Molecular cloning of a novel water channel from rice: its products expression in Xenopus oocytes and involvement in chilling tolerance

Plant Science 154 (2000) 43 – 51 www.elsevier.com/locate/plantsci

Molecular cloning of a novel water channel from rice: its products expression in Xenopus oocytes and involvement in chilling tolerance Le-gong Li 1, Shi-fang Li 2, Yan Tao, Yoshichika Kitagawa * Laboratory of Plant Genetic Engineering, Biotechnology Institute, Akita Prefectural Uni6ersity, Ogata, Akita 010 -0444, Japan Received 15 March 1999; received in revised form 28 October 1999; accepted 1 December 1999

Abstract Water channel proteins, aquaporins, play a fundamental role in transmembrane water movements in plants. We isolated rice cDNA, rwc1, by screening a rice (Oryza sati6a cv. Josaeng Tongil) cDNA library using a conserved motif of aquaporins. Like other aquaporin genes, rwc1 encodes a 290-residue protein with six putative transmembrane domains. The derived amino acid sequence of RWC1 shows high homology with PIP1 (plasma membrane intrinsic protein 1) subfamily members, which suggest it is localized in the plasma membrane. Injection of its cRNA into Xenopus oocytes increased the osmotic water permeability of the oocytes 2–3 times. Northern analysis showed that rice aquaporin genes are expressed in rice seedling leaves and roots, but that it disappeared from the root 6 h after osmotic stress began and that the transcript level remained low for about 24 h, then recovered. The time course of rice aquaporin gene-expression under osmotic stress was correlated with time course of turgor transition in plant. On the other hand, the levels of rice aquaporin gene-transcripts in leaves under chilling and recovery temperature depend on the pretreatment of mannitol for short time. This variation of the transcripts shown that rice aquaporin genes may play an important role in response to water stress-induced chilling tolerance. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rice; Water channel; Water stress; Chilling tolerance; Xenopus oocyte

1. Introduction Transmembrane water flow is a fundamental process of life. Although water permeability as a biophysical feature of cell membrane, the molecular pathway of transmembrane water movement remained unknown until the discovery of aquapor The nucleotide sequence data reported appeared in EMBL, GenBank and DDBJ Nucleotide sequence Database under the accession number AB009665. * Corresponding author. E-mail address: [email protected] (Y. Kitagawa) 1 Permanent address: Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shanghai 200032, People’s Republic of China. 2 Present address: Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 2 West Yuanmingyuan Road, Beijing 100094, China. Le-gong Li and Shi-fang Li contributed equally to this work.

ins [1]. The recent discovery that plants express numerous aquaporins in both the plasma membrane and the tonoplast has changed our view of how plant cells regulate transmembrane water movement [2,3]. Water channel proteins (aquaporins) belong to the major intrinsic protein (MIP) superfamily that permit the passage of specific molecules through biological membranes. Since the first aquaporin (AQP1) was identified in human erythrocytes [1], many more have been isolated from various organisms, including bacteria, plants, and animals [3]. In plants, many MIP genes have been isolated. They are encoded by several gene families and are hydrophobic integral membrane proteins that range in apparent molecular mass from 23 to 31 kDa [4]. Since g-tonoplast intrinsic protein (g-TIP) was first recognized as a plant aquaporin [5], more different genes have

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been identified [6]. Their products are located in the tonoplast and plasma membrane. Sequence comparisons have shown a high homology between plant aquaporins; all published sequences are clearly of TIP or plasma membrane intrinsic protein (PIP) members. These relationships even extend to the PIP1 and PIP2 subfamilies, originally introduced by Kammerloher et al. [7]; easily classified as PIP1 or PIP2 based on specific arrays of amino acids at the N- and C-termini. Although DNA sequences with high homology to the aquaporin genes have been identified in several plant species [4], the water permeability and function haven’t been determined, only a few of the gene products have been characterized [6– 11]. Their roles in response to various physiological and stress conditions warrants further research. Here we report the isolation of the first water channel gene to be identified in rice, the activity of its water permeability, and the possible role in response to osmotic and chilling stress.

2. Materials and methods

2.1. Plant material, osmotic and chilling treatments Wasetoitsu (Oryza sati6a cv. Josaeng Tongil) is a less chilling-tolerant hybrid rice variety developed in the Republic of Korea. Seedlings and culture cells were prepared as described previously [12]. For treatment with mannitol and NaCl, rice seedlings at the three leaf-stage were transferred to fresh liquid Hoagland’s medium [5 mM Ca(NO3)2, 5 mM KNO3, 2 mM MgSO4, 0.025 mM FeSO4EDTA in tap water] containing 0.25 M mannitol or 0.15 NaCl, at 25°C. For chilling, after mannitol treatment the seedling was washed with tap water, and transferred to fresh Hoagland’s medium, incubated for 24 h at 4°C and then transferred to 25°C for 24 h.

2.2. cDNA library construction and screening Total RNA was extracted from leaves that had been treated for 30 min with 0.5 M mannitol. A cDNA library was constructed in the NotI site of lgt10. Aliquots of the cDNA were used as a template for the polymerase chain reaction (PCR) with designed primers corresponding to the se-

quences surrounding the Asn-Pro-Ala motif of aquaporins [5]. A PCR product of 393 bp was subcloned into the pT7Blunt vector (Novergen, USA) and sequenced on both strands using ABI373 Applied Biosystem (USA). This DNA fragment was radioactive labeled with [a 32P]dCTP by random DNA labeling (Takara, Japan) and was used as a hybridization probe to screen 3 × 105 plaques from the cDNA library. A positive plaque was isolated and the insert of the phage DNA was subcloned into a pBluescript KS+ according to the manufacturer’s protocol (Stratagene, USA). The subcloned cDNA, rwc1, was sequenced and analyzed using the GENETYX Version 8 (Software Development Co., Japan).

2.3. Northern analysis Total RNA was prepared as described by KogaBan et al. [13], and 5 mg of total RNA was fractionated on a 1.2% denaturing formaldehyde agarose gel. The RNA was transferred to a Hybond™-N+ membrane (Amersham, UK) using 200SSC [14]. Prehybridization and hybridization were performed using a rapid-hybridization buffer, as suggested by the manufacturer (Amersham). The probe was the full-length rwc1 sequence labeled with 32P by the random primer method (Takara, Japan).

2.4. Competiti6e PCR Transcription levels of rwc1 were specifically determined by the competitive PCR method [15]. Competitor lambda RNA (448 bases) against rice actin mRNA (accession number X15863 [16]) was made from a lambda DNA fragment placed in between the rice actin 5% primer (5%-AGAGCTACGAGCTTCCTGATGGAC-3%) and 3% primer (5% - GAGAGATGCCAAGATGGATCCTCC - 3%) regions. A SP6 promoter (5%-ATTTAGGTGACACTATAGAATAC-3%), induced upstream of the 5% actin primer, was used to synthesize RNA using SP6 polymerase according to the manufacture’s protocol (Takara, Japan). Competitor lambda RNA (530 bases) against rwc1 mRNA was prepared in the same manner from a lambda DNA fragment that was spaced by rwc1 5% (5%-ATCTACAACAAGGACCATGCCTGGA-3%) and rwc1 3% (5% - ATTACACGATTGAGTTGTTCAGGGT - 3%) primer, and placed downstream of the SP6 pro-

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moter. Using the rwc1 or actin-primers, fragment sizes of 333 bp for actin and 280 bp for rwc1 are obtained by RT-PCR using total RNA. The reaction mixture contained 10 ng of sample RNA, 105 –109 copies of competitor lambda RNA, 10 pmol of random 9 mer primer, 1 U of AMV reverse transcriptase, 4 U of RNase inhibitor, and 4 mM dNTP mixture in 4 ml of PCR buffer (10 mM Tris – HCl, pH 8.3, 50 mM KCl and 5 mM MgCl2). cDNA synthesize was allowed to proceed for 30 min at 42°C, followed by denaturation for 5 min at 99°C. The 4 ml cDNA solution was then mixed with 16 ml of PCR reaction mixture containing 1 – 4 pmol each of the particular 5% and 3% primers, and 0.5 U of Taq polymerase in PCR buffer. Samples were amplified by 30 cycles at 94°C for 0.5 min, 55°C for 0.5 min, and 72°C for 1 min. An aliquot of each reaction mixture was subjected to electrophoresis on 3% NuSive agarose gel.

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(Nanoject; Drummond). The oocytes were incubated for 2 days at 18°C in Barth’s buffer before water permeability measurements. Individual oocytes were transferred from Barth’s buffer (200 mosm) to a three-fold dilution of Barth’s buffer with distilled water (70 mosm) at 20°C. Oocyte swelling was checked using an area Colony Analyzer (CA-7, Toyo Sottuki, Japan). The area covered by the oocytes was measured at 5-s intervals; the total cell volume was calculated from the cell area. The relative oocyte volume (V/V0) was calculated from the relative oocyte area (A/A0) using the equation V/V0 =(A/A0)3/2. The osmotic water permeability coefficient (Pf, cm/s) was calculated from oocyte surface area (S= 0.045 cm2), initial oocyte volume (V0 =9× 10 − 4 cm), water volume (Vw =18 cm3/mol), and the initial rate of oocyte swelling, d(V/V0)/dt, using the equation Pf=V0d(V/V0)/dt[SVw(osmout −osmin)], where osmout is 70 mosm and osmin is 200 mosm.

2.5. In 6itro synthesis of rwc1 cRNA The rwc1 gene which was cut from the plasmid with BamHI was inserted into the BglII site of the Xenopus expression construct pXbG/ev-1; a pSP64T-derived pBluescript-type vector into which Xenopus b-globin 5% and 3% untranslated sequences were inserted [1]. Recombination of the plasmid was checked by sequencing. Capped RNA transcripts of rwc1 were synthesized in vitro using T3 RNA polymerase and linearized recombinant plasmids containing rwc1 cDNA after digestion with BamHI (Stratagene). After ethanol precipitation, the synthesis products were suspended in diethylpyrocarbonate-treated H2O at a final concentration of 1 mg/ml.

2.6. Microinjection of cRNA into oocytes and water permeability analysis Mature oocytes (1.2–1.3 mm diameter, stage V and VI) were isolated from adult Xenopus la6is [17,18] and stored in Barth’s buffer containing Na-penicillin (10 mg/ml) and streptomycin (10 mg/ ml). The follicular cell layer was removed by incubation with 2 mg/ml collagenase (Boehringer, Germany) in Barth’s buffer for 2.0–2.5 h at 25°C with gentle continuous agitation. Defolliculated oocytes were injected with 46 nl of cRNA (1 mg/ml) or water using an automatic injector

3. Results and discussion

3.1. Isolation of rwc1 from the rice cDNA library We used the most conserved sequences of the aquaporins to design our PCR primers, and used the cDNA library as the template. The 393-bp product showed high sequence homology to PIP1c from Arabidopsis, and it was used to probe the same cDNA library. The 1.1-kb fragment of rwc1 contained a 49-bp 5%-untranslated sequence preceding an initiation site consensus sequence. An 870-bp open reading frame was followed by a 3%-untranslated sequence containing a polyadenylation consensus sequence (accession no. AB009665).

3.2. Deduced structure of RWC1 and homology comparisons Analysis of the Genbank database showed that rwc1 is a novel member of the aquaporin family. The open reading frame encodes a polypeptide of 290 amino acids. Alignment of the deduced amino acid sequence of RWC1 with the sequences of mammalian (AQP2) and plant aquaporins (Fig. 1A) showed that RWC1 has residues known to be highly conserved in each of the transmembrane

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Fig. 1. (Continued)

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domains as well as the functionally important B and E loops. Hydropathy analysis indicated that RWC1 has six putative bilayer-spanning domains and five connecting loops, of which loops B and E were also hydrophobic (Fig. 1b). Both hydrophobic loops, which may form opposite leaflets, contained the NPA motif (Asn-Pro-Ala) that is present in all aquaproins (Fig. 1b), however RWC1 did not contain the cysteine residue that confers mercury sensitivity to AQP1 [19]. In addition, the phosphorylation site seen in PM28A (Ser-274) [20] was not identified in RWC1; however, the predicted topology of RWC1 contained some distinctive features. One putative cAMP or cGMP-dependent protein kinase phosphorylation motif (RKLS) was identified at residues 128 – 131. Two consensus sequences (SER, SSK) for protein kinase C phosphorylation were present near the N-terminal region (Fig. 1a), which was longer than the corresponding domain in E. coli [21] and mammalian aquaporins; some domains may have functions that are specific to plants. Nucleotide sequence comparisons between rwc1 and other genes analysis are shown in Fig. 1C. Related sequences were detected in human, Arabidopsis and spinach genes. The phylogenetic analysis indicates that rwc1 belongs to the MIP-related genes and is more closely related to PIP1 of Ara-

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bidopsis than to PM28A of spinach. We have classified rwc1 as a member of the PIP1 subfamily. The deduced amino acid sequence of RWC1 differs significantly from that of g-TIP, which is located in the tonoplast, so RWC1 may be located in plasma membrane.

3.3. Rice aquaporin genes response to osmotic 6ariation and in6ol6e into water stress-induced chilling tolerance The expression of rice aquaporin genes was determined by Northern blotting using total RNA isolated from leaves and roots of rice seedlings at the three-leaf stage and full-length rwc1 as the probe. Rice aquaporin-mRNAs were detected in both leaves and roots (Fig. 2a and b). When the seedlings were treated with 0.25 M mannitol or 0.15 M NaCl, the transcript level changed. Transcript level in root had declined drastically 6 h after the stress began and remained low for about 24 h. After that period, the transcript level of aquaporin genes increased again to at least the pre-stress level (Fig. 2A and B). The response of rice aquaporin genes to osmotic stress is similar to response of MipA and MipC genes in ice plants [11]; expression of aquaporin genes is suppressed under osmotic stress. Beside aquaporin gene-signal, there are several signals in Fig. 2A and B, but

Fig. 1. Aquaporin gene comparison and the structure of RWC1 polypeptide product. (a) Comparison of the amino acid sequence of RWC1 and those of other aquaporins. Two conserved NPA motif repeats are boxed. Putative bilayer-spanning domains (TM1-6) and connecting loops (A–E) are indicated by solid line. Consensus sequences for phosphorylation sites are indicated by italics and underlining. (b) Proposed membrane topology based on our sequence hydropathy analysis and the hourglass model of aquaporins [24], six bilayer-spanning domains and five connecting loops (A – E). The selected residues are identified, NPA motifs in the first and second aqueous hemichannels, and special motifs (RKLS etc.). (c) The evolutionary tree of the aquaporins. The PIP1 and PIP2 subfamilies are indicated.

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Fig. 2. Northern blot analysis of rice aquaporin-mRNA, stress response and tissue specificity. Total RNA fractions were prepared from leaves and roots, and then aliquots (5 mg) of them were applied to northern analysis. The gels were stained with ethidium bromide to detect rRNAs (indicated below). (A) Mannitol treatment. 1, control; 2, 0.25 M mannitol for 1 h; 3, 0.25 M mannitol for 3 h; 4, 0.25 M mannitol for 6 h; 5, 0.25 M mannitol for 24 h; 6, 0.25 M mannitol for 48 h; 7, 0.25 M mannitol for 72 h. (B) NaCl treatment. 1, control; 2, 0.15 M NaCl for 1 h; 3, 0.15 M NaCl for 3 h; 4, 0.15 M NaCl for 6 h; 5, 0.15 M NaCl for 24 h; 6, 0.15 M NaCl for 48 h; 7, 0.15 M NaCl for 72 h. (C) Chilling treatment (leaf). 1, control; 2, 4°C for 24 h; 3, 4°C for 24 h, then transferred to 25°C for 24 h; 4, 0.25 M mannitol for 3 h; 5, 0.25 M mannitol for 3 h, then incubated at 4°C for 24 h; 6, 0.25 M mannitol for 3 h, then incubated at 4°C for 24 h and transferred to 25°C for 24 h; 7, 0.25 M mannitol for 6 h; 8, 0.25 M mannitol for 6 h, then incubated at 4°C for 24 h; 9, 0.25 M mannitol for 6 h, then incubated at 4°C for 24 h and transferred to 25°C for 24 h.

not in Fig. 2C. It is presumed that washing condition of RNA blot of Fig. 2A and B was not optimal.

In previous reports, we showed that rice seedlings of chilling sensitive varieties (Wasetoitsu) could acquire chilling tolerance when first exposed

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to 0.25 – 0.5 M mannitol over a short time before chilling treatment [12]. When Wasetoitsu seedlings pretreated with 0.25 M mannitol for 3 and 6 h were incubated at 4°C for 24 h and then transferred to 25°C for 1 day, the expression of the aquaporin genes in the leaves showed fluctuation. Mannitol pretreatment for short time is helpful to down-regulate the expression of the aquaporin genes under chilling stress, and improve its expression during the period in recovering (Fig. 2C). When the pretreatment time with mannitol are increased, the transcription levels of the aquaporin genes in leaves are to be lower at low temperature,

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but the transcription levels are recovered significantly after the seedling transferred to 25°C (Fig. 2C). As we know, water channel is a bi-directional pore for water efflux and influx. The driving forces behind water movement are hydraulic or osmotic in nature [2]. One aspect of the chilling harm to plant is lost water. At low temperature, lower expression of aquaporin genes is beneficial to keep a suitable status of water; a fast recovery is also necessary for adaptation to standard physiological process. Characterization of the expression of aquaporin genes is just right to match the plant request. It is presumed that aquaporin genes may play an important role in response to water-stress induced chilling tolerance. The multiplicity of MIP genes are in plants as reported before [22], it leads us to presume that many similar genes exist in rice; some may be precisely regulated by water stress, whereas other are probably not affected. In this Northern experiment we could not determine the specific expression of rwc1, because we used full-length rwc1 as the probe. Next we determined the specific expression of rwc1 in rice roots under osmotic stress and chilling stress by competitive PCR using specific primers for the 3%-untranslated rwc1 region. The agarose gel results are shown in Fig. 3A, and the calculated numbers of rwc1 transcripts per actin transcript are shown in Fig. 3B. The specific expression of rwc1 mRNA under osmotic and chilling stress is essentially similar to the results of Northern hybridization shown in Fig. 2C, and suggests that rwc1 in particular is involved in responses to water stress-induced chilling tolerance.

3.4. Analysis of the water transport function of RWC1

Fig. 3. Determination of rwc1 mRNA expressed under chilling stress by competitive PCR. Wasetoitsu seedling were treated for 24 h at (1) 25°C, (2) 4°C, and (3) 4°C and then 25°C. The roots of another seedlings were treated for 1 h in 0.5 M mannitol and then treated for 24 h at (4) 25°C, (5) 4°C and (6) 4 and then 25°C. Total RNA fractions were prepared from roots and then aliquots (10 ng) of them were assayed to competitive PCR. (A) Data of agarose gel electrophoresis, (B) calculated munbers of rwc1 transcripts per actin transcripts from the data of agarose gel electrophoresis.

The water channel activity of RWC1 was assayed by injecting rwc1 complementary RNA (cRNA) into Xenopus oocytes. A cRNA that was synthesized in vitro from the cDNA, which had been cloned in vector pXbG/ev-1. cRNA of aqp2 [23] and rwc1 synthesized using pXbG/ev-l, were injected into oocytes. After 3 days of incubation in isotonic Barth’s buffer, the oocytes were transferred to a hypotonic buffer (1/3 Barth’s buffer), and the relative increase in cell volume, a consequence of water uptake, was measured [17]. The results indicate that RWC1 has water channel

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Acknowledgements We thank Dr K. Fushimi and M. Kuwahara of Tokyo Medical and Dental University for kindly providing the AQP2 gene and pXbG/ev-1 vector. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (Japan), for Special Scientific Research on Agriculture, Forestry and Fisheries, and by Grants from the Sumitomo Foundation (Japan) and Ciba-Geigy Foundation (Japan) for the Promotion of Science; and National nature and science foundation: 39670074 (China). References Fig. 4. Expression of rwc1 cRNA and cell membrane permeability. Oocytes were injected with cRNA orF water as indicated. Oocyte swelling was as described in Section 2. Values are the mean9 S.D. of 7–10 oocytes (stippled bars).

activity in oocytes (Fig. 4). Our osmotic water permeability indicated that the RWC1 protein led to a swelling rate 2–3 times higher than that of water-injected oocytes. However, the water permeability of the oocytes injected with rwc1 cRNA was not as great as that of oocytes injected with aqp2 cRNA. Why did RWC1 show weaker water channel activity? There are several possible reasons. One is that the phosphorylation sites and the other residues of the carboxyl-terminal cytoplasmic domain of RWC1 do not clarify function as restraints, as they do in animals. The second possibility is that, as for MIP in animals, a specific membrane environment may be required to improve the translation of the rwc1 mRNA [24]. Aquaporins belong to the MIP superfamily. Functional diversification may have produced extant complexity of the MIP members. The same superfamily members may play different roles in response to the different requirements of specialized physiological processes of species and tissues [25]. We expect that the gene complexity of plants reflects different metabolite transport activities or different adaptations to stress challenges.

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