Gimeno, Teresa E.Campany, Courtney E.Drake, John E.Barton, Craig V MTjoelker, Mark GUbierna, NereaMarshall, John D.2022-10-170028-646Xhttp://hdl.handle.net/1885/275551Photosynthetic water-use efficiency (WUE) describes the link between terrestrial carbon (C) and water cycles. Estimates of intrinsic WUE (iWUE) from gas exchange and C isotopic composition (δ13C) differ due to an internal conductance in the leaf mesophyll (gm) that is variable and seldom computed. We present the first direct estimates of whole-tree gm, together with iWUE from whole-tree gas exchange and δ13C of the phloem (δ13Cph). We measured gas exchange, online 13C-discrimination, and δ13Cph monthly throughout spring, summer, and autumn in Eucalyptus tereticornis grown in large whole-tree chambers. Six trees were grown at ambient temperatures and six at a 3°C warmer air temperature; a late-summer drought was also imposed. Drought reduced whole-tree gm. Warming had few direct effects, but amplified drought-induced reductions in whole-tree gm. Whole-tree gm was similar to leaf gm for these same trees. iWUE estimates from δ13Cph agreed with iWUE from gas exchange, but only after incorporating gm. δ13Cph was also correlated with whole-tree 13C-discrimination, but offset by −2.5 ± 0.7‰, presumably due to post-photosynthetic fractionations. We conclude that δ13Cph is a good proxy for whole-tree iWUE, with the caveats that post-photosynthetic fractionations and intrinsic variability of gm should be incorporated to provide reliable estimates of this trait in response to abiotic stress.The WTC experiment was supported by a grant from the Australian Research Council (DP140103415) awarded to MGT and JED. JDM was supported by the KA Wallenberg Foundation (#2015.0047) during writing. TEG was supported by the Spanish Ministry of Science (grant PHLISCO, PID2019- 107817RB-I00). The complete data set can be downloaded from https://doi.org/10.6084/m9.figshare.13234310.v2application/pdfen-AU© 2020 The Authors New Phytologist © 2020 New Phytologist Foundationcarbon stable isotopedrought, Eucalyptusphloemphotosynthesisrespirationwarmingwhole-tree chamberWhole-tree mesophyll conductance reconciles isotopic and gas-exchange estimates of water-use efficiency202010.1111/nph.170882021-11-28