Background

Chlorophylls (Chls) are the most abundant organic pigment molecules on Earth. As components of photosynthesis machinery, Chls absorb light energy that is mostly used in photosynthesis. However, free Chl and most of its biosynthetic intermediates are particularly destructive once they become excited after light absorption [1]. These excited porphyrin molecules can interact with the surrounding ground-state oxygen molecule, leading to the highly reactive singlet oxygen (¹O₂) [2], which causes photooxidative damage and eventually programmed cell death (PCD) [3]. Thereby, the plant exerts strict control over tetrapyrrole biosynthesis [4,5]. In plants, Chl biosynthesis halts at the step of protochlorophyllide (Pchlide) generation due to the inhibitory effect of the FLU protein and resumes upon light illumination when Pchlide is photo-reduced to Chlide [6]. The Arabidopsis flu mutant over-accumulates Pchlide in the chloroplast in darkness and generates ¹O₂ when transferred to light due to the photosensitizing activity of the Pchlide molecules [7]. In the flu mutant, the level of generated ¹O₂ after dark-to-light shift is positively correlated with the amount of accumulated Pchlide, i.e., the duration of dark treatment [8]. However, when flu mutants are grown under continuous light, only a very low level of ¹O₂ is produced since Pchlide is immediately used for Chl biosynthesis, and the flu mutant grows exactly like the wild-type plants. These properties of the flu mutant make it an ideal tool for controlled generation of ¹O₂, and the exploration of this mutant has led to the identification of at least two chloroplastic ¹O₂-induced retrograde signaling pathways: the ¹O₂-EX1 pathway and ¹O₂-SAFE1 pathway [9–13].

Since Pchlide is a critical indicator of ¹O₂ level, precise quantification of its content is necessary not only for studies of flu-related mutants but also of plants suffering from environmental stresses [14–16]. In the etiolated flu seedlings, Pchlide accumulation can be directly visualized by its characteristic red fluorescence under blue light. However, Pchlide accumulation can hardly be detected in dark-incubated green leaves due to the interference of pigments, especially Chls, and is generally quantified using a fluorescence spectrophotometer [13,17] or high-performance liquid chromatography (HPLC) [11,18]. Compared with the former, HPLC-based Pchlide quantification is more accurate and reliable. Here, we provide a simple procedure for Pchlide extraction from Arabidopsis thaliana seedlings and a detailed workflow for Pchlide separation and detection using a fluorescence detector-equipped HPLC system, based on our recent publication [11]. Pchlide (DV-Pchlide) quantification is achieved based on fluorescence peak area and an experimentally derived standard curve.