Molecular analysis of the chloroplast Cu/Zn-SOD gene(AhCSD2)in peanut

Xiurong Zhang,Qian Wan,Fengzhen Liu*,Kun Zhang,Aiqing Sun,Bing Luo, Li Sun,Yongshan Wan**

State Key Laboratory of Crop Biology,Shandong Key Laboratory of Crop Biology,Shandong Agricultural University,Tai'an 271018,China

Molecular analysis of the chloroplast Cu/Zn-SOD gene(AhCSD2)in peanut

Xiurong Zhang,Qian Wan,Fengzhen Liu*,Kun Zhang,Aiqing Sun,Bing Luo, Li Sun,Yongshan Wan**

State Key Laboratory of Crop Biology,Shandong Key Laboratory of Crop Biology,Shandong Agricultural University,Tai'an 271018,China

A R T I C L E I N F O

Article history:

Received 18 December 2014

Received in revised form

26 February 2015

Accepted 10 March 2015

Available online 19 April 2015

Peanut

SOD activity

AhCSD2 gene

Bioinformatics analysis

Drought resistance

Superoxide dismutase(SOD,EC 1.15.1.1)plays a key role in response to drought stress,and differences in SOD activity changes among cultivars are important under drought conditions.We obtained the full-length DNA of the chloroplast Cu/Zn-SOD gene(AhCSD2) from 11 allotetraploid cultivars and 5 diploid wild species in peanut.BLAST search against the peanut genome showed that the AhCSD2 genes gCSD2-1 and gCSD2-2 are located at the tops of chromosome A03(A genome)and B03(B genome),respectively,and both contain 8 exons and 7 introns.Nucleotide sequence analyses indicated that gCSD2-2 sequences were identical among all the tested cultivars,while gCSD2-1 sequences showed allelic variations. The amino acid sequences deduced from gCSD2-1 and gCSD2-2 both contain a chloroplast transit peptide and are distinguished by 6 amino acid(aa)residue differences.The other 2 aa residue variations in the mature peptide regions give rise to three-dimensional structure changes of the protein deduced from the genes gCSD2-1 and gCSD2-2.Sequences analyses of cultivars and wild species showed that gCSD2-2 of Arachis hypogaea and gAipCSD2(Arachis ipaensis)are identical,and despite the abundant polymorphic loci between gCSD2-1 of A. hypogaea and sequences from A genome wild species,the deduced amino acid sequence of AhCSD2-1(A.hypogaea)is identical to that of AduCSD2(Arachis duranensis),whereas AcoCSD2(Arachis correntina)and AcaCSD2(Arachis cardenasii)both have 2 aa differences in the transit peptide region compared with AhCSD2-1(A.hypogaea).Based on the Peanut Genome Project,promoter prediction revealed many stress-related cis-acting elements within the potential promoter regions(pp-A and pp-B).pp-A contains more binding sites for drought-associated transcriptional factors than pp-B.We hypothesize that the marked changes in SOD activity in different cultivars under drought stress are tightly regulated by transcription factors through transcription and expression of AhCSD2 genes.

?2015 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Plants have developed a delicate dynamic balance system for generation and scavenging of endogenous reactive oxygen species(ROS)during their normal development[1,2].However, many adverse stress conditions,such as drought and heat,can disrupt such coordination by inducing excessive accumulation of ROS.Oxidative stress results in damage to cellular macromolecules such as proteins,nucleic acids,membrane lipids, and carbohydrates[1,2].As one of the key enzymes in the antioxidant protection system,superoxide dismutase(SOD,EC 1.15.1.1)has been suggested to play an important role in detoxifying ROS-induced stresses[3].Based on its metal cofactors,SOD can be categorized into three forms:copper/zinc-(Cu/ Zn-SOD),manganese-(Mn-SOD),and iron-(Fe-SOD).Cu/Zn-SOD is located mainly in the chloroplast and cytoplasm[3].The chloroplast,the photosynthetic organelle,is one of the primary sites for ROS generation in plants.Oxidative damage caused by excessive ROS in the chloroplast can destroy the membrane system and molecular structure of chlorophyll and ultimately undermine the photoreaction center[4,5].ROS have important functions in regulating enzymatic and non-enzymatic antioxidants at both transcriptional and translational levelsand act asa regulator in the network of signal transduction pathways of biosynthesis of hormones associated with stress[2,5–7].They play a dual role as a damaging toxic element and a positive signal transduction molecule.Thus,the chloroplast has evolved a high-efficiency antioxidant defense mechanism against adversity,especially oxidative damage caused by drought and heat.

Encoded by a single nuclear gene(CSD2)[8],the precursor of chloroplast Cu/Zn-SOD in Arabidopsis thaliana is synthesized and transported into the chloroplast by an N-terminal transit peptide[9].To date,chloroplast Cu/Zn-SOD genes from several plant species have been isolated,characterized,and used to enhance resistance against oxidative stress[10,11]. Peanut(Arachis hypogaea L.)is one of the most important edible oil crops and is grown in more than 100 countries,with an annual production of 34.1 million Mg[12].Water deficiency is the main constraint to plant growth and productivity,and drought is the primary factor limiting peanut yield and quality [12].In a recent study,traits of drought resistance and sensitivity were largely cultivar dependent and different peanut genotypes responded to drought stress differently[13].Clearly,breeding drought-resistant peanut varieties is important and possible. Several other studies have indicated that drought-resistant varieties show higher SOD activity than drought-sensitive cultivars under drought conditions[14–16].In general,high SOD activity is a great advantage in response to drought stress [14–16].Chloroplast Cu/Zn-SOD is one of the most important antioxidant enzymes present in plant leaves[17].Continuous cultivar selection for improvement in yield and photosynthetic features may have facilitated the evolution of the high-efficiency antioxidant system in the chloroplast[18].

Previous studies of SOD in peanut have focused mainly on changes in physiological activity under stress[12–16].The cytoplasmic Cu/Zn-SOD gene has been cloned and analyzed in four peanut cultivars[19],but cDNA and genomic DNA sequences of chloroplast Cu/Zn-SOD AhCSD2 and AhCSD2's molecular biological analysis have not been reported.It is widely believed that Arachis duranensis(AA)and Arachis ipaensis(BB)are the ancestors of A.hypogaea,which arose by natural hybridization followed by spontaneous chromosome doubling[20–22].The completion of the Peanut Genome Project(PGP)[23]in A. duranensis and A.ipaensis provides a valuable data resource for deducing gene and evolutionary processes in cultivated peanut and for breeding new varieties with high yield,quality,and resistance against biotic and abiotic stresses.

The objectives of the present study were(1)to clone and sequence the AhCSD2 gene from tetraploid(A.hypogaea) cultivars with different SOD activity and drought resistance and five diploid wild species in section Arachis,(2)to determine the physical location of the AhCSD2 gene in the peanut genome, (3)to identify allelic polymorphisms of AhCSD2 among cultivars and diploid wild species,and(4)to identify and analyze potential promoter sequences of AhCSD2 based on the Peanut Genome Project[23].The ultimate goal of this study is to provide a basis for understanding the molecular mechanism of SOD activity and the AhCSD2 gene in response to drought stress.

2.Materials and methods

2.1.Materials and growth conditions

Five diploid wild species and 11 allotetraploid cultivars were used as experimental materials(Tables 1 and 2).The wild species were grown in the greenhouse,and the cultivars were grown in 2013 and 2014 in the test field of the Agricultural Experiment Station of Shandong Agricultural University (36.15°N,117.15°E),Tai'an,China.Field experiments for cultivars were conducted in a randomized complete block design with three replications.Two water management treatments were assigned in main plots,consisting of standard watering and drought stress imposition by withholding water until harvest.The field capacity of the test field was 21.39%in 2013 and 20.97%in 2014.The standard treatment maintained the relative water content(RWC)at 70±5%.For drought stress treatment,irrigation was withheld to hold RWC at 45±5%.Soil water content(%)was calculated as(M–Ms)/Ms×100%,where M is the weight of wet soil and Ms is the weight of dry soil. RWC was determined by soil water content combined with a CNC503DR neutron probe(Beijing Nuclear Instrument Factory China National Nuclear Company,Beijing,China)every 5 days at soil depths of 0–20 cm to determine whether irrigation was providing sufficient water.Plot size was 8.28 m2with a 4.6 m row length.The distance between rows was 40 cm and the spacing between plants was 20 cm.

2.2.SOD activity assay and drought resistance identification

At the stress stage in 2013 and 2014,in both flowering and pegging periods,samples of cultivar leaves were used for SOD activity analysis.For SOD extraction,0.5 g samples of frozen leaves were quickly ground in liquid N2into fine powder.The SOD extraction protocol and SOD activity assays using nitro blue tetrazolium(NBT)were based on Jiang and Zhang[24]. SOD activity assay was determined by NBT photoreduction inhibition(one unit of SOD activity=the amount of enzyme required to inhibit 50%of the NBT reduction,with sample absorbance determined at 560 nm against a blank).

Table 1-SOD activity and yield drought resistance of tested peanut cultivars.

Yield of every plot was collected for drought resistance evaluation.Drought resistance was measured as drought resistance coefficient(DC)according to a previous study[25]. DC of each variety was calculated as Yd/Yp,where Ydis the yield of each plot under stress and Ypis the yield under the standard treatment.

2.3.Genomic DNA and RNA extraction and first-strand cDNA synthesis

Young leaves from seedlings of all cultivars and wild species were sampled and stored at–80°Cfor DNA and RNA extraction. Genomic DNA of each sample was extracted using a Plant Genomic DNA Kit(TIANGEN Biotech Company,Beijing,China), while total RNA was isolated according to the instructions for the RNAprep pure Plant Kit(TIANGEN)and the manufacturer's optimized method.For cDNA synthesis,2 μg of total RNA of each sample was used for reverse transcription,using the Quantscript RT Kit(TIANGEN).

2.4.Isolation,cloning,and sequencing of AhCSD2 cDNA and genomic DNA

Arabidopsis thaliana(GenBank:NM_128379)CSD2 cDNA was used as a probe to detect peanut ESTs.The identified ESTs were assembled into a cDNA sequence containing an openreading frame(ORF)of 624 bp.The ORF was predicted by ORF Finder in NCBI(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). A search of amino acid sequence similarity was performed with DNAMAN(Lynnon Biosoft Company,Vaudreuil,Canada) and Blastp(http://blast.ncbi.nlm.nih.gov/Blast.cgi).The deduced AhCSD2 amino acid sequence showed high identity with other known chloroplast Cu/Zn-SODs,which contain typical ion binding sites for Cu2+and Zn2+.Specific primers CSD2-F(5′-CTC TGC GGC ATT ATC ACA-3′)and CSD2-R(5′-GGA AAA GCC ATA GAA GGT-3′)were designed using PrimerPrimer 5.0(Premier Biosoft Company,California,USA)combined with Oligo 6.0(http://www.oligo.net/)software to identify the splicing sequence.Primers were synthesized by Shanghai Biological Engineering Company,Shanghai,China.

Table 2-AhCSD2 sequence labels of Arachis germplasm samples.

The PCR protocol for full-length AhCSD2 cDNA amplification was as follows:pre-denaturation at 94°C for 5 min,followed by denaturation at 95°C for 30 s,annealing at 54°C for 30 s,and elongation at 72°C for 40 s(35 cycles),and post-elongation at 72°Cfor7 min.The PCR protocol for AhCSD2DNA amplification was as follows:pre-denaturation at 94°C for 5 min,followed by denaturation at 95°C for 30 s,annealing at 54°C for 30 s,and elongation at 72°C for 2 min(35 cycles);and post-elongation at 72°C for 10 min.The amplified products present in the 1.0% (w/v)agarose gels were extracted with a TIANgel Midi Purification Kit(TIANGEN),cloned into the pEASY-T1 Vector according to previous study[26],and then sequenced and analyzed by BGI Sequencing Technology Company,Beijing,China.The dNTPs,Taq E,and DL2000 Marker were purchased from TaKaRa Biotechnology Company,Dalian,China.

2.5.Bioinformatics analysis of AhCSD2

Sequence alignment was performed with DNAMAN and intron analysis was performed with Splign(http://www.ncbi.nlm.nih. gov/sutils/splign/splign.cgi).The molecular weights(Mw)and isoelectric points(pI)of deduced amino acid sequences were analyzed with ProtParam of ExPASy[27](http://web.expasy.org/ protparam/).The protein secondary structures were analyzed with SOPMA(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? page=npsa_sopma.html).Conserved domains were identified with CD-Search(http://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi),and the prediction of 3D structure and of transit peptides was performed with Swiss-Model[28,29]and iPSORT [30](http://ipsort.hgc.jp/),respectively.Protein sequences of different species were obtained from the GenBank database. Peanut genome sequences were obtained from PeanutBase (http://peanutbase.org/genomes)[23].The online software Place[31](http://www.dna.affrc.go.jp/PLACE/)and Plantcare[32] (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) were used to predict cis-acting elements in potential promoter sequences of AhCSD2.

3.Results

3.1.SOD activity and drought resistance of peanut cultivars

Data from field tests were collected in 2013 and 2014.As shown in Table 1,changes in trends of SOD activity and drought resistance of cultivars were similar during the two years,aside from some small differences.SOD activity change differences among tested cultivars are significant in response to drought stress.Based on the yield drought resistance study,the DC values of the 11 cultivars were also significantly different. For example,the SOD activity of Shanhua 11,Jihua 4,and Rugaoxiyangsheng showed increases significantly higher than those of other varieties under drought conditions,while these cultivars showed higher DC(Table 1).In contrast,the DC values of ICG6848,Baisha 1016 and Krapt.st.16 were significantly lower than those of other varieties,and their SOD activity decreased markedly under drought stress in comparison with the standard treatment(Table 1).SOD activities under stress and their changes were significantly and positively correlated with DC(r＞0.65,P＜0.01)(Table 3),strongly suggesting that SOD activity has a close relationship with drought resistance in peanut.All the above results were detected in the two years (Tables 1 and 3).Thus,SOD plays a crucial role in responding to drought-associated stresses.

3.2.Cloning and sequencing of AhCSD2

To perform the AhCSD2 cloning and sequence analyses, genomic DNA and cDNA transcribed from RNA were amplified by PCR using the specific primer pair CSD2-F/CSD2-R.The amplification of all the samples'cDNA resulted in a single band with the expected size of 750 bp.The amplification of genomic DNA generated an intense band 2400 bp in size. These bands were extracted from the agarose gels,cloned and sequenced.Two cDNA sequences,namely,rCSD2-1 and rCSD2-2,were obtained from each cultivar and showed very high identity with the EST splicing sequence(98.43%and 99.87%,respectively),whereas one sequence was obtained from each wild species(Table 2).The sequenced genomic DNA amplification products of the 11 cultivars also showed 2 sequences,named as gCSD2-1 and gCSD2-2,whereas those of the 5 wild species had only one sequence each(Table 2).

3.3.Physical location of AhCSD2 in the peanut genome

The position of AhCSD2 genes in the full peanut genome, determined by sequence alignment usingg CSD2-1 and gCSD2-2,is located at the tops of chromosome A03 and B03, respectively.Sequence alignment showed that gCSD2-2 and the corresponding region within B03 share 100%identity. Aradu.A03 is 135.1 Mb and Araip.B03 136.1 Mb in length[23]. BLAST of gAduCSD2 against Aradu.A03 showed that the gene is located between 747,247 and 744,856 bp.In Araip.B03, gAipCSD2 is located between 2,760,316 and 2,757,888 bp.BLAST of rCSD2-1 and rCSD2-2 against the peanut genome database showed that AhCSD2 genes contain 8 exons and 7 introns.

Table 3-Correlation coefficient between SOD activities and DC in peanut cultivars.

3.4.Polymorphism analyses of AhCSD2 nucleotide sequences

3.4.1.AhCSD2 sequence analyses

Taking Shanhua 11 as an example,cDNA sequences,rSh11CSD2-1 and rSh11CSD2-2,are 764 bp in full-length containing a 3′UTR of 87 bp,a 5′UTR of 53 bp,and an ORF of 624 bp that encodes 207 amino acid(aa)residues.The two genomic DNA counterparts, gSh11CSD2-1andgSh11CSD2-2,are2395 bpand2428 bpinlength, respectively,and detailed Splign analyses revealed that each of these genomic DNA sequences contains 8 exons and 7 introns (Fig.1-A),whose shearing modes follow the GT/AG rules. Sequence alignment showed that the exons of rSh11CSD2-1 and rSh11CSD2-2 are identical to those of gSh11CSD2-1 and gSh11CSD2-2,respectively.The average length of these exons is 96 bp,with the longest located between 1 and 291 bp and the shortest located between 1230 and 1261 and 1261–1292 bp, respectively(Fig.1-A).The two genomic DNA sequences share 96.54%identity,with a total of 84 site differences(Fig.1-B),of which 9 single-nucleotide differences are present in the ORF and another 2 in the 3′UTR and 5′UTR regions.Interestingly, intronic polymorphisms are abundant:gSh11CSD2-1 has deletions of 8 bp(391–398 bp)and 24 bp(544–567 bp)in intron I and another deletion of 2 bp(2081–2082 bp)in intron VII(Fig.1-B).

3.4.2.Allelic polymorphism analyses of AhCSD2 in peanut cultivars

To investigate the differences among AhCSD2 sequences within the 11 cultivars,we aligned them.We found that the sequences of gCSD2-2 are completely conserved,whereas gCSD2-1 showed 99.98%identity,with 3 single-nucleotide polymorphisms(SNPs).It is interesting to note that the 3 SNPs detected in the gCSD2-1 sequences in these samples occurred only in introns.Based on the similarity of the distributions of the 3 SNPs in gCSD2-1 among these cultivars,Shanhua 9, Shanhua 11,Jihua 4,Fenghua 2,Baisha 1016,ICG6848,and Krapt.st.16 belong to group a1,whereas Nongda 818 andRugaoxiyangsheng belong in the a2 group on the basis of their T deletion at 1220 bp and a transition from G to A at 1523 bp. The a3 group consists of Lanna1 and Lipudahuasheng,both of which have a Tdeletion at 1515 bpcompared with gSh11CSD2-1.

Fig.3-The predicted 3D structure of the chloroplast Cu/Zn-SOD(AhCSD2)in Arachis hypogaea compared with that in tomato (Solanum lycopersicum L.).(A)Structure of chloroplast Cu/Zn-SOD in tomato;(B)3D structure of AhCSD2;(C)binding sites of Cu2+; (D)binding sites of Zn2+;(E)the disulfide bond;(F)the salt bridge.

3.4.3.Polymorphism analyses of AhCSD2 in peanut cultivars with wild species

Sequences analyses strongly indicated that the gCSD2-1 and gCSD2-2 are derived from the A and B genomes,respectively, of cultivated peanut(A.hypogaea).The gCSD2-2 sequences of A.hypogaea and gAipCSD2 of A.ipaensis from the B genome are identical,while the gCSD2-1 sequence shares identity of 97.45% with gAduCSD2 of A.duranensis,96.84%with gAcaCSD2 of Arachis cardenasii,95.65% with gAcoCSD2 of Arachis correntina,and95.28% with gAviCSD2 of Arachis villosa,respectively.Polymorphism analyses showed that sequences from the 4 wild A genome diploid species have abundant polymorphic loci compared with gSh11CSD2-1(Fig.1-B),with 15 single-nucleotide differences in the exon region and 12 in the ORF(Fig.1-B).The intronic polymorphism suggested that introns I,VI,and VII have abundant variant sites(Fig.1-B).

3.5.Molecular characteristics of deduced AhCSD2 amino acid sequences

3.5.1.Analyses of AhCSD2 in A.hypogaea

The deduced amino acid sequences of AhCSD2-1 and AhCSD2-2, based on the rCSD2-1 and rCSD2-2 genes,both consist of 207 aa. The two sequences show 96.14% identity,with 8 aa differences,6 of which occur in the transit peptide region(Fig.2).Given that there is no clear transmembrane structure,AhCSD2 is likely a non-secreted protein.Based on the predicted subcellular localization,it is most probably located in the chloroplast,based on its high prediction value of 9.8.The predicted chloroplast transit peptides of AhCSD2-1 and AhCSD2-2 both span 1–53 aa(Fig.2). We speculate that the AhCSD2 precursor is synthesized in the cytoplasm and then transported into the chloroplast to exercise a biological function.Analyses of the predicted mature AhCSD2-1 and AhCSD2-2 peptides(designated as Ah-1 and Ah-2,both spanning 54–207 aa)revealed that their MW is 15.7 kDa.The pI of Ah-1 is 5.15 and that of Ah-2 is 5.32. Secondary structure prediction indicated that the mature Ah-1 peptide consists of 11.69%α helices,37.66%extended strands, 11.04%β turns,and 39.61%random coils,while Ah-2 consists of 14.01%α helices,33.33%extended strands,9.18%β turns,and 43.48%random coils.

Next,we compared the deduced peanut AhCSD2 sequences with the known chloroplast Cu/Zn-SOD sequences of many other plants.Sequence alignment analyses revealed that the AhCSD2 in Arachis shows very high(61–82%)identity with proteins of most other plants.These are highly conserved proteins,especially at the active sites,which show two motifs(Fig.2).Motif I belongs to the PS00087 group(GFHLHEYGDTT)and is a Cu2+binding region,whereas motif II belongs to the PS00332 group (GNAGGRLACGVV)and is a disulfide bond formation site.These sites are essential for catalysis.Furthermore,the AhCSD2 proteins harbor the standard ion binding sites of copper(Cu2+) and zinc(Zn2+)constituting an active enzyme center,similar to other members of the Cu/Zn-SOD family.As is shown in Fig.2, the N-terminal transit peptides harbor most of the variations in chloroplast Cu/Zn-SOD sequences from different plants,while the mature protein sequences are highly conservative.

Fig.4-Predicted 3D structure differences between the two classes(Ah-1 and Ah-2)of mature peptide.(A)Amino acid residues and H-bonds contained in the region of Asn155 as the center with radius of 6 ? in class Ah-1;(B)amino acid residues and H-bonds contained in the region of Lys155 as the center with radius of 6 ? in class Ah-2;(C)structure of class Ah-1 with Ser160 as the center with radius of 6 ?;(D)structure of class Ah-2 with Thr160 as the center with radius of 6 ?.

An X-ray crystal structure study of Kitagawa[33]showed that chloroplast Cu/Zn-SOD is a homodimer,with each subunit binding a Cu2+and a Zn2+.The use of the tomato chloroplast Cu/Zn-SOD(PDB id:1SRD;Fig.3-A)as a template allowed us to construct the AhCSD2's three-dimensional(3D)structure. The 3D structure showed each subunit consists of eight anti-parallel β strands that constitute a flattened cylinder,in addition to two short loops(Fig.3-B).The Cu2+binding sites were located at His99,His101,His116,and His173(Fig.3-C) and the Zn2+binding sites at His116,His124,His133,and Asp136(Fig.3-D).In addition,a disulfide bond between Cys110 and Cys199(Fig.3-E)and a salt bridge between Arg132 and Asp154(Fig.3-F)may be associated with the stabilization of the spatial structures.

3.5.2.Analysis of AhCSD2 in peanut cultivars with wild species Although within the coding regions of AhCSD2 there are loci polymorphic between cultivars and wild species,the deduced amino acid sequences are highly conserved.Alignment showed that AhCSD2-2 of A.hypogaea shares 100%identity with AipCSD2 of A.ipaensis and AviCSD2 of Arachis villosa. AhCSD2-1 is identical to AduCSD2 of A.duranensis.AcoCSD2 of A.correntina and AcaCSD2 of A.cardenasii both have 2 aa differences(Thr17→Pro17,Phe32→Leu32)within the transit peptide region compared with AhCSD2-1,whereas the mature peptide regions are completely conserved(Fig.2).Thus,the protein sequences can be divided into two classes based on the mature peptide sequences:classes Ah-1 and Ah-2.

In the two classes of mature peptides,Ah-1 and Ah-2,two amino acid variant sites were identified at the end of the fourth β strand.Asn155 of Ah-1 formed H-bonds with Thr82 and Asp154 (Fig.4-A).Lys155 of Ah-2 formed no H-bond with any other residue,owing to the positively charged tail extending towards the outside(Fig.4-B).The difference between Ser160 in Ah-1 and Thr160 in Ah-2 did not result in an obvious spatial change (Fig.4-C,D).

3.6.Potential promoter sequence analysis of AhCSD2

Based on the genome sequence,we obtained the potential promoter sequences upstream of the 5′end of gCSD2 from A.duranensis(AA)and A.ipaensis(BB),which are 1500 bp in length and named pp-A and pp-B,respectively.Sequence alignment showed that the two sequences share only 52.23%identity,with abundant polymorphic loci.cis-acting element prediction showed that typical eukaryotic promoter elements including CAAT-box and TATA-box were found in pp-A and pp-B(Fig.5).Both contained many cis-acting elements responding to light,hormone,drought,heat,frost, and other environmental stresses(Fig.5).These findings strongly suggest that AhCSD2 plays a key role in response to drought and many other stresses in peanut.Further analysis showed that pp-A contains 2 dehydration-responsive elements(DREs)and 3 low temperature-responsive elements (LTREs),none of which are found in pp-B(Fig.5 and Table 4). pp-A also contains 8 binding sites for MYB,10 for MYC,and 8 for WRKY,which are all important drought-related transcription factors,while pp-B contains 5 binding sites for MYB,4 for MYC,and 4 for WRKY(Table 4).

4.Discussion

Compared with other crops,peanut is considered to be drought tolerant,but yield loss and poor quality caused by drought remains the primary issue that needs to be addressed[12].As one of the key enzymes in the antioxidant protection system, SOD plays a crucial role in responding to drought-associated stresses.In peanut,Cu/Zn-SOD comprises a major proportion of SOD isozymes and is one of the most important antioxidant enzymes in leaves[17].Sequence alignment analyses revealed that the chloroplast Cu/Zn-SOD of different plants share very high identity,especially in the two conserved motifs that are essential for catalysis(Fig.2).These results also illustrate the conservation and importance of chloroplast Cu/Zn-SOD in the evolution of plants.Studies of SOD and related genes will provide us with valuable information for understanding the mechanism of SOD response to drought and other environmental stresses.

It is widely believed that A.duranensis(AA)and A.ipaensis (BB)are the ancestors of A.hypogaea,which was likely derived from natural hybridization followed by spontaneous chromosome doubling[20–22].The present study revealed that the AhCSD2 sequences of gCSD2-1 and gCSD2-2 are derived from respectively theA and B genomes of A.hypogaea.The gCSD2-2 is identical to gAipCSD2 of A.ipaensis(BB).Despite DNA sequencedifferences among peanut cultivars and A.duranensis(AA),the deduced AhCSD2-1 and AduCSD2 sequences are completely identical(Fig.2).The results indicate that cultivars obtained these valuable genes from their wild ancestors by inheritance. Allelic polymorphism analyses in this study showed that the gCSD2-2 sequences from the B genome in all cultivars are identical,while the gCSD2-1 sequences from the A genome show some nucleotide differences.Based on the 3 SNPs in the intronic region in the A genome,cultivars of var.vulgaris,var. fastigiata,and irregular type all belong to the a1group,and those of var.hypogaea and var.hirsute to the a2 and a3 groups.These results provide molecular evidence for morphological classifications in the cultivated peanut.

Table 4-Details and function of some predicted cis-acting elements in potential promoter sequences of AhCSD2.

In the two AhCSD2 genes of cultivars,gCSD2-1 and gCSD2-2, polymorphic sites were abundant,in addition to the variations within ORFs(Fig.1).Differences in 2 aa residues were detected in the Ah-1 and Ah-2 mature peptides based on the deduced sequence alignment analyses(Fig.2).It is possible that such variations result in changes in protein spatial configuration (Fig.4),thereby changing their biological functions and enzyme activity.Similar conclusions have been reached by two independent studies of FAD2[34,35].Chloroplast transit peptide variants showed different protein-transferring efficiencies, even though they were all capable of transporting exogenous proteins into chloroplasts,ultimately improving plant oxidation resistance[36].Our results suggest that 6 aa differences distinguish the transit peptides of AhCSD2-1 and AhCSD2-2 (Fig.2).However,their transferring efficiencies and effects on SOD activity remain to be further investigated.

Previous studies have revealed that drought-tolerant varieties showed higher SOD activity than drought-sensitive ones,so that high SOD activity can be considered a drought-tolerance indicator[14–16].In the present study,drought resistances of the tested 11 cultivars,including both drought-tolerant and -sensitive ones,measured by DC were significantly different. SOD activities and their changes differed significantly between cultivars with different drought resistance(Table 1).Thesimilar change trends of SOD activity and drought resistance of cultivars in the two years also suggest that different varieties respond to drought stress differently and that the traits of SOD activity and drought resistance are largely genotype dependent. A study has proven this assertion[13].The significant positive correlation between SOD activity and DC of cultivars(Table 3) also suggests that SOD,as one of the key enzymes of protective antioxidant system,plays an important role under drought stress in peanut.

However,the coding sequences and the deduced amino acid sequences of AhCSD2 gene among the tested cultivars with significantly different drought resistances are highly conserved.We accordingly investigated the regulatory region of the AhCSD2 gene.Interestingly,promoter prediction of gCSD2-1 and gCSD2-2 within A and B genome(pp-A and pp-B) identified many stress-associated cis-acting elements(Fig.5). Thus,the AhCSD2 genes are likely to be regulated by many transcriptional factors,such as DREB,MYB,MYC,and WRKY, through the ABA-dependent or-independent pathway in response to various biological and abiotic stresses.The binding sites of transcriptional factors indicated that AhCSD2 in peanut also plays a key role in response to many other stresses besides drought.We hypothesized that the changes of SOD activity in different cultivars under drought are highly regulated by transcription factors through transcriptional and expressional level of the AhCSD2 gene.Our current experiments have recently confirmed this point and will be reported elsewhere.

Further analysis showed that PP-A harbors 2 DREs and 3 LTREs,unusually,and contains even more binding sites for MYB, MYC,and WRKY,which have been proven to be important in response to drought stress,than pp-B(Table 3).We accordingly hypothesized that the 2 wild species,A.duranensis and A.ipaensis, have different regulatory pathways from gCSD2.In view of the 3D structure differences between AhCSD2-1 and AhCSD2-2,we may speculate that the two genes from cultivated peanut,gCSD2-1 and gCSD2-2,also have a different gene regulation pathway from AhCSD2.Whether the SOD activity differences are regulated by promoter activity of AhCSD2 in different peanut varieties because of the polymorphic loci in promoter regions is currently under experimental study.

Acknowledgments

We thank Dr.Jiping Zhao(Ball Horticultural Company,USA) for his constructive comments and English improvement of this manuscript.We appreciate the assistance and dedication of Professor Huifang Jiang(Oil Crops Research Institute, Chinese Academy of Agricultural Sciences)for supplying the five wild species A.correntina,A.villosa,A.duranensis, A.cardenasii,and A.ipaensis.The authors acknowledge financial support by the Natural Science Foundation of China(NSFC) (31201167),the earmarked fund for China Agriculture Research System(CARS-14),the Peanut Seed Industry Project in Shandong province of China,and the earmarked fund for Agriculture Research System in Shandong province of China.

[1]C.Bowler,M.V.Montagu,D.Inze,Superoxide dismutase and stress tolerance,Annu.Rev.Plant Biol.43(1992)83–116.

[2]S.S.Gill,N.Tuteja,Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants,Plant Physiol.Biochem.48(2010)909–930.

[3]C.H.Huang,W.Y.Kuo,C.Weiss,T.L.Jinn,Copper chaperone-dependent and-independent activation of three copper-zinc superoxide dismutase homologs localized in different cellular compartments in Arabidopsis,Plant Physiol. 158(2012)737–746.

[4]H.Wang,T.Feng,X.Peng,M.Yan,X.Tang,Up-regulation of chloroplastic antioxidant capacity is involved in alleviation of nickel toxicity of Zea mays L.by exogenous salicylic acid, Ecotoxicol.Environ.Saf.72(2009)1354–1362.

[5]H.Li,J.Cai,F.Liu,D.Jiang,T.Dai,W.Cao,Generation and scavenging of reactive oxygen species in wheat flag leaves under combined shading and waterlogging stress,Funct. Plant Biol.39(2012)71–81.

[6]R.Mittler,S.Vanderauwera,M.Gollery,F.Van Breusegem, Reactive oxygen gene network of plants,Trends Plant Sci.9 (2004)490–498.

[7]Y.Ishibashi,T.Tawaratsumida,K.Kondo,S.Kasa,M. Sakamoto,N.Aoki,S.H.Zheng,T.Yuasa,M.Iwaya-Inoue, Reactive oxygen species are involved in gibberellin/abscisicacid signaling in Barley aleurone cells,Plant Physiol.158 (2012)1705–1714.

[8]D.J.Kliebenstein,R.A.Monde,R.L.Last,Superoxide dismutase in Arabidopsis:an eclectic enzyme family with disparate regulation and protein localization,Plant Physiol.118(1998) 637–650.

[9]H.M.Li,C.C.Chiu,Protein transport into chloroplasts,Annu. Rev.Plant Biol.61(2010)157–180.

[10]E.Apostolova,M.Rashkova,N.Anachkov,I.Denev,V. Toneva,I.Minkov,G.Yahubyan,Molecular cloning and characterization of cDNAs of the superoxide dismutase gene family in the resurrection plant Haberlea rhodopensis,Plant Physiol.Biochem.55(2012)85–92.

[11]Y.S.Kim,H.S.Kim,Y.H.Lee,M.S.Kim,H.W.Oh,K.W.Hahn,H. Joung,J.H.Jeon,Elevated H2O2production via overexpression of a chloroplastic Cu/ZnSOD gene of lily(Lilium oriental hybrid‘Marco Polo’)triggers ethylene synthesis in transgenic potato,Plant Cell Rep.27(2008)973–983.

[12]H.D.Upadhyaya,Variability for drought resistance related traits in the mini core collection of peanut,Crop Sci.4(2005) 1432–1440.

[13]T.Girdthai,S.Jogloy,N.Vorasoot,C.Akkasaeng,S. Wongkaew,C.Holbrook,A.Patanothai,Associations between physiological traits for drought tolerance and aflatoxin contamination in peanut genotypes under terminal drought, Plant Breed.129(2010)693–699.

[14]H.F.Jiang,X.P.Ren,The effect on SOD activity and protein content in groundnut leaves by drought stress,Acta Agron. Sin.30(2004)169–174(in Chinese with English abstract).

[15]G.Naik,Physiological and biochemical responses of groundnut genotypes to drought stress,World J.Sci.Technol. 1(2011)60.

[16]G.H.Li,K.Zhang,F.Z.Liu,D.D.Liu,Y.S.Wan,Leaf physiological traits at pod-setting stage in peanut cultivars with different drought resistance,Chin.J.Appl.Ecol.25(2014) 1988–1996(in Chinese with English abstract).

[17]I.Juszczak,M.Baier,The strength of the miR398-Csd2-CCS1 regulon is subject to natural variation in Arabidopsis thaliana, FEBS Lett.586(2012)3385–3390.

[18]L.Rizhsky,H.Liang,R.Mittler,The water-water cycle is essential for chloroplast protection in the absence of stress, J.Biol.Chem.278(2003)38921–38925.

[19]X.R.Zhang,F.Z.Liu,Y.J.Yu,K.Zhang,Y.S.Wan,Cloning and allelic polymorphism analysis of Cu/Zn-SOD gene(AhCSD1) in Peanut(Arachis hypogaea L.),Mol,Plant Breed.11(2013) 1068–1081(in Chinese with English abstract).

[20]A.P.Fávero,C.E.Simpson,J.F.Valls,N.A.Vello,Study of the evolution of cultivated peanut through crossability studies among Arachis ipaensis,A.duranensis,and A.hypogaea,Crop Sci.46(2006)1546–1552.

[21]A.Hilliker,M.D.Burow,C.E.Simpson,M.W.Faries,J.L.Starr, A.H.Paterson,Molecular biogeographic study of recently described B-and A-genome Arachis species,also providing new insights into the origins of cultivated peanut,Genome 52 (2009)107–119.

[22]M.C.Moretzsohn,E.G.Gouvea,P.W.Inglis,S.C.Leal-Bertioli, J.F.Valls,D.J.Bertioli,A study of the relationships of cultivated peanut(Arachis hypogaea)and its most closely related wild species using intron sequences and microsatellite markers,Ann.Bot.111(2013)113–126.

[23]PGP.Peanut Genome Project,http://peanutbase.org/.

[24]M.Jiang,J.Zhang,Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves,J.Exp.Bot.53(2002) 2401–2410.

[25]G.H.Li,K.Zhang,F.Z.Liu,D.D.Liu,Y.S.Wan,Morphological and physiological traits of leaf in different drought resistant peanut cultivars,Sci.Agric.Sin.47(2014)644–654(in Chinese with English abstract).

[26]J.Y.Dong,Y.S.Wan,F.Z.Liu,Sequence analysis of Δ9-stearoyl-ACP desaturase gene(SAD)in Peanut,Acta Agron. Sin.38(2012)1167–1177(in Chinese with English abstract).

[27]E.Gasteiger,C.Hoogland,A.Gattiker,M.R.Wilkins,R.D. Appel,A.Bairoch,Protein identification and analysis tools on the ExPASy server,in:John M.Walker(Ed.),The Proteomics Protocols Handbook,Humana Press,Totowa,New Jersey 2005,pp.571–607.

[28]T.Schwede,J.Kopp,N.Guex,M.C.Peitsch,SWISS-MODEL: an automated protein homology-modeling server,Nucleic Acids Res.31(2003)3381–3385.

[29]K.Arnold,L.Bordoli,J.Kopp,T.Schwede,The SWISS-MODEL workspace:a web-based environment for protein structure homology modeling,Bioinformatics 22(2006)195–201.

[30]H.Bannai,Y.Tamada,O.Maruyama,K.Nakai,S.Miyano, Extensive feature detection of N-terminal protein sorting signals,Bioinformatics 18(2002)298–305.

[31]K.Higo,Y.Ugawa,M.Iwamoto,T.Korenaga,Plant cis-acting regulatory DNA elements(PLACE)database,Nucleic Acids Res.27(1999)297–300.

[32]L.Magali,D.Patrice,T.Ger,M.Kathleen,M.Yves,V.P.Yves, R.Pierre,R.Stephane,PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences,Nucleic Acids Res. 30(2002)325–327.

[33]Y.Kitagawa,N.Tanaka,Y.Hata,M.Kusunoki,G.P.Lee,Y. Katsube,K.Asada,S.Aibara,Y.Morita,Three-dimensional structure of Cu,Zn-superoxide dismutase from spinach at 2.0 ? resolutions,J.Biochem.109(1991)477–485.

[34]S.Jung,G.Powell,K.Moore,A.Abbott,The high oleate trait in the cultivated peanut(Arachis hypogaea L.):II.Molecular basis and genetics of the trait,Mol.Gen.Genet.263(2000) 806–811.

[35]M.Patel,S.Jung,K.Moore,G.Powell,C.Ainsworth,A.Abbott, High-oleate peanut mutants result from a MITE insertion into the FAD2 gene,Theor.Appl.Genet.108(2004)1492–1502.

[36]E.M.Bruch,G.L.Rosano,E.A.Ceccarelli,Chloroplastic Hsp100 chaperones ClpC2 and ClpD interact in vitro with a transit peptide only when it is located at the N-terminus of a protein, BMC Plant Biol.12(2012)57.

*Corresponding author.Tel.:+86 13854849601.

**Corresponding author.Tel.:+86 13805386663.

E-mail addresses:liufz@sdau.edu.cn(F.Liu),yswan@sdau.edu.cn(Y.Wan).

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science,CAAS.

http://dx.doi.org/10.1016/j.cj.2015.03.006

2214-5141/?2015 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).