Journal of Experimental Botany, Vol. 52, No. 363, pp. 1953-1957,
October 1, 2001
© 2001 Oxford University Press
Original Papers |
The tobacco plasma membrane aquaporin NtAQP1
Universität Würzburg, Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
Received 12 February 2001; Accepted 30 April 2001
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Abstract |
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 Top Abstract IntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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This paper gives a summary of a project to characterize a tobacco aquaporin. The cDNA and gene, including the 5' upstream region, for the tobacco aquaporin NtAQP1 has been isolated and the encoded protein characterized. The significance of promoter regions for an abscisic acid- and gibberellic acid-induced gene expression could be restricted to a region between -1450 and -1112 upstream of the transcription start point by transient transformation of a bicistronic vector into tobacco protoplasts. NtAQP1 expression in tobacco plants was found to be elevated in flowers, stems and roots. In roots, the protein was detected close to xylem vessels in pitch-like structures. Studies with a NtAQP1-GFP fusion indicated a plasma membrane location. For a functional analysis, the cDNA was expressed in Xenopus oocytes. NtAQP1 was found to be a heavy metal-insensitive aquaporin with additional permeability for glycerol. Mutation of a threonine at position 233 to a cysteine transformed NtAQP1 into a heavy metal-sensitive aquaporin.
Key words: Aquaporin, gene expression, Nicotiana tabaccum.
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Introduction |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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About 10 years ago the molecular basis of an increased water permeability for certain human cells was characterized (Preston and Agre, 1991
). The identified protein showed a good sequence homology to a bovine lens fibre membrane protein (Gorin et al., 1984), which was the first member of the so-called major intrinsic protein family (MIP) (Reizer et al., 1993). Heterologous expression of the human protein in Xenopus leavis oocytes revealed a significant increase in cellular water permeability, which confirmed its function as a water-permeable pore (Preston et al., 1992). Accordingly, the protein was named aquaporin 1 (AQP1). Using the data obtained by protein crystal analysis (Murata et al., 2000; Fu et al., 2000) a molecular mechanism for the AQP1 water selectivity and the E. coli glycerol facilitator (GlpF) was suggested.
Since the MIP superfamily was founded, a high number of aquaporins has been detected in many organisms including plants (Ludevid et al., 1992; Kaldenhoff et al., 1993). Although a transport capacity for water and small solutes could be obtained by heterologous expression in Xenopus laevis oocytes (Daniels et al., 1994; Biela et al., 1999; Gerbeau et al., 1999) and stopped flow analysis of membrane vesicles (Maurel et al., 1997; Niemietz and Tyerman, 1997), the function of aquaporins in plant physiology and plant water transport at the cellular and whole plant level is still a matter of debate. Indirect indications relevant to these questions were provided by gene expression analysis using mRNA steady-state quantification, in situ localization or aquaporin-promoter reporter genes as well as protein localization studies using immunological techniques (Barrieu et al., 1998; Chaumont et al., 1998; Kaldenhoff et al., 1995, 1996; Ludevid et al., 1992; Otto and Kaldenhoff, 2000).
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The Arabidopsis thaliana aquaporin PIP1b |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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In order to provide direct in planta evidence for the aquaporin function of the Arabidopsis thaliana plasma membrane intrinsic protein 1b (PIP1b), anti-sense pip1b-constructs were used for transformation. Expression of the anti-sense pip1b-mRNA resulted in a dramatic decrease in PIP1b and the closely related PIP1c aquaporin expression (Kaldenhoff et al., 1998
). The physiological consequences of aquaporin reduction became evident after measurement of cellular water permeability of protoplasts. The average water permeability, as determined by a newly developed swelling assay, was decreased by a factor of about 3–4 in comparison to control plants. This observation could be confirmed by an automatic counter device, which was modified for the determination of increasing protoplast size in hypo-osmotic conditions (Grote et al., 1999). Besides the changes at the cellular level, the anti-sense plants developed a larger, more branched root system, which was apparently a reaction to the reduced cellular water permeability. It was interpreted as a compensation reaction towards the internal water stress. Since the analysis of Arabidopsis genome sequence data revealed the existence of more than 30 aquaporin homologues (Weig et al., 1997; Johansson et al., 2000), the question arises how the repression of one or two aquaporins (i.e. PIP1b, PIP1c) could have such a profound effect. Using RNase-protection experiments it could clearly be demonstrated that PIP1b was the predominant aquaporin-mRNA in mature plants (Grote et al., 1998) and thus the explanation appears very simple: The anti-sense construct has targeted to the major aquaporin and therefore consequences on cellular water permeability and root morphology were induced. |
Tobacco as a model system |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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Despite all its advantages, the model plant Arabidopsis offers some small but crucial limitations, which led to the decision to continue the aquaporin-studies with another plant. One of the major reasons was the overall small size which limits the interpretation of many data obtained by physiological and biophysical experimental techniques. Among numerous possible plants, tobacco was selected for future work on plasma membrane aquaporins because it is large enough, for example, for water flux or root pressure measurements, it is easy to transform and several tissue culture cell lines are available.
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Molecular characterization of the tobacco aquaporin NtAQP1 |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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Consequently, a PIP1b-homologue was isolated from a tobacco cDNA library and functionally characterized after heterologous expression in Xenopus oocytes (Biela et al., 1999
). This tobacco aquaporin, named NtAQP1 (Nicotiana tabacum aquaporin 1), turned out to be a functional water channel. In contrast to many other aquaporins characterized so far, it was also, and to a comparable extent with the E. coli glycerol facilitator GlpF (Sweet et al., 1990; Maurel et al., 1994), permeable for glycerol (Biela et al., 1999). In addition, expression of the protein increased the oocyte membrane permeability for urea (Eckert et al., 1999). Patch clamp analysis of the respective oocytes clearly demonstrated that NtAQP1 is not permeable for ions (Biela et al., 1999).
As an outcome of successful collaborations, aquaporin genes from Samanea saman, Allium cepa, Lotus japonicus, Medicago truncatula (Krajinski et al., 2000), and Lycopersicon esculentum (Werner et al., 2001) were isolated and functionally characterized in oocytes expressing the respective protein. Unexpectedly, the water permeability (Pf value) was consistently low for specific aquaporins and always high for certain others. These differences rely on unknown structural characteristics which might affect water-transport function or protein-incorporation into the oocyte membrane.
In former experiments, high Pf-values were detected when aquaporins with a short N-terminus and an elongated loop A were expressed in oocytes (Fig. 1). These elements seem to contribute to the degree of water permeability. Generally, the water permeability mediated by these aquaporin-subtypes was sensitive to heavy metals such as mercury. NtAQP1 belongs to those aquaporins inducing only a moderate heavy metal-insensitive water permeability. It possesses a comparably long N-terminus and a short loop A. In an attempt to find the structural basis of the different Pf-values, the coding sequence was modified in a way that the N-terminus was deleted or additional amino acids were inserted into the hydrophilic loop A. Close to the pore-site a cysteine was introduced at a position similar to that of heavy metal-sensitive aquaporins (Fig. 1). With the exception of the latter, which leads to a heavy metal-sensitive water flux, none of these modifications induced any differences, i.e. an increased water permeability of oocyte membranes (Fig. 2). Experiments with proteins that have multiple modifications as well as a quantification of aquaporin incorporation-rates into the oocyte membrane are currently in progress.
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TIP and A. thaliana PIP2b. +Hg indicates the presence of 500 µM HgCl2 during the swelling assay. Control refers to water-injected oocytes. Loop A: NtAQP1 protein modified by a five amino acid insertion (AGGDV) into loop A (see Fig. 1
As already discussed by the authors in detail (Kaldenhoff and Eckert, 1999
; Eckert et al., 1999) expression and testing in the heterologous oocyte system could yield some indication about the functional characteristics of an aquaporin, but the molecular and physiological function in the plant remains to be analysed independently.
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Expression and localization of NtAQP1 |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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Gene-expression in a specific cell type, only at certain developmental stages or in defined physiological conditions could sometimes indicate the function of the encoded protein. A good example in the case of water channel genes is given by the human aquaporin-family (Agre et al., 1998
).
The indication for tissue-specific expression of the tobacco NtAQP1 was obtained by northern analysis with RNA from different tissues (Fig. 3). The studies revealed a low NtAQP1-mRNA concentration in stems and leaves, and a high concentration in flowers and roots. Extraction of RNA from different flower organs and subsequent NtAQP1 Northern analysis revealed the highest expression in petals.
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A detailed analysis of the NtAQP1 expression in a given plant organ was achieved by whole mount in situ hybridization and in situ immunolocalization in blots and tissue cross-sections (Otto and Kaldenhoff, 2000
). By whole mount in situ hybridization NtAQP1-mRNA was detected in the root tip, including the elongation and differentiation zones. At these sites high cellular water permeability and water flux is necessary for the young growing and developing cells. Immunolocalization in lignified roots could detect the presence of the protein in cells close to the tracheary system. In this case the loading or unloading of the vessel would be facilitated by the function of NtAQP1. Electron microscopy analysis of root sections support this suggestion, since the protein is detected close to tracheary elements (Fig. 4A). It is noted that the signal was not evenly distributed, but concentrated in certain areas that resemble pitch-like structures (Fig. 4B).
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Cross-sections of younger parts of the stem show expression of the protein in the developing xylem vessels and companion cells of the internal phloem. In older parts NtAQP1 is concentrated at the outer xylem border and in cells of the internal phloem. A low but overall expression of the protein was detected in leaves. In leaf cross-sections the aquaporin was concentrated in cells of spongy parenchyma with clear accumulation around the stomatal cavity. The function in this tissue can on the one hand be related to high water fluxes caused by transpiration. On the other hand, it can be speculated that if NtAQP1 would be a CO2-facilitating aquaporin, similar to the human AQP1 (Nakhoul et al., 1998
), it might also be a membrane transport mechanism for CO2.
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Promoter-activity |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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Since it is known from the studies on the PIP1b promoter regulation that certain aquaporins are dramatically regulated at the level of transcription (Kaldenhoff et al., 1996
) a 
1500 bp promoter region of NtAQP1 was isolated. Subsequently, tobacco was transformed with a NtAQP1-promoter glucuronidase construct and the reporter gene expression was monitored in different plant tissues under diverse growth conditions. Taken together, the NtAQP1 promoter activity confirms the data obtained by mRNA and protein expression analysis, which mainly excludes an expression-regulation beside the transcriptional level. |
Regulation of NtAQP1 promotor |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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Sequence analysis of the NtAQP1 5' upstream region and comparison with known cis-acting regulatory DNA elements revealed the presence of elements that activate transcription depending on development or the phytohormones gibberrellic acid (TAACAAA, Skriver et al., 1991
) and abscisic acid (CTAACCA, Abe et al., 1997), respectively. The activity of the elements was analysed by protoplast transformation with a bicistronic construct. Luciferase was expressed under the control of the CaMV 35S promoter and the second cistron, coding for GUS, was regulated by the NtAQP1 promoter, respectively, promoter deletions. First results indicate that a region between -1450 and -1112, which encompasses the ABA- as well as the GA-responsive elements is responsible for the phytohormone dependent gene regulation (Fig. 5).
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Subcellular localization of NtAQP1 |
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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In order to get an idea about the distribution of NtAQP1 in a complete plant cell a construct including a translational fusion between NtAQP1 and green fluorescence protein (GFP) was introduced into tobacco protoplasts by PEG-mediated transient transformation (Maas et al., 1995
) and the GFP-specific fluorescence visualized (Fig. 6). It was detected in the plasma membrane and not yet identified vesicles. These possibly reflect the routing of NtAQP1 from the synthesis site at the endoplasmic reticulum to the integration site in the plasma membrane.
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Notes |
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1 To whom correspondence should be addressed. Fax: +49 931 8886158. E-mail: kaldenhoff{at}botanik.uni.wuerzburg.de
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TopAbstractIntroductionThe Arabidopsis thaliana...Tobacco as a model...Molecular characterization of...Expression and localization of...Promoter-activityRegulation of NtAQP1 promotorSubcellular localization of...References |
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