Development of a Matrix-Assisted Laser Desorption Ionization High Resolution Mass Spectrometry Method for the Quantification of Camalexin and Scopoletin in Arabidopsis thaliana

Abstract

Rationale

Understanding plant defense mechanisms against pathogens is essential for enhancing agricultural productivity and crop protection. This study focuses on the quantification of camalexin and scopoletin, two critical phytoalexins in Arabidopsis thaliana, using mass spectrometry techniques. Precise measurement of these compounds provides insights into plant resistance and supports agricultural research.

Methods

Camalexin and scopoletin were quantified using matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS). The matrix and solvent conditions were optimized to maximize sensitivity and accuracy. MS/MS experiments confirmed compound identification with high mass accuracy (mass error < 5 ppm). The method was validated through comparative analysis of wild-type (WT) and mutant Arabidopsis lines, using internal standards and multiple replicates to ensure precision and reliability.

Results

The method exhibited high linearity for scopoletin (R² = 0.9992) and camalexin (R² = 0.9987) across concentration ranges of 0.16–5 and 0.31–5 μM, respectively. Limits of detection (LOD) were 0.16 μM for camalexin and 0.04 μM for scopoletin, with limits of quantification (LOQ) at 0.2 μM and 0.08 μM, respectively. Samples analysis demonstrated reliable quantification in WT and mutant lines, with significant reductions in camalexin and scopoletin levels observed in the atwrky33-2 and atmyb15-1 mutants, respectively. Additionally, the method detected sub-physiological concentrations, confirming its sensitivity and robustness for low-level detection.

Conclusions

This study presents a validated, precise, and accurate MALDI-HRMS method for the quantification of camalexin and scopoletin in Arabidopsis thaliana. The approach not only enhances understanding of plant defense mechanisms but also offers potential applications for biotechnological and agricultural research, especially for investigating genetic variations and stress-induced phytoalexin production.