Chemical composition and digestibility of Trifolium exposed to elevated ozone and carbon dioxide in a free-air (FACE) fumigation system

1. Tropospheric ozone (O 3 ) and carbon dioxide (CO 2 ) are significant drivers of plant growth and chemical composition. We hypothesized that exposure to elevated concentrations of O 3 and CO 2 , singly and in combination, would modify the chemical composition of Trifolium and thus alter its digestibility and nutritive quality for ruminant herbivores. 2. We tested our hypothesis by collecting samples of Red Clover ( Trifolium pratense ) and White Clover ( Trifolium repens ) from the understoreys of Trembling Aspen ( Populus tremuloides )–Sugar Maple ( Acer saccharum ) communities that had been exposed since 1998 to ambient air, elevated CO 2 , elevated O 3 or elevated CO 2 + O 3 at the Aspen FreeAir CO 2 and O 3 Enrichment (FACE) site located near Rhinelander, WI, USA. Foliage samples were analysed for (1) concentrations of N, total cell wall constituents, lignin and soluble phenolics; and (2) in vitro dry-matter digestibility (IVDMD) and in vitro cell-wall digestibility (IVCWD) using batch cultures of ruminal micro-organisms. 3. Significant air-treatment effects were observed for lignin concentration, IVDMD and IVCWD, and between Red and White Clover for all dependent variables. No air treatment × clover species interactions were detected. 4. Exposure to elevated O 3 resulted in increased concentration of lignin and decreased IVDMD and IVCWD compared with exposure to ambient air, and the response was similar regardless of whether plants had been coexposed to elevated CO 2 . Exposure to elevated CO 2 alone did not affect chemical composition or in vitro digestibility, nor did it ameliorate the negative effect of elevated O 3 on these determinants of nutritive quality for ruminant herbivores. 5. In contrast to recent reports of a protective effect of elevated CO 2 against growth reduction in plants under O 3 stress, our results indicate that elevated CO 2 would not be expected to ameliorate the negative impact of elevated O 3 on nutritive quality of Trifolium under projected future global climate scenarios.