Using a free-air CO2 enrichment (FACE)\ud experiment, poplar trees (Populus · euramericana clone\ud I214) were exposed to either ambient or elevated [CO2]\ud from planting, for a 5-year period during canopy\ud development, closure, coppice and re-growth. In each\ud year, measurements were taken of stomatal density\ud (SD, number mm2) and stomatal index (SI, the\ud proportion of epidermal cells forming stomata). In\ud year 5, measurements were also taken of leaf stomatal\ud conductance (gs, lmol m2 s1), photosynthetic CO2\ud fixation (A, mmol m2 s1), instantaneous water-use\ud efficiency (A/E) and the ratio of intercellular to\ud atmospheric CO2 (Ci:Ca). Elevated [CO2] caused\ud reductions in SI in the first year, and in SD in the first\ud 2 years, when the canopy was largely open. In following\ud years, when the canopy had closed, elevated\ud [CO2] had no detectable effects on stomatal numbers\ud or index. In contrast, even after 5 years of exposure to\ud elevated [CO2], gs was reduced, A/E was stimulated,\ud and Ci:Ca was reduced relative to ambient [CO2].\ud These outcomes from the long-term realistic field conditions of this forest FACE experiment suggest\ud that stomatal numbers (SD and SI) had no role in\ud determining the improved instantaneous leaf-level\ud efficiency of water use under elevated [CO2]. We propose\ud that altered cuticular development during canopy\ud closure may partially explain the changing response of\ud stomata to elevated [CO2], although the mechanism\ud for this remains obscure
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