Stomatal responses of differently CO2-acclimated plants to natural and experimental CO2 gradients

Stomata, the microscopic pores in the epidermis of plant leaves, facilitate gas exchange between the leaf interior and the external environment and thus play a pivotal role in the global carbon, water, and energy cycles. Their development and function are influenced by atmospheric CO₂ levels. However, the mechanisms and the extent of this influence remain incompletely understood. Understanding the stomatal responses to variations in atmospheric CO₂ is essential for predicting their impact on future plant productivity or the role of vegetation as a carbon sink. We investigated the stomatal responses - specifically stomatal aperture and stomatal frequency - of C₃ plants acclimated to either ambient (approximately 40 Pa) or reduced (approximately 30 Pa) partial pressure of CO₂ (pCO₂). Stomatal responses were assessed both under natural conditions along an altitudinal gradient and under controlled experimental conditions with defined CO₂ levels (30 vs. 42 Pa). Our results showed that, under experimental conditions, stomatal frequency decreased with increasing CO₂ levels in both plant groups, regardless of their acclimation history. Furthermore, plants exposed to reduced pCO₂ exhibited smaller stomatal apertures compared to those grown at ambient pCO₂. Above-ground fresh biomass was higher at ambient CO₂ levels than at reduced levels. Interestingly, no significant difference in stomatal frequency was observed between plants that were acclimated and grown under reduced pCO₂ (at 2,970 m a.s.l.) and those under ambient pCO₂ (540 m). These results support the hypothesis that the inverse stomatal response to CO₂ - i.e., a reduction in stomatal frequency with increasing CO₂ levels - is a general physiological pattern in C₃ plants in the range of 30-42 Pa. However, this response pattern can be overridden by specific (locally occurring) environmental conditions such as low humidity or elevated temperatures. Furthermore, the findings are consistent with the optimal stomatal theory, which postulates that plants regulate stomatal conductance, including stomatal frequency and aperture, to maximize photosynthesis while minimizing water loss to achieve optimal water-use efficiency.