JXB Advance Access originally published online on October 10, 2005
Journal of Experimental Botany 2005 56(421):2971-2981; doi:10.1093/jxb/eri294
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RESEARCH PAPER |
Differential responses of maize MIP genes to salt stress and ABA
1Insitute of Biochemical Plant Pathology, GSF—National Research Center for Environment and Health, D-85764 Neuherberg, Germany
2Julius-von-Sachs-Institute of Biological Sciences, University of Würzburg, D-97092 Würzburg, Germany
* To whom correspondence should be addressed. Fax: +49 89 3187 2726. E-mail: schaeffner{at}gsf.de
Received 6 April 2005; Accepted 21 August 2005
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Abstract |
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Salt stress is known to reduce root hydraulic conductivity and growth. To examine a concomitant regulation of aquaporins, the expression of the maize MIP gene family in response to NaCl was analysed by DNA array hybridization. Plants responded differentially to 100 versus 200 mM NaCl treatments. Leaf water content was reduced rapidly and persistently after the application of 200 mM NaCl in contrast to 100 mM NaCl. Endogenous ABA strongly accumulated in roots after 2 h; it remained at a highly elevated level for 48 h after the addition of 200 mM NaCl, but rapidly declined in plants treated with 100 mM NaCl, indicating an early recovery from water deficit. Interestingly, 2 h after the addition of 100 mM NaCl, when maize regained the osmotic potential allowing water uptake, three highly expressed, specific isoforms ZmPIP1;1, ZmPIP1;5, and ZmPIP2;4 were transiently induced. They were preferentially transcribed in the outer root tissue suggesting a role in cellular water transport. None of the ZmTIP genes was altered. By contrast, after the addition of 200 mM NaCl these responses were missing. Instead, multiple ZmPIP and ZmTIP genes were repressed by 200 mM NaCl after 24 h. After 48 h, deregulations were overridden in both cases indicating homeostasis. ABA (1 µM) exogenously applied to the roots transiently induced ZmPIP2;4 similar to 100 mM NaCl as well as ZmPIP1;2. Thus, the early induction of ZmPIP2;4 by NaCl may be mediated by ABA. Previously, an increase in root hydraulic conductivity had been observed upon ABA application. By contrast, 100 µM ABA led to a complete, possibly non-specific repression of all detected ZmPIP and ZmTIP genes after 24 h.
Key words: ABA, aquaporins, expression analyses, major intrinsic proteins, water stress, Zea mays
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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Plant water relations are disturbed by drought or high salinity due to the lowered water potential in the environment hampering water uptake or favouring loss of water from plants. Plants have evolved several mechanisms to cope with these stresses. Closing of stomata reduces transpirational losses of water from leaves. The uptake and the biosynthesis of compatible solutes and the regulation of membrane permeability and transport are further measures that either hold back water within the plant or facilitate further uptake of water in the roots (Zhu, 2001
; Sharp et al., 2004). According to the composite water flux model, water passes plant organs by three pathways, the apoplastic, symplastic, and transcellular pathways. The latter two routes cannot be experimentally distinguished and are combined as the cell-to-cell pathway. However, since both require transport of water across cellular membranes, integral membrane proteins of the major intrinsic protein (MIP) family are supposed to play an important role. Several MIPs have been shown to act as aquaporins facilitating the permeation of water across membranes driven by differences in water potential (Johansson et al., 2000; Baiges et al., 2002; Maurel et al., 2002; Tyerman et al., 2002). Structural investigation of the mammalian AQP1 prototype uncovered elements which are involved in forming the water channel and determining its specificity (Sui et al., 2001; de Groot et al., 2003). In addition to water, individual MIP members may mediate the movement of small non-charged molecules, such as glycerol, urea, CO2, or formaldehyde (Tyerman et al., 2002; Gaspar et al., 2003; Uehlein et al., 2003). Furthermore, a role as osmotic or turgor sensors has been suggested for MIPs (Hill et al., 2004).
Plant MIPs are classified into four groups, PIPs (plasma membrane intrinsic proteins), TIPs (tonoplast intrinsic proteins), NIPs (nodulin-26 like intrinsic proteins), and SIPs (small and basic intrinsic proteins) (Johanson et al., 2001). PIPs are further separated into two, highly homologous subgroups, PIP1 and PIP2 (Schäffner, 1998). Genome projects have revealed 35 MIPs in Arabidopsis thaliana and at least 32 members in maize. So far, 13 PIPs split into six PIP1 and seven PIP2, 12 TIPs, four NIPs, and three SIPs were identified in Zea mays (Chaumont et al., 2001; cDNA accession no. AF057183). Eight maize isoforms have been examined for water channel activity by heterologous expression in Xenopus oocytes. ZmTIP1;1, ZmTIP2;3 (Chaumont et al., 1998; Lopez et al., 2004) as well as ZmPIP2;1, ZmPIP2;4, and ZmPIP2;5 (Chaumont et al., 2000; Fetter et al., 2004) have high activity, whereas ZmPIP1;1, ZmPIP1;2, and ZmPIP1;5 had no or very low activity, at least in this system (Chaumont et al., 2000; Gaspar et al., 2003). However, Fetter et al. (2004) could show that co-expression of ZmPIP1 and ZmPIP2 members may synergistically enhance water permeability in Xenopus oocytes.
The involvement of MIPs in water relations in planta has been approached by antisense strategies targeting PIP1 and PIP2 members as well as a single isoform, AtPIP2;2, by analysing a knock-out mutant (Martre et al., 2002; Javot et al., 2003; see below). On the transcriptional level, various reports demonstrated the responsiveness of plant MIPs to drought, low temperature, high salinity, light, pathogens, or hormonal stimuli. Thus, aquaporin genes that are up- or down-regulated by drought and salt stress have been pinpointed in Arabidopsis, rice, sunflower, cauliflower, wheat, barley, and maize (Kawasaki et al., 2001; Seki et al., 2002a; Maathuis et al., 2003; Martinez-Ballesta et al., 2003; Wang et al., 2003; Jang et al., 2004). Suppression of MIPs may be interpreted as a measure to reduce loss of water from cells, to reduce water uptake into cells, and/or to decrease water fluxes through tissues. Reports on the transgenic manipulation of different PIP1 isoforms in Arabidopsis or tobacco were, in part, in agreement with these roles, although seemingly similar genetic alterations lead to contrasting results (Martre et al., 2002; Siefritz et al., 2002; Aharon et al., 2003). These findings underscore that plant responses to water stress require complex, differential, and specific regulations of water permeability and, importantly, the consideration of isoform-specific roles (Luu and Maurel, 2005).
The phytohormone abscisic acid (ABA) is an important signal molecule regulating physiological processes and, in particular, responses to environmental stresses such as drought and salinity (Hose et al., 2002; Sharp et al., 2004). ABA accumulates in plants suffering from water deficit and triggers the expression of several ABA-responsive genes (Jia et al., 2002; Himmelbach et al., 2003). It stimulates and maintains root development and growth under stress, increases root hydraulic conductivity, and affects stomatal opening. ABA may also regulate the expression of individual MIPs in Arabidopsis, barley, radish, tomato, Brassica napus, and Craterostigma plantagineum (Fray et al., 1994; Mariaux et al., 1998; Gao et al., 1999; Hoth et al., 2002; Seki et al., 2002b; Suga et al., 2002). Hose et al. (2000) have also shown that ABA transiently induced the water conductivity of roots and the water permeability of cortex cells in maize. Wan et al. (2004) measured the root hydraulic conductivity of maize root cortical cells using the cell-pressure probe and concluded that ABA stabilizes aquaporins during high rates of water flow through the pores, which results in increased conductivity.
To assess the contribution of the complete set of maize MIP genes in response to salt stress and ABA and to identify isoform-specific roles, the transcriptional regulation of the complete maize MIP family was approached using gene-specific target sequences that eliminate the problem of cross-hybridization when using larger cDNA fragments.
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Materials and methods |
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Plant material and treatment
Seeds of maize (Zea mays L. cv. Helix, Kleinwanzlebener Saatzucht AG, Einbeck, Germany) were immersed for 2 h in distilled water and germinated on wetted paper for 4 d at 24 °C. Seedlings were transferred to continuously aerated hydroponic medium (Gibeaut et al., 1997
) and grown for 7 d under 16 h light at photosynthetically active radiation of 180–230 µmol m–2 s–1, temperature was controlled between 22 °C and 25 °C and relative humidity ranged from 45–60%. ABA or high-salinity stress treatments were carried out. For ABA treatments, 1 µM or 100 µM ABA was added to the hydroponic medium. Sampling times were 1, 6, and 24 h. For high-salinity stress, 100 mM or 200 mM NaCl was applied to the hydroponic solution, which resulted in an increase of the osmolality from 14 mOsm to 200 mOsmol kg–1 and 373 mOsmol kg–1, respectively. Accordingly, the osmotic pressure changed from 0.04 MPa to 0.50 MPa and 0.92 MPa. After 2, 9, 24, and 48 h of treatment probes were harvested and frozen in liquid nitrogen for further analyses. The osmotic potential of the cell sap of maize roots was determined cryoscopically using the Osmomat 030 osmometer (Gonotec, Berlin, Germany).
Cortex and stele tissues from 10–50 mm zone of primary roots were separated with the help of two pairs of tweezers. One pair of tweezers was used to hold the central, stelar tissue, whereas the second one allowed the outer, cortical tissue to be torn off (Hose, 2000). Control via a microscope showed that the separation occurred right at the endodermis, which was attached to the cortical layers.
Leaf water status was assayed by measuring the fresh weight and dry weight of leaves of NaCl-treated (100 and 200 mM) and control seedlings (treated seedling by 100 mM and 200 mM NaCl and control). Dry weight was obtained after drying the leaves at 104 °C for 3 h.
Nucleic acids
Total RNA was prepared using the TRIZOL reagent (Invitrogen, Karlsruhe, Germany) and then purified using the RNA-Teasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. After an initial photometric determination of total RNA concentration, ethidium bromide fluorescence of ribosomal bands on the electrophoresis-gel was used for the control of total RNA integrity and the adjustment of equal RNA amounts. Isolation of genomic DNA was performed according to Fabri and Schäffner (1994).
DNA array design and production
To produce gene-specific probes 3'-UTR sequences were selected from ZmMIP cDNA sequences (Chaumont et al., 2001; cDNA accession AF057183) and Rab17 as a known ABA-responsive control gene (Kizis and Pagès, 2002) and amplified by PCR from maize genomic DNA as a template. All PCR fragments were cloned into pGEM-Teasy plasmid vector (Promega, Madison, USA) and verified by sequencing. The specificity of MIP gene targets was checked by BLAST analyses at http://www.ncbi.nlm.nih.gov/BLAST/ (see below; Supplementary Table 1 can be found at JXB online). Three human cDNAs SP1, SP3, and SP4 (TGF-Beta, accession M60316; HLA class I histocompatibility antigen, M11886; Brachyury, AJ001699) were used as negative, non-hybridizing control genes. For normalization, their target sequences were spotted as well (see below).
The inserts were amplified by PCR using plasmid DNA as templates and oligonucleotides directed to flanking vector sequences. Fragments were concentrated by filtration using filter plates (Millipore, Eschborn, Germany), controlled by gel electrophoresis, and subsequently spotted onto Hybond N+ nylon membranes (Pharmacia Amersham, Freiburg, Germany) in duplicate (MicroGrid II, BioRobotics, Cambridge, UK). Each array was hybridized with 33P-labelled oligonucleotide 5'-TAATACGACTCACTATAGGG recognizing the flanking vector sequences of the PCR products. These signals served as loading controls and were used for normalization according to Thimm et al. (2001) (see below).
DNA array expression analyses
For hybridizations, 10 µg total RNA was spiked with a single batch of in vitro transcribed RNA from human genes SP1 (100 pg), SP3 (50 pg), SP4 (50 pg) (NASC Arabidopsis Stock Centre, UK), and labelled with 2.1 MBq [
-33P]-dATP (42.9 kBq pmol–1) by oligo-dT primed reverse transcription according to the manufacturer (Strip-EZTM RT, Ambion, Huntingham, UK). Heat-denatured purified probes were hybridized for at least 18 h in 5x SSC, 5x Denhardt's buffer, 0.5% SDS, and 100 µg ml–1 ssDNA at 65 °C. Washing involved a final step at 0.2x SSC/0.1% SDS at 68 °C for 30 min. Primary data were obtained by exposure of imaging plates and reading using a phosphoimager (Fuji FLA3000); software for evaluation was ArrayVision 5.1 (InterFocus, Mering, Germany).
For further processing of data, the local background measured within each 3x3 subgrid was subtracted from the value of each individual signal. These values were divided by the mean of the signal measured for three internal standard transcripts (SP1, SP3, and SP4) for a first normalization regarding labelling by reverse transcription and exposure. These values were divided by individual reference hybridization values (T7 oligonucleotide see above) in order to compensate for differences in the amount of spotted DNA probes (Thimm et al., 2001). Next, the mean for each gene was calculated from the duplicate spots. Final ‘gene activities’ were determined as the mean of three biological replicates. In order to compare two different scenarios, the gene activities of individual experiments from treated versus untreated samples were divided to result in a relative gene activity or ‘expression ratio’. Means and standard deviations were calculated from three independent biological replicates. The result was subjected to t-test; P-values were calculated to assess the statistical relevance of changes (Precht and Kraft, 1993). Changes were classified as inductions with varying extent: ratio 
2.0, P 
0.05; ratio=1.67–2.0, P 0.05; ratio 2.0, 0.05<P0.1. Reductions are indicated by: ratio <0.5, P 0.05; ratio <0.5, 0.05 <P0.10; ratio=0.50–0.60, P 0.05.
Quantitative RT-PCR
For real-time RT-PCR, total RNA was incubated with RNase-free DNase at 37 °C for 15 min, purified by phenol-chloroform, precipitated by ethanol and dissolved in DEPC-treated water. The concentration and integrity was controlled as described above.
For cDNA synthesis 2 µg total RNA, 106 copies pAW109 (GeneAmp PCR system, Applied Biosystems, Darmstadt, Germany), 0.5 µg oligo (dT)15 were incubated in 12 µl at 70 °C for 10 min and chilled on ice. Then, an 8 µl cocktail was added, resulting in final concentrations of 1x first strand buffer, 500 µM dNTP Mix (Roche, Mannheim, Germany), 0.01 M DTT, and 25 U RNase inhibitor and 200 U Superscript II enzyme (Gibco, Karlsruhe, Germany). The mixture was incubated at 42 °C for 60 min followed by 15 min at 70 °C. The cDNA solution was stored at –20 °C or directly used for quantitative PCR. For each RNA sample a reaction without reverse transcription was performed to control contamination by genomic DNA. For real-time PCR analysis 4 µl of 1:50 diluted cDNA samples were used in 25 µl reactions containing 0.5 µM gene-specific primers (Supplementary Table 1 can be found at JXB online) and 12.5 µl Absolute QPCR SYBR Green ROX Mix (ABgene, Surrey, UK) in an Applied Biosystems PE-7700 (Applied Biosystems, Darmstadt, Germany). The program was as follows: 15 min at 95°C for DNA polymerase activation, 40 cycles of 20 s at 95 °C, 30 s at 55 °C, and 45 s at 72 °C. Data collection was performed at 72 °C for ZmPIP1;1 and ZmPIP1;2 or at 81 °C for ZmPIP1;5, ZmPIP2;4, and pAW109 according to the melting curves of the respective products. A control experiment without cDNA was included. The program for analysis of melting curves consisted of 10 s at 95 °C, 30 s at 60 °C, heating to 90 °C at a rate of 0.2 °C s–1, and data were continuously collected. The data were analysed according to the 2–
Ct method (Pfaffl et al., 2002). Externally added pAW109 was used for normalization.
Determination of ABA content
Freeze-dried tissue samples were homogenized and extracted in 80% methanol. Extracts were passed through a Sep Pak C18-cartridge. Methanol was removed under reduced pressure and the aqueous residue partitioned three times against ethyl acetate at pH 3.0. The ethyl acetate of the combined organic fractions was evaporated under reduced pressure. The residue was taken up in TBS-buffer (TRIS-buffered saline; 150 mM NaCl, 1 mM MgCl2, 50 mM TRIS; pH 7.8) and subjected to an immunological ABA assay (ELISA) as described earlier (Peuke et al., 1994). The accuracy of the ELISA was verified for maize in earlier investigations (Hartung et al., 1994).
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Results |
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Gene-specific DNA targets for maize MIP gene expression analysis
In order to analyse the expression profile of MIP genes in maize, a DNA array was established that harbours probes for 11 ZmPIP, 12 ZmTIP, three ZmSIP, and four ZmNIP genes (see Materials and methods). In particular, among subfamily members, there are high DNA sequence similarities. Within the coding regions of ZmPIP, ZmTIP, ZmSIP, and ZmNIP subfamilies, the sequence similarity varies from 59.2% to 99.3%, 56.6% to 99.6%, 47.7% to 81.4%, and 51.7% to 92.6%, respectively. Therefore, 110–350 bp fragments were PCR-amplified as targets for hybridization originating from the more divergent 3'-UTR sequences compared with maize cDNA sequences by BLAST analyses (see Materials and methods). Nineteen target sequences did not detect any other homologous maize genes. Ten additional target sequences exhibited considerable homologies to other ZmMIP members, however, encompassing only short stretches of up to 67 nt at 88% homology (Supplementary Table 1 can be found at JXB online). This level of homology does not hamper specific detection since cross-hybridizations was not detectable under the stringency conditions used (Supplementary Fig. 1 can be found at JXB online), which is in accordance with other reports (Xu et al., 2001
). The target sequences of ZmTIP3;1 and ZmTIP3;2 showing homologies of 84% over 98 nt might weakly cross-hybridize. Finally, two pairs of highly homologous genes, ZmTIP2;3/ZmTIP2;4 and ZmPIP1;3/ZmPIP1;4
, were not discernible; therefore a single target detecting both was designed (Supplementary Table 1 can be found at JXB online; see Materials and methods).
Abundance of MIP transcripts in unstressed maize roots
To determine the expression of maize MIP members in unstressed roots using the maize MIP array, RNA was isolated from 11-d-old roots of hydroponically cultivated plants. Although signals derived from different hybridization targets cannot be compared in a strict, quantitative manner, it was evident that MIP genes were transcribed in roots at very different levels. By far, ZmPIP1;5 and ZmTIP1;1 showed the highest expression. In addition, ZmPIP1;1, ZmPIP2;1, ZmPIP2;4, ZmPIP2;5, ZmPIP2;6, ZmTIP2;1, ZmTIP2;2, and ZmTIP2;4 were highly transcribed in roots (Fig. 1A). All other MIP members were expressed at low levels or not detected at all (Fig. 1A). Considering the sensitivity of the hybridization (Supplementary Fig. 1D can be found at JXB online) the latter genes are not significantly transcribed in unstressed maize roots or restricted to a specific, numerically minor cell type obviating their detection.
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Furthermore, it was interesting to assess whether ZmMIPs were differentially expressed in different root cell layers. The zone 10–50 mm from tip of primary roots of 6-d-old seedlings was separated into inner and outer tissue at the endodermis. The outer layers contained epidermis, cortex, and the endodermis, the inner tissue consisted of the stele (Supplementary Fig. 2 can be found at JXB online). Interestingly, DNA array hybridization revealed that several highly expressed ZmMIP members had higher abundance in the outer cell layers than in the stele. There was no difference for ZmPIP1;2, ZmPIP1;3, and ZmPIP2;6 (Fig. 1B).
Effect of salt stress on water content of maize leaves and root osmotic potential
Application of NaCl to the root system of maize plants exerts a strong water stress onto the plants. The reactions of 11-d-old, hydroponically grown maize seedlings towards two different salt concentrations were compared by determining the water content of their leaves and assessing the osmotic potential of the root cells.
Application of 100 mM NaCl slightly, but not significantly decreased the water content of leaves in comparison to control plants (Fig. 2A). By contrast, 200 mM NaCl rapidly and significantly reduced the water content of leaves in comparison with both control and 100 mM NaCl treatment (P <0.05) (Fig. 2A). However, after 1 d, leaves appeared turgid in both cases indicating a recovery from stress and regaining water homeostasis. For the severe treatment with 200 mM NaCl, a constant, but lower water content was established.
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After 2 h of salt stress, the osmotic potential of the root cells was strongly reduced (Fig. 2B). In the case of 100 mM NaCl, the osmotic potential was already slightly lower than the surrounding medium. After 24 h, a further reduction was observed. Although the reduction of the osmotic potential was even stronger after 2 h of 200 mM NaCl, it did not reach the level of the medium. Yet, after 24 h, the external and internal osmotic potential was also roughly equal in this case.
Effect of NaCl on gene expression of maize MIP genes
NaCl strongly influences plant water relations (Hasegawa et al., 2000; Zhu, 2001; Munns, 2002; Fig. 2). Therefore, the expression of maize MIP genes encoding aquaporins in roots of 11-d-old seedlings was assayed after the administration of 100 mM and 200 mM NaCl to the hydroponic solution. In addition to the differential impact on leaf water content and root osmotic potential the stress- and ABA-responsive maize gene Rab17 was, at most, weakly induced by 100 mM NaCl at 24 h, but significantly up-regulated by 200 mM NaCl at 9, 24, and 48 h (Table 1).
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Interestingly, the transcriptional responses of ZmPIP and ZmTIP genes were also different for both NaCl concentrations. Two hours after the addition of 100 mM, but not of 200 mM NaCl, a transient induction of the highly expressed PIP isoforms ZmPIP1;1 and ZmPIP2;4 was observed; in addition, the most highly expressed maize PIP gene in roots, ZmPIP1;5, showed a weak, yet significant induction (1.6-fold, P-value 0.004) (Table 1). Quantitative RT-PCR confirmed these results (Fig. 3A-D). Although these inductions were weak, it has to be stressed that the respective ZmPIP members were already expressed at high levels in unstressed maize roots. None of the ZmTIP genes was significantly altered 2 h after the application of NaCl.
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The expression of the MIP genes to both NaCl concentrations was not significantly altered after 9 h. However, the situation changed completely 24 h after NaCl addition. With 100 mM NaCl added, no significant inductions or suppressions were detected, except a weak up-regulation of ZmTIP2;4. However, multiple ZmPIP and ZmTIP transcripts were reduced by 200 mM NaCl after 24 h (Table 1). The repressed genes included both less highly and more highly transcribed members of the PIP1 and PIP2 subfamilies, ZmPIP1;2, ZmPIP1;5, ZmPIP1;6, ZmPIP2;4, ZmPIP2;5, and ZmPIP2;6, as well as most ZmTIP genes. Interestingly, the highly expressed ZmPIP1;1 and ZmPIP2;1 were not affected. After 48 h, transcriptional deregulation of all ZmPIP and ZmTIP genes was basically abolished at both NaCl concentrations, indicating recovery and homeostasis in both cases. Nevertheless, the ABA-responsive marker Rab17 continued to be enhanced in the 200 mM NaCl treatment. The differential responses of ZmPIP1;1, ZmPIP1;2, ZmPIP1;5 and ZmPIP2;4 to 200 mM NaCl after 24 h were confirmed by quantitative RT-PCR (Fig. 3A–D).
Data for ZmSIP and ZmNIP were collected as well (Supplementary Table 2 can be found at JXB online), but they are not discussed here because of their low expression, which renders expression ratios less certain.
Effect of salt stress on ABA concentration
ABA is known to accumulate in response to drought and salt stress. Since differential reactions to two NaCl concentrations were observed for leaf water content, root osmotic potential, and ZmMIP transcription in roots, ABA levels in roots were determined in parallel (see Material and methods). Indeed, salt stress significantly induced the amount of ABA in maize roots (Fig. 4). After 2 h, ABA accumulated to identical levels both in 100 mM and in 200 mM NaCl experiments. However, at later time points there was a clear difference between both treatments. ABA levels were strongly reduced to values 2–3-fold above controls in roots treated with 100 mM NaCl, whereas they remained at very high levels, up to 17-fold, throughout the whole period examined after the application of 200 mM NaCl; nevertheless, a clear decline was also observed after 48 h (Fig. 4).
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Effect of ABA on the expression of MIP genes
Since NaCl increased the ABA concentration in roots, whether the transcriptional reaction of ZmMIP genes to NaCl was mediated by ABA would be interesting. Therefore, 11-d-old, hydroponically grown maize seedlings were subjected to 1 µM ABA for 1, 6, and 24 h. Previous and parallel analyses had shown that internal ABA concentrations rapidly increased by a factor of 3–8 after the external addition of 1 µM ABA, which is in the range observed here after NaCl treatment (Fig. 4). Furthermore, considering a possible delay of ABA biosynthesis after NaCl application compared with the direct effect of exogenously added ABA, the first two time points in the ABA experiments were chosen earlier than in the NaCl experiments. Transcription was analysed using the ZmMIP DNA array.
After 1 h treatment with 1 µM ABA only ZmPIP1;2 and ZmPIP2;4 were specifically induced (P <0.05) (Table 1). In addition, ZmPIP1;1 showed a weak, but less significant enhancement of transcription (1.68 fold; P=0.052). Quantitative RT-PCR corroborated the induction of ZmPIP1;2 and ZmPIP2;4 (Fig. 3E–H). For ZmPIP2;4 this reaction was reminiscent of the early response to NaCl (Table 1). After 6 h and 24 h treatment by 1 µM ABA, only a weak enhancement of ZmPIP1;2 was persistent (Table 1), whereas no other ZmMIP transcripts significantly deviated from the control levels. Importantly, none of the ZmTIP genes showed a significant alteration of gene expression during the whole treatment.
Instead of 1 µM ABA, several studies reported by other laboratories used up to 100 µM ABA in order to provoke more drastic changes in gene expression, although such a high concentration may also lead to non-hormonal effects. Indeed, when 100 µM ABA was applied, a completely altered situation was apparent, indicated by the total repression of all ZmPIP and ZmTIP genes after 24 h (Table 1; Fig. 3E–H). At earlier time points, no consistent changes attributable to ABA could be detected (data not shown).
Semi-quantitative RT-PCR analyses of the known ABA-responsive genes Rab17 and Dbf1 served as controls, showing that the plants investigated responded to both ABA concentrations showing similar kinetics and ABA-sensitivity as described previously (Kizis and Pagès, 2002; Supplementary Fig. 3 can be found at JXB online).
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Expression of MIP in unstressed maize roots
A DNA array harbouring gene-specific target sequences has been used to assess the ZmMIP members that are expressed in maize roots. Six PIP and four TIP members were highly transcribed in roots indicating that these isoforms will be most important in roots. Nevertheless, other members of the MIP complement, including NIP and SIP isoforms that were detected only at low levels, could fulfil roles in specialized cell types or under certain conditions. This result was in accordance with the analyses of expressed sequence tags that revealed higher numbers for ZmPIP1;1, ZmPIP1;5, ZmPIP2;1, and ZmPIP2;4 among the PIP members (Chaumont et al., 2001
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Furthermore, it was interesting to find that several of the highly expressed ZmMIP members were not evenly expressed across the root, but prevailed in the outer cell layers. The highly expressed ZmPIP2;6 was expressed at the same level in both the outer layers and the stele. The higher expression of major plasma membrane and tonoplast aquaporins could indicate an important role in transcellular water transport across the epidermis, cortex, and endodermis, or alternative functions in these cells (see below). Nevertheless, these findings might be at variance with other studies that indicated high(er) expression in the vascular tissue. However, it has to be noted that the endodermis, which may harbour vascular-expressed MIP members, was included in the outer layers after tissue separation (Supplementary Fig. 2 can be found at JXB online). Furthermore, several studies on maize PIP or TIP genes comply with these results. In particular, Barrieu et al. (1998) showed that ZmTIP1;1 is highly expressed in root epidermis and endodermis as well as in xylem parenchyma and the pericycle; Gaspar et al. (2003) found the highly transcribed ZmPIP1;5 in all root cells with strong expression in the epidermis. In addition, in situ visualization may underestimate the expression in the larger cells of the cortex layer. Similar observations and considerations apply to ZmPIP2;5 (Fetter et al., 2004).
Differential responses to salt stress
Differential responses of maize seedlings were observed after adding 100 mM or 200 mM NaCl to the medium. These salt concentrations increase the osmotic pressure from 0.014 to 0.200 and 0.373 Osmol kg–1, respectively, leading to a reduction in the external water potential from –0.035 MPa to –0.50 and –0.92 MPa. In the control medium, the water potential of the root cells, which is composed of an osmotic pressure of 0.47 MPa and a turgor in the range of 0.5–0.7 MPa (Fig. 2; Rodriguez et al., 1997), will be close to equilibrium with the external solution.
Thus, in the case of 100 mM NaCl added to the medium, a complete loss of turgor could already account for most of the difference in water potential (0.46 MPa) since the cellular osmotic potential is around –0.47 MPa (Fig. 2B). An immediate consequence of this osmotic effect is the sudden reduction in growth of the roots and leaves (Frensch and Hsiao, 1994; Rodriguez et al., 1997; Munns, 2002). In addition to the sole efflux of water, measures to lower the osmotic potential are initated in order to regain turgor and the capability of water uptake. The synthesis of compatible solutes as well as the uptake of Na+ and Cl– is involved in the osmoregulation, although both ions are toxic at high concentrations (Azaizeh et al., 1992; Frensch and Hsiao, 1994; Rodriguez et al., 1997; Amtmann and Sanders, 1999; Uozumi et al., 2000; Munns, 2002; Essah et al., 2003; Wang et al., 2003). Within 2 h after the addition of 100 mM NaCl, the root osmotic pressure was increased to 0.55 MPa (Fig. 2), similar to that described by Rodriguez et al. (1997).
Coincidently, three highly expressed ZmPIP isoforms (ZmPIP1;1, ZmPIP1;5, ZmPIP2;4) were specifically induced at this stage; all three genes showed higher expression in the outer cell layers of unstressed maize roots (Table 1; Fig. 1B). Therefore, the transient induction of these specific PIP isoforms indicates that they may be involved in cellular water uptake in the outer cell layers facilitated by the appropriate decrease in cellular osmotic potential. Commencing water homeostasis after 2 h treatment with 100 mM NaCl is also supported by the observation that the water content of the leaves was not different from that of control plants (Fig. 2). Later, a further reduction in the cellular osmotic potential (Fig. 2B; Frensch and Hsiao, 1994; Rodriguez et al., 1997) may supersede the requirement for enhanced aquaporin expression.
The observed transient induction of maize PIP members was reminiscent of the salt-sensitive rice line IR29 (Kawasaki et al., 2001). Treatment with 150 mM NaCl transiently induced two rice PIP members after 3 h. Interestingly, the rice genes aligned to the transiently enhanced maize ZmPIP1;1 and ZmPIP2;4 (Supplementary Fig. 4 can be found at JXB online). Thus, they may reflect orthologous genes, having an isoform-specific and conserved function in maize and in the salt-sensitive rice cultivar. This correlation, as well as the specific and focused response of individual PIP members, emphasizes the importance of isoform-specific roles of individual MIP members as already proposed by Luu and Maurel (2005).
Maize plants responded in a different way after the severe shock with 200 mM NaCl. After 2 h the decrease of the root osmotic potential was not sufficient to allow the cellular uptake of water (intracellular –0.75 versus external –0.92 MPa; Fig. 2). Thus, in this scenario, even further shrinking of cells and plasmolysis is possible until the cellular osmoticum roughly equalled the external concentration after 24 h (Fig. 2; Munns, 2002). In accordance with this situation, under the severe 200 mM NaCl conditions a transient induction of aquaporins at 2 h was not observed.
In contrast to the treatment with 100 mM NaCl, 200 mM NaCl significantly reduced the water content of leaves at 2 h. This lowered water content persisted at a constant level, indicating that stressed plants manifested a new water balance. Furthermore, the reduced water status and continued stress was also reflected by the high level of root ABA, which lasted for at least 24 h, indicating a continued reduction of root cell volume during this period as suggested by Jia et al. (2001).
However, 24 h after the addition of 200 mM NaCl most ZmPIPs and ZmTIPs were repressed in a much less specific manner, which may be correlated with a prolonged retardation of growth and sustained reduction of water flux due to a diminished transpiration. There is previous physiological evidence that recovery of growth of maize roots, at least at a strongly reduced rate, is retarded with increasing osmotic stress. In addition, the root growth zone was reduced with high salt stress (Frensch and Hsiao, 1994, 1995; Neumann et al., 1994; Sharp et al., 2004). In Arabidopsis thaliana, a relatively severe stress of 100 mM NaCl strongly reduced hydraulic conductivity and AtPIP1 transcription after 24 h (Martínez-Ballesta et al., 2003). In contrast to most other PIPs, the highly expressed ZmPIP1;1 and ZmPIP2;1 were not affected, which again argues for isoform-specific roles of the aquaporins.
Nevertheless, even in the case of 200 mM NaCl, both the stabilized water content of maize leaves and the significant reduction of ABA content in roots after 48 h implies that water homeostasis was established. Accordingly, ZmMIP transcription recovered back to control levels.
Wang et al. (2003) found that seven ZmPIPs and two ZmTIPs were repressed after 12 h treatment with 150 mM NaCl in maize roots. Since 150 mM NaCl also constitutes a more severe osmotic stress, with an osmotic potential exceeding the magnitude of the cellular turgor as discussed above, these results were in agreement with their analyses
. Several analyses of salt-induced transcriptional changes have been performed in other plants, in particular with Arabidopsis. However, apparently contradictory results have been reported, which might be attributed to different developmental and growth conditions (Pih et al., 1997; Kreps et al., 2002; Martinez-Ballesta et al., 2003; Jang et al., 2004). Nevertheless, there are also consistent results like the transient induction of AtPIP1;4 (Pih et al., 1999; Kreps et al., 2002, Jang et al., 2004), which are reminisent of the induction of ZmPIP1;2.
Another interesting precedent also distinguished the early reaction towards lower versus higher NaCl concentrations. Elevated Ca2+ is an immediate early signal that may also trigger gene expression in response to NaCl (Kiegle et al., 2000). Lynch et al. (1989) observed a dependence of cytosolic calcium on the concentration of NaCl, which resulted in a differentiation similar to this study's analyses. Up to 90 mM, NaCl did not significantly increase intracellular calcium, whereas between 90 and 120 mM NaCl a sharp rise was provoked that was sustained at 150 and 180 mM NaCl.
ABA regulation
The transient induction of ZmPIP members by 100 mM NaCl could be mediated by ABA. Indeed, exposure to 1 µM ABA transiently induced ZmPIP2;4 and another member, ZmPIP1;2. Thus, the salt-induction of ZmPIP2;4 was correlated to its induction by ABA, whereas an ABA-independent pathway seems to activate the salt enhancement of ZmPIP1;1 and ZmPIP1;5. However, the same ABA level was determined after 2 h for the 200 mM NaCl experiment, and it was not associated with the transcriptional induction of any of the ZmPIP members. Therefore, either other factors overruled the ABA regulation of ZmPIP2;4 in the 200 mM NaCl experiment or the ABA-dependence of ZmPIP2;4 induction is more complex and can sense the ABA concentration and/or its changes. Indeed, ABA was further enhanced after 200 mM NaCl application in contrast to 100 mM NaCl.
Interestingly, a short-term treatment of maize roots with low concentrations of ABA induced cellular and root hydraulic conductance (Hose et al., 2000; Wan et al., 2004). Thus, the transcriptional level of two specific PIP isoforms, ZmPIP2;4 and ZmPIP1;2, could be involved in this hormone-dependent up-regulation, besides potential, as yet unknown, regulation at the protein level.
Previous reports also indicated positive or negative effects of ABA on the transcription of aquaporin genes, which did not give a consistent view (Weig et al., 1997; Hoth et al., 2002; Seki et al., 2002b; Jang et al., 2004). However, in all these analyses high, unphysiological concentrations of ABA up to 100 µM had been applied. Although such concentrations may facilitate the discovery of truly regulated genes, unspecific pharmaceutical effects can not be excluded. Indeed, 100 µM ABA massively and apparently unspecifically repressed most ZmMIP genes in roots after 24 h (Table 1).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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The comparison of the effects of two different NaCl concentrations on the expression of maize MIP genes revealed differential and gene-specific effects. This emphasizes isoform-specific functions in relation to tissue water permeation or growth as well as differential responses to varying extents of the same abiotic stressor. However, these findings may also mean that there are different roles for aquaporins than those related to the simple permeation of water or other uncharged molecules through cellular membranes. Hill et al. (2004)
recently challenged the importance of a ‘simple permeation hypothesis’. They suggested an additional function of aquaporins as osmotic or turgor sensors. Regarding the transiently induced ZmPIP isoforms, a putative role or participation as such sensors would mean that they were induced under conditions when the osmotic potential was lowered to a level allowing water uptake and turgor re-establishment. Conversely, a widespread suppression of these functions would be provoked after 24 h under severe salt stress, although only specific isoforms could be involved in sensing. However, in these cases, as well as in other reports, there is currently no experimental verification of such an additional role of aquaporins. |
Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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There are four figures and three tables which constitute supplementary data to this manuscript. These can be found at JXB online.
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Acknowledgements |
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We thank Bianca Röger, Würzburg for excellent technical assistance in determining ABA and Olivier da Ines for critical discussions. The work was supported by the DFG (Scha 454/7-1 and Ha 963/11-1) and by the DFG Schwerpunktprogramm SPP1108 (Scha 454/8-1).
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Footnotes |
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ZmPIP1;3 and ZmPIP1;4 may indeed correspond to the same gene (Chaumont et al., 2001). 
However, Wang et al. (2003) used a selection of ten cDNA clones (>400 bp) that allowed cross-hybridization and thus could not obviate the detection of altered transcription from homologous members.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
|---|
Aharon R, Shahak Y, Wininger S, Bendov R, Kapulnik Y, Galili G. 2003. Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress. The Plant Cell 15, 439–447.
Amtmann A, Sanders D. 1999. Mechanisms of Na+ uptake by plant cell. Advances in Botanical Research 29, 75–112.
Azaizeh H, Gunse B, Steudle E. 1992. Effects of NaCl and CaCl2 on water transport across root cells of maize (Zea mays L.) seedlings. Plant Physiology 99, 886–894.
Baiges I, Schäffner AR, Affenzeller MJ, Mas A. 2002. Plant aquaporins. Physiologia Plantarum 115, 175–182.[CrossRef][Medline]
Barrieu F, Chaumont F, Chrispeels MJ. 1998. High expression of the tonoplast aquaporin ZmTIP1 in epidermal and conducting tissues of maize. Plant Physiology 117, 1153–1163.
Chaumont F, Barrieu F, Herman EM, Chrispeels MJ. 1998. Characterization of a maize tonoplast aquaporin expressed in zones of cell division and elongation. Plant Physiology 117, 1143–1152.
Chaumont F, Barrieu F, Jung R, Chrispeels MJ. 2000. Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity. Plant Physiology 122, 1025–1034.
Chaumont F, Barrieu F, Wojcik E, Chrispeels MJ, Jung R. 2001. Aquaporins constitute a large and highly divergent protein family in maize. Plant Physiology 125, 1206–1215.
De Groot BL, Frigato T, Helms V, Grubmüller H. 2003. The mechanism of proton exclusion in the aquaporin-1 water channel. Journal of Molecular Biology 333, 279–293.[CrossRef][Web of Science][Medline]
Essah PA, Davenport R, Tester M. 2003. Sodium influx and accumulation in Arabidopsis. Plant Physiology 133, 307–318.
Fabri CO, Schäffner AR. 1994. An Arabidopsis thaliana RFLP mapping set to localize mutations to chromosomal regions. The Plant Journal 5, 149–156.
Fetter K, Van Wilder V, Moshelion M, Chaumont F. 2004. Interactions between plasma membrane aquaporins modulate their water channel activity. The Plant Cell 16, 215–228.
Fray RG, Wallace A, Grierson D, Lycett GW. 1994. Nucleotide sequence and expression of ripening and water stress-related cDNA from tomato with homology to the MIP class of membrane channel proteins. Plant Molecular Biology 24, 539–543[CrossRef][Web of Science][Medline]
Frensch J, Hsiao TC. 1994. Transient responses of cell turgor and growth of maize roots as affected by changes in water potential. Plant Physiology 104, 247–254.[Abstract]
Frensch J, Hsiao TC. 1995. Rapid response of the yield threshold and turgor regulation during adjustment of root growth to water stress in Zea mays. Plant Physiology 108, 303–312.[Abstract]
Gao YP, Young L, Bonham-Smith P, Gusta LV. 1999. Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions. Plant Molecular Biology 40, 635–644.[CrossRef][Web of Science][Medline]
Gaspar M, Bousser A, Sissoeff I, Roche O, Hoarau J, Mahe A. 2003. Cloning and characterization of ZmPIP1;5b, an aquaporin transporting water and urea. Plant Science 165, 21–31.[CrossRef]
Gibeaut DM, Hulett J, Cramer GR, Seemann JR. 1997. Maximal biomass of Arabidopsis thaliana using a simple, low-maintenance hydroponic method and favorable environmental conditions. Plant Physiology 115, 317–319.[CrossRef][Web of Science][Medline]
Hartung W, Zhang J, Davies WJ. 1994. Does abscisic acid play a stress physiological role in maize plants growing in heavily compacted soil? Journal of Experimental Botany 45, 221–226.
Hasewaga PM, Bressan RA, Zhu J-K, Bohnert HJ. 2000. Plant cellular and molecular response to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51, 263–499.
Hill AE, Shachar-Hill B, Shachar-Hill Y. 2004. What are aquaporins for? Journal of Membrane Biology 197, 1–32.[CrossRef][Web of Science][Medline]
Himmelbach A, Yang Y, Grill E. 2003. Relay and control of abscisic acid signaling. Current Opinion in Plant Biology 6, 470–479.[CrossRef][Web of Science][Medline]
Hose E. 2000. Untersuchungen zum radialen Abscisinsäure- und Wassertransport in Wurzeln von Helianthus annuus L. und Zea mays L. PhD thesis, University of Würzburg, Germany.
Hose E, Sauter A, Hartung W. 2002. Abscisic acid in roots: biochemistry and physiology. In: Waisel Y, Eshel A, Kafkafi U, eds. Plant roots: the hidden half, 3rd edn. New York: Marcel Dekker, 435–448.
Hose E, Steudle E, Hartung W. 2000. Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes. Planta 211, 874–882.[CrossRef][Web of Science][Medline]
Hoth S, Morgante M, Sanchez J-P, Hanafey MK, Tingey SV, Chua N-H. 2002. Genome-wide expression profiling in Arabidopsis thaliana reveals new targets of abscisic acid and largely impaired gene regulation in the abi1-1 mutant. Journal of Cell Science 115, 4891–4900.
Jang JY, Kim DG, Kim YO, Kim JS, Kang H. 2004. An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Molecular Biology 54, 713–725.[CrossRef][Web of Science][Medline]
Javot H, Lauvergeat V, Martin F, et al. 2003. Role of a single aquaporin isoform in root water uptake. The Plant Cell 15, 509–522.
Jia W, Wang Y, Zhang S, Zhang J. 2002. Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots. Journal of Experimental Botany 53, 2201–2206.
Jia W, Zhang J, Liang J. 2001. Initiation and regulation of water deficit-induced abscisic acid accumulation in maize leaves and roots: cellular volume and water relations. Journal of Experimental Botany 52, 295–300.
Johanson U, Karlsson M, Johansson I, Gustavsson S, Sjövall S, Fraysse L, Weig A, Kjellbom P. 2001. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiology 126, 1358–1369.
Johansson I, Karlsson M, Johanson U, Larsson C, Kjellbom P. 2000. The role of aquaporins in cellular and whole plant water balance. Biochimica et Biophysica Acta 1465, 324–342.[Medline]
Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K, Galbraith D, Bohnert HJ. 2001. Gene expression profiles during the initial phase of salt stress in rice. The Plant Cell 13, 889–905.
Kiegle E, Moore CA, Haseloff J, Tester MA, Knight MR. 2000. Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root. The Plant Journal 23, 267–278.[CrossRef][Web of Science][Medline]
Kizis D, Pagès M. 2002. Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway. The Plant Journal 30, 679–689.[CrossRef][Web of Science][Medline]
Kreps JA, Wu Y, Chang H-S, Zhu T, Wang X, Harper JF. 2002. Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiology 130, 2129–2141.
Lopez F, Bousser A, Sissoëff I, Hoarau J, Mahé A. 2004. Characterization in maize of ZmTIP2;3, a root-specific tonoplast intrinsic protein exhibiting aquaporin activity. Journal of Experimental Botany 55, 539–541.
Luu DT, Maurel C. 2005. Aquaporins in a challenging environment: molecular gears for adjusting plant water status. Plant, Cell and Environment 28, 85–96.[CrossRef]
Lynch J, Polito VS, Läuchli A. 1989. Salinity stress increases cytoplasmic Ca2+ activity in maize root protoplast. Plant Physiology 90, 1271–1274.
Maathuis F, Filatov V, Herzyk P, et al. 2003. Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress. The Plant Journal 35, 675–692.[CrossRef][Web of Science][Medline]
Mariaux JB, Bockel, Salamini F, Bartels D. 1998. Desiccation- and abscisic acid-responsive genes encoding major intrinsic proteins (MIPs) from the resurrection plant Craterostigma plantagineum. Plant Molecular Biology 38, 1089–1099.[CrossRef][Web of Science][Medline]
Martinez-Ballesta MC, Aparicio F, Pallas V, Martinez V, Carvajal M. 2003. Influence of saline stress on root hydraulic conductance and PIP expression in Arabidopsis. Journal of Plant Physiology 160, 689–697.[CrossRef][Web of Science][Medline]
Martre P, Morillon R, Barrieu F, North GB, Nobel PS, Chrispeels MJ. 2002. Plasma membrane aquaporins play a significant role during recovery from water deficit. Plant Physiology 130, 2101–2110.
Maurel C, Javot H, Lauvergeat V, Gerbeau P, Tournaire C, Santoni, V, Heyes J. 2002. Molecular physiology of aquaporins in plants. International Review of Cytology 215, 105–148.[Web of Science][Medline]
Munns R. 2002. Comparative physiology of salt and water stress. Plant, Cell and Environment 25, 239–250.[CrossRef][Medline]
Neumann PM, Azaizeh H, Leon D. 1994. Hardening of root cell walls: a growth inhibitory response to salinity stress. Plant, Cell and Environment 17, 303–309.[CrossRef]
Peuke AD, Jeschke WD, Hartung W. 1994. The uptake and flow of C, N and ions between roots and shoots in Ricinus communis L. III. Long-distance transport of abscisic acid depending on nitrogen nutrition and salt stress. Journal of Experimental Botany 45, 741–747.
Pfaffl MW, Horgan GW, Dempfle L. 2002. Relative expression software tool (REST©) for group-wise comparison and statistical analysis if relative expression results in real-time PCR. Nucleic Acids Research 30, e36.
Pih KT, Kabilan V, Lim JH, Kang SG, Piao HL, Jin JB, Hwang I. 1999. Characterization of two new channel protein genes in Arabidopsis. Molecular Cells 9, 84–90.
Precht VM, Kraft M. 1993. Biostatitik 2. P11. R. München, Germany: Oldenbourg Verlag GmbH.
Rodriguez HG, Roberts JKM, Jordan WR, Drew MC. 1997. Growth, water relations and accumulation of organic and inorganic solutes in roots of maize seedlings during salt stress. Plant Physiology 113, 881–893.[Abstract]
Schäffner AR. 1998. Aquaporin function, structure, and expression: are there more surprises to surface in water relations? Planta 204, 131–139.[CrossRef][Web of Science][Medline]
Seki M, Ishida J, Narusaka M, et al. 2002b. Monitoring the expression pattern of around 7000 Arabidopsis genes under ABA treatments using full-length cDNA microarray. Functional and Integral Genomics 2, 282–291.
Seki M, Narusaka M, Ishida J, et al. 2002a. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal 31, 279–292.[CrossRef][Web of Science][Medline]
Siefritz F, Tyree MT, Lovisolo C, Schubert A, Kaldenhoff R. 2002. PIP1 plasma membrane aquaporins in tobacco: from cellular effects to function in plants. The Plant Cell 14, 869–876.
Sharp RE, Poroyko V, Hejlek LG, Spollen WG, Springer GK, Bohnert HJ, Nguyen HT. 2004. Root growth maintenance during water deficits: physiology to functional genomics. Journal of Experimental Botany 55, 2343–2351.
Suga S, Komatsu S, Maeshima M. 2002. Aquaporin isoforms responsive to salt and water stresses and phytohormones in radish seedlings. Plant Cell Physiology 43, 1229–1237.
Sui H, Han B-G, Lee JK, Walian P, Jap BK. 2001. Structural basis of water-specific transport through the AQP1 water channel. Nature 414, 872–878.[CrossRef][Medline]
Thimm O, Essigmann B, Kloska S, Altmann T, Buckhout TJ. 2001. Response of Arabidopsis to iron deficiency stress as revealed by microarray analysis. Plant Physiology 127, 1030–1043.
Tyerman SD, Niemietz CM, Bramley H. 2002. Plant aquaporins: multifunctional water and solute channels with expanding roles. Plant, Cell and Environment 25, 173–194.[CrossRef][Medline]
Uehlein N, Lovisolo C, Siefritz F, Kaldenhoff R. 2003. The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature 425, 734–737.[CrossRef][Medline]
Uozumi N, Kim EJ, Rubio F, Yamaguchi T, Muto S, Tsuboi A, Bakker EP, Nakamura T, Schroeder JI. 2000. The Arabidopsis HKT1 gene homolog mediates inward Na+ currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant Physiology 122, 1249–1259.
Wan X, Steudle E, Hartung W. 2004. Reversible inhibition of water channels (aquaporins) in cortical cells of young corn roots by mechanical stimuli (pressure pulses): effects of ABA and HgCl2. Journal of Experimental Botany 55, 411–422.
Wang H, Miyazaki S, Kawai K, Deyholos M, Galbraith D, Bohnert H. 2003. Temporal progression of gene expression responses to salt shock in maize roots. Plant Molecular Biology 52, 873–891.[CrossRef][Web of Science][Medline]
Weig A, Deswarte C, Chrispeels MJ. 1997. Major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group. Plant Physiology 114, 1347–1357.[Abstract]
Xu W, Bak S, Decker A, Paquette SM, Feyereisen R, Galbraith DW. 2001. Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana. Gene 272, 61–74.[CrossRef][Web of Science][Medline]
Zhu JK. 2001. Cell signaling under salt, water and cold stresses. Current Opinion in Plant Biology 4, 401–406.[CrossRef][Web of Science][Medline]
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