants, respectively (not detectable in controls).