A high-throughput fluorescence-based microplate reader assay to quantify total flavonol levels in plant tissues

Area of Science:

• Plant Science
• Biochemistry
• Metabolomics

Background:

• Flavonols are plant metabolites crucial for growth, development, and stress response, offering human health benefits due to antioxidant properties.
• Current quantification methods like HPLC and LC-MS are time-consuming and resource-intensive.
• Diphenylboric acid 2-aminoethyl ester (DPBA) is used for microscopy-based visualization but not typically for high-throughput quantification.

Purpose of the Study:

• To develop a rapid, high-throughput, fluorescence-based microplate reader assay for flavonol quantification in plant extracts.
• To provide a simpler, more efficient alternative to existing time-consuming and resource-intensive methods.

Main Methods:

• A fluorescence-based assay using the flavonol-specific dye DPBA to form fluorescent complexes with flavonols in plant extracts.
• Optimization of extraction efficiency, validation for sensitivity, accuracy, and reproducibility.
• Comparison of results with High-Performance Liquid Chromatography (HPLC) measurements.

Main Results:

• The assay demonstrated consistency with HPLC measurements.
• Flavonol levels were quantified in various tomato tissues (reproductive vs. vegetative), across cultivars, and between species.
• Variations in tomato fruit skin flavonol content were observed across different cultivars, indicating potential for genetic studies.

Conclusions:

• The developed assay is a simple, robust, and scalable tool for screening flavonol levels.
• It is suitable for applications in plant metabolism, environmental physiology, breeding, and metabolic engineering.
• The assay facilitates high-throughput analysis, accelerating research in these fields.