Karina Alleva

AtTIP1;3 and AtTIP5;1, the only highly expressed Arabidopsis pollen‐specific aquaporins, transport water and urea

Pollination includes processes where water and/or solute movements must be finely regulated, suggesting participation of aquaporins. Using information available from different transcriptional profilings of Arabidopsis thaliana mature pollen, we showed that the only aquaporins that are selectively and highly expressed in mature pollen are two TIPs: AtTIP1;3 and AtTIP5;1. Pollen exhibited a lower number and more exclusive type of aquaporin expressed genes when compared to other single cell transcriptional profilings. When characterized using Xenopus oocyte swelling assays, AtTIP1;3 and AtTIP5;1 showed intermediate water permeabilities. Although they displayed neither glycerol nor boric acid permeability they both transported urea. In conclusion, these results suggest a function for AtTIP1;3 and AtTIP5;1 as specific water and urea channels in Arabidopsis pollen.

TIP5;1 is an aquaporin specifically targeted to pollen mitochondria and is probably involved in nitrogen remobilization in Arabidopsis thaliana

In plant sexual reproduction, water and solute movement are tightly regulated, suggesting the involvement of aquaporins. We previously identified TIP5;1 and TIP1;3 as the only Arabidopsis aquaporin genes that are selectively and highly expressed in mature pollen, and showed that they can transport both water and urea when expressed in Xenopus oocytes. Here, we show that TIP5;1 has unusual characteristics, as its water transport activity is regulated by pH. Analysis of the water transport activity of a mutant version of TIP5;1 (TIP5;1-H131A) and amino acid alignment with other plant aquaporins regulated by pH suggested that a conserved motif is involved in pH sensing. GFP-TIP5;1 is located in the mitochondria of pollen tubes. The single mutants tip1;3 and tip5;1, as well as the tip1;3 tip5;1 double mutant, are fertile, but all mutants had shorter than normal pollen tubes when germinated in vitro in the absence of exogenous nitrogen. Thus, we propose that TIP5;1 and TIP1;3 are involved in nitrogen recycling in pollen tubes of Arabidopsis thaliana.

Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression.

The plant plasma membrane barrier can express aquaporins (PIP1 and PIP2) that show two intriguing aspects: (1) the potential of modulating whole membrane water permeability by co-expression of both types, which have recently been distinguished for showing a different capacity to reach the plasma membrane; and (2) the faculty to reduce water permeation through the pore after cytosolic acidification, as a consequence of a gating process. Our working hypothesis is that these two key features might enhance plasticity of the membrane water transport capacity if they jointly trigger any cooperative interaction. In previous work, we proved by biophysical approaches that the plasma membrane of the halophyte Beta vulgaris storage root presents highly permeable aquaporins that can be shut down by acidic pH. Root Beta vulgaris PIPs were therefore subcloned and expressed in Xenopus oocytes. Co-expression of BvPIP1;1 and BvPIP2;2 not only enhances oocyte plasma membrane water permeability synergistically but also reinforces pH inhibitory response from partial to complete shut down after cytosolic pH acidification. This pH dependent behavior shows that PIP1–PIP2 co-expression accounts for a different pH sensitivity in comparison with PIP2 expression. These results prove for the first time that PIP co-expression modulates the membrane water permeability through a pH regulatory response, enhancing in this way membrane versatility to adjust its water transfer capacity.

Heteromerization of PIP aquaporins affects their intrinsic permeability

Significance Aquaporins are known for their capacity to increase transcellular water exchange. In plants, a highly conserved group known as plasma membrane intrinsic proteins (PIP) affects the adjustment of not only membrane water permeability but also overall plant hydraulic conductivity. An experimental design combined with a mathematical modeling approach allowed us to explore the interplay of channel gating, membrane translocation, and channel stoichiometric arrangement of a pair of PIP1 and PIP2 aquaporins. We dissect the individual contribution of each PIP, showing that (i) PIP1 has a high water transport capacity when coexpressed with PIP2, (ii) PIP2 water permeability is enhanced if it physically interacts with PIP1, and (iii) the PIP1–PIP2 interaction results in the formation of heterotetramers with random stoichiometric arrangement. The plant aquaporin plasma membrane intrinsic proteins (PIP) subfamily represents one of the main gateways for water exchange at the plasma membrane (PM). A fraction of this subfamily, known as PIP1, does not reach the PM unless they are coexpressed with a PIP2 aquaporin. Although ubiquitous and abundantly expressed, the role and properties of PIP1 aquaporins have therefore remained masked. Here, we unravel how FaPIP1;1, a fruit-specific PIP1 aquaporin from Fragaria x ananassa, contributes to the modulation of membrane water permeability (Pf) and pH aquaporin regulation. Our approach was to combine an experimental and mathematical model design to test its activity without affecting its trafficking dynamics. We demonstrate that FaPIP1;1 has a high water channel activity when coexpressed as well as how PIP1–PIP2 affects gating sensitivity in terms of cytosolic acidification. PIP1–PIP2 random heterotetramerization not only allows FaPIP1;1 to arrive at the PM but also produces an enhancement of FaPIP2;1 activity. In this context, we propose that FaPIP1;1 is a key participant in the regulation of water movement across the membranes of cells expressing both aquaporins.

Genome-wide data (ChIP-seq) enabled identification of cell wall-related and aquaporin genes as targets of tomato ASR1, a drought stress-responsive transcription factor

BackgroundIdentifying the target genes of transcription factors is important for unraveling regulatory networks in all types of organisms. Our interest was precisely to uncover the spectrum of loci regulated by a widespread plant transcription factor involved in physiological adaptation to drought, a type of stress that plants have encountered since the colonization of land habitats 400 MYA. The regulator under study, named ASR1, is exclusive to the plant kingdom (albeit absent in Arabidopsis) and known to alleviate the stress caused by restricted water availability. As its target genes are still unknown despite the original cloning of Asr1 cDNA 20 years ago, we examined the tomato genome for specific loci interacting in vivo with this conspicuous protein.ResultsWe performed ChIP followed by high throughput DNA sequencing (ChIP-seq) on leaves from stressed tomato plants, using a high-quality anti-ASR1 antibody. In this way, we unraveled a novel repertoire of target genes, some of which are clearly involved in the response to drought stress. Many of the ASR1-enriched genomic loci we found encode enzymes involved in cell wall synthesis and remodeling as well as channels implicated in water and solute flux, such as aquaporins. In addition, we were able to determine a robust consensus ASR1-binding DNA motif.ConclusionsThe finding of cell wall synthesis and aquaporin genes as targets of ASR1 is consistent with their suggested role in the physiological adaptation of plants to water loss. The results gain insight into the environmental stress-sensing pathways leading to plant tolerance of drought.

A fruit-specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry (Fragaria × ananassa) fruit ripening

Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry (Fragaria xananassa) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.

Prediction of Aquaporin Function by Integrating Evolutionary and Functional Analyses

Aquaporins (AQPs) are a family of channel proteins, which transport water and/or small solutes across cell membranes. AQPs are present in Bacteria, Eukarya, and Archaea. The classical AQP evolution paradigm explains the inconsistent phylogenetic trees by multiple transfer events and emphasizes that the assignment of orthologous AQPs is not possible, making it difficult to integrate functional information. Recently, a novel phylogenetic framework of eukaryotic AQP evolution showed congruence between eukaryotic AQPs and organismal trees identifying 32 orthologous clusters in plants and animals (Soto et al. Gene 503:165–176, 2012). In this article, we discuss in depth the methodological strength, the ability to predict functionality and the AQP community perception about the different paradigms of AQP evolution. Moreover, we show an updated review of AQPs transport functions in association with phylogenetic analyses. Finally, we discuss the possible effect of AQP data integration in the understanding of water and solute transport in eukaryotic cells.

Cloning, functional characterization, and co-expression studies of a novel aquaporin (FaPIP2;1) of strawberry fruit

In strawberry, the putative participation of aquaporins should be considered during fruit ripening. Furthermore, the availability of different firmness cultivars in this non-climacteric fruit is a very useful tool to determine their involvement in softening. In a previous work, the cloning of a strawberry fruit-specific aquaporin, FaPIP1;1, which showed an expression profile associated with fruit ripening was reported. Here, FaPIP2;1, an aquaporin subtype of PIP2 was cloned and its functional characterization in Xenopus oocytes determined. The FaPIP2;1 gene encodes a water channel with high water permeability (Pf) that is regulated by cytosolic pH. Interestingly, the co-expression of both FaPIP subtypes resulted in an enhancement of water permeability, showing Pf values that exceeds their individual contribution. The expression pattern of both aquaporin subtypes in two cultivars with contrasting fruit firmness showed that the firmer cultivar (Camarosa) has a higher accumulation of FaPIP1 and FaPIP2 mRNAs during fruit ripening when compared with the softer cultivar (Toyonoka). In conclusion, not only FaPIP aquaporins showed an expression pattern associated with fruit firmness but it was also shown that the enhancement of water transfer through the plasma membrane is coupled to the presence/absence of the co-expression of both subtypes.

Tonoplast vesicles of Beta vulgaris storage root show functional aquaporins regulated by protons

Background information. Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re‐evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation.

Aquaporins: Another piece in the osmotic puzzle

Osmolarity not only plays a key role in cellular homeostasis but also challenges cell survival. The molecular understanding of osmosis has not yet been completely achieved, and the discovery of aquaporins as molecular entities involved in water transport has caused osmosis to again become a focus of research. The main questions that need to be answered are the mechanism underlying the osmotic permeability coefficients and the extent to which aquaporins change our understanding of osmosis. Here, attempts to answer these questions are discussed. Critical aspects of the state of the state of knowledge on osmosis, a topic that has been studied since 19th century, are reviewed and integrated with the available information provided by in vivo, in vitro and in silico approaches.