The cause of reduced leaf-level transpiration under elevated CO2 remains largely elusive. Here, we assessed stomatal, hydraulic, and morphological adjustments in a long-term experiment on Aleppo pine (Pinus halepensis) seedlings germinated and grown for 22-40 months under elevated (eCO2; c. 860 ppm) or ambient (aCO2; c. 410 ppm) CO2. We assessed if eCO2-triggered reductions in canopy conductance (gc) alter the response to soil or atmospheric drought and are reversible or lasting due to anatomical adjustments by exposing eCO2 seedlings to decreasing [CO2]. To quantify underlying mechanisms, we analyzed leaf abscisic acid (ABA) level, stomatal and leaf morphology, xylem structure, hydraulic efficiency, and hydraulic safety. Effects of eCO2 manifested in a strong reduction in leaf-level gc (-55%) not caused by ABA and not reversible under low CO2 (c. 200 ppm). Stomatal development and size were unchanged, while stomatal density increased (+18%). An increased vein-to-epidermis distance (+65%) suggested a larger leaf resistance to water flow. This was supported by anatomical adjustments of branch xylem having smaller conduits (-8%) and lower conduit lumen fraction (-11%), which resulted in a lower specific conductivity (-19%) and leaf-specific conductivity (-34%). These adaptations to CO2 did not change stomatal sensitivity to soil or atmospheric drought, consistent with similar xylem safety thresholds. In summary, we found reductions of gc under elevated CO2 to be reflected in anatomical adjustments and decreases in hydraulic conductivity. As these water savings were largely annulled by increases in leaf biomass, we do not expect alleviation of drought stress in a high CO2 atmosphere.
© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.