Low CO2 condition was considered a preconditioning or selection pressure for C4 evolution. However, it remains elucidated how low CO2 condition contribute to the evolutionary assembly of the C4 pathway. We conducted a systematic transcriptomics and metabolomics analysis under short-term low CO2 condition and found that Arabidopsis grown under this condition showed increased expression of most genes encoding C4- related enzymes and transporters. Low CO2 condition increased NH4+ content in leaves; as expected, photorespiratory and ammonia refixing pathways were enhanced. Furthermore, we found that compared to low CO2 condition, in vitro treatment with NH4+ induced a similar pattern of changes in C4 related genes and genes involved in ammonia refixation. This supports that increased expression of C4 genes induced by low CO2 condition can supply carbon skeleton for ammonia recycling. This study provides new insight into the regulatory preconditioning which may have facilitated the evolution of C4 photosynthesis under low atmospheric CO2 environments.
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