Abstract
Homeostasis between the cytoplasmic plant hormone salicylic acid (SA) and its’ inactive, vacuolar storage forms, SA-2-O-β-D-glucoside (SAG) and SA-β-D-Glucose Ester (SGE), regulates the fine-tuning of defense responses to biotrophic pathogens in Arabidopsis thaliana. This protocol describes a simplified, optimized procedure to extract and quantify free SA and total hydrolyzable SA in plant tissues using a classical HPLC-based method.
Keywords: Salicylic acid, SA-glucoside, Defense hormone, Arabidopsis thaliana, HPLC
Background
SA (2-hydroxybenzoic acid) is a plant hormone, which is synthesized in the chloroplast in response to pathogen attack. It is then exported to the cytoplasm, where it establishes both local and systemic-acquired resistance (SAR). In a generalized scheme, plant resistance to biotrophic pathogens is thought to be mediated through SA signaling, whereas resistance to necrotrophic pathogens is controlled by jasmonic acid (JA) and ethylene (ET). SA and JA/ET signaling pathways interact antagonistically. SA accumulation to high concentrations is toxic and leads to cell- and tissue damage. Most pathogen-induced SA is thus glycosylated by UDP-glucosyltransferases (UGTs) to form hydrophilic, non-toxic SAG and SGE (Noutoshi et al., 2012; George Thompson et al., 2017). SAG and SGE are then sequestered in vacuoles, where they form reusable sources for hydrolysis to active SA. Increasing amounts of total SA (SA + SAG/SGE) in plant tissues thus reflect SA synthesis as a response to biotrophic pathogen attack. However, the amplitude of defense responses in infected plant tissues is determined by the amount of available cytoplasmic, unconjugated SA. To evaluate both the onset of SA-dependent defense responses and their amplitude, it is essential to quantify free and conjugated SA, respectively. This article describes a method for measuring conjugated and unconjugated SA levels in phase-partitioned extracts from A. thaliana seedlings. It is based on a protocol established for SA analysis in cucumber leaves (Meuwly and Métraux, 1993), which we optimized and downscaled for convenient, routine use.
Materials and Reagents
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Data analysis
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Acknowledgments
This protocol was modified from previously published works (Meuwly and Métraux, 1993; Quentin et al., 2016). The work was supported by the French Government (National Research Agency, ANR) through the ‘Investments for the Future’ LABEX SIGNALIFE [program reference #ANR-11-LABX-0028-01]. The authors declare no conflicts of interest or competing interests.
References
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Dear lingling xu,What is your question ? How to perform the calibartion curve ? if yes : you should read point E of the protocol everything is explained there, for the calibration curves with SALICYLATE from NA. Best regards
Dear Ihab Nazar Al-AbasiTFA is used to acidify the solution and protonate the carboxyl group of SA (and other organic acids), thereby discharging the group and making the molecules accessible for reverse phase chromatography.Best regards
Hello, if you have respected the starting proportion between the quantity of fresh materail and the volume of solvent at the beggining of extraction (2 extractions of the ground material) normally the quantity of residues obtained (of green color) after evaporation of the solvents, and speed vac less than 30 min must recover in the acidic MeOH. Try to add first 10 μL of 100% MeOH on the residues and then add 90 μL H2O containing 0.1% TFA, vortex 1 min.