.............................................................. The tobacco aquaporin NtAQP1 is a membrane CO 2 pore with physiological functions Norbert Uehlein 1 *, Claudio Lovisolo 2 *, Franka Siefritz 1 & Ralf Kaldenhoff 1 1 Institute of Botany, Schnittspahnstrasse 3, D-64287 Darmstadt, Germany 2 Department of Arboriculture and Pomology, University of Turin, I-10095 Grugliasco, Italy * These authors contributed equally to this work ............................................................................................................................................................................. Aquaporins, found in virtually all living organisms, are mem- brane-intrinsic proteins that form water-permeable complexes. The mammalian aquaporin AQP1 has also shown CO 2 per- meability when expressed heterologously in Xenopus oocytes 1 , although whether this is a biochemical curiosity or of physio- logical significance is a matter of debate 2,3 . Here we report that, in the same expression system, a CO 2 permeability comparable to that of the human AQP1 is observed for the tobacco plasma membrane aquaporin NtAQP1. NtAQP1 facilitates CO 2 mem- brane transport in the homologous plant system at the cellular level, and has a significant function in photosynthesis and in stomatal opening. NtAQP1 overexpression heightens membrane permeability for CO 2 and water, and increases leaf growth. The results indicate that NtAQP1-related CO 2 permeability is of physiological importance under conditions where the CO 2 gra- dient across a membrane is small, as is the case between the atmosphere and the inside of a plant cell. CO 2 gas exchange is of pivotal significance, not only in animals but also in plants. CO 2 is the substrate for photosynthetic carbon fixation and in its reduced form, as a sugar compound, is the basis for life. However, the key CO 2 -reducing enzyme Rubisco has a relatively low affinity for this substrate, and the CO 2 gradient between the atmosphere and the plant is ,400 times less than that between animal lungs and the atmosphere. Nevertheless, the CO 2 exchange rates in mammalian lungs and plants are similar 4 . Consequently, a low resistance to CO 2 diffusion in plants can be expected and a slight variation of this resistance—for example, by a change of membrane CO 2 transport rate—should immediately affect reactions that are limited by CO 2 availability, such as the photosynthesis rate. Owing to the high sequence conservation of aquaporins, it is possible that a plant aquaporin with a CO 2 permeability comparable to the human AQP1 could exist, and facilitation of CO 2 membrane transport could explain the incon- sistency between the small CO 2 concentration gradients and CO 2 exchange rates in plants. In fact, the plant aquaporin NtAQP1 does share a high sequence similarity with AQP1 and functionally important amino-acid residues in the pore region are identical 5 . Whether the CO 2 permeability of the human AQP1 is just a biophysical curiosity or has physiological relevance was analysed by comparing transgenic AQP1-null mice with controls 2 . It was found that CO 2 blow-off by the lung was independent of AQP1 expression, and the CO 2 permeability of AQP1 thus appeared irrelevant for CO 2 permeation in mice 3 . Because the existence of unstirred layers or perfusion-limited conditions might have masked the contribution of AQP1 to CO 2 permeability, concerns were raised against this interpretation 6 . Using the technique of Nakhoul et al. 1 , we show here that the plant aquaporin NtAQP1, when expressed in Xenopus oocytes, has CO 2 permeability features similar to those of the human AQP1. Furthermore, in contrast to findings in animal systems, the physio- logical significance of aquaporin CO 2 permeability can be attributed to NtAQP1 from the data provided in this report. For the determination of CO 2 membrane transport rates, Xeno- pus oocytes were injected with a solution of carbonic anhydrase, an enzyme that accelerates the conversion of CO 2 to HCO 3 2 . In these oocytes, CO 2 membrane transport rather than the conversion reaction is rate limiting for HCO 3 2 accumulation 1 . As a result, CO 2 transport into the cells caused a decrease in intracellular pH (pH i ) in controls injected with carbonic anhydrase only, and also in NtAQP1-expressing/carbonic anhydrase oocytes (Fig. 1). From the rates of pH i decrease, we found that the CO 2 uptake rates of controls and the initial CO 2 uptake rates of oocytes overexpressing NtAQP1 were, respectively, 45% higher (n ¼ 10) and in a range comparable to those of the human AQP1. Therefore, in addition to its per- meability to water and glycerol 7 , NtAQP1 is also a CO 2 transporter in the oocyte expression system. Results from heterologous expression can be transferred to the homologous system only with many restrictions. For analysis of NtAQP1 function on CO 2 transport in the homologous system, NtAQP1 antisense tobacco lines with a severe impairment in NtAQP1 expression were used 8 , as well as plants with an NtAQP1 coding region under the control of a tetracycline-inducible promotor 9 (TET-NtAQP1). Thus, plants with a relatively low (antisense NtAQP1 lines) or high (TET- Figure 1 Representative kinetics of the pH decrease in a Xenopus oocyte injected with carbonic anhydrase only (dotted line) or with additional expression of NtAQP1. Initial rates for the oocyte overexpressing NtAQP1 indicate a 45% increase in CO 2 plasma membrane permeability. Figure 2 Equal amounts of total RNA (10 mg) were loaded on a gel, blotted and hybridized to a gene-specific probe for NtAQP1 or to 28S-rRNA as loading control. RNA was isolated from controls or NtAQP1-transformed lines: Nicotiana tabacum var. samsun transformed with a 35S-GUS construct for comparison with antisense lines constructed in the same variety (Control 1) and transformed with a 35S-NtAQP1 antisense construct (Antisense) 11 ; or N. tabacum, Ho ¨ 20.20 transformed with a 35S-tetracycline-repressor construct and a construct containing an NtAQP1 gene under control of a tetracycline- repressor-regulated promoter. Plants were treated with tetracycline (TET expression) or irrigated with water. After application of tetracycline to Ho ¨ 20.20 lines without the NtAQP1 construct (Control 2), expression of NtAQP1 was comparable to that of the watered plants mentioned above. letters to nature NATURE | VOL 425 | 16 OCTOBER 2003 | www.nature.com/nature 734 © 2003 Nature Publishing Group