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A cotton gene encodes a tonoplast aquaporin that is involved in cell tolerance to cold stress
Gene 438 (2009) 26–32
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Gene j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e n e
A cotton gene encodes a tonoplast aquaporin that is involved in cell tolerance to cold stress Deng-Di Li 1, Fu-Ju Tai 1,2, Ze-Ting Zhang, Yang Li, Yong Zheng, Yan-Feng Wu, Xue-Bao Li ⁎ Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, HuaZhong Normal University, Wuhan 430079, China
a r t i c l e
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Article history: Received 20 October 2008 Received in revised form 8 February 2009 Accepted 12 February 2009 Available online 17 March 2009 Received by G. Theissen Keywords: Cotton (Gossypium hirsutum) Gene expression TIP Cold stress Cell tolerance
a b s t r a c t To enhance the survival probability in cold stress, plant cells often increase their cold- and freezing-tolerance in response to low, nonfreezing temperatures by expressing some cold-related genes. In present study, a cotton gene encoding tonoplast intrinsic protein (TIP) was isolated from a cotton seedling cDNA library, and designated as GhTIP1;1. GFP fluorescent microscopy indicated that GhTIP1;1 protein was localized to the vacuolar membrane. Assay on GhTIP1;1 expression in Xenopus laevis oocytes demonstrated that GhTIP1;1 protein displayed water channel activity and facilitated water transport to the cells. At normal conditions, GhTIP1;1 transcripts were predominantly accumulated in roots and hypocotyls, but less abundance in other tissues of cotton. The GhTIP1;1 expression was dramatically up-regulated in cotyledons, but down-regulated in roots within a few hours after cotton seedlings were cold-treated. Overexpression of GhTIP1;1 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival probability, suggesting that the GhTIP1;1 protein is involved in cell freezing-tolerance.
1. Introduction Temperature is one of the important factors which affect plant growth and development. Cold stress together with other abiotic stresses such as drought and soils with changing salt and nutrient concentrations represent the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Bray et al., 2000). Many crop species are sensitive to low temperatures and grow poorly after exposure to a cold period. It is generally considered that low-temperature stress causes reduction in photosynthesis, inhibition in energy generation and material synthesis, but increase in respiration and consumption of plants. Such damages may result in plant death (Guo et al., 2004). Nevertheless, many plants have the ability to sense low temperature and respond by activating mechanisms that lead to an increase in
Abbreviations: cDNA, DNA complementary to mRNA; cRNA, complementary RNA; GhTIP, cotton gene for translation tonoplast intrinsic protein; AtTIP, tonoplast intrinsic protein of Arabidopsis; AQY, yeast gene for translation aquaporin; CBF, C-box binding factor; DREB1, dehydration-responsive element binding-1; CFU, cloning-forming units; ABA, abscisic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; UTR, untranslated region; ORF, open reading frame; bp, base pair; kDa, kilodalton; dCTP, deoxycytidine triphosphate; eGFP, enhanced green fluorescent protein. ⁎ Corresponding author. Tel./Fax: +86 27 67862443. E-mail address: [email protected] (X.B. Li). 1 These authors contributed equally to this work. 2 Present address: Department of Plant Science, Henan Agricultural University, Zhengzhou 450002, China. 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.02.023
© 2009 Elsevier B.V. All rights reserved.
freezing-tolerance, an adaptive response known as cold acclimation (Thomashow, 1999). Freezing-tolerance is a multigenic trait. Previous studies revealed that a lot of genes (such as the genes encoding CBF and DREB1 transcription factors) are involved in cold acclimation (Thomashow, 2001). Overexpression of CBF or DREB1 resulted in increase in plant tolerance to cold stress (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Pino et al., 2008). Except for transcription factors, many downstream proteins, such as aquaporins (AQPs), also play important roles in response to cold stress (Tanghe et al., 2002; Aroca et al., 2005). AQPs which belong to a large superfamily known as MIPs (major intrinsic proteins) are a family of small (23–34 kDa) integral membrane proteins that transport water and small neutral solutes across the cellular membrane (Park and Saier, 1996; Maurel, 1997; Tyerman et al., 1999; Tyerman et al., 2002). So far, genome projects have revealed that Arabidopsis thaliana contains 35 genes encoding aquaporins (Chrispeels et al., 1999; Johanson et al., 2001), and maize has at least 33 AQP genes (Chaumont et al., 2001). In plants, AQPs are divided into four clades: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs) (Chaumont et al., 2001; Johanson et al., 2001). All of four kinds of plant AQPs contain the highly conserved structures: six membrane-spanning alpha helices (H1 to H6), linked by five short loops (loop A to loop E) with N and C termini always facing the cytosol. Two highly conserved asparagineproline-alanine (NPA) motifs locate in loop B and E which form two short helices and dip into the membrane from opposite sides, playing
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a key role in formation of water-selective channel (Chaumont et al., 2001). Plant AQPs play important roles in seed germination, cell elongation, stoma movement, and in response to stresses. Expression patterns of all 13 Arabidopsis PIP genes under several abiotic stresses (such as drought, cold, high salinity and ABA) were investigated, indicating that all these genes were up- or downregulated under different environmental stimuli (Jang et al., 2004). In Baker's yeast and Candida albicans, there is a clear correlation between AQP expression and freezing-tolerance. Overexpression of AQY1 and AQY2 in Baker's yeast or overexpression AQY1 in C. albicans improved freezing-tolerance of the cells. Deletion of the respective genes, the cells became more sensitive to freezing (Tanghe et al., 2002; Tanghe et al., 2005). Aroca et al. (2005) also found that AQP is necessary to respond to chilling injury. In the chilling-tolerant genotype of maize, the recovery of root hydraulic conductance (Lo) is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of AQPs. Cotton, including Upland cotton (Gossypium hirsutum) and Sea Island cotton (Gossypium barbadense), is the main source of natural fibers used in textile industry worldwide. In most districts of China, temperature in spring is still low and unstable. Cotton seedlings often encountered low temperature which caused growth retardation, even death, in spring season. Therefore, it is both biological and agricultural importance to understand the molecular mechanism of cotton coldtolerance. Although some progress has been made in understanding the phenomenon of plant cold acclimation in recent years, the roles of AQP genes in cold-tolerance of plants (especially cotton) still remain largely unknown so far. In present study, a cold-induced gene GhTIP1;1 (access number in GenBank: FJ384629) was isolated from cotton. The GhTIP1;1 protein was localized in the vacuolar membrane and displayed high water transport activity in cells. The experimental results also revealed that GhTIP1;1 gene was preferentially expressed in roots and hypocotyls, and overexpression of GhTIP1;1 gene in yeast enhanced the freezing-tolerance of the transformed cells. 2. Materials and methods 2.1. Plant materials Cotton (G. hirsutum cvs. Xuzhou 142, Coker 312, Emian No. 9 and No. 10) seeds were surface-sterilized with 70% (v/v) ethanol for 1 min and 10% (v/v) H2O2 for 2 h, followed by washing with sterile water. The sterilized seeds were germinated on half-strength Murashige and Skoog (1/2MS) medium under a 16 h-light/8 h-dark cycle at 28 °C for 5–6 days. Roots, cotyledons and hypocotyls were collected from sterile seedlings for RNA isolation. Other tissues, such as leaves, stems, petals, anthers, ovules and fibers, were derived from cotton plants grown in a greenhouse. All tissues were collected 2–3 h after light onset since some plant aquaporins display a diurnal expression pattern, with a peak of expression at 2–4 h after the beginning of the light period (Hachez et al., 2006). For cold treatment, 4–5 day-old seedlings grown at normal conditions were transferred to 4 °C with a 24 h photoperiod. The parallel control plants were placed at 28 °C with a 24 h photoperiod. The samples from stress-treated and control plants were collected and immediately frozen in liquid nitrogen and stored at −70 °C. 2.2. Construction of cotton seedling cDNA library and isolation of GhTIP1;1 cDNA Total RNA was extracted from 5–6 day-old cotton seedlings using the method as described previously (Li et al., 2002). Poly(A)+ mRNA was prepared from a pool of seedling total RNA by using an mRNA
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purification kit (Qiagen). Complementary DNA was synthesized and cloned into the EcoR I–Xho I sites of the ZAP express vector and packaged by using a ZAP-cDNA Gigapack Gold III cloning kit (Stratagene) according to the manufacturer's instruction. More than 4000 cDNA clones were randomly selected from the cDNA library for sequencing. Over 30 cDNA clones (including GhTIP1;1) with potential involvement in cold acclimation were selected by analyzing the sequence data. For each candidate, its expression level was detected in cotton tissues grown under normal condition and cold stress by Northern blot and RT-PCR analyses. 2.3. Quantitative RT-PCR analysis Real-time quantitative reverse transcriptase (RT)-PCR was performed using a method described earlier (Li et al., 2005). The cotton polyubiquitin gene (GhUBI1, access number in GenBank: EU604080) was used as a standard control in the RT-PCR reactions. The primer sequences were as follows: GhTIP1;1: up-chain primer, 5′NTACACAGTGTACGCCACAGCCGTTb3′, down-chain primer, 5′N TCACATTTACAGCCCTGATGCTGGTb3′; GhUBI1: up-chain primer, 5′NCTGAATCTTCGCTTTCACGTTATCb3′, down-chain primer, 5′N GGGATGCAAATCTTCGTGAAAACb3′. The data of real-time RT-PCR are mean values and standard errors of three independent experiments with three biological replicates. 2.4. DNA and protein sequence analysis Nucleotide and amino acid sequences were analyzed using DNAstar (DNAstar Inc). The peptide sequences were aligned with the ClustalW program (http://www.ebi.ac.uk), and phylogenetic analysis was employed to investigate the evolutionary relationships among the aquaporins. A minimum evolution tree was generated in MEG3.1. A bootstrap analysis with 1000 replicates was performed to assess the statistical reliability of the tree topology. 2.5. RNA Gel–Blot analysis 3′-untranslated region (3′-UTR) fragment (285 bp, + 1 to +285 downstream stop codon) of GhTIP1;1 cDNA was prepared as a probe by PCR amplification. RNA samples (20 μg per lane) from cotyledons and roots of cold-treated cotton seedlings were separated on 1.2% (w/v) agarose-formaldehyde gels (55 V) for 4–5 h and transferred onto Hybond-N+ nylon membranes by capillary blotting. Genespecific probe was labeled with α-32P-dCTP using the Random Primer DNA Labeling Kit (Ver.2, TAKARA, Dalian, China). RNA Northern-blot hybridization was performed as described previously (Li et al., 2002). The membrane was exposed to X-film (Eastman Kodak, Rochester, NY) with two intensifying screens at −70 °C for 1–3 d. 2.6. In vitro complementary RNA synthesis The cDNA of GhTIP1;1 was subcloned into pGEM-7Z vector using the restriction sites EcoR I and Xba I. Capped cRNA transcripts were synthesized in vitro with mMACHINE SP6 Kit (Ambion) with Xba I linearized vector. 2.7. Oocyte preparation, cRNA injection, and osmotic water permeability assay Xenopus laevis oocytes of stages V and VI were isolated and defolliculated by digestion at room temperature for 1 h with 2 mg/ml collagenase A (Sigma) in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 5 mM Hepes-NaOH, pH 7.4, 220 mosm/kg). 50 nl volume of in vitro transcripts (50 ng) of the target gene, using the same volume of distilled water as negative
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2.9. Overexpression of GhTIP1;1 in fission yeast
control, was injected into the oocytes, and then the oocytes were incubated at 19 °C for 48 h in ND96 solution supplemented with 10 μg/ml penicillin and 10 μg/ml streptomycin. To measure the osmotic water permeability coefficient (Pf), a single oocyte was transferred to 5-fold diluted ND96 solution. Change in the oocyte volume was monitored at room temperature with a microscope video system by taking digital images at 30 s intervals. Oocyte volume (V) was calculated from the measured area of each oocyte. The osmotic permeability coefficient (Pf) was calculated for the first 5 min using the formula Pf = V0[d(V0 / V) / dt] / [S0 × VW(Osmin − Osmout)], with an initial volume (V0) of 9 × 10− 4 cm3, an initial oocyte surface area (S0) of 0.045 cm2, and a molar volume of water (VW) of 18 cm3/mol (Zhang and Verkman, 1991).
The coding sequences of GhTIP1;1 gene was cloned into yeast vector pREP5N with Sal I/Not I sites. Afterwards, the constructed vector and pREP5N empty vector were transferred into yeast (Schizosaccharomyces pombe) cells by electroporation (Bio-Rad MicroPulser Electroporation Apparatus) according to the manufacturer's instructions. Transformants were then selected on minimal medium (MM) supplemented with 75 mg/l adenine and uracil at 29 °C. Ten colonies of each transformant were randomly picked out to grow in liquid MM with 2 μM thiamine, which represses the nmt-1 promoter activity, until mid-log phase in a shaker (220 rev min− 1, 29 °C). Subsequently, the yeast cells were harvested and washed three times with MM containing no thiamine to de-repress the promoter, and then incubated in the same thiamine-free medium for 20 h (220 rev min− 1, 29 °C). 10 μl of the cultured cells was transferred to 10 ml fresh thiamine-free MM medium and grew to exponential phase (OD600 = 0.4 to 0.6). Afterwards, 2 ml of the cultured cells was divided into 10 equal portions. Five aliquots (200 μl each) were kept at 4 °C, and additional five aliquots were frozen at − 20 °C. After 48 h, the five thawed test samples were diluted at 4 °C and stained with 0.1% methylene blue, which stained the dead cells into blue colour, for 5 min. The control samples (nonfrozen cells) were also stained with methylene blue. Cell survival probability was determined as the percentage of CFU of frozen samples relative to the control samples.
2.8. Subcellular localization of GhTIP1;1 protein
3. Results
To construct GhTIP1;1:eGFP vector, the ORF sequence of eGFP gene was cloned into pBluescript II SK+ vector to obtain an intermediate construct pSK-eGFP. Subsequently, the GhTIP1;1 ORF (without the stop codon) was cloned into the pSK-eGFP vector at a position upstream of the eGFP gene. The constructed GhTIP1;1:eGFP fusion gene was cloned into pBI121 vector at BamH I/Sac I sites, replacing the GUS gene. Cotton hypocotyl explants were transformed with Agrobacterium tumefaciens harboring the GhTIP1;1:eGFP vector, according to the method described previously (Li et al., 2002). After subculturing for 2– 3 months, the stably transformed cells (calli) were selected on MS selective medium. Subsequently, fluorescence microscopy was performed on a SP5 Meta confocal laser microscope (Leica, Germany). Transformed cells were plasmolyzed in 4% NaCl solution for 20 min, and then examined with a filter set for GFP fluorescence (488 nm for excitation and 506–538 nm for emission). SP5 software (Leica, Germany) was employed to record and process the digital images taken.
3.1. GhTIP1;1 expression is induced by cold stress
Fig. 1. RNA Gel–Blot analysis of GhPIP1;1 expression in cold-treated roots and cotyledons of cotton. Upper panel: autoradiograph of RNA hybridization; bottom panel: RNA gel before being transferred to membrane showing equal loading of RNAs. C, control (untreated roots or cotyledons); 4 h, 12 h and 24 h refer to the roots or cotyledons coldtreated (4 °C) for 4 h, 12 h, 24 h, respectively.
To isolate the genes involved in response to cold stress, more than 4000 cDNA clones were randomly selected from a cotton cDNA library for sequencing. Over 30 cDNA clones, including one cDNA (designated as GhTIP1;1, access number in GenBank: FJ384629) encoding tonoplast intrinsic protein (TIP), with potential involvement in cold acclimation were selected for further study. Quantitative RT-PCR was performed to find out which candidates were markedly up-regulated under cold stress. Among the candidate genes, the expression level of GhTIP1;1 was three-fold higher in cotyledons of the cold-treated seedlings than in those of the control plants (data not shown). To further confirm that GhTIP1;1 expression is induced by cold stress, a time course of mRNA accumulation in roots and cotyledons was examined by Northern blot analysis. When 4–5 day-old seedlings were treated at 4 °C cold stress for 4–24 h, the level of GhTIP1;1 mRNA was gradually increased in cotyledons, whereas its expression activity was obviously decreased in roots (Fig. 1).
Fig. 2. Analysis of GhTIP1;1 expression in cotton tissues as well as in root development. Relative values of the GhTIP gene expressions in cotton are shown as percentage of GhUBI1 expression activity (see Materials and methods). Error bars represent standard deviation. (A) GhTIP1;1 expression in cotton different organs or tissues. R, roots; H, hypocotyls; C, cotyledons; L, leaves; P, petals; A, anthers; O, ovules; F, fibers. (B) Expression pattern of GhTIP1;1 during roots development. 3d, 6d, 9d and 14d refer to 3, 6, 9, 14 day-old roots, respectively.
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relatively low in ovules and fibers. In addition, a further analysis concerning the expression patterns of GhTIP1;1 in root development was also performed by quantitative RT-PCR (Fig. 2B). At early stage of root development (3 day-old), high levels of the gene products were detected in roots. As roots further developed, the expression activity of GhTIP1;1 was gradually declined to much low levels in 14 day-old roots. 3.3. GhTIP1;1 structure and phylogenetic analyses
Fig. 3. Phylogenetic relationship of cotton TIPs with Arabidopsis TIP proteins. The minimum evolution tree was constructed in MEGA3.1 from 1000 bootstrap replicates. Accession numbers of the known proteins of Arabidopsis in GenBank are as follows: AtTIP1;1 (P25818), AtTIP1;2 (Q41963), AtTIP1;3 (NP 192056), AtTIP2;1 (Q41951), AtTIP2;2 (NP 193465), AtTIP2;3 (NP 199556), AtTIP3;1 (P26587), AtTIP3;2 (O22588), AtTIP4;1 (O82316), AtTIP5;1 (NP 190328), The accession numbers of the known cotton TIPs are as follows: GhγTIP1 (ABR68795) and GhδTIP (AAB04557).
3.2. Expression pattern of GhTIP1;1 gene in cotton tissues To study the expression pattern of GhTIP1;1 gene in cotton, mRNA levels of GhTIP1;1 in cotton different organs or tissues were quantified by real-time RT-PCR (Fig. 2A). The results showed that the transcripts of GhTIP1;1 were accumulated at relatively high level in roots and hypocotyls, and the moderate activity of GhTIP1;1 was detected in cotyledons, leaves, petals and anthers, whereas its expression level was
The isolated GhTIP1;1 cDNA is 1104 bp in length, including 756 bp of open reading frame (ORF), a short 5′-UTR and 285 bp 3′-UTR. GhTIP1;1 gene encodes 251 amino acids of a TIP. Sequence analysis showed that GhTIP1;1 protein contains the MIP family signature sequence HVNPAVTFG, and shares 90% identity with bobTIP26-1 (AAB51393) from Brassica oleracea var. botrytis and 88% identity with AtTIP1;1 (P25818) from A. thaliana, indicating that GhTIP1;1 belongs to TIP subfamily. To investigate the evolutionary relationship of GhTIP1;1, all 10 Arabidopsis TIP proteins and 2 cotton TIPs were selected from GenBank for phylogenetic analysis. As shown in the phylogenetic tree (Fig. 3), 13 tonoplast aquaporins split into two groups which can be further divided into four subgroups. The two members of TIP3 (AtTIP3;1 and AtTIP3;2) subgroup inhabit a single branch, while AtTIP4;1 locates at the clade based to TIP2 subgroup. GhTIP1;1 clusters with the other members of TIP1 subgroup except AtTIP1;2, which forms another single branch together with AtTIP5;1. In addition to GhTIP1;1, two other TIP proteins, GhγTIP1 (AAB04557) and GhδTIP (ABR68795) were also identified from cotton (G. hirsutum) (Ferguson et al., 1997; Liu et al., 2008). GhTIP1;1 shares 78% identity with GhγTIP1 that is a member of TIP1 subgroup, and is relatively low similarity with GhδTIP (67% identity) which belongs to TIP2 subgroup. A more detailed analysis concerning the amino acid sequences was performed by multiple alignments (Fig. 4). The peptide sequences of bobTIP26-1(AAB51393) and AtTIP1;1 (P25818) which show highest
Fig. 4. Comparison of the predicted amino acid sequence of GhTIP1;1 with the other known TIP proteins. Amino acid sequences are aligned by ClustalW software. The conserved amino acid residues in all proteins are highlighted in grey and six transmembrane-helix (H1 to H6) are shown in box. Overstriking letters refer to the most highly conserved amino acid sequences of MIP. Accession numbers of these known proteins in GenBank are as follows: AtTIP1;1 (P25818), bobTIP26-1 (AAB51393), GhγTIP1 (ABR68795) and GhδTIP (AAB04557).
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Fig. 5. Assay on functional expression of GhTIP1;1 in Xenopus oocytes. (A) Increase in relative volume of oocytes injected with GhTIP1;1 cRNA after transfer to hypoosmotic medium (see Materials and methods). (B) Pf-values of oocytes injected with GhTIP1;1 cRNA, using water as control. ⁎⁎Independent t-tests demonstrated that there was very significant difference between the Pf-values of oocytes injected with water and GhTIP1;1 cRNA (t-test for equality of means, P value b 0.01).
similarity with GhTIP1;1 as well as the other two cotton TIP proteins were selected out from GenBank for analysis. The results of multiple alignments indicated that the structure and the amino acid sequences of TIP proteins are highly conserved. They all contain six transmembrane helixes (H1 to H6) and five loops. 52.8% residues are identical in the five proteins, and 71.3% residues are identical in all selected TIP1 proteins. Both N-terminus and C-terminus of the TIPs are very short and less conserved (Fig. 4). 3.4. Functional analysis of GhTIP1;1 in X. laevis oocytes To test the protein activity, capped sense cRNA of GhTIP1;1 gene was injected into X. laevis oocytes. After incubation for 2 days for cRNA translation and the target proteins to cell membrane, the oocytes were transferred to a hypoosmotic solution, and the increase in area was measured in time and used to calculate the increase in volume. This change in volume was used to calculate relative water permeability (Pf) of the oocytes. The experimental results are presented in Fig. 5. The oocytes expressed GhTIP1;1 protein showed a Pf of 29.02 ± 6.81 µm/s (n = 30) which was about three-fold higher than that of the controls injected with water (Pf = 9.47 ± 5.30 µm/s, n = 30). The above data demonstrated that GhTIP1;1 protein possess water transport capacity. 3.5. Subcellular localization of GhTIP1;1 in cotton cells To investigate the subcelluar localization of GhTIP1;1, green fluorescent protein (GFP) fused to C-terminal of the GhTIP1;1 (see Materials and methods) was expressed in cotton calli under the control of a cauliflower mosaic virus 35S promoter. After incubated in 4% NaCl for 20 min, the transformed cells with GhTIP1;1:eGFP expression were plasmolyzed completely. GhTIP1;1 fused to eGFP confined the fluorescence obviously to the periphery of the central
vacuole, but the other parts of the transformed cells showed weak or no fluorescence (Fig. 6). These data indicated that GhTIP1;1 protein is most likely localized in the tonoplast membrane of cotton cells. 3.6. Overexpression of GhTIP1;1 enhances yeast cell tolerance to cold stress To investigate whether GhTIP1;1 protein plays an important role in cold acclimation, GhTIP1;1 was ectopically expressed in fission yeast. We constructed GhTIP1;1 into pREP5N vector, placing the gene under the control of an nmt-1 promoter which is activated in the absence of exogenous thiamine. Ten transformed yeast cell lines with pREP5NGhTIP1;1 vector and the control lines containing empty vector (pREP5N) were randomly selected for figuring out cell survival probability under cold stress (at −20 °C for 48 h), calculated as percentage of the same cell lines incubated at 4 °C. After frozen for 48 h, there were obviously more cells remaining alive in the transformed cell lines with GhTIP1;1 expression in induction medium than those of the same lines suppressed GhTIP1;1 expression in noninduction medium (Fig. 7C and D). By contrast, there were few alive cells in the control cells harboring the empty pREP5N vector either grown in induction medium or grown in non-induction medium (Figs. 7A and B). Statistical analysis revealed that the survival probability of the transformed cells with GhTIP1;1 expression was 15.18 ± 6.05%, whereas the CFU of control cells was around 9 ± 4.12% (Fig. 7E). The results indicated that overexpression of GhTIP1;1 gene enhanced freezing-tolerance of the host cells. 4. Discussion In present study, a cold-induced gene GhTIP1;1 was isolated from cotton. Sequence analysis showed that the GhTIP1;1 protein contains
Fig. 6. Subcellular localization of GhTIP1;1 protein in transformed cotton cells. eGFP fused to the C-terminal of GhTIP1;1 was expressed in transformed cotton cells. The cells were plasmolyzed in 4% NaCl solution for 20 min. GFP fluorescence signals were mainly detected in tonoplast membranes of cotton cells harboring the GhTIP1;1:eGFP reporter construct. GFP-derived fluorescence (left), transmission image (middle), and fluorescence superimposed over the transmission image (right) are shown.
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Fig. 7. Overexpression of GhTIP1;1 enhances yeast cell tolerance to cold stress. (A–D) Yeast cells harboring empty plasmid (pREP5N, A and B) or constructed vector (pREP5N-GhTIP1;1, C and D) were cultured in non-induction medium (A and C) or induction medium (B and D) to exponential phase (OD600 = 0.4 to 0.6). Then the cells were frozen at − 20 °C for 48 h and thawed at 4 °C before dyed with 0.1% methylene blue for 5 min. The cells dyed blue were dead. Scale bars, 10 μm. (E) Survival probability of frozen cells compared to non-frozen cells (stored at 4 °C) is expressed as % CFU. Experiments were repeated three times, and mean values and standard deviations are depicted. ⁎Independent t-tests demonstrated that there was significant difference in survival rate between the yeast cells harboring GhTIP1;1 and control (t-test for equality of means, P value b 0.05).
the MIP family signature sequence HVNPAVTFG and two “NPA” motifs (Fig. 4). In addition, it also constitutes six transmembrane helixes and five loops, liking the known TIP members. Comparing with PIP proteins, TIP protein usually has a shorter N-terminus (Schäffner, 1998; Liu et al., 2008). The N-terminus of GhTIP1;1 is also very short and contains the conserved sequence MX2–5(R/.)X4GX3(D/E)X6–7(R/K) which unambiguously identified GhTIP1;1 as a member of TIP subfamily. Phylogenetic analysis (Fig. 3) showed that GhTIP1;1 has a more close evolutional relationship with TIP1s (74–88% identity) than other TIP subgroups (38–64% identity). It shares a low homology (32–34% identity) with cotton PIP proteins. These data strongly indicate that the isolated GhTIP1;1 gene belongs to TIP1 subgroup. Overexpression of TIPs in Xenopus oocytes revealed that plant TIP1 homologs can function as water channels. Arabidopsis TIP1;1 (previously named as Atγ-TIP or Atγ-TIP1) is the first TIP protein identified with high water channel activity which increased 6- to 8-fold osmotic permeability of the oocytes (Maurel et al.,1993). OsTIP1;2, a TIP1 member from rice, also possesses water transport activity (Li et al., 2008). Similarly, cotton GhTIP1;1 protein displayed the functional water channel which increased 3- to 4-fold osmotic permeability of the X. laevis oocytes (Fig. 5), demonstrating that it is also a functional TIP protein in cotton. TIPs are integral membrane proteins in the tonoplast, but subcellular localization of TIP isoforms in plant exhibits their high complexity. Previous study showed that distinct types of vacuole can
coexist in the same cell and are equipped with specific combinations of TIP isoforms. TIP1 homologues are preferentially associated with the large lytic vacuoles, while TIP2 members usually localize in the vacuoles accumulating vegetative storage proteins (Jauh et al., 1999). However, in the isolated Arabidopsis leaf protoplasts, which seem to exist as a separate entity, YFP (yellow fluorescent protein) tagged all AtTIPs (α-, γ- and δ-TIPs) to the tonoplast of the large central vacuole (Hunter et al., 2007). In this study, GhTIP1;1 fused to GFP also confined fluorescence obviously to the tonoplast of the large central vacuole in cotton callus cells (Fig. 6). As the cells of cotton callus are the dedifferentiated cells which exist as single entities, like the above Arabidopsis leaf protoplasts, the subcellular localization of endogenous GhTIP1;1 protein in developing tissues may be more complex than that in the transformed calli (cells) of cotton. GhTIP1;1 and GhγTIP1, two members of cotton TIP1 gene subfamily, share high sequence similarity both at nucleotide and amino acid levels. But they exhibit completely different expression patterns in cotton. GhγTIP1 was preferentially expressed from 5 to 15 DPA fiber (Liu et al., 2008), whereas GhTIP1;1 mainly accumulated in roots and hypocotyls (Fig. 2). Furthermore, the GhTIP1;1 expression was regulated during root development. It showed high expression level in young roots and gradually declined to much low levels in mature roots. Their different expression patterns suggest that the two genes might be involved in different physiological processes. GhγTIP1 was
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thought to play a role in fiber cell expansion, whereas GhTIP1;1 may be involved in development of roots and hypocotyls of cotton. Response or adaptation to cold in plants is a complex process that involves the additive properties of many gene products (Thomashow, 2001). In Arabidopsis, only AtPIP2;5 was up-regulated by cold treatment, but the other PIP genes were all depressed by cold stress (Jang et al., 2004). Two RcPIP2 genes were also down-regulated by freeze stress (Peng et al., 2008), whereas in maize roots, the abundance of both PIP1 and PIP2 proteins increased (Aroca et al., 2005). In present study, GhTIP1;1 expression was regulated in response to cold stress. Intriguingly, GhTIP1;1 was up-regulated in cotyledons, but downregulated in roots by cold stress within a few hours (Fig. 1). These data may suggest that there are two different modulation mechanisms to regulate gene expression in cotton roots and cotyledons. Plants appear to express a surprisingly high number of aquaporin homologues. There are 35 members of aquaporin family in Arabidopsis (Johanson et al., 2001). Cotton is an allotetraploid which may have a large number of aquaporin isovariants. So many homologous proteins make it difficult to investigate the role of a single aquaporin protein in plants. Thus, we chose a relative simple system, yeast cell, to check whether there is a correlation between GhTIP1;1 expression and cold resistance. The results showed that overexpression of GhTIP1;1 in fission yeast (S. pombe) significantly increased the survival probability of yeast cells under freezing stress (Fig. 7). Likewise, previous studies also suggested that there is a correlation between AQP expression and freezing-tolerance of the yeast (Tanghe et al., 2002, 2005). A rapid and osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation which often results in cell damage (Tanghe et al., 2002). Although the mechanism of plants response to cold stress may be different from that of yeast cells response to freeze stress, our data in yeast cells suggest that GhTIP1;1 may function in cotton responding to cold stress. An increase in activity or abundance of aquaporins can protect plants from cold damage to a certain extent (Aroca et al., 2005). Thus, the facts that GhTIP1;1 was up-regulated significantly by cold stress in cotton seedlings and its expression in yeast enhanced the freeze-resistance of the cells throw some lights into understanding how plant cope with cold stress. Acknowledgements This work was supported by National Natural Science Foundation of China (Grant no. 30871317) and National Program for Basic Research (973) of China (Grant no. 2004CB117304).
References Aroca, R., Amodeo, G., Fernández-Illescas, S., Herman, E.M., Chaumont, F., Chrispeels, M.J., 2005. The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots. Plant Physiol. 137, 341–353. Bray, E.A., Bailey-Serres, J., Weretilnyk, E., 2000. Responses to abiotic stresses. In: Gruissem, W., Buchannan, B., Jones, R. (Eds.), Responses to Abiotic Stresses. American Society of Plant Physiologists, Rockville, MD, pp. 1158–1249. Chaumont, F., Barrieu, F., Wojcik, E., Chrispeels, M.J., Jung, R., 2001. Aquaporins constitute a large and highly divergent protein family in maize. Plant Physiol. 125, 1206–1215.
Chrispeels, M.J., Crawford, N.M., Schroeder, J.I., 1999. Proteins for transport of water and mineral nutrients across the membranes of plant cells. Plant Cell 11, 661–675. Ferguson, D.L., Turley, R.B., Kloth, R.H., 1997. Identification of a δ-TIP cDNA clone and determination of related A and D genome subfamilies in Gossypium species. Plant Mol. Biol. 34, 111–118. Guo, Z.W., Li, X.L., Gao, D.S., Duan, C.G., 2004. Advance in the mechanism of biochemistry and molecular biology in response to cold stress of plant. Chinese J. Eco-Agriculture 12, 54–57 (in Chinese). Hachez, C., Moshelion, M., Zelazny, E., Cavez, D., Chaumont, F., 2006. Localization and quantification of plasma membrane aquaporin expression in maize primary root: a clue to understanding their role as cellular plumbers. Plant Mol. Biol. 62, 305–323. Hunter, P.R., Craddock, C.P., Di Benedetto, S., Roberts, L.M., Frigerio, L., 2007. Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells. Plant Physiol. 145, 1371–1382. Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O., Thomashow, M.F., 1998. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science 280, 104–106. Jang, J.Y., Kim, D.G., Kim, Y.O., Kim, J.S., Kang, H., 2004. An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Mol. Biol. 54, 713–725. Jauh, G.Y., Philips, T.E., Rogers, J.C., 1999. Tonoplast intrinsic protein isoforms as markers for vacuolar functions. Plant Cell 11, 1867–1882. Johanson, U., et al., 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 Physiol. 126, 1358–1369. Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., Shinozaki, K., 1999. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stressinducible transcription factor. Nat. Biotechnol. 17, 287–291. Li, X.B., Cai, L., Cheng, N.H., Liu, J.W., 2002. Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber. Plant Physiol. 130, 666–674. Li, X.B., Fan, X.P., Wang, X.L., Cai, L., Yang, W.C., 2005. The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell 17, 859–875. Li, G.W., et al., 2008. Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice. J. Plant Physiol. 165, 1879–1888. Liu, D., Tu, L., Wang, L., Li, Y., Zhu, L., Zhang, X., 2008. Characterization and expression of plasma and tonoplast membrane aquaporins in elongating cotton fibers. Plant Cell Rep. 27, 1385–1394. Maurel, C., 1997. Aquaporins and water permeability of plant membranes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 399–429. Maurel, C., Reizer, J., Schroeder, J.I., Chrispeels, M.J., 1993. The vacuolar membrane protein γ-TIP creates water specific channels in Xenopus oocytes. EMBO J. 12, 2241–2247. Park, J.H., Saier Jr., M.H., 1996. Phylogenetic characterization of the MIP family of transmembrane channel proteins. J. Membr. Biol. 153, 171–180. Peng, Y., Arora, R., Li, G., Wang, X., Fessehaie, A., 2008. Rhododendron catawbiense plasma membrane intrinsic proteins are aquaporins, and their over-expression compromises constitutive freezing tolerance and cold acclimation ability of transgenic Arabidopsis plants. Plant Cell Environ. 31, 1275–1289. Pino, M.T., et al., 2008. Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-associated physiological modifications in potato. Plant Cell Environ. 31, 393–406. Schäffner, A.R., 1998. Aquaporin function, structure, and expression: are there more surprises to surface in water relations? Planta 204, 131–139. Tanghe, A., Van Dijck, P., Dumortier, F., Teunissen, A., Hohmann, S., Thevelein, J.M., 2002. Aquaporin expression correlates with freeze tolerance in Baker's yeast, and overexpression improves freeze tolerance in industrial strains. Appl. Environ. Microbiol. 68, 5981–5989. Tanghe, A., Carbrey, J.M., Agre, P., Thevelein, J.M., Van Dijck, P., 2005. Aquaporin expression and freeze tolerance in Candida albicans. Appl. Environ. Microbiol. 71, 6434–6437. Thomashow, M.F., 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant physiol. Plant Mol. Biol. 50, 571–599. Thomashow, M.F., 2001. So what's new in the field of plant cold acclimation? Lots! Plant Physiol. 125, 89–93. Tyerman, S.D., Bohnert, H.J., Maurel, C., Steudle, E., Smith, J.A.C., 1999. Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. J. Exp. Bot. 50, 1055–1071. Tyerman, S.D., Niemietz, C.M., Bramley, H., 2002. Plant aquaporins: multifunctional water and solute channels with expanding roles. Plant Cell Environ. 25, 173–194. Zhang, R.B., Verkman, A.S., 1991. Water and urea permeability properties of Xenopus oocytes: expression of mRNA from toad urinary bladder. Am. J. Physiol. 260, C26–C34.
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Gene j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e n e
A cotton gene encodes a tonoplast aquaporin that is involved in cell tolerance to cold stress Deng-Di Li 1, Fu-Ju Tai 1,2, Ze-Ting Zhang, Yang Li, Yong Zheng, Yan-Feng Wu, Xue-Bao Li ⁎ Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, HuaZhong Normal University, Wuhan 430079, China
a r t i c l e
i n f o
Article history: Received 20 October 2008 Received in revised form 8 February 2009 Accepted 12 February 2009 Available online 17 March 2009 Received by G. Theissen Keywords: Cotton (Gossypium hirsutum) Gene expression TIP Cold stress Cell tolerance
a b s t r a c t To enhance the survival probability in cold stress, plant cells often increase their cold- and freezing-tolerance in response to low, nonfreezing temperatures by expressing some cold-related genes. In present study, a cotton gene encoding tonoplast intrinsic protein (TIP) was isolated from a cotton seedling cDNA library, and designated as GhTIP1;1. GFP fluorescent microscopy indicated that GhTIP1;1 protein was localized to the vacuolar membrane. Assay on GhTIP1;1 expression in Xenopus laevis oocytes demonstrated that GhTIP1;1 protein displayed water channel activity and facilitated water transport to the cells. At normal conditions, GhTIP1;1 transcripts were predominantly accumulated in roots and hypocotyls, but less abundance in other tissues of cotton. The GhTIP1;1 expression was dramatically up-regulated in cotyledons, but down-regulated in roots within a few hours after cotton seedlings were cold-treated. Overexpression of GhTIP1;1 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival probability, suggesting that the GhTIP1;1 protein is involved in cell freezing-tolerance.
1. Introduction Temperature is one of the important factors which affect plant growth and development. Cold stress together with other abiotic stresses such as drought and soils with changing salt and nutrient concentrations represent the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Bray et al., 2000). Many crop species are sensitive to low temperatures and grow poorly after exposure to a cold period. It is generally considered that low-temperature stress causes reduction in photosynthesis, inhibition in energy generation and material synthesis, but increase in respiration and consumption of plants. Such damages may result in plant death (Guo et al., 2004). Nevertheless, many plants have the ability to sense low temperature and respond by activating mechanisms that lead to an increase in
Abbreviations: cDNA, DNA complementary to mRNA; cRNA, complementary RNA; GhTIP, cotton gene for translation tonoplast intrinsic protein; AtTIP, tonoplast intrinsic protein of Arabidopsis; AQY, yeast gene for translation aquaporin; CBF, C-box binding factor; DREB1, dehydration-responsive element binding-1; CFU, cloning-forming units; ABA, abscisic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; UTR, untranslated region; ORF, open reading frame; bp, base pair; kDa, kilodalton; dCTP, deoxycytidine triphosphate; eGFP, enhanced green fluorescent protein. ⁎ Corresponding author. Tel./Fax: +86 27 67862443. E-mail address: [email protected] (X.B. Li). 1 These authors contributed equally to this work. 2 Present address: Department of Plant Science, Henan Agricultural University, Zhengzhou 450002, China. 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.02.023
© 2009 Elsevier B.V. All rights reserved.
freezing-tolerance, an adaptive response known as cold acclimation (Thomashow, 1999). Freezing-tolerance is a multigenic trait. Previous studies revealed that a lot of genes (such as the genes encoding CBF and DREB1 transcription factors) are involved in cold acclimation (Thomashow, 2001). Overexpression of CBF or DREB1 resulted in increase in plant tolerance to cold stress (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Pino et al., 2008). Except for transcription factors, many downstream proteins, such as aquaporins (AQPs), also play important roles in response to cold stress (Tanghe et al., 2002; Aroca et al., 2005). AQPs which belong to a large superfamily known as MIPs (major intrinsic proteins) are a family of small (23–34 kDa) integral membrane proteins that transport water and small neutral solutes across the cellular membrane (Park and Saier, 1996; Maurel, 1997; Tyerman et al., 1999; Tyerman et al., 2002). So far, genome projects have revealed that Arabidopsis thaliana contains 35 genes encoding aquaporins (Chrispeels et al., 1999; Johanson et al., 2001), and maize has at least 33 AQP genes (Chaumont et al., 2001). In plants, AQPs are divided into four clades: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs) (Chaumont et al., 2001; Johanson et al., 2001). All of four kinds of plant AQPs contain the highly conserved structures: six membrane-spanning alpha helices (H1 to H6), linked by five short loops (loop A to loop E) with N and C termini always facing the cytosol. Two highly conserved asparagineproline-alanine (NPA) motifs locate in loop B and E which form two short helices and dip into the membrane from opposite sides, playing
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a key role in formation of water-selective channel (Chaumont et al., 2001). Plant AQPs play important roles in seed germination, cell elongation, stoma movement, and in response to stresses. Expression patterns of all 13 Arabidopsis PIP genes under several abiotic stresses (such as drought, cold, high salinity and ABA) were investigated, indicating that all these genes were up- or downregulated under different environmental stimuli (Jang et al., 2004). In Baker's yeast and Candida albicans, there is a clear correlation between AQP expression and freezing-tolerance. Overexpression of AQY1 and AQY2 in Baker's yeast or overexpression AQY1 in C. albicans improved freezing-tolerance of the cells. Deletion of the respective genes, the cells became more sensitive to freezing (Tanghe et al., 2002; Tanghe et al., 2005). Aroca et al. (2005) also found that AQP is necessary to respond to chilling injury. In the chilling-tolerant genotype of maize, the recovery of root hydraulic conductance (Lo) is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of AQPs. Cotton, including Upland cotton (Gossypium hirsutum) and Sea Island cotton (Gossypium barbadense), is the main source of natural fibers used in textile industry worldwide. In most districts of China, temperature in spring is still low and unstable. Cotton seedlings often encountered low temperature which caused growth retardation, even death, in spring season. Therefore, it is both biological and agricultural importance to understand the molecular mechanism of cotton coldtolerance. Although some progress has been made in understanding the phenomenon of plant cold acclimation in recent years, the roles of AQP genes in cold-tolerance of plants (especially cotton) still remain largely unknown so far. In present study, a cold-induced gene GhTIP1;1 (access number in GenBank: FJ384629) was isolated from cotton. The GhTIP1;1 protein was localized in the vacuolar membrane and displayed high water transport activity in cells. The experimental results also revealed that GhTIP1;1 gene was preferentially expressed in roots and hypocotyls, and overexpression of GhTIP1;1 gene in yeast enhanced the freezing-tolerance of the transformed cells. 2. Materials and methods 2.1. Plant materials Cotton (G. hirsutum cvs. Xuzhou 142, Coker 312, Emian No. 9 and No. 10) seeds were surface-sterilized with 70% (v/v) ethanol for 1 min and 10% (v/v) H2O2 for 2 h, followed by washing with sterile water. The sterilized seeds were germinated on half-strength Murashige and Skoog (1/2MS) medium under a 16 h-light/8 h-dark cycle at 28 °C for 5–6 days. Roots, cotyledons and hypocotyls were collected from sterile seedlings for RNA isolation. Other tissues, such as leaves, stems, petals, anthers, ovules and fibers, were derived from cotton plants grown in a greenhouse. All tissues were collected 2–3 h after light onset since some plant aquaporins display a diurnal expression pattern, with a peak of expression at 2–4 h after the beginning of the light period (Hachez et al., 2006). For cold treatment, 4–5 day-old seedlings grown at normal conditions were transferred to 4 °C with a 24 h photoperiod. The parallel control plants were placed at 28 °C with a 24 h photoperiod. The samples from stress-treated and control plants were collected and immediately frozen in liquid nitrogen and stored at −70 °C. 2.2. Construction of cotton seedling cDNA library and isolation of GhTIP1;1 cDNA Total RNA was extracted from 5–6 day-old cotton seedlings using the method as described previously (Li et al., 2002). Poly(A)+ mRNA was prepared from a pool of seedling total RNA by using an mRNA
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purification kit (Qiagen). Complementary DNA was synthesized and cloned into the EcoR I–Xho I sites of the ZAP express vector and packaged by using a ZAP-cDNA Gigapack Gold III cloning kit (Stratagene) according to the manufacturer's instruction. More than 4000 cDNA clones were randomly selected from the cDNA library for sequencing. Over 30 cDNA clones (including GhTIP1;1) with potential involvement in cold acclimation were selected by analyzing the sequence data. For each candidate, its expression level was detected in cotton tissues grown under normal condition and cold stress by Northern blot and RT-PCR analyses. 2.3. Quantitative RT-PCR analysis Real-time quantitative reverse transcriptase (RT)-PCR was performed using a method described earlier (Li et al., 2005). The cotton polyubiquitin gene (GhUBI1, access number in GenBank: EU604080) was used as a standard control in the RT-PCR reactions. The primer sequences were as follows: GhTIP1;1: up-chain primer, 5′NTACACAGTGTACGCCACAGCCGTTb3′, down-chain primer, 5′N TCACATTTACAGCCCTGATGCTGGTb3′; GhUBI1: up-chain primer, 5′NCTGAATCTTCGCTTTCACGTTATCb3′, down-chain primer, 5′N GGGATGCAAATCTTCGTGAAAACb3′. The data of real-time RT-PCR are mean values and standard errors of three independent experiments with three biological replicates. 2.4. DNA and protein sequence analysis Nucleotide and amino acid sequences were analyzed using DNAstar (DNAstar Inc). The peptide sequences were aligned with the ClustalW program (http://www.ebi.ac.uk), and phylogenetic analysis was employed to investigate the evolutionary relationships among the aquaporins. A minimum evolution tree was generated in MEG3.1. A bootstrap analysis with 1000 replicates was performed to assess the statistical reliability of the tree topology. 2.5. RNA Gel–Blot analysis 3′-untranslated region (3′-UTR) fragment (285 bp, + 1 to +285 downstream stop codon) of GhTIP1;1 cDNA was prepared as a probe by PCR amplification. RNA samples (20 μg per lane) from cotyledons and roots of cold-treated cotton seedlings were separated on 1.2% (w/v) agarose-formaldehyde gels (55 V) for 4–5 h and transferred onto Hybond-N+ nylon membranes by capillary blotting. Genespecific probe was labeled with α-32P-dCTP using the Random Primer DNA Labeling Kit (Ver.2, TAKARA, Dalian, China). RNA Northern-blot hybridization was performed as described previously (Li et al., 2002). The membrane was exposed to X-film (Eastman Kodak, Rochester, NY) with two intensifying screens at −70 °C for 1–3 d. 2.6. In vitro complementary RNA synthesis The cDNA of GhTIP1;1 was subcloned into pGEM-7Z vector using the restriction sites EcoR I and Xba I. Capped cRNA transcripts were synthesized in vitro with mMACHINE SP6 Kit (Ambion) with Xba I linearized vector. 2.7. Oocyte preparation, cRNA injection, and osmotic water permeability assay Xenopus laevis oocytes of stages V and VI were isolated and defolliculated by digestion at room temperature for 1 h with 2 mg/ml collagenase A (Sigma) in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 5 mM Hepes-NaOH, pH 7.4, 220 mosm/kg). 50 nl volume of in vitro transcripts (50 ng) of the target gene, using the same volume of distilled water as negative
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2.9. Overexpression of GhTIP1;1 in fission yeast
control, was injected into the oocytes, and then the oocytes were incubated at 19 °C for 48 h in ND96 solution supplemented with 10 μg/ml penicillin and 10 μg/ml streptomycin. To measure the osmotic water permeability coefficient (Pf), a single oocyte was transferred to 5-fold diluted ND96 solution. Change in the oocyte volume was monitored at room temperature with a microscope video system by taking digital images at 30 s intervals. Oocyte volume (V) was calculated from the measured area of each oocyte. The osmotic permeability coefficient (Pf) was calculated for the first 5 min using the formula Pf = V0[d(V0 / V) / dt] / [S0 × VW(Osmin − Osmout)], with an initial volume (V0) of 9 × 10− 4 cm3, an initial oocyte surface area (S0) of 0.045 cm2, and a molar volume of water (VW) of 18 cm3/mol (Zhang and Verkman, 1991).
The coding sequences of GhTIP1;1 gene was cloned into yeast vector pREP5N with Sal I/Not I sites. Afterwards, the constructed vector and pREP5N empty vector were transferred into yeast (Schizosaccharomyces pombe) cells by electroporation (Bio-Rad MicroPulser Electroporation Apparatus) according to the manufacturer's instructions. Transformants were then selected on minimal medium (MM) supplemented with 75 mg/l adenine and uracil at 29 °C. Ten colonies of each transformant were randomly picked out to grow in liquid MM with 2 μM thiamine, which represses the nmt-1 promoter activity, until mid-log phase in a shaker (220 rev min− 1, 29 °C). Subsequently, the yeast cells were harvested and washed three times with MM containing no thiamine to de-repress the promoter, and then incubated in the same thiamine-free medium for 20 h (220 rev min− 1, 29 °C). 10 μl of the cultured cells was transferred to 10 ml fresh thiamine-free MM medium and grew to exponential phase (OD600 = 0.4 to 0.6). Afterwards, 2 ml of the cultured cells was divided into 10 equal portions. Five aliquots (200 μl each) were kept at 4 °C, and additional five aliquots were frozen at − 20 °C. After 48 h, the five thawed test samples were diluted at 4 °C and stained with 0.1% methylene blue, which stained the dead cells into blue colour, for 5 min. The control samples (nonfrozen cells) were also stained with methylene blue. Cell survival probability was determined as the percentage of CFU of frozen samples relative to the control samples.
2.8. Subcellular localization of GhTIP1;1 protein
3. Results
To construct GhTIP1;1:eGFP vector, the ORF sequence of eGFP gene was cloned into pBluescript II SK+ vector to obtain an intermediate construct pSK-eGFP. Subsequently, the GhTIP1;1 ORF (without the stop codon) was cloned into the pSK-eGFP vector at a position upstream of the eGFP gene. The constructed GhTIP1;1:eGFP fusion gene was cloned into pBI121 vector at BamH I/Sac I sites, replacing the GUS gene. Cotton hypocotyl explants were transformed with Agrobacterium tumefaciens harboring the GhTIP1;1:eGFP vector, according to the method described previously (Li et al., 2002). After subculturing for 2– 3 months, the stably transformed cells (calli) were selected on MS selective medium. Subsequently, fluorescence microscopy was performed on a SP5 Meta confocal laser microscope (Leica, Germany). Transformed cells were plasmolyzed in 4% NaCl solution for 20 min, and then examined with a filter set for GFP fluorescence (488 nm for excitation and 506–538 nm for emission). SP5 software (Leica, Germany) was employed to record and process the digital images taken.
3.1. GhTIP1;1 expression is induced by cold stress
Fig. 1. RNA Gel–Blot analysis of GhPIP1;1 expression in cold-treated roots and cotyledons of cotton. Upper panel: autoradiograph of RNA hybridization; bottom panel: RNA gel before being transferred to membrane showing equal loading of RNAs. C, control (untreated roots or cotyledons); 4 h, 12 h and 24 h refer to the roots or cotyledons coldtreated (4 °C) for 4 h, 12 h, 24 h, respectively.
To isolate the genes involved in response to cold stress, more than 4000 cDNA clones were randomly selected from a cotton cDNA library for sequencing. Over 30 cDNA clones, including one cDNA (designated as GhTIP1;1, access number in GenBank: FJ384629) encoding tonoplast intrinsic protein (TIP), with potential involvement in cold acclimation were selected for further study. Quantitative RT-PCR was performed to find out which candidates were markedly up-regulated under cold stress. Among the candidate genes, the expression level of GhTIP1;1 was three-fold higher in cotyledons of the cold-treated seedlings than in those of the control plants (data not shown). To further confirm that GhTIP1;1 expression is induced by cold stress, a time course of mRNA accumulation in roots and cotyledons was examined by Northern blot analysis. When 4–5 day-old seedlings were treated at 4 °C cold stress for 4–24 h, the level of GhTIP1;1 mRNA was gradually increased in cotyledons, whereas its expression activity was obviously decreased in roots (Fig. 1).
Fig. 2. Analysis of GhTIP1;1 expression in cotton tissues as well as in root development. Relative values of the GhTIP gene expressions in cotton are shown as percentage of GhUBI1 expression activity (see Materials and methods). Error bars represent standard deviation. (A) GhTIP1;1 expression in cotton different organs or tissues. R, roots; H, hypocotyls; C, cotyledons; L, leaves; P, petals; A, anthers; O, ovules; F, fibers. (B) Expression pattern of GhTIP1;1 during roots development. 3d, 6d, 9d and 14d refer to 3, 6, 9, 14 day-old roots, respectively.
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relatively low in ovules and fibers. In addition, a further analysis concerning the expression patterns of GhTIP1;1 in root development was also performed by quantitative RT-PCR (Fig. 2B). At early stage of root development (3 day-old), high levels of the gene products were detected in roots. As roots further developed, the expression activity of GhTIP1;1 was gradually declined to much low levels in 14 day-old roots. 3.3. GhTIP1;1 structure and phylogenetic analyses
Fig. 3. Phylogenetic relationship of cotton TIPs with Arabidopsis TIP proteins. The minimum evolution tree was constructed in MEGA3.1 from 1000 bootstrap replicates. Accession numbers of the known proteins of Arabidopsis in GenBank are as follows: AtTIP1;1 (P25818), AtTIP1;2 (Q41963), AtTIP1;3 (NP 192056), AtTIP2;1 (Q41951), AtTIP2;2 (NP 193465), AtTIP2;3 (NP 199556), AtTIP3;1 (P26587), AtTIP3;2 (O22588), AtTIP4;1 (O82316), AtTIP5;1 (NP 190328), The accession numbers of the known cotton TIPs are as follows: GhγTIP1 (ABR68795) and GhδTIP (AAB04557).
3.2. Expression pattern of GhTIP1;1 gene in cotton tissues To study the expression pattern of GhTIP1;1 gene in cotton, mRNA levels of GhTIP1;1 in cotton different organs or tissues were quantified by real-time RT-PCR (Fig. 2A). The results showed that the transcripts of GhTIP1;1 were accumulated at relatively high level in roots and hypocotyls, and the moderate activity of GhTIP1;1 was detected in cotyledons, leaves, petals and anthers, whereas its expression level was
The isolated GhTIP1;1 cDNA is 1104 bp in length, including 756 bp of open reading frame (ORF), a short 5′-UTR and 285 bp 3′-UTR. GhTIP1;1 gene encodes 251 amino acids of a TIP. Sequence analysis showed that GhTIP1;1 protein contains the MIP family signature sequence HVNPAVTFG, and shares 90% identity with bobTIP26-1 (AAB51393) from Brassica oleracea var. botrytis and 88% identity with AtTIP1;1 (P25818) from A. thaliana, indicating that GhTIP1;1 belongs to TIP subfamily. To investigate the evolutionary relationship of GhTIP1;1, all 10 Arabidopsis TIP proteins and 2 cotton TIPs were selected from GenBank for phylogenetic analysis. As shown in the phylogenetic tree (Fig. 3), 13 tonoplast aquaporins split into two groups which can be further divided into four subgroups. The two members of TIP3 (AtTIP3;1 and AtTIP3;2) subgroup inhabit a single branch, while AtTIP4;1 locates at the clade based to TIP2 subgroup. GhTIP1;1 clusters with the other members of TIP1 subgroup except AtTIP1;2, which forms another single branch together with AtTIP5;1. In addition to GhTIP1;1, two other TIP proteins, GhγTIP1 (AAB04557) and GhδTIP (ABR68795) were also identified from cotton (G. hirsutum) (Ferguson et al., 1997; Liu et al., 2008). GhTIP1;1 shares 78% identity with GhγTIP1 that is a member of TIP1 subgroup, and is relatively low similarity with GhδTIP (67% identity) which belongs to TIP2 subgroup. A more detailed analysis concerning the amino acid sequences was performed by multiple alignments (Fig. 4). The peptide sequences of bobTIP26-1(AAB51393) and AtTIP1;1 (P25818) which show highest
Fig. 4. Comparison of the predicted amino acid sequence of GhTIP1;1 with the other known TIP proteins. Amino acid sequences are aligned by ClustalW software. The conserved amino acid residues in all proteins are highlighted in grey and six transmembrane-helix (H1 to H6) are shown in box. Overstriking letters refer to the most highly conserved amino acid sequences of MIP. Accession numbers of these known proteins in GenBank are as follows: AtTIP1;1 (P25818), bobTIP26-1 (AAB51393), GhγTIP1 (ABR68795) and GhδTIP (AAB04557).
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Fig. 5. Assay on functional expression of GhTIP1;1 in Xenopus oocytes. (A) Increase in relative volume of oocytes injected with GhTIP1;1 cRNA after transfer to hypoosmotic medium (see Materials and methods). (B) Pf-values of oocytes injected with GhTIP1;1 cRNA, using water as control. ⁎⁎Independent t-tests demonstrated that there was very significant difference between the Pf-values of oocytes injected with water and GhTIP1;1 cRNA (t-test for equality of means, P value b 0.01).
similarity with GhTIP1;1 as well as the other two cotton TIP proteins were selected out from GenBank for analysis. The results of multiple alignments indicated that the structure and the amino acid sequences of TIP proteins are highly conserved. They all contain six transmembrane helixes (H1 to H6) and five loops. 52.8% residues are identical in the five proteins, and 71.3% residues are identical in all selected TIP1 proteins. Both N-terminus and C-terminus of the TIPs are very short and less conserved (Fig. 4). 3.4. Functional analysis of GhTIP1;1 in X. laevis oocytes To test the protein activity, capped sense cRNA of GhTIP1;1 gene was injected into X. laevis oocytes. After incubation for 2 days for cRNA translation and the target proteins to cell membrane, the oocytes were transferred to a hypoosmotic solution, and the increase in area was measured in time and used to calculate the increase in volume. This change in volume was used to calculate relative water permeability (Pf) of the oocytes. The experimental results are presented in Fig. 5. The oocytes expressed GhTIP1;1 protein showed a Pf of 29.02 ± 6.81 µm/s (n = 30) which was about three-fold higher than that of the controls injected with water (Pf = 9.47 ± 5.30 µm/s, n = 30). The above data demonstrated that GhTIP1;1 protein possess water transport capacity. 3.5. Subcellular localization of GhTIP1;1 in cotton cells To investigate the subcelluar localization of GhTIP1;1, green fluorescent protein (GFP) fused to C-terminal of the GhTIP1;1 (see Materials and methods) was expressed in cotton calli under the control of a cauliflower mosaic virus 35S promoter. After incubated in 4% NaCl for 20 min, the transformed cells with GhTIP1;1:eGFP expression were plasmolyzed completely. GhTIP1;1 fused to eGFP confined the fluorescence obviously to the periphery of the central
vacuole, but the other parts of the transformed cells showed weak or no fluorescence (Fig. 6). These data indicated that GhTIP1;1 protein is most likely localized in the tonoplast membrane of cotton cells. 3.6. Overexpression of GhTIP1;1 enhances yeast cell tolerance to cold stress To investigate whether GhTIP1;1 protein plays an important role in cold acclimation, GhTIP1;1 was ectopically expressed in fission yeast. We constructed GhTIP1;1 into pREP5N vector, placing the gene under the control of an nmt-1 promoter which is activated in the absence of exogenous thiamine. Ten transformed yeast cell lines with pREP5NGhTIP1;1 vector and the control lines containing empty vector (pREP5N) were randomly selected for figuring out cell survival probability under cold stress (at −20 °C for 48 h), calculated as percentage of the same cell lines incubated at 4 °C. After frozen for 48 h, there were obviously more cells remaining alive in the transformed cell lines with GhTIP1;1 expression in induction medium than those of the same lines suppressed GhTIP1;1 expression in noninduction medium (Fig. 7C and D). By contrast, there were few alive cells in the control cells harboring the empty pREP5N vector either grown in induction medium or grown in non-induction medium (Figs. 7A and B). Statistical analysis revealed that the survival probability of the transformed cells with GhTIP1;1 expression was 15.18 ± 6.05%, whereas the CFU of control cells was around 9 ± 4.12% (Fig. 7E). The results indicated that overexpression of GhTIP1;1 gene enhanced freezing-tolerance of the host cells. 4. Discussion In present study, a cold-induced gene GhTIP1;1 was isolated from cotton. Sequence analysis showed that the GhTIP1;1 protein contains
Fig. 6. Subcellular localization of GhTIP1;1 protein in transformed cotton cells. eGFP fused to the C-terminal of GhTIP1;1 was expressed in transformed cotton cells. The cells were plasmolyzed in 4% NaCl solution for 20 min. GFP fluorescence signals were mainly detected in tonoplast membranes of cotton cells harboring the GhTIP1;1:eGFP reporter construct. GFP-derived fluorescence (left), transmission image (middle), and fluorescence superimposed over the transmission image (right) are shown.
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Fig. 7. Overexpression of GhTIP1;1 enhances yeast cell tolerance to cold stress. (A–D) Yeast cells harboring empty plasmid (pREP5N, A and B) or constructed vector (pREP5N-GhTIP1;1, C and D) were cultured in non-induction medium (A and C) or induction medium (B and D) to exponential phase (OD600 = 0.4 to 0.6). Then the cells were frozen at − 20 °C for 48 h and thawed at 4 °C before dyed with 0.1% methylene blue for 5 min. The cells dyed blue were dead. Scale bars, 10 μm. (E) Survival probability of frozen cells compared to non-frozen cells (stored at 4 °C) is expressed as % CFU. Experiments were repeated three times, and mean values and standard deviations are depicted. ⁎Independent t-tests demonstrated that there was significant difference in survival rate between the yeast cells harboring GhTIP1;1 and control (t-test for equality of means, P value b 0.05).
the MIP family signature sequence HVNPAVTFG and two “NPA” motifs (Fig. 4). In addition, it also constitutes six transmembrane helixes and five loops, liking the known TIP members. Comparing with PIP proteins, TIP protein usually has a shorter N-terminus (Schäffner, 1998; Liu et al., 2008). The N-terminus of GhTIP1;1 is also very short and contains the conserved sequence MX2–5(R/.)X4GX3(D/E)X6–7(R/K) which unambiguously identified GhTIP1;1 as a member of TIP subfamily. Phylogenetic analysis (Fig. 3) showed that GhTIP1;1 has a more close evolutional relationship with TIP1s (74–88% identity) than other TIP subgroups (38–64% identity). It shares a low homology (32–34% identity) with cotton PIP proteins. These data strongly indicate that the isolated GhTIP1;1 gene belongs to TIP1 subgroup. Overexpression of TIPs in Xenopus oocytes revealed that plant TIP1 homologs can function as water channels. Arabidopsis TIP1;1 (previously named as Atγ-TIP or Atγ-TIP1) is the first TIP protein identified with high water channel activity which increased 6- to 8-fold osmotic permeability of the oocytes (Maurel et al.,1993). OsTIP1;2, a TIP1 member from rice, also possesses water transport activity (Li et al., 2008). Similarly, cotton GhTIP1;1 protein displayed the functional water channel which increased 3- to 4-fold osmotic permeability of the X. laevis oocytes (Fig. 5), demonstrating that it is also a functional TIP protein in cotton. TIPs are integral membrane proteins in the tonoplast, but subcellular localization of TIP isoforms in plant exhibits their high complexity. Previous study showed that distinct types of vacuole can
coexist in the same cell and are equipped with specific combinations of TIP isoforms. TIP1 homologues are preferentially associated with the large lytic vacuoles, while TIP2 members usually localize in the vacuoles accumulating vegetative storage proteins (Jauh et al., 1999). However, in the isolated Arabidopsis leaf protoplasts, which seem to exist as a separate entity, YFP (yellow fluorescent protein) tagged all AtTIPs (α-, γ- and δ-TIPs) to the tonoplast of the large central vacuole (Hunter et al., 2007). In this study, GhTIP1;1 fused to GFP also confined fluorescence obviously to the tonoplast of the large central vacuole in cotton callus cells (Fig. 6). As the cells of cotton callus are the dedifferentiated cells which exist as single entities, like the above Arabidopsis leaf protoplasts, the subcellular localization of endogenous GhTIP1;1 protein in developing tissues may be more complex than that in the transformed calli (cells) of cotton. GhTIP1;1 and GhγTIP1, two members of cotton TIP1 gene subfamily, share high sequence similarity both at nucleotide and amino acid levels. But they exhibit completely different expression patterns in cotton. GhγTIP1 was preferentially expressed from 5 to 15 DPA fiber (Liu et al., 2008), whereas GhTIP1;1 mainly accumulated in roots and hypocotyls (Fig. 2). Furthermore, the GhTIP1;1 expression was regulated during root development. It showed high expression level in young roots and gradually declined to much low levels in mature roots. Their different expression patterns suggest that the two genes might be involved in different physiological processes. GhγTIP1 was
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thought to play a role in fiber cell expansion, whereas GhTIP1;1 may be involved in development of roots and hypocotyls of cotton. Response or adaptation to cold in plants is a complex process that involves the additive properties of many gene products (Thomashow, 2001). In Arabidopsis, only AtPIP2;5 was up-regulated by cold treatment, but the other PIP genes were all depressed by cold stress (Jang et al., 2004). Two RcPIP2 genes were also down-regulated by freeze stress (Peng et al., 2008), whereas in maize roots, the abundance of both PIP1 and PIP2 proteins increased (Aroca et al., 2005). In present study, GhTIP1;1 expression was regulated in response to cold stress. Intriguingly, GhTIP1;1 was up-regulated in cotyledons, but downregulated in roots by cold stress within a few hours (Fig. 1). These data may suggest that there are two different modulation mechanisms to regulate gene expression in cotton roots and cotyledons. Plants appear to express a surprisingly high number of aquaporin homologues. There are 35 members of aquaporin family in Arabidopsis (Johanson et al., 2001). Cotton is an allotetraploid which may have a large number of aquaporin isovariants. So many homologous proteins make it difficult to investigate the role of a single aquaporin protein in plants. Thus, we chose a relative simple system, yeast cell, to check whether there is a correlation between GhTIP1;1 expression and cold resistance. The results showed that overexpression of GhTIP1;1 in fission yeast (S. pombe) significantly increased the survival probability of yeast cells under freezing stress (Fig. 7). Likewise, previous studies also suggested that there is a correlation between AQP expression and freezing-tolerance of the yeast (Tanghe et al., 2002, 2005). A rapid and osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation which often results in cell damage (Tanghe et al., 2002). Although the mechanism of plants response to cold stress may be different from that of yeast cells response to freeze stress, our data in yeast cells suggest that GhTIP1;1 may function in cotton responding to cold stress. An increase in activity or abundance of aquaporins can protect plants from cold damage to a certain extent (Aroca et al., 2005). Thus, the facts that GhTIP1;1 was up-regulated significantly by cold stress in cotton seedlings and its expression in yeast enhanced the freeze-resistance of the cells throw some lights into understanding how plant cope with cold stress. Acknowledgements This work was supported by National Natural Science Foundation of China (Grant no. 30871317) and National Program for Basic Research (973) of China (Grant no. 2004CB117304).
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