Biological nitrogen fixation maintains carbon/nitrogen balance and photosynthesis at elevated CO₂

Understanding crop responses to elevated CO 2 is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO 2 due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO 2 due to their ability to exchange carbon for nitrogen. Here, the effects of biological nitrogen fixation (BNF) on the physiological and gene expression responses to elevated CO 2 were examined at multiple nitrogen levels by comparing alfalfa mutants incapable of nitrogen fixation to wild‐type. Elemental analysis revealed a role for BNF in maintaining shoot carbon/nitrogen (C/N) balance under all nitrogen treatments at elevated CO 2 , whereas the effect of BNF on biomass was only observed at elevated CO 2 and the lowest nitrogen dose. Lower photosynthetic rates at were associated with the imbalance in shoot C/N. Genome‐wide transcriptional responses were used to identify carbon and nitrogen metabolism genes underlying the traits. Transcription factors important to C/N signalling were identified from inferred regulatory networks. This work supports the hypothesis that maintenance of C/N homoeostasis at elevated CO 2 can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2‐like) GLK ortholog.