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

Using a mutant screen, we identified trehalose 6-phosphate phosphatase 1 (TSPP1) as a functional enzyme dephosphorylating trehalose 6-phosphate (Tre6P) to trehalose in Chlamydomonas reinhardtii. The tspp1 knock-out results in reprogramming of the cell metabolism via altered transcriptome. As a secondary effect, tspp1 also shows impairment in 1O2-induced chloroplast retrograde signalling. From transcriptomic analysis and metabolite profiling, we conclude that accumulation or deficiency of certain metabolites directly affect 1O2-signalling. 1O2-inducible GLUTATHIONE 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, recovers 1O2-signalling and GPX5 expression in otherwise aconitate-deficient tspp1. Genes encoding known essential components of chloroplast-to-nucleus 1O2-signalling, PSBP2, MBS, and SAK1, show decreased transcript levels in tspp1, which also can be rescued by exogenous application of aconitate. We demonstrate that chloroplast retrograde signalling involving 1O2 depends on mitochondrial and cytosolic processes and that the metabolic status of the cell determines the response to 1O2.

Interactions Between Carbon Metabolism and Photosynthetic Electron Transport in a Chlamydomonas reinhardtii Mutant Without CO2 Fixation by RuBisCO.

A Chlamydomonas reinhardtii RuBisCO-less mutant, ΔrbcL, was used to study carbohydrate metabolism without fixation of atmospheric carbon. The regulatory mechanism(s) that control linear electron flow, known as "photosynthetic control," are amplified in ΔrbcL at the onset of illumination. With the aim to understand the metabolites that control this regulatory response, we have correlated the kinetics of primary carbon metabolites to chlorophyll fluorescence induction curves. We identify that ΔrbcL in the absence of acetate generates adenosine triphosphate (ATP) via photosynthetic electron transfer reactions. Also, metabolites of the Calvin Benson Bassham (CBB) cycle are responsive to the light. Indeed, ribulose 1,5-bisphosphate (RuBP), the last intermediate before carboxylation by Ribulose-1,5-bisphosphate carboxylase-oxygenase, accumulates significantly with time, and CBB cycle intermediates for RuBP regeneration, dihydroxyacetone phosphate (DHAP), pentose phosphates and ribose-5-phosphate (R5P) are rapidly accumulated in the first seconds of illumination, then consumed, showing that although the CBB is blocked, these enzymes are still transiently active. In opposition, in the presence of acetate, consumption of CBB cycle intermediates is strongly diminished, suggesting that the link between light and primary carbon metabolism is almost lost. Phosphorylated hexoses and starch accumulate significantly. We show that acetate uptake results in heterotrophic metabolism dominating phototrophic metabolism, with glyoxylate and tricarboxylic acid (TCA) cycle intermediates being the most highly represented metabolites, specifically succinate and malate. These findings allow us to hypothesize which metabolites and metabolic pathways are relevant to the upregulation of processes like cyclic electron flow that are implicated in photosynthetic control mechanisms.