The evolution of stomatal traits along the trajectory toward C4 photosynthesis

Stomatal traits evolve with the evolution of C4 photosynthesis. C-4 photosynthesis optimizes plant carbon and water relations, allowing high photosynthetic rates with low stomatal conductance. Stomata have long been considered a part of the C-4 syndrome. However, it remains unclear how stomatal traits evolved along the path from C-3 to C-4. Here, we examined stomata in the Flaveria genus, a model used for C-4 evolutionary study. Comparative, transgenic, and semi-in vitro experiments were performed to study the molecular basis that underlies the changes of stomatal traits in C-4 evolution. The evolution from C-3 to C-4 species is accompanied by a gradual rather than an abrupt change in stomatal traits. The initial change appears near the Type I intermediate stage. Co-evolution of the photosynthetic pathway and stomatal traits is supported. On the road to C-4, stomata tend to be fewer in number but larger in size and stomatal density dominates changes in anatomical maximum stomatal conductance (g(smax)). Reduction of FSTOMAGEN expression underlies decreased g(smax) in Flaveria and likely occurs in other C-4 lineages. Decreased g(smax) contributes to the increase in intrinsic water-use efficiency in C-4 evolution. This work highlights the stomatal traits in the current C-4 evolutionary model. Our study provides insights into the pattern, mechanism, and role of stomatal evolution along the road toward C-4.