Stomatal responses of C₃, C₃‐C₄ and C₄Flaveria species to light and intercellular CO₂ concentration: implications for the evolution of stomatal behaviour

Stomatal function mediates physiological trade‐offs associated with maintaining a favourable H 2 O balance in leaf tissues while acquiring CO 2 as a photosynthetic substrate. The C 3 and C 4 species appear to have different patterns of stomatal response to changing light conditions, and variation in this behaviour may have played a role in the functional diversification of the different photosynthetic pathways. In the current study, we used gain analysis theory to characterize the stomatal conductance response to light intensity in nine different C 3 , C 4 and C 3 ‐C 4 intermediate species Flaveria species. The response of stomatal conductance ( g s ) to a change in light intensity represents both a direct (related to a change in incident light intensity, I ) and indirect (related to a change in intercellular CO 2 concentration, C i ) response. The slope of the line relating the change in g s to C i was steeper in C 4 species, compared with C 3 species, with C 3 ‐C 4 species having an intermediate response. This response reflects the greater relative contribution of the indirect versus direct component of the g s versus I response in the C 4 species. The C 3 ‐C 4 species, Flaveria floridana , exhibited a C 4 ‐like response whereas the C 3 ‐C 4 species, Flaveria sonorensis and Flaveria chloraefolia , exhibited C 3 ‐like responses, similar to their hypothesized position along the evolutionary trajectory of the development of C 4 photosynthesis. There was a positive correlation between the relative contribution of the indirect component of the g s versus I response and water use efficiency when evaluated across all species. Assuming that the C 3 ‐C 4 intermediate species reflect an evolutionary progression from fully expressed C 3 ancestors, the results of the current study demonstrate an increase in the contribution of the indirect component of the g s versus I response as taxa evolve toward the C 4 extreme. The greater relative contribution of the indirect component of the stomatal response occurs through both increases in the indirect stomatal components and through decreases in the direct. Increases in the magnitude of the indirect component may be related to the maintenance of higher water use efficiencies in the intermediate evolutionary stages, before the appearance of fully integrated C 4 photosynthesis.