Abstract
BackgroundWhen photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed.ResultsMutants with altered GPXH response were isolated from UV-mutagenized cells containing a GPXH-arylsulfatase reporter gene construct. Out of 5500 clones tested, no mutant deficient in GPXH induction was isolated, whereas several clones showed constitutive high GPXH expression under normal light conditions. Many of these GPXH overexpressor (gox) mutants exhibited higher resistance to oxidative stress conditions whereas others were sensitive to high light intensities. Interestingly, most gox mutants produced increased singlet oxygen levels correlating with high GPXH expression. Furthermore, different patterns of altered photoprotective parameters like non-photochemical quenching, carotenoid contents and α-tocopherol levels were detected in the various gox mutants.ConclusionsScreening for mutants with altered GPXH expression resulted in the isolation of many gox mutants with increased singlet oxygen production, showing the relevance of controlling the production of this ROS in photosynthetic organisms. Phenotypic characterization of these gox mutants indicated that the mutations might lead to either stimulated triplet chlorophyll and singlet oxygen formation or reduced detoxification of singlet oxygen in the chloroplast. Furthermore, changes in multiple protection mechanisms might be responsible for high singlet oxygen formation and GPXH expression, which could either result from mutations in multiple loci or in a single gene encoding for a global regulator of cellular photoprotection mechanisms.
Highlights
When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast
To identify components affecting 1O2-induced gene expression in C. reinhardtii, a mutant screen was performed using the GPXH-arylsulfatase (GPXH-ARS) reporter gene construct pYSn1 to search for clones with altered GPXH response [21]
A total number of 5500 clones were analyzed for their GPXHARS expression under medium light (ML) condition of 80 μmol photons m-2 s-1 in the presence or absence of 1 μM neutral red (NR)
Summary
When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. Excess light can lead to the increased production of reactive oxygen species (ROS) which can damage cellular components such as lipids, proteins and DNA. Increased levels of these xanthophylls together with the protonation of specific pigment-binding antenna proteins cause a conformational change of PSII into a high quenching state where excess light energy is dissipated as heat [5]. Two LHCSR3 genes have been found to be involved in NPQ in Chlamydomonas reinhardtii indicating that other unidentified components might function in photoprotection and prevention of 1O2 formation in photosynthetic organisms [10]
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