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

Understanding crop responses to elevated CO₂ is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO₂ due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO₂ 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 CO₂ 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 CO₂, whereas the effect of BNF on biomass was only observed at elevated CO₂ 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 CO₂ can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2-like) GLK ortholog.