Chloroplast signaling involves mechanisms to relay information from chloroplasts to the nucleus, in order to change nuclear gene expression in response to environmental cues. Besides reactive oxygen species (ROS) produced under stress conditions, changes in the reduction/oxidation state of photosynthetic electron transfer components or coupled compounds in the stroma and the accumulation of photosynthesis-derived metabolites are likely origins of chloroplast signals. We attempted to investigate the origin of the signals from chloroplasts in mature Arabidopsis leaves by differentially modulating the redox states of the plastoquinone pool and components on the reducing side of photosystem I, as well as the rate of CO₂ fixation, while avoiding the production of ROS by excess light. Differential expression of several nuclear photosynthesis genes, including a set of Calvin cycle enzymes, was recorded. These responded to the stromal redox conditions under prevailing light conditions, but were independent of the redox state of the plastoquinone pool. The steady-state CO₂ fixation rate was reflected in the orchestration of the expression of a number of genes encoding cytoplasmic proteins, including several glycolysis genes and the trehalose-6-phosphate synthase gene, and also the chloroplast-targeted chaperone DnaJ. Clearly, in mature leaves the redox state of the compounds on the reducing side of photosystem I are of greater importance in light-dependent modulation of nuclear gene expression than the redox state of the plastoquinone pool, particularly at early signaling phases. It also became apparent that photosynthesis-mediated generation of metabolites or signaling molecules is involved in the relay of information from chloroplast to nucleus.