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

The protochlorophyllide (Pchlide) level is a crucial indicator of plant fitness. Precise quantification of Pchlide content is necessary not only in studies of <i>flu</i>-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 <i>Arabidopsis thaliana</i> 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.