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

Abstract

Background Flavonols are plant specialized metabolites that regulate plant growth, development, and stress responses. Due to their antioxidant activity, they also confer nutritional benefits to human health. Quantification of flavonols in plant tissues typically relies on chromatographic methods such as HPLC or LC-MS, or on microscopy-based approaches using diphenylboric acid 2-aminoethyl ester (DPBA) staining to visualize flavonols in plant tissues. These methods are time-consuming and resource intensive. Here, we present a rapid, high-throughput, fluorescence-based microplate reader assay for flavonol quantification, in which the flavonol-specific dye DPBA is added to plant extracts to form fluorescent complexes. Results The assay was optimized for extraction efficiency and validated for sensitivity, accuracy, and reproducibility. It also shows consistency with HPLC measurements. We demonstrate its utility by quantifying flavonol levels in different tomato tissues, across different cultivars, and even between plant species. Our assay showed that reproductive tissue in tomato plants has higher flavonol levels than vegetative tissue. Also, we found variation in flavonol levels in tomato fruit skin across different laboratory and commercial cultivars, suggesting that our approach shows promise for use in genome-wide association studies to identify genetic factors underlying variation in flavonol levels. Lastly, we measured flavonol levels in dry leaves of different plants used for brewed beverages. Conclusion In conclusion, the assay represents a simple, robust, and scalable flavonol screening tool for studies in plant metabolism, environmental physiology, breeding, and metabolic engineering.

Plant phenotyping relevance

植物組織中のフラボノール量を定量する高スループット測定法を開発し、感度・精度・再現性およびHPLCとの一致を検証している。単なる代謝測定ではなく、植物育種・代謝研究向けのスクリーニングツールとして方法自体が中心である。

Code and data availability

The article describes a DPBA fluorescence microplate assay for flavonol quantification, but no public phenotype dataset, image/sensor data, analysis code, or trained model is deposited. The Data availability statement says all data are in the article/supplement and available only from the corresponding author onrequest

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