Molecular determinants involved in regulation of NAD(P)-ME of two members of the Panicoideae clade, Setaria itálica and Panicum virgatum.

CALACE, PAULA; ANDREO, CARLOS S; GERRARD WHEELER, MARIEL C.; SAIGO, MARIANA

Salta, Argentina

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

C4 photosynthesis has evolved to increase the photosynthetic efficiency in conditions where photorespiration would be enhanced. This was achieved through the introduction of a series of anatomical and biochemical features that allow the concentration of CO2 around Rubisco. In most C4 species, this is achieved through compartmentalizing the initial and definitive CO2 assimilation processes into two discrete cell types, namely mesophyll (M) and bundle sheath (BS) cells. As part of this mechanism a compound of four carbon atoms is transported from M to BS cells. Setaria italica (foxtail millet) and Panicum virgatum (switchgrass) are two members of the Panicoideae clade used for feed and biofuel production. Despite their evolutionary closeness, they have been classified into different C4 subtypes as they posseses a NADP or NAD-dependent malic enzyme as major malate decarboxylase within chloroplasts or mitochondria of BS cells, respectively. In this study we made progresses in the kinetic and structural characterization that distinguishes each decarboxylase enzyme operating in both grasses. Furthemore, we carried out a comparative analysis in order to identify the isoforms involved in the C4 cycle and to characterize the differences between the photosynthetic and the non-photosynthetic versions. These results were compared with those of other species previously studied by our group. To gain insight into the physiological roles of NAD(P)-MEs, the activity of recombinants enzymes in the malate decarboxylation direction was evaluated in the presence of different metabolites. The general analysis of the NAD-MEs responses shows some similarities with what was reported for the Arabidopsis enzymes. The NAD-ME type β (Seita.2G322000 and Pavir.2KG446000) responded positively to the presence of Krebs cycle intermediates such as citrate, fumarate, succinate and oxaloacetate and the NAD-ME type α (Seita.9G200600 and Pavir.9KG132400) were strongly activated by both Acetyl-CoA and CoA and by F1,6BP and PEP, both metabolites markers of the decrease in pyruvate glycolytic supply. These regulations agree with the functioning of NAD-ME as pyruvate-contributing enzymes when there is a decrease in glycolysis and a high intermediate content of the Krebs Cycle. Both the NADP-MEs of Setaria and P. virgatum studied showed inhibition by malate at pH 7, a characteristic linked to a photosynthetic function. In this work two other characteristics were found that would link NADP-ME of P. virgatum to photosynthesis. First, activation by ATP would imply that NADP-ME would be more active in the presence of light, at which time tilacoidal electron transport would boost ATP synthesis. On the other hand, aspartate inhibition would indicate that in the presence of large amounts of aspartate from CMs, the main decarboxylase would be mitocondrial NAD-ME.