Although responsiveness of N₂-fixing plants to elevated CO₂ conditions have been analyzed in previous studies, important uncertainties remain in relation to the effect enhanced CO₂ in nodule proteomic profile and its implication in leaf responsiveness. The aim of our study was to deepen our understanding of the relationship between leaf and nodule metabolism of N₂-fixing alfalfa plants after longterm exposure to elevated CO₂. After 30-day exposure to elevated CO₂, plants showed photosynthetic down-regulation with reductions in the light-saturated rate of CO₂ assimilation (Asat) and the maximum rate of rubisco carboxylation (Vcmax). Under elevated CO₂ conditions, the rubisco availability limited potential photosynthesis by around 12 %, which represented the majority of the observed fall in Vcmax. Photosynthetic down-regulation has been associated with decreased N availability even if those plants are capable to assimilate N₂. Diminishment in shoot N demand (as reflected by the lower rubisco and leaf N content) suggests that the lower aboveground N requirements affected negatively nodule performance. In this condition, specific nodule activity was reduced due to an effect on nodule metabolism that manifested as a lower amount of nitrogenase reductase. Moreover, the nodule proteomic approach also revealed that nodule functioning was altered simultaneously in various enzyme quantity apart from nitrogenase. At elevated CO₂, the tricarboxylic acid cycle was also altered with a reduced amount of isocitrate synthase protein. The nodule proteome analysis also revealed the relaxation of the antioxidant system as shown by a decline in the amount of catalase and isoflavone reductase protein.
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