The productivity of crops is largely determined by how efficiently they convert carbon dioxide (CO₂) into organic molecules through the process of photosynthesis. The exchange of gases between the atmosphere and the leaf intercellular spaces is facilitated by stomata, tiny pores found on the leaf epidermis. These pores act as gateways, allowing CO₂ to enter the leaf while water vapor exits through transpiration. The regulation of stomatal behavior results in a trade-off between CO2 uptake and water loss in higher plants.

While genetic and biochemical analysis of stomatal regulation has focused heavily on the C₃ species Arabidopsis thaliana, it is well-known that stomatal regulation can vary between different species and different photosynthetic types. Therefore, this PhD project aimed to investigate stomatal regulation in C₃ and C₄ plants, comparing their responses to changes in light and CO2. To control for potential confounding effects not related to photosynthetic type, three phylogenetically-controlled species from Cleomaceae, Flaveria, and Alloteropsis were used in the study.

Chapter focused on the response to blue and red light in C₃ and C₄ species. The study found that stomatal opening was highly responsive to blue light in a red light background in C₃ dicot species of Cleomaceae and Flaveria, which confirmed previous observations in Arabidopsis. However, stomatal conductance in C₄ dicot species was not responsive to the same treatment. In addition, stomatal opening in C₃ and C₄ species responded significantly to red light, while the intercellular CO₂ was kept constant. However, this response appeared to be largely species-specific, rather than associated with photosynthetic pathway.

The next chapter investigated stomatal regulation by [CO₂]. To distinguish responses linked to photosynthesis vs responses driven by mitochondrial respiration, CO2 responses were assessed in darkness and under illumination with red light similar to growing conditions. Whereas the C₃ species showed significant opening responses to sub-ambient CO2 in darkness, C₄s were invariable. In contrast, the opening response to sub-ambient CO2 under illumination was stronger in the C₄ species. Together with the differences in blue light response in Chapter 2, this may suggest that stomatal opening in the C₄ species relies more on guard cell photosynthesis and less on mitochondrial respiration, compared to the C₃ species.

Finally, in the last chapter of the thesis, using NADP-ME antisense lines of C₄ F. bidentis. It was hypothesized that perturbing the C₄ cycle, resulting in reduced carbon flux into the bundle sheath cells, may alter stomatal regulation. The study found that mutant plants had significantly increased operating C_i compared to control plants. When C_i was kept the same as the control plants a significant increase in stomatal opening was observed in the mutant lines, suggesting that disruption of the C₄ cycle disrupted stomatal sensitivity to CO2. Further exploration of molecular factors known to be involved in the guard cell CO2 response was performed, but turned out inconclusive.

Altogether, the work in this thesis shows that while stomatal regulation in response to CO2 and light has remained qualitatively similar to the ancestral C₃ pathway, quantitative shifts in sensitivity may have supported further optimization of CO2 assimilation and watervuse efficiency following evolution of the C₄ pathway.