Using a mutant screen, we identified trehalose 6-phosphate phosphatase 1 (TSPP1) as a functional enzyme dephosphorylating trehalose 6-phosphate (Tre6P) to trehalose inChlamydomonas reinhardtii. Thetspp1knock-out results in reprogramming of the cell metabolism via altered transcriptome. As a secondary effect,tspp1also shows impairment in1O2-induced chloroplast retrograde signalling. From transcriptomic analysis and metabolite profiling, we conclude that accumulation or deficiency of certain metabolites directly affect1O2-signalling.1O2-inducibleGLUTATHIONE PEROXIDASE 5(GPX5) gene expression is suppressed by increased content of fumarate and 2-oxoglutarate, intermediates in the tricarboxylic acid cycle (TCA cycle) in mitochondria and dicarboxylate metabolism in the cytosol, but also myo-inositol, involved in inositol phosphate metabolism and phosphatidylinositol signalling system. Application of another TCA cycle intermediate, aconitate, recovers1O2-signalling andGPX5expression in otherwise aconitate-deficienttspp1. Genes encoding known essential components of chloroplast-to-nucleus1O2-signalling, PSBP2, MBS, and SAK1, show decreased transcript levels intspp1, which also can be rescued by exogenous application of aconitate. We demonstrate that chloroplast retrograde signalling involving1O2depends on mitochondrial and cytosolic processes and that the metabolic status of the cell determines the response to1O2.