Trees (2002) 16:94–99 DOI 10.1007/s00468-001-0154-2
O R I G I N A L A RT I C L E
Taro Takemura · Nobutaka Hanagata · Zvy Dubinsky Isao Karube
Molecular characterization and response to salt stress of mRNAs encoding cytosolic Cu/Zn superoxide dismutase and catalase from Bruguiera gymnorrhiza Received: 10 July 2001 / Accepted: 12 November 2001 / Published online: 23 January 2002 © Springer-Verlag 2002
Abstract To analyze the potential of the active oxygenscavenging system of the cytosol in leaves of saltstressed Bruguiera gymnorrhiza, we isolated a fulllength cDNA encoding a 153-amino-acid sequence of cytosolic Cu/Zn superoxide dismutase (SOD) and a partial cDNA encoding catalase. Northern blot analyses showed that the transcript level of cytosolic Cu/Zn-SOD increased after 1 and 5 days NaCl treatment, but no significant change occurred in the expression of the catalase gene. The transcript of cytosolic Cu/Zn-SOD was also induced by mannitol treatment. This suggests that the increase in cytosolic Cu/Zn-SOD 1 day after NaCl treatment is a response to osmotic stress. After 5 days treatment with NaCl, the transcript level of cytosolic Cu/ZnSOD increased in young and mature leaves rather than in old leaves. Expression of the cytosolic Cu/Zn-SOD gene was induced by exogenous abscisic acid, while the catalase gene was induced by application of 2-chloroethylphosphonic acid, which is a generator of ethylene. The results from this study suggest that salt stress leads to the generation of superoxide in the cytosol and that the oxygen-scavenging system in the cytosol contributes to the salt tolerance capacity of B. gymnorrhiza. Keywords Bruguiera gymnorrhiza · Catalase · Cytosolic superoxide dismutase · Salt tolerance
T. Takemura · N. Hanagata (✉) · I. Karube Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan e-mail:
[email protected] Tel.: +81-426-37-4517 Z. Dubinsky Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Present address: N. Hanagata, Katayanagi Advanced Research Lab., Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan
Introduction Most plants suffer from oxidative stress via the formation of active oxygen species when exposed to extreme environmental conditions. Plant cells possess protective mechanisms against oxidative damage. According to Smirnoff (1993), the mechanisms can be divided into two types. One includes superoxide dismutase (SOD), catalase, peroxidases, ascorbate and α-tocopherol, which react with active oxygen. The other includes glutathione (GSH), glutathione reductase (GR), ascorbate and dehydroascorbate reductases, which regenerate oxidized antioxidants. Superoxide (O2–) is generated from electron transport activities diverted from their normal course when the plant is exposed to osmotic stress (Smirnoff 1993). The superoxide is thought to be generated mainly in mitochondria or chloroplasts and causes formation of hydrogen peroxide and the hydroxyl radical (·OH) by successive univalent reduction of oxygen. These reactive species lead to lipid peroxidation and damage to proteins and nucleic acids. Superoxide dismutase catalyzes dismutation of superoxide to hydrogen peroxide and oxygen (Fridovich 1986) and contributes to minimizing hydroxyl radicals formed by the Haber-Weiss or Fenton reactions. Hydrogen peroxide formed by SOD is subsequently decomposed by catalase in the cytosol (Anderson and Beardall 1991) and ascorbate peroxidase in the chloroplast (Asada 1992). Plants generally have three SOD isozymes: Cu/Zn-SOD in the cytosol and chloroplasts, Mn-SOD in mitochondria and Fe-SOD in chloroplasts; however, some plants lack Fe-SOD (Bowler et al. 1994). Recently, it was suggested that salt stress gives rise to oxidative stress in plant cells and increased activities of antioxidant enzymes (Gossett et al. 1994; Hernandez et al. 1995; Olmos et al. 1994; Sehmer et al. 1995). In the mangrove plant, Bruguiera gymnorrhiza, Takemura et al. (2000) showed an increase in the activities of total SOD and catalase after plants were transferred from freshwater to high salinity. Under salt stress, the
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sequence of physiological events is thought to be increased salinity, water stress by osmotic unbalance, reduction of stomatal opening, decrease in CO2 supply, and decrease in photosynthesis. A decrease in CO2 supply obliges chloroplasts to synthesize superoxide by reduction of oxygen. Van Camp et al. (1996) reported that oxidative stress tolerance was enhanced in transgenic tobacco which overexpressed SOD in chloroplasts. However, it is still not clear whether antioxidant enzymes in the cytosol contribute to increase in salt tolerance. In this study, we present changes in expression of two genes in salt-stressed B. gymnorrhiza which produce antioxidant enzymes in the cytosol, cytosolic Cu/ZnSOD and catalase.
94°C, 60 s at 54°C and 90 s at 72°C. The design of gene-specific primers (5′- for 5′ RACE; 5′- for 3′ RACE) was based on the nucleotide sequence obtained from the RT-PCR product. 5′ RACE was carried out with the 5′ RACE system for Rapid Amplification of cDNA Ends, Version 2.0 (GIBCO-BRL). 3′ RACE was conducted with NotI-(dT)18 and the gene-specific primer. These PCR products were linked into the pGEM-T Easy Vector (Promega) and sequenced using a DNA autosequencer SQ-5500 (Hitachi Co., Tokyo, Japan). A catalase cDNA was obtained from an expressed sequence tag (EST) collection. A cDNA library was constructed with poly(A)+ RNA from leaves of 4-month-old B. gymnorrhiza treated with 500 mM NaCl solution using a SMART cDNA Library Construction Kit (Clonetech Laboratories) following the protocol supplied. Northern blot analysis
Materials and methods Source of plants Seeds of B. gymnorrhiza were collected on Iriomote Island, Okinawa, Japan in September 1999. Two or three viviparous seeds were planted in a pot containing vermiculite. The pot, with six small holes in the side and three in the bottom, was placed in a container containing freshwater and the plants were grown at 25°C. The photon irradiance at the top of the leaves was maintained at approximately 500 µmol m–2 s–1. The photoperiod was 12 h light per day. After 6 months, several pots were transferred into another container containing either 500 mM NaCl solution or 1 M mannitol. For phytohormone treatments, 100 µM solutions of abscisic acid (ABA), methyl jasmonate (MJ) or 2-chloroethylphosphonic acid (CEPA) were spread on the leaves of freshwatergrown plants. After 24 h, the leaves were harvested for RNA extraction.
Total RNA (20 µg) was subjected to formaldehyde-agarose gel (1%) electrophoresis and blotted to Hybond-XL (Amersham Pharmacia Biotech). The cDNA probe was labeled by the random primer method with α-32P dCTP using the Megaprime DNA labeling system (Amersham Pharmacia Biotech). The membrane was hybridized with the probe at 42°C for 16 h in 0.01% (w/v) salmon sperm DNA, 0.1% SDS, 0.75 M NaCl, 50 mM NaH2PO4 2H2O, 6.25 mM EDTA, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% Ficoll and 10% dextran sulfate with 50% deionized formamide. The membrane was washed with 2×SSC, 0.1% SDS at 42°C for 15 min, and once more in 1× SSC, 0.1% SDS at 50°C for 15 min (20×SSC contained 333 mM NaCl and 33 mM C6H5O7Na3.2H2O). The membrane was exposed to Hyperfilm-MP (Fujifilm) for 96 h and developed. All the measurements were repeated twice at least.
Results and discussion Nucleotide and amino acid sequences
Extraction of RNA One or two leaves (1–5 g fresh weight) were collected and immediately frozen in liquid nitrogen. The frozen leaves were ground into a powder with a mortar and pestle. The powder was then incubated at 65°C for 1 h in 25 ml extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 25 mM EDTA, 2 M NaCl, 0.05% spermidine and 4% 2-mercaptoethanol). The suspension was centrifuged at 2,500 g for 10 min. An equal volume of chloroform/ isoamyl alcohol (24:1, v/v) was added to the supernatant, which was then centrifuged at 2,500 g for 5 min. RNA was purified with 2 M LiCl at –20°C for 12–16 h, then collected by centrifugation at 18,000 g for 20 min. Purified RNA was resuspended in 500 µl SSTE buffer (1 M NaCl, 0.5% SDS, 1 mM EDTA and 10 mM Tris-HCl pH 8.0) and precipitated with ethanol at –20°C for 2–3 h. The RNA was recovered by centrifugation at 18,000 g for 30 min, rinsed with 1 ml 80% ethanol and suspended in diethyl pyrocarbonate-treated water (5 µg/µl). Cloning of cytosolic Cu/Zn-SOD and catalase cDNAs The nucleotide sequence of cytosolic Cu/Zn-SOD was determined by a combination of reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) (Frohman et al. 1988). Single-stranded cDNA was synthesized from total RNA using the NotI-(dT)18 primer. To isolate the cDNA fragment encoding cytosolic Cu/Zn-SOD, RT-PCR was conducted using degenerated primers (forward: 5′-GCIYTIGGIGAYACIACIAAYGGITG–3′; reverse: 5′-CCYTGIARICCDATDATICCRCAIGC–3′ encoding the conserved amino acid sequences ALGDTTNGC and ACGIIGLQG, respectively). The PCR included 30 cycles of 30 s at
Although the nucleotide sequence of the RT-PCR product (314 bp) had homology with other sequences of Cu/Zn-SOD genes, this approach did not result in the isolation of full-length clones. To determine the sequences of the 3′-and 5′ regions, 3′-and 5′ RACE was performed using gene-specific primers based on the nucleotide sequence obtained from the RT-PCR product. The PCR products from 3′- and 5′ RACE were 362 bp and 380 bp, respectively. The nucleotide sequence of the entire Cu/Zn-SOD cDNA, determined by assembling these sequences, is shown in Fig. 1A. In the unbroken reading frame of the cDNA, the first ATG codon is at nucleotide position 140 and the open reading frame terminates with a TAA stop codon at position 599, thereby coding for 153 amino acids. The cDNA probably encodes a cytosolic Cu/Zn-SOD, since a signal peptide is not recognizable in the putative amino acid sequence. The 3′-untranslated region includes the putative polyadenylation signal 27 bp upstream of the poly(A) tail. The initiation site for translation corresponds with the consensus sequence AACAATGG reported for plants (Lutcke et al. 1987). The nucleotide sequence of the fulllength cDNA of cytosolic Cu/Zn-SOD in B. gymnorrhiza has been deposited in the DDBJ/EMBL database under accession number of AB062752. The putative amino
96 Fig. 1A, B Nucleotide and amino acid sequences of the cytosolic Cu/Zn-SOD cDNA. A Nucleotide and deduced amino acid sequences of cytosolic Cu/Zn-SOD isolated from Bruguiera gymnorrhiza (accession number: AB062752). Bold letters indicate binding sites of the primers in RT-PCR. Underlining and double underlining show the nucleotide sequences of primers used in 3′-and 5′ RACE-PCR, respectively. The shaded box is the putative polyadenylation signal. B Comparison of the amino acid sequences of cytosolic Cu/Zn-SOD from B. gymnorrhiza with those of Populus tremuloides, Zea mays, Ipomoea batatas and Mesembryanthemum crystallinum. Asterisks indicate amino acids that are conserved between different sequences. The copper- and zinc-binding sites are shaded
acid sequence of Cu/Zn-SOD cDNA in B. gymnorrhiza showed more than 80% identity with that of other plants (Fig. 1B). Deduced copper- and zinc-binding sites were found in a histidine at positions 45, 47, 62, 70, 79 and 119, and aspartic acid at position 82. According to Getzoff et al. (1988), five conserved glycine residues at 43, 60, 81, 137 and 140 are involved in maintaining the structure of the active site. Aspartic acid at 123 forms hydrogen bonds to histidine at 45 and 70, which are binding sites for copper and zinc, respectively. The positively charged arginine at 142 provides a binding pocket above the catalytic copper ion and stabilizes the negatively charged superoxide (Tainer et al. 1983). Two cys-
teine residues at 56 and 145 form the intermolecular disulfide bridge. A cDNA encoding catalase was found from our EST collection of B. gymnorrhiza grown under 500 mM NaCl. The partial nucleotide sequence of the 3′ region and the putative amino acid sequence was determined (Fig. 2). Northern blot analyses After the 500 mM NaCl treatment, all the leaves wilted within 1 day then quickly recovered. Northern blot ana-
97 Fig. 2 Partial nucleotide and deduced amino acid sequences of catalase in B. gymnorrhiza
lyses were performed using cDNAs of cytosolic Cu/ZnSOD and catalase as probes. The transcript level of cytosolic Cu/Zn-SOD in the first and second leaves from the top increased after 1 and 5 days NaCl treatment (Fig. 3). However, the transcript level of catalase in these leaves did not change significantly after NaCl treatment. We previously reported that the Na+ concentration in leaves increased following NaCl treatment and reached a steady level of approximately 400 mM 3 days after the treatment (Takemura et al. 2000). This suggests that the plant suffers from osmotic stress for several days after the treatment, is then released from the osmotic stress but suffers from ion stress by accumulation of Na+ after 3 days. Therefore, it is considered that expression of cytosolic Cu/Zn-SOD after 1 day was induced by osmotic stress and the expression after 5 days was induced by ion stress. The catalase gene was not induced by either stress. After mannitol treatment, which provided the same osmotic pressure as 500 mM NaCl, the leaves also wilted within 1 day but did not recover (data not shown). After a 1-day mannitol treatment, cytosolic Cu/Zn-SOD was expressed in the top leaves, but the transcript level of catalase did not change significantly (Fig. 4). Thus osmotic stress appears to induce cytosolic Cu/Zn-SOD but not expression of the catalase gene. The transcript level of cytosolic Cu/Zn-SOD in B. gymnorrhiza increased 5 days after NaCl treatment and then decreased (Fig. 3).
Fig. 3 Expression of the transcripts of cytosolic Cu/Zn-SOD and catalase after treatment with 500 mM NaCl. RNA was extracted from the first or second leaves from the top. Total RNA (20 µg) was loaded in each lane. A probe of 18 S rRNA was used as a loading and transfer control
It is reported that plants have a mechanism for compartmentalizing salt into vacuoles (Matoh et al. 1987). Thus, the decrease in NaCl after 7 days treatment may be attributable to compartmentalization of Na+ into vacuoles. The expression of these genes at different leaf positions was compared (Fig. 5). The transcript level of cytosolic Cu/Zn-SOD did not differ with leaf position in unstressed control plants, but increased in the first, second and fourth leaves from the top after 5 days NaCl treat-
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Fig. 4 Expression of the transcripts of cytosolic Cu/Zn-SOD and catalase after treatment with 1 M mannitol. Left lane Control; right lane first or second leaves from the top, 1 day after treatment
Fig. 6 Expression of cytosolic Cu/Zn-SOD and catalase transcripts after treatment with phytohormones. RNA was extracted from the first or second leaves of plants exposed for 24 h to 100 µM abscisic acid (ABA), methyl jasmonate (MJ) or 2-chloroethylphosphonic acid (CEPA)
Fig. 5A, B Expression of transcripts of cytosolic Cu/Zn-SOD and catalase in leaves at different positions from the top of the plant. A Expression without treatment. B Expression after treatment with 500 mM NaCl. The samples were extracted 5 days after treatment
ment. The expression of the cytosolic Cu/Zn-SOD gene was suppressed in the sixth leaf after NaCl treatment. This suggests that ion stress actively induces expression of cytosolic Cu/Zn-SOD in young and mature leaves rather than in old leaves. The expression pattern of the catalase gene at different leaf positions after 5 days NaCl treatment was similar to that of the unstressed control plant, suggesting that the catalase gene is not induced by ion stress. The transcript of catalase was strongly expressed in the fourth leaves of both unstressed and stressed plants. The effects of phytohormones on the expression of cytosolic Cu/Zn-SOD and catalase genes were examined (Fig. 6). Application of ABA increased the transcript level of cytosolic Cu/Zn-SOD, but suppressed expression of the catalase gene. However, application of the ethylene generator CEPA increased the transcript level of catalase, but did not affect expression of the cytosolic Cu/Zn-SOD gene. Both transcripts were down-regulated by exogenous MJ. Guan and Scandalios (1998) reported that one of two highly conserved Cu/Zn-SOD genes in Zea mays was regulated by ABA. While Lee et al. (1999) showed that the cytosolic Cu/Zn-SOD gene of cassava was induced by wounding stress and application of MJ, which is a known mediator of wounding stress. These findings imply that expression of the cytosolic Cu/Zn-SOD gene is regulated in a species-specific man-
ner. Expression of the catalase gene in B. gymnorrhiza may be regulated by ethylene. In a previous paper (Takemura et al. 2000), we showed increase in catalase activity in leaves of B. gymnorrhiza after NaCl treatment. However, no significant change occurred in expression of the catalase gene. Several isozymes of catalase have been isolated from Nicotiana plumbaginifolia (Willekens et al. 1994), maize (Polidoros and Scandalios 1997) and Arabidopsis thaliana (Frugoli et al. 1996). It is likely that different isozymes contribute to the active oxygen-scavenging system of the cytosol resulting from salt stress in the leaves of B. gymnorrhiza. The results of this study indicate that cytosolic Cu/ZnSOD is expressed to cope with both osmotic and ion stresses in the cytosol of B. gymnorrhiza leaves. The importance of chloroplastic SODs and other chloroplastic antioxidant enzymes under various environmental stress conditions has been pointed out, as the chloroplast is easily damaged by oxidative stress due to excess electrons in photosystems. Our results suggest that salt stress causes the generation of superoxide anions in the cytosol. There are, however, few reports about the effect of environmental stresses on the generation of active oxygen species in the cytosol. One possible mechanism is a leak of active oxygen species from chloroplasts and/or mitochondria. Further study is now required on the generation of active oxygen species in the cytosol during salt stress.
99 Acknowledgements We thank Ms. Minako Kaga and Mr. Akio Hayashi for their technical assistance. An Endowed Chair in Environmental Biotechnology funded by the EBARA Corporation, Japan supported this work.
References Anderson JW, Beardall J (1991) Molecular activities of plant cells: an introduction to plant biochemistry. Blackwell, Oxford Asada K (1992) Ascorbate peroxidase: a hydrogen peroxide-scavenging enzyme in plants. Physiol Plant 85:235–241 Bowler C, Van CW, Van MM, Inze D (1994) Superoxide dismutase in plants. Crit Rev Plant Sci 13:199–218 Fridovich I (1986) Biological effects of the superoxide radical. Arch Biochem Biophys 247:1–11 Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002 Frugoli JA, Zhong HH, Nuccio ML, McCourt P, McPeek MA, Thomas TL, McClung CR (1996) Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh. Plant Physiol 112:327–336 Getzoff ED, Tainer JA, Lerner RA, Geysen HM (1988) The chemistry and mechanism of antibody binding to protein antigens. Adv Immunol 43:1–98 Gossett DR, Millhollon EP, Lucas MC (1994) Antioxidant response to NaCl stress in salt-tolerant and salt-sensitive cultivars of cotton. Crop Sci 34:706–714 Guan L, Scandalios JG (1998) Two structurally similar maize cytosolic superoxide dismutase genes, Sod4 and Sod4A, differentially respond to ABA and high osmoticum. Plant Physiol 117:217–224 Hernandez JA, Olmos E, Corpas FJ, Sevilla F, Del RLA (1995) Salt-induced oxidative stress in chloroplasts of pea plants. Plant Sci 105:151–167
Lee HS, Kim KY, You SH, Kwon SY, Kwak SS (1999) Molecular characterization and expression of a cDNA encoding copper/ zinc superoxide dismutase from cultured cells of cassava (Manihot esculenta Crantz). Mol Gen Genet 262:807–14 Lutcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differs in plants and animals. EMBO J 6:43–8 Matoh T, Watanabe J, Takahashi E (1987) Sodium, potassium, chloride, and betaine concentrations in isolated vacuoles from salt-grown Atriplex gmelini leaves. Plant Physiol 84: 173–177 Olmos E, Hernandez JA, Sevilla F, Hellin E (1994) Induction of several antioxidant enzymes in the selection of a salt-tolerant cell line of Pisum sativum. J Plant Physiol 144:594–598 Polidoros AN, Scandalios JG (1997) Response of the maize catalases to light. Free Radic Biol Med 23:497–504 Sehmer L, Alaoui SB, Dizengremel P (1995) Effect of salt stress on growth and on the detoxifying pathway of pedunculate oak seedlings (Quercus robur L.). J Plant Physiol 147:144– 151 Smirnoff N (1993) Tansley Review No. 52: The role of active oxygen in the response of plants to water deficit and desiccation. New Phytol 125:27–58 Tainer JA, Getzoff ED, Richardson JS, Richardson DC (1983) Structure and mechanism of copper, zinc superoxide dismutase. Nature 306:284–7 Takemura T, Hanagata N, Sugihara K, Baba S, Karube I, Dubinsky Z (2000) Physiological and biochemical responses to salt stress in the mangrove, Bruguiera gymnorrhiza. Aquat Bot 68:15–28 Van Camp W, Capiau K, Van Montagu M, Inze D, Slooten L (1996) Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts. Plant Physiol 112:1703–1714 Willekens H, Villarroel R, Van MM, Inze D, Van CW (1994) Molecular identification of catalases from Nicotiana plumbaginifolia (L.). FEBS Lett 352:79–83
