Quantification of Protochlorophyllide (Pchlide) Content in Arabidopsis Seedlings Using a High-Performance Liquid Chromatography (HPLC) System.

Abstract

The protochlorophyllide (Pchlide) level is a crucial indicator of plant fitness. Precise quantification of Pchlide content is necessary not only in studies of flu -related mutants that over-accumulate Pchlide in the dark but also for research on plants suffering from environmental stresses. Due to its low content and interference of chlorophylls, quantitative determination of Pchlide content is a challenge. Here, we describe an optimized protocol for Pchlide extraction from Arabidopsis thaliana seedlings and subsequent analysis using high-performance liquid chromatography (HPLC) coupled with fluorescence detection. Divinyl-Protochlorophyllide (DV-Pchlide, the major form of Pchlide in plants) quantification is achieved by interpolating fluorescence peak areas against an experimentally derived standard curve. This protocol provides a reliable workflow for Pchlide quantification, facilitating the deciphering of the underlying mechanism of plant environmental resilience. Key features • This method adopts acetone as a solvent for both Pchlide extraction and HPLC run. • This protocol adopts a gradient HPLC system equipped with a fluorescence detector. • This protocol applies an experimentally derived standard calibration curve using synthetic DV-Pchlide.

Plant phenotyping relevance

植物のフィットネスや環境ストレス状態に関連するPchlide量を、抽出・HPLC・蛍光検出・標準曲線で定量する手法を中心に提示しており、単なる生物学的実験のルーチン測定ではない。

Code and data availability

This is a Bio-protocol methods article describing an HPLC-based Pchlide quantification protocol. The text contains no public phenotype/trait datasets, no deposited plant images or sensor data, no author analysis code or workflows, and no trained models. The only data shown are example standard-curve peak areas embedded

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