ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 6, с. 874-883
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
ISOLATION OF GRAPE PEROXIREDOXIN GENE RESPONDING
TO ABIOTIC STRESSES1 © 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 Prxll 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 VvPrxll 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.
Keywords: Vitis vinifera — abiotic stress — antioxidants — oxidative stress — peroxiredoxin
DOI: 10.7868/S0015330315050085
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
1 This text was submitted by the authors in English.
Abbreviations: APX — ascorbate peroxidase; CAT — catalase; CTAB — cetyltrimethylamonium bromide; DHAR — dehy-droascorbate 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
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 Prxll C gene contained a large number of putative regulatory elements and respond to a wide variety of environmental signals. Moreover, we revealed that Prxll 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 ^mol 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'-ataggatc-cATGGCTCCGATTGCAGTTGG-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 Deoxynu-cleotidyl Transferase ("Fermentas", Germany). Amplifications were performed using prxR (5'-actctc-gagTCAGATAGCTTTGAGGATGTC- 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 dideox-ynucleotide 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 |M 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 |M 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 |mol 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 |M CuSO4, 100 |M CoCl2, 100 |M CdCl2, 100 |M AlCl3, 100 |M ABA, and 100 |M SA (salicylic acid). The leaf slices were incubated at 21°C and a photon flu-ence rate of 120 ^M quanta/(m2 s) for 4 h.
Semi-quantitative RT-PCR. Semi-quantitative RT-PCR was performed using 5 ^g of DNase I-treat-ed 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
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