Fig. 1: Generation and initial characterisation of the mutant impaired in 1O2-signalling. | Communications Biology

Fig. 1: Generation and initial characterisation of the mutant impaired in 1O2-signalling.

From: Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas reinhardtii cell

Fig. 1

a Arylsulfatase (ARS2) assay for selection of the strain expressing GPX5-ARS2 reporter gene in a 1O2-inducible manner. The mutant selected for further applications was named signalling Reporter (sigRep). WT and chlD-1/GUN4 do not carry GPX5-ARS2 and were used as negative controls in ARS2 assay. b Expression kinetics of cytosolic (GPX5cyt) and chloroplast GPX5 (GPX5cp) in sigRep compared to WT upon exposure to light; the values were calculated as a fold change normalised to WT in dark (2-ΔΔCt, WT = 1). c Expression of GPX5-ARS2 in sigRep positively correlates with time of exposure to light. WT was used as a negative control. d ARS2 activity assay following mutagenesis of sigRep. Screen was performed to identify mutants not expressing GPX5-ARS2 compared to sigRep in the same conditions. The mutant selected for further applications was named genomes uncoupled Singlet Oxygen Signalling 1 (gunSOS1). WT and chlD-1/GUN4 were used as negative controls. e TSPP1 (Cre12.g497750) gene model. The insertion (bleR) was identified in the first exon of 3241 bp TSPP1. f Expression of TSPP1 in gunSOS1 compared to sigRep. g Expression of GPX5cyt and GPX5cp in gunSOS1 compared to sigRep. h Expression of GPX5-ARS2 in gunSOS1 compared to sigRep; WT was used as a negative control (ND, not detectable). For f–h (TSPP1, GPX5cyt, GPX5cp, and GPX5-ARS2) mRNA was determined 2 h after transfer from dark to light. Transcript analyses were performed by qRT-PCR on biological triplicates, values were calculated either as a fold change (2-ΔΔCt; normalised to the mean of Ctexp-Ctref of WT) or relative transcript level (normalised against a reference gene, calculated as 2ΔCt). For b and c, horizontal bars represent the calculated mean (n = 3), vertical error bars represent calculated ±SD; significant differences were calculated using two-tailed Student’s t-test and are indicated by asterisks (non-significant not shown), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. For f-h, error bars indicate calculated ±SD; one-way ANOVA, pair-wise comparison with the Tukey’s post-hoc test (non-significant not shown), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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