Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes

 Recently, it has been shown that water fluxes across biological membranes occur not only through the lipid bilayer but also through specialized water-conducting proteins, the so called aquaporins. In the present study, we investigated in young and mature leaves of Brassica napus L. the expression and localization of a vacuolar aquaporin homologous to radish γ-tonoplast intrinsic protein/vacuolar-membrane integral protein of 23 kDa (TIP/VM 23). In-situ hybridization showed that these tonoplast aquaporins are highly expressed not only in developing but also in mature leaves, which export photosynthates. No substantial differences could be observed between different tissues of young and mature leaves. However, independent of the developmental stage, an immunohistochemical approach revealed that the vacuolar membrane of bundle-sheath cells contained more protein cross-reacting with antibodies raised against radish γ-TIP/VM 23 than the mesophyll cells. The lowest labeling was detected in phloem cells. We compared these results with the distribution of plasma-membrane aquaporins cross-reacting with antibodies detecting a domain conserved among members of the plasma-membrane intrinsic protein 1 (PIP1) subfamily. We observed the same picture as for the vacuolar aquaporins. Furthermore, a high density of gold particles labeling proteins of the PIP1 group could be observed in plasmalemmasomes of the vascular parenchyma. Our results indicate that γ-TIP/VM 23 and PIP1 homologous proteins show a similar expression pattern. Based on these results it is tempting to speculate that bundle-sheath cells play an important role in facilitating water fluxes between the apoplastic and symplastic compartments in close proximity to the vascular tissue. Received: 23 December 1999 / Accepted: 3 June 2000     

Aquaporin isoforms responsive to salt and water stresses and phytohormones in radish seedlings

Aquaporins in the plasma and vacuolar membranes play a key role in the intercellular and intracellular water transport in plants. First, we quantitated the absolute amounts for mRNAs of eight aquaporin isoforms in hypocotyls of radish seedlings. Then, we investigated the effects of salt and water stresses (150 mM NaCl, 300 mM mannitol and 20% polyethylene glycol) and phytohormones (gibberellic acid, abscisic acid and brassinolide) on the mRNA and protein levels of aquaporins in the plasma membrane (RsPIP1-1, 1-2, 1-3, 2-1, 2-2 and 2-3) and vacuolar membrane (RsTIP1-1 and 2-1). The mRNA and protein levels of RsTIP1-1, RsTIP2-1, RsPIP1-1, RsPIP1-2 and RsPIP1-3 were comparatively constant. In contrast, mannitol treatment altered the mRNA levels of RsPIP2-1, RsPIP2-2 and RsPIP2-3 in roots. Immunoblot analysis showed that the RsPIP2-1 protein level was increased by NaCl treatment and decreased by treatment with mannitol and polyethylene glycol. Gibberellic acid and abscisic acid suppressed the levels of mRNAs of RsPIP2-1, RsPIP2-2 and RsPIP2-3 and the protein level of RsPIP2-1 in roots. On the other hand, the protein levels of RsPIP1-group members and RsTIPs were scarcely changed by these phytohormones. In the case of hypocotyls and cotyledons, the mRNA and protein levels of eight isoforms were not markedly affected by any treatment. These results indicate that aquaporins in the root, especially the RsPIP2 group, may be a stress responsive type of aquaporin at least in the protein level.     

Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family. A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M_r of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patterns are discussed with respect to the probable function of the gene products. Received: 27 March 1997 / Accepted: 20 May 1997     

Differences in aquaporin levels among cell types of radish and measurement of osmotic water permeability of individual protoplasts

We investigated tissue- and cell-specific accumulation of radish aquaporin isoforms by immunocytochemical analysis. In taproots, the plasma membrane aquaporins (RsPIP1 and RsPIP2) were accumulated at high levels in the cambium, while the tonoplast aquaporin (RsTIP) was distributed in all tissues. The three isoforms were highly accumulated in the central cylinder of seedling roots and hypocotyls, and rich in the vascular tissue of the petiole of mature plants. The results suggest that RsPIP1 and RsPIP2 are abundant in the cells surrounding the sieve tube of the radish plant. The swelling rate of protoplasts in a hypotonic solution was determined individually by a newly established method to compare the osmotic water permeability of different cell types. All cells of the cortex and endodermis in seedlings showed a high water permeability of more than 300 microm s(-1). There was no marked difference in the values between the root endodermis and cortex protoplasts, although the RsPIP level was lower in the cortex than in the endodermis. This inconsistency suggests two possibilities: (1) a low level of aquaporin is enough for high water permeability and (2) the water channel activity of aquaporin in the tissues is regulated individually. The uneven distribution of aquaporins in tissues is discussed along with their physiological roles.     

Low aquaporin content and low osmotic water permeability of the plasma and vacuolar membranes of a CAM plant Graptopetalum paraguayense: comparison with radish.

Aquaporin facilitates the osmotic water transport across biomembranes and is involved in the transcellular and intracellular water flow in plants. We immunochemically quantified the aquaporin level in leaf plasma membranes (PM) and tonoplast of Graptopetalum paraguayense, a Crassulacean acid metabolism (CAM) plant. The aquaporin content in the Graptopetalum tonoplast was approximately 1% of that of radish. The content was calculated to be about 3 microg mg(-1) of tonoplast protein. The level of PM aquaporin in Graptopetalum was determined to be less than 20% of that of radish, in which an aquaporin was a major protein of the PM. The PM aquaporin was detected in the mesophyll tissue of Graptopetalum leaf by tissue print immunoblotting. The osmotic water permeability of PM and tonoplast vesicles prepared from both plants was determined with a stopped-flow spectrophotometer. The water permeability of PM was lower than that of the tonoplast in both plants. The Graptopetalum PM vesicles hardly showed water permeability, although the tonoplast showed a relatively high permeability. The water permeability changed depending on the assay temperature and was also partially inhibited by a sulfhydryl reagent. Furthermore, measurement of the rate of swelling and shrinking in different mannitol concentrations revealed that the protoplasts of Graptopetalum showed low water permeability. These results suggest that the low content of aquaporins in PM and tonoplast is one of the causes of the low water permeability of GRAPTOPETALUM: The relationship between the water-storage function of succulent leaves of CAM plants and the low aquaporin level is also discussed.     

Expression of a cauliflower tonoplast aquaporin tagged with GFP in tobacco suspension cells correlates with an increase in cell size

In plants, vacuoles are essential organelles that undergo dynamic volume changes during cell growth due to rapid and high flow of water through tonoplast water-carrying channels composed of integral proteins (tonoplast aquaporins). The tonoplast BobTIP26-1 from cauliflower has previously been shown to be an efficient active aquaporin in Xenopus leavis oocytes. In this study we used tobacco (Nicotiana tabacum cv. Wisconsin 38) suspension cells to examine the effect of BobTIP26-1 expression. In order to follow the intracellular localisation of the protein in real time, the gfp sequence was fused downstream to the BobTIP26-1 coding region. The fusion protein BobTIP26-1::GFP is less active than BobTIP26-1 by itself when expressed in Xenopus oocytes. Nevertheless, this fusion protein is well targeted to the tonoplast of the plant suspension cell when expressed via Agrobacterium co-cultivation. A complex tonoplast labelling is shown when young vacuolated cells are observed. The expression of the fusion protein does not affect the growth rate of the cells but increases their volume. We postulate that the increase in cell volume is triggered by the fusion protein allowing vacuolar volume increase.     

Characterization of the major integral protein of vacuolar membrane

The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N′-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H⁺-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane.     

Aquaporins and cell growth

This review covers the data concerning the relationship between cell growth and aquaporins in the cell membranes, the plasmalemma and tonoplast. Genes of aquaporins, water channel-forming proteins, are actively expressed before the onset and during cell elongation, thus providing accumulation of aquaporin protein and higher membrane hydraulic conductivity. As a result, an additional water uptake favors cell vacuolation and elongation. The review gives information on all growing plant organs. In actively dividing plant cells, only plasmalemma aquaporins are synthesized, whereas in elongating cells, tonoplast aquaporins are synthesized as well. The review includes also the findings of aquaporin research after growth completion.     

Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site.

The membranes of plant and animal cells contain aquaporins, proteins that facilitate the transport of water. In plants, aquaporins are found in the vacuolar membrane (tonoplast) and the plasma membrane. Many aquaporins are mercury sensitive, and in AQP1, a mercury-sensitive cysteine residue (Cys-189) is present adjacent to a conserved Asn-Pro-Ala motif. Here, we report the molecular analysis of a new Arabidopsis aquaporin, delta-TIP (for tonoplast intrinsic protein), and show that it is located in the tonoplast. The water channel activity of delta-TIP is sensitive to mercury. However, the mercury-sensitive cysteine residue found in mammalian aquaporins is not present in delta-TIP, or in gamma-TIP, a previously characterized mercury-sensitive tonoplast aquaporin. Site-directed mutagenesis was used to identify the mercury-sensitive site in these two aquaporins as Cys-116 and Cys-118 for delta-TIP and gamma-TIP, respectively. These mutations are at a conserved position in a presumed membrane-spanning domain not previously known to have a role in aquaporin mercury sensitivity. Comparing the tissue expression patterns of delta-TIP with gamma-TIP and alpha-TIP showed that the TIPs are differentially expressed.     

水孔蛋白在细胞延长、盐胁迫和光合作用中的作用

水孔蛋白属于一个高度保守的、能够进行跨生物膜水分运输的通道蛋白MIP家族。水孔蛋白作为膜水通道,在控制细胞和组织的水含量中扮演重要角色。本研究的重点是属于PIP亚家族的GhPIP1;2和属于TIP亚家族的γTIP1在植物细胞延长中的作用。使用特异基因探针的Northern杂交和实时荧光PCR技术证明GhPIP1;2和GhγTIP1主要在棉花纤维延长过程中显著表达,且最高表达量在开花后5d。在细胞延长过程中,GhPIP1;2和GhγTIP1表达显著,表明它们在促使水流迅速进入液泡这一过程中扮演重要角色。而且也研究了盐胁迫植物中钙离子对水孔蛋白的影响。分别或一起用NaCl或CaCl2处理原生质体或细胞质膜。结果发现在盐胁迫条件下,水渗透率值在原生质体和质膜颗粒中都下降了,同时PIP1水孔蛋白的含量也下降了,表明NaCl对水孔蛋白的功能和含量有抑制作用。同时也观察了Ca2+的两种不同的作用。感知胁迫的胞质中游离钙离子浓度的增加可能导致水孔蛋白的关闭。而过剩的钙离子将导致水孔蛋白的上游调控。同时实验已经证明大麦的一类水孔蛋白-HvPIP2;1有更高的水和CO2转移率。本研究的目标是确定负责转运水和CO2的关键水孔蛋白...     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.     

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.     

Yeast water channels: an overview of orthodox aquaporins

In yeast, the presence of orthodox aquaporins has been first recognized in Saccharomyces cerevisiae, in which two genes (AQY1 and AQY2) were shown to be related to mammal and plant water channels. The present review summarizes the putative orthodox aquaporin protein sequences found in available genomes of yeast and filamentous fungi. Among the 28 yeast genomes sequenced, most species present only one orthodox aquaporin, and no aquaporins were found in eight yeast species. Alignment of amino acid sequences reveals a very diverse group. Similarity values vary from 99% among species within the Saccharomyces genus to 34% between ScAqy1 and the aquaporin from Debaryomyces hansenii. All of the fungal aquaporins possess the known characteristic sequences, and residues involved in the water channel pore are highly conserved. Advances in the establishment of the structure are reviewed in relation to the mechanisms of selectivity, conductance and gating. In particular, the involvement of the protein cytosolic N‐terminus as a channel blocker preventing water flow is addressed. Methodologies used in the evaluation of aquaporin activity frequently involve the measurement of fast volume changes. Particular attention is paid to data analysis to obtain accurate membrane water permeability parameters. Although the presence of aquaporins clearly enhances membrane water permeability, the relevance of these ubiquitous water channels in yeast performance remains obscure.     

Overexpression of the tonoplast aquaporin AtTIP5;1 conferred tolerance to boron toxicity in Arabidopsis

Boron (B) toxicity to plants is responsible for low crop productivity in many regions of the world. Here we report a novel and effective means to alleviate the B toxicity to plants under high B circumstance. Functional characterization of AtTIP5;1, an aquaporin gene, revealed that overexpression of AtTIP5; 1(OxAtTIP5;1) in Arabidopsis significantly increased its tolerance to high B toxicity. Compared to wild-type plants, OxAtTIP5;1 plants exhibited longer hypocotyls, accelerated development, increased silique production under high B treatments. GUS staining and quantitative RT-PCR(qRT-PCR) results demonstrated that the expression of AtTIP5;l was induced by high B concentration treatment. Subcellular localization analysis revealed that the AtTIP5; 1-GFP fusion protein was localized on the tonoplast membrane, which was consistent with the prediction based on bioinformatics. Taken together, our results suggest that AtTIP5;I is involved in B transport pathway possibly via vacuolar compartmentation for B, and that overexpression of AtTIP5;1 in plants may provide an effective way to overcome the problem resulting from high B concentration toxicity.     

Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds

Two integral membrane proteins, MP28 and MP23, were found in protein bodies isolated from pumpkin (Cucurbita sp.) seeds. Molecular characterization revealed that both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. The predicted 29 kDa precursor to MP23 includes six putative membrane-spanning domains, and the loop between the first and second transmembrane domains is larger than that of MP28. The N-terminal sequence of the mature MP23 starts from residue 66 in the first loop, indicating that an N-terminal 7 kDa fragment that contains one transmembrane domain is post-translationally removed. During maturation of pumpkin seeds, mRNAs for MP28 and MP23 became detectable in cotyledons at the early stage, and their levels increased slightly until a rapid decrease occurred at the late stage. This is consistent with the accumulation of the 29 kDa precursor and MP28 in the cotyledons at the early stage. By contrast, MP23 appeared at the late stage simultaneously with the disappearance of the 29 kDa precursor. Thus, it seems possible that the conversion of the 29 kDa precursor to the mature MP23 might occur in the vacuoles after the middle stage of seed maturation. Both proteins were localized immunocytochemically on the membranes of the vacuoles at the middle stage and the protein bodies at the late stage. These results suggest that both MP28 and the precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, and then the precursor is converted to the mature MP23 at the late stage. These two TIPs might have a specific function during the maturation of pumpkin seeds.     

Regulation of plant aquaporin activity

Accumulating evidence indicates that aquaporins play a key role in plant water relations. Plant aquaporins are part of a large and highly divergent protein family that can be divided into four subfamilies according to amino acid sequence similarity. As in other organisms, plant aquaporins facilitate the transcellular movement of water, but, in some cases, also the flux of small neutral solutes across a cellular membrane. Plant cell membranes are characterized by a large range of osmotic water permeabilities, and recent data indicate that plant aquaporin activity might be regulated by gating mechanisms. The factors affecting the gating behaviour possibly involve phosphorylation, heteromerization, pH, Ca2+, pressure, solute gradients and temperature. Regulation of aquaporin trafficking may also represent a way to modulate membrane water permeability. The aim of this review is to integrate recent molecular and biophysical data on the mechanisms regulating aquaporin activity in plant membranes and to relate them to putative changes in protein structure.     

Cell-specific expression of the mercury-insensitive plasma-membrane aquaporin NtAQP1 from Nicotiana tabacum

 The aquaporin NtAQP1 from Nicotiana tabacum L. is insensitive to heavy-metal ions. In addition to water, the transport of urea or glycerol is facilitated by this plasma-membrane-located water channel. Northern hybridization and whole-mount in situ hybridization revealed a high steady-state level of NtAQP1-RNA in roots, a decreased content in shoots and a low content in leaves. By immunolocalization with an antibody targeted to the N-terminus of the aquaporin, the localization of NtAQP1-protein at sites of expected high water transport rates from and to the apoplast or symplast could be demonstrated. The specific pattern of NtAQP1 distribution in petioles strongly indicates a transcellular movement of water. Received: 12 August 1999 / Accepted: 27 December 1999     

蚕豆保卫细胞质膜水通道蛋白的鉴定及其在气孔运动中的作用

水通道或水通道蛋白是水分运动的主要通道.以RD28 cDNA和RD28抗体为探针证明了蚕豆(Vicia fabaL.)保卫细胞中存在水通道蛋白,并以气孔运动为指标,结合抗体和抑制剂处理证明水通道蛋白是水分运动的主要通道.研究表明编码质膜水通道蛋白的RD28转录体在叶片保卫细胞、叶肉细胞和维管束中高表达,尤以保卫细胞中最多;荧光免疫染色和Confocal显微镜观察表明,RD28抗体反应主要位于保卫细胞质膜.进一步采用RD28抗体和水通道蛋白抑制剂--HgCl2 (25μmol/L)处理可抑制壳梭孢素(FC)、光照诱导的气孔开放和原生质体体积膨胀以及ABA诱导的气孔关闭,但这种抑制作用可以被水通道抑制剂的逆转剂β-巯基乙醇(ME)逆转.表明蚕豆保卫细胞中存在水通道蛋白并参与蚕豆保卫细胞的运动过程.