Cell wall thickness has phylogenetically consistent effects on the photosynthetic nitrogen-use efficiency of terrestrial plants

Leaf photosynthetic nitrogen-use efficiency (PNUE) diversified significantly among C-3 species. To date, the morpho-physiological mechanisms and interrelationships shaping PNUE on an evolutionary time scale remain unclear. In this study, we assembled a comprehensive matrix of leaf morpho-anatomical and physiological traits for 679 C-3 species, ranging from bryophytes to angiosperms, to comprehend the complexity of interrelationships underpinning PNUE variations. We discovered that leaf mass per area (LMA), mesophyll cell wall thickness (T-cwm), Rubisco N allocation fraction (P-R), and mesophyll conductance (g(m)) together explained 83% of PNUE variations, with P-R and g(m) accounting for 65% of those variations. However, the P-R effects were species-dependent on g(m), meaning the contribution of P-R on PNUE was substantially significant in high-g(m) species compared to low-g(m) species. Standard major axis (SMA) and path analyses revealed a weak correlation between PNUE and LMA (r(2) = 0.1), while the SMA correlation for PNUE-T-cwm was robust (r(2) = 0.61). P-R was inversely related to T-cwm, paralleling the relationship between g(m) and T-cwm, resulting in the internal CO2 drawdown being only weakly proportional to T-cwm. The coordination of P-R and g(m) in relation to T-cwm constrains PNUE during the course of evolution.