Rewiring of primary metabolism for ammonium recycling under short-term low CO₂ treatment - its implication for C₄ evolution

The dramatic decrease in atmospheric CO₂ concentration during Oligocene was proposed as directly linked to C₄ evolution. However, it remains unclear how the decreased CO₂ concentration directly facilitate C₄ evolution, besides its role as a selection pressure. We conducted a systematic transcriptomics and metabolomics analysis under short-term low CO₂ condition and found that Arabidopsis grown under this condition showed 1) increased expression of most genes encoding C₄-related enzymes and transporters; 2) increased expression of genes involved in photorespiration and pathways related to carbon skeleton generation for ammonium refixation; 3) increased expression of genes directly involved in ammonium refixation. Furthermore, we found that in vitro treatment of leaves with NH₄ ⁺ induced a similar pattern of changes in C₄ related genes and genes involved in ammonium refixation. These data support the view that Arabidopsis grown under short-term low CO₂ conditions rewired its metabolism to supply carbon skeleton for ammonium recycling, during which process the expression of C₄ genes were up-regulated as a result of a hitchhiking process. This study provides new insights into the adaptation of the C₃ model plant Arabidopsis under low CO₂ conditions and suggests that low CO₂ can facilitate the evolution of C₄ photosynthesis beyond the commonly assumed role of being a selection pressure.