Tobacco aquaporin NtAQP1 and human aquaporin hAQP1 contribute to single cell photosynthesis in Synechococcus.

BACKGROUND INFORMATION: Aquaporins are H2O-permeable membrane protein pores. However, some aquaporins are also permeable to other substances such as CO2. In higher plants, overexpression of such aquaporins has already led to an enhanced photosynthetic performance due to improved CO2 mesophyll conductance. In this work, we investigated the effects of such aquaporins on unicellular photosynthetically active organisms, specifically cyanobacteria. RESULTS: Overexpression of aquaporins NtAQP1 or hAQP1 that might have a function to improve CO2 membrane permeability lead to increased photosynthesis rates in the cyanobacterium Synechococcus sp. PCC7002 as concluded by the rate of evolved O2. A shift in the Plastoquinone pool state of the cells supports our findings. Water permeable aquaporins without CO2 permeability, such as NtPIP2;1, do not have this effect. CONCLUSIONS AND SIGNIFICANCE: We conclude that also in single cell organisms like cyanobacteria, membrane CO2 conductivity could be rate limiting and CO2-porins reduce the respective membrane resistance. We could show that besides the tobacco aquaporin NtAQP1 also the human hAQP1 most likely functions as CO2 diffusion facilitator in the photosynthesis assay.

Aquaporins and membrane diffusion of CO2 in living organisms.

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.

T-DNA insertion in aquaporin gene AtPIP1;2 generates transcription profiles reminiscent of a low CO2 response.

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.

Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays.

Stomata, the microscopic pores on the surface of the aerial parts of plants, are bordered by two specialized cells, known as guard cells, which control the stomatal aperture according to endogenous and environmental signals. Like most movements occurring in plants, the opening and closing of stomata are based on hydraulic forces. During opening, the activation of plasma membrane and tonoplast transporters results in solute accumulation in the guard cells. To re-establish the perturbed osmotic equilibrium, water follows the solutes into the cells, leading to their swelling. Numerous studies have contributed to the understanding of the mechanism and regulation of stomatal movements. However, despite the importance of transmembrane water flow during this process, only a few studies have provided evidence for the involvement of water channels, called aquaporins. Here, we microdissected Zea mays stomatal complexes and showed that members of the aquaporin plasma membrane intrinsic protein (PIP) subfamily are expressed in these complexes and that their mRNA expression generally follows a diurnal pattern. The substrate specificity of two of the expressed ZmPIPs, ZmPIP1;5 and ZmPIP1;6, was investigated by heterologous expression in Xenopus oocytes and yeast cells. Our data show that both isoforms facilitate transmembrane water diffusion in the presence of the ZmPIP2;1 isoform. In addition, both display CO2 permeability comparable to that of the CO2 diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes.

The grapevine root-specific aquaporin VvPIP2;4N controls root hydraulic conductance and leaf gas exchange under well-watered conditions but not under water stress.

We functionally characterized the grape (Vitis vinifera) VvPIP2;4N (for Plasma membrane Intrinsic Protein) aquaporin gene. Expression of VvPIP2;4N in Xenopus laevis oocytes increased their swelling rate 54-fold. Northern blot and quantitative reverse transcription-polymerase chain reaction analyses showed that VvPIP2;4N is the most expressed PIP2 gene in root. In situ hybridization confirmed root localization in the cortical parenchyma and close to the endodermis. We then constitutively overexpressed VvPIP2;4N in grape 'Brachetto', and in the resulting transgenic plants we analyzed (1) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, (2) whole-plant, root, and leaf ecophysiological parameters, and (3) leaf abscisic acid content. Expression of transgenic VvPIP2;4N inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. Under well-watered conditions, transgenic plants showed higher stomatal conductance, gas exchange, and shoot growth. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. The leaf component of total plant hydraulic resistance was low and unaffected by overexpression of VvPIP2;4N. Upon water stress, the overexpression of VvPIP2;4N induced a surge in leaf abscisic acid content and a decrease in stomatal conductance and leaf gas exchange. Our results show that aquaporin-mediated modifications of root hydraulics play a substantial role in the regulation of water flow in well-watered grapevine plants, while they have a minor role upon drought, probably because other signals, such as abscisic acid, take over the control of water flow.

Impacts of Radio-Frequency Electromagnetic Field (RF-EMF) on Lettuce (Lactuca sativa)-Evidence for RF-EMF Interference with Plant Stress Responses.

The increased use of wireless technology causes a significant exposure increase for all living organisms to radio frequency electromagnetic fields (RF-EMF). This comprises bacteria, animals, and also plants. Unfortunately, our understanding of how RF-EMF influences plants and plant physiology remains inadequate. In this study, we examined the effects of RF-EMF radiation on lettuce plants (Lactuca sativa) in both indoor and outdoor environments using the frequency ranges of 1890-1900 MHz (DECT) at 2.4 GHz and 5 GHz (Wi-Fi). Under greenhouse conditions, RF-EMF exposure had only a minor impact on fast chlorophyll fluorescence kinetics and no effect on plant flowering time. In contrast, lettuce plants exposed to RF-EMF in the field showed a significant and systemic decrease in photosynthetic efficiency and accelerated flowering time compared to the control groups. Gene expression analysis revealed significant down-regulation of two stress-related genes in RF-EMF-exposed plants: violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP). RF-EMF-exposed plants had lower Photosystem II's maximal photochemical quantum yield (FV/FM) and non-photochemical quenching (NPQ) than control plants under light stress conditions. In summary, our results imply that RF-EMF might interfere with plant stress responses and reduced plant stress tolerance.

The Arabidopsis thaliana aquaporin AtPIP1;2 is a physiologically relevant CO2 transport facilitator.

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.

The aquaporin TcAQP1 of the desert truffle Terfezia claveryi is a membrane pore for water and CO(2) transport.

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.

Molecular convergence of the parasitic plant species Cuscuta reflexa and Phelipanche aegyptiaca.

The parasitic plant species Cuscuta reflexa and Phelipanche aegyptiaca have independently developed parasitism, the former parasitizing on shoots and the latter attaching to roots. Regardless of these differences, the two species use similar organs, termed haustoria, to attach to the host plant. In this study, we show that this morphological similarity can be extended to the molecular level. An attAGP-promoter from Solanum lycopersicum, which is activated by Cuscuta infections, was also induced after infection by P. aegyptiaca. Furthermore, we show by validation of transcriptome sequencing data that the Phelipanche orthologue of a haustorium-specific Cuscuta gene, which codes for a cysteine proteinase, was activated in the early stages of Phelipanche invasion. Inhibition of the Phelipanche cysteine proteinase was achieved by 35S- or attAGP-promoter-driven expression of its intrinsic inhibitory polypeptide. A reduction in P. aegyptiaca infection rates during experiments in flower pots and in an in vitro polybag system in comparison to controls was recorded.

The Arabidopsis aquaporin PIP1;2 rules cellular CO(2) uptake.

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.

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.

Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions.

BACKGROUND: Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. RESULTS: One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events. CONCLUSIONS: The study provides new information about molecular events during the parasitic plant--host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants.

The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions.

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.

Metabolic engineering of ketocarotenoids biosynthetic pathway in Chlamydomonas reinhardtii strain CC-4102.

In Chlamydomonas reinhardtii, ketocarotenoid biosynthesis is limited to the diploid zygospore stage. In this study, we attempted to engineer the ketocarotenoid pathway into Chlamydomonas haploid vegetative green cells by overexpressing the key enzyme ß-carotene ketolase (CrBKT). We chose strain CC-4102 for the approach; competitive pathways, α-carotene biosynthesis and xanthophyll cycle are silenced in this strain. Driven by the strong constitutive HSP70/RBCS2 promoter CrBKT overexpression resulted in the production of canthaxanthin, the ketolation product from ß-carotene as well as a drastic reduction in the chlorophyll concentration. Intriguingly, these phenotypes could only be detected from lines transformed and grown heterotrophically in the dark. Once exposed to light, these transformants lost the aforementioned phenotypes as well as their antibiotic resistance. This phenomenon is in agreement with the fact that we were unable to recover any canthaxanthin-producing line among light-selected transformants.

Resistances along the CO2 diffusion pathway inside leaves.

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.

Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System.

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Aquaporin PIP2;1 affects water transport and root growth in rice (Oryza sativa L.).

Aquaporins are key proteins in regulating water transport, plant growth and development. In this study, we investigated the function of plasma membrane intrinsic proteins (PIPs) in both yeast (Saccharomyces cerevisiae) and rice (Oryza sativa cv. Nipponbare). Three OsPIP1s (OsPIP1;1, OsPIP1;2 and OsPIP1;3) and four OsPIP2s (OsPIP2;1, OsPIP2;3, OsPIP2;4 and OsPIP2;5) were successfully amplified and expressed in yeast. Overexpression of OsPIP2s, especially OsPIP2;1, increased yeast membrane water permeability (Pf). Root hydraulic conductivity (Lpr) was decreased by approximately four-fold in OsPIP2; 1 RNAi knock-down plants, resulting in a decrease in OsPIP2;1 expression levels of 70% and 50% in line 3 and line 4, respectively, compared to the wild type (WT) plants. No significant differences in the photosynthetic rate, transpiration rate, mesophyll conductance and chloroplast CO2 concentration were observed between WT and OsPIP2; 1 RNAi plants. Higher stomatal conductance and intercellular CO2 concentrations were observed in line 3 plants than in WT plants. In addition, lower root total length, surface area, root volume and fewer root tips were found in the RNAi plants than in the WT plants. Finally, the RNAi plants were more sensitive to drought stress. The results indicate that PIP2; 1 plays an important role in the regulation of water transport and plant growth.

Aquaporins and plant water balance.

The impact of aquaporin function on plant water balance is discussed. The significance of these proteins for root water uptake, water conductance in the xylem, including embolism refilling and the role of plant aquaporins in leaf physiology, is described. Emphasis is placed on certain aspects of water stress reactions and the correlation of aquaporins to abscisic acid as well as on the relation of water and CO2 permeability in leaves.

Aquaporins and plant leaf movements.

BACKGROUND: Plant leaf movements can be mediated by specialized motor organs, the pulvini, or can be epinastic (i.e. based on different growth velocities of the adaxial and abaxial halves of the leaf). Both processes are associated with diurnally regulated increases in rates of membrane water transport, which in many cases has been shown to be facilitated by aquaporins. Rhythmic leaf movements are known from many plant species, but few papers deal with the involvement of aquaporins in such movements. SCOPE: Many details of the architecture and function of pulvini were worked out by Ruth Satter and co-workers using Samanea saman as a model organism. More recently a contribution of aquaporins to pulvinar movement in Samanea was demonstrated. Another model plant to study pulvinus-mediated leaf movements is Mimosa pudica. The contribution of both plasma membrane- and tonoplast-localized aquaporins to the seismonastic leaf movements in Mimosa was analysed. In tobacco, as an example of epinastic leaf movement, it was shown that a PIP1 aquaporin family member is an important component of the leaf movement mechanism.

Functional aquaporin diversity in plants.

Due to the fact that most plants are immobile, a rapid response of physiological processes to changing environmental conditions is essential for their survival. Thus, in comparison to many other organisms, plants might need a more sophisticated tuning of water balance. Among others, this is reflected by the comparable large amount of aquaporin genes in plant genomes. So far, aquaporins were shown to be involved in many physiological processes like root water uptake, reproduction or photosynthesis. Their classification as simple water pores has changed according to their molecular function into channels permeable for water, small solutes and/or gases. An adjustment of the corresponding physiological process could be achieved by regulation mechanisms. Concerning aquaporins these range from posttranslational modification, molecular trafficking to heteromerization of aquaporin isoforms. The aim of this review is to underline the function of the four plant aquaporin family subclasses with regard to the substrate specificity, regulation and physiological relevance.