Zeaxanthin Accumulation in the Absence of a Functional Xanthophyll Cycle Protects Chlamydomonas reinhardtii from Photooxidative Stress

Abstract

Xanthophylls participate in light harvesting and are essential in protecting the chloroplast from photooxidative damage. To investigate the roles of xanthophylls in photoprotection, we isolated and characterized extragenic suppressors of the npq1 lor1 double mutant of Chlamydomonas reinhardtii , which lacks zeaxanthin and lutein and undergoes irreversible photooxidative bleaching and cell death at moderate to high light intensities. Here, we describe three suppressor strains that carry point mutations in the coding sequence of the zeaxanthin epoxidase gene, resulting in the constitutive accumulation of zeaxanthin in a range of concentrations. The presence of zeaxanthin in these strains was sufficient to prevent photooxidative damage in the npq1 lor1 background. The size of the light-harvesting antenna in the suppressors decreased in high light in a manner that was proportional to the relative content of zeaxanthin, with the strain having the most zeaxanthin showing a severe reduction in levels of the major light-harvesting complex II proteins in high light. We show that the effect of constitutive zeaxanthin on light harvesting is not the main cause of increased photoprotection, because in the absence of zeaxanthin, a strain with a smaller light-harvesting antenna showed only minor protection against photobleaching in high light. Furthermore, the zeaxanthin-accumulating suppressors were able to tolerate higher levels of exogenous reactive oxygen than their parental strain under conditions that did not affect light harvesting. Our results are consistent with an antioxidant role of zeaxanthin in the quenching of singlet oxygen and/or free radicals in the thylakoid membrane in vivo.

A Mechanism of Nonphotochemical Energy Dissipation, Independent from PsbS, Revealed by a Conformational Change in the Antenna Protein CP26

“…Exposure to excess light, in addition to short-term energy dissipation, induces ROS synthesis, leading to upregulation of many genes (Rossel et al, 2002) and growth retardation (Wagner et al, 2004). Although these effects might, in principle, be relevant to the phenotypes we observed in high light conditions, we do not think these long term effects are responsible for the reduced growth of npq2 because this phenotype is most evident in very low light, a condition that is unlikely to promote ROS formation; moreover, zeaxanthin accumulation has been shown to further decrease the photooxidative damage (Baroli et al, 2003). Putative carotenoid cleaving enzymes have been shown to be involved in the synthesis of signal molecules affecting branching in Arabidopsis, implying that accumulation of its substrate might affect plant development (Sorefan et al, 2003).…”

“…Fluorescence quenching in LHC proteins has been reported as a result of binding of Zea versus Viola to the inner allosteric site L2 (Formaggio et al, 2001), whereas binding to the external site V1 (Caffarri et al, 2001) did not change the fluorescence yield of the complexes. The photoprotection effect of zeaxanthin in npq2 would then result from two components: (1) the increased singlet chlorophyll excited state dissipation suggested from the decreased fluorescence yield and slow growth and (2) the previously reported antioxidant effect of zeaxanthin released into the thylakoid membranes (Havaux and Kloppstech, 2001;Havaux and Niyogi, 1999;Baroli et al, 2003). This latter mechanism is clearly distinct from the fluorescence quenching effect described here because chlorophyll-carotenoid energy transfer requires van der Vaals distance between chromophores (Dexter, 1953), whereas xanthophylls bound to the site V1 cannot even operate energy transfer to chlorophyll (Caffarri et al, 2001), a process active at much longer distances (Forster, 1965).…”

Minor Antenna Proteins CP24 and CP26 Affect the Interactions between Photosystem II Subunits and the Electron Transport Rate in Grana Membranes of Arabidopsis

“…CP26 is a component of the PSII antenna in the most ancient green algae species in which photoprotection is mainly performed through Zea synthesis (Baroli et al, 2003) independently from qE (Ledford et al, 2007), which is strongly decreased in green algae with respect to higher plants (Finazzi et al, 2004). We propose that CP26, here shown to be largely responsible for qI, is specialized in Zea-mediated photoprotection (Dall'Osto et al, 2005).…”

Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta)

“…Twenty microliters of each extract was separated by HPLC on a Waters Spherisorb 1 5 mm ODS2 4.6 Â 250 mm analytical column (Waters, Milford, MA, USA) according to the method described by Ref. [14]. Pigments were eluted at a flow rate of 1.2 ml min À1 with a linear gradient from 100% solvent A (acetonitrile:methanol:0.1 M Tris-HCl, pH 8.0 [84:2:14]) to 100% solvent B (methanol:ethyl acetate [68:32]) for 15 min, followed by 20 min of solvent B.…”