Differential Expression of Copper-Zinc Superoxide Dismutase Gene of Polygonum sibiricum Leaves, Stems and Underground Stems, Subjected to High-Salt Stress

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5′ untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3′ untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1–16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO3. The different mRNA levels’ expression of PS-CuZnSOD show the gene’s different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.


Introduction
Even when plants grow and develop under natural conditions, they are inevitably affected by environmental stresses due to their immobility [1]. This could lead to the production of a lot of reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide (H 2 O 2 ), hydroxyl radical and singlet oxygen, which are harmful to intercellular components such as DNA, protein and membrane lipids. High active oxygen species (AOS) levels initiate signaling responses that include enzyme activation, gene expression, cell apoptosis and cellular damage [2]. Organisms employ an antioxidant defense system to protect themselves against the toxic effects caused by such a signal. Superoxide dismutases (superoxide: superoxide oxidoreductase, EC 1.15.1.1; SOD) are the first line of defense against the oxidative stresses by catalyzing reactive oxygen molecules to hydrogen peroxide that is consequently converted to water by catalase [3].
Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygenrespiring organisms [4]. SODs [5,6], which is closely related to anti-aging and resistance to stress in plants [6][7][8][9]. Iron SOD has been found in prokaryotes, in algae and in some higher plant chloroplasts [10]; Manganese SOD is found in prokaryotes and mitochondria; and a fourth with the coupled Ni (II/III) at the active site (Ni-SOD), which is found in the Streptomyces genus [11]. Copper-zinc superoxide dismutase can be divided into two forms; one is in cytosolic and the other is in chloroplastic isoenzymes. Copper-zinc superoxide dismutase in cytosolic is found mainly in cases of induced adverse environment [12,13]. Superoxide dismutase (SOD) was first isolated from bovine red blood cells by Mann and Keilin in 1938. Until now, copper-zinc superoxide dismutase has been cloned in several species of plants including rice, corn, and others [13][14][15][16][17][18][19][20][21]. The transgenic plants with overexpression of SOD gene in tobacco and alfalfa could resist cold stress, and markedly enhance antioxidant capacities [22][23][24].
Polygonum sibiricum Laxm. is a Dicotyledoneae Polygonaceae perennial herb. It grows in wetland, near the riverbank on saline and alkaline land. As one of the minority important halophytes grown in salinity-alkalinity areas, it is considered to be a promising species as a potential genetic resource of genetic transformation, and also can be employed as an experimental system for conducting research on salt resistance.
Salinity-alkalinity stress is one of the main abiotic stresses that restrict the development of agriculture worldwide. Compared to other abiotic salt stresses, there are limited studies on carbonate stress, though the main salt of soil, NaHCO 3 , has a severe effect on plants. The aim of the present study is to clone copper-zinc superoxide dismutase, which is located in the cytosolic in P. sibiricum Laxm., and to present the nucleotide sequence of copper-zinc superoxide dismutase, comparing its sequence with other known SODs from other species; and to evaluate this copper-zinc superoxide dismutase expression in leaves, stems and underground stems, when P. sibiricum Laxm. was induced under salinity-alkalinity stress. It is hoped to clarify the effects of PS-CuZnSOD in salinity-alkalinity resistance, forming a good basis for further study on the mechanisms of salinity-alkalinity stress tolerance in P. sibiricum Laxm.

cDNA Cloning, Sequencing and Bioinformatics Analysis of PS-CuZnSOD
In order to isolate cDNA encoding for copper-zinc superoxide dismutase, PCR reactions were performed using primers and total cDNA of plant leaves. Products of amplification were cloned and sequenced. Computer analysis, using the BLAST algorithm, confirmed that the selected sequence corresponded to a copper-zinc superoxide dismutase. The full-length copper-zinc superoxide dismutase cDNA fragment of P. sibiricum Laxm. was obtained by overlapping two cDNA fragments.

Tissue Expression of PS-CuZnSOD
Copper-zinc superoxide dismutase expressed in each organ of P. sibiricum Laxm. is shown in Figure 4. In a RT-PCR study, specific primers (SOD-F: 5'-AGTGCGGGAGTTAGTGG-3' and SOD-R: 5'-CGATGCTCGTCTTCTGG-3') were used to amplify a 203 bp fragment with cDNA from leaves, stems and underground stems, organs using 18S as a positive control. The RT-PCR showed that the CuZnSOD was detected in leaves, stems and underground stems. In leaves, the increase of the copper-zinc superoxide dismutase mRNA expression level reached its peak in 24 hours after 3% NaHCO 3 stress, and gradually decreased ( Figure 4B). In stems, the increase of the copper-zinc superoxide dismutase mRNA expression reached its peak in 72 hours after salinity-alkalinity stress ( Figure 4C). That is, in leaves and stems they were up-regulated and then down-regulated during 3% NaHCO 3 stress. Contrastingly, the copper-zinc superoxide dismutase transcripts were fluctuated and down-regulated after 3% NaHCO 3 stress in underground stems' organs ( Figure 4D). Tissue distribution of PS-CuZnSOD mRNA was ubiquitous in all the tissues examined in this study, which is not surprising since the expression of copper-zinc superoxide dismutase in a wide range of cell types has already been found previously. Based on experimental exposures to 3% NaHCO 3 , P. sibiricum Laxm. SOD mRNA was apparently affected by the durations of 3% NaHCO 3 stress. mRNA expression of PS-CuZnSOD was durations-dependent in general; the saturation of expression (reaching maximum level) was observed in stems at 72 h based on the RT-PCR. There were some different responses of PS-CuZnSOD to 3% NaHCO 3 exposure in every organ (or tissue) from our data. We deduce that tuning expression of copper-zinc superoxide dismutase mRNA may be used to change copper-zinc superoxide dismutase activity and in turn modulate plant growth under the salinity-alkalinity stress. The different expression mode of copper-zinc superoxide dismutase in leaves, stems and underground stems might be the reason P. sibiricum Laxm. has higher efficiency and economy in antioxidant and resistance to stress in plants. However, the specific factors underlying the regulatory mechanism have not been clearly understood. Our results may provide the basis for future investigations of copper-zinc superoxide dismutase roles in salinity-alkalinity stress development in different organs.

RNA Isolation from P. sibiricum Laxm. and Reverse Transcription (RT)
Total RNA was extracted using a phenol sodium dodecyl sulfate extraction/LiCl precipitation procedure [25].

Obtaining 3' and 5' Regions by RACE
To isolate the complete 5' and 3' regions of this gene, the rapid amplification of cDNA ends (RACE) method was used. First-strand cDNA synthesis was performed using Smart TM RACE cDNA amplification kit (Clontech). Previously we obtained the PS-CuZnSOD 3' EST sequences from the P. sibiricum Laxm. cDNA Library [26]. According to the EST, two specific primers were designed on the basis of the SOD 3'UTR for 5'-RACE. SODZnN: 3'-AGAACCAAACAGACCAAAACAAG-5', SODZn: 3'-CTCATAACATAAGGAAAGAAAGGG-5'. The 5' fragment PCR was then carried out according to the manufacturer's instructions (Clontech Kit). Next, the fragments were cloned and sequenced. A pair of specific primers was designed to amplify the ORF of PS-CuZnSOD gene (SOD-A: 5'-GATTACAGCCAATTTCAATAC-3', SOD-S: 5'-CTCTTACAACAAGGGGTTC-3').

Subcloning
The PCR fragments were subjected to electrophoresis on 0.8% agarose gel for length differentiation, and amplified cDNA fragments were cloned into the pGEM-T Easy vector following the instructions provided (Promega, Madison, WI, U.S.). Recombinant bacteria were identified by blue/white screening and confirmed by PCR. Plasmids containing the insert were purified (Promega minipreps) and used as a template for DNA sequencing.

Nucleotide Sequence Analysis
The fragments were linked by the soft Bio-Edit CAP contig assembly program. The PS-CuZnSOD gene sequence was analyzed and compared using the BLAST P and ORF search programs with GenBank database search. The multiple sequence alignment of PS-CuZnSO gene was created by Clustal W analysis program, signal-peptide site was predicted by Signal P3.0, the SOD protein MW and pI were computed by ProtParam [27], and disulfide connectivity was predicted by SCRATCH Protein Predictor [28].

Statistical Analysis
A multiple comparisons (Duncan's) test was conducted to compare significant differences in PS-CuZnSOD expression between leaves, stems and underground stems, using the SPSS software. A significant level of p = 0.05 was chosen.

Conclusions
In plants, several enzymes like SODs are involved in the release of AOSs. It is known that SOD catalyzes the rapid two-step dismutation of the toxic superoxide anion to molecular oxygen and hydrogen peroxide, through alternating reduction and oxidation of the active-site metal ion [29]. SODs play an important role in antioxidant defense pathways in response to oxidative stress [10]. In this work, the full length of a PS-CuZnSOD gene was isolated from P. sibiricum Laxm. by the rapid amplification of cDNA ends method. Analysis of the nucleotide sequence revealed that the Expression analysis by real-time quantitative PCR revealed that PS-CuZnSOD gene is expressed in leaves, stems and underground stems. In leaves and stems it was up-regulated and then down-regulated during 3% NaHCO 3 stress. On the contrary, the copper-zinc superoxide dismutase transcripts fluctuated and were down-regulated after 3% NaHCO 3 stress in underground stem organs. That is, under 3% NaHCO 3 stress, PS-CuZnSOD gene expression can be induced differently. It indicates that there are different express modes in leaves, stems and underground stems. We presume that PS-CuZnSOD genes in leaves and stems play important roles in the process of induction by salinity-alkalinity resistance, and the down-regulation of PS-CuZnSOD genes may be related to the effects of H 2 O 2 -products of PS-CuZnSOD. Additionally, it seems that the PS-CuZnSOD gene does not function in salinity-alkalinity resistance in underground stem organs. Currently, we do not know the exact role PS-CuZnSOD genes play in P. sibiricum Laxm. resistance to salinity-alkalinity stress. However, differential mRNA expression of some genes in P. sibiricum Laxm. seems to be either "protective" or cause "division of labor" [30][31][32][33][34]. So far, no reports have been shown that PS-CuZnSOD genes are differentially expressed during the salinity-alkalinity stress processes. We propose that the PS-CuZnSOD "division of labor" in different organs may play an important role in P. sibiricum Laxm. resistance to salinity-alkalinity stress. Our results may provide the basis for future investigations of PS-CuZnSOD roles in P. sibiricum Laxm. resistance to salinity-alkalinity stress.