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Junko Sakurai, Fumiyoshi Ishikawa, Tomoya Yamaguchi, Matsuo Uemura, Masayoshi Maeshima, Identification of 33 Rice Aquaporin Genes and Analysis of Their Expression and Function, Plant and Cell Physiology, Volume 46, Issue 9, September 2005, Pages 1568–1577, https://doi.org/10.1093/pcp/pci172
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Abstract
Plant aquaporins form a large protein family including plasma membrane-type (PIPs) and tonoplast-type aquaporins (TIPs), and facilitate osmotic water transport across membranes as a key physiological function. We identified 33 genes for aquaporins in the genome sequence of rice (Oryza sativa L. cv. Nipponbare). We investigated their expression levels in leaf blades, roots and anthers of rice (cv. Akitakomachi) using semi-quantitative reverse transcription–PCR (RT–PCR). At both early tillering (21 d after germination) and panicle formation (56 d) stages, six genes, including OsPIP2;4 and OsPIP2;5, were expressed predominantly in roots, while 14 genes, including OsPIP2;7 and OsTIP1;2, were found in leaf blades. Eight genes, such as OsPIP1;1 and OsTIP4;1, were evenly expressed in leaf blades, roots and anthers. Analysis by stopped-flow spectrophotometry revealed high water channel activity when OsPIP2;4 or OsPIP2;5 were expressed in yeast but not when OsPIP1;1 or OsPIP1;2 were expressed. Furthermore, the mRNA levels of OsPIP2;4 and OsPIP2;5 showed a clear diurnal fluctuation in roots; they showed a peak 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness. The mRNA levels of 10 genes including OsPIP2;4 and OsPIP2;5 markedly decreased in roots during chilling treatment and recovered after warming. The changes in mRNA levels during and after the chilling treatment were comparable with that of the bleeding sap volume. These results suggested the relationship between the root water uptake and mRNA levels of several aquaporins with high water channel activity, such as OsPIP2;4 and OsPIP2;5.
Introduction
Aquaporins are membrane proteins that facilitate water transport across the membranes in various microorganisms, animals and plants (King et al. 2004). Intensive research has revealed the involvement of aquaporins in plant growth and water relationships (see reviews by Maurel et al. 2002, Tyerman et al. 2002). Plant aquaporins are distributed in various plant tissues relating to water transport, cell differentiation and enlargement. Some aquaporins were also expressed in motor cells (Moshelion et al. 2002), guard cells (Kaldenhoff et al. 1995, Sarda et al. 1997) and reproductive organs (Dixit et al. 2001, O’Brien et al. 2002, Bots et al. 2005a, Bots et al. 2005b). Studies using mutant lines of aquaporins clearly demonstrated the involvement of aquaporins in plant–water relationships; for example, Ma et al. (2004) showed that reduction of TIP1;1 expression levels in Arabidopsis thaliana using the RNA interference method caused death of the plant, in the case of serious damage. In addition, gene expression and protein accumulation of aquaporins in response to various environmental stresses and hormones have been reported in several plants (Maurel et al. 2002, Tyerman et al. 2002). Furthermore, it has been reported recently that some types of plant aquaporin could transport not only water but also various small molecules such as glycerol, urea (Maurel et al. 2002, Gaspar et al. 2003, Liu et al. 2003), ammonia (Niemietz and Tyerman 2000, Loqué et al. 2005) and CO2 (Uehlein et al. 2003, Hanba et al. 2004). These studies are developing our knowledge on the physiological meanings of aquaporins in plants.
Recent genome sequencing projects have revealed that aquaporins constitute a large gene family in plants; A. thaliana and Zea mays have 35 and 33 genes, respectively (Chaumont et al. 2001, Johanson et al. 2001). These aquaporins have been classified into four major subfamilies referred to as plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nod26-like intrinsic proteins (NIPs) and small and basic intrinsic proteins (SIPs). Although recent studies provided information on the physiological function of several of those aquaporin members, the number of aquaporins examined in detail is still limited.
We have been conducting a series of experiments to elucidate the physiological relevance of aquaporins in rice, which is an important major crop. Although several aquaporin genes have already been identified and characterized in rice (Liu et al. 1994, Malz and Sauter 1999, Li et al. 2000, Lian et al. 2004, Takahashi et al. 2004), a complete set of rice aquaporins is unclear. In the present study, we identified 33 genes for aquaporins in the rice genome sequence and investigated their expression profiles in rice plant organs. We also studied the expression profile in roots during a light/dark cycle and chilling treatment. Furthermore, we expressed typical members of rice PIPs in yeast cells and determined the water channel activity of the membrane accumulating these aquaporins by stopped-flow spectrophotometry. From these results, we discuss the function of aquaporins in rice roots.
Results
Identification and nomenclature of rice aquaporin genes
We surveyed the genes for aquaporins in the genome database of rice (cv. Nipponbare). These genes were selected from a large number of the candidate sequences including both genome and cDNA sequences. The overlapping sequences were removed and finally 33 genes were selected, as shown in Table 1. Phylogenetic analysis of rice aquaporins was conducted with those of a monocotyledonous plant, Z. mays (Chaumont et al. 2001) as shown in Fig. 1. The aquaporins identified in rice were systematically named according to the phylogenetic relationship with the documented aquaporins in A. thaliana (Johanson et al. 2001) and Z. mays (Chaumont et al. 2001).
OsPIP1;2 was identified only from the cDNA sequence (AK098849, see Table 1, and AK058323, AK065188 and AK104736). There was no genome sequence identical to those cDNAs. However, the genomic DNA AL606687 contained a fairly similar sequence; it had a single base insertion and a few base substitutions when compared with AK098849. Thus, we named AL606687 and AK098849 as the same aquaporin gene OsPIP1;2.
Although several other genes encode the partial sequences of aquaporins (data not shown), they were considered to be non-functional pseudo-genes. This is mainly because they lacked some parts of characteristic sequences conserved in the aquaporin family, such as a transmembrane domain, when we analyzed their transmembrane topologies (data not shown).
Phylogenetic analysis of rice aquaporins
We classified the rice aquaporins into four subfamilies as same as those of Z. mays (Chaumont et al. 2001) and A. thaliana (Johanson et al. 2001) (Fig. 1). Rice had 11 PIPs, 10 TIPs, 10 NIPs and two SIPs. The OsPIP members had a relatively high sequence similarity of 58.5–92.7%, while the members of the OsTIP, OsNIP and OsSIP subfamilies had a somewhat variable sequence similarity. For example, the sequence similarity among the OsTIP members was 35.8–72.0%.
The OsPIP subfamily was divided into two groups, PIP1 and PIP2 (Fig. 1). OsPIP2;8 had a unique sequence and formed a long branch in the tree. However, we provisionally assigned OsPIP2;8 as a member of the PIP2 group because its sequence was similar to that of OsPIP2 members but not OsPIP1 members.
The OsTIP subfamily consisted of five groups, OsTIP1, OsTIP2, OsTIP3, OsTIP4 and OsTIP5, which had two, two, two, three and one member, respectively. The OsNIP subfamily consisted of four groups, OsNIP1, OsNIP2, OsNIP3 and OsNIP4, which had four, two, three and one member, respectively. The OsSIP subfamily had only two members and their sequence similarity is low (33%).
Organ-specific expression of rice aquaporin genes
The mRNA levels of 32 aquaporin genes were quantified by reverse transcription–PCR (RT–PCR) and compared in some organs (Fig. 2). The expression profiles varied with aquaporins. The mRNA levels of OsPIP1;3, OsPIP2;3, OsPIP2;4, OsPIP2;5, OsTIP2;1 and OsNIP2;1 were higher in roots than in leaf blades. In particular, OsPIP2;3, OsPIP2;4 and OsPIP2;5 were expressed predominantly in roots at both early tillering and panicle formation stages. In contrast, higher levels of the mRNA of 14 aquaporin genes (OsPIP2;7, OsPIP2;8, OsTIP1;2, OsTIP3;1, OsTIP3;2, OsTIP4;2, OsTIP4;3, OsTIP5;1, OsNIP1;1, OsNIP1;2, OsNIP1;4, OsNIP3;2, OsNIP3;3 and OsNIP4;1) were detected in leaf blades than in roots. Several genes (OsPIP1;1, OsPIP1;2, OsPIP2;1, OsPIP2;2, OsPIP2;6, OsTIP2;2, OsTIP4;1 and OsSIP1;1) were expressed almost equally in both roots and leaf blades.
The expression profile of some genes, such as OsTIP1;1, OsNIP2;2, OsNIP3;1 and OsSIP2;1, differed with the stages, i.e. early tillering or panicle formation. For example, OsTIP1;1 was highly expressed in roots at the early tillering stage; however, the level decreased to 43% at the panicle formation stage. Interestingly, the relative mRNA levels of OsPIP1;1, OsTIP4;2, OsTIP5;1 and OsSIP2;1 were also abundant in anthers in the heading stage.
Osmotic water permeabilities of OsPIP1;1, OsPIP1;2, OsPIP2;4 and OsPIP2;5
To measure the osmotic water transport activity of rice aquaporins by the yeast expression system, we expressed them in Saccharomyces cerevisiae strain BJ5458, which lacks vacuolar proteases, to avoid degradation of translation products. It has been confirmed that this strain lacks functional aquaporins (Suga and Maeshima 2004).
Rice aquaporins OsPIP1;1, OsPIP1;2, OsPIP2;4 and OsPIP2;5 were tagged with a c-myc epitope at the N-termini (Fig. 3A). This c-myc epitope has been reported to have no effect on water channel activity of recombinant aquaporin (Suga and Maeshima 2004). Protein accumulation in the yeast membranes was detected at the calculated molecular sizes of monomers (31–32 kDa) and dimers (63–69 kDa) by immunobloting with an anti-myc antibody (Fig. 3B). The expressed rice aquaporins in yeast were also detected by the anti-PAQs antibody, which recognizes most PIP members in radish (Ohshima et al. 2001) (Fig. 3C). This antibody showed only qualitative information, because the immunoreactivity of the antibody depends on the amino acid sequences of aquaporin antigens. No immunostained band was observed in the membranes prepared from yeast cells expressing the vacant vector.
The osmotic water permeability of membrane vesicles was measured with a stopped-flow light scattering spectrophotometer. The swelling rate of vesicles in the hypotonic solution was monitored as a decrease in the scattered light intensity (Fig. 3D). The vacant vector showed a slow influx of water into the membrane vesicles and we thought that this was the basal activity of the yeast membrane preparation. Fig. 3E shows the relative stimulation of osmotic water permeability of yeast membranes on the basis of the expressed amount of aquaporin. Considering the protein expression levels, the myc-tagged OsPIP1;1 and OsPIP1;2 did not stimulate water permeability much compared with myc-OsPIP2;4 and OsPIP2;5.
Diurnal variations of aquaporin mRNA levels in roots
The mRNA levels in roots under 12 h light/12 h dark conditions were determined by RT–PCR. In this experiment, we selected several aquaporins that were expressed at relatively high levels in the roots to analyze the relationship between mRNA levels and the water uptake from the roots. Interestingly, the mRNA levels of OsPIP1;2, OsPIP1;3, OsPIP2;3, OsPIP2;4, OsPIP2;5, OsTIP1;2 and OsTIP2;1 showed a clear diurnal change with a large amplitude (Fig. 4). These aquaporins reached the maximum level in the daytime and the basal level at midnight. In particular, the mRNA levels of OsPIP2;4 and OsPIP2;5, which had high water channel activity (Fig. 3D), showed a peak 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness.
Effect of chilling treatment on bleeding sap volume and the aquaporin mRNA levels in roots
The bleeding sap is an exudate of the xylem and its volume is highly correlated with root hydraulic conductance. The bleeding sap volume of rice plants decreased immediately after incubation at 4°C and then recovered 12 h after warming at 25°C with continuous light (Fig. 5A). In a similar manner, 4°C treatment caused a decrease in the transcript level of 10 aquaporin genes: OsPIP1;1, OsPIP1;2, OsPIP2;1, OsPIP2;2, OsPIP2;3, OsPIP2;4, OsPIP2;5, OsPIP2;6, OsTIP1;1 and OsTIP2;2 (Fig. 5B). OsPIP1;2, OsPIP2;4 and OsPIP2;5 decreased markedly after 24 h at 4°C, while the mRNA levels of OsPIP1;1, OsPIP2;1, OsPIP2;2, OsPIP2;3, OsPIP2;6, OsTIP1;1 and OsTIP2;2 decreased slowly (Fig. 5B, C). The decreased mRNA levels of most aquaporins were recovered 9 h after warming (Fig. 5B, C). It is noteworthy that the OsPIP1;3 mRNA level increased by 60% during the chilling treatment (Fig. 5C).
Discussion
Thirty-three rice aquaporin genes
We identified 33 rice aquaporin genes from the database obtained by the rice genome sequencing project (Table 1). The number of rice aquaporin genes was comparable with those in Z. mays and A. thaliana (33 and 35 genes, respectively). There were some differences between rice and other plant aquaporins. First, there were only 11 rice PIP members, which was less than those of Z. mays and A. thaliana (13 members in both) (Chaumont et al. 2001, Johanson et al. 2001). This is because rice had only three PIP1 members, while Z. mays and A. thaliana had six and five members, respectively. Secondly, rice had unique aquaporin members: OsPIP2;7, OsPIP2;8 and OsNIP4;1. Although these three members showed low sequence identity to other plant aquaporins (Fig. 1), they had two sets of the common Asn-Pro-Ala motif and six transmembrane domains. These results suggest that OsPIP2;7, OsPIP2;8 and OsNIP4;1 might have their own characteristic functions in rice.
Several rice aquaporin genes have already been registered in the databases. Although we identified most of them in this study (Table 1), we could not identify three genes, RWC1 (Li et al. 2000), RWC3 (Lian et al. 2004) and OsPIP2a (Malz and Sauter 1999). This may be due to the difference in the cultivar of rice used for gene analysis. For example, RWC1 identified in the japonica cultivar Josaeng Tongli (Wasetoitsu) (Li et al. 2000) was slightly different from OsPIP1;1 in the cultivar Nipponbare. We considered that RWC1 in Josaeng Tongli corresponds to OsPIP1;1 in Nipponbare. Similarly, RWC3 and OsPIP2a may correspond to OsPIP1;3 and OsPIP2;1, respectively. On the other hand, OsPIP1a (Malz and Sauter 1999), γTIP1 (Liu et al. 1994), OsTIP1, OsTIP2, OsTIP3 (Takahashi et al. 2004) and γMIP1 (Liu et al. 1994) were identical to the rice aquaporin genes identified in this study (Table 1), although some of them were cloned from other cultivars. These results indicated that there could be minor differences in some aquaporin sequences among rice cultivars.
Organ specificity of rice aquaporins
The expression profile of aquaporin genes in different organs and growth stages should provide information on their physiological roles. Most genes showed clear organ specificity, which was maintained over a long period of growth from early tillering to panicle formation stages (Fig. 2). This tendency was seen in other developmental stages, such as the maximum tillering and heading stage (data not shown). Therefore, the organ specificity may be tightly related to the physiological function in each organ.
The transcript levels of OsTIP3;1, OsTIP3;2, OsTIP4;2 and OsTIP5;1 and most OsNIP genes were estimated to be extremely low judging from the number of cycles of RT–PCR needed to detect those mRNAs (Fig. 2). In addition, no cDNA clones corresponding to OsPIP2;3, OsNIP1;2, OsNIP1;4, OsNIP3;1, OsNIP3;2 and OsNIP3;3 were identified in the database (Table 1). These results indicate that the expression of these genes might be extremely low or limited to specific tissues in rice plants.
OsPIP2;4 and OsPIP2;5 have high water channel activity
By the yeast heterologous expression system and stopped-flow spectrophotometric analysis, we found that OsPIP2;4 and OsPIP2;5 had significant osmotic water channel activity (Fig. 3E). In contrast, OsPIP1;1 and OsPIP1;2 showed no significant activity. Judging from the high expression of OsPIP2;4 and OsPIP2;5 in roots (Fig. 2), both OsPIP2 members might play a crucial role as water channels in roots.
The low and high water channel activities in PIP1 and PIP2 members, respectively, were reported for other plants (Chaumont et al. 2000, Moshelion et al. 2002, Fetter et al. 2004, Suga and Maeshima 2004). Suga and Maeshima (2004) demonstrated that the valine residue in loop E of radish PIP2 members is essential for the water channel activity. Radish PIP1 members have an isoleucine residue at the corresponding site. Similarly, isoleucine and valine residues were conserved in all members of the OsPIP1 and OsPIP2 group, respectively, except for OsPIP2;8. We should examine whether the members of OsPIP1 group facilitate the transport of other substrates and whether their water channel function is regulated by post-translational modification.
Response to chilling treatment and diurnal variation of rice aquaporins
One of the aims of the present work is to examine the involvement of rice aquaporins in water uptake from the roots under low temperature conditions. Therefore, we analyzed the relationship between bleeding sap volume, which is highly correlated with root hydraulic conductivity, and mRNA levels of rice aquaporins during chilling treatment. The change in the bleeding sap volume during and after chilling treatment was closely correlated with changes in the expression of aquaporin genes, especially genes for functional water channels, such as OsPIP2;4 and OsPIP2;5 (Fig. 5A, B, C). The down-regulation of PIP gene expression in roots was also reported for Z. mays (Aroca et al. 2005) and for rice (Li et al. 2000). On the other hand, Aroca et al. (2005) reported that the protein levels of Z. mays PIP members increased during chilling treatment. Therefore, further studies, such as analysis at the protein level, should be conducted to understand the physiological function of OsPIP2;4 and OsPIP2,5 in water uptake during chilling treatment.
We also found a diurnal variation in the mRNA levels of rice aquaporin in roots. OsPIP2;4 and OsPIP2;5 mRNA levels in roots varied diurnally with a large amplitude (Fig. 4). The diurnal changes in the mRNAs and proteins have also been reported for aquaporins of other plants such as Lotus japonicus (Henzler et al. 1999), Hordeum vulgare (Katsuhara et al. 2003) and Z. mays (Lopez et al. 2003, Lopez et al. 2004). Lopez et al. (2003) revealed that the protein levels of Z. mays ZmPIP2 members, but not ZmPIP1s, in roots were correlated closely with the diurnal variation in root water flux. ZmPIP2-1 and ZmPIP2-5 showed high water channel activities (Lopez et al. 2003), while ZmPIP1 members had low water channel activities (Chaumont et al. 2000, Gaspar et al. 2003). Therefore, Lopez et al. (2003) concluded that ZmPIP2 members might contribute to diurnal water transport in roots. These observations in conjunction with the present results underline the importance of members of the PIP2 group for diurnal water movement in plant roots.
Materials and Methods
Identification and phylogenetic analysis of rice aquaporin genes
Aquaporin genes from the genome sequence (O. sativa L. cv. Nipponbare) were identified by BLAST searches on the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/) and the Rice Genome Research Program (RGP; http://rgp.dna.affrc.go.jp/) based on the sequence similarity with aquaporins of A. thaliana (Johanson et al. 2001) and Z. mays (Chaumont et al. 2001). The phylogenetic analysis was conducted for their deduced amino acid sequences using the Clustal W program (Thompson et al. 1994) and the results were displayed using the TreeView program (Page 1996). The transmembrane topology of the rice aquaporins was predicted by the ConPred II method (Arai et al. 2004, http://bioinfo.si.hirosaki-u.ac.jp/~ConPred2/).
Plant materials and growth conditions
Rice (cv. Akitakomachi) seeds were germinated in the dark for 3 d at 25°C and grown in a growth chamber under 12 h light/12 h dark (light period; 450 µmol s–1 m–2) and at day/night temperatures of 25/20°C at a relative humidity of 75%. Plants were grown in tap water for the first 5 d, and then were supplied continuously with fresh culture solution at a slow rate. The culture solution contained 10 ppm nitrogen (NH4NO3), phosphorus (NaH2PO4), potassium (K2SO4), calcium (CaCl2), magnesium (MgSO4), 0.4 ppm iron (Fe(III)-EDTA) and 0.1 ppm manganese (MnCl2), pH 5.0. Leaf blades and roots were harvested from 21-day-old plants (early tillering stage) and 56-day-old plants (panicle formation stage). Leaf blades were harvested from all tillers of 21-day-old plants and from the upper three leaves of 56-day-old plants. Roots were collected from the apical half of the roots. Anthers were harvested from 67-day-old plants (heading stage, a few days before anthesis). For analysis of diurnal change in the levels of aquaporin mRNAs, total RNA fractions were obtained from 16-day-old plants.
Chilling treatment of plants and measurements of bleeding sap volume
Sixteen-day-old rice plants cultivated under 12 h light/12 h dark conditions were placed in a chamber set at 4°C without light under 100% relative humidity for 96 h to avoid the stresses of drought and light. Then plants were moved to a chamber set at 25°C with continuous light. The bleeding sap was collected into the cotton for 12 h from the stem cut off 3–4 cm above the soil surface at 25°C in continuous light to avoid the effect of diurnal variation of water uptake from roots. The bleeding sap volume was calculated from the increase in the weight of the cotton covered with parafilm. For analysis of aquaporin mRNAs, plants were chilled for 72 h and then moved to the continuous light chamber at 25°C.
RNA extraction and semi-quantitative RT–PCR
Tissues of rice plants were frozen in liquid nitrogen and ground in a mortar with a pestle. RNA was extracted from frozen powder of the tissue with the RNeasy Plant Mini kit (Qiagen K.K., Tokyo, Japan). For RT–PCR, the first strand cDNA was synthesized using ReverTra Ace (Toyobo Co., Ltd., Osaka, Japan). PCR was performed using AmpliTaq GOLD (ABI, Foster City, CA, USA) and the primers listed in Table 2 (Supplementary data). All primers were designed based on the sequences of the 3′-untranslated regions in each aquaporin gene and to have similar Tm values (59.3 ± 1.2°C). The PCR conditions used were 94°C for 30 s, 58°C for 30 s and 72°C for 1 min. In the case of some aquaporins, the annealing temperature was set at 61°C to prevent the amplification of non-specific PCR products. The reaction was repeated for 16–36 cycles to obtain an appropriate amount of DNA. The conditions and cycle numbers were determined to avoid the saturation of DNA amplification. The obtained DNA was subjected to agarose gel electrophoresis and stained with ethidium bromide. The signal intensity of the stained bands was photographed by a charge-coupled device (CCD) camera and analyzed by the NIH Image program (http://rsb.info.nih.gov/nih-image). The fact that there was no contamination of genomic DNA in the cDNA samples was confirmed by PCR using the primer sets listed in Table 2 (Supplementary data).
Expression of rice aquaporin genes in yeast
EcoRI–SalI or EcoRI–PvuII fragments of rice aquaporin cDNA (OsPIP1;1 and OsPIP1;2, EcoRI–SalI; OsPIP2;4 and OsPIP2;5, EcoRI–PvuII) were amplified by RT–PCR with gene-specific primers (Table 2, Supplementary data) and LA Taq (TAKARA SHUZO Co., LTD., Kyoto, Japan) using total RNA of the rice roots as a template. Reverse primers included an additional 3′-non-coding region (8–17 bases), because the open reading frames of rice aquaporins were quite similar to each other. The obtained fragments were inserted into the yeast expression vector myc-pKT10 (Tanaka et al. 1990, Suga and Maeshima 2004) (see Fig. 3A). This vector includes a c-myc epitope sequence at the down-stream region of the GAPDH promoter. After confirming the DNA sequences, the obtained plasmid was introduced into S. cerevisiae strain BJ5458, which is deficient in major vacuolar proteinases and functional aquaporins (Suga and Maeshima 2004).
Transformed yeast, which was selected using URA3 (orotidine-5′-phosphate decarboxylase), was grown in AHCW/Glc plates [0.17% yeast nitrogen base without amino acid, 0.5% ammonium sulfate (Difco), 1% casamino acid, 0.002% adenine sulfate, 0.002% tryptophan, 50 mM potassium phosphate, pH 5.5, 2% glucose and 2% agar] as described previously (Nakanishi et al. 2001, Suga and Maeshima 2004). Accumulation of the transformed aquaporins was confirmed by immunoblotting using anti-myc (9E10) (Nacalai Tesque Inc., Osaka, Japan) and anti-PAQs antibodies, which recognize most isoforms of PIP1s and PIP2s of radish (Ohshima et al. 2001). The expression level of each aquaporin protein was calculated from the signal intensity of the stained bands by anti-myc antibody (Fig. 3C) using the NIH Image program.
Determination of the osmotic water permeability of membranes
The osmotic water permeability of membranes was measured by a stopped-flow spectrophotometer (model SX18MV, Applied Photophysics, Surrey, UK) as described previously (Ohshima et al. 2001, Suga and Maeshima 2004). Yeast membrane vesicles (0.5 mg ml–1) containing each rice aquaporin in a 0.45 M mannitol solution were quickly mixed with an equal volume of 0.1 M mannitol solution. The membrane suspension medium contained 0.45 M mannitol, 90 mM KCl, 1 mM EDTA and 20 mM Tris–HCl, pH 7.2. The light-scattering assay was carried out at 10°C for 7–12 times and the average was calculated for each membrane sample. The initial rate constants were calculated from the lines between 0 and 10 ms. The relative fold stimulation of osmotic water permeabilities was determined by the ratio of the initial rate constant of each aquaporin to that of vector control on the basis of the expressed amount of protein (ratio to that of myc-OsPIP1;1).
Supplementary material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oupjournals.org.
Acknowledgments
We are grateful to Drs. Masumi Okada and Mari Murai for their valuable discussions throughout this work. We also thank Katsuhiro Nakayama for his technical advice. This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan to J.S. (No. 17780196) and M.M. (13CE2005 and 14COEA04).
Name | Genome sequence accession No. | cDNA clone accession No. | Notes | |
Gene | Protein a | |||
OsPIP1;1 | AP005108 | BAD28398 | AK061769 | Identical to OsPIP1a (AJ224327), similar to RWC1 (AB09665) |
OsPIP1;2 | (AL606687) b | AK098849 | ||
OsPIP1;3 | AP004026 | BAD22920 | AK102174 | Similar to RWC3 (AB029325) |
OsPIP2;1 | AP003802 | BAC15868 | AK072519 | Similar to OsPIP2a (AF062393) |
OsPIP2;2 | AP006168 | BAD23735 | AK061782 | |
OsPIP2;3 | AL662958 | CAD41442 | ||
OsPIP2;4 | AP004668 | BAC16113 | AK072632 | |
OsPIP2;5 | AP004668 | BAC16116 | (AK107700) c | |
OsPIP2;6 | AL731636 | CAE05002 | AK061312 | |
OsPIP2;7 | AP006149 | BAD46581 | AK109439 | |
OsPIP2;8 | AC092263 | AAP44741 | AK109024 | |
OsTIP1;1 | AC090485 | AAK98737 | AK058322 | Identical to OsγTIP1 (D22534) |
OsTIP1;2 | AP003627 | BAB63833 | AK111747 | Identical to OsTIP1 (AB114829) |
OsTIP2;1 | AP005289 | BAD25765 | (AK064728) c | Identical to OsTIP2 (AB114830) |
OsTIP2;2 | AP004784 | BAD61899 | AK099141 | |
OsTIP3;1 | AC023240 | AAG13544 | AK111931 | Identical to OsTIP3 (AB114828) |
OsTIP3;2 | AL663019 | CAE05657 | AK108116 | |
OsTIP4;1 | AC145396 | AAS98488 | AK060193 | |
OsTIP4;2 | AP001550 | BAA92993 | AK099190 | |
OsTIP4;3 | AP001550 | BAA92991 | (AK069192) c | |
OsTIP5;1 | AL663000 | AK070602 | ||
OsNIP1;1 | AP004070 | BAD27715 | AK068806 | Identical to γMIP1 (D17443) |
OsNIP1;2 | AP003105 | BAD73177 | ||
OsNIP1;3 | AC135918 | AAV44140 | (AK062320) c | |
OsNIP1;4 | AP003682 | BAD53665 | ||
OsNIP2;1 | AP005297 | BAD16128 | AK069842 | |
OsNIP2;2 | AP003569 | BAD37471 | AK112022 | |
OsNIP3;1 | AC068924 | AAG13499 | ||
OsNIP3;2 | AP005467 | BAC99758 | ||
OsNIP3;3 | AP005467 | BAC65382 | ||
OsNIP4;1 | AP003219 | BAB61180 | AK106825 | |
OsSIP1;1 | AP003047 | BAB32914 | AK109424 | |
OsSIP2;1 | AC119748 | (AK071190) c |
Name | Genome sequence accession No. | cDNA clone accession No. | Notes | |
Gene | Protein a | |||
OsPIP1;1 | AP005108 | BAD28398 | AK061769 | Identical to OsPIP1a (AJ224327), similar to RWC1 (AB09665) |
OsPIP1;2 | (AL606687) b | AK098849 | ||
OsPIP1;3 | AP004026 | BAD22920 | AK102174 | Similar to RWC3 (AB029325) |
OsPIP2;1 | AP003802 | BAC15868 | AK072519 | Similar to OsPIP2a (AF062393) |
OsPIP2;2 | AP006168 | BAD23735 | AK061782 | |
OsPIP2;3 | AL662958 | CAD41442 | ||
OsPIP2;4 | AP004668 | BAC16113 | AK072632 | |
OsPIP2;5 | AP004668 | BAC16116 | (AK107700) c | |
OsPIP2;6 | AL731636 | CAE05002 | AK061312 | |
OsPIP2;7 | AP006149 | BAD46581 | AK109439 | |
OsPIP2;8 | AC092263 | AAP44741 | AK109024 | |
OsTIP1;1 | AC090485 | AAK98737 | AK058322 | Identical to OsγTIP1 (D22534) |
OsTIP1;2 | AP003627 | BAB63833 | AK111747 | Identical to OsTIP1 (AB114829) |
OsTIP2;1 | AP005289 | BAD25765 | (AK064728) c | Identical to OsTIP2 (AB114830) |
OsTIP2;2 | AP004784 | BAD61899 | AK099141 | |
OsTIP3;1 | AC023240 | AAG13544 | AK111931 | Identical to OsTIP3 (AB114828) |
OsTIP3;2 | AL663019 | CAE05657 | AK108116 | |
OsTIP4;1 | AC145396 | AAS98488 | AK060193 | |
OsTIP4;2 | AP001550 | BAA92993 | AK099190 | |
OsTIP4;3 | AP001550 | BAA92991 | (AK069192) c | |
OsTIP5;1 | AL663000 | AK070602 | ||
OsNIP1;1 | AP004070 | BAD27715 | AK068806 | Identical to γMIP1 (D17443) |
OsNIP1;2 | AP003105 | BAD73177 | ||
OsNIP1;3 | AC135918 | AAV44140 | (AK062320) c | |
OsNIP1;4 | AP003682 | BAD53665 | ||
OsNIP2;1 | AP005297 | BAD16128 | AK069842 | |
OsNIP2;2 | AP003569 | BAD37471 | AK112022 | |
OsNIP3;1 | AC068924 | AAG13499 | ||
OsNIP3;2 | AP005467 | BAC99758 | ||
OsNIP3;3 | AP005467 | BAC65382 | ||
OsNIP4;1 | AP003219 | BAB61180 | AK106825 | |
OsSIP1;1 | AP003047 | BAB32914 | AK109424 | |
OsSIP2;1 | AC119748 | (AK071190) c |
a The protein accession numbers were obtained from the result of annotation analysis in the database.
b AL606687 encoded a gene which had a high homology to OsPIP1;2 (see Results).
c cDNA clones in parentheses did not contain full-length genes.
Name | Genome sequence accession No. | cDNA clone accession No. | Notes | |
Gene | Protein a | |||
OsPIP1;1 | AP005108 | BAD28398 | AK061769 | Identical to OsPIP1a (AJ224327), similar to RWC1 (AB09665) |
OsPIP1;2 | (AL606687) b | AK098849 | ||
OsPIP1;3 | AP004026 | BAD22920 | AK102174 | Similar to RWC3 (AB029325) |
OsPIP2;1 | AP003802 | BAC15868 | AK072519 | Similar to OsPIP2a (AF062393) |
OsPIP2;2 | AP006168 | BAD23735 | AK061782 | |
OsPIP2;3 | AL662958 | CAD41442 | ||
OsPIP2;4 | AP004668 | BAC16113 | AK072632 | |
OsPIP2;5 | AP004668 | BAC16116 | (AK107700) c | |
OsPIP2;6 | AL731636 | CAE05002 | AK061312 | |
OsPIP2;7 | AP006149 | BAD46581 | AK109439 | |
OsPIP2;8 | AC092263 | AAP44741 | AK109024 | |
OsTIP1;1 | AC090485 | AAK98737 | AK058322 | Identical to OsγTIP1 (D22534) |
OsTIP1;2 | AP003627 | BAB63833 | AK111747 | Identical to OsTIP1 (AB114829) |
OsTIP2;1 | AP005289 | BAD25765 | (AK064728) c | Identical to OsTIP2 (AB114830) |
OsTIP2;2 | AP004784 | BAD61899 | AK099141 | |
OsTIP3;1 | AC023240 | AAG13544 | AK111931 | Identical to OsTIP3 (AB114828) |
OsTIP3;2 | AL663019 | CAE05657 | AK108116 | |
OsTIP4;1 | AC145396 | AAS98488 | AK060193 | |
OsTIP4;2 | AP001550 | BAA92993 | AK099190 | |
OsTIP4;3 | AP001550 | BAA92991 | (AK069192) c | |
OsTIP5;1 | AL663000 | AK070602 | ||
OsNIP1;1 | AP004070 | BAD27715 | AK068806 | Identical to γMIP1 (D17443) |
OsNIP1;2 | AP003105 | BAD73177 | ||
OsNIP1;3 | AC135918 | AAV44140 | (AK062320) c | |
OsNIP1;4 | AP003682 | BAD53665 | ||
OsNIP2;1 | AP005297 | BAD16128 | AK069842 | |
OsNIP2;2 | AP003569 | BAD37471 | AK112022 | |
OsNIP3;1 | AC068924 | AAG13499 | ||
OsNIP3;2 | AP005467 | BAC99758 | ||
OsNIP3;3 | AP005467 | BAC65382 | ||
OsNIP4;1 | AP003219 | BAB61180 | AK106825 | |
OsSIP1;1 | AP003047 | BAB32914 | AK109424 | |
OsSIP2;1 | AC119748 | (AK071190) c |
Name | Genome sequence accession No. | cDNA clone accession No. | Notes | |
Gene | Protein a | |||
OsPIP1;1 | AP005108 | BAD28398 | AK061769 | Identical to OsPIP1a (AJ224327), similar to RWC1 (AB09665) |
OsPIP1;2 | (AL606687) b | AK098849 | ||
OsPIP1;3 | AP004026 | BAD22920 | AK102174 | Similar to RWC3 (AB029325) |
OsPIP2;1 | AP003802 | BAC15868 | AK072519 | Similar to OsPIP2a (AF062393) |
OsPIP2;2 | AP006168 | BAD23735 | AK061782 | |
OsPIP2;3 | AL662958 | CAD41442 | ||
OsPIP2;4 | AP004668 | BAC16113 | AK072632 | |
OsPIP2;5 | AP004668 | BAC16116 | (AK107700) c | |
OsPIP2;6 | AL731636 | CAE05002 | AK061312 | |
OsPIP2;7 | AP006149 | BAD46581 | AK109439 | |
OsPIP2;8 | AC092263 | AAP44741 | AK109024 | |
OsTIP1;1 | AC090485 | AAK98737 | AK058322 | Identical to OsγTIP1 (D22534) |
OsTIP1;2 | AP003627 | BAB63833 | AK111747 | Identical to OsTIP1 (AB114829) |
OsTIP2;1 | AP005289 | BAD25765 | (AK064728) c | Identical to OsTIP2 (AB114830) |
OsTIP2;2 | AP004784 | BAD61899 | AK099141 | |
OsTIP3;1 | AC023240 | AAG13544 | AK111931 | Identical to OsTIP3 (AB114828) |
OsTIP3;2 | AL663019 | CAE05657 | AK108116 | |
OsTIP4;1 | AC145396 | AAS98488 | AK060193 | |
OsTIP4;2 | AP001550 | BAA92993 | AK099190 | |
OsTIP4;3 | AP001550 | BAA92991 | (AK069192) c | |
OsTIP5;1 | AL663000 | AK070602 | ||
OsNIP1;1 | AP004070 | BAD27715 | AK068806 | Identical to γMIP1 (D17443) |
OsNIP1;2 | AP003105 | BAD73177 | ||
OsNIP1;3 | AC135918 | AAV44140 | (AK062320) c | |
OsNIP1;4 | AP003682 | BAD53665 | ||
OsNIP2;1 | AP005297 | BAD16128 | AK069842 | |
OsNIP2;2 | AP003569 | BAD37471 | AK112022 | |
OsNIP3;1 | AC068924 | AAG13499 | ||
OsNIP3;2 | AP005467 | BAC99758 | ||
OsNIP3;3 | AP005467 | BAC65382 | ||
OsNIP4;1 | AP003219 | BAB61180 | AK106825 | |
OsSIP1;1 | AP003047 | BAB32914 | AK109424 | |
OsSIP2;1 | AC119748 | (AK071190) c |
a The protein accession numbers were obtained from the result of annotation analysis in the database.
b AL606687 encoded a gene which had a high homology to OsPIP1;2 (see Results).
c cDNA clones in parentheses did not contain full-length genes.
Abbreviations
- NIP
Nod26-like intrinsic protein
- PIP
plasma membrane intrinsic protein
- RT–PCR
reverse transcription–PCR
- SIP
small and basic intrinsic protein
- TIP
tonoplast intrinsic protein
References
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