Gas-tight triblock-copolymer membranes are converted to CO2 permeable by insertion of plant aquaporins.

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.

Aquaporin tetramer composition modifies the function of tobacco aquaporins.

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.

Function of Nicotiana tabacum aquaporins as chloroplast gas pores challenges the concept of membrane CO2 permeability.

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.

Characterization of plant aquaporins.

Plants have been reported to contain a large set of aquaporins (38 for Arabidopsis), which has been divided into four subfamilies on the basis of similarities in their amino acid sequences. They belong to the large superfamily of major intrinsic proteins (MIP), which was the basis for the nomenclature PIP, TIP, and NIP, also indicating the subcellular localization plasma membrane, tonoplast, and nodule of the respective founding member. The fourth subfamily of small and basic intrinsic proteins is not well characterized so far. The increasing number of reports dealing with various aspects of plant aquaporins is starting to advance our understanding of aquaporin biology in plants. Fundamental questions include: what is the basic function of the different plant aquaporins, what is their primary substrate, and what is the consequence of function/malfunction of a particular aquaporin for the overall function of the plant? Biochemical and biophysical techniques can be employed to get information on the basic functional characteristics of plant aquaporins. An impressive set of techniques has been used to study aquaporin function on molecular, subcellular, and cellular levels in plants, as well as in heterologous expression systems. The physiological role of aquaporins in plants is much less well understood, but reports unraveling the physiological role of aquaporins, mainly employing genetic techniques and functional measurement on the whole plant level, are emerging. The goal of this chapter is to give an overview on the applied methods, together with some exemplary findings.

Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo.

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).

The plasma membrane aquaporin NtAQP1 is a key component of the leaf unfolding mechanism in tobacco.

Epinastic leaf movement of tobacco is based on differential growth of the upper and lower leaf surface and is distinct from the motor organ-driven mechanism of nyctinastic leaf movement of, for example, mimosa species. The epinastic leaf movement of tobacco is observed not only under diurnal light regimes but also in continuous light, indicating a control by light and the circadian clock. As the transport of water across membranes by aquaporins is an important component of rapid plant cell elongation, the role of the tobacco aquaporin Nt aquaporin (AQP)1 in the epinastic response was studied in detail. In planta NtAQP1-luciferase (LUC) activity studies, Northern and Western blot analyses demonstrated a diurnal and circadian oscillation in the expression of this plasma membrane intrinsic protein (PIP)1-type aquaporin in leaf petioles, exhibiting peaks of expression coinciding with leaf unfolding. Cellular water permeability of protoplasts isolated from leaf petioles was found to be high in the morning, i.e. during the unfolding reaction, and low in the evening. Moreover, diurnal epinastic leaf movement was shown to be reduced in transgenic tobacco lines with an impaired expression of NtAQP1. It is concluded that the cyclic expression of PIP1-aquaporin represents an important component of the leaf movement mechanism.

Plasma membrane aquaporins in the motor cells of Samanea saman: diurnal and circadian regulation.

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.