Simultaneous determination of coenzyme Q and plastoquinone redox states in the coral–Symbiodinium symbiosis during thermally induced bleaching
Introduction
There is growing concern over increasing ocean temperatures that threaten the health of coral reefs by disrupting the mutualistic partnership between reef-building corals (Cnidaria: Scleractinia) and their dinoflagellate (Symbiodinium sp.) partners causing coral bleaching (Hoegh-Guldberg and Bruno, 2010). Coral bleaching (loss of algal symbionts and/or pigments), which can affect entire reef systems over large areas (“mass-bleaching” events), has been consistently linked to high solar irradiance in combination with elevated maximum temperatures attributed to global climate change (Eakin et al., 2009). The consensus is that coral bleaching is a response to acute oxidative stress whereby excessive levels of reactive oxygen species (ROS) overwhelm the antioxidant defence capacity of the symbiosis (Lesser, 2011, Weis, 2008). Regardless of the primary impact sites of damage, coral bleaching is attributed to ROS formation by the electron transport chains (ETC) of coral mitochondria and Symbiodinium chloroplasts (Buxton et al., 2012, Weis, 2008). In addition to the indispensable roles of coenzyme Q (CoQ; ubiquinone) and plastoquinone (PQ) in electron transport for ATP production, their reduced forms ubiquinol (CoQH2) and plastoquinol (PQH2) have an important antioxidant function within mitochondrial (Ernster and Forsmark-Andrée, 1993), cellular (Bentinger et al., 2007) and thylakoid (Nowicka and Kruk, 2012) membranes. The CoQ pool redox state has been used as a sensitive plasma biomarker of oxidative stress in human disease (Yamamoto et al., 1998) ageing (Wada et al., 2007), and has been demonstrated to play a role in the hepatic response of fish exposed to polycyclic aromatic hydrocarbons (Hasbi et al., 2011). To investigate the significance of host and symbiont redox poise during bleaching, an analytical procedure was developed for simultaneous analysis of the CoQ and PQ pool redox states in corals. While similar methods have been reported for the determination of CoQ and PQ in the chloroplasts of Arabidopsis thaliana (Martinis et al., 2011, Yoshida et al., 2010), high performance liquid chromatography mass-spectrometry (LC–MS) methods applicable to invertebrate-algal symbiosis have not previously been available. In addition, as a proof of concept, the novel method described here, is used to monitor changes in the redox states of host CoQ and symbiont PQ in the scleractinian coral Acropora millepora (Ehrenberg, 1834) during bleaching induced by experimental thermal stress.
Section snippets
Reagents
Ubiquinone-9 (CoQ9), ubiquinone-10 (CoQ10), sodium borohydride, EDTA, formic acid, methanol (MeOH), ethanol (EtOH), isopropanol (IPA), hexane and ethyl acetate (EtOAc) were purchased from Sigma Aldrich (St. Louis, MO, USA). All solvents used were HPLC grade. Plastoquinone-9 (PQ9) was a kind gift of Professor Ewa Swiezewska from the Polish Academy of Sciences, Poland.
Collection and preparation of coral and Symbiodinium samples
A. millepora colonies roughly 50 cm in diameter containing type C2 Symbiodinium (ITS1 terminology, see below) were collected from
Results
Tandem mass spectrometry with standards provided unequivocal identification of both redox forms of CoQ10 (ubiquinone-10 and ubiquinol-10) and PQ9 (plastoquinone-9 and plastoquinol-9) in coral extracts. Host CoQ10 could have potentially been contaminated with CoQ10 from Symbiodinium as the length of the hydrophobic side chain of CoQ – the number of isoprenoid units – is species specific (Bentinger et al., 2007); however, analysis of cultured (type C1) and freshly isolated Symbiodinium (type C2)
Discussion
In this study, we present the first direct measurements of the redox states of CoQ and PQ in coral–Symbiodinium symbiosis based on quantitative LC–MS analysis. The method was found to be not only highly sensitive, but also ion-selective with respect to CoQ and PQ isoforms (Fig. 1), and could easily be adapted to measure the CoQ and PQ redox states in other cnidarians, as well as other systems (algae, higher plants, other animals), providing an alternative to existing methods. Previous analyses
Acknowledgements
This project was funded by the Australian Institute of Marine Science and the AIMS@JCU program of James Cook University. The research was performed under GBRMPA permit number G08/25734.1. [SS]
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