AbstractUsing 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.