ISOLATION OF GRAPE PEROXIREDOXIN GENE RESPONDING

TO ABIOTIC STRESSES 1

© 2015 R. Haddad, R. H. Japelaghi

Department of Agricultural Biotechnology, Faculty of Engineering and Technology, Imam Khomeini International University, Qazvin, Islamic Republic of Iran

Received November 22, 2014

Peroxiredoxins (Prxs) are peroxidases that reduce hydrogen peroxide (H2O2) and various alkyl hydroperoxides and act as reductants. A full-length cDNA encoding for a Prx polypeptide was isolated and cloned from grape (Vitis vinifera L. cv. Askari) berries. The cDNA was 773 nucleotides in length with a deduced amino acid of 162 residues, possessing one conserved cysteine, which belongs to the type II Prx C. The calculated molecular mass and the predicted isoelectric point of the deduced polypeptide are 17.26 kD and 5.15, respectively. The deduced protein sequence showed a high similarity to PrxII C from other plants, in particular from cotton (Gossypium hirsutum), poplar (Populus trichocarpa), and Citrus sP. The in silico analysis of the promoter region of grape Prx demonstrated the presence of a number of potential cis-acting elements to respond to environmental signals, suggesting that VvPrxII C may respond to a variety of environmental signals, including dehydration, heat, heavy metals, light, pathogens, wounding, and plant hormones. The grape Prx gene was also analyzed for its expressional response to abiotic stress, oxidative stress, and antioxidants application. The results revealed a highly induced response to abiotic stress conditions due to the presence of different putative regulatory elements in its promoter.

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1 This text was submitted by the authors in English.

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Abbreviations: APX  ascorbate peroxidase; CAT  catalase; CTAB  cetyltrimethylamonium bromide; DHAR  dehydroascorbate reductase; GST  glutathione-S-transferase; HSE  heat stress responsive element; MBS  MYB binding site; MRE  metal responsive element; MYB  myeloblastosis; ORF  open reading frame; POX  peroxidase; Prx  peroxiredoxin; RWC  relative water content; SA  salicylic acid; SOD  superoxide dismutase; Trx  thioredoxin.

Corresponding author: Raheem Haddad. Department of Agricultural Biotechnology, Faculty of Engineering and Technology, Imam Khomeini International University, Qazvin, Islamic Republic of Iran; fax: +98281 33901175, e-mail: raheemhaddad@yahoo.co.uk

Keywords: Vitis vinifera  abiotic stress  antioxidants  oxidative stress  peroxiredoxin

 

INTRODUCTION

Peroxiredoxins (Prxs) catalyze the reduction of hydrogen peroxide (H2O2) and various alkyl hydroperoxides via catalytic cysteine and thiol-containing proteins that act as reductants [1]. The importance of these enzymes is underlined by their presence in all groups of organisms, high abundance and involvement in multiple cellular processes such as antioxidant defense, H2O2-mediated cellular signaling [1] and molecular chaperones [2]. In plants, based on amino acid sequence similarities and specific structural features, mainly the number and position of conserved Cys residues, the Prxs can be divided into four different classes: (1) 2-Cys Prx; (2) Prx Q; (3) Prx II, which all contain two catalytic Cys residues in distinct sequence environment, and (4) 1-Cys Prx with one conserved Cys residue only [3]. A phylogenetic distance analysis suggests that 2-Cys Prx, Prx Q, and 1-Cys Prx are related proteins, whereas the group of Prx II is likely to have evolved independently [4].

The first Prxs were identified from bacteria such as Salmonella typhimurium and Escherichia coli [5]. Since then, they have been isolated from many organisms. For example, there are at least five Prx isoforms in yeast (Saccharomyces cerevisiae) and six isoforms characterized in mammalian cells [6]. The Prx family has been also intensively studied in different plants. In Arabidopsis thaliana genome there are 10 ORFs (open reading frames) with sequence similarity to Prxs that can be assigned to the four well-known groups of Prxs: two ORFs code for 2-Cys Prx, one  for 1-Cys Prx, one  for Prx Q, and six  ORFs for Prx II [3, 4]. One of the reasons explaining this multiplicity is the multiple subcellular localization of these proteins in different organisms including the chloroplasts, cytosol, peroxisomes, mitochondria, and possibly the nucleus [3].

The Prxs have been also reported to be involved in response to abiotic stress conditions, such as divergent light, drought, heat, salinity, heavy metals, and plant hormones [711]. Kim et al. [2] reported that dual functions of Chinese cabbage 2-Cys Prx acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying by its structural changes. In addition, overexpression of Prx genes in tall fescue (Festuca arundinacea) [12] and yeast (S. cerevisiae) [11] increased tolerance to abiotic stress conditions compared with the control plants. Moreover, studies indicated that expression levels of Prx genes are differentially modified under different stress conditions [4, 10], suggesting unique roles for Prxs in plants during various aspects of the plant life cycle.

We described here the isolation and characterization of a Prx of the type C from grape berry and showed that this gene differentially expressed in different organs. Using in silico analysis, we indicated that the promoter region of PrxII C gene contained a large number of putative regulatory elements and respond to a wide variety of environmental signals. Moreover, we revealed that PrxII C gene is differentially induced upon abiotic stress conditions.

 

MATERIALS AND METHODS

Plant materials and extraction of total RNA. One-year-old cuttings with different organs of grape (Vitis vinifera L. cv. Askari) including berries, leaves, clusters, petioles, stems, tendrils, buds, roots, and seeds, were prepared from plants in the field collection of the Grape Research Station, Takistan-Qazvin, Iran, during the 2010 field season. The cuttings were grown in pots containing soil culture with a 16-h light period at 21°C, 50% humidity, and a photon fluence rate of 120 µmol quanta/(m2 s) in a greenhouse, and different grape organs were immediately frozen in liquid nitrogen at the time of collection and then stored at –80°C until extraction. Total RNA was also extracted from different grape organs by cetyltrimethylamonium bromide (CTAB) method [13].

3′- and 5′-RACE reactions. For 3′-RACE reaction, first strand cDNA was synthesized using 3′-RACE primer as the initiation primer and amplifications were performed using prxF (5′-ataggatccATGGCTCCGATTGCAGTTGG-3′), 3′-RACE (5′-tatggatccgagctcctcgagT18-3′), and adaptor primer (5′-TATGGATCCGAGCTCCTCGAG-3′) primers [14]. For 5′-RACE reaction, the reverse transcription reaction was also carried out using oligo (dT)18 primer as the initiation primer and RNA was degraded by RNase H. A homopolymeric C-tail was added to the 5′ end of the purified cDNA by Terminal Deoxynucleotidyl Transferase (“Fermentas”, Germany). Amplifications were performed using prxR (5′-actctcgagTCAGATAGCTTTGAGGATGTC-3′), 5′-RACE (5′-tatggatccgagctcctcgagG15-3′), and adaptor primer primers [14]. The oligonucleotides primers were designed based on the available expressed sequence tag (EST FQ437352), identified with the BLAST program (http://www.ncbi.nlm.nih.gov).

Cloning and sequencing. The PCR products were purified by AccuPrep Gel Purification kit (“Bioneer”, South Korea) and subcloned into the pTG19-T vector (“Vivantis”, Malaysia) according to the manufacturer’s instructions. The nucleotide sequence of the inserts was also determined in both directions by dideoxynucleotide sequencing (“Bioneer”).

Abiotic stress treatments. To investigate the response of the different grape Trx h genes to various stresses, the cuttings were treated with abiotic stimuli in three independent replicates. Progressive water deficit was applied by withholding watering for 10–12 days and leaf RWC (relative water content) was determined as described by Pruvot et al. [15]. For salt stress, the cuttings were treated with different concentrations of NaCl, including 50, 100, 150, 200, 250, and 300 mM. The cuttings were gradually exposed salt stress during 57 days and then the youngest fully expanded leaves were harvested after a period of approximately 7 days. Control and abiotic treated cuttings were held under a 16-h light period at 21°C, 50% humidity, and a photon fluence rate of 120 µM quanta/(m2 s) in a greenhouse. Heat stress was also applied by exposing the cuttings to a temperature of 40°C for 6, 12, 18, 24, 48, and 72 h under a 16-h light period, 50% humidity, and a photon fluence rate of 120 µM quanta/(m2 s) in a chamber growth (“Grouc”, Iran). For recovery, the cuttings were held in under a 16-h light period at 21°C, 50% humidity, and a photon fluence rate of 120 µmol quanta/(m2 s) in a greenhouse for 7 days.

Oxidative stress treatments. For the different oxidative stress treatments, 3 to 4 youngest fully expanded leaves from one-year-old cuttings were pooled and cut into 1 cm diameter leaf slices. After vacuum infiltration with distilled water, the leaf slices were suspended in effector solutions (pH 56) including 10 mM H2O2, 1 mM diamide, 100 µM CuSO4, 100 µM CoCl2, 100 µM CdCl2, 100 µM AlCl3, 100 µM ABA, and 100 µM SA (salicylic acid). The leaf slices were incubated at 21°C and a photon fluence rate of 120 µM quanta/(m2 s) for 4 h.

Semi-quantitative RT-PCR. Semi-quantitative RT-PCR was performed using 5 µg of DNase I-treated total RNA for first-strand synthesis of cDNA and about 1/20 of the reverse transcription reaction was used for RT-PCR with specific primers for distinct VvPrx cDNA. PCR amplifications were performed in a thermal cycler programmed with the following temperature parameters: 3 min at 94°C, followed by 35 cycles of 30 s at 94°C, 1 min at 58°C, 30 s at 72°C, and the final extension of 5 min at 72°C. The grape actin gene (VvAct2) was used as an internal control to normalize each sample for variations in the amounts of RNA used. To control for possible genomic DNA contamination, parallel reactions were carried out where reverse transcriptase activity was inactivated by incubation at 95°C. A negative control lacking a template was included for each set of RT-PCR reactions. Reactions were performed in triplicates. Amplification products were separated by agarose gel electrophoresis and quantified using ImageJ software (W.S. Rasband; 1997–2007; National Institutes of Health; http://rsb.info.nih.gov/ij). Signal intensities were normalized with respect to VvAct2 from the same sample.

Sequence analysis and molecular modeling. The properties of deduced amino acid sequence were estimated using ProtParam (http://www.expasy.ch/tools/protparam.html) program and its subcellular localization prediction was performed by using a combination of three programs, TargetP (http://www.cbs.dtu.dk/services/TargetP/), iPSORT (http://ipsort.hgc.jp/), and YLOC (http://www-bs.informatik.uni-tuebingen.de/Services/YLoc/). Promoter region of the grape Prx gene was obtained from the Phytozome website (http://www.phytozome.net) and searched to find regulatory elements controlling several types of plant stress responses using PlantCare software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The three-dimensional (3D) structure of deduced amino acid sequence was determined by I-TASSER (http://zhang.bioinformatics.ku.edu/I-TASSER) and superimposition analysis of the 3D models of grape Prx and its templates, such as human (Homo sapiens) Prx V protein (PDB ID code 3mngA), poplar (Populus tremula) PrxII C (PDB ID code 1tp9A), and PsPrxII F (PDB ID code 2pwjA) from pea (Pisum sativum) was done using 3-dimensional Structural Superposition (3d-SS) service (http://cluster.physics.iisc.ernet.in/3dss/severalinput.html).

Phylogenetic analysis. Protein sequences were aligned using the ClustalW2 at European Bioinformatics Institute (http://www.abi.ac.uk) using default parameters. Neighbor Joining tree was constructed with MEGA4.1 Beta 2 software [16] and bootstrap analysis with 1000 replicates was also conducted in order to obtain confidence levels for the branches. The NCBI and SwissProt accession numbers for the sequences described and mentioned in this study are as follows: Arabidopsis (Arabidopsis thaliana): At-2CPrx A (At3g11630), At-2CPrx B (At5g06290), At-1CPrx (At1g48130), AtPrx Q (At3g26060), AtPrxII A (At1g65990), AtPrxII B (At1g65980), AtPrxII C (At1g65970), AtPrxII D (At1g60740), AtPrxII E (At3g52960); (A. lyrata): AlPrxII A (EFH64732); castor oil (Ricinus communis): Rc-2CPrx A (EEF32207), RcPrx Q (EEF46226), RcPrxII C (EEF48227), RcPrxII E (EEF33787), RcPrxII F (EEF45351); Chlamydomonas reinhardtii (EDP07991); Citrus sP.: CitrusPrxII C (ABL67649); cotton (Gossypium hirsutum): GhPrxII C (ACJ11720); Escherichia coli (EDV66373); human (H. sapiens): Prx V (P30044); maize (Zea mays): Zm-1CPrx (DAA63833); pea (P. sativum): PsPrxII F (CAG30523); poplar (Populus trichocarpa): Pt-2CPrx A (EEF05155), PtPrx Q (EEE71975), PtPrxII C (EEF03570), PtPrxII E (ABK92641), PtPrxII F (EEE99739); (P. tremula): PtrPrxII C (AAL90751); soybean (Glycine max): Gm-2CPrx A (XP_003535806), Gm-1CPrx (XP_003531110), GmPrxII F (ACU14896).

 

RESULTS AND DISCUSSION

Cloning and sequence analysis of grape Prx gene

 

One EST sequence (NCBI GenBank accession no. FQ437352) corresponding to a putative full-length Prx cDNA was identified using the BLASTn program. In order to characterize the full-length cDNA, 3′ and 5′ RACE reactions were performed and the PCR products were cloned into pTG19-T plasmid vector to generate the pTG19-Prx plasmid. The grape Prx cDNA (submitted at NCBI GenBank under accession no. KC195963) was 773 nucleotides long and contained a single ORF of 489 bp coding for a polypeptide of 162 amino acid residues. The calculated molecular mass and the predicated isoelectric point of the deduced polypeptide sequence were 17.26 kD and 5.15, respectively.

A phylogenetic tree was constructed using the grape Prx and Prx sequences from other plants (fig. 1a). The tree is divided into two major clusters. One cluster contains type II Prxs including A, B, C, D, E, and F that show different subcellular localizations. The PrxsII E and F possess an N-terminus extension that presumably codes for a chloroplastic or mitochondrial transit peptide [3]. Nevertheless, expression activity has not been observed for PrxII A and D, indicating that the two Prxs might be pseudogenes [4, 7]. In general, PrxII B and C do not possess N-terminus extensions and are thus probably not routed to mitochondria, chloroplasts, or to vacuolar and extracellular compartments [1]. The second cluster has three main branches, including the nuclear 1-Cys Prxs, the chloroplastic Prxs Q, and the chloroplastic 2-Cys Prxs [3], which are further subdivided into A and B types. The grape Prx displays strict identity with type II Prx C sequences from other plants, such as cotton (G. hirsutum; GhPrxII C, 89%), poplar (P. trichocarpa; PtPrxII C, 89%), Citrus sP. (CiPrxII C, 88%), and castor oil (R. communis; RcPrxII C, 86%). In contrast, the grape Prx shares lower degree of identity to Prx of human, C. reinhardtii, and E. coli with 40, 51, and 30%, respectively. The absence of an N-terminal extension and of identified signals as Ser-Lys-Leu for the peroxisomes [1] suggest that the subcellular localization of the grape Prx might be the cytosol and the interrogation of the three programs, TargetP, iPSORT, and YLOC, support this proposal. The amino acid sequences comparison of PrxII C from plants reveals the presence of one Cys residue (Cys51 in the grape Prx) that is strictly conserved with the surrounding consensus sequence: P44GAFTPTCS52 (fig. 1b). Site-directed mutagenesis has shown that this residue is the catalytic one, whereas the second Cys residue (Cys76) is not essential for PrxII C activity [17]. Nevertheless, an important difference is the presence of an additional Cys in human and C. reinhardtii Prxs in positions 152 and 151, respectively, and two atypical Cys residues (51 and 100 positions) in E. coli Prx. The grape and all other plant sequences do not possess these additional Cys residues. The grape sequence isolated in this study shows only one Cys (Cys51) and it is strongly associated with the type II Prx C from plants. A predicted 3D structure was determined for VvPrxII C by applying I-TASSER simulation. VvPrxII C and all Prxs are organized in a fold similar to Trx (thioredoxin) with a central pleated β-sheet surrounded by α-helices [18]. VvPrxII C contains five α-helices and seven β-sheets with secondary structural elements: β1β2α1β3α2β4α3β5α4β6β7α5 (fig. 2a). VvPrxII C 3D structure matches almost perfectly with Prx crystal structures from human (H. sapiens), poplar (P. tremula), and pea (P. sativum) (fig. 2b).

 

The grape Prx promoter contains regulatory elements

in response to environmental signals

To obtain insight into the regulation of grape Prx gene by abiotic stresses, its promoter region was obtained from the Phytozome website and looked for relevant cis-acting elements by PlantCare software. The analysis of the promoter region of grape Prx showed that a number of potential cis-acting elements to respond to environmental signals were present (table). A heat stress responsive element (HSE) was present at position 1274. However, a functional heat shock element contains at least three basic repeats that must be arranged in alternating orientations [19]. Thus, the HSE motif in VvPrxII C promoter no appears to be functional. Four putative basic motifs (CAACTG) of MYB binding site (MBS) involved in drought inducibility were located at positions 169, 810, 925, and 1209. The MBS element is essential for water stress-induced gene expression but is not under the regulation of ABA [20]. Therefore, it seems that VvPrxII C gene may be induced by the ABA-independent pathway. An metal responsive element (MRE)-like sequence (TGCAGAC), similar to the conserved core MRE sequence involved in heavy metal-induced expression of metallothionein genes in animals [21], was located at position 233. A putative TCA-element (382) thought to be involved in SA response was also found [22]. Lastly, a W-box was located in promoter region of the grape Prx (799), indicating its possible implication in response to pathogens. Nevertheless, mutational experiments were exhibited that at least two copies of the W-box are necessary to perform the WRKY6-dependent gene activation [23]. The WRKY proteins comprise a family of plant-specific zinc-finger-type factors implicated in the regulation of genes associated with pathogen defense [23]. These putative regulatory elements suggest that the promoter region of the grape Prx may respond to a variety of environmental signals, including dehydration, heat, heavy metals, light, pathogens, wounding, and plant hormones. The sequence also contains a putative TATA box at 44 relative to the transcriptional start site and a putative CAAT box at 219. The other potential cis-acting elements to respond to environmental signals in the promoter of grape Prx are indicated in the table.

 

Expression analysis of VvPrxII C in different grape organs

The pattern of expression of the VvPrxII C gene was analyzed in different organs of grape by semi-quantitative RT-PCR. The expression level of VvPrxII C gene was also compared with the expression of VvAct2 gene that is used as an internal control in the PCR reaction. With the exception petiole, the grape Prx gene was expressed in all organs (fig. 3). Studying of the number of ESTs coding for VvPrxII C in the NCBI GenBank database also revealed around 113 ESTs at most grape organs, such as berry, leaf, cluster, stem, root, seed, bud, and flower, suggesting a high level of expression for this gene in grape. However, no EST coding for VvPrxII C was found in petiole and tendril organs in the NCBI EST sequence database. Based on the number of identified ESTs in different organs, VvPrxII C thus appears to be transcribed at high levels in berries (41 hits) and leaves (19 hits) tissues, and at low levels in stems (4 hits) and clusters (7 hits).

The highest levels of expression were detected in berry, leaf, and tendril organs, whereas the lowest amount of grape Prx transcripts is related to clusters, stems, and buds. Similarly, the expression profiles of four Prx genes were analyzed in Tamarix hispida roots, stems, and leaves under normal growing conditions. The results indicated that these genes were expressed in all organs but with different expression patterns than each other [10]. In poplar (P. trichocarpa), Rouhier et al. [1] reported that the type C Prx gene is efficiently transcribed and translated in plant. The northern-blot experiment revealed that transcription level of this gene was high in leaves, lower in roots, and weak in stems and the western-blot experiment confirmed the results of the northern-blot experiment.

 

Expression of Prx gene under abiotic stress conditions

To analyze the response of grape Prx gene to abiotic stress, the grape cuttings were treated under drought, salt, and heat stresses in three independent replicates and expression analysis was performed by semi-quantitative RT-PCR. Under the abiotic stress, the expression of grape Prx gene was clearly induced (fig. 4). In response to drought stress (fig. 4a), the transcript amounts of VvPrxII C were detected at a low level in leaves from 90 to 70% RWC, but at much higher level in plants subjected to severe deficit (60% RWC). The transcript abundances considerably increased at RWC around 55% and followed to a relatively dramatic decrease upon very severe stress conditions (50% RWC). When wilted plants (50% RWC) were rewatered for one week, plants recovered a leaf RWC close to 95%, and level of transcripts decreased to control level. The VvPrxII C was also up-regulated under salt stress (fig. 4b). The transcript levels of VvPrxII C gene remarkably increased after incubation with 150 mM NaCl and then there was a severe decreased at 300 mM NaCl. The presence of MBS in promoter region of VvPrxII C gene is demonstrated that induction of its expression under salinity or drought may transcriptionally not be controlled by ABA, and it may be induced by the ABA-independent pathway [20]. The up-regulation of Prx genes during drought and high salt stress was also observed in wheat (Triticum aestivum) [8] and T. hispida [10].

Similar to the drought and salt stresses, the grape Prx was also induced upon heat treatment (fig. 4c). The transcripts of VvPrxII C gradually increased to 48 h and then slightly decreased to 72 h of heat treatment. It has been indicated that hydrogen peroxide is rapidly accumulated upon high temperature, among other stresses, and may act as an important signaling molecule [24]. The up-regulation of several enzymes involved in redox homeostasis in response to oxidative stress generated by heat, including SOD (superoxide dismutase), CAT (catalase), DHAR (dehydroascorbate reductase), POX (peroxidase), and GST (glutathione-S-transferase) was reported [25]. Kim et al. [12] reported that overexpression an Arabidopsis 2-Cys Prx in transgenic tall fescue (F. arundinacea) plants confers tolerance against heat stress and protects leaves from oxidative damage probably due to chaperon activity. Kim et al. [11] also showed that heterologous expression of salt-induced 2-Cys Prx from rice (Oryza sativa) increased heat stress tolerance and fermentation capacity in the transgenic yeast (S. cerevisiae) cells. Therefore, under heat stress, it seems that induction of VvPrxII C expression may be exposed by ROS.

 

Expression of Prx gene under oxidative stress

To test whether the steady-state transcript amounts respond to oxidative stress, various treatments were used including chemical inducers, heavy metals, and hormonal treatments to trigger production of ROS. The effectors were applied to leaf slices by incubation in effector solution after infiltration to ensure fast and homogenous application (fig. 5). VvPrxII C showed a strong increase in the transcript amount with H2O2, heavy metals, and hormonal treatments, whereas its transcript level was almost unaffected by the diamide (fig. 5). H2O2 is a compound with directly oxidizing properties, whereas diamide acts indirectly as oxidative stressor by depletion of the cellular thiol pool [7]. Reactive oxygen species and peroxides are known to be inducers which via Keap1/Nrf2 signaling pathway up-regulate the transcription of antioxidant and associated enzymes including SOD, CAT, Prx, glutathione peroxidase, thioredoxin reductase, and glutathione reductase [26]. The high up-regulation of grape Prx under H2O2 treatment thus suggests that VvPrxII C may be implicated in response to oxidative stress and be involved in antioxidant defense. Similarly, in Arabidopsis, PrxII C gene also showed a general strong increase in the transcript amount with H2O2 than other Prxs [7].

The accumulating evidence suggests that heavy metals and plant hormones including ABA and SA induce ROS production and cause oxidative stress in plants [9]. Contreras-Porcia et al. [9] have reported that the activities of antioxidant enzymes APX (ascorbate peroxidase), Prx, Trx, and GST increased in response to copper excess, indicating that the marine alga (Ulva compressa) was under oxidative stress. Studies also proposed that the expression of Prx genes increased under heavy metal treatments [10, 27]. In Arabidopsis, Cd application increased the mitochondrial PrxII F content and demonstrated a principal role for this gene in antioxidant defense and potential redox signaling in plant cells [27]. The ABA-induced expression of Prx genes was also reported in T. hispida [10]. It seems that the induction of VvPrxII C expression by heavy metals and SA was probably a direct response to them due to the presence of cis-acting elements (MRE and TCA-element) in the its promoter region no by ROS. In contrast, the activation of VvPrxII C by ABA as well as the absence of ABA-responsive element (ABRE) in the VvPrxII C promoter are likely in favor of an activation of expression mediated by ROS.

 

Antioxidants effects on Prx gene expression

Ascorbate is the most abundant, powerful and water soluble antioxidant acts to prevent or minimize the damage caused by ROS in plants. It occurs in all plant tissues, usually being highest in mature leaves with fully developed chloroplast and highest chlorophyll. Proline is also a potent antioxidant, a potential inhibitor of programmed cell death, and a ROS scavenger [28]. To test antioxidants effects on VvPrxII C gene expression, leaf slices were suspended in media supplemented with ascorbate or proline. VvPrxII C was largely affected by antioxidants and its transcript amounts increased upon external application of antioxidants (fig. 6). In Arabidopsis, after addition of ascorbate, the transcript levels of chloroplastic Prx genes declined, whereas PrxII C gene showed high level of mRNAs [9]. A growing body of evidence displays that abiotic stress conditions significantly increased the ascorbate and proline contents in plants [28]. It strongly proposes that abiotic stress causes increased generation of ROS and induced the effect of an oxidative stress condition. Ascorbate and proline are considered as ROS scavengers because of their ability to donate electrons in a number of enzymatic and non-enzymatic reactions. Ascorbate is directly involved in the Apx-mediated detoxification of H2O2 and indirectly  of lipid peroxides [29]. Proline has been also proposed to act as an osmoprotectant, a protein stabilizer, a metal chelator, an inhibitor of lipid peroxidation, and OH and 1O2 scavenger [30]. Furthermore, Prx proteins are known as alternative enzymes in H2O2 and lipid peroxide reduction. These data indicate that antioxidants can regulate expression of Prx genes and may communicate with each other in the cell redox regulation. However, further studies are needed to understand the potential role of antioxidants in regulation of Prx genes expression.

In conclusion, Prx gene, designated VvPrxII C, was isolated from grape berry organs of Iranian cultivar, called Askari. The constructed phylogenetic tree revealed that the grape Prx belongs to type II Prx C and contains a high degree of identity with type II Prx C form other plants. A modeling analysis indicated that VvPrxII C shares a common structure with Trxs in which the central β-sheets surrounded by flanking α-helices. The grape Prx gene was also found to be highly induced under abiotic stress conditions, oxidative stress, and antioxidants application. Collectively, the specific expression in almost all tissues, responses to environmental stress conditions, and specially antioxidants-induced regulation of expression suggest that VvPrxII C could play a critical role in the cell redox regulation.

We thank M. Sc. A. Alborzian (Imam Khomeini International University, Qazvin, Iran) for technical assistance.

This work was supported by research grant from the Iranian Imam Khomeini International University.

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The potential cis-acting regulatory elements identified in the promoter region of VvPrxII C gene using PlantCare software

 

 

FIGURE CAPTIONS

 

Fig. 1. Phylogenetic tree and multiple alignment of VvPrxII C and Prxs from different sources.

a – phylogenetic tree of VvPrxII C and Prxs from other plants using MEGA4.1 Beta 2 software. The phylogenetic tree is divided into two major clusters, including type II Prx and 2Cys Prx with 1Cys Prx and Prx Q; b – multiple sequence alignment of VvPrxII C and Prx sequences from different organisms. The protein sequence deduced from the VvPrxII C gene was aligned with its homologs and human Prx V, C. reinhardtii Prx, and Prx protein from E. coli using ClustalW2. The typical Cys residues in Prx sequences and atypical Cys residues in Prxs of human, Chlamydomonas, and E. coli are shown by black triangles and squares, respectively. The consensus sequence of PGAFTPTCS is also indicated in black box. Accession numbers are given in materials and methods.

 

Fig. 2. Three-dimensional models and conserved residue prediction for VvPrxII C.

a – cartoon display of the three-dimensional structure of VvPrxII C. The α-helices, β-sheets, and coiled coil regions are marked as α, β, and cc, respectively. The cysteine, initiation methionine, and end isoleucine residues are indicated as C51, M1, and I162 spheres, respectively. The consensus sequence of P44GAFTPT50 is also showed by sticks; b – superimposition of 3D model of VvPrxII C (1) and the top three templates of: human Prx V protein (2; PDB ID code 3mngA), poplar PrxII C (3; PDB ID code 1tp9A), and PsPrxII F from pea (4; PDB ID code 2pwjA) using 3d-SS (3 Dimensional Structural Superposition) service.

 

Fig. 3. Expression of the VvPrxII C gene in different grape organs by semi-quantitative RT-PCR.

RT-PCR analysis was performed using gene specific (VvPrxII C) and reference gene (VvAct2) primers in berry, leaf, cluster, petiole, stem, tendril, bud, root, and seed organs. One representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.

 

Fig. 4. Expression analysis of the VvPrxII C gene in youngest fully expanded leaves in response to drought, salinity, and heat by semi-quantitative RT-PCR.

a – expression analysis of VvPrxII C gene upon water deficit and after rewatering. Progressive water deficit was applied by withholding watering for 10–12 days and leaf RWC was determined ranged from 90 to 50%; b – expression analysis of VvPrxII C gene under salt stress. For salt stress, different concentrations of NaCl were used, including 50, 100, 150, 200, 250, and 300 mM; c – expression analysis of VvPrxII C gene upon high temperature and after recovery. The heat stress was applied by exposing the cuttings to a temperature of 40°C for 6, 12, 18, 24, 48, and 72 h in a chamber growth. The grape cuttings were treated under heat stress in three independent replicates and the semi-quantitative RT-PCR was performed using gene specific (VvPrxII C) and reference gene (VvAct2) primers in leaf tissue. One representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.

 

Fig. 5. Effect of oxidative stressors on the expression of VvPrxII C gene in grape leaf slices.

Leaf slices were incubated in the presence of H2O2 (10 mM), diamide (1 mM), CuSO4 (100 µM), CoCl2 (100 µM), CdCl2 (100 µM), AlCl3 (100 µM), ABA (100 µM), and SA (100 µM) as mediators of oxidative stress for 4 h before total RNA extraction. The experiments were carried out in three independent replicates and one representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.

 

Fig. 6. Effect of exogenous application of antioxidants on VvPrxII C gene expression of grape leaf slices.

Leaf slices were incubated in the presence of 50 mM ascorbate and proline for 2, 4, 6, and 8 h before total RNA extraction. The experiments were carried out in three independent replicates and one representative gel is shown from three independent replicates. Relative band intensities were normalized to the VvAct2 band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions.