The interplay of light and oxygen in the reactive oxygen stress response of Chlamydomonas reinhardtii dissected by quantitative mass spectrometry

Abstract

Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox(II) was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O₂ and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.

Fig. 1.

Experimental setup. The interlinked experimental setup is defined by four treatments: treatment 1 (LL, AN), treatment 2 (HL, AR), treatment 3 (LL, AR), and treatment 4 (HL, AN). HL: high light, 200 μE/m⁻²/s⁻¹, or LL, low light 20–30 μE/m⁻²/s⁻¹, AR, normal atmosphere (80% N₂, 20% O₂, 500 ppm CO₂), and AN, anaerobic atmosphere (99.9995% N₂, 500 ppm CO₂). Two opposite cultures were metabolically labeled with ¹⁵N. Samples were treated for 5 h in minimal medium (HSM).

Fig. 2.

Fluorescence measurements and immunoblot analysis. A, PAM fluorescence yield measurements of cells after 5 h under treatments 1–4 (NPQ is indicated by the gray double arrow). B, Western blot (ECL) analysis using specific antibodies for LHCSR3 and CF1 (loading control). Samples for the labeled (¹⁵N) and unlabeled (¹⁴N) heterotrophic starting cultures (t = −1 h), after 1 h regeneration in HSM and photoautotrophic growth conditions (t = 0 h) and after 5 h treatment under treatments 1–4 (see Fig. 1) (t = 5 h) are shown. 40 μg per lane were loaded.

Fig. 3.

Venn diagram of peptides identified by different search engines. Venn diagram of the peptides identified (PEP < = 0.05) by the three search engines used: OMSSA (yellow), X! Tandem (red) and SEQUEST (blue). Peptide identifications for all three biological replicates and technical replicates (99 LC-MS/MS runs) were taken into account.

Fig. 4.

Node map and communities obtained by pyGCluster. Selected communities of the clustered proteins are shown (Nodemap illustrated as backdrop; full map can be found in supplemental Fig. S13). The heat map represents the ratios of the proteins ranges from black to green to yellow indicating an up-regulation and from black to red to purple indicating a down-regulation. The standard deviation is represented by the size of the box, the smaller the box, the higher the standard deviation of the protein ratio (see legend). For each protein in the heat maps, the JGI4 Augustus 10.2 tag, community protein frequencies (obCoFreq), the path difference (superscript indicate which ratio was added via path difference calculation) and a manually annotated name for protein description/classification are listed. Only clusters with a frequency higher than 1% in at least one DLC were considered and grouped into communities.

Fig. 5.

Comparison to transcriptome and proteome studies. Comparison of proteome data to transcriptome and proteome studies illustrated as heat map. Protein or DNA sequences from ROS communities (3860 and 13558) and light induced community (12472) were blasted (1E-20 cutoff) against the reference genomes/proteomes/EST library of C. reinhardtii or A. thaliana. Shown are the protein ratios from the study presented here (a, columns 1–4), ROS studies investigating A. thaliana by op den Camp et al. (15) (b, columns 6–8), Ramel et al. (23), (c, column 9) Lundquist et al. (24), (d, columns 10–11) ROS and light studies investigating C. reinhardtii by Ledford et al. (56), (e, column 12) by Fischer et al. (55), (e, columns 13–14) by Duanmu et al. (53) (g, column 15). Shown are JGI4 Augustus 10.2 tags or manually annotated protein names or domains.