research article kvlea , a new isolated late embryogenesis...

Research ArticleKvLEA a New Isolated Late EmbryogenesisAbundant Protein Gene from Kosteletzkya virginicaResponding to Multiabiotic Stresses

Xiaoli Tang12 Hongyan Wang123 Liye Chu4 and Hongbo Shao15

1Key Laboratory of Coastal Biology amp Bioresources Utilization Yantai Institute of Coastal Zone Research (YIC)Chinese Academy of Sciences (CAS) Yantai 264003 China2University of Chinese Academy of Sciences Beijing 100049 China3Yantai Academy of China Agricultural University Yantai 264670 China4The Central Laboratory Jiangsu Academy of Agricultural Sciences Nanjing 210014 China5Jiangsu Key Laboratory for Bioresources of Saline Soils Provincial Key Laboratory of Agrobiology Institute of Agro-BiotechnologyJiangsu Academy of Agricultural Sciences Nanjing 210014 China

Correspondence should be addressed to Hongbo Shao shaohongbochu126com

Received 14 January 2016 Accepted 3 March 2016

Academic Editor Byoung R Jeong

Copyright copy 2016 Xiaoli Tang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

TheLEAproteins are a kind of hydrophilic proteins playingmain functions in desiccation toleranceHowever their importance as akind of stress proteins in abiotic stress is being clarified little by little In this study we isolated cloned and identified the firstKvLEAgene in Kosteletzkya virginica Bioinformatic analysis showed that the protein encoded by this gene had common properties of LEAproteins and the multiple sequences alignment and phylogenetic analysis further showed that this protein had high homology withtwo Arabidopsis LEA proteins Gene expression analysis revealed that this gene had a higher expression in root and it was inducedobviously by salt stress Moreover the transcripts of KvLEA were also induced by other abiotic stresses including drought hightemperature chilling and ABA treatment Among these abiotic stresses ABA treatment brought about the biggest changes to thisgene Collectively our research discovered a novel LEA gene and uncovered its involvement in multiabiotic stresses in K virginicaThis research not only enriched studies on LEA gene in plant but also would accelerate more studies on K virginica in the future

1 Introduction

Plants are continuously subjected to environmental stressessuch as drought high salinity cold chilling and freezingalong their life cycles These abiotic stresses can negativelyaffect plants in growth and development leading to enor-mous losses in agricultural production throughout the world[1 2] For survival plants have developed a variety ofanatomical and morphological features which may alleviatethe damage of these adverse conditions Both the activation ofstress responsive genes and the synthesis of resistant proteinstrigger a series of physiological and metabolism responsesto environmental stresses [3ndash5] The late embryogenesisabundant (LEA) protein is one of the many resistant proteinswhich were synthesized under adverse conditions [6 7]

The researches on LEA proteins began with the discoveryof the first LEA protein which was discovered in cotton seedsmore than three decades ago [8] From then on the studieson LEA proteins have never stopped So far we have knownthat these proteins are highly hydrophilic and intrinsicallydisordered with low proportions of cysteine and tryptophanand high proportions of glycine glutamate lysine andthreonine [9 10] According to the amino acid sequences andconserved motifs the LEA proteins are generally categorizedinto different groups in different plants [11] For instancethere are as many as 51 LEA proteins in Arabidopsis andthey are divided into nine groups including LEA-1 LEA-2LEA-3 LEA-4 LEA-5 SMP PvLEA18 dehydrin and AtM[7] However according to the PFAM database LEA proteinswere grouped into eight families dehydrin LEA 1 LEA 2

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 9823697 10 pageshttpdxdoiorg10115520169823697

2 BioMed Research International

LEA 3 LEA 4 LEA 5 LEA 6 and SMP without the AtMgroup [12] The LEAPdb which was a public database of LEAgene sequences gave a more detailed classification with 12nonredundant groups [13]

As the researches on LEA move along the importanceof LEA proteins is reported by more and more studiesTheir synthesis in large amount during the late stages ofseed development enables the maturing seeds to acquire thedesiccation tolerance Importantly by means of stabilizingproteins nucleic acids and cell membranes LEA proteinsare proved to be the common components associated withtolerance to various stresses [14 15] For example the maizeLEA3 gene was discovered to be induced by ABA andhyperosmolarity [16] and afterwards it was proved to beable to reduce cell shrinkage effects under dehydration [17]Similarly a novel LEA genewas identified inwheat whichwasinduced by freezing tolerance [18] The reduced expressionof CeLEA1 a LEA-like protein from Caenorhabditis elegansled to reduced survival under desiccation and osmotic andheat stresses [19] On the other hand ectopic expressions ofLEA in various organisms also gave the evidence for theirfunctions in stress protection [9 20 21] The transgenicwheat and rice with heterogeneous expression of LEA genefrom barley acquired enhanced drought and salt tolerance[22 23] Overexpressing NtLEA7-3 in Arabidopsis endowedthe transgenic plants with enhanced resistance to colddrought and salt stresses [24] In Arabidopsis the SAG21was discovered to enhance the tolerance against bacterialpathogen (Pst DC3000) and oxidative stress [25] The LEAprotein Rab28 which is localized in the nucleus in maizeembryo can enhance transgenic maize tolerance to droughtstress [26] Therefore the LEA proteins are involved not onlyin water deficit but also in abiotic stresses It is one of the keyfactors for plants to respond to adverse environment

K virginica is a newly introduced species due to itsamazing salt stress resistance and potential economic value[27] It was reported that this plant was able to maintainnormal growth and development under adverse conditionswith 03 to 25 sodium salt [28] Thus it must possesshighly efficient physiological biochemical and molecularmechanisms to do this Therefore we are interested in itsspecial mechanisms under salt stress in the hope of uncover-ing novel resistance mechanisms in it However the geneticinformation on it is very scarce As far as we know thereare almost no reports about the molecular mechanism ofsalt resistance on K virginica except for one report coveringthe gene expression analysis on K virginica seedlings bycDNA-amplified fragment length polymorphism (cDNA-AFLP) technology [29]

Based on the diverse biological functions of LEA proteinsas stated before we wondered whether LEA also had similarroles in K virginica Therefore this study aimed to cloneisolate identify and explore the possible functions of a LEAgene in K virginica With the help of transcriptome analysisof K virginica under salt stress in our recent research wegot the core sequence of the gene [30] RACE technologywas adopted to clone the whole sequence of this geneThe biological software such as ProParam InterProScan 5BLASTp and CLUSTALWX was used to do the sequence

prediction and analysis Phylogenetic tree was constructedto analyze which subgroup it may belong to FurthermoreqPCR assays were carried out to detect the gene expressionpatterns under abiotic stressesThis study about the first LEAgene inK virginica further enriched the research informationon LEA gene Meanwhile it also laid a good foundation formolecular studies in K virginica

2 Material and Methods

21 Plant Materials and Growth Conditions The seeds of Kvirginica used in this study were coming from theK virginicagrowing in Yellow River Delta Shandong Province ChinaThe collected seeds were disinfected with 70 alcohol solu-tion for 30 s and 01 mercuric chloride solution for 10minand then washed thoroughly with sterile water six timesThe surface-sterilized seeds were laid on wet filter paper forgermination and then the germinating seeds were planted invermiculite in plastic pots watered with Hoaglandrsquos solution[31] The seedlings were cultivated under conditions of 16 hlight8 h dark and the temperature was kept at 25∘C20∘Crespectively in day and night in artificial climatic chambers(Huier China) The humidity was kept at 60 and theseedlings were watered every 3 days with nutrition solutionTwo-week-old and homogeneous seedlings were chosen fordifferent samplings and six homogeneous seedlings at leastwere contained in each sample However the samples fortissue expression pattern detection came from the differentcorresponding tissues respectively All the samples werefrozen with liquid nitrogen and stored at minus80∘C

22 Cloning and Sequence Analysis of KvLEA For genecloning total RNA was extracted with RNAiso Plus (TaKaRatotal RNA extraction reagent) according to the opera-tion steps Agilent 2100 BioAnalyzer (Agilent Technolo-gies CA USA) was used for the detection of the RNAquality and quantity The full length of KvLEA was ampli-fied by RACE according to the user manual (SMARTRACE cDNA Amplification Kit) The gene-specific primers(GSPs) (forward CCTCCTGCTCCTGGCTCTTA reverseGGTGGCTGAAGGGACGGAGTA) and the nested primers(NGSPs) (forward CCTGGCTCTTACTACCAACGACreverse AAGCAGACGGCATCCGAAAC) were designedbased on the cDNA core sequence coming from our previoustranscriptome sequencing data which had been deposited inTranscriptome Shotgun Assembly (TSA) Sequence Databasewith the accession number GCJL00000000 [30] The PCRproducts were cloned into pEASY-T5 Zero Cloning Vector(Transgene China) after purification from gels for sequen-cingThe physical and chemical parameters of KvLEAproteinwere analyzed with ProParam InterProScan 5 (httpwwwebiacukToolspfaiprscan5) and the Sequence Manipula-tion Suite (httpwwwbio-softnetsms) The BLASTp wasperformed at NCBI (National Center for Biotechnologyfor Information httpblastncbinlmnihgovBlastcgi) [32]Multiple sequence alignment was carried out among thededuced KvLEA protein and all the eighteen LEA 4 proteinsinArabidopsiswithCLUSTALWXprogramThephylogenetic

BioMed Research International 3

tree was produced based on the multiple sequence alignmentusing MEGA 60 software by Neighbor-Joining method [33]

23 Multiabiotic Stress Treatments The two-week-old Kvirginica seedlings grown in plastic pots (six seedlings at leastin each pot) were selected for our research For heat andchilling stress treatments the homogeneous seedlings weretransferred to different artificial climatic chambers (HuierChina) at 42∘C and 4∘C respectively For high salinitydrought and ABA stress treatments the plants were irrigatedwith nutrient solution with 300mM NaCl 15 PEG 6000and 100 120583M ABA respectively The handling times for allthe treatments were 0 2 6 12 and 24 hours The controlsample was kept at normal growth conditions and irrigatedwith nutrition solution The control and treated sampleswere all removed from the matrix and washed carefully aftertreatments Then the seedlings were soaked up cautiouslyand quick-frozen in liquid nitrogen and stored at minus80∘C foruse

24 RNA Isolation and Gene Expression Analysis The totalRNAwas extracted from samples with RNAiso Plus (TaKaRatotal RNA extraction reagent) The first-strand DNA wassynthesized with TransScriptOne-Step gDNA Removal andcDNA Synthesis SuperMix (TransGen China) The qPCRwas performed with the cDNA diluted at a certain ratio astemplate and the gene-specific qPCR primers were designedwith the primer-design software Beacon Designer 70 Theprimers are 51015840-TAGTAAGAGCCAGGAGCAGGAG and31015840-TTGAACTCGCCAGCCTTATCTC The primers of theinternal control gene KvEF1-120572 are 51015840-TCAATGAGCCAA-AGAGG and 31015840-CAACACGACCAACAGGA

The qPCR was conducted on the instrument ABI Prism7500 FAST (Applied Biosystems Foster City CA) and thereaction was performed using SYBR Green Real-TimeSelected Master Mix (Applied Biosystems by Life Technolo-gies) according to the user manual The reaction volumewas 20120583L including 2120583L diluted cDNA template 10 120583L 2xSYBR Master Mix and 200 nM of each primer The reactionprocesses were as follows (1) initial denaturation at 95∘C for2min 40 cycles of 15 s at 95∘C for denaturation of templateand 1min at 60∘C for annealing and extensionThe detectionof the fluorescence signal was conducted at the temperaturebetween 60∘C and 90∘C The primer specificity was ensuredbeforehand by the typical melting curve and amplificationplot as well as the sole product in RT-PCR The cDNAtemplate was diluted to guarantee that the Ct values of all thesamples were located in the appropriate scope Each samplewas performed in triplicate and three biological replicateswere performed to decrease the error

25 Statistical Analysis The relative expression level wascalculated with 2minusΔΔCt method Namely the ΔCt values werethe difference of the Ct values between target gene and theinternal control gene (ΔCt = targetCt minus EFCt) ΔΔCt valueswere the difference of the ΔCt values between sample ΔCtand control ΔCt (ΔΔCt = sampleΔCt minus controlΔCt) Themean value and standard error (SD) of the three biological

replicates were calculated through Microsoft Excel 2007Plotting was performed with SigmaPlot 120

3 Results

31 Isolation and Sequence Analysis of KvLEA Gene Thefull length of KvLEA gene was determined by 51015840 and 31015840rapid amplification of cDNA ends (RACE) technology Thetotal sequence of the gene is 1359 bp along with a 184 bp51015840 untranslated region (UTR) and a 359 bp 31015840 UTR Hencethe open reading frame (ORF) of KvLEA is 816 bp andencodes a protein with 271 amino acids (Figure 1(a)) Thesequence information of this gene had been presented toNCBI database and the GenBank accession number wasKP688391 The KvLEA protein was predicted to be 2979 kDmolecular mass and the isoelectric point (pI) was 761 Thegrand average of hydropathicity (GRAVY) and the instabil-ity index of it were minus1292 and 2849 respectively (httpwebexpasyorgprotparam) Meanwhile the amino acidsequence of KvLEA protein is rich in alanine (17) lysine(15) and glutamic acid (11) while lacking cysteine histi-dine tryptophan and arginine Thus the percentage of thehydrophilic amino acid in it is reaching up to 7085 ahydrophilic protein According to Kyte-Doolittle hydropa-thy plot [34] KvLEA protein also should be hydrophilicexcept for a few hydrophobic residues at its N-terminus(Figure 1(b))

With the deduced amino acid sequence a BLASTpsearch was conducted at NCBI database (httpblastncbinlmnihgovBlastcgi)The results indicated thatKvLEApro-tein contained three LEA 4 (accession pfam02987) super-family conserved motif repeats and the locations of the threerepeat motifs were 107ndash150 bp (119864-value 464119890 minus 05) 152ndash194 bp (119864-value 337119890 minus 04) and 191ndash234 bp (119864-value 455119890 minus04) respectively (Figure 1(c)) implying that this LEA proteinmight belong to LEA 4 group which was the largest groupamong the nine groups in Arabidopsis The motif analysis byInterProScan 5 (httpwwwebiacukToolspfaiprscan5)also showed a similar result with four repeats of LEA 4(accession pfam02987) superfamily conserved motifs (Sup-plemental S1 in Supplementary Material available onlineat httpdxdoiorg10115520169823697) We also analyzedthe transmembrane domain of the protein at the same timeThe prediction indicated that it had no transmembranedomain staying with the same character of the other LEA 4proteins (Supplemental S2) To further verify whether KvIEAbelonged to the LEA 4 type LEA gene a phylogenetic treewas constructed with the predicted sequence of KvLEAprotein and all the eighteen LEA proteins belonging to LEA 4group in Arabidopsis (Figure 1(d)) The result revealed thatKvLEA should be a LEA 4 group member sharing highgenetic homology with twoArabidopsis genes At2g36640 andAt3g53040 in the LEA 4 group

32 KvLEA Expression Profiles under Normal and Salt StressTreatment As soon as we acquired the full gene sequence wedetermined the tissue expression profiles of this gene at firstRNA was extracted from root stem and leaf respectively


CA A A AA A A A A AA A A AA A A A AT TT T T T T T TG GG GG G G G G G G G G G G G G G G GGC C C C C C C CC C1

DPAQRAAAARKKGQMM TE E1

A AA A AA A A A A A A A A A A A A A AA AA AAG GG G G G GC C C C C CC C C CCC G G G G G G GT T T T T TT T T61

EKKAGDRERNNADRLE YUUR20

ARLMSGIUGPREQQQDWE LP40

EEEE QQSSGUUDKAHRUGF K60

TT C C C C C CA A AA A A AG G G G G G G G GG G G G G G G GC CA A A A AAT T TT T T TT T T TT T T T T T T T T121

G GG G CGA A A A A A A A A A A AG G G AG A T TG G G G CA A A AA A A A A AG G G G G GTT T T T TCT T T T T T241

T T T T T T T T T T TC C C C C C C C CCA A A A A A A A A A A A A A AAAAG GG G G G G G G GG G G G GG G G G GG421

A AAAC DDDD KKKK FEGA ERR100

T T TT T T T TC C C C C C C CCGA AG GG G G G G G G G GG G G G G G GGG GA A A A A A A A A A AA A AAAAA A481

T T DK KQ E Y YA S ET T T TK KAQ120

T T T T T T TT TC C C C C C C CC C C C CCA G G G G GG G G G G GGA A AA A A A A A A A A A A A A A A A A AA A A541

A A GG G G G GG G G G GG G G G G G GCCCCCCCCCTT T T TT T T T TT T TA A A AAA A A A A A A A AA A A A601

GA A A A A A A A AA A AAA A A A A A A A ATT TTTT TTTG CC C C CC C CCG G G G G C G G G G G G G G G G GG661

G G G GG G GG GG G GG G G G G G G G G G G GG AAAAAAA AAAAAAAAA AAAAAC C C C C C C CTTTTTT721

G G G G G G GG GGG GG GG G G G AAAAAAAAAAAAAAAAAA TT TT T T T T T TCCCCCCCAAAAAA A781

G CA A A A A A A A A A A A A A AA A C C C CC C CA G GG GG G GG G G G GG G GG G G G G TTTTTTTTTTTT T841

D D DE E Y Y KK KMG R GA TT T A A200

D S S S D SK KL LG K G EA N E A A A220

SIFDMAAA KR KKG A ESTQTG240

D S RD EMA K K UT T lowast260

GG G G G G GG G GG G G G GG GA A A A A A A A AA A A A A A A A AAGC CCC C C C C C C C C C CCT T T T T T T T901

A A A AAA A A A A A A A A AAG G G G G G G G G GGC C C C C C C C C C C C C CT T T T T T T T T T TT T TT TAAT961

A A A AA A A A A A A AA AA AG G G G G G G G G G GA A A A A AC G GGC C C C C C CT T T T T T T TT TT T T T TT1021

A A A A A A A A A A A A A A A A A A A AG GG G G G G G G G G GC C C C C G G G G G G G G G GG CT T T T T T T T T T T1081

A A A AA A A A AA A AA AA AG GG G G G G G G G G G GG A GG AG G GGC C CC C C C C CT T T TT TT T T T T TT1141

AA A A A A A A A AA AAAAG G G G G G GG G G G G G G G G G A A AC C C C C C CT T T T T T T T T T T TT GGT GT1201

A A A A A A A A A A AA G G G G GG GG G GGG GGC C C C C C CT T TT T T T T T G GG G GT T T T T T TT TT T TT1261

AA A AG G G GC C GC C C CT T T TT T T T TT TT T TT T CTTTT T T1321

S UD DK KKE E EY YS KA A A AA G160

EEEE Y KKKKK N AAA U AAATG180

S U DD KK E YYST KKQGG A A AS140

G GGG G G G G G G G G G G G G G G G G G G G CCCTT TT T T TC C C C C C CC CA AA A A A A A A AA A A A AT T T301

AAAA G RRSF D T DD KK MEWTT80

T T T T T T T T TC C C C C C CC CCG GA A A A AA A A A A A A A A A A A AAG G G G G G GG G G G G G G G GG G GG361

G GG G G G G G G G G G GG G GG G G G G G G GC C C CC C C C CT TTA A AA AC C CA A A A A A A AA A A A AAT T181

(a)

Hydrophobic

Hydrophilic

HphobKyte and Doolittle

minus35

minus3

minus25

minus2

minus15

minus1

minus05

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LEA_4 LEA_4LEA_4

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Figure 1 Continued


At2G037409797

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8379

4498

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40

9928

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99KvLEA

At2G03850

At2G42530At2G42540

At2G36640At3G53040

At2G42560At3G02480

At4G13560At1G52690

At3G15670At1G72100

At4G13230At4G21020

At5G44310At3G17520

At2G18340At4G36600

(d)

Figure 1 Sequence hydrophilic repeat motifs and phylogenetic analysis of KvLEA (a) The nucleotide and predicted amino acid sequencesof KvLEA gene The initiation codon and termination codon were highlighted with red underline The encoded amino acids were listedcorrespondingly to the ORF (b) Hydrophilic analysis of KvLEA The analysis was conducted according to Kyte-Doolittle hydropathy plotThe section above zero stands for the hydrophobic part of the sequence and the section below zero represents the hydrophilic part (c)The BLASTp search of the predicted amino acid sequence The red boxes show the category and position of the conserved motifs (LEA 4accession pfam02987) (d) Phylogenetic tree of KvLEA and eighteen LEA 4 proteins in Arabidopsis The phylogenetic tree was constructedusingMEGA60 byNeighbor-Joiningmethod based on themultiple sequence alignment of the nineteen LEA proteinsThe protein sequencesof theArabidopsis LEA genes were acquired from the Arabidopsis Information Resource (TAIR) database (httpwwwarabidopsisorg)Thegene IDs and names of the proteins were listed in Supplemental S3

and the determination of the expressions was carried outby qPCR The expression pattern (Figure 2(a)) showed thatKvLEA tended to be expressed in root with priority Theexpression level in root is more than 50 times that in leafimplying that it might play an important role in root

On account that KvLEA gene was obtained by screen-ing our transcriptome sequencing data (accession num-ber GCJL00000000) [30] of K virginica which displayedapparent transcripts accumulation under salt treatment westudied this gene under salt stress in detail We detectedthe conditions of the expression under different salt stressduration and different salt concentrations In keeping withthe transcriptome results the gene indeed was induced bysalt stress in different degrees under different salt conditionsWith the increase of the salt stress intensities the responsesstrengthened gradually (Figure 2(b)) 300 and 400mMNaClled to pronounced elevations in gene expression comparedwith 100 and 200mM treatments As shown in Figure 2(b)400mM NaCl triggered the most pronounced surge inexpression at about 16-fold increase With regard to thedifferent duration of salt stresses the accumulations ofKvLEAgene kept at a high level all the time from 2 h to 24 h

(Figure 2(c)) The transcripts accumulated a lot immediatelyafter 2 h treatment and kept steadily high in the followingtime Therefore KvLEA should play an important role insalt tolerance and contribute to the high salt resistance of Kvirginica

33 Expression Patterns of KvLEA under Various Abiotic StressTreatments Previous researches have reported that someLEA proteins participated in various abiotic stresses such ashigh temperature [21] high salinity [35 36] osmotic stress[36] and chilling stress [24] Then we wondered whetherour KvLEA protein was also involved in these abiotic stressesso we also examined the performances of KvLEA geneunder these conditions The results showed that KvLEA generesponded to drought heat chilling andABA treatments tooUnder drought stress treatment the KvLEA transcripts wereinduced and the accumulation increased progressively withtime (Figure 3(a)) while the responses of KvLEA to droughtstress were weak The difference could be regarded as notsignificant or weak (generally expression ratio ge 2 or le05was deemed as the boundary of the significance) Comparedto drought treatment heat stress triggered more obvious


60

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Leaf Stem Root(a)

0 100 200 300Concentration of NaCl (mM)

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(c)

Figure 2 The expression patterns of KvLEA gene in different tissues and under salt stress (a) The expression levels of KvLEA in differenttissues Root stem and leaf were collected from seedlings growing under normal condition and total RNA was extracted from themrespectivelyThe expression quantity in leaf which was the smallest among three samples was set as the referenceThe expression levels of theother twowere the relative expression levels compared to leaf (b)The transcripts accumulation ofKvLEA under different NaCl concentrations(0 100 200 300 and 400mM)The 0mMNaCl treatment was the control sample treated with the same nutrition solution without any NaCl(c) The transcripts accumulation of KvLEA at different time of salt stresses (0 2 6 12 and 24 h after salt treatment) The 0 h treatment wasthe corresponding control sample The samples of NaCl treatment were the whole seedlings Three biological replicates were performed andthe mean values of the three biological replicates were used for plotting Error bar indicates standard error (SD 119899 = 3)

accumulation to KvLEA transcripts and the accumulationappeared to be the highest at 2 h after treatment Howevercontrary to drought stress the transcripts under heat stressdecreased with time (Figure 3(b)) The responses of KvLEAto chilling stress were interesting It only displayed distinctresponse at 6 h after treatment yet there were no differencesat other time points (Figure 3(c)) In addition 100120583M ABAcaused the most obvious accumulations to this gene amongall the abiotic stresses especially at 6 h with 44-fold increase(Figure 3(d))

4 Discussion

To adapt to the ever-changing ambient conditions higherplants have developed complicated resistance mechanismsalong with their evolutionary history The existence of LEA

proteins is a good example Generally these proteins sharesome common properties including high hydrophilicityrepeat motifs and low sequence complexity [37 38] Inthe present study we isolated and cloned the first LEAgene in K virginica In the context of previous studies thisresearch also analyzed the sequence information of KvLEAgene at first Similar to the common properties of mostLEA proteins KvLEA protein also has high hydrophilicityand contains repeat motifs It is composed of high per-centage of hydrophilic amino acids and the percentage ofthe hydrophilic amino acids is as much as 7085 Boththe value of the grand average of hydropathicity (GRAVY)and the analysis of Kyte-Doolittle hydropathy plot verify itshydrophily at the same time (Figure 1(b)) On the otherhand the BLASTp search (Figure 1(c)) and the InterProScan5 prediction (Supplemental S1) proved the existence of therepeat motifs in KvLEA protein


Drought

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(d)

Figure 3 Transcript accumulation ofKvLEA in response to different abiotic stresses (a) 15 (wv) PEG 6000 treatment (b) high temperature42∘C treatment (c) 4∘Cchilling treatment and (d) 100 120583MABA treatmentTheheat and the chilling stresses were achieved in artificial climaticchambers Drought and ABA treatments were performed through irrigating sufficient nutrition solutions with corresponding concentrationsof PEG and ABA meanwhile the control sample was watered only with nutrition solution Three biological replicates were carried out forevery stress treatment and the mean values were used for the expression analysis Expression analysis was carried out with ΔΔCt methodError bar indicates SD (119899 = 3)

On account of the function researches LEA proteins areproposed to have a broad influence on abiotic stress responsesin plants (Sun et al 2013) For instance dehydrin CaDHN1made great contributions to multiple abiotic stresses inCapsicum annuum L [39] In potato the expressions of fiveStLEA genes were induced by both salt and drought stresses[40]Moreover the CaLEA1 in pepper acted inABA signalingregulation drought and salt stress responses [41] In ourKvLEA expression analysis the results indicated a similarcondition Its expression is also strongly induced by saltstress under different NaCl concentrations and at differenttime points With the increase of the salt concentrations theresponses enhanced gradually and 300 and 400mM treat-ment led to obvious responses (Figure 2(b)) On the bases ofour previous researches on K virginica 100 to 200mMNaClwhich was already drastic salt stress for common plant hadlittle effect on it and only when the concentration reached300mM or more did the influence begin to emerge [42ndash44]

That is why we chose 300mM for different time gradienttreatments and this reflected the strong salt resistance of Kvirginica at the same time Under 300mM condition theresponse to NaCl was rapid and the expression level reachedhigh value immediately after 2 h treatment (Figure 2(c))though from 2 h to 12 h the accumulation of the transcriptsdeclined a little while from 12 h to 24 h the expression rose upagain indicating that there might be a feedback adjustmentbetween the salt stress and the plant resistance The mixperformances were also discovered in previous research onother species under the same conditions For instance theZmLEA3 inmaize displayed two peaks during 48 h treatmentThe accumulation reached a peak at 24 h and then reducedfrom 24 h to 36 h while from 36 h to 48 h the accumulationrose up again reaching a new peak [10] In tobacco the samepresentation also appeared while the difference was thatthe mix performances were downregulated [45] Thereforemore in-depth studies are needed to uncover the complicated


mechanisms underlying the KvLEA gene and the salt resis-tance in K virginica

The responses of KvLEA gene to abiotic stresses showedus that it could be induced by a variety of abiotic stresseswhile the response of KvLEA to drought stress was weak(Figure 3(a)) We speculated that there may be other LEAproteins in charge of responding to drought stress in Kvirginica and there may also be many other LEA proteinshaving key functions in various abiotic stresses In additionaccording to the gene expression analysis the gene alsoresponded to heat (Figure 3(b)) and chilling (Figure 3(c))Although many of the reported LEA genes were discoveredto be expressed highly in seeds there were several LEAgenes responding to temperature fluctuation For examplethe Arabidopsis LEA genes At2g42530 and At2g42540 werecold responsive genes thus they were called COR15A (coldresponsive 15A) and COR15B [46]

The most amusing performance appeared in ABA treat-ment (Figure 3(d)) ABA is a key phytohormone which canbe involved in kinds of abiotic stresses On the one hand theabiotic stresses such as salt drought and cold stresses canlead to the production of ABA On the other hand the accu-mulated ABA can in return affect the expressions of manyabiotic responsive genes and lead to stress responses [47 48]In this study the exogenous applied ABA caused an obviousaccumulation to this gene (Figure 3(d)) Furthermore theexpression of this gene reached more than 40-fold increaseafter 6 h treatment illustrating that there may be complicatedcorrelations between KvLEA gene and ABA in K virginicaLately a proteomic analysis on maize seeds revealed that thedeficiency of ABA in ABA-deficit mutant decreased the accu-mulation of several LEA proteins [49] And the transgenicArabidopsis overexpressing pepper CaLEA1 gene exhibitedsupersensitivity to ABA [41] Therefore more researches areneeded to uncover the specific correlation between ABA andKvLEA in K virginica in the future Importantly transgenictechnology should be employed to study the functions of thegene in depth

5 Conclusion

In conclusion our present study isolated cloned and identi-fied the first LEA gene from K virginica The deduced aminoacid sequences of this gene showed that it was a typical LEAprotein possessingmany common properties of LEA proteinsand had high homology with Arabidopsis LEA 4 genesAt2g36640 and At3g53040 The tissue expression patternanalysis showed that this gene displayed a higher expressionlevel in root In spite of that most LEA genes are induced bydesiccation and large amount of LEA proteins is synthesizedto improve desiccation tolerance while the LEA genes alsohave many other known and unknown important functionsOur research revealed that KvLEA gene was induced not onlyby salt stress but also by a variety of other abiotic stressesespecially for ABA treatment in K virginica

Competing Interests

The authors declare no conflict of interests

Authorsrsquo Contributions

Xiaoli Tang Hongyan Wang and Hongbo Shao designedthe experiment Xiaoli Tang performed the experiment andanalyzed the data with Liye Chu Xiaoli Tang and HongboShao wrote the paper All authors read and agreed on the finalpaper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (41171216) the National Basic ResearchProgram of China (2013CB430403) Shuangchuang TalentPlan of Jiangsu Province Jiangsu Autonomous Innova-tion of Agricultural Science amp Technology [CX(15)1005]Jiangsu Key Laboratory for Bioresources of Saline Soils(JKLBS2014002) and Yantai Double-Hundred Talent Plan(XY-003-02)

References

[1] R Munns and M Tester ldquoMechanisms of salinity tolerancerdquoAnnual Review of Plant Biology vol 59 pp 651ndash681 2008

[2] X Tang X Mu H Shao H Wang and M Brestic ldquoGlobalplant-responding mechanisms to salt stress physiological andmolecular levels and implications in biotechnologyrdquo CriticalReviews in Biotechnology vol 35 no 4 pp 425ndash437 2015

[3] L Xiong K S Schumaker and J-K Zhu ldquoCell signaling duringcold drought and salt stressrdquo Plant Cell vol 14 no 1 pp S165ndashS183 2002

[4] K Yamaguchi-Shinozaki and K Shinozaki ldquoTranscriptionalregulatory networks in cellular responses and tolerance todehydration and cold stressesrdquo Annual Review of Plant Biologyvol 57 pp 781ndash803 2006

[5] J You W Zong H Hu X Li J Xiao and L Xiong ldquoA STRESS-RESPONSIVE NAC1-regulated protein phosphatase gene riceProtein Phosphatase18 modulates drought and oxidative stresstolerance through abscisic acid-independent reactive oxygenspecies scavenging in ricerdquo Plant Physiology vol 166 no 4 pp2100ndash2114 2014

[6] N Bies-Etheve P Gaubier-Comella A Debures et al ldquoInven-tory evolution and expression profiling diversity of the LEA(late embryogenesis abundant) protein gene family inArabidop-sis thalianardquoPlantMolecular Biology vol 67 no 1-2 pp 107ndash1242008

[7] M Hundertmark and D K Hincha ldquoLEA (late embryogenesisabundant) proteins and their encoding genes in Arabidopsisthalianardquo BMC Genomics vol 9 article 118 2008

[8] L Dure III and G A Galau ldquoDevelopmental biochemistry ofcottonseed embryogenesis and germinationrdquo Plant Physiologyvol 68 no 1 pp 187ndash194 1981

[9] A Candat G Paszkiewicz M Neveu et al ldquoThe ubiquitousdistribution of late embryogenesis abundant proteins across cellcompartments in Arabidopsis offers tailored protection againstabiotic stressrdquo Plant Cell vol 26 no 7 pp 3148ndash3166 2014

[10] Y Liu L Wang X Xing et al ldquoZmLEA3 a multifunctionalgroup 3 LEA protein from maize (Zea mays L) is involved inbiotic and abiotic stressesrdquo Plant and Cell Physiology vol 54 no6 pp 944ndash959 2013


[11] M Battaglia Y Olvera-Carrillo A Garciarrubio F Camposand A A Covarrubias ldquoThe enigmatic LEA proteins and otherhydrophilinsrdquo Plant Physiology vol 148 no 1 pp 6ndash24 2008

[12] R D Finn A Bateman J Clements et al ldquoPfam the proteinfamilies databaserdquo Nucleic Acids Research vol 42 no 1 ppD222ndashD230 2014

[13] G Hunault and E Jaspard ldquoLEAPdb a database for the lateembryogenesis abundant proteinsrdquo BMC Genomics vol 11 no1 article 221 2010

[14] M Battaglia and A A Covarrubias ldquoLate EmbryogenesisAbundant (LEA) proteins in legumesrdquo Frontiers in Plant Sci-ence vol 4 article 190 2013

[15] F Jia S Qi H Li et al ldquoOverexpression of late embryoge-nesis abundant 14 enhances arabidopsis salt stress tolerancerdquoBiochemical andBiophysical ResearchCommunications vol 454no 4 pp 505ndash511 2015

[16] E BThomann J Sollinger CWhite and C J Rivin ldquoAccumu-lation of group 3 late embryogenesis abundant proteins in zeamays embryos Roles of abscisic acid and the viviparous-1 geneproductrdquo Plant Physiology vol 99 no 2 pp 607ndash614 1992

[17] I Amara A Odena E Oliveira et al ldquoInsights into maize LEAproteins from proteomics to functional approachesrdquo Plant andCell Physiology vol 53 no 2 pp 312ndash329 2012

[18] K Sasaki N K Christov S Tsuda and R Imai ldquoIdentificationof a novel LEA protein involved in freezing tolerance in wheatrdquoPlant and Cell Physiology vol 55 no 1 pp 136ndash147 2014

[19] T Z Gal I Glazer and H Koltai ldquoAn LEA group 3 familymember is involved in survival of C elegans during exposureto stressrdquo FEBS Letters vol 577 no 1-2 pp 21ndash26 2004

[20] M Wang P Li C Li et al ldquoSiLEA14 a novel atypical LEAprotein confers abiotic stress resistance in foxtail milletrdquo BMCPlant Biology vol 14 no 1 article 290 2014

[21] X Zhang S Lu C Jiang et al ldquoRcLEA a late embryogenesisabundant protein gene isolated fromRosa chinensis confers tol-erance toEscherichia coli andArabidopsis thaliana and stabilizesenzyme activity under diverse stressesrdquoPlantMolecular Biologyvol 85 no 4 pp 333ndash347 2014

[22] D Xu X Duan B Wang B Hong T-H D Ho and R WuldquoExpression of a late embryogenesis abundant protein geneHVA1 from barley confers tolerance to water deficit and saltstress in transgenic ricerdquo Plant Physiology vol 110 no 1 pp249ndash257 1996

[23] E Sivamani A Bahieldin J M Wraith et al ldquoImprovedbiomass productivity and water use efficiency under waterdeficit conditions in transgenic wheat constitutively expressingthe barley HVA1 generdquo Plant Science vol 155 no 1 pp 1ndash92000

[24] Y-PGai X-L JiW Lu X-JHanG-D Yang andC-C ZhengldquoA novel late embryogenesis abundant like protein associatedwith chilling stress inNicotiana tabacum cv bright yellow-2 cellsuspension culturerdquoMolecularampCellular Proteomics vol 10 no11 Article ID M111010363 2011

[25] F M Salleh K Evans B Goodall et al ldquoA novel function fora redox-related LEA protein (SAG21AtLEA5) in root develop-ment and biotic stress responsesrdquo Plant Cell and Environmentvol 35 no 2 pp 418ndash429 2012

[26] I Amara M Capellades M D Ludevid M Pages and AGoday ldquoEnhanced water stress tolerance of transgenic maizeplants over-expressing LEA Rab28 generdquo Journal of PlantPhysiology vol 170 no 9 pp 864ndash873 2013

[27] H Wang X Tang C Shao H Shao and H Wang ldquoMolecularcloning and bioinformatics analysis of a new plasmamembraneNa+H+ antiporter gene from the halophyte Kosteletzkya vir-ginicardquoThe ScientificWorld Journal vol 2014 Article ID 1416757 pages 2014

[28] G Zhou Y Xia B L Ma C Feng and P Qin ldquoCulture ofseashore mallow under different salinity levels using plasticnutrient-rich matrices and transplantationrdquo Agronomy Journalvol 102 no 2 pp 395ndash402 2010

[29] Y-Q Guo Z-Y Tian G-Y Qin et al ldquoGene expression ofhalophyte Kosteletzkya virginica seedlings under salt stress atearly stagerdquo Genetica vol 137 no 2 pp 189ndash199 2009

[30] X Tang H Wang C Shao and H Shao ldquoGlobal geneexpression ofKosteletzkya virginica seedlings responding to saltstressrdquo PLoS ONE vol 10 no 4 Article ID e0124421 2015

[31] D R Hoagland and D I ArnonTheWater-Culture Method forGrowing Plants without Soil vol 347 of California AgriculturalExperiment Station Circular California Agricultural Experi-ment Station Publications 1950

[32] A Marchler-Bauer S Lu J B Anderson et al ldquoCDD aConserved Domain Database for the functional annotation ofproteinsrdquo Nucleic Acids Research vol 39 no 1 pp D225ndashD2292011

[33] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[34] J Kyte and R F Doolittle ldquoA simple method for displayingthe hydropathic character of a proteinrdquo Journal of MolecularBiology vol 157 no 1 pp 105ndash132 1982

[35] Y Liu Y Zheng Y Zhang W Wang and R Li ldquoSoybean PM2protein (LEA3) confers the tolerance of Escherichia coli andstabilization of enzyme activity under diverse stressesrdquo CurrentMicrobiology vol 60 no 5 pp 373ndash378 2010

[36] S-C Park Y-H Kim J C Jeong et al ldquoSweetpotato lateembryogenesis abundant 14 (IbLEA14) gene influences ligni-fication and increases osmotic- and salt stress-tolerance oftransgenic callirdquo Planta vol 233 no 3 pp 621ndash634 2011

[37] M-D Shih F A Hoekstra and Y-I C Hsing ldquoLate embryo-genesis abundant proteinsrdquo in Advances in Botanical ResearchJ-C Kader andM Delseny Eds vol 48 pp 211ndash255 AcademicPress 2008

[38] A Tunnacliffe D Hincha O Leprince and D Macherel ldquoLEAproteins versatility of form and functionrdquo in Dormancy andResistance in Harsh Environments E Lubzens J Cerda and MClark Eds pp 91ndash108 Springer Berlin Germany 2010

[39] R-G Chen H Jing W-L Guo et al ldquoSilencing of dehydrinCaDHN1 diminishes tolerance to multiple abiotic stresses inCapsicum annuum Lrdquo Plant Cell Reports vol 34 no 12 pp2189ndash2200 2015

[40] S Charfeddine M N Saıdi M Charfeddine and R Gargouri-Bouzid ldquoGenome-wide identification and expression profilingof the late embryogenesis abundant genes in potato withemphasis on dehydrinsrdquoMolecular Biology Reports vol 42 no7 pp 1163ndash1174 2015

[41] C W Lim S Lim W Baek and S C Lee ldquoThe pepper lateembryogenesis abundant protein CaLEA1 acts in regulatingabscisic acid signaling drought and salt stress responserdquo Physi-ologia Plantarum vol 154 no 4 pp 526ndash542 2015

[42] C-J Ruan H Li Y-Q Guo et al ldquoKosteletzkya virginica anagroecoengineering halophytic species for alternative agricul-tural production in Chinarsquos east coast ecological adaptation


and benefits seed yield oil content fatty acid and biodieselpropertiesrdquo Ecological Engineering vol 32 no 4 pp 320ndash3282008

[43] H Wang X Tang H Wang and H Shao ldquoPhysiologicalresponses of Kosteletzkya virginica to coastal wetland soilrdquo TheScientific World Journal vol 2015 Article ID 354581 9 pages2015

[44] H Wang X Tang H Wang and H-B Shao ldquoProline accu-mulation and metabolism-related genes expression profiles inKosteletzkya virginica seedlings under salt stressrdquo Frontiers inPlant Science vol 6 article 792 2015

[45] Y Liu L Wang S Jiang J Pan G Cai and D Li ldquoGroup5 LEA protein ZmLEA5C enhance tolerance to osmotic andlow temperature stresses in transgenic tobacco and yeastrdquo PlantPhysiology and Biochemistry vol 84 pp 22ndash31 2014

[46] P L Steponkus M Uemura R A Joseph S J Gilmour andM F Thomashow ldquoMode of action of the COR15a gene on thefreezing tolerance of Arabidopsis thalianardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 95 no 24 pp 14570ndash14575 1998

[47] K Nakashima and K Yamaguchi-Shinozaki ldquoABA signaling instress-response and seed developmentrdquo Plant Cell Reports vol32 no 7 pp 959ndash970 2013

[48] A S Raghavendra V K Gonugunta A Christmann and EGrill ldquoABA perception and signallingrdquo Trends in Plant Sciencevol 15 no 7 pp 395ndash401 2010

[49] XWu F Gong L Yang X Hu F Tai andWWang ldquoProteomicanalysis reveals differential accumulation of small heat shockproteins and late embryogenesis abundant proteins betweenABA-deficient mutant vp5 seeds and wild-type Vp5 seeds inmaizerdquo Frontiers in Plant Science vol 5 article 801 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


LEA 3 LEA 4 LEA 5 LEA 6 and SMP without the AtMgroup [12] The LEAPdb which was a public database of LEAgene sequences gave a more detailed classification with 12nonredundant groups [13]

As the researches on LEA move along the importanceof LEA proteins is reported by more and more studiesTheir synthesis in large amount during the late stages ofseed development enables the maturing seeds to acquire thedesiccation tolerance Importantly by means of stabilizingproteins nucleic acids and cell membranes LEA proteinsare proved to be the common components associated withtolerance to various stresses [14 15] For example the maizeLEA3 gene was discovered to be induced by ABA andhyperosmolarity [16] and afterwards it was proved to beable to reduce cell shrinkage effects under dehydration [17]Similarly a novel LEA genewas identified inwheat whichwasinduced by freezing tolerance [18] The reduced expressionof CeLEA1 a LEA-like protein from Caenorhabditis elegansled to reduced survival under desiccation and osmotic andheat stresses [19] On the other hand ectopic expressions ofLEA in various organisms also gave the evidence for theirfunctions in stress protection [9 20 21] The transgenicwheat and rice with heterogeneous expression of LEA genefrom barley acquired enhanced drought and salt tolerance[22 23] Overexpressing NtLEA7-3 in Arabidopsis endowedthe transgenic plants with enhanced resistance to colddrought and salt stresses [24] In Arabidopsis the SAG21was discovered to enhance the tolerance against bacterialpathogen (Pst DC3000) and oxidative stress [25] The LEAprotein Rab28 which is localized in the nucleus in maizeembryo can enhance transgenic maize tolerance to droughtstress [26] Therefore the LEA proteins are involved not onlyin water deficit but also in abiotic stresses It is one of the keyfactors for plants to respond to adverse environment

K virginica is a newly introduced species due to itsamazing salt stress resistance and potential economic value[27] It was reported that this plant was able to maintainnormal growth and development under adverse conditionswith 03 to 25 sodium salt [28] Thus it must possesshighly efficient physiological biochemical and molecularmechanisms to do this Therefore we are interested in itsspecial mechanisms under salt stress in the hope of uncover-ing novel resistance mechanisms in it However the geneticinformation on it is very scarce As far as we know thereare almost no reports about the molecular mechanism ofsalt resistance on K virginica except for one report coveringthe gene expression analysis on K virginica seedlings bycDNA-amplified fragment length polymorphism (cDNA-AFLP) technology [29]

Based on the diverse biological functions of LEA proteinsas stated before we wondered whether LEA also had similarroles in K virginica Therefore this study aimed to cloneisolate identify and explore the possible functions of a LEAgene in K virginica With the help of transcriptome analysisof K virginica under salt stress in our recent research wegot the core sequence of the gene [30] RACE technologywas adopted to clone the whole sequence of this geneThe biological software such as ProParam InterProScan 5BLASTp and CLUSTALWX was used to do the sequence

prediction and analysis Phylogenetic tree was constructedto analyze which subgroup it may belong to FurthermoreqPCR assays were carried out to detect the gene expressionpatterns under abiotic stressesThis study about the first LEAgene inK virginica further enriched the research informationon LEA gene Meanwhile it also laid a good foundation formolecular studies in K virginica

2 Material and Methods

21 Plant Materials and Growth Conditions The seeds of Kvirginica used in this study were coming from theK virginicagrowing in Yellow River Delta Shandong Province ChinaThe collected seeds were disinfected with 70 alcohol solu-tion for 30 s and 01 mercuric chloride solution for 10minand then washed thoroughly with sterile water six timesThe surface-sterilized seeds were laid on wet filter paper forgermination and then the germinating seeds were planted invermiculite in plastic pots watered with Hoaglandrsquos solution[31] The seedlings were cultivated under conditions of 16 hlight8 h dark and the temperature was kept at 25∘C20∘Crespectively in day and night in artificial climatic chambers(Huier China) The humidity was kept at 60 and theseedlings were watered every 3 days with nutrition solutionTwo-week-old and homogeneous seedlings were chosen fordifferent samplings and six homogeneous seedlings at leastwere contained in each sample However the samples fortissue expression pattern detection came from the differentcorresponding tissues respectively All the samples werefrozen with liquid nitrogen and stored at minus80∘C

22 Cloning and Sequence Analysis of KvLEA For genecloning total RNA was extracted with RNAiso Plus (TaKaRatotal RNA extraction reagent) according to the opera-tion steps Agilent 2100 BioAnalyzer (Agilent Technolo-gies CA USA) was used for the detection of the RNAquality and quantity The full length of KvLEA was ampli-fied by RACE according to the user manual (SMARTRACE cDNA Amplification Kit) The gene-specific primers(GSPs) (forward CCTCCTGCTCCTGGCTCTTA reverseGGTGGCTGAAGGGACGGAGTA) and the nested primers(NGSPs) (forward CCTGGCTCTTACTACCAACGACreverse AAGCAGACGGCATCCGAAAC) were designedbased on the cDNA core sequence coming from our previoustranscriptome sequencing data which had been deposited inTranscriptome Shotgun Assembly (TSA) Sequence Databasewith the accession number GCJL00000000 [30] The PCRproducts were cloned into pEASY-T5 Zero Cloning Vector(Transgene China) after purification from gels for sequen-cingThe physical and chemical parameters of KvLEAproteinwere analyzed with ProParam InterProScan 5 (httpwwwebiacukToolspfaiprscan5) and the Sequence Manipula-tion Suite (httpwwwbio-softnetsms) The BLASTp wasperformed at NCBI (National Center for Biotechnologyfor Information httpblastncbinlmnihgovBlastcgi) [32]Multiple sequence alignment was carried out among thededuced KvLEA protein and all the eighteen LEA 4 proteinsinArabidopsiswithCLUSTALWXprogramThephylogenetic








3 Results










































(a)

Hydrophobic

Hydrophilic


minus35

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

15

Scor

e


(b)1


Superfamilies

LEA_4 LEA_4LEA_4


50 100 150 200 250 271

(c)

Figure 1 Continued


At2G037409797

33

8379

4498

5497

40

9928

18

4767

02

99KvLEA

At2G03850

At2G42530At2G42540

At2G36640At3G53040

At2G42560At3G02480

At4G13560At1G52690

At3G15670At1G72100

At4G13230At4G21020

At5G44310At3G17520

At2G18340At4G36600

(d)







60

50

4

Relat

ive e

xpre

ssio

n le

vel

10

Leaf Stem Root(a)


400

Relat

ive e

xpre

ssio

n le

vel

0

2

4

6

8

10

12

14

16

18

(b)

0

Relat

ive e

xpre

ssio

n le

vel


240

2

4

6

8

10

12

14

16

(c)



4 Discussion




Drought

0 2

Relat

ive e

xpre

ssio

n le

vel


00

05

10

15

20

25

(a)

Heat

Relat

ive e

xpre

ssio

n le

vel


200

1

2

3

4

5

6

7

(b)

Chilling


0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n le

vel

(c)

ABA


20

60

50

406

10

Relat

ive e

xpre

ssio

n le

vel

(d)








5 Conclusion


Competing Interests




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








3 Results










































(a)

Hydrophobic

Hydrophilic


minus35

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

15

Scor

e


(b)1


Superfamilies

LEA_4 LEA_4LEA_4


50 100 150 200 250 271

(c)

Figure 1 Continued


At2G037409797

33

8379

4498

5497

40

9928

18

4767

02

99KvLEA

At2G03850

At2G42530At2G42540

At2G36640At3G53040

At2G42560At3G02480

At4G13560At1G52690

At3G15670At1G72100

At4G13230At4G21020

At5G44310At3G17520

At2G18340At4G36600

(d)







60

50

4

Relat

ive e

xpre

ssio

n le

vel

10

Leaf Stem Root(a)


400

Relat

ive e

xpre

ssio

n le

vel

0

2

4

6

8

10

12

14

16

18

(b)

0

Relat

ive e

xpre

ssio

n le

vel


240

2

4

6

8

10

12

14

16

(c)



4 Discussion




Drought

0 2

Relat

ive e

xpre

ssio

n le

vel


00

05

10

15

20

25

(a)

Heat

Relat

ive e

xpre

ssio

n le

vel


200

1

2

3

4

5

6

7

(b)

Chilling


0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n le

vel

(c)

ABA


20

60

50

406

10

Relat

ive e

xpre

ssio

n le

vel

(d)








5 Conclusion


Competing Interests




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































(a)

Hydrophobic

Hydrophilic


minus35

minus3

minus25

minus2

minus15

minus1

minus05

0

05

1

15

Scor

e


(b)1


Superfamilies

LEA_4 LEA_4LEA_4


50 100 150 200 250 271

(c)

Figure 1 Continued


At2G037409797

33

8379

4498

5497

40

9928

18

4767

02

99KvLEA

At2G03850

At2G42530At2G42540

At2G36640At3G53040

At2G42560At3G02480

At4G13560At1G52690

At3G15670At1G72100

At4G13230At4G21020

At5G44310At3G17520

At2G18340At4G36600

(d)







60

50

4

Relat

ive e

xpre

ssio

n le

vel

10

Leaf Stem Root(a)


400

Relat

ive e

xpre

ssio

n le

vel

0

2

4

6

8

10

12

14

16

18

(b)

0

Relat

ive e

xpre

ssio

n le

vel


240

2

4

6

8

10

12

14

16

(c)



4 Discussion




Drought

0 2

Relat

ive e

xpre

ssio

n le

vel


00

05

10

15

20

25

(a)

Heat

Relat

ive e

xpre

ssio

n le

vel


200

1

2

3

4

5

6

7

(b)

Chilling


0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n le

vel

(c)

ABA


20

60

50

406

10

Relat

ive e

xpre

ssio

n le

vel

(d)








5 Conclusion


Competing Interests




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


At2G037409797

33

8379

4498

5497

40

9928

18

4767

02

99KvLEA

At2G03850

At2G42530At2G42540

At2G36640At3G53040

At2G42560At3G02480

At4G13560At1G52690

At3G15670At1G72100

At4G13230At4G21020

At5G44310At3G17520

At2G18340At4G36600

(d)







60

50

4

Relat

ive e

xpre

ssio

n le

vel

10

Leaf Stem Root(a)


400

Relat

ive e

xpre

ssio

n le

vel

0

2

4

6

8

10

12

14

16

18

(b)

0

Relat

ive e

xpre

ssio

n le

vel


240

2

4

6

8

10

12

14

16

(c)



4 Discussion




Drought

0 2

Relat

ive e

xpre

ssio

n le

vel


00

05

10

15

20

25

(a)

Heat

Relat

ive e

xpre

ssio

n le

vel


200

1

2

3

4

5

6

7

(b)

Chilling


0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n le

vel

(c)

ABA


20

60

50

406

10

Relat

ive e

xpre

ssio

n le

vel

(d)








5 Conclusion


Competing Interests




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


60

50

4

Relat

ive e

xpre

ssio

n le

vel

10

Leaf Stem Root(a)


400

Relat

ive e

xpre

ssio

n le

vel

0

2

4

6

8

10

12

14

16

18

(b)

0

Relat

ive e

xpre

ssio

n le

vel


240

2

4

6

8

10

12

14

16

(c)



4 Discussion




Drought

0 2

Relat

ive e

xpre

ssio

n le

vel


00

05

10

15

20

25

(a)

Heat

Relat

ive e

xpre

ssio

n le

vel


200

1

2

3

4

5

6

7

(b)

Chilling


0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n le

vel

(c)

ABA


20

60

50

406

10

Relat

ive e

xpre

ssio

n le

vel

(d)








5 Conclusion


Competing Interests




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Drought

0 2

Relat

ive e

xpre

ssio

n le

vel


00

05

10

15

20

25

(a)

Heat

Relat

ive e

xpre

ssio

n le

vel


200

1

2

3

4

5

6

7

(b)

Chilling


0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n le

vel

(c)

ABA


20

60

50

406

10

Relat

ive e

xpre

ssio

n le

vel

(d)








5 Conclusion


Competing Interests




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusion


Competing Interests




Acknowledgments


References




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article kvlea , a new isolated late embryogenesis...

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