Regulation of Arabidopsis leaf hydraulics involves light-dependent phosphorylation of aquaporins in veins.

The water status of plant leaves depends on the efficiency of the water supply, from the vasculature to inner tissues. This process is under hormonal and environmental regulation and involves aquaporin water channels. In Arabidopsis thaliana, the rosette hydraulic conductivity (Kros) is higher in darkness than it is during the day. Knockout plants showed that three plasma membrane intrinsic proteins (PIPs) sharing expression in veins (PIP1;2, PIP2;1, and PIP2;6) contribute to rosette water transport, and PIP2;1 can fully account for Kros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant was sufficient to restore Kros. In addition, a positive correlation, in both wild-type and PIP2;1-overexpressing plants, was found between Kros and the osmotic water permeability of protoplasts from the veins but not from the mesophyll. Thus, living cells in veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses showed that light-dependent regulation of Kros is linked to diphosphorylation of PIP2;1 at Ser-280 and Ser-283. Expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at these two sites is necessary for Kros enhancement under darkness. These findings establish how regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics.

Auxin regulates aquaporin function to facilitate lateral root emergence.

Aquaporins are membrane channels that facilitate water movement across cell membranes. In plants, aquaporins contribute to water relations. Here, we establish a new link between aquaporin-dependent tissue hydraulics and auxin-regulated root development in Arabidopsis thaliana. We report that most aquaporin genes are repressed during lateral root formation and by exogenous auxin treatment. Auxin reduces root hydraulic conductivity both at the cell and whole-organ levels. The highly expressed aquaporin PIP2;1 is progressively excluded from the site of the auxin response maximum in lateral root primordia (LRP) whilst being maintained at their base and underlying vascular tissues. Modelling predicts that the positive and negative perturbations of PIP2;1 expression alter water flow into LRP, thereby slowing lateral root emergence (LRE). Consistent with this mechanism, pip2;1 mutants and PIP2;1-overexpressing lines exhibit delayed LRE. We conclude that auxin promotes LRE by regulating the spatial and temporal distribution of aquaporin-dependent root tissue water transport.

A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis.

Aquaporins are channel proteins that facilitate the transport of water across plant cell membranes. In this work, we used a combination of pharmacological and reverse genetic approaches to investigate the overall significance of aquaporins for tissue water conductivity in Arabidopsis (Arabidopsis thaliana). We addressed the function in roots and leaves of AtPIP1;2, one of the most abundantly expressed isoforms of the plasma membrane intrinsic protein family. At variance with the water transport phenotype previously described in AtPIP2;2 knockout mutants, disruption of AtPIP1;2 reduced by 20% to 30% the root hydrostatic hydraulic conductivity but did not modify osmotic root water transport. These results document qualitatively distinct functions of different PIP isoforms in root water uptake. The hydraulic conductivity of excised rosettes (K(ros)) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased K(ros) by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of K(ros) by approximately 13% and approximately 25% in rosettes from plants grown in the light or under prolonged (11-18 h) darkness, respectively. Prolonged darkness enhanced the transcript abundance of several PIP genes, including AtPIP1;2. Mutant analysis showed that, under prolonged darkness conditions, AtPIP1;2 can contribute to up to approximately 20% of K(ros) and to the osmotic water permeability of isolated mesophyll protoplasts. Therefore, AtPIP1;2 can account for a significant portion of aquaporin-mediated leaf water transport. The overall work shows that AtPIP1;2 represents a key component of whole-plant hydraulics.

Stimulus-induced downregulation of root water transport involves reactive oxygen species-activated cell signalling and plasma membrane intrinsic protein internalization.

The water uptake capacity of plant roots (i.e. their hydraulic conductivity, Lp(r)) is determined in large part by aquaporins of the plasma membrane intrinsic protein (PIP) subfamily. In the present work, we investigated two stimuli, salicylic acid (SA) and salt, because of their ability to induce an accumulation of reactive oxygen species (ROS) and an inhibition of Lp(r) concomitantly in the roots of Arabidopsis plants. The inhibition of Lp(r) by SA was partially counteracted by preventing the accumulation of hydrogen peroxide (H(2)O(2)) with exogenous catalase. In addition, exogenous H(2)O(2) was able to reduce Lp(r) by up to 90% in <15 min. Based on the lack of effects of H(2)O(2) on the activity of individual aquaporins in Xenopus oocytes, and on a pharmacological dissection of the action of H(2)O(2) on Lp(r), we propose that ROS do not gate Arabidopsis root aquaporins through a direct oxidative mechanism, but rather act through cell signalling mechanisms. Expression in transgenic roots of PIP-GFP fusions and immunogold labelling indicated that external H(2)O(2) enhanced, in <15 min, the accumulation of PIPs in intracellular structures tentatively identified as vesicles and small vacuoles. Exposure of roots to SA or salt also induced an intracellular accumulation of the PIP-GFP fusion proteins, and these effects were fully counteracted by co-treatment with exogenous catalase. In conclusion, the present work identifies SA as a novel regulator of aquaporins, and delineates an ROS-dependent signalling pathway in the roots of Arabidopsis. Several abiotic and biotic stress-related stimuli potentially share this path, which involves an H(2)O(2)-induced internalization of PIPs, to downregulate root water transport.

The response of Arabidopsis root water transport to a challenging environment implicates reactive oxygen species- and phosphorylation-dependent internalization of aquaporins.

Aquaporins, which facilitate the diffusion of water across biological membranes, are key molecules for the regulation of water transport at the cell and organ levels. We recently reported that hydrogen peroxide (H(2)O(2)) acts as an intermediate in the regulation of Arabidopsis root water transport and aquaporins in response to NaCl and salicylic acid (SA).1 Its action involves signaling pathways and an internalization of aquaporins from the cell surface. The present addendum connects these findings to another recent work which describes multiple phosphorylations in the C-terminus of aquaporins expressed in the Arabidopsis root plasma membrane.2 A novel role for phosphorylation in the process of salt-induced relocalization of AtPIP2;1, one of the most abundant root aquaporins, was unraveled. Altogether, the data delineate reactive oxygen species (ROS)-dependent signaling mechanisms which, in response to a variety of abiotic and biotic stresses, can trigger phosphorylation-dependent PIP aquaporin intracellular trafficking and root water transport downregulation.

Expression and inhibition of aquaporins in germinating Arabidopsis seeds.

Extensive and kinetically well-defined water exchanges occur during germination of seeds. A putative role for aquaporins in this process was investigated in Arabidopsis. Macro-arrays carrying aquaporin gene-specific tags and antibodies raised against aquaporin subclasses revealed two distinct aquaporin expression programs between dry seeds and young seedlings. High expression levels of a restricted number of tonoplast intrinsic protein (TIP) isoforms (TIP3;1 and/or TIP3;2, and TIP5;1) together with a low expression of all 13 plasma membrane aquaporin (PIP) isoforms was observed in dry and germinating materials. In contrast, prevalent expression of aquaporins of the TIP1, TIP2 and PIP subgroups was induced during seedling establishment. Mercury (5 microM HgCl(2)), a general blocker of aquaporins in various organisms, reduced the speed of seed germination and induced a true delay in maternal seed coat (testa) rupture and radicle emergence, by 8-9 and 25-30 h, respectively. Most importantly, mercury did not alter seed lot homogeneity nor the seed germination developmental sequence, and its effects were largely reversed by addition of 2 mM dithiothreitol, suggesting that these effects were primarily due to oxidation of cell components, possibly aquaporins, without irreversible alteration of cell integrity. Measurements of water uptake in control and mercury-treated seeds suggested that aquaporin functions are not involved in early seed imbibition (phase I) but would rather be associated with a delayed initiation of phase III, i.e. water uptake accompanying expansion and growth of the embryo. A possible role for aquaporins in germinating seeds and more generally in plant tissue growth is discussed.

Analysis of the genomic response of a wine yeast to rehydration and inoculation.

We used DNA microarrays to study the transcriptome of a wine yeast before and after rehydration and during the first hours following inoculation of a synthetic must. There was a substantial transcriptional remodeling during this period, including 1,874 genes regulated more than threefold. Dried yeasts displayed an expression profile typical of respiratory-grown cells starved for nitrogen and carbon and which had been highly stressed. During rehydration, many genes involved in biosynthetic pathways, in transcription or in protein synthesis were coordinately induced while genes subject to glucose repression were down-regulated. The transcriptional response was very rapid indicating that yeast quickly recovered the capacity to sense environmental signals and to respond appropriately. Our data show that genes involved in the general stress response were repressed during rehydration while acid stress specific genes were induced probably in response to organic acid accumulation. The glycolytic genes and acid stress-responsive genes were simultaneously and transiently repressed after inoculation into the fermentation medium suggesting that regulation of glycolytic genes may correspond to an adjustment to the energetic needs of the cells. Surprisingly, inoculation into the must did not trigger a stress response despite the high concentrations of sugars.