Gating of aqùaporins by light and reactive oxygen species in leaf parenchyma cells of the midrib of Zea mays

Changes of the water permeability aqùaporin (AQP) activity of leaf cells were investigated in response to different light regimes (low versus high). Using a cell pressure probe, hydraulic properties (half-time of water exchange, T1/2 ∞ 1/water permeability) of parenchyma cells in the midrib tissue of maize (Zea mays L.) leaves have been measured. A new perfusion technique was applied to excised leaves to keep turgor constant and to modify the environment around cells by perfusing solutions using a pressure chamber. In response to low light (LL) of 200 μmol m−2 s−1, T1/2 decreased during the perfusion of a control solution of 0.5 mM CaCl2 by a factor of two. This was in line with earlier results from leaf cells of intact maize plants at a constant turgor. In contrast, high light (HL) at intensities of 800 μmol m−2 s−1 and 1800 μmol m−2 s−1 increased the T1/2 in two-thirds of cells by factors of 14 and 35, respectively. The effects of HL on T1/2 were similar to those caused by H2O2 treatment in the presence of Fe2+, which produced ·OH (Fenton reaction; reversible oxidative gating of aquaporins). Treatments with 20 mM H2O2 following Fe2+ pre-treatments increased the T1/2 by a factor of 30. Those increased T1/2 values could be partly recovered, either when the perfusion solution was changed back to the control solutuion or when LL was applied. 3mM of the antioxidant glutathione also reversed the effects of HL. The data suggest that HL could induce reactive oxygen species (ROS) such as ·OH, and they affected water relations. The results provide evidence that the varying light climate adjusts water flow at the cell level; that is, water flow is maximized at a certain light intensity and then reduced again by HL. Light effects are discussed in terms of an oxidative gating of aquaporins by ROS

Protein kinase SnRK2. 4 is a key regulator of aquaporins and root hydraulics in Arabidopsis

Soil water uptake by roots is a key component of plant water homeostasis contributing to plant growth and survival under ever-changing environmental conditions. The water transport capacity of roots (root hydraulic conductivity; Lpr ) is mostly contributed by finely regulated Plasma membrane Intrinsic Protein (PIP) aquaporins. In this study, we used natural variation of Arabidopsis for the identification of quantitative trait loci (QTLs) contributing to Lpr . Using recombinant lines from a biparental cross (Cvi-0 x Col-0), we show that the gene encoding class 2 Sucrose-Non-Fermenting Protein kinase 2.4 (SnRK2.4) in Col-0 contributes to >30% of Lpr by enhancing aquaporin-dependent water transport. At variance with the inactive and possibly unstable Cvi-0 SnRK2.4 form, the Col-0 form interacts with and phosphorylates the prototypal PIP2;1 aquaporin at Ser121 and stimulates its water transport activity upon coexpression in Xenopus oocytes and yeast cells. Activation of PIP2;1 by Col-0 SnRK2.4 in yeast also requires its protein kinase activity and can be counteracted by clade A Protein Phosphatases 2C. SnRK2.4 shows all hallmarks to be part of core abscisic acid (ABA) signaling modules. Yet, long-term (>3 h) inhibition of Lpr by ABA possibly involves a SnRK2.4-independent inhibition of PIP2;1. SnRK2.4 also promotes stomatal aperture and ABA-induced inhibition of primary root growth. The study identifies a key component of Lpr and sheds new light on the functional overlap and specificity of SnRK2.4 with respect to other ABA-dependent or independent SnRK2s

Aquaporin gene expression and apoplastic water flow in bur oak (Quercus macrocarpa) leaves in relation to the light response of leaf hydraulic conductance

It has previously been shown that hydraulic conductance in bur oak leaves (Quercus macrocarpa Michx.), measured with the high pressure flow meter technique (HPFM), can significantly increase within 30 min following exposure to high irradiance. The present study investigated whether this increase could be explained by an increase in the cell-to-cell pathway and whether the response is linked to changes in the transcript level corresponding to aquaporin genes. Four cDNA sequences showing high similarity to members of the aquaporin gene family from other plant species were characterized from bur oak leaves and the expression levels of these cDNA sequences were examined in leaves by quantitative real-time PCR (QRT-PCR). No change was found in the relative transcript abundance corresponding to these four putative aquaporin genes in leaves with light-induced high hydraulic conductance (exposed to high irradiance) compared to leaves with low hydraulic conductance (exposed to low irradiance). However, in sun leaves that were exposed to different light levels prior to leaf collection (full sunlight, shade, and covered with aluminium foil for 16 h), the relative transcript levels of two of the putative aquaporin genes increased several-fold in shaded leaves compared to the sun-exposed or covered leaves. When the leaves were pressure-infiltrated with the apoplastic tracer dye trisodium 3-hydroxy-5,8,10-pyrenetrisulphonate (PTS3, 0.02%), there was no change in the PTS3 concentration of leaf exudates collected in ambient light or in high irradiance, but there was a small apoplastic acidification. There was also no change in PTS3 concentration between the leaves infiltrated under high irradiance with 0.02% PTS3 or with 0.1 mM HgCl2 in 0.02% PTS3. The results suggest that the putative aquaporin genes that were identified in the present study probably do not play a role in the light responses of hydraulic conductance at the transcript level, but they may function in regulating water homeostasis in leaves adapted to different light conditions. In addition, it is shown that high irradiance induced changes in the pH of the apoplast and that there does not appear to be a significant shift to the cell-to-cell mediated water transport in bur oak leaves exposed to high irradiance as measured by the apoplastic tracer dye

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots

Water uptake by seminal and adventitious roots in relation to whole-plant water flow in barley (Hordeum vulgare L.)

Prior to an assessment of the role of aquaporins in root water uptake, the main path of water movement in different types of root and driving forces during day and night need to be known. In the present study on hydroponically grown barley (Hordeum vulgare L.) the two main root types of 14- to 17-d-old plants were analysed for hydraulic conductivity in dependence of the main driving force (hydrostatic, osmotic). Seminal roots contributed 92% and adventitious roots 8% to plant water uptake. The lower contribution of adventitious compared with seminal roots was associated with a smaller surface area and number of roots per plant and a lower axial hydraulic conductance, and occurred despite a less-developed endodermis. The radial hydraulic conductivity of the two types of root was similar and depended little on the prevailing driving force, suggesting that water uptake occurred along a pathway that involved crossing of membrane(s). Exudation experiments showed that osmotic forces were sufficient to support night-time transpiration, yet transpiration experiments and cuticle permeance data questioned the significance of osmotic forces. During the day, 90% of water uptake was driven by a tension of about –0.15 MPa

Developmental pattern of aquaporin expression in barley (Hordeum vulgare L.) leaves

Aquaporins are multifunctional membrane channels which belong to the family of major intrinsic proteins (MIPs) and are best known for their ability to facilitate the movement of water. In the present study, earlier results from microarray experiments were followed up. These experiments had suggested that, in barley (Hordeum vulgare L.), aquaporin family members are expressed in distinct patterns during leaf development. Real-time PCR and in situ hybridization were used to analyse the level and tissue-distribution of expression of candidate aquaporins, focusing on plasma membrane and tonoplast intrinsic proteins (PIPs, TIPs). Water channel function of seven aquaporins, whose transcripts were the most abundant and the most variable, was tested through expression in yeast and, in part, through expression in oocytes. All PIP1 and PIP2 subfamily members changed in expression during leaf development, with expression being much higher or lower in growing compared with mature tissue. The same applied to those TIPs which were expressed at detectable levels. Specific roles during leaf development are proposed for particular aquaporins

Algal MIPs, high diversity and conserved motifs

<p>Abstract</p> <p>Background</p> <p>Major intrinsic proteins (MIPs) also named aquaporins form channels facilitating the passive transport of water and other small polar molecules across membranes. MIPs are particularly abundant and diverse in terrestrial plants but little is known about their evolutionary history. In an attempt to investigate the origin of the plant MIP subfamilies, genomes of chlorophyte algae, the sister group of charophyte algae and land plants, were searched for MIP encoding genes.</p> <p>Results</p> <p>A total of 22 MIPs were identified in the nine analysed genomes and phylogenetic analyses classified them into seven subfamilies. Two of these, Plasma membrane Intrinsic Proteins (PIPs) and GlpF-like Intrinsic Proteins (GIPs), are also present in land plants and divergence dating support a common origin of these algal and land plant MIPs, predating the evolution of terrestrial plants. The subfamilies unique to algae were named MIPA to MIPE to facilitate the use of a common nomenclature for plant MIPs reflecting phylogenetically stable groups. All of the investigated genomes contained at least one <it>MIP </it>gene but only a few species encoded MIPs belonging to more than one subfamily.</p> <p>Conclusions</p> <p>Our results suggest that at least two of the seven subfamilies found in land plants were present already in an algal ancestor. The total variation of MIPs and the number of different subfamilies in chlorophyte algae is likely to be even higher than that found in land plants. Our analyses indicate that genetic exchanges between several of the algal subfamilies have occurred. The PIP1 and PIP2 groups and the Ca²⁺gating appear to be specific to land plants whereas the pH gating is a more ancient characteristic shared by all PIPs. Further studies are needed to discern the function of the algal specific subfamilies MIPA-E and to fully understand the evolutionary relationship of algal and terrestrial plant MIPs.</p

Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

Atomic-resolution X-ray crystallography, functional analyses, and molecular dynamics simulations suggest a novel mechanism for the regulation of water flux through the yeast Aqy1 water channel

Exploring the three PIPs and three TIPs of grapevine for transport of water and atypical substrates through heterologous expression in aqy-null yeast

Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (Pf) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl2, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl2. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO2, H2O2 and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H2O2, suggesting the contribution of Vitis aquaporins in the transport of these speciesinfo:eu-repo/semantics/publishedVersio