Characterization of differences in N assimilation and deficiency responses in C3 and C4 Cleome species The evolution of C4 photosynthesis led to increased carbon assimilation rates and plant growth. In contrast to C3 plants, which use Rubisco for primary CO2 fixation in all photosynthetic cell types, in C4 plants carbon is initially fixed in mesophyll cells (MCs) by PEP carboxylase (PEPC). Subsequent decarboxylation of the resulting C4 acid takes place in the bundle sheath cells (BSCs). This increases the local CO2 concentration around Rubisco, which minimizes its oxygenation reaction reducing photorespiration and thereby improving photosynthetic efficiency. Similarly, in most C4 plants the reduction of nitrate and nitrite is restricted to MCs, while the further assimilation of reduced nitrogen into the amino acids glutamate and glutamine mainly takes place in the BSCs. Changes in the expression pattern of glycine decarboxylase (GDC) lead to the restriction of photorespiration and therefore serine production to BSCs. Balancing of metabolic routes is therefore necessary to maintain homeostasis of not only carbon but also nitrogen (N) metabolism. These differences in N requirements of the two photosynthetic mechanisms pose the question of how N assimilation pathways differ between C3 and C4 species and whether these changes are a consequence or prerequisite of C4 evolution. Characterization of mineral nutrition traits revealed clear differences in the N metabolism of C3 and C4 Cleome species under N deficiency conditions, indicating a higher N deficiency tolerance in the C4 species. The addition of NH4+ was shown to offset the reduction of biomass and photosynthetic efficiency in the C3 but not the C4 Cleome species.