Specificity of the accumulation of mRNAs and proteins of the plasma membrane and tonoplast aquaporins in radish organs

Plant aquaporins occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We cloned cDNAs for plasma-membrane aquaporins (PAQ1, 1b, 1c, 2, 2b, and 2c) of radish (Raphanus sativus L.). The amino acid sequences of the PAQs showed on average 63% sequence identity. Their sequences were 23% identical to those of tonoplast aquaporins (γ- and δ-VM23). A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In other organs, the mRNA level of each isoform varied according to the organ. In petals, stamens, pistils and sepals of flowers, the levels of PAQ1, 1b, 1c and γ-VM23 mRNAs were high, and mRNAs of all aquaporins except for δ-VM23 were detected at high levels in pericarps. The protein levels of aquaporins on the basis of the membrane protein were determined by immunoblotting. Proteins PAQ1 and VM23 were detected in every organ except for the mature petiole. The PAQ2 protein level was especially high in green cotyledons and leaves, but was extremely low in seedling cotyledons and hypocotyls. Proteins PAQ1, PAQ2 and VM23 were highly accumulated in growing pericarps, but not in the immature seeds. These results indicate that the gene expression of the aquaporin isoforms was individually regulated in an organ- and tissue-specific manner, and that the amounts of aquaporin protein, especially PAQ2, are regulated in certain tissues at the translational level and by the rate of protein turnover. Received: 10 February 2000 / Accepted: 30 June 2000     

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     

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.     

The turnover of cell surface proteins of carrot protoplasts

The covalent modification of cell surface proteins with N-hydroxysuccinimide esters of biotin was used to develop a strategy for following the turnover of proteins on the surface of carrot (Daucus carota L.) protoplasts. A biotinylation/internalisation assay was established which enabled the turnover of cell surface proteins to be examined by biochemical and immunocytochemical techniques. The detection of biotinylated proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that a variety of proteins on the surface of the protoplasts were covalently modified. Immunolocalisation of biotinylated proteins in protoplasts directly after their derivatisation, demonstrated that the proteins were initially restricted to the cell surface. Incubation of biotinylated protoplasts at 25 °C for 1 h resulted in the detection of biotin-labelled proteins on the cell surface and intracellularly. A small proportion of these proteins was associated with coated pits, the Golgi apparatus and vacuolar compartments. Biochemical analysis of internalised proteins revealed that a polypeptide of approximate M_r 100 000 was internalised by the protoplasts. Immunolabelling of a biotinylated protein of M_r 100 000 by an antibody raised against an isoform of a tobacco plasma-membrane H⁺-ATPase, strongly suggests that the plasma-membrane H⁺-ATPase is internalised by carrot protoplasts. The implications of these results are discussed within the context of endocytosis in plants. Received: 13 July 1998 / Accepted: 11 November 1998     

A γ-TIP cross-reacting protein is abundant in the cortex of soybean N2-fixing nodules

The distribution and abundance of tonoplast intrinsic protein (γ-TIP), a putative aquaporin which is abundant in the tonoplast of osmoregulated pulvinus motor cells, were determined in nodules of Glycine max (L.) Merr. using chemical fixation and immunolocalization. This protein was highly expressed in the tonoplast of the inner cortical cells of the nodules but poorly expressed in the vascular transfer cells and in infected cells. It is concluded that the differentiation of the inner cortical cells of the nodules like that of pulvinus motor cells, is accompanied by an increased expression of γ-TIP. This result is consistent with our previous hypothesis that a reversible exchange of intercellular water by the inner cortical cells plays a role in the regulation of nodule conductance to O₂ diffusion, and hence in subsequent N₂-fixing activity. Received: 7 February 1998 / Accepted: 22 May 1998     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I. High osmotic water permeability in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) root cells as measured by a new method

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P ( f1)) and VM (P ( f2)), as well as the bulk osmotic water permeability of a protoplast (P ( f(bulk))) isolated from radish (Raphanus sativus) roots. The values of P ( f(bulk)) and P ( f2) were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P ( f1) was calculated from P ( f(bulk)) and P ( f2) by using the 'three-compartment model', which describes the theoretical relationship between P ( f1), P ( f2) and P ( f(bulk)) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 mum s(-1), indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P ( f1) and P ( f2) can be measured accurately in individual higher plant cells.     

Increased Expression of Vacuolar Aquaporin and H+-ATPase Related to Motor Cell Function in Mimosa pudica L

Mature motor cells of Mimosa pudica that exhibit large and rapid turgor variations in response to external stimuli are characterized by two distinct types of vacuoles, one containing large amounts of tannins (tannin vacuole) and one without tannins (colloidal or aqueous vacuole). In these highly specialized cells we measured the abundance of two tonoplast proteins, a putative water-channel protein (aquaporin belonging to the [gamma]-TIPs [tonoplast intrinsic proteins]) and the catalytic A-subunit of H+-ATPase, using either high-pressure freezing or chemical fixation and immunolocalization. [gamma]-TIP aquaporin was detected almost exclusively in the tonoplast of the colloidal vacuole, and the H+-ATPase was also mainly localized in the membrane of the same vacuole. Cortex cells of young pulvini cannot change shape rapidly. Development of the pulvinus into a motor organ was accompanied by a more than 3-fold increase per length unit of membrane in the abundance of both aquaporin and H+-ATPase cross-reacting protein. These results indicate that facilitated water fluxes across the vacuolar membrane and energization of the vacuole play a central role in these motor cells.     

Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves: localization, relative proportion and their role in ammonium assimilation or nitrogen transport

 Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C₄ plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively, were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1 and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed. Received: 25 January 2000 / Accepted: 30 March 2000     

Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways

Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV.     

Drought, abscisic acid and transpiration rate effects on the regulation of PIP aquaporin gene expression and abundance in Phaseolus vulgaris plants

BACKGROUND AND AIMS: Drought causes a decline of root hydraulic conductance, which aside from embolisms, is governed ultimately by aquaporins. Multiple factors probably regulate aquaporin expression, abundance and activity in leaf and root tissues during drought; among these are the leaf transpiration rate, leaf water status, abscisic acid (ABA) and soil water content. Here a study is made of how these factors could influence the response of aquaporin to drought. METHODS: Three plasma membrane intrinsic proteins (PIPs) or aquaporins were cloned from Phaseolus vulgaris plants and their expression was analysed after 4 d of water deprivation and also 1 d after re-watering. The effects of ABA and of methotrexate (MTX), an inhibitor of stomatal opening, on gene expression and protein abundance were also analysed. Protein abundance was examined using antibodies against PIP1 and PIP2 aquaporins. At the same time, root hydraulic conductance (L), transpiration rate, leaf water status and ABA tissue concentration were measured. KEY RESULTS: None of the treatments (drought, ABA or MTX) changed the leaf water status or tissue ABA concentration. The three treatments caused a decline in the transpiration rate and raised PVPIP2;1 gene expression and PIP1 protein abundance in the leaves. In the roots, only the drought treatment raised the expression of the three PIP genes examined, while at the same time diminishing PIP2 protein abundance and L. On the other hand, ABA raised both root PIP1 protein abundance and L. CONCLUSIONS: The rise of PvPIP2;1 gene expression and PIP1 protein abundance in the leaves of P. vulgaris plants subjected to drought was correlated with a decline in the transpiration rate. At the same time, the increase in the expression of the three PIP genes examined caused by drought and the decline of PIP2 protein abundance in the root tissues were not correlated with any of the parameters measured.     

PIP1 Aquaporins Are Concentrated in Plasmalemmasomes of Arabidopsis thaliana Mesophyll

The PIP1 subfamily of water channel proteins (aquaporins) constitute about 1% of the plasma membrane (PM) proteins from Arabidopsis thaliana leaves. Immunogold electron microscopy has confirmed their localization at the PM of mesophyll cells. Very high labeling density at PM invaginations known as plasmalemmasomes was observed. Therefore, we suggest that these subcellular structures are involved in water transport between the apoplast and the vacuole.     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I. High osmotic water permeability in radish ( Raphanus sativus ) root cells as measured by a new method

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P _f1) and VM (P _f2), as well as the bulk osmotic water permeability of a protoplast (P _f(bulk)) isolated from radish (Raphanus sativus) roots. The values of P _f(bulk) and P _f2 were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P _f1 was calculated from P _f(bulk) and P _f2 by using the ‘three-compartment model’, which describes the theoretical relationship between P _f1, P _f2 and P _f(bulk) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 μm s⁻¹, indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P _f1 and P _f2 can be measured accurately in individual higher plant cells. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. It includes four appendices, four tables and two figures. Mari Murai-Hatano and Tsuneo Kuwagata contributed equally to the paper. An erratum to this article is available at .     

Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar α-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8–5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and β-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-β-1,3-glucanase and mature β-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar α-mannosidase, chitinase and β-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and β-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment. Received: 3 September 1997 / Accepted: 30 October 1997     

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.     

Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues. Before flower anthesis, anther and pollen dehydration takes place before the release of mature pollen at dehiscence. Aquaporins represent a class of proteins that mediates the movement of water over cellular membranes. Aquaporins of the plasmamembrane PIP2 family are expressed in tobacco (Nicotiana tabacum) anthers and may therefore be involved in the movement of water in this organ. To gain more insight into the role these proteins may play in this process, we have analyzed their localization using immunolocalizations and generated plants displaying RNA interference of PIP2 aquaporins. Our results indicate that PIP2 protein expression is modulated during anther development. Furthermore, in tobacco PIP2 RNA interference plants, anther dehydration was slower, and dehiscence occurred later when compared with control plants. Together, our results suggest that aquaporins of the PIP2 class are required for efficient anther dehydration prior to dehiscence.     

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P _os) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P _os values were in the range 200–1000 μm s⁻¹. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar P _os values for red beet and onion were reduced by 1 mM HgCl₂, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl₂-treated onion vacuoles, the reduced P _os (56 μm s⁻¹) had a low activation energy (approx. 6 kJ mol⁻¹), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low. Received: 18 February 1999 / Accepted: 21 June 1999     

Methylation of aquaporins in plant plasma membrane

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.     

Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75 kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17 kDa), at 12 to 24 h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24 h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins. Received October 9, 2001 Accepted January 18, 2002