SummaryLeaf mesophyll conductance to CO 2 (g m ) has been recognized to be finite and variable, rapidly adapting to environmental conditions. The physiological basis for fast changes in g m is poorly understood, but current reports suggest the involvement of protein-facilitated CO 2 diffusion across cell membranes. A good candidate for this could be the Nicotiana tabacum L. aquaporin NtAQP1, which was shown to increase membrane permeability to CO 2 in Xenopus oocytes. The objective of the present work was to evaluate its effect on the in vivo mesophyll conductance to CO 2 , using plants either deficient in or overexpressing NtAQP1. Antisense plants deficient in NtAQP1 (AS) and NtAQP1 overexpressing tobacco plants (O) were compared with their respective wild-type (WT) genotypes (CAS and CO). Plants grown under optimum conditions showed different photosynthetic rates at saturating light, with a decrease of 13% in AS and an increase of 20% in O, compared with their respective controls. CO 2 response curves of photosynthesis also showed significant differences among genotypes. However, in vitro analysis demonstrated that these differences could not be attributed to alterations in Rubisco activity or ribulose-1,5-bisphosphate content. Analyses of chlorophyll fluorescence and on-line 13 C discrimination indicated that the observed differences in net photosynthesis (A N ) among genotypes were due to different leaf mesophyll conductances to CO 2 , which was estimated to be 30% lower in AS and 20% higher in O compared with their respective WT. These results provide evidence for the in vivo involvement of aquaporin NtAQP1 in mesophyll conductance to CO 2 .
Photosynthesis is often limited by the rate of CO 2 diffusion from the atmosphere to the chloroplast. The primary resistances for CO 2 diffusion are thought to be at the stomata and at photosynthesizing cells via a combination resulting from resistances of aqueous solution as well as the plasma membrane and both outer and inner chloroplast membranes. In contrast with stomatal resistance, the resistance of biological membranes to gas transport is not widely recognized as a limiting factor for metabolic function. We show that the tobacco (Nicotiana tabacum) plasma membrane and inner chloroplast membranes contain the aquaporin Nt AQP1. RNA interference-mediated decreases in Nt AQP1 expression lowered the CO 2 permeability of the inner chloroplast membrane. In vivo data show that the reduced amount of Nt AQP1 caused a 20% change in CO 2 conductance within leaves. Our discovery of CO 2 aquaporin function in the chloroplast membrane opens new opportunities for mechanistic examination of leaf internal CO 2 conductance regulation.
Summary A new aquaporin from Nicotiana tabacum (cv. Samsun) was characterized. It shares sequence homology to the Arabidopsis thaliana PIP1 protein family. By two‐phase partitioning and immunoblot analysis, plasma membrane localization could be demonstrated. The corresponding mRNA is highly abundant in roots and flowers, while it is rarely expressed in leaves and stems. Functional expression in Xenopus oocytes revealed that NtAQP1 can mediate glycerol transport in addition to water flow. However, NtAQP1 is impermeable for Na + , K + and Cl – ions. The water permeability and selectivity could not be modulated by addition of mercurials or the activity of cAMP‐dependent protein kinases.
Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO 2 -triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO 2 -related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO 2 -triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO 2 -related transport processes, a structure built up by the tetramer is the basis of this function.Water moves across biological membranes by diffusion. In most living organisms, the rate of water diffusion can be increased via pore-forming transmembrane proteins, the socalled aquaporins. These consist of six-membrane-spanning helices; N and C termini of the proteins face the cytosol. The helix-connecting loops B and E are themselves short helices that dip into the membrane from opposite sides and form the water-conducting channel (1, 2). Aquaporin monomers can assemble into tetramers (3, 4). Evidence for their function as water transport facilitators was observed by results from experiments using heterologous expression systems, such as Xenopus laevis oocytes (5). Despite this initially detected function, a facilitated membrane transport for glycerol or volatile substances like CO 2 or NH 3 was postulated (6 -9). Plant aquaporins were subdivided into protein groups according to the cellular location in which they have been initially detected and on the basis of sequence similarities (10). Accordingly, the PIPs (plasma membrane-intrinsic proteins) were split into two major groups, the PIP1 and PIP2 aquaporins. Compared with PIP1 proteins, PIP2 proteins have a shorter N-terminal extension and a longer C terminus (11)(12)(13)(14). In general, plant PIP2 proteins have been shown to facilitate membrane water transport in heterologous expression systems; however, PIP1 proteins display low or no activity in this respect (8,11,(15)(16)(17)(18). For the human AQP1 as well as for the tobacco PIP1 aquaporin NtAQP1, an increased cellular acidification rate under CO 2 -enriched buffer was obtained in oocytes expressing the respective aquaporin in addition to a carbonic anhydrase (9,19,20). The observations on NtAQP1, together with the fact that plants deficient in NtAQP1 express...
Leaf-moving organs, remarkable for the rhythmic volume changes of their motor cells, served as a model system in which to study the regulation of membrane water fluxes. Two plasma membrane intrinsic protein homolog genes, SsAQP1 and SsAQP2, were cloned from these organs and characterized as aquaporins in Xenopus laevis oocytes. Osmotic water permeability (P(f)) was 10 times higher in SsAQP2-expressing oocytes than in SsAQP1-expressing oocytes. SsAQP1 was found to be glycerol permeable, and SsAQP2 was inhibited by 0.5 mM HgCl(2) and by 1 mM phloretin. The aquaporin mRNA levels differed in their spatial distribution in the leaf and were regulated diurnally in phase with leaflet movements. Additionally, SsAQP2 transcription was under circadian control. The P(f) of motor cell protoplasts was regulated diurnally as well: the morning and/or evening P(f) increases were inhibited by 50 microM HgCl(2), by 2 mM cycloheximide, and by 250 microM phloretin to the noon P(f) level. Our results link SsAQP2 to the physiological function of rhythmic cell volume changes.
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