Molecular cloning and characterization of genes ...

ARTICLE IN PRESS Journal of Plant Physiology 166 (2009) 1646—1659

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Molecular cloning and characterization of genes encoding Pennisetum glaucum ascorbate peroxidase and heat-shock factor: Interlinking oxidative and heat-stress responses Ramesha A. Reddy, Bhumesh Kumar, Palakolanu Sudhakar Reddy, Rabi N. Mishra, Srikrishna Mahanty, Tanushri Kaul, Suresh Nair, Sudhir K. Sopory, Malireddy K. Reddy International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India Received 30 January 2009; received in revised form 2 April 2009; accepted 2 April 2009

KEYWORDS Ascorbate peroxidase; cDNA library; Electrophoretic mobility-shift assay; Heat-shock element; Heat-shock transcription factor

Summary The recent genetic and biochemical studies reveal a considerable overlap among cellular processes in response to heat and oxidative stress stimuli in plants suggesting an intimate relationship between the heat-shock response and oxidative stress responses. Pennisetum glaucum (Pg) seedlings were exposed to heat stress (42 1C for 0.5, 1.0 and 24 h) and a mixture of RNA from all the heat stressed seedlings was used to prepare cDNA. Full-length cDNA clones encoding for cytoplasmic ascorbate peroxidase 1 (PgAPX1) and heat-shock factor (PgHSF) were isolated by screening heat stress-specific cDNA library using corresponding EST sequences as radioactive probes. These full-length cDNAs were expressed in E. coli and their recombinant proteins were purified to near homogeneity. The recombinant PgAPX1 preferred ascorbate but did not accept guaiacol as a reducing substrate. Overexpression of PgAPX1 protects E. coli cells against methyl viologen-induced oxidative stress. Sequence analysis of PgAPX1 promoter identified a number of putative stress regulatory cis-elements including a heat-shock element (HSE). Heat-shock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Gel shift analysis and competition with specific and non-specific unlabeled DNA probes showed a specific interaction between HSE of PgAPX1 and the PgHSF protein. Expression analysis of PgHSF in Pennisetum showed maximum increase in transcript level in response to heat stress within 30 min of exposure and

Abbreviations: APX, ascorbate peroxidase; HSE, heat-shock element; HSF, heat-shock transcription factor; Pg, Pennisetum glaucum; ROI, reactive oxygen intermediates. Corresponding author. Tel.: +91 11 26741242; fax: +91 11 26742316. E-mail address: [email protected] (M.K. Reddy). 0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2009.04.007

ARTICLE IN PRESS Co-ordinated regulation of ascorbate peroxidase and heat-shock factor

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slowed down at subsequent time points of heat stress, indicating a typical characteristic of HSF in terms of early responsiveness. Expression of PgAPX1 significantly increased under heat-stress condition; however, the maximum expression observed at 24 h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42 1C) being maximum at 24 h and these trends are in conformity with the expression pattern of PgAPX1. Expression patterns and interactive specificity of HSF with HSE (PgAPX1) suggest a probable vital interlink in heat and oxidative stress signaling pathways that plays a significant role in comprehending the underlying mechanisms in plant abiotic stress tolerance. & 2009 Elsevier GmbH. All rights reserved.

Introduction Reactive oxygen intermediates (ROIs) such as hydrogen peroxide (H2O2), superoxide radicals (O 2 ), hydroxyl radicals (OH ) and singlet oxygen 1 ( O2) are continuously produced in plants as byproducts of various metabolic pathways localized in different sub-cellular compartments like chloroplasts, mitochondria and peroxisomes (Asada, 2006). The intracellular ROI production increases when plants are exposed to various environmental stresses and this excessive production of ROIs exceeds the detoxification capacity of the plant causing oxidative damage to proteins, DNA, lipids and other macromolecules (Møller et al., 2007). Among ROIs, H2O2 is the most stable form that acts as an oxidant and plays an important role in integrated cellular response against biotic as well as abiotic stress adaptations in plants (Mullineaux et al., 2006). However, being stable oxygen radical, H2O2 has a tendency to accumulate to toxic levels unless removed. Plants have evolved efficient scavenging system to get rid of excess H2O2, thus preventing oxidative stress damage. Ascorbate peroxidase (APX) together with superoxide dismutase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase constitute the ascorbate–glutathione cycle, which is considered the major defense system for effective deactivation of ROIs in multiple reduction/ oxidation reactions and protect the plant cells from oxidative damage. Based on the molecular and enzymatic properties, APX belongs to the class I peroxidase and has high specificity for ascorbate as an electron donor to reduce H2O2 to H2O and O2. This enzyme is very unstable in the absence of ascorbate and looses its activity within minutes particularly in the case of chloroplastic and mitochondrial isoforms (Leonardis et al., 2000). APX is encoded by multi-gene families and its different isoforms are classified according to their sub-cellular localization. Soluble isoforms are found in cytosol and chloroplast stroma, while

membrane-bound isoforms are found in peroxisomes, glyoxysomes and chloroplast thylakoids (D’arcy-Lameta et al., 2006). According to the phylogenetic analysis based on the deduced amino acid sequences of different isoforms of higher plant APXs, these can be grouped into four evolutionarily related classes: cytoplasmic APX1, cytoplasmic APX2, chloroplastic APX and membrane-bound APX (Teixeira et al., 2006). These four groups show 50–70% sequence homology with each other and 70–90% homology within each group across different plant species because the functional amino acid residues that are related to active site architecture, heme and ascorbate binding regions are evolutionarily conserved among all the isoforms. In some plants, the stromal and thylakoid membrane-bound isoforms of APX is encoded by single gene with alternate splicing of two exons at the 30 terminus of the gene (Yoshimura et al., 2002). The molecular mass of chloroplastic and membrane-bound APX are relatively larger than that of cytoplasmic soluble APX isoforms because of N-terminus extension of transit peptide and/or C-terminus extension of membrane spanning regions. The changes in transcript level and/or the enzymatic activity of APX in higher plants have been reported to be positively correlated with the tolerance to environmental stresses. Drought resistant maize shows a greater induction of APX activity as compared to its drought sensitive variety (Pastori and Trippi, 1992). Similarly, the salt tolerant pst1 mutant of Arabidopsis expresses high levels of APX and SOD activity as compared to the wild type when grown on NaCl containing medium (Tsugane et al., 1999). Plants showing reduced APX activity due to ascorbate deficiency (VeljovicJovanovic et al., 2001) or due to antisense RNA of ¨ var and Ellis, 1997) show more sensitivity to APX (O environmental stresses than their corresponding controls. The simultaneous monitoring of the expression of each isoform of APX in response to various environmental stresses in spinach indicates that the cytoplasmic APX up-regulates in response

ARTICLE IN PRESS 1648 to various stresses as compared to the chloroplastic isoforms (Yoshimura et al., 2000). The stress-specific expression of cytoplasmic APX is responsible for regulating the H2O2 concentration in the cytosol fraction which functions as a secondary messenger and subsequently plays a central role in stress signaling for co-ordinated expression of stress responsive genes including the expression of APX itself. An intimate relationship between the heat-shock response and oxidative stress response has been suggested (Suzuki and Mittler, 2006) and the heatshock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Pennisetum glaucum (Pg), a forage and grain crop of arid and semi-arid regions of the world and known as a stress-tolerant species has been used for the present study. In the present manuscript, we studied the interaction between heat-shock transcription factor (PgHSF) protein and HSE element of ascorbate peroxidase (PgAPX1) as both were found to be up-regulated in a heat-stressspecific cDNA library of Pennisetum. The PgAPX1 was cloned, expressed in E. coli and recombinant protein was purified and characterized. To verify that the interaction between HSF and HSE sequence in the PgAPX1 promoter is functional, we cloned and expressed the PgHSF in E. coli and purified the recombinant protein and performed electrophoretic mobility-shift assays. Relative changes in transcript levels of both PgAPX1 and PgHSF were monitored under heat-stress conditions to ascertain a potential correlation between the expression levels of HSF and APX1.

Materials and methods Plant materials and stress treatments Pearl millet [Pennisetum glaucum (L) R. Br] seeds were surface sterilized and grown in plastic pots filled with vermiculite under greenhouse conditions (14/10 h light/dark cycle at temperature 3072 1C). One set of 12 d-old seedlings was exposed to heat stress (42 1C) for the 0.5, 1.0 and 24 h and another set of seedlings was maintained under control conditions. After heat stress, leaves were excised, snap frozen in liquid nitrogen and stored at 80 1C until RNA extraction for subsequent cDNA library construction or real-time PCR analysis.

cDNA library construction Total RNA was isolated from Pennisetum leaves according to Chomczynski and Sacchi (1987) and

R.A. Reddy et al. poly(A) RNA was purified after annealing it to 50 biotinylated oligo-dT(18) primer and subsequently immobilizing it on streptavidin-linked paramagnetic beads followed by magnetic separation (Mishra et al., 2005). Five micrograms of mRNA was used to synthesize the cDNA library in UNIZAP vector using a cDNA synthesis kit (Stratagene, USA) following the manufacturer’s protocol.

Isolation of PgAPX1 cDNA clone One of the EST clones (CD726440) from our P. glaucum heat-stress responsive EST database (Mishra et al., 2007) that showed maximum homology to APX1 was used as a DNA probe for screening the Pennisetum cDNA library by plaque hybridization method. The cDNA insert of positive plaques was excised into pBluescript SK+ phagemid by in vivo excision following manufacturer’s instructions. The cDNA insert was completely sequenced (Acc#EF495352).

Expression of PgAPX1 in E. coli and purification of recombinant protein For cloning PgAPX1 cDNA in the pET-28a(+) expression vector, we designed two specific oligonucleotides, one for the N-terminus region, 50 CGAGTCCATATGGCGAAGTGCTACCCGAC-30 and the other for the C-terminus region, 50 -CTAGGATCCTTATGCATCAGCGAACCCCAG-30 based upon the complete cDNA sequence of PgAPX1 (Acc#EF495352). The 50 and 30 untranslated regions in the cDNA were removed and two restriction sites (NdeI at the translation initiation site and a BamHI site just downstream of the translation termination codon) were introduced. Using these primers the complete coding sequence for the PgAPX1 was PCR amplified (150 ng of each primer, 200 mM dNTPs, 2.5 units Taq DNA polymerase and 1 Taq buffer in a 50 mL reaction; 94 1C 1 min; 55 1C 1 min and 72 1C 1 min; 30 cycles) using total cDNA as template. The amplified product was gel purified, digested with NdeI and BamHI and cloned into pET-28a(+) expression vector. The sequences adjoining the 50 and 30 ends of the cloned segment were confirmed by sequencing. This construct resulted in the expression of PgAPX1 polypeptide with additional hexa-histidine tag at the N-terminus. The recombinant pET-28a(+) plasmid with PgAPX1 cDNA were transformed into BL21 (DE3) cells, and grown in LB-medium supplemented with 50 mg/mL kanamycin at 37 1C. When absorbance at 600 nm of the culture reached a value of 0.6, isopropyl b-D-thiogalactopyranoside (IPTG) was

ARTICLE IN PRESS Co-ordinated regulation of ascorbate peroxidase and heat-shock factor added at a final concentration of 1 mM and the culture was grown for another 4 h. The recombinant APX1 protein was purified near to homogeneity by Ni-NTA column chromatography following the manufacturer’s instructions (Qiagen, Germany).

Oxidative stress tolerance of E. coli Overnight cultures of E. coli BL21 (DE3) cells transformed with pET-28a(+) (vector control) or with pET-APX plasmids were grown in fresh LBmedium containing 50 mg/mL kanamycin. When the absorbance at 600 nm reached a value of 0.15, varying concentrations of methyl viologen (0.05–0.4 mM) and 0.4 mM IPTG were added simultaneously and the cultures were kept at 37 1C for 12 h in a shaking incubator and cell growth was monitored by measuring the absorbance at 600 nm.

Characterization of recombinant PgAPX1 activity APX activity was estimated by measuring the amount of ascorbate oxidized using extinction coefficient for ascorbate (e290 ¼ 2800 M1 cm1) (Nakano and Asada, 1981). Enzyme activity was calculated in terms of units mg1 protein min1 (1 unit: amount of enzyme required for the oxidation of 1 mmole of ascorbate in 1 min). The oxidation of alternate substrates was measured by replacing ascorbate with 20 mM pyrogallol or 10 mM guaiacol. Reaction was initiated by the addition of 0.1 mM H2O2 and substrate oxidation was followed by the decrease in the absorbance at 430 nm for pyrogallol and 470 for guaiacol. Amount of substrate oxidation was calculated using extinction coefficient for pyrogallol (e430 ¼ 2470 M1 cm1) and guaiacol (e470 ¼ 5570 M1 cm1) and Km was calculated for each substrate. To determine the pH optima for the maximum PgAPX1 activity, different buffers, viz., 50 mM sodium acetate (pH 4.0–5.0), MES (pH 5.5–6.5) and Tris (pH 7.0–90) were used and the rates of ascorbate oxidation was estimated under the standard assay condition. To estimate the thermo-stability of recombinant PgAPX1, it was pre-incubated at different temperatures (0, 15, 25, 37, 45 and 55 1C) for 30 and 60 min then the rates of ascorbate oxidation under the standard assay condition were estimated. To identify the potent PgAPX1 inhibitors, varying amounts of inhibitors such as salicylic acid or hydroxylamine were supplemented in standard reaction mixture. The Ki (amount of inhibitor required to reduce the APX activity to 50% of its optimum activity) value was calculated for each inhibitor.

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Cloning of the 50 -flanking region of the PgAPX1 gene Genomic DNA was isolated from P. glaucum leaves using the CTAB method (Lodhi et al., 1994). The 50 flanking region of the PgAPX1 gene was PCR amplified according to Reddy et al. (2008) using a gene specific primer (50 -CTTCTCGACGGCCTCCTGGTACTC-30 ), synthesized in antisense orientation and the T7 primer. PCR was carried out using 150 ng each of the gene specific and T7 primers along with 200 mM of each dNTPs and 2.5 units of Taq DNA polymerase with 20 ng of P. glaucum genomic DNA as template in a 50 mL reaction. PCR conditions were 94 1C, 1 min; 55 1C, 1 min and 72 1C, 1 min for 30 cycles. The amplification product was gel purified and cloned into TopoTA vector (Invitrogen). The insert DNA was completely sequenced (Acc#EU492461).

Cloning of full-length cDNA encoding for HSF from P. glaucum We used radio-labeled P. glaucum EST clone (CD725302) that showed sequence homology with heat-shock factor (HSF) (Mishra et al., 2007) as a DNA probe and screened the Pennisetum cDNA library by the plaque hybridization method as mentioned earlier and the cDNA insert of the positive clone was sequenced (Acc#EU492460).

Expression of PgHSF in E. coli and purification of recombinant protein The PgHSF cDNA was cloned into NheI and NotI sites in the pET-28a(+) expression vector using sequence specific primers (50 -GGCTAGCAATGGAGGCGGGCGGCGGG-30 and GATGCGGCCGCGGAATCATACAAGTTGAGA-30 ) and the complete coding sequence for the PgHSF from the Pennisetum cDNA was amplified by PCR as mentioned earlier. The sequences adjoining the 50 and 30 ends of the cloned segment were confirmed by sequencing. This construct resulted in the expression of PgHSF polypeptide with additional hexa-histidine tag at the N-terminus. The recombinant pET-28a(+) plasmid with PgHSF cDNA were transformed into BL21 (DE3) cells and the over expression of recombinant PgHSF was induced with 1 mM IPTG as described earlier. The recombinant heat-shock factor was purified to near homogeneity on a NiNTA column following the manufacturer’s instructions (Qiagen, Germany).

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DNA probes and gel mobility-shift assay Two pairs of partially complementary oligonucleotides were synthesized for Wild (W-HSE1 and WHSE2) and mutated (M-HSE1 and M-HSE2) HSEs based on the sequence information from the PgAPX1 promoter (Table 1). One microgram of each of these partially complementary oligonucleotides were annealed separately by incubating at 72 1C for 30 min for filling complementary strand using Taq DNA polymerase in 1 Taq buffer containing 200 mM dNTPs (without dCTP) and 1 mL of [a32P]dCTP for both wild and mutated HSE separately. The radio-labeled double stranded oligonucleotide probes (W-HSE and M-HSE) were purified using Sephadex G50 spun column. The protein–DNAbinding reactions were carried out using labeled DNA probe (20,000 cpm), 1 mg of poly dI-dC and 0.3–1.0 mg of recombinant PgHSF were incubated in binding buffer (50 mM Tris–HCl, pH 7.3, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5% sucrose and 5% glycerol) in a total volume of 20 mL then the samples were incubated for 20 min at room temperature (Storozhenko et al., 1998). Competition assay was carried out using 10 , 5 unlabeled homologous probe or 50 non-specific double stranded oligonucleotide probe and reaction mix was incubated for 10 min at room temperature prior to adding labeled probe. For

Table 1.

heterologous competition, non-specific unlabeled probe was used in the same concentration as for homologous competition. The DNA–protein complexes were fractionated on 1.3% agarose gel in 0.5 TBE and 5% glycerol buffer.

Real-time PCR analysis of PgAPX and PgHSF transcripts The real-time quantitative PCR amplification of either PgAPX1 and/or PgHSF along with Pg-b tubulin as an internal housekeeping standard was performed with specific oligonucleotide primers (Table 2), using the first strand cDNA synthesized from RNA samples collected from 12-d-old Pennisetum seedlings exposed to heat stress (42 1C for 0.5, 1.0 and 24 h) along with control seedlings, in the presence of SYBR-GreenR using Icycler (BioRad, USA). At the end of the PCR cycles, the products were put through a melt curve analysis to check the specificity of PCR amplification. The experiments were repeated at least five times independently and the data were averaged. The relative change in expression levels of PgAPX1 and PgHSF transcripts in response to heat stress was estimated using REST software (Pfaffl et al., 2002) with b-tubulin as a reference gene.

Oligonucleotide sequence of wild type and mutated heat-shock factor binding sequences.

Name

Nucleotide sequence

W-HSE1 W-HSE2 M-HSE1 M-HSE2

50 -CGCCCTACGATAAGCTTACCTTTTTCCAGTCC-30 50 -GAGGACGCGGAGGACTGGAAAAAGGTAAGC-30 50 -CGCCCTACGAGAAGCTTCCCTTTTTCCAGTCCTCCGCGTCCTC-30 50 -GCGGGATGCTCTTCGAAGGGAAAAAGGTCAGGAGGCGCAGGAG-30

W-HSE (double stranded)

50 -CGCCCTACGATAAGCTTACCTTTTTCCAGTCCTCCGCGTCCTC-30 30 -GCGGGATGCTATTCGAATGGAAAAAGGTCAGGAGGCGCAGGAG-50

M-HSE (double stranded)

50 -CGCCCTACGAGAAGCTTCCCTTTTTCCAGTCC-30 50 -GAGGACGCGGAGGACTGGAAAAAGGGAAGC-30

HSE region is underlined and the mutated nucleotides are shown in bold.

Table 2.

Gene specific primer sequences used for real-time PCR analysis.

Primer name

Primer sequence (50 –30 )

PgAPX forward PgAPX reverse PgHSF forward PgHSF reverse PG-b tubulin forward Pg-b tubulin reverse

GTCGCCTTCCTGATGCTAC AAGTTCCTTGAAGTAAGAGTTGTC TGAGGGAGGCGAAGATATGATAC GAAGAAGAATGTGGAACAGATGAC ACGATATACCACCACCACCAC CGGACGAAAGGACCTCACC


In gel activity of APX The extraction for the enzymes was done as suggested by Larkindale and Huang (2004). Leaves (0.25 g) were harvested and ground to a fine powder in liquid nitrogen. The ground powder was homogenized in 1.5 mL of cold phosphate buffer (100 mM, pH 7.0) containing 1% polyvinylpyrrolidone (PVP), 2 mM L-ascorbate and 1 mM EDTA and centrifuged at 4 1C for 15 min at 10,000g. Samples were electrophoresed using PAGE under non-reducing, non-denaturing conditions at 4 1C according to the method suggested by Laemmli (1970). Electrode buffer containing 2 mM ascorbate was used and the gel was pre-run for 30 min before the samples were loaded. After electrophoresis, gels were incubated for 15 min at room temperature in 100 mM potassium phosphate (pH 6.4) containing 4 mM ascorbate and 4 mM H2O2. The gel was washed with distilled water and stained for 10 min at room temperature with a solution of 0.125 M HCl containing 3 mM ferricyanide and 3.5 mm ferrichloride. After development of achromatic bands against the dark blue background, gel was photographed using a white illumination tray.

Sequence analysis Most of the routine sequence (DNA and protein) analyses were performed using MacVector (v 9.0.2; Mac Vector Inc., USA). Homology searches were done using BLAST.

Results and discussion Isolation and characterization of full-length cDNA encoding for PgAPX A full-length cDNA clone was isolated by screening cDNA library (subtracted against heat stress) using PgAPX1 EST (Acc#CD726440) as a radioactive probe. PgAPX1 (Acc#Acc#EF495352) was 1080 bp in size and contained an open reading frame of 753 bp with a 87 bp 50 and 240 bp 30 untranslated regions (UTRs) which encoded for a protein of 250 amino acids (Figure 1A). This protein had an apparent molecular weight of 27.5 kDa with an estimated isoelectric point of 5.62. The homology search was carried out using the deduced amino acids sequence of PgAPX1 against the translated non-redundant nucleotide database which showed an overall 85–93% sequence identity with APX1 isolated from Zea mays (BT016458), Hordeum vulgare (AJ006358), Oryza sativa (Acc#AY254495), Gossypium hirsutum (Acc#EF432582) and Arachis hypo-

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gaea (Acc#EF165068). PgAPX1 contained only a common core catalytic region without any organelle-specific N-terminus transit peptide sequence or a C-terminus transmembranous region found in membrane-bound APX isoforms, suggesting that PgAPX1 is a cytosolic soluble APX. There are more than one cytoplasmic APX isoforms that exist in plant systems (Panchuk et al., 2002; Shigeoka et al., 2002). Phylogenetic analysis based on the deduced amino acid sequences of PgAPX with amino acid sequences of other plant APXs revealed that PgAPX belongs to the cytoplasmic APX1 evolutionary lineage (Figure 1B). The catalytic region of PgAPX1 consisted of two typical functional domains. First is an active site domain, with a conserved histidine (His-42) that acts as an acid–base catalyst in the reaction between H2O2 and the APX and the other site is a heme-binding domain (Figure 1A). The amino acid residues (His-42, Arg-38, Glu-65, His-163 and Asp-208) that are actively participating in the hydrogen-bonding network in the active site geometry observed in all classes of peroxidases were also found in the PgAPX1. The amino acid Trp-179 that is conserved in PgAPX1 similar to that of all other class I peroxidases whereas, in class II and class III peroxidases, Trp-179 is replaced by phenylalanine. The peptide segments 69–72 (XANX), 131–135 (LPDAX), and 171–175 (ERSGF/W) that form the surface of the active site entrance channel near the heme edge and assumed to be essential for the interaction of ascorbate with APX is also conserved in PgAPX with identical spatial distribution. The key basic amino acid residues (Arg-172 and Lys-30) involved in binding of the (anionic) ascorbate molecule to the active site of APX (Sharp et al., 2003), is also conserved. The structural characteristics of PgAPX discussed above suggest that it belongs to the class I, soluble cytosolicAPX1.

Oxidative stress tolerance in E. coli Methyl viologen, a redox-cycling agent, widely used as a source of superoxide radical in a variety of experimental systems, was used to create oxidative stress in E. coli by supplementing with different concentrations of methyl viologen (0.05–0.4 mM) in the culture medium. Growth of bacterial cells decreased with increasing concentration of methyl viologen in untransformed E. coli. To check if oxidative stress tolerance can be improved by increasing the levels of H2O2 scavenging enzymes as reported earlier (Sohal et al., 1995), we over-expressed PgAPX1 in E. coli. The growth of E. coli over-expressing PgAPX1 showed


R.A. Reddy et al.

1

MAKCY PTVSAEYQ EAVEKARRKLRALIAEKSCAP LMLRLAWHSAG

46

T FDVSTKTGGP FGTM KNPA E QAHGANAGLDIAVRMLEPVKEEFPI

91

LSYA DLYQLAGVVAVEVTGGPE I PFHPGREDKPQPPPEG RLPDAT

136

KGSDHLRQVFGKOMGLSDQDIVALSGGHTLGRCHKERSGFEGPWT

181

RN PLVFDNSYFKEL LTGDKEGLLQLPS D KTLLSDPVFRPLV EK YA

225

ADEKAFFDDYKEAHLRLSELGFADA 0.081 0.013

Arabidopsis thaliana (CAA42168) 0.074

0.007 0.034

Zea mays (CAA84406) 0.040

Lycopersicon esculentum (AAZ77771)

0.040

Nicotiana tabacum (BAA12918)

0.083

0.011

Glycine max (BAC92739) 0.050

0.033

0.050

Pennisetum glaucum (EF495352) Oryza sativa (P93404)

Bacterial growth at A600 nm

1

2

97

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2.0

66

1.6 45 1.2 33

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20

Concentration of methyl viologen (mM)

Figure 1. Deduced amino acid sequences of PgAPX1 (A). The active site domain is shown in ‘bold and italics’ and the haem-binding domain is shown in ‘bold’. The amino acid residues that actively participate in hydrogen bonding network in the active site geometry are underlined. (B) Phylogenetic analysis of APX from different sources. (C) Oxidative stress tolerance of E. coli cells transformed with PgAPX1. (D) SDS-PAGE showing the purified recombinant PgAPX1(lane 2) and the molecular weight markers (lane 1).

increased growth compared to untransformed cells at different concentrations of methyl viologen (Figure 1C). Methyl viologen enhances the production of superoxide radicals which can be converted into H2O2 by the action of superoxide dismutases. Insufficient scavenging of the H2O2 results in its accumulation in untransformed E. coli while in PgAPX1 transformed E. coli, efficient scavenging of H2O2 might be responsible for the improved growth of the cells.

Purification and characterization of recombinant PgAPX1 Different isoforms of APX have been identified in plants and all these isoforms are expressed within

the same cell. In addition, multiple genes exist for each isoform in plants and they are expressed differentially in response to environmental and/or developmental cues. Therefore, it is very difficult to purify and characterize these different isoforms of APX from plant extracts. In order to obtain large amount of highly purified APX, we have utilized the heterologous system for high-level expression of PgAPX1 in E. coli. The majority of the recombinant protein was in the soluble fraction of the E. coli lysate. The recombinant PgAPX protein was purified to near homogeneity (Figure 1D) from clarified E. coli lysate by passing through Ni-NTA agarose beads, as the recombinant protein possessed a hexa-histidine tag at its N-terminus. The activity of recombinant PgAPX1 was estimated by measuring the amount of oxidized


80

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LT50 = 40.1°°C

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pH optimum 6.6

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LT50 = 28.2°C

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M1 cm1) (Figure 2). The recombinant PgAPX1 did not accept guaiacol as a reducing substrate (Figure 2E) whereas APX from pea and potato were reported to accept guaiacol as a reducing substrate

Residual activity (%)


ascorbate using extinction coefficient for ascorbate (e290 ¼ 2800 M1 cm1) and also the oxidation of other electron donor substrates like pyrogallol (e430 ¼ 2470 M1 cm1) and guaiacol (e470 ¼ 5570

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Figure 2. Biochemical characterization of recombinant PgAPX1. Effect of pH (A), temperature (B), different substrates, ascorbate (C), pyrogallol (D) and guaiacol (E), and inhibitors salicylic acid (F) and hydroxylamine (G) on PgAPX1 activity.


R.A. Reddy et al.

(Shigeoka et al., 2002). The Km value for ascorbate was found to be 70 mM (Figure 2C) while it was 248 mM for pyrogallol (Figure 2D) suggesting that the recombinant protein preferred ascorbate as reducing substrate. The PgAPX1 enzyme is acted within a pH range of 6.0–7.0 with maximum activity at 6.6 (Figure 2A). Enzyme activity reduced to 50% when the pH of the reaction was below pH 5.6 or above pH 7.8. Recombinant PgAPX1 lost 50% of its activity when pre-incubated at 28 1C for 60 min or 40 1C for 30 min before estimating the oxidation of ascorbate under the standard assay condition (Figure 2B). The PgAPX1 enzyme activity was inhibited by the general peroxidase inhibitors, which interact with the heme group of the APX. We assayed the PgAPX1 activity in the presence of salicylic acid and found that the peroxidase activity was inhibited by salicylic acid with a Ki value of 190.5 mM (Figure 2F). Another compound, hydroxylamine inhibited the PgAPX1 activity more effectively with a Ki value of 30.8 mM (Figure 2G). Salicylic acid plays an important role in plant defense against pathogen attack by inhibiting H2O2 scavenging enzymes and thereby promoting hypersensivity response (Durner and Klessig, 1995).

TACTC-30 ) synthesized in the antisense orientation. Approximately, a 0.85 kb region of genomic DNA fragment was PCR amplified and the sequence analysis showed 131 nucleotides (77 nucleotides in the 50 UTR region and 54 nucleotides in the translated region of the cDNA) that overlap with PgAPX1 cDNA sequence. In silico analysis of this promoter sequence identified a number of putative stress regulatory cis-elements like heat-shock element (HSE) for interacting with HSF, GC-motifs for anoxic stress responsive expression, Sp1 and G-box motifs for light responsive expression, TGACGmotif for methyl jasmonate responsive expression, LTR element for low-temperature responsive expression and TCA element for salicylic acid responsive expression (Figure 3) in addition to core sequences of ABRE elements for ABA responsive expression and MYB transcription factor binding regions were also found. Sequence comparison of APX promoter from Pennisetum with other plant APX promoters identified one common motif with high homology to HSE. The promoter analysis as well as over-expression studies of HSFs in Arabidopsis suggest that the HSE motif present in APX1 promoter interact with HSFs (Panchuk et al., 2002).

Isolation and analysis of 50 flanking region of PgAPX1 gene

Isolation and characterization of full-length cDNA encoding for HSF from P. glaucum

We have cloned the promoter of the PgAPX1 by PCR-based directional genome walking (Reddy et al., 2008) using P. glaucum genomic DNA and a specific primer (50 -CTTCTCGACGGCCTCCTGG-

In order to verify that the HSE sequence identified in the PgAPX1 promoter is functional to interact with HSF, we cloned PgHSF, expressed it in E. coli and purified the recombinant protein. The

Figure 3. The 50 flanking genomic region of PgAPX1 gene. The coding sequence of PgAPX1 is shown in lower case and the 50 UTR region is shown in italics. The arrow indicates the transcriptional start site. Putative regulatory elements Tata-box, HSE, GC-motifs, Sp1-motif, TGACF-motif, G-Box, LTR-motif and TCA-motif are shown by underlines.


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full-length PgHSF cDNA clone was isolated by screening heat stress-specific cDNA library with its EST clone (CD725302) as a radioactive probe. The complete nucleotide sequence of the cDNA is 1822 bp with an open reading frame of 1308 bp flanked by 177 bp 50 and 337 bp 30 UTRs. The deduced protein is composed of 435 amino acids (Figure 4A) with an apparent molecular weight of 48.7 kDa and an estimated isoelectric point of 5.14. However, this recombinant protein migrated slower in SDS-PAGE due to its low isoelectric point and appears to be a 60 kDa protein (Figure 4B) as suggested by Vickers et al. (2004). The amino acid sequence analysis revealed that the encoded protein has high homology with a class-A HSF of O. sativa homolog (HSF9) and has all the fingerprint motifs that are characteristics of a class-A HSFs. It contains a DNA-binding domain (11–105 amino acids) that is most conserved among eukaryotic HSFs and a helix–turn–helix motif (H2–T–H3) that allows the specific recognition and binding of the palindromic (nGAAn/nTTCn) sequences of HSEs. Downstream of the DNA-binding domain, a hydrophobic repeat HR-A/B region (118–184 amino acids) that comprises the oligomerization domain was also present. It is thought that the primary role of the HR-A/B is to provide hydrophobic surfaces for HSF trimerization. Towards the C-terminus of the protein there is an AHA motif (DGFWEQFLTE) enriched with aromatic, hydrophobic and acidic amino acids. The presence of an AHA domain in PgHSF indicates that it is likely a transcriptional activator (Doring et al., 2000). PgHSF contains both a nuclear localization signal

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Figure 4. The deduced amino acid sequences of PgHSF (A). The DNA-binding site is shown in ‘bold’; the oligomerization domain is ‘italics’; the nuclear localization region is underlined; nuclear export signal is ‘double underline’ and the transcriptional activation domain is shown in ‘bold and underline’. (B) SDS-PAGE showing the purified recombinant PgHSF (lane 2) and the molecular weight markers (lane 1).

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Nonspecific DNA Unlabelled HSE PgHSF protein (µ µg) Lane number

Figure 5. Analysis of in vitro interaction between PgHSF with wild and mutated HSE sequences of PgAPX1 promoter by electrophoretic mobility-shift assay. Purified recombinant PgHSF is mixed with a radioactive labeled synthetic DNA fragment containing the 43-nucleotide sequence that encompass HSE motif from PgAPX1 promoter (wild type as well as mutated) and the DNA–protein complexes were analyzed by 1.3% agarose gel electrophoresis followed by autoradiography. Lanes 1–4 and 9–11 wild-type HSE (W-HSE), and lanes 5–8 mutated HSE (M-HSE).


In vitro interaction of PgHSF with HSE motif of PgAPX Pennisetum HSF was expressed in E. coli and recombinant protein was purified to near homogeneity. In vitro analysis of HSE–HSF binding has been carried out by electrophoretic mobility-shift assay. Radio-labeled double stranded 43-mer synthetic oligonucleotide (W-HSE) encompassing the HSE sequence (Table 1) was incubated with an increasing amount of recombinant PgHSF protein and was run on 1.3% agarose gel. Results showed that in the presence of the PgHSF, there was a shift in the band and intensity of the shifted band was dependent of the PgHSF concentration (Figure 5, lanes 2–4). To check the specificity of the interaction, mutated HSE (M-HSE) was used in place of WHSE which led to non-binding of PgHSF as shown by an absence of shift (lanes 6–8). This shows that the replacement of G base in the nGAAn basic HSE motif has a deleterious effect on the HSF binding in vitro (Fernandes et al., 1994). Although in vivo HSF binds to HSE as a trimer, trimerization only increased the affinity of HSF to DNA. However, the monomeric HSF or the DNA-binding domain of the HSF was also able to specifically bind to the HSE DNA sequences in vitro (Flick et al., 1994). The presence of two different DNA–protein complexes (Figure 5) suggested that probably the other higher oligomeric forms also interact with the HSE sequences and show different DNA–protein complexes in the gel mobility-shift assay. Gel shift analysis and competition with specific and nonspecific unlabeled DNA probes, showed that the HSE–HSF complexes were insensitive to increasing amounts of non-specific competitor nucleic acids, indicating a specific interaction between HSE and the PgHSF (Figure 5, lanes 9–11). The specific interaction of PgHSF with HSE present in the PgAPX promoter may regulate the expression of APX transcript as reported in Arabidopsis (Storozhenko et al., 1998).

Co-ordinated expression of PgHSF and PgAPX1 transcript under high temperature The electrophoretic mobility-shift assay conducted with the recombinant PgHSF protein along with the HSE of APX promoter suggests that PgHSF may have a regulatory role in PgAPX1 gene expression. Recent genetic and biochemical studies also suggest an intimate relationship between the

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(KKRRLP) as well as a nuclear export signal (TEKLGHL) (Figure 4A).

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Figure 6. Analysis of relative transcript levels of PgHSF and PgAPX1 under heat stress (42 1C) condition (A). Standard error bars are shown. In gel PgAPX1 protein activity (B).

heat-shock response and oxidative stress response (Suzuki and Mittler, 2006) and the steady-state level of HSFs not only increases in response to heat stress but also in response to oxidative stress. To verify the correlation between these two responses, we monitored the co-expression of PgHSF along with PgAPX1 under heat stress (42 1C) at various time points using real-time PCR. After 30 min of heat stress, the PgHSF transcript increased by 10.6 folds compared to that of unstressed plant whereas increase in PgAPX1 transcript was only 2 folds. After 1 h heat stress, expression level of PgHSF was found to be 6.3 fold as compared to control plants while it was 2.5 folds in case of PgAPX1. However, this trend was reversed at 24 h of heat stress where PgAPX1 transcript was found to be 7 folds while it was 2.1 folds only in case of PgHSF (Figure 6A). The upregulation of PgHSF within 30 min of heat stress is characteristic of a typical heat-shock transcriptional factor. The relative expression level of PgAPX1 initially showed slow up-regulation but later peaked at 24 h stress. In the light of the results obtained from the interaction experiment between HSF and HSE (Figure 5), it can be inferred

ARTICLE IN PRESS Co-ordinated regulation of ascorbate peroxidase and heat-shock factor that during early period of heat stress, PgHSF transcript increases and in due course HSF protein binds to HSE of APX and activates its transcription as can be seen at 24 h heat-stress point (Figure 6A). To support the above findings, we also checked in gel activity of APX protein in the soluble fraction of Pennisetum leaves under heat stress (42 1C). Results showed that APX activity was up-regulated in response to heat stress as compared to unstressed plants and the up-regulation was maximum at 24 h time point (Figure 6B). These results are well in tune with the transcript analysis (Figure 6A) where the maximum up-regulation in transcription of PgAPX1 was also found to be at 24 h time point. Together, we can sum up that PgHSF protein interacts with HSE of PgAPX1 leading to its upregulation under heat stress as can be seen at the APX activity level. There is a considerable overlap in cellular processes during response to heat and oxidative stress stimuli. Transcriptome analysis of Arabidopsis has revealed the involvement of factors other than classical heat-stress responsive genes in thermotolerance (Kotak et al., 2007). The induction of heat-stress responsive expression of target defensive genes is attributed to HSE present in their promoter region. HSEs are the binding sites for HSFs for modulated expression of target defensive genes in response to heat stress. The differential up-regulation and/or activation of HSFs occur not only in response to heat but also in response to oxidative stress and other abiotic stresses. Volkov et al. (2006) reported an accumulation of H2O2 in Arabidopsis cell culture under heat-stress conditions and suggested that H2O2 is involved in HSF activation during the early phase of heat stress. The accumulation of H2O2 stimulates the HSFs from an inactive monomer to an active trimer in a reversible redox-regulated manner (Miller and Mittler, 2006). Further, it was demonstrated in animal systems that the two-cysteine residues located within and near the DNA-binding domain of HSF participate in intra-molecular disulfide bond formation in a redox-regulated reversible manner. The conformational changes induced by these non-native disulfide bonds in inactive monomeric HSFs can serve as a signal for the formation of an active trimeric form that induced an oxidative stress-mediated heat-shock response (Ahn and Thiele, 2003). HSFs play a dual role in mediating the overlapping cellular processes in response to heat and/or oxidative stress stimuli. Compared with other eukaryotes, the plant HSF family shows a striking multiplicity with more than 20 members (von Koskull-Do ¨ring et al., 2007). Despite many conserved features, members of the

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HSF family show a strong diversification of spatial and temporal expression patterns and functions (Baniwal et al., 2004). HSF-dependent gene expression is not limited to known stress genes involved in protection from proteotoxic effects but is also known to co-regulate other pathways and mechanisms dealing with a broader range of physiological adaptations to stress (Busch et al., 2005) which need to be understood in depth and will be the focus of future research.

Conclusions cDNA clones that encode for APX1 and HSF has been isolated from heat stress-specific cDNA library of P. glaucum. The nucleotide sequence of PgAPX1 and its deduced amino acid sequence analysis revealed that PgAPX is a member of cytosolic, soluble, class I peroxidase. The heterologous expression of this cDNA clone in E. coli produced enzymatically active recombinant APX protein, which was purified to near homogeneity. The biochemical characterization of this recombinant protein further demonstrated the enzymatic properties similar to typical APX from other plants. Sequence analysis of 50 -flanking region of the PgAPX1 gene revealed the presence of one HSE site. In vitro study confirmed specific interaction between PgHSF protein and HSE of PgAPX1. Further, relative transcript analysis of PgHSF and PgAPX1 under heat stress suggests that the HSE motifs may be responsible for stress-specific expression of PgAPX1 by interacting with PgHSF. Results together indicate that HSFs play a central role in mediating overlapping cellular processes especially those that involve oxidative stress.

Acknowledgements Grant from Department of Biotechnology (Government of India) and fellowship to Ramesha from CSIR (India) is acknowledged.

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