Low nitrogen supply decreases the mesophyll conductance and limits the effects of elevated CO2 on photosynthesis in winter wheat across multiple generations

Although the interaction between elevated CO₂ ( e [CO₂]) and nitrogen (N) availability on photosynthesis has been extensively studied within single generations, their interplay across multiple generations remains poorly understood. This study investigated the photosynthetic responses of wheat ( Triticum aestivum L.) to atmospheric CO₂ concentration (400 μmol mol −1 for Control vs. 800 μmol mol −1 for e [CO 2 ]) and soil N supply (0.5 vs. 1.5 g N pot⁻¹) in plants from the first (F1) and fourth (F4) generations of a lineage exposed to these conditions. We quantitatively assessed the leaf photosynthetic limitations imposed by stomatal conductance ( g ₛ), mesophyll conductance ( g ₘ), and biochemistry ( V c,max ) during the grain filling stage. Compared to the strong effects of CO₂ concentrations and N levels, changes attributable to generational exposure were minor. Under e [CO₂] and sufficient N, both generations exhibited enhanced chloroplast CO₂ concentration and net photosynthesis rate without declines in g ₘ. Low N under e [CO₂] significantly reduced g ₘ (68–73%) and net photosynthesis rate (32–41%), with the magnitude of reduction being similar between F1 and F4 plants, indicating that the immediate N status had a far greater impact than transgenerational exposure. Photosynthesis was primarily limited by biochemistry (57%) under e [CO₂] with adequate N, but shifted to a strong co-limitation by g ₘ (38%) and biochemistry (44%) under low N, a pattern consistent in both generations. Path analysis confirmed that N availability had the most substantial effect on the net photosynthesis rate (path coefficient = 0.95), whereas generational effects were minimal. Multivariate analysis showed apparent clustering by CO₂ and N treatments, but complete overlap between generational groups. We conclude that the positive effect of e [CO₂] on wheat photosynthesis is strongly influenced by soil N availability within the timeframe of four generations. The photosynthetic apparatus showed high plasticity to current conditions, with limited modification by transgenerational effects. These results highlight that improving nitrogen management is essential for maintaining wheat photosynthetic performance and, by extension, potential yield, in future high-CO₂ environments. This underscores the continued primacy of nitrogen management for securing crop productivity in a changing climate. • Low nitrogen under elevated CO₂ severely reduces mesophyll conductance (gₘ). • Photosynthesis becomes co-limited by gₘ and biochemistry under low N at high CO₂. • Nitrogen status dominates photosynthetic response over four generations. • Photosynthetic traits show high plasticity with limited transgenerational influence. • Sustained photosynthetic benefits under elevated CO₂ require adequate N management.