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Results from CO2 diffusion studies and characterization of Arabidopsis thaliana aquaporin AtPIP1;2 T-DNA insertion lines support the idea that specific aquaporins facilitate the diffusion of CO2 through biological membranes. However, their function as CO2 diffusion facilitators in plant physiology is still a matter of debate. Assuming that a lack of AtPIP1;2 causes a characteristic transcriptional response, we compared data from a AtPIP1;2 T-DNA insertion line obtained by Illumina sequencing, Affymetrix chip analysis and quantitative RT-PCR to the transcriptome of plants grown under drought stress or under low CO2 conditions. The plant reaction to the deficit of AtPIP1;2 was unlike drought stress responses but comparable with that of low CO2 conditions. In addition, we observed a phenotype characteristic to plants grown under low CO2 . The findings support the hypothesis that the AtPIP1;2 function in plant physiology is not to facilitate water but CO2 diffusion., (© 2015 John Wiley & Sons Ltd.)

Black lipid bilayers, a general model system for biomembranes were studied for diffusion rates of small molecules such as water or CO2 using advanced analysis techniques and cell free synthesized proteins. We provide evidence that by simple insertion of proteins or sterols the diffusion rates of water or those of CO2 decrease. Insertion of cell free synthesized water permeable aquaporins restored water diffusion rates as well as insertion of CO2-facilitating aquaporins the CO2 diffusion. Insertion of water or CO2 impermeable proteins decreased the respective diffusion rates. Therefore, for normal high cellular CO2 diffusion rates specific aquaporins are mandatory.

Background: Determination of CO2 diffusion rates in living cells revealed inconsistencies with existing models about the mechanisms of membrane gas transport. Mainly, these discrepancies exist in the determined CO2 diffusion rates of bio-membranes, which were orders of magnitudes below those for pure lipid bilayers or theoretical considerations as well as in the observation that membrane insertion of specific aquaporins was rescuing high CO2 transport rates. This effect was confirmed by functional aquaporin protein analysis in heterologous expression systems as well as in bacteria, plants and partly in mammals., Scope of Review: This review summarizes the arguments in favor of and against aquaporin facilitated membrane diffusion of CO2 and reports about its importance for the physiology of living organisms., Major Conclusions: Most likely, the aquaporin tetramer forming an additional fifth pore is required for CO2 diffusion facilitation. Aquaporin tetramer formation, membrane integration and disintegration could provide a mechanism for regulation of cellular CO2 exchange. The physiological importance of aquaporin mediated CO2 membrane diffusion could be shown for plants and cyanobacteria and partly for mammals., General Significance: Taking the mentioned results into account, consequences for our current picture of cell membrane transport emerge. It appears that in some or many instances, membranes might not be as permeable as it was suggested by current bio-membrane models, opening an additional way of controlling the cellular influx or efflux of volatile substances like CO2. This article is part of a Special Issue entitled Aquaporins., (© 2013.)

The membrane CO(2) flux into Arabidopsis mesophyll cells was studied using a scanning pH microelectrode. Arabidopsis thaliana mesophyll cells were exposed to photosynthesis-triggering light intensities, which induced cellular CO(2) uptake. Data obtained on a AtPIP1;2 T-DNA insertion line indicated that under these conditions, cellular CO(2) transport was not limited by unstirred layer effects but was dependent on the expression of the aquaporin AtPIP1;2. Complementation of the AtPIP1;2 knockout restored membrane CO(2) transport levels to that of controls. The results provide new arguments for the ongoing debate about the validity of the lipid bilayer model system and the Meyer - Overton rule for cellular gas transport. In conclusion, we suggest a modified model of molecular gas transport mechanisms in living cells., (© 2011 Blackwell Publishing Ltd.)

Plants provide an excellent system to study CO(2) diffusion because, under light saturated conditions, photosynthesis is limited by CO(2) availability. Recent findings indicate that CO(2) diffusion in leaves can be variable in a short time range. Mesophyll CO(2) conductance could change independently from stomata movement or CO(2) fixing reactions and it was suggested that, beside others, the membranes are mesophyll CO(2) conductance limiting components. Specific aquaporins as membrane intrinsic pore proteins are considered to have a function in the modification of membrane CO(2) conductivity. Because of conflicting data, the mechanism of membrane CO(2) diffusion in plants and animals is a matter of a controversy vivid debate in the scientific community. On one hand, data from biophysics are in favor of CO(2) diffusion limiting mechanisms completely independent from membrane structure and membrane components. On the other, there is increasing evidence from physiology that a change in membrane composition has an effect on CO(2) diffusion., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Terfezia claveryi is a hypogeous mycorrhizal fungus belonging to the so-called "desert truffles," with a good record as an edible fungus and of considerable economic importance. T. claveryi improves the tolerance to water stress of the host plant Helianthemum almeriense, for which, in field conditions, symbiosis with T. claveryi is valuable for its survival. We have characterized cDNAs from T. claveryi and identified a sequence related to the aquaporin gene family. The full-length sequence was obtained by rapid amplification of cDNA ends and was named TcAQP1. This aquaporin gene encoded a functional water-channel protein, as demonstrated by heterologous expression assays in Saccharomyces cerevisiae. The mycorrhizal fungal aquaporin increased both water and CO(2) conductivity in the heterologous expression system. The expression patterns of the TcAQP1 gene in mycelium, under different water potentials, and in mycorrhizal plants are discussed. The high levels of water conductivity of TcAQP1 could be related to the adaptation of this mycorrhizal fungus to semiarid areas. The CO(2) permeability of TcAQP1 could be involved in the regulation of T. claveryi growth during presymbiotic phases, making it a good candidate to be considered a novel molecular signaling channel in mycorrhizal fungi.

We demonstrate that membranes consisting of certain triblock-copolymers were tight for CO₂. Using a novel approach, we provide evidence for aquaporin facilitated CO₂ diffusion. Plant aquaporins obtained from heterologous expression were inserted into triblock copolymer membranes. These were employed to separate a chamber with a solution maintaining high CO₂ concentrations from one with depleted CO₂ concentrations. CO₂ diffusion was detected by measuring the pH change resulting from membrane CO₂ diffusion from one chamber to the other. An up to 21 fold increase in diffusion rate was determined. Besides the supply of this proof of principle, we could provide additional arguments in favour of protein facilitated CO₂ diffusion to the vivid on-going debate about the principles of membrane gas diffusion in living cells.

Cellular exchange of carbon dioxide (CO₂) is of extraordinary importance for life. Despite this significance, its molecular mechanisms are still unclear and a matter of controversy. In contrast to other living organisms, plants are physiologically limited by the availability of CO₂. In most plants, net photosynthesis is directly dependent on CO₂ diffusion from the atmosphere to the chloroplast. Thus, it is important to analyze CO₂ transport with regards to its effect on photosynthesis. A mutation of the Arabidopsis thaliana AtPIP1;2 gene, which was characterized as a non-water transporting but CO₂ transport-facilitating aquaporin in heterologous expression systems, correlated with a reduction in photosynthesis under a wide range of atmospheric CO₂ concentrations. Here, we could demonstrate that the effect was caused by reduced CO₂ conductivity in leaf tissue. It is concluded that the AtPIP1;2 gene product limits CO₂ diffusion and photosynthesis in leaves., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

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.

CO(2) faces a series of resistances while diffusing between the substomatal cavities and the sites of carboxylation within chloroplasts. The absence of techniques to measure the resistance of individual steps makes it difficult to define their relative importance. Resistance to diffusion through intercellular airspace differs between leaves, but is usually of minor importance. Leaves with high photosynthetic capacity per unit leaf area reduce mesophyll resistance by increasing the surface area of chloroplasts exposed to intercellular airspace per unit leaf area, S(c). Cell walls impose a significant resistance. Assuming an effective porosity of the cell wall of 0.1 or 0.05, then cell walls could account for 25% or 50% of the total mesophyll resistance, respectively. Since the fraction of apoplastic water that is unbound and available for unhindered CO(2) diffusion is unknown, it is possible that the effective porosity is <0.05. Effective porosity could also vary in response to changes in pH or cation concentration. Consequently, cell walls could account for >50% of the total resistance and a variable proportion. Most of the remaining resistance is imposed by one or more of the three membranes as mesophyll resistance can be altered by varying the expression of cooporins. The CO(2) permeability of vesicles prepared from chloroplast envelopes has been reduced by RNA interference (RNAi) expression of NtAQP1, but not those prepared from the plasma membrane. Carbonic anhydrase activity also influences mesophyll resistance. Mesophyll resistance is relatively insensitive to the manipulation of any step in the pathway because it represents only part of the total and may also be countered by pleiotropic compensatory changes. The parameters in greatest need of additional measurements are S(c), mesophyll cell wall thickness, and the permeabilities of the plasma membrane and chloroplast envelope.

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.

The effects of short-term (minutes) variations of CO2 concentration on mesophyll conductance to CO2 (gm) were evaluated in six different C3 species by simultaneous measurements of gas exchange, chlorophyll fluorescence, online carbon isotope discrimination and a novel curve-fitting method. Depending on the species, gm varied from five- to ninefold, along the range of sub-stomatal CO2 concentrations typically used in photosynthesis CO2-response curves (AN)-Ci curves; where AN is the net photosynthetic flux and Ci is the CO2 concentrations in the sub-stomatal cavity), that is, 50 to 1500 micromol CO2 mol(-1) air. Although the pattern was species-dependent, gm strongly declined at high Ci, where photosynthesis was not limited by CO2, but by regeneration of ribulose-1,5-bisphosphate or triose phosphate utilization. Moreover, these changes on gm were found to be totally independent of the velocity and direction of the Ci changes. The response of gm to Ci resembled that of stomatal conductance (gs), but kinetic experiments suggested that the response of gm was actually faster than that of gs. Transgenic tobacco plants differing in the amounts of aquaporin NtAQP1 showed different slopes of the gm-Ci response, suggesting a possible role for aquaporins in mediating CO2 responsiveness of gm. The importance of these findings is discussed in terms of their effects on parameterization of AN-Ci curves.

The measurement of the response of net photosynthesis to leaf internal CO2 (i.e. A-Ci curves) is widely used for ecophysiological studies. Most studies did not consider CO2 exchange between the chamber and the surrounding air, especially at the two extremes of A-Ci curves, where large CO2 gradients are created, leading to erroneous estimations of A and Ci. A quantitative analysis of CO2 leakage in the chamber of a portable open gas exchange system (Li-6400, LI-COR Inc., NE, USA) was performed. In an empty chamber, the measured CO2 leakage was similar to that calculated using the manufacturer's equations. However, in the presence of a photosynthetically inactive leaf, the magnitude of leakage was substantially decreased, although still significant. These results, together with the analysis of the effects of chamber size, tightness, flow rate, and gasket material, suggest that the leakage is larger at the interface between the gaskets than through the gaskets. This differential leakage rate affects the parameterization by photosynthesis models. The magnitude of these errors was assessed in tobacco plants. The results showed that leakage results in a 10% overestimation of the leaf maximum capacity for carboxylation (Vc,max) and a 40% overestimation of day respiration (Rl). Using the manufacturer's equations resulted in larger, non-realistic corrections of the true values. The photosynthetic response to CO2 concentrations at the chloroplast (i.e. A-Cc curves) was significantly less affected by leakage than A-Ci curves. Therefore, photosynthetic parameterization can be improved by: (i) correcting A and Ci values for chamber leakage estimated using a photosynthetically inactive leaf; and (ii) using A-Cc instead of A-Ci curves.

Leaf 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).

Aquaporins, found in virtually all living organisms, are membrane-intrinsic proteins that form water-permeable complexes. The mammalian aquaporin AQP1 has also shown CO2 permeability when expressed heterologously in Xenopus oocytes, although whether this is a biochemical curiosity or of physiological significance is a matter of debate. Here we report that, in the same expression system, a CO2 permeability comparable to that of the human AQP1 is observed for the tobacco plasma membrane aquaporin NtAQP1. NtAQP1 facilitates CO2 membrane 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 CO2 and water, and increases leaf growth. The results indicate that NtAQP1-related CO2 permeability is of physiological importance under conditions where the CO2 gradient across a membrane is small, as is the case between the atmosphere and the inside of a plant cell.