Flow-Batch Sample Preparation for Fractionation of the Stress Signaling Phytohormone Salicylic Acid in Fresh Leaves.

Thiago L Marques,Lidiane C Nunes,Milton K Sasaki,Fábio R P Rocha

doi:10.1155/2020/8865849

Abstract

Salicylic acid (SA) is an important stress signaling phytohormone and plays an essential role in physiological processes in plants. SA fractionation has been carried out batchwise, which is not compatible with the high analytical demand in agronomical studies and increases susceptibility to analytical errors. In this context, a novel flow-batch sample preparation system for SA fractionation on fresh plant leaves was developed. It was based on microwave-assisted extraction with water and conversion of the conjugated species to free SA by alkaline hydrolysis. Free and total SA were quantified by fluorimetry after separation by sequential injection chromatography in a C18 monolithic column. The proposed procedure is directly applicable to plant leaves containing up 16 mg kg−1 SA, with a limit of detection of 0.1 mg kg−1 of SA, coefficient of variation of 3.0% (n = 10), and sampling rate of 4 samples h−1. The flow-batch sample preparation system was successfully applied to SA fractionation in sugarcane, corn, and soybean leaves without clogging or increasing in backpressure. The proposed approach is simple, less time-consuming, and more environmentally friendly in comparison to batchwise procedures.

Highlights

Salicylic acid (SA) is an important phytohormone in plants, which acts as stress signaling, as well as on seed germination, seedling formation, plant respiration, legumes nodulation, fruits production, and defense against pathogens [1,2,3]
Chromatographic techniques are usually applied for this task, including high performance liquid chromatography (HPLC) with detection by mass spectrometry (MS) [6], tandem mass spectrometry (MS/MS) [7], fluorimetry [8] or spectrophotometry [9], ultra high performance liquid chromatography coupled to MS/MS [4], or gas chromatography (GC) coupled to MS [1, 10]. ese alternatives are relatively expensive and sample preparation involves several steps, which increase sample handling and susceptibility to analytical errors [4, 6, 7]
General Aspects. e fractionation method exploited in this work is based on the measurement of the intrinsic fluorescence of salicylate ion

Summary

Introduction

Salicylic acid (SA) is an important phytohormone in plants, which acts as stress signaling, as well as on seed germination, seedling formation, plant respiration, legumes nodulation, fruits production, and defense against pathogens [1,2,3]. Microwave-assisted flow-batch extraction was carried out with 2 mL of deionized water introduced in the tube by the peristaltic pump of the SIA system (30 s, 4 mL min−1). Multivariate optimization was carried out to maximize free and total SA peak areas, which are indicative of the efficiency of analyte extraction and hydrolysis.

Results

Conclusion