Revisiting Why Plants Become N Deficient Under Elevated CO₂: Importance to Meet N Demand Regardless of the Fed-Form

An increase in plant biomass under elevated CO₂ (eCO₂) is usually lower than expected. N-deficiency induced by eCO₂ is often considered to be a reason for this. Several hypotheses explain the induced N-deficiency: (1) eCO₂ inhibits nitrate assimilation, (2) eCO₂ lowers nitrate acquisition due to reduced transpiration, or (3) eCO₂ reduces plant N concentration with increased biomass. We tested them using C₃ (wheat, rice, and potato) and C₄ plants (guinea grass, and Amaranthus) grown in chambers at 400 (ambient CO₂, aCO₂) or 800 (eCO₂) μL L^-1 CO₂. In most species, we could not confirm hypothesis (1) with the measurements of plant nitrate accumulation in each organ. The exception was rice showing a slight inhibition of nitrate assimilation at eCO₂, but the biomass was similar between the nitrate and urea-fed plants. Contrary to hypothesis (2), eCO₂ did not decrease plant nitrate acquisition despite reduced transpiration because of enhanced nitrate acquisition per unit transpiration in all species. Comparing to aCO₂, eCO₂ remarkably enhanced water-use efficiency, especially in C₃ plants, decreasing water demand for CO₂ acquisition. As our results supported hypothesis (3) without any exception, we then examined if lowered N concentration at eCO₂ indeed limits the growth using C₃ wheat and C₄ guinea grass under various levels of nitrate-N supply. While eCO₂ significantly increased relative growth rate (RGR) in wheat but not in guinea grass, each species increased RGR with higher N supply and then reached a maximum as no longer N was limited. To achieve the maximum RGR, wheat required a 1.3-fold N supply at eCO₂ than aCO₂ with 2.2-fold biomass. However, the N requirement by guinea grass was less affected by the eCO₂ treatment. The results reveal that accelerated RGR by eCO₂ could create a demand for more N, especially in the leaf sheath rather than the leaf blade in wheat, causing N-limitation unless the additional N was supplied. We concluded that eCO₂ amplifies N-limitation due to accelerated growth rate rather than inhibited nitrate assimilation or acquisition. Our results suggest that plant growth under higher CO₂ will become more dependent on N but less dependent on water to acquire both CO₂ and N.