Multi-Year Leaf-Level Response to Sub-Ambient and Elevated Experimental CO2 in Betula nana

The strong link between stomatal frequency and CO2 in woody plants is key for understanding past CO2 dynamics, predicting future change, and evaluating the significant role of vegetation in the hydrological cycle. Experimental validation is required to evaluate the long-term adaptive leaf response of C3 plants to CO2 conditions; however, studies to date have only focused on short-term single-season experiments and may not capture (1) the full ontogeny of leaves to experimental CO2 exposure or (2) the true adjustment of structural stomatal properties to CO2, which we postulate is likely to occur over several growing seasons. We conducted controlled growth chamber experiments at 150 ppmv, 450 ppmv and 800 ppmv CO2 with woody C3 shrub Betula nana (dwarf birch) over two successive annual growing seasons and evaluated the structural stomatal response to atmospheric CO2 conditions. We find that while some adjustment of leaf morphological and stomatal parameters occurred in the first growing season where plants are exposed to experimental CO2 conditions, amplified adjustment of non-plastic stomatal properties such as stomatal conductance occurred in the second year of experimental CO2 exposure. We postulate that the species response limit to CO2 of B. nana may occur around 400-450 ppmv. Our findings strongly support the necessity for multi-annual experiments in C3 perennials in order to evaluate the effects of environmental conditions and provide a likely explanation of the contradictory results between historical and palaeobotanical records and experimental data.