An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization

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Plant Physiology and Biochemistry 46 (2008) 794e804 www.elsevier.com/locate/plaphy

Research article

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization Kumaresan Kavitha, Gayatri Venkataraman, Ajay Parida* M.S. Swaminathan Research Foundation, Third Cross Street, Taramani Institutional Area, Chennai 600113, Tamilnadu, India Received 30 October 2007 Available online 28 May 2008

Abstract APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4 kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)10-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616 bp corresponding to the 50 upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Ascorbate peroxidase; Avicennia marina; Green fluorescent protein; Hydrogen peroxide; Peroxisome; Subcellular localization

1. Introduction Reactive oxygen species (ROS; e.g. O 2 and H2O2) are generated as by-products of plant cellular metabolism. At lower concentrations, ROS can serve as signaling molecules in plant redox signal transduction [45]. Under stress conditions, ROS overproduced in plant cells can damage cellular components including DNA, proteins and membrane lipids [18]. Plants have evolved efficient anti-oxidant systems that can protect them from the damaging effects of oxidative stress [1]. These Abbreviations: AD, arbitrary degenerate; APX, ascorbate peroxidase; cAPX, cytosolic APX; EST, expressed sequence tags; pAPX, peroxisomal APX; pER, peroxisomal endoplasmic reticulum; sAPX, stromal APX; TAILPCR, thermal asymmetric interlaced PCR; tAPX, thylakoid APX; TSS, translation start site; SOE-PCR, splicing by overlap extension-PCR. * Corresponding author. Tel.: þ91 044 2254 1229/2698/1698; fax: þ91 044 2254 1319. E-mail address: [email protected] (A. Parida). 0981-9428/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.05.008

mechanisms employ ROS scavenging enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) as well as low molecular weight anti-oxidants such as ascorbic acid, GSH and phenolic compounds [1]. APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants [35]. This class I peroxidase catalyzes the conversion of H2O2 to H2O and O2 using ascorbate as the specific electron donor [1]. The reduced ascorbate is oxidized back in a series of reactions catalyzed by monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) known as the HalliwelleAsada cycle and is one of the most important anti-oxidant systems in plants. Distinct APX isoforms are known to exist in plants and these are classified according to their subcellular location as: (a) soluble isoforms found in the cytosol (cAPX) [9] and stroma of plastids (sAPX) [10]; and (b) membrane-bound isoforms present in the peroxisomes (pAPX) [11] and associated

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with the thylakoid membrane of chloroplasts (tAPX) [10]. The different isoforms of APX (showing distinct subcellular localization) possess distinct biochemical properties such as molecular mass, substrate specificity, ideal pH for activity and stability in the absence of ascorbate [11]. Thus, when compared to the cytosolic and peroxisomal isoforms, the chloroplastic enzymes are more labile and are rapidly inactivated in the absence of ascorbate [11,35]. Considering the specific distribution, roles of the APX isoenzymes and the potential for active oxygen species production in each organelle, it seems likely that the APX isoenzymes are expressed by distinct regulatory mechanisms. The transcript of cAPX is dramatically induced during the hypersensitive response of tobacco plants infected with TMV [19] and in spinach in response to high light stress, methyl viologen and abscisic acid [43]. Three rice APX genes, including the cytosolic (OsAPX2) and chloroplastic (OsAPX7 and OsAPX8) isoforms, show an altered transcript level in response to salt stress [38]; the up-regulation of cAPX in response to high and low temperature in rice has also been reported [32]. Peroxisomal APX has been reported from pumpkin, cotton, spinach, castor bean endosperm, sunflower, pea, cucumber cotyledons and Arabidopsis [35]. Biochemically, pAPX shows very high donor specificity for ascorbate (0.5 mM) and no activity in the presence of other electron donors such as NADPH, NADH and cytochrome c. Cyanide and azide strongly inhibit its activity as do thiol reagents. The latter effect can be attributed to the presence of a metal prosthetic group and a sulfhydryl group in pAPX that participate in enzymatic reactions. In comparison to the extensive data available on the functions of cytosolic and chloroplastic APXs, relatively little is known about the function of pAPX. Jimenez et al. [12] report a decrease in the activities of pAPX and MDHAR in dark induced senescent leaves of pea. Peroxisomal fractions isolated from Lycopersicon pennellii (Lpa; salt tolerant wild tomato subjected to 100 mM NaCl) show increased SOD, APX, MDHAR and catalase activity as compared to salt sensitive Lycopersicon esculentum [22]. The salt-induced increase in the anti-oxidant enzyme activities in Lpa plants confers cross-tolerance towards enhanced peroxisomal reactive oxygen species production imposed by 3-amino-1,2,4-triazole (3-AT), an inhibitor of peroxisomal catalase. A remarkable increase in the transcript level of a pAPX isolated from barley (HvAPX1; Hordeum vulgare cv. Haruna-nijyo) in response to heat, salt and abscisic treatment is seen and over-expression in Arabidopsis confers tolerance to heat stress [34]. Over-expression of Arabidopsis APX3 (AtAPX3), a putative pAPX, in transgenic tobacco plants leads to increased tolerance against 3-AT [41] and a higher seed production under moderate drought conditions in tobacco plants [42]. These data indicate that the increased level of APX in peroxisomal membrane appears to increase stress tolerance in plants. In this study, we report the isolation and characterization of a cDNA from Avicennia marina coding for a peroxisomal APX (Am-pAPX1). The Am-pAPX1 transcript was found to be induced by salinity, high light, H2O2 and iron overload.

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The genomic clone of Am-pAPX1 was sequenced and found to contain nine exons. APX activity of the Am-pAPX1 ORF was examined by over-expression in E. coli. Am-pAPX1 was found to utilize ascorbate as the most effective natural electron donor as compared to guiacol and pyrogallol and activity was inhibited by azide and excess H2O2, properties typical of peroxisomal ascorbate peroxidases. Finally, using a GFP fusion, we show Am-pAPX1 targeting to peroxisomes when transformed stably into tobacco. 2. Materials and methods 2.1. Sequencing of full length APX cDNA Random EST sequencing of the A. marina cDNA library [17] lead to the identification of an EST that showed homology to peroxisomal APX (EST No: BM173238). This EST was sequenced completely using BigDye terminator method (ABI 310, Applied Biosystems). 2.2. Plant material and growth conditions A. marina seedling growth conditions were according to Mehta et al. [17]. One month old seedlings (four leaf stage) were used in this study. Stress conditions imposed on A. marina seedlings {Light (500 mEm2 s1), salinity (500 mM NaCl), H2O2 (90 mM) and iron overload (1000 mM; iron III citrate)} are as described by Jithesh et al. [13]. 2.3. RNA isolation and Northern blotting Total RNA from frozen leaf tissue was isolated according to Chomczynski and Sacchi [3]. Total RNA (20 mg/lane) was fractionated on a 1.5% denaturing formaldehyde-agarose gel, transferred to nylon membrane (Hybond-N, Amersham). Aqueous hybridization was carried out at 65  C with 32Plabeled Am-pAPX1 (full length) as probe. The blots were washed sequentially with 2 SSC, 0.1% SDS; 1 SSC, 0.1% SDS at 65  C for 150 each and exposed to Kodak BioMax MS film. 2.4. Isolation of the Am-pAPX1 genomic clone Primers corresponding to different regions of Am-pAPX1 cDNA were used to generate overlapping genomic fragments. Fragments of size 290 bp, 2.20 kb and 772 bp were amplified using primer pairs (APX-UTR-F: 50 -TCAGACTACCTCAAG GAGATCGA-30 /APX Tail 1R: 50 -CGAACCGTTCGGACCAC CAGT-30 ), (APX-INT1F: 50 -GGAGTATAATTCGTTAGGT GGC-30 /APX 2R: 50 -ATCAGACAAATCCATCCGGTAA-30 ) and (APX 1F: 50 -GGGAGGTGTTTTACCGGATGGAT-30 / APX UTR-R: 50 -CAAGCAAGCATGCGCCAACAAGT-30 ) respectively. The PCR reactions were analyzed on an agarose gel, specific fragments eluted and cloned in TA vector (MBI, Fermentas). The overlapping clones were sequenced completely and compared with the Am-pAPX1 cDNA sequence.

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2.5. Isolation of the 50 upstream sequence of Am-pAPX1 A 1616 bp fragment corresponding to the 50 upstream region of Am-pAPX1 was isolated using TAIL-PCR with cycling conditions modified according to Sessions et al. [33]. In the primary reaction, primers APX-GSP1-R: 50 -CGAACCGT TCGGACCACCAGT-30 ) and [(AD1: 50 -NGTCGASWGAN AWGAA-30 ) or (AD2: 50 -TGWGNAGSANCASAGA-30 ) or (AD3: 50 -WGTGNAGWANCANAGA-30 ) or (AD4: 50 -STT GNTASTNCTNTGC-30 )] were used. The primary PCR product was diluted 40-fold and used in the secondary reaction with APX-GSP2-R: 50 -GGAGAGATTAGAAAATACGCCAATC-30 and AD (1e4) primers. The secondary product was diluted 10-fold for the tertiary reaction, using APXGSP3-R: 50 -GGCGAGCCTTCTCGATCTCCT-30 primer and AD (1e4) primers. The products of the primary, secondary and tertiary reactions were analyzed on a 0.8% agarose gel. The fragment exhibiting a difference in size consistent with nested gene specific-primer positions was selected for cloning in a TA vector and sequenced completely. 2.6. Over-expression of Am-pAPX1 in E. coli The open reading frame of Am-pAPX1 was amplified from the cDNA using APX EXP-F (50 -CGCGGATCCATGCTTCGATTGGCGTGGCATG-30 ) and APX EXP-R primers (50 CCCAAGCTTCTATTTCATCTTTTTCCTGACTTCG-30 ) that were designed to incorporate BamHI and HindIII sites respectively. The amplified product was digested with the same enzymes and cloned in the E. coli expression vector, pET 28a (Novagen; pET28a-Am-pAPX1) and transformed into E. coli BL21 (DE3). Cultures of E. coli cells carrying pET28a-AmpAPX1 or vector control (pET28a) were grown at 37  C in LB medium containing 50 mg/ml kanamycin to an A600 ¼ 0.5 and induced with 1 mM IPTG. The cells were pelleted after 2 h of induction, suspended in 25 mM TriseCl (pH 7.5), 5% glycerol, 1 mM EDTA, 2 mM ascorbic acid, 250 mM PMSF and sonicated (LabSonics, output 1500 psi, 3  30 s). The supernatant was separated by high-speed centrifugation and used for peroxidase assays with different reducing substrates such as ascorbate, guaiacol and pyrogallol and/or inhibitor (sodium azide). The amount of protein in the soluble extract was estimated according to Bradford method. Polyclonal antibodies were raised against E. coli expressing AmpAPX1 (Bangalore Genei, India). 2.7. Measurement of peroxidase activity APX activity was determined spectrophotometrically by the method of Nishikawa et al. [27]. The assay mixture contained 50 mM PBS (pH 7.0), 1 mM EDTA, 0.5 mM L-ascorbic acid and soluble E. coli extracts (25 mg total protein; Am-pAPX1 or pET28a). The reaction was initiated with the addition of 0.1 mM H2O2. Enzyme activity was determined by measuring the decrease in absorbance of ascorbate at 290 nm (3290/ 2.8 mM1 cm1) over 3 min at 25  C. Ascorbate was replaced by guaiacol (20 mM) or pyrogallol (20 mM) as the reducing

substrate and peroxidase activity in the E. coli extracts measured according to Lu et al [16]. Detection of in gel APX activity was according to Mittler and Zilinskas [20]. 2.8. Isolation of crude plant extracts A. marina leaf tissue (isolated from seedlings subjected to salinity, excess light, dark, H2O2, iron overload or iron limiting conditions for 6 h each) was ground in liquid nitrogen with a mortar and pestle, suspended in homogenization buffer {1:4 w/v; 25 mM TriseCl (pH 7.8), 5 mM ascorbate 0.1 mM EDTA, 5% (w/v) glycerol and 250 mM PMSF}. The homogenate was centrifuged at 12,000  g for 200 and the supernatant used for the APX assay as described above. 2.9. Construction of GFP-(Ala)10-Am-pAPX1 fusion and transformation into tobacco The GFP-(Ala)10-Am-pAPX1 fusion construct was assembled by SOE-PCR. The clone for GFP (mGFP6) was obtained from Dr. Mark Curtis (Institute of Plant Biology, Zurich, Switzerland) as part of a plasmid, pMDC 83. The GFP fragment was cloned in pBSSK II with a HindIII site at the 50 end and a BamHI site at the 30 end. GFP FWD: 50 -CTAGTCT AGAATGAGTAAAGGAGAAGAACTTTTCA-30 and ALASOE-GFP-R: 50 -AGCGGCTGCAGCTGCGGCAGCTGCGG CTGCTTTGTATAGTTCATCCATGCCATG-30 ) were used to amplify the 700 bp GFP fragment using Pfu Turbo (Stratagene) while ALA SOE-APX-F: 50 -GCTGCCGCAGCTGC AGCCGCTATGGCGAAAGTTGTCGTCGACT-30 and APX ORF R: 50 -CGCGGATCCCTATTTCATCTTTTTCCTGAC TTCG-30 ) were used to amplify the Am-pAPX1 ORF (858 bp). The GFP and Am-pAPX1 fragments were mixed in a 1:1 molar ratio, heated to 72  C and a third PCR was performed using primers GFP FWD and APX ORF R The resultant 1.65 kb SOEed product was gel eluted and cloned in pBSSK II (SmaI digested). The fusion product was sequenced to confirm an in-frame fusion, excised using BamHI and XbaI and cloned in pCAMBIA 1301 driven by the 2 35S Cauliflower mosaic promoter (pGFP-(Ala)10-Am-pAPX1). pGFP-(Ala)10-Am-pAPX1 was mobilized into Agrobacterium tumefaciens strain LBA4404 and was used to transform Nicotiana tabacum cv. Petit Havana. Preliminary selection of transformed plants was done using GUS staining. Independently transformed lines were screened for GFP fluorescence by fluorescence microscopy (Nikon, Optiphot-2). One brightly fluorescing line was used for raising suspension cells as described by Reynolds and Murashige [31] and used for confocal imaging. Total protein was isolated from suspension cells as well as from leaf extracts of the pGFP-(Ala)10-Am-pAPX1 transformed line and used for immunoblotting (25 mg) with Anti-Am-pAPX1 antibodies. 2.10. Confocal laser scanning microscopy analysis For microscopic analysis, epidermal leaf peels taken from the GFP-(Ala)10-Am-pAPX1 expressing line were mounted

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in water as were suspension cells prepared from the same line. The confocal laser scanning microscope, LSM 510 META (Zeiss), equipped with a water immersion lens (63) was used for imaging. Excitation and emission wavelengths used for GFP were 488 and 509 nm (BP 505e530 filter) respectively. A low laser setting (8%) was sufficient to visualize GFP fluorescence. Chlorophyll autofluorescence was imaged using 488 nm for excitation and 633 nm (BP 630e700 filter) for emission. The green and red autofluorescence were collected in separate channels. For guard cells, single images were recorded while for suspension cells optical sections (fluorescence as well light microscopic images) were recorded at 1.4 mm intervals (n ¼ 63). All images were processed using the Zeiss LSM Image Browser 3.2 (Zeiss Corporation, Germany).

This EST was sequenced completely (EU024941) and was found to be 1143 bp in length with an ORF of 858 bp coding for a 31.4 kDa protein and the cDNA was named Am-pAPX1. The deduced amino acid sequence of Am-pAPX1 was compared with those of other plant peroxisomal APXs available at the NCBI database. Am-pAPX1 displayed highest homology (85%) to a pAPX from Capsicum annuum (AAL35365) (Fig. 1). The APX active site signature (A region) is present at positions 30e42 (APIMLRLAWHDA), together with the proximal heme-ligand motif (H region) between residues 151e160 (DIVALSGGH) [39]. A putative membrane spanning signal was detected at positions 259e278 (LVQSAVGVAVAATVV ILSYL) using PSORT followed by a C-terminal putative peroxisomal targeting signal (RKKMK), also seen in other pAPXs.

3. Results

3.2. Expression of Am-pAPX1 in response to salinity/ oxidative stress

3.1. Isolation and sequencing of cDNA encoding a peroxisomal ascorbate peroxidase An EST (BM173238) showing homology to peroxisomal APX was identified from the A. marina cDNA library [17].

The changes in the transcript level of Am-pAPX1 under different stress conditions viz., salinity, excess light, H2O2 application and iron overload are shown in Fig. 2AeD. Under salt stress, a gradual increase in the transcript level was observed,

Fig. 1. Alignment (ClustalW) of the deduced amino acid sequence of Am-pAPX1 from Avicennia marina with pAPXs from other plant species. Capsicum annuum (AAL35365), Curcubita cv. Kurokawa Amakuri (BAB64351), Gossypium hirsutum (AAB52954), Vigna unguiculata (AAS46016). The asterisks (*) indicate conserved amino acid sequence while (:) indicate conserved amino acid replacements. The horizontal lines depict the conserved APX active site (A), the proximal heme-ligand motif (H), the transmembrane domain (TMD) and the peroxisomal targeting signal (PTS).

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Fig. 2. Expression analysis of Am-pAPX1 under abiotic stress. A. marina seedlings were exposed to (A) light stress (500 mEm2 s1); (B) NaCl (500 mM); (C) H2O2 stress (90 mM); (D) Fe(III) citrate (1000 mM) and total RNA isolated at various time points from leaves. Equal aliquots (20 mg) of total RNA were electrophoresed in a formaldehyde-agarose gel, transferred to Hybond Nþ membrane and probed with 32P labeled Am-pAPX1. C, control unstressed plants, hw, hours after withdrawal.

peaking at 12 h and dropping at 24 and 48 h. Upon withdrawal of NaCl, the Am-pAPX1 transcript level was seen to drop further. A steady increase in the transcript level of Am-pAPX1 in response to high light intensity was observed from 0.5 to 24 h. With hydrogen peroxide application, Am-pAPX1 was strongly expressed at 6 h and was not sustained thereafter. When derooted A. marina plantlets were supplied with iron citrate [13], a rapid accumulation of APX transcript was observed from 0.5 to 6 h and the transcript level declined after iron withdrawal. 3.3. Effect of salinity and oxidative stress on total APX activity in A. marina leaves The effect of dark acclimation, prolonged light treatment, salinity, hydrogen peroxide, iron deficit and overload (at 6 h) on total APX activity was examined in leaves of A. marina (Fig. 3). Upon shifting dark acclimatized seedlings to light, a 26.73% higher APX activity was seen in the leaf extracts. With NaCl and H2O2 treatments, leaves of A. marina showed 15.01% and 10.56% higher APX activity compared to

APX activity (µmol/min/mg protein)

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Fig. 3. (A) Total APX activity measured in leaf extracts of A. marina seedlings exposed to dark and light (500 mEm2 s1) conditions. (B) Total APX activity measured in leaf extracts of control (unstressed) A. marina seedlings and those exposed to NaCl (500 mM) and H2O2 (90 mM) for 6 h. (C) Total APX activity measured in leaf extracts of A. marina seedlings (shoots) acclimatized in medium lacking [Fe()] and those transferred to medium containing iron (III) citrate (1000 mM) [Fe(þ)]. Values represent  SE of three measurements, each from three independent experiments.

unstressed control. A. marina shoots exposed to iron overload showed 10.59% higher activity as compared to iron limited plants. 3.4. Genomic organization of Am-pAPX1 The genomic clone of Am-pAPX1 is 3942 bp in length (Accession No. EU025130), 2799 bp longer than the Am-pAPX1 cDNA. Alignment of Am-pAPX1 (cDNA) with its genomic clone revealed the presence of eight introns (Fig. 4). The size of the introns varied between 83 and 740 bp while the size of the exon varied between 41 and 416 bp. For all the exons, the introneexon splice junctions were readily identifiable and conformed to the consensus sequence GT at the donor site and AG at the acceptor site. The position of the introns (region corresponding to Am-pAPX1 ORF) in the Am-pAPX1 genomic clone appears to be conserved when compared with APX3 from A. thaliana.

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Fig. 4. Nucleotide and deduced amino acid sequence of Am-pAPX1. Sequences shown in uppercase indicate exons and the 50 untranslated region of Am-pAPX1 while sequences shown in lowercase indicate introns and the 50 upstream region of Am-pAPX1. The numbering of 50 upstream region of Am-pAPX1 is relative to the TSS of Am-pAPX1. The putative regulatory regions referred to in Table 1 have been underlined.

3.5. Isolation and characterization of the 50 upstream region of Am-pAPX1 Using TAIL-PCR, a 1616 bp fragment 50 upstream of Am-pAPX1 was isolated in the tertiary reaction using

AD1 and APX-GSP3-R primers (EU024942). Putative cis-acting elements present in the 50 upstream region of Am-pAPX1 were identified using the program PLACE [8]. A putative TATA box is located 92 bp upstream of the translation start site (TSS) of Am-pAPX1 while a putative

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Fig. 4. (continued).

CCAAT box is present 36 bp upstream from the TATA box. The 50 upstream region of Am-pAPX1 contains several other putative regulatory elements that are shown in Table 1.

3.6. Expression of Am-pAPX1 in BL21 (DE3) cells APX activity in the soluble fraction of E. coli cells expressing pAm-pAPX1 was dependent on the presence of ascorbate

Table 1 Promoter elements identified in the 50 untranslated region of the Am-pAPX1 gene Category Light regulated

Cis-acting element

GATABOX GT1-consensus I-box Cold/freeze/dehydration MYC element Heat shock element CCAAT box ABRE ABRE element Ethylene responsive element ERE element Copper response element CURE (copper response element) Elicitor responsive element ELRE (elicitor responsive element)

Sequence

Position

GATA 283, 548, 570, 601, 805, 978, 1346 GRWAAW 45, 313, 314, 548, 557, 570, 616, 625, 978, 1602 GATAA 548, 570, 601, 978 CANNTG/CACATG 1195, 1322, 1551, 1592, 1194 CCAAT 128, 149, 1303, 1593 MACGYGB 1295 AWTTCAAA 930, 343 GTAC 1082, 1256 TTGACC 410

Positions of the cis-acting elements are indicated with respect to the initiation codon (ATG) of Am-pAPX1. A description of the cis-elements is available at http:// www.dna.affrc.go.jp/PLACE.

K. Kavitha et al. / Plant Physiology and Biochemistry 46 (2008) 794e804 Table 2 Relative APX activity in E. coli cells expressing Am-pAPX1 with different reducing substrates Reducing substrates and inhibitors in assay

Relative activity (%)*

Ascorbate Guaiacol Pyrogallol Sodium azide

100 9.8 8.6 0

line) with anti-Am-pAPX1 antibody could detect an additional 58 kDa protein along with the endogenous 31 kDa APX protein (Fig. 5E) seen in control suspension cells. 4. Discussion A. marina grows in anoxic soils in coastal areas with high salinity, often under conditions of high temperature and light and may serve as a good model to study anti-oxidant responses involving ROS scavengers [13]. These conditions affect the photosynthetic rate in two ways: high water deficiency and low stomatal conductance leading to an excess of excitation energy [2,36]. All members of the APX gene family, irrespective of the isoforms they encode, are ultimately associated with general cellular metabolism, stress response(s), signaling processes or the development of chloroplasts. The deduced amino acid sequence of Am-pAPX1 shows high similarity to pAPXs from Capsicum, Cucurbita and tomato, including the presence of a putative membrane spanning signal at the C-terminus of the protein and a peroxisomal targeting signal at the C-terminus (RKKMK). The peroxisomal targeting signal consists of a transmembrane domain rich in valine and alanine, followed by a positively charged domain containing five amino acid residues [25]. The in vivo subcellular localization of Am-pAPX1 was investigated by the stable expression of its GFP fusion in tobacco cv. Petit Havana and suspension cells prepared from the same. The GFP fluorescence of tobacco leaf peels expressing GFP(Ala)10-Am-pAPX1 was observed in the rim of spherical organelles dispersed in the cytosol of both guard cells as well as trichomes (not shown) in different optical planes using low laser settings. This has also been reported by Sparkes et al. [37] for the AtPEX10-YFP fusion, a peroxisomal membrane protein. In suspension cells expressing GFP-(Ala)10-Am-pAPX1, the green fluorescence was additionally associated with a reticular network around the nuclei of suspension cells and this was seen for cotton pAPX and rice pAPX3 [24,38]. Immunocytochemical analysis of Arabidopsis (expressing pumpkin pAPX) revealed pAPX localization on peroxisomal membranes and also in unknown membranous structure(s) in cotyledons [28].

*Enzyme activities determined spectrophotometrically as described in Section 2. Activity with ascorbate as the reducing substrate was defined as 100% and activity with other substrates was defined relative to ascorbate.

as the electron donor. Ascorbate was the preferred substrate (100%) as compared to Guaiacol (9.8%) or Pyrogallol (8.6%) (Table 2). This ascorbate-dependent APX activity was completely inhibited by sodium azide (10 mM). Western blot analysis of E. coli soluble cell extracts expressing AmpAPX1 with anti-cucumber pAPX antibody could detect a 31 kDa protein as did anti-Am-pAPX1 antibodies (Fig. 5B,C). Native-PAGE analysis revealed an additional band of approximately 31 kDa in soluble extracts prepared from induced cells and faintly in uninduced cells whereas no APX activity was detectable in control (pET 28a; Fig. 5D). 3.7. Intracellular localization of GFP-(Ala)10-Am-pAPX1 fusion Transgenic tobacco lines stably expressing GFP-(Ala)10Am-pAPX1 and suspension cells prepared from the same were analyzed by confocal laser scanning microscopy. GFP(Ala)10-Am-pAPX1 located to punctate spherical structures similar in size to plant peroxisomes (0.5e1.5 mm) in guard cells (Fig. 6EeH). Low laser settings of the microscope allowed more precise identification of the distribution of the GFP fluorescence as being towards the rim of some of these spherical structures. In suspension cultures, GFP fluorescence was detected in a reticular/circular structure around the nucleus and in spherical spots distributed diffusely throughout the periphery of the cytosol (Fig. 6AeD). Western blot analysis of the suspension cells (prepared from a GFP-(Ala)10-Am-pAPX1 expressing

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Fig. 5. (A) SDS-PAGE (12%) analysis of total E. coli cell extracts expressing Am-pAPX1. A 31 kDa protein is seen in induced cell extracts. (B & C) Western blot analysis of soluble E. coli cell extracts expressing Am-pAPX1 with anti-cucumber pAPX and anti-Am-pAPX1 antibodies respectively. (D) In-gel APX activity (8% native-PAGE) detected in the soluble fraction of E. coli cells expressing Am-pAPX1. P-pET 28a at 2 h of IPTG induction; U, uninduced cells expressing AmpAPX1, I, induced cells expressing Am-pAPX1 at 2 h of IPTG induction. (E) Western blot analysis of tobacco suspension cells prepared from GFP-(Ala)10Am-pAPX1 expressing and control (un-transformed) plants. A 58 kDa protein was detected in GFP-(Ala)10-Am-pAPX1 expressing cells (T) while endogenous APX is (w31 kDa) detected in both untransformed (C) and GFP-(Ala)10-Am-pAPX1 expressing cells.

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Fig. 6. Subcellular localization of GFP-(Ala)10-Am-pAPX1 in suspension cells and guard cells of transgenic tobacco. Confocal imaging of GFP-(Ala)10-Am-pAPX1 expressing transgenic tobacco suspension cells reveals GFP fluorescence to be localized in reticular/circular (pER) around the nucleus (n) and in spherical spots distributed diffusely throughout the periphery of the cytosol (indicated by white arrows). Images of the same cell were recorded at an interval of 1.4 mm (n ¼ 63); (AeC) represent image numbers 24, 27 and 39 respectively and (d) is a light microscopic image of the same suspension cell. Bar in (AeD) ¼ 20 mm. in guard cells of transgenic tobacco lines expressing GFP-(Ala)10-Am-pAPX1, green fluorescence is localized to punctate, spherical structures, similar in size to plant peroxisomes (indicated by white arrows). Chlorophyll autofluorescence (red) is also visible in the sections. Images of the same cell were recorded at intervals of 1.0 mm (n ¼ 23); (EeG) represent image numbers 9, 15 and 18 respectively and (h) is a light microscopic image of the cell. Bar in (EeH) ¼ 10 mm.

These membranous structures have been described as a sub-domain of the endoplasmic reticulum known as peroxisomal endoplasmic reticulum (pER) that contributes to peroxisome biogenesis [15,25]. Western blot analysis of suspension cells expressing GFP-(Ala)10-Am-pAPX1 with anti-Am-pAPX1

antibody could detect an additional 58 kDa protein along with the endogenous 31 kDa APX protein seen in control suspension cells. A 31 kDa protein was observed in E. coli cells transformed with pAm-pAPX1 upon induction with IPTG. Western blot

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analysis with anti-cucumber pAPX and anti-Am-pAPX1 antibodies detected the same size protein band. Activity studies indicated that ascorbate was the preferred substrate as compared to guaiacol and pyrogallol, with activity being inhibited in the presence of sodium azide. This is similar to spinach glyoxysomal APX that utilizes ascorbate as its most effective electron donor [11]. Salt stress imposed oxidative stress transiently up-regulated Am-pAPX1 levels in A. marina at 12 h of NaCl treatment, declining upon increased exposure to salinity. The accumulation of ROS during salt stress is mainly attributed to the inhibition of photosynthesis and a decline in CO2 fixation [6]. The effect of light-mediated oxidative stress on transcript levels of AmpAPX1 was analyzed in this study. Am-pAPX1 was highly expressed in leaves when dark-adapted A. marina seedlings were transferred to light. The transcript level of Hv-pAPX1 from barley is reported to increase in response to heat, salt and abscisic acid treatments [34]. AtAPX3 transcript levels were shown to increase slightly with cold, UV and treatments with H2O2 and paraquat [44]. VupAPX shows an increased transcript level in a desiccation-sensitive cowpea cultivar (1183), in response to rapid water loss and ABA treatment as compared to a desiccation-tolerant cultivar (EPACE-1) [4]. With H2O2 application, Am-pAPX1 transcript was expressed strongly at 6 h of treatment, the level rapidly declining thereafter. pAPXs are proposed to be type II (NcytosoleCmatrix) integral membrane proteins, located mainly on the cytosolic face of the membrane [15,23,24]. Under stress conditions, more H2O2 is generated and this may readily diffuse into the cytosol from the microbody matrix. Therefore, pAPX, located on the cytosolic face of the membrane may play an important role in removing H2O2 and protecting cells from oxidative damage. Excess free iron is thought to harm plant cells by enhancing the intracellular production of ROS [5]. APX is an iron containing enzyme and therefore different signals may be involved in iron-mediated induction of pAPX. Here, we show that the expression of Am-pAPX1 is specifically induced in response to iron overload and the transcript level drops upon withdrawal of iron from the medium. APX mRNA abundance has been reported to increase in response to excess iron in cotyledons of Brassica napus, bean and Arabidopsis [5,30,40]. In A. marina, total APX activity increased with salt (15%), light (26%), iron overload (10.5%) and H2O2 (10.5%) treatments at 6 h. APX activity is found to increase along with activities of other anti-oxidant enzymes like catalase, SOD, and GSH reductase in response to various environmental stress factors, suggesting that the components of ROS-scavenging systems are co-regulated [35]. MDHAR activity in A. marina leaf extracts also showed a marked increase in response to H2O2 treatment and iron overload but not to other stress treatments (data not shown). In Bruguiera parviflora, a mangrove species, ascorbate-mediated H2O2 degradation was enhanced 2.5 fold when plants were exposed to salt stress in the first week and 4.5 fold at 45 days of salt exposure [29]. Cytosolic fractions obtained from NaCl-tolerant pea cultivar cv. Granada show an increase in the activities of APX, GR, MDHAR, Mnsuperoxide dismutase (Mn-SOD) and DHAR while CuZnSOD activity remained constant. In contrast, in NaCl-sensitive

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pea (cv. Challis), salinity did not produce significant changes in APX, MDHAR and GR activities. Only DHAR activity was induced in cv. Challis, whereas soluble CuZn-SOD activity decreased by about 35% [7]. A differential increase in the activities of APX and CAT was reported by Mittova et al. [22] in leaf peroxisomes of salt-tolerant tomato (Lpa) plants. The genomic organization of Am-pAPX1 consists of nine exons interrupted by eight introns. The position of introns relative to the open reading frame is similar to that seen for the genomic sequences of At-cAPX1 [14], Pea cAPX1 [21] and AtpAPX3 [26] and differs from them in that it lacks an intron in the 50 UTR. OsAPX3 and OsAPX4, both putative pAPXs, show a similar genomic organization with nine exons and eight introns [39]. Am-pAPX1, (like other pAPXs), contains an intron that separates exons 2 and 3 and is absent in cAPXs. In addition, pAPXs have a final large exon (coding for the transmembrane domain and the peroxisomal targeting signal). In silico analysis of the 50 upstream region of Am-pAPX1 revealed the presence of a putative TATA box 92 bp upstream of the TSS of Am-pAPX1 and a putative CCAAT box 36 bp upstream from the TATA box. Putative cis-acting elements present in the 50 upstream region of Am-pAPX1 were identified using the PLACE program [8]. Potential regulatory sequences were identified in the promoter region including light regulatory elements (GATA, GT1 and I-boxes), dehydration responsive elements (MYB and MYC), heat, ABREs, EREs, CuREs and elicitor responsive elements. The precise contribution of these elements towards regulation of Am-pAPX1 expression needs further analysis. Excess reduction of the photosynthetic electron transport chain causes the generation of active oxygen species such as singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radical. The data above suggests that oxidative stress induced increase in the transcript of Am-pAPX1 and increase in total APX activity in leaves of A. marina might have a role in mitigating the deleterious effect of oxidative stress brought about by light, salt, H2O2 and iron overload. Acknowledgments This work was supported by a grant from the Department of Biotechnology, Government of India. Kumaresan Kavitha is a recipient of a grant for a Postdoctoral Program in Biotechnology and Life Sciences from the Department of Biotechnology, Government of India. The authors would like to thank Dr. Richard Trelease, Arizona State University, for a generous gift of the anti-cucumber pAPX antibody. The authors would also like to thank Ms Nandini Rangarajan, CCMB, Hyderabad for help with confocal imaging. References [1] K. Asada, The waterewater cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 601e639. [2] M.C. Ball, G.D. Farquhar, Photosynthetic and stomatal responses of the Grey mangrove Avicennia marina, to transient salinity conditions, Plant Physiol. 74 (1984) 7e11.

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