Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.

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.

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 (P f ) 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.T he plasma membrane (PM) is the first barrier that limits water exchange in plant cells. The rate of its water transport capacity is mainly associated with aquaporins. Among the seven aquaporin subfamilies described in the plant kingdom, only plasma membrane intrinsic proteins (PIP) and some members of the nodulin-26-like intrinsic proteins (NIP) and X intrinsic proteins (XIP) subfamilies have been shown to be preferentially localized at the PM (1, 2). Of these, PIP aquaporins appear to have a large role in controlling membrane water permeability, whereas NIP and XIP have been mainly described as solute transporters (2-4). Plant PIP aquaporins represent a conserved subfamily that has been historically divided into two subgroups due to their differences in primary structure, PIP1 and PIP2. Interestingly, PIP aquaporins compose ∼40% of the total aquaporin set, and the PIP1 and PIP2 ratio among different species is relatively constant (5-12). Fig. S1 shows the distribution of all aquaporin genes present in plants whose genome has been completely sequenced and analyzed. Antisense inhibition experiments on Arabidopsis thaliana PIP1 and PIP2 have suggested that the two subgroups of aquaporins contribute to root or leaf hydraulic conductivity in the same way (13). In several plant species, members of the PIP1 and PIP2 subgroups were shown to be coexpressed in the same cell type (14-17).Although PIP1 are as ubiquitous as PIP2, the functional properties of each type of channel are different. PIP2 are very well described as a homotetramer with high water transport activity (18, 19) and a gating mechanism unequivocally associated with specific and conserved amino acid motifs triggered by cytosolic acidification (20-22), phosphorylation (23, 24), or divalent cation concentration (22). In contrast, PIP1 have shown complex heterogeneity in water and solute transpor...

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.

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.

SUMMARYIn 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.

Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was experimentally demonstrated in planta and in heterologous systems. The condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis indicated that MsAACT1 expression is highly increased in roots and leaves under cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the MVA pathway, resulted in a decrease in squalene production, antioxidant activity, and the survival of stressed plants. As expected, the presence of mevastatin did not change chlorophyll and carotenoid levels, isoprenoids synthesized via the plastidial MVA-independent pathway. The addition of vitamin C suppressed the sensitive phenotype of plants challenged with mevastatin, suggesting a critical function of the MVA pathway in abiotic stress-inducible antioxidant defence. MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable with empty vector transformed plants and enhanced production of squalene without altering the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity in salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in isoprenoid biosynthesis involved in abiotic stress adaptation.

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