Abstract
Hydroxylation of chlorophyll catabolites at the so-called C32 position (Hauenstein et al., 2016) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the substrate epi-pFCC (epi-primary Fluorescent Chlorophyll Catabolite) (Mühlecker et al., 2000) to epi-pFCC-OH.
Keywords: TIC55 (Translocon at the inner chloroplast membrane 55 kDa), Chlorophyll breakdown, PAO/phyllobilin pathway, Senescence, Chlorophyll catabolites, Phyllobilins
Background
During leaf senescence and fruit ripening, light-absorbing chlorophylls are degraded to non-fluorescent catabolites to prevent oxidative damage. The chlorophyll breakdown pathway (PAO/phyllobilin pathway) consists of consecutive steps catalyzed by several enzymes and the final degradation products, called phyllobilins, are ultimately stored in the vacuole (Kräutler, 2016). epi-primary Fluorescent Chlorophyll Catabolite (epi-pFCC) is the first non-phototoxic intermediate. After its formation in the chloroplast, side-chain modifications of epi-pFCC can occur, most of which take place outside the chloroplast. One of these modifications, however, is the hydroxylation of the C32 position (Figure 1) catalyzed by the inner chloroplast envelope enzyme TIC55, a member of the family of ferredoxin (Fd)-dependent non-heme oxygenases. TIC55 contains a Rieske and a mononuclear iron-binding domain and was shown to require a Fd reducing system as well as molecular oxygen for its hydroxylating activity. Here we describe an in vitro enzyme assay for TIC55, which was used to characterize the epi-pFCC hydroxylating enzyme activity from red pepper chromoplasts.Figure 1. Outline of the pathway of chlorophyll breakdown, highlighting the TIC55-catalyzed reaction from epi-pFCC to epi-pFCC-OH. The circle shows the C32 position, the site of hydroxylation.
Materials and Reagents
Equipment
Procedure
Data analysis
Data was analyzed by LC-MS/MS as described (Christ et al., 2016). Figure 3 shows an example of the time-dependent formation of epi-pFCC-OH from epi-pFCC using the here described hydroxylation assay. Figure 4 shows MS/MS experiments of both epi-pFCC and epi-pFCC-OH that are characteristic for both compounds and help for their identification in LC-MS/MS experiments. Ideally, assays are performed with a minimum of three replicates to allow for statistical analysis of the results. Figure 3. Time dependent formation of epi-pFCC-OH. The formation of epi-pFCC-OH over a time course of 60 min was measured by LC-MS. Shown are extracted ion chromatograms for the masses 629 m/z (epi-pFCC) and 645 m/z (epi-pFCC-OH). Figure 4. MS/MS fractionation pattern of epi-pFCC (629 m/z; bottom) and its hydroxylated form (645 m/z; top). See Table 1 for MS/MS characteristics of both compounds. Table 1. Differences between epi-pFCC and epi-pFCC-OH in mass, chemical formula and three characteristic MS/MS fragments
Recipes
Acknowledgments
This work was supported by the Swiss National Science Foundation (grant # 31003A_149389/1). This protocol was adapted from previous work (Hauenstein et al., 2016).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.