The C4 cycle and beyond: diverse metabolic adaptations accompany dual-cell photosynthetic functions in Setaria

C-4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C-4 acids, which are transported to the BS cells. There, C-4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C-4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C-4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C-4 carbon shuttle. Secondly, we compared the proteomes of M and BS compartments and found 825 differentially accumulated proteins that could support different metabolic scenarios. Most interestingly, we found evidence of metabolic strategies to insulate the C-4 core avoiding the leakage of intermediates by either up-regulation or down-regulation of chloroplastic, mitochondrial, and peroxisomal proteins. Overall, the results presented in this work provide novel data concerning the complexity of C-4 metabolism, uncovering future lines of research that will undoubtedly contribute to the expansion of knowledge on this topic.