Research ArticleExpression Profiles of 12 Late Embryogenesis Abundant ProteinGenes from Tamarix hispida in Response to Abiotic Stress

Caiqiu Gao12 Yali Liu3 Chao Wang1 Kaimin Zhang1 and Yucheng Wang1

1 State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University 26 Hexing Road Harbin 150040 China2 Key Laboratory of Forest Genetics amp Biotechnology Nanjing Forestry University Ministry of Education Nanjing 210037 China3Northeast Forestry University Library 26 Hexing Road Harbin 150040 China

Correspondence should be addressed to Yucheng Wang ychngwangyahoocom

Received 23 February 2014 Revised 21 June 2014 Accepted 21 June 2014 Published 10 July 2014

Academic Editor Hongbo Shao

Copyright copy 2014 Caiqiu Gao 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

Twelve embryogenesis abundant protein (LEA) genes (namedThLEA-1 to -12) were cloned from Tamarix hispida The expressionprofiles of these genes in response toNaCl PEG and abscisic acid (ABA) in roots stems and leaves ofT hispidawere assessed usingreal-time reverse transcriptase-polymerase chain reaction (RT-PCR) TheseThLEAs all showed tissue-specific expression patternsin roots stems and leaves under normal growth conditions However they shared a high similar expression patterns in the rootsstems and leaves when exposed to NaCl and PEG stress FurthermoreThLEA-1 -2 -3 -4 and -11 were induced by NaCl and PEGbutThLEA-5 -6 -8 -10 and -12 were downregulated by salt and drought stresses Under ABA treatment someThLEA genes suchasThLEA-1 -2 and -3 were only slightly differentially expressed in roots stems and leaves indicating that they may be involved inthe ABA-independent signaling pathway These findings provide a basis for the elucidation of the function of LEA genes in futurework

1 Introduction

Adverse environmental conditions such as salt drought andhigh temperatures severely limit the growth and geograph-ical distribution of many plants At the same time plantshave evolved many mechanisms to tolerate these adverseconditions [1ndash5] and the regulation of gene expression iscritical for these processes [6] Late embryogenesis abun-dant (LEA) genes play important roles in stress toleranceespecially in drought stress In wheat there are severalgenes which are responsible for drought stress tolerance andproduce different types of enzymes and proteins for instancelate embryogenesis abundant (LEA) protein responsive toabscisic acid (Rab) rubisco helicase proline glutathione-S-transferase (GST) and carbohydrates during drought stress[7]The LEAproteins to be firstly identifiedwere from cottonand they were highly synthesized during the later stages ofembryogenesis [8]

To date hundreds of LEAs have been cloned from dif-ferent plant species [9] Some LEA genes are highly inducedby various abiotic or biotic stress treatments including salt

[10 11] drought or osmotic stress [12 13] heat [14] cold[15 16] andwounding [17] In addition the expression ofLEAgenes are also modulated by abscisic acid (ABA) [10 18] andethylene [17] Transformed plants that overexpress LEA genesshow improved tolerance to salt stress [19 20] water deficitor drought conditions [10 21] and cold [16 22] but they arehypersensitive to ABA [23]

However to date the true functions of LEAs are not fullyunderstood Therefore analyzing the expression patterns ofLEA genes will be beneficial to understanding their rolein stress responses Tamarix hispida a woody halophyte iswidely distributed in drought-stricken areas and saline orsaline-alkali soil in Central Asia and China This species ishighly tolerant to salt drought and high temperature whichmakes it the desirable tree special to study the salt tolerancemechanism and clone the genes involved in salt tolerance

In the present study 12 ThLEA genes were cloned fromT hispida and the expression patterns of these genes weredetermined in response to salt (NaCl) drought (PEG) andabscisic acid (ABA) treatments in the roots stems and leavesby using real-time reverse transcriptase-polymerase chain

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 868391 9 pageshttpdxdoiorg1011552014868391

2 The Scientific World Journal

Table 1 Primers used for quantitative real-time RT-PCR analysis

Gene GenBank accession number Forward primer (51015840-31015840) Reverse primer (51015840-31015840)ThLEA-1 KF801660 CAGCGAAGTTTGGATGGAATG ACCTGTCGCCAATCAGAAGATThLEA-2 KF801661 CACACAATAGAAACCGTAGAG CCTCCATGGTCTCCTTAACTThLEA-3 KF801662 CGAGATCCTTGGTGGAGCTCG TCACGTATGTGTCATGACTACThLEA-4 KF801663 GTGGCTATCTCGATTCGTAGT GCAACAGACACCAACCAAGATThLEA-5 KF801664 GTTCGCAGCAGGAAAGAGCAG ACTCCGTCAACACGGTACTACTThLEA-6 KF801665 GTTGAGGATGTGGATATCAAG CGAGTTCATAATCGATATCCThLEA-7 KF801666 TCATGATATTGGTGAGAAG AGTGGTATCTTAACAGTCTCTThLEA-8 KF801667 GATGACGACACATGATGAAGC AGCCTCCACAGTCCCTTCCGTThLEA-9 KF924555 GTGGTTGTGGAGATGAACGGAG GGTAGCAGCATCATCAGCAATGThLEA-10 KF924556 AGAGTCAGCAACCGATACAGC TCTCTGCCTTGCTGATTCCTCThLEA-11 KF924557 ACGGAGGAAGCTAGGCACGAAG CTGCTGCGTCCTTGTATTCCThLEA-12 KF924558 CAAGATGCAGGAGGTGGTGAT ATCTGCTATTCTTCTGCCTGTActin FJ618517 AAACAATGGCTGATGCTG ACAATACCGTGCTCAATAGG120572-Tubulin FJ618518 CACCCACCGTTGTTCCAG ACCGTCGTCATCTTCACC120573-Tubulin FJ618519 GGAAGCCATAGAAAGACC CAACAAATGTGGGATGCT

reaction (RT-PCR) Our study may provide the fundamentdata for studying the function of LEA genes

2 Materials and Methods

21 Plant Materials and Treatments Seedlings of T hisp-ida were grown in pots with a mixture of turf peat andsand (2 1 vv) in a greenhouse at 70ndash75 relative humiditylightdark cycle of 1410 h and the temperature of 24∘C Two-month-old seedlingswere used for experimental analysesTheseedlings were watered at the roots with one of the followingsolutions 04M NaCl 20 (wv) PEG

6000 or 100 120583M ABA

for 0 3 6 9 12 and 24 h In controls the seedlings werewatered with the same volume of fresh water Followingthese treatments the leaves stems and roots from at least 20seedlings were harvested and pooled frozen immediately inliquid nitrogen and stored at minus80∘C for RNA preparationThree samples were used for real-time RT-PCR biologicalrepeats

22 Cloning of the LEA Genes from T hispida In a previousstudy 11 cDNA libraries of T hispida treated with NaHCO

3

were constructed using Solexa technology which were the8 libraries respectively from roots of T hispida treatedwith NaHCO

3for 0 12 24 and 48 h (2 libraries were

built from each treatment as biological replication) and 3libraries from leaves of T hispida treated with NaHCO

3

for 0 12 and 24 h In total 94359 nonredundant unigenesof gt200 nt in length were generated using the SOAP denovo software Subsequently these unigenes were searchedagainst the Nr database and the Swiss-Prot database usingblastx (httpblastncbinlmnihgov) with a cut-off 119864-valueof 10minus5 [24 25] The unigenes representing the ldquoLEArdquo geneswere queried and identified from these libraries according tothe functional annotation Then primers for the LEA geneswere designed according to these sequences RT-PCR andresequencing were used to confirm the LEA sequences

23 Sequence Alignments and Phylogenetic Analysis Firstlythe open reading frames (ORFs) of all the LEA unigenes(named as ThLEA-1 to -12) were identified using the ORFfinder tools from the NCBI (httpwwwncbinlmnihgovgorfgorfhtml) Subsequently the LEAs with complete ORFswere subjected to molecular weight (MW) and isoelec-tronic point (pI) predictions using the Compute pIMwtool (httpwwwexpasyorgtoolsprotparamhtml) Subcel-lular location predictions for these genes were undertakenusing the Target 11987511 Server (httpwwwcbsdtudkser-vicesTargetP) All the LEA proteins from T hispida and51 Arabidopsis LEA proteins were subjected to phylogeneticanalysis by constructing a phylogenetic tree by the neighbor-joining (NJ) method in ClustalX

24 RNA Extraction and Reverse Transcription (RT) TotalRNA was isolated from the roots stems and leaves usingthe CTAB method Total RNA was digested with DNaseI (Promega) to remove any residual DNA Approximately1 120583g of total RNA was reverse transcribed to cDNA in a10 120583L volume using oligodeoxythymidine and 6-bp randomprimers following the PrimeScript RT reagent kit (TaKaRa)protocol The synthesized cDNAs were diluted 10-fold withsterile water and used as templates for real-time RT-PCR

25 Quantitative Real-Time RT-PCR Real-time RT-PCR wascarried out in an Opticon machine (Biorad Hercules CA)using a real-time PCR MIX Kit (SYBR Green as the fluo-rescent dye TOKOBO) and the primers used are shown inTable 1 The alpha tubulin (FJ618518) beta tubulin (FJ618519)and actin (FJ618517) genes were used as internal controls(reference genes) to normalize the total RNA amount presentin each reaction The reaction mixture (20120583L) contained10 120583L of SYBR Green Real-Time PCR Master Mix (Toyobo)05 120583M of forward and reverse primer and 2 120583L of cDNAtemplate (equivalent to 20 ng of total RNA)The amplificationwas performed using the following cycling parameters 94∘C

The Scientific World Journal 3

Table 2 Characteristics of the 8ThLEAs from T hispida that had full ORFs

Gene name Deduced number of amino acids MW (kDa) pI Predicted subcellular localizationlowast

ThLEA-1 584 6540 818 OtherThLEA-2 435 4670 867 SecretedThLEA-3 462 5131 802 ChloroplastThLEA-4 212 2355 960 SecretedThLEA-5 114 1229 554 OtherThLEA-6 151 1631 497 OtherThLEA-7 318 3531 475 OtherThLEA-8 171 1749 802 OtherlowastSubcellular localization was predicted from protein sequence analysis using the targetP algorithm

for 30 s followed by 45 cycles of 94∘C for 12 s 60∘C for30 s 72∘C for 40 s and then at 81∘C for 1 s for plate readingA melting curve was generated for each sample at the endof each run to assess the purity of the amplified productReal-time RT-PCR was carried out in triplicate to ensurethe reproducibility of the results The expression levels werecalculated from the threshold cycle according to 2minusΔΔCt [26]

3 Results

31 Characterization of ThLEAs In total 12 unique ThLEAs(ThLEA-1-8 respectively with the GenBank numbersKF801660 to KF801667 ThLEA-9-12 respectively withthe GenBank numbers KF924555 to KF924558) that areexpressed differentially in response to at least one stress wereidentified from the T hispida cDNA libraries Among theseeight ThLEA proteins had complete ORFs that encodeddeduced polypeptides of 114ndash584 amino acids with predictedmolecular masses of 1229ndash654 kDa and pIs of 475ndash96(Table 2) Subcellular locations were predicted from proteinsequence analysis using the targetP algorithm The resultssuggested that ThLEA-3 may be located in the chloroplastThLEA-2 and -4 may be secreted proteins and the locationsof the other fiveThLEA genes with full ORFsmay be classifiedas ldquootherrdquo

Hunault and Jaspard [9] identified 51 LEA proteinencoding genes in Arabidopsis genome and classified theminto nine distinct groups (LEA-1 -2 -3 -4 -5 Atm SMPdehydrin and PvLEA18) According to this classification thephylogenetic relationships showed that ThLEA-1 -3 -6 and-7 are in the LEA-2 group and ThLEA-2 -4 -9 -10 -11 and-12 are in the LEA-1 group while ThLEA-8 and -5 are in theAtm and LEA-5 groups respectively (Figure 1)

32 Relative Transcript Abundances of ThLEAs under NormalGrowth Conditions In order to study the tissue specificity ofThLEA gene expression the relative transcript abundancesof the ThLEA genes in T hispida roots stems leaves andseeds under normal growth conditions were monitored byreal-time RT-PCR The transcript level of the actin genewas assigned as 100 while the transcript levels of ThLEAgenes were plotted relative to transcript level of actin gene(Table 3) The results indicated thatThLEA-4 is the gene withthe highest expression in stems and leaves and in the roots

Table 3 Relative transcript abundance of the 12ThLEAs in differenttissues of T hispidaThe transcript levels of the 12ThLEA genes wereplotted relative to expression of the actin gene Transcript levels ofthe actin gene in roots stems leaves and seeds were all assigned as100

Gene Relative abundanceRoots Stems Leaves Seeds

ThLEA-1 68 116 362 10ThLEA-2 53 264 121 230ThLEA-3 82 232 648 4ThLEA-4 1881 3656 3555 161ThLEA-5 15 02 03 20ThLEA-6 01 14 07 3ThLEA-7 08 195 279 10ThLEA-8 85 08 15 770ThLEA-9 177 617 262 180ThLEA-10 1983 20 51 1240ThLEA-11 1618 41 237 6050ThLEA-12 99 01 01 1220

only ThLEA-10 had greater expression while the transcriptlevels of ThLEA-5 and -6 were very low in all four tissues(roots stems leaves and seeds) The expressions ofThLEA-8and -12 were very high in seeds while those were very lowin the other three tissues In the roots the transcript levelof ThLEA-6 was lowest and only 01 (equivalent to 11000of actin) Meanwhile in stems and leaves the transcriptlevel of ThLEA-12 was lowest and less than 11000 of actinMoreover the transcript level of the ThLEA genes highlydiffered among each other and also strongly differed amongthe four tissues For instance ThLEA-10 -11 and -12 wereexpressed mainly in the roots and seeds and their transcriptlevels were all exceeded 1220-fold in the seedsThLEA-2 and -9were expressedmainly in the stems and seeds whileThLEA-1 -3 and -7 showed relatively high transcript level in theleaves

33 The Expression Profiles of ThLEAs in Response to NaClPEG and ABA Treatments

331 NaCl Stress The transcript patterns of all of theThLEAgenes were quite similar among the roots stems and leaves

4 The Scientific World Journal

ThLEA-7ThLEA-6At1g01470At2g46140At1g54410At2g44060ThLEA-1ThLEA-3At3g53770At4g15910At1g02820At4g02380At1g03120At5g27980At3g22490At3g22500At5g53260At5g53270ThLEA-4At1g72100ThLEA-2At4g38410At1g76180At1g20440At1g20450At2g21490At4g39130At3g50970At3g50980At5g66400At2g33690At2g23110At2g23120ThLEA-9At3g17520At3g02480At4g13560At1g52690At3g15670ThLEA-11At2g18340At4g36600ThLEA-12At4g21020At5g44310At4g13230ThLEA-10At2g36640At3g53040At2g42560At2g03740At2g03850At2g42530At2g42540At1g32560At2g35300At5g06760ThLEA-8At2g41260At2g41280ThLEA-5At2g40170At3g51810

LEA-2

SMP

LEA-3

LEA-4

Dehydrin

PvLEA18

LEA-4

LEA-1

AtM

LEA-5

Figure 1 Phylogenetic tree of the 12 ThLEAs and 51 Arabidopsis LEAs based on sequence alignments of the predicted proteins All the 12ThLEA proteins from T hispida and 51 Arabidopsis LEA proteins were subjected to phylogenetic analysis by constructing a phylogenetic treeby the neighbor-joining (NJ) method in ClustalX According to the classification of Hunault and Jaspard [9] the LEA proteins were classifiedinto nine groups

under NaCl stress Furthermore ThLEA-2 -3 -4 and -11were induced (gt2-fold) during NaCl stress period ThLEA-7was also upregulated during most treatment period and sixThLEA genes reached their peak expression level in the leavesat 24 h of stress The most highly induced gene wasThLEA-1which was induced 1596-fold HoweverThLEA-5 -6 -8 -10

or -12 were highly downregulated at almost each stress timepoint Except for ThLEA-5 the other four downregulatedgenes reached lowest transcript levels at 9 h in stems andleaves Interestingly distinct from these genes the transcriptpattern of ThLEA-9 was different among the three tissuesIn the roots it was induced at 3 h but downregulated at the

The Scientific World Journal 5

minus8minus6minus4minus2

02468

10Th

LEA-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2Re

lativ

e exp

ress

ion

leve

l

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10

minus5

0

5

10

15

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes

Relat

ive e

xpre

ssio

n le

vel

3h6h9h

12h24h

(c)

Figure 2 Expression analysis of the 12ThLEAs in response to 04M NaCl stress Relative expression level = transcription level under stresstreatmenttranscription level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

other time points while in stems and leaves it was mainlyupregulated Similar with the other upregulated ThLEAsThLEA-9 reached greatest expression at 24 h in the leaves(Figure 2)

332 PEG Stress Consistent with NaCl stress the transcriptpatterns of theseThLEA genes under PEG stress were dividedinto two distinct groups (Figure 3) One groupwas composedof ThLEA-1 -2 -3 -4 and -11 and these genes were mainlyupregulated The second group includedThLEA-5 -6 -8 -9-10 and -12 that were largely downregulated during the PEGtreatment period

333 ABA Treatment After application of exogenous ABAThLEA-1 -2 and -3 were only slightly differentially regulatedin the roots stems and leaves indicating that they may beregulated via the ABA-independent signaling pathway How-ever the genes that were downregulated by PEG andor NaClstress were also highly downregulated by ABA treatmentIn contrast ThLEA-4 -7 and -11 were mainly upregulatedespecially ThLEA-4 whose transcript levels in the stem at6 h were increased by 38-fold compared with the control(Figure 4)

4 Discussion

In the present study 12 ThLEA genes were identified fromTamarix RNA-seq data base and the transcript abundanceof each gene was assessed in normal growth conditions byreal-time RT-PCR Furthermore NaCl and PEG were usedto simulate salinity and drought conditions respectively andthe expression levels of theThLEA genes were investigated inroots stems and leaves in response to these stress environ-ments

Many studies have shown that members of the plant LEAfamily displayed tissue-specific expression and are involvedin various processes in plant growth and development Forexample in Arabidopsis 22 of the 51 LEA genes (43)showed high expression levels (relative expression gt10) inthe nonseed organs in the absence of a stress or hormonetreatment [27] The OsLEA3-2 gene is not expressed invegetative tissues under normal conditions and it is thoughtto play an important role in the maturation of the embryo[10] Moreover the transcripts of MsLEA3-1 were stronglyenriched in leaves compared with roots and stems of maturealfalfa plants [20] Expression profiles of the 12ThLEA genesin different tissues of T hispida were determined undernormal growth conditions and this showed that most of

6 The Scientific World Journal

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10minus8minus6minus4minus2

02468

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 3 Expression analysis of the 12ThLEAs in response to 20 PEG6000 stress Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

the ThLEAs were expressed in various organs and tissuesHowever they displayed different expression levels betweenthe different tissue types and this may reflect the complexityof functions performed by this gene family [28] Among the12 ThLEAs ThLEA-10 -11 and -12 were mainly expressed inthe roots suggesting that these genes may play roles in rootstress responseThLEA-2 and -9 were highly expressed in thestem while ThLEA-1 -3 and -7 may play important roles inthe leaves as these genes are highly and specifically expressedin leaves Moreover ThLEA-4 has very high expression levelin the roots stems and leaves suggesting that it might playimportant roles in all of these tissues The expression levelsof ThLEA-5 -6 -8 and -12 were very low in each of thetissues examined indicating that these genes may play arelatively unimportant role in these tissues under normalgrowth conditions

There is increasing evidence suggesting that LEA genesare associated with abiotic stress tolerance particularly inplant responses to dehydration salt and cold stresses [18 29]In Arabidopsis of the 22 genes highly expressed in nonseedtissues 12 were induced by more than 3-fold in response tocold drought and salt stresses [27] In sweet potato plantsIbLEA14 expressionwas strongly induced by dehydration andNaCl [30] In the present study half of the 12 ThLEA genes

(ThLEA-1 -2 -3 -4 -7 and -11) were induced under saltand drought stress indicating that these genes might playimportant roles in response to salt and drought stresses in Thispida Bies-Etheve et al [31] reported that LEA genes fromthe same group do not show identical expression profilesand regulation of LEA genes that show apparently similarexpression patterns does not systematically involve the sameregulatory pathway Consistent with this the six upregulatedThLEAs in T hispida belong to two LEAs groups (LEA-2and LEA-4) while LEAs in the same group (such as LEA-4)showed completely different expression patterns

There are ABA-dependent and ABA-independent regula-tory networks controlling plant responses to abiotic stressesLEA genes are often considered as being ABA-regulated andprevious studies have shown that some ThLEA genes canbe induced by exogenous ABA [10 30] However our studyshows that someThLEA genes includingThLEA-1 -2 and -3were highly induced by abiotic stresses but were not regulatedby ABA Consistent with this Bies-Etheve et al [31] reportedthat most of the LEA genes in Arabidopsis seedlings showno response at all following exogenous ABA treatment Hun-dertmark and Hincha [27] deduced the presence of differentsignal transduction pathways in different Arabidopsis tissues(vegetative plants and seeds) by comparing the expression of

The Scientific World Journal 7

minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus15

minus10

minus5

0

5

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus15minus12minus9minus6minus3

036

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 4 Expression analysis of the 12ThLEAs in response to 100 120583MABA treatment Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

LEA genes after ABA treatment In contrast we found thatexpression patterns were highly similar in the three tissuesexamined after ABA treatment Expression analyses showedthat nine ThLEA genes in three tissues of T hispida wereupregulated or downregulated by ABA which suggests thatthese ThLEA genes are regulated by ABA-dependent stressresistance pathways These findings demonstrate that theThLEA genes involved in salt and drought stress resistancein T hispida are probably regulated by two different pathways(ABA-dependent andABA-independent) Further studies arerequired to elucidate the functions of theThLEAs in responseto the abiotic stress and to fully characterize the signalingpathways that regulate their expression

5 Conclusion

In summary we identified 12 ThLEA genes from T hispidawhich belong to three groups The LEA-1 group includesThLEA-2 -4 -9 -10 -11 and -12 proteins the LEA-2 groupcontains ThLEA-1 ThLEA-3 ThLEA-6 and ThLEA-7 andThLEA-8 and -5 are in the Atm and LEA-5 groups TheThLEA genes displayed tissue-specific expression patternsunder normal growth conditionsThLEA-10 -11 and -12were

expressed mainly in the roots and seeds ThLEA-2 and -9were preferentially expressed in the stems and seeds whileThLEA-1 -3 and -7 showed high transcript level in the leavesFurthermore real-time RT-PCR analysis showed that theThLEA genes were regulated by salt and drought stressesThLEA-1 -2 -3 -4 -7 and -11 were mainly induced by saltand drought stress ButThLEA-5 -6 -8 -9 -10 and -12 werelargely downregulated during the NaCl and PEG treatmentAfter the application of exogenous ABA the expression levelsof all ThLEA genes (except for the ThLEA-1 -2 and -3) wereobviously different under the ABA treatment Our studieswill contribute to a better understanding of the functions ofThLEA genes involved in stress response in T hispida

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Caiqiu Gao and Yali Liu have the same contribution to thispaper

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Nucleic AcidsJournal of

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Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 The Scientific World Journal

Table 1 Primers used for quantitative real-time RT-PCR analysis

Gene GenBank accession number Forward primer (51015840-31015840) Reverse primer (51015840-31015840)ThLEA-1 KF801660 CAGCGAAGTTTGGATGGAATG ACCTGTCGCCAATCAGAAGATThLEA-2 KF801661 CACACAATAGAAACCGTAGAG CCTCCATGGTCTCCTTAACTThLEA-3 KF801662 CGAGATCCTTGGTGGAGCTCG TCACGTATGTGTCATGACTACThLEA-4 KF801663 GTGGCTATCTCGATTCGTAGT GCAACAGACACCAACCAAGATThLEA-5 KF801664 GTTCGCAGCAGGAAAGAGCAG ACTCCGTCAACACGGTACTACTThLEA-6 KF801665 GTTGAGGATGTGGATATCAAG CGAGTTCATAATCGATATCCThLEA-7 KF801666 TCATGATATTGGTGAGAAG AGTGGTATCTTAACAGTCTCTThLEA-8 KF801667 GATGACGACACATGATGAAGC AGCCTCCACAGTCCCTTCCGTThLEA-9 KF924555 GTGGTTGTGGAGATGAACGGAG GGTAGCAGCATCATCAGCAATGThLEA-10 KF924556 AGAGTCAGCAACCGATACAGC TCTCTGCCTTGCTGATTCCTCThLEA-11 KF924557 ACGGAGGAAGCTAGGCACGAAG CTGCTGCGTCCTTGTATTCCThLEA-12 KF924558 CAAGATGCAGGAGGTGGTGAT ATCTGCTATTCTTCTGCCTGTActin FJ618517 AAACAATGGCTGATGCTG ACAATACCGTGCTCAATAGG120572-Tubulin FJ618518 CACCCACCGTTGTTCCAG ACCGTCGTCATCTTCACC120573-Tubulin FJ618519 GGAAGCCATAGAAAGACC CAACAAATGTGGGATGCT

reaction (RT-PCR) Our study may provide the fundamentdata for studying the function of LEA genes

2 Materials and Methods

21 Plant Materials and Treatments Seedlings of T hisp-ida were grown in pots with a mixture of turf peat andsand (2 1 vv) in a greenhouse at 70ndash75 relative humiditylightdark cycle of 1410 h and the temperature of 24∘C Two-month-old seedlingswere used for experimental analysesTheseedlings were watered at the roots with one of the followingsolutions 04M NaCl 20 (wv) PEG

6000 or 100 120583M ABA

for 0 3 6 9 12 and 24 h In controls the seedlings werewatered with the same volume of fresh water Followingthese treatments the leaves stems and roots from at least 20seedlings were harvested and pooled frozen immediately inliquid nitrogen and stored at minus80∘C for RNA preparationThree samples were used for real-time RT-PCR biologicalrepeats

22 Cloning of the LEA Genes from T hispida In a previousstudy 11 cDNA libraries of T hispida treated with NaHCO

3

were constructed using Solexa technology which were the8 libraries respectively from roots of T hispida treatedwith NaHCO

3for 0 12 24 and 48 h (2 libraries were

built from each treatment as biological replication) and 3libraries from leaves of T hispida treated with NaHCO

3

for 0 12 and 24 h In total 94359 nonredundant unigenesof gt200 nt in length were generated using the SOAP denovo software Subsequently these unigenes were searchedagainst the Nr database and the Swiss-Prot database usingblastx (httpblastncbinlmnihgov) with a cut-off 119864-valueof 10minus5 [24 25] The unigenes representing the ldquoLEArdquo geneswere queried and identified from these libraries according tothe functional annotation Then primers for the LEA geneswere designed according to these sequences RT-PCR andresequencing were used to confirm the LEA sequences

23 Sequence Alignments and Phylogenetic Analysis Firstlythe open reading frames (ORFs) of all the LEA unigenes(named as ThLEA-1 to -12) were identified using the ORFfinder tools from the NCBI (httpwwwncbinlmnihgovgorfgorfhtml) Subsequently the LEAs with complete ORFswere subjected to molecular weight (MW) and isoelec-tronic point (pI) predictions using the Compute pIMwtool (httpwwwexpasyorgtoolsprotparamhtml) Subcel-lular location predictions for these genes were undertakenusing the Target 11987511 Server (httpwwwcbsdtudkser-vicesTargetP) All the LEA proteins from T hispida and51 Arabidopsis LEA proteins were subjected to phylogeneticanalysis by constructing a phylogenetic tree by the neighbor-joining (NJ) method in ClustalX

24 RNA Extraction and Reverse Transcription (RT) TotalRNA was isolated from the roots stems and leaves usingthe CTAB method Total RNA was digested with DNaseI (Promega) to remove any residual DNA Approximately1 120583g of total RNA was reverse transcribed to cDNA in a10 120583L volume using oligodeoxythymidine and 6-bp randomprimers following the PrimeScript RT reagent kit (TaKaRa)protocol The synthesized cDNAs were diluted 10-fold withsterile water and used as templates for real-time RT-PCR

25 Quantitative Real-Time RT-PCR Real-time RT-PCR wascarried out in an Opticon machine (Biorad Hercules CA)using a real-time PCR MIX Kit (SYBR Green as the fluo-rescent dye TOKOBO) and the primers used are shown inTable 1 The alpha tubulin (FJ618518) beta tubulin (FJ618519)and actin (FJ618517) genes were used as internal controls(reference genes) to normalize the total RNA amount presentin each reaction The reaction mixture (20120583L) contained10 120583L of SYBR Green Real-Time PCR Master Mix (Toyobo)05 120583M of forward and reverse primer and 2 120583L of cDNAtemplate (equivalent to 20 ng of total RNA)The amplificationwas performed using the following cycling parameters 94∘C

The Scientific World Journal 3

Table 2 Characteristics of the 8ThLEAs from T hispida that had full ORFs

Gene name Deduced number of amino acids MW (kDa) pI Predicted subcellular localizationlowast

ThLEA-1 584 6540 818 OtherThLEA-2 435 4670 867 SecretedThLEA-3 462 5131 802 ChloroplastThLEA-4 212 2355 960 SecretedThLEA-5 114 1229 554 OtherThLEA-6 151 1631 497 OtherThLEA-7 318 3531 475 OtherThLEA-8 171 1749 802 OtherlowastSubcellular localization was predicted from protein sequence analysis using the targetP algorithm

for 30 s followed by 45 cycles of 94∘C for 12 s 60∘C for30 s 72∘C for 40 s and then at 81∘C for 1 s for plate readingA melting curve was generated for each sample at the endof each run to assess the purity of the amplified productReal-time RT-PCR was carried out in triplicate to ensurethe reproducibility of the results The expression levels werecalculated from the threshold cycle according to 2minusΔΔCt [26]

3 Results

31 Characterization of ThLEAs In total 12 unique ThLEAs(ThLEA-1-8 respectively with the GenBank numbersKF801660 to KF801667 ThLEA-9-12 respectively withthe GenBank numbers KF924555 to KF924558) that areexpressed differentially in response to at least one stress wereidentified from the T hispida cDNA libraries Among theseeight ThLEA proteins had complete ORFs that encodeddeduced polypeptides of 114ndash584 amino acids with predictedmolecular masses of 1229ndash654 kDa and pIs of 475ndash96(Table 2) Subcellular locations were predicted from proteinsequence analysis using the targetP algorithm The resultssuggested that ThLEA-3 may be located in the chloroplastThLEA-2 and -4 may be secreted proteins and the locationsof the other fiveThLEA genes with full ORFsmay be classifiedas ldquootherrdquo

Hunault and Jaspard [9] identified 51 LEA proteinencoding genes in Arabidopsis genome and classified theminto nine distinct groups (LEA-1 -2 -3 -4 -5 Atm SMPdehydrin and PvLEA18) According to this classification thephylogenetic relationships showed that ThLEA-1 -3 -6 and-7 are in the LEA-2 group and ThLEA-2 -4 -9 -10 -11 and-12 are in the LEA-1 group while ThLEA-8 and -5 are in theAtm and LEA-5 groups respectively (Figure 1)

32 Relative Transcript Abundances of ThLEAs under NormalGrowth Conditions In order to study the tissue specificity ofThLEA gene expression the relative transcript abundancesof the ThLEA genes in T hispida roots stems leaves andseeds under normal growth conditions were monitored byreal-time RT-PCR The transcript level of the actin genewas assigned as 100 while the transcript levels of ThLEAgenes were plotted relative to transcript level of actin gene(Table 3) The results indicated thatThLEA-4 is the gene withthe highest expression in stems and leaves and in the roots

Table 3 Relative transcript abundance of the 12ThLEAs in differenttissues of T hispidaThe transcript levels of the 12ThLEA genes wereplotted relative to expression of the actin gene Transcript levels ofthe actin gene in roots stems leaves and seeds were all assigned as100

Gene Relative abundanceRoots Stems Leaves Seeds

ThLEA-1 68 116 362 10ThLEA-2 53 264 121 230ThLEA-3 82 232 648 4ThLEA-4 1881 3656 3555 161ThLEA-5 15 02 03 20ThLEA-6 01 14 07 3ThLEA-7 08 195 279 10ThLEA-8 85 08 15 770ThLEA-9 177 617 262 180ThLEA-10 1983 20 51 1240ThLEA-11 1618 41 237 6050ThLEA-12 99 01 01 1220

only ThLEA-10 had greater expression while the transcriptlevels of ThLEA-5 and -6 were very low in all four tissues(roots stems leaves and seeds) The expressions ofThLEA-8and -12 were very high in seeds while those were very lowin the other three tissues In the roots the transcript levelof ThLEA-6 was lowest and only 01 (equivalent to 11000of actin) Meanwhile in stems and leaves the transcriptlevel of ThLEA-12 was lowest and less than 11000 of actinMoreover the transcript level of the ThLEA genes highlydiffered among each other and also strongly differed amongthe four tissues For instance ThLEA-10 -11 and -12 wereexpressed mainly in the roots and seeds and their transcriptlevels were all exceeded 1220-fold in the seedsThLEA-2 and -9were expressedmainly in the stems and seeds whileThLEA-1 -3 and -7 showed relatively high transcript level in theleaves

33 The Expression Profiles of ThLEAs in Response to NaClPEG and ABA Treatments

331 NaCl Stress The transcript patterns of all of theThLEAgenes were quite similar among the roots stems and leaves

4 The Scientific World Journal

ThLEA-7ThLEA-6At1g01470At2g46140At1g54410At2g44060ThLEA-1ThLEA-3At3g53770At4g15910At1g02820At4g02380At1g03120At5g27980At3g22490At3g22500At5g53260At5g53270ThLEA-4At1g72100ThLEA-2At4g38410At1g76180At1g20440At1g20450At2g21490At4g39130At3g50970At3g50980At5g66400At2g33690At2g23110At2g23120ThLEA-9At3g17520At3g02480At4g13560At1g52690At3g15670ThLEA-11At2g18340At4g36600ThLEA-12At4g21020At5g44310At4g13230ThLEA-10At2g36640At3g53040At2g42560At2g03740At2g03850At2g42530At2g42540At1g32560At2g35300At5g06760ThLEA-8At2g41260At2g41280ThLEA-5At2g40170At3g51810

LEA-2

SMP

LEA-3

LEA-4

Dehydrin

PvLEA18

LEA-4

LEA-1

AtM

LEA-5

Figure 1 Phylogenetic tree of the 12 ThLEAs and 51 Arabidopsis LEAs based on sequence alignments of the predicted proteins All the 12ThLEA proteins from T hispida and 51 Arabidopsis LEA proteins were subjected to phylogenetic analysis by constructing a phylogenetic treeby the neighbor-joining (NJ) method in ClustalX According to the classification of Hunault and Jaspard [9] the LEA proteins were classifiedinto nine groups

under NaCl stress Furthermore ThLEA-2 -3 -4 and -11were induced (gt2-fold) during NaCl stress period ThLEA-7was also upregulated during most treatment period and sixThLEA genes reached their peak expression level in the leavesat 24 h of stress The most highly induced gene wasThLEA-1which was induced 1596-fold HoweverThLEA-5 -6 -8 -10

or -12 were highly downregulated at almost each stress timepoint Except for ThLEA-5 the other four downregulatedgenes reached lowest transcript levels at 9 h in stems andleaves Interestingly distinct from these genes the transcriptpattern of ThLEA-9 was different among the three tissuesIn the roots it was induced at 3 h but downregulated at the

The Scientific World Journal 5

minus8minus6minus4minus2

02468

10Th

LEA-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2Re

lativ

e exp

ress

ion

leve

l

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10

minus5

0

5

10

15

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes

Relat

ive e

xpre

ssio

n le

vel

3h6h9h

12h24h

(c)

Figure 2 Expression analysis of the 12ThLEAs in response to 04M NaCl stress Relative expression level = transcription level under stresstreatmenttranscription level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

other time points while in stems and leaves it was mainlyupregulated Similar with the other upregulated ThLEAsThLEA-9 reached greatest expression at 24 h in the leaves(Figure 2)

332 PEG Stress Consistent with NaCl stress the transcriptpatterns of theseThLEA genes under PEG stress were dividedinto two distinct groups (Figure 3) One groupwas composedof ThLEA-1 -2 -3 -4 and -11 and these genes were mainlyupregulated The second group includedThLEA-5 -6 -8 -9-10 and -12 that were largely downregulated during the PEGtreatment period

333 ABA Treatment After application of exogenous ABAThLEA-1 -2 and -3 were only slightly differentially regulatedin the roots stems and leaves indicating that they may beregulated via the ABA-independent signaling pathway How-ever the genes that were downregulated by PEG andor NaClstress were also highly downregulated by ABA treatmentIn contrast ThLEA-4 -7 and -11 were mainly upregulatedespecially ThLEA-4 whose transcript levels in the stem at6 h were increased by 38-fold compared with the control(Figure 4)

4 Discussion

In the present study 12 ThLEA genes were identified fromTamarix RNA-seq data base and the transcript abundanceof each gene was assessed in normal growth conditions byreal-time RT-PCR Furthermore NaCl and PEG were usedto simulate salinity and drought conditions respectively andthe expression levels of theThLEA genes were investigated inroots stems and leaves in response to these stress environ-ments

Many studies have shown that members of the plant LEAfamily displayed tissue-specific expression and are involvedin various processes in plant growth and development Forexample in Arabidopsis 22 of the 51 LEA genes (43)showed high expression levels (relative expression gt10) inthe nonseed organs in the absence of a stress or hormonetreatment [27] The OsLEA3-2 gene is not expressed invegetative tissues under normal conditions and it is thoughtto play an important role in the maturation of the embryo[10] Moreover the transcripts of MsLEA3-1 were stronglyenriched in leaves compared with roots and stems of maturealfalfa plants [20] Expression profiles of the 12ThLEA genesin different tissues of T hispida were determined undernormal growth conditions and this showed that most of

6 The Scientific World Journal

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10minus8minus6minus4minus2

02468

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 3 Expression analysis of the 12ThLEAs in response to 20 PEG6000 stress Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

the ThLEAs were expressed in various organs and tissuesHowever they displayed different expression levels betweenthe different tissue types and this may reflect the complexityof functions performed by this gene family [28] Among the12 ThLEAs ThLEA-10 -11 and -12 were mainly expressed inthe roots suggesting that these genes may play roles in rootstress responseThLEA-2 and -9 were highly expressed in thestem while ThLEA-1 -3 and -7 may play important roles inthe leaves as these genes are highly and specifically expressedin leaves Moreover ThLEA-4 has very high expression levelin the roots stems and leaves suggesting that it might playimportant roles in all of these tissues The expression levelsof ThLEA-5 -6 -8 and -12 were very low in each of thetissues examined indicating that these genes may play arelatively unimportant role in these tissues under normalgrowth conditions

There is increasing evidence suggesting that LEA genesare associated with abiotic stress tolerance particularly inplant responses to dehydration salt and cold stresses [18 29]In Arabidopsis of the 22 genes highly expressed in nonseedtissues 12 were induced by more than 3-fold in response tocold drought and salt stresses [27] In sweet potato plantsIbLEA14 expressionwas strongly induced by dehydration andNaCl [30] In the present study half of the 12 ThLEA genes

(ThLEA-1 -2 -3 -4 -7 and -11) were induced under saltand drought stress indicating that these genes might playimportant roles in response to salt and drought stresses in Thispida Bies-Etheve et al [31] reported that LEA genes fromthe same group do not show identical expression profilesand regulation of LEA genes that show apparently similarexpression patterns does not systematically involve the sameregulatory pathway Consistent with this the six upregulatedThLEAs in T hispida belong to two LEAs groups (LEA-2and LEA-4) while LEAs in the same group (such as LEA-4)showed completely different expression patterns

There are ABA-dependent and ABA-independent regula-tory networks controlling plant responses to abiotic stressesLEA genes are often considered as being ABA-regulated andprevious studies have shown that some ThLEA genes canbe induced by exogenous ABA [10 30] However our studyshows that someThLEA genes includingThLEA-1 -2 and -3were highly induced by abiotic stresses but were not regulatedby ABA Consistent with this Bies-Etheve et al [31] reportedthat most of the LEA genes in Arabidopsis seedlings showno response at all following exogenous ABA treatment Hun-dertmark and Hincha [27] deduced the presence of differentsignal transduction pathways in different Arabidopsis tissues(vegetative plants and seeds) by comparing the expression of

The Scientific World Journal 7

minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus15

minus10

minus5

0

5

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus15minus12minus9minus6minus3

036

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 4 Expression analysis of the 12ThLEAs in response to 100 120583MABA treatment Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

LEA genes after ABA treatment In contrast we found thatexpression patterns were highly similar in the three tissuesexamined after ABA treatment Expression analyses showedthat nine ThLEA genes in three tissues of T hispida wereupregulated or downregulated by ABA which suggests thatthese ThLEA genes are regulated by ABA-dependent stressresistance pathways These findings demonstrate that theThLEA genes involved in salt and drought stress resistancein T hispida are probably regulated by two different pathways(ABA-dependent andABA-independent) Further studies arerequired to elucidate the functions of theThLEAs in responseto the abiotic stress and to fully characterize the signalingpathways that regulate their expression

5 Conclusion

In summary we identified 12 ThLEA genes from T hispidawhich belong to three groups The LEA-1 group includesThLEA-2 -4 -9 -10 -11 and -12 proteins the LEA-2 groupcontains ThLEA-1 ThLEA-3 ThLEA-6 and ThLEA-7 andThLEA-8 and -5 are in the Atm and LEA-5 groups TheThLEA genes displayed tissue-specific expression patternsunder normal growth conditionsThLEA-10 -11 and -12were

expressed mainly in the roots and seeds ThLEA-2 and -9were preferentially expressed in the stems and seeds whileThLEA-1 -3 and -7 showed high transcript level in the leavesFurthermore real-time RT-PCR analysis showed that theThLEA genes were regulated by salt and drought stressesThLEA-1 -2 -3 -4 -7 and -11 were mainly induced by saltand drought stress ButThLEA-5 -6 -8 -9 -10 and -12 werelargely downregulated during the NaCl and PEG treatmentAfter the application of exogenous ABA the expression levelsof all ThLEA genes (except for the ThLEA-1 -2 and -3) wereobviously different under the ABA treatment Our studieswill contribute to a better understanding of the functions ofThLEA genes involved in stress response in T hispida

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Caiqiu Gao and Yali Liu have the same contribution to thispaper

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 3

Table 2 Characteristics of the 8ThLEAs from T hispida that had full ORFs

Gene name Deduced number of amino acids MW (kDa) pI Predicted subcellular localizationlowast

ThLEA-1 584 6540 818 OtherThLEA-2 435 4670 867 SecretedThLEA-3 462 5131 802 ChloroplastThLEA-4 212 2355 960 SecretedThLEA-5 114 1229 554 OtherThLEA-6 151 1631 497 OtherThLEA-7 318 3531 475 OtherThLEA-8 171 1749 802 OtherlowastSubcellular localization was predicted from protein sequence analysis using the targetP algorithm

for 30 s followed by 45 cycles of 94∘C for 12 s 60∘C for30 s 72∘C for 40 s and then at 81∘C for 1 s for plate readingA melting curve was generated for each sample at the endof each run to assess the purity of the amplified productReal-time RT-PCR was carried out in triplicate to ensurethe reproducibility of the results The expression levels werecalculated from the threshold cycle according to 2minusΔΔCt [26]

3 Results

31 Characterization of ThLEAs In total 12 unique ThLEAs(ThLEA-1-8 respectively with the GenBank numbersKF801660 to KF801667 ThLEA-9-12 respectively withthe GenBank numbers KF924555 to KF924558) that areexpressed differentially in response to at least one stress wereidentified from the T hispida cDNA libraries Among theseeight ThLEA proteins had complete ORFs that encodeddeduced polypeptides of 114ndash584 amino acids with predictedmolecular masses of 1229ndash654 kDa and pIs of 475ndash96(Table 2) Subcellular locations were predicted from proteinsequence analysis using the targetP algorithm The resultssuggested that ThLEA-3 may be located in the chloroplastThLEA-2 and -4 may be secreted proteins and the locationsof the other fiveThLEA genes with full ORFsmay be classifiedas ldquootherrdquo

Hunault and Jaspard [9] identified 51 LEA proteinencoding genes in Arabidopsis genome and classified theminto nine distinct groups (LEA-1 -2 -3 -4 -5 Atm SMPdehydrin and PvLEA18) According to this classification thephylogenetic relationships showed that ThLEA-1 -3 -6 and-7 are in the LEA-2 group and ThLEA-2 -4 -9 -10 -11 and-12 are in the LEA-1 group while ThLEA-8 and -5 are in theAtm and LEA-5 groups respectively (Figure 1)

32 Relative Transcript Abundances of ThLEAs under NormalGrowth Conditions In order to study the tissue specificity ofThLEA gene expression the relative transcript abundancesof the ThLEA genes in T hispida roots stems leaves andseeds under normal growth conditions were monitored byreal-time RT-PCR The transcript level of the actin genewas assigned as 100 while the transcript levels of ThLEAgenes were plotted relative to transcript level of actin gene(Table 3) The results indicated thatThLEA-4 is the gene withthe highest expression in stems and leaves and in the roots

Table 3 Relative transcript abundance of the 12ThLEAs in differenttissues of T hispidaThe transcript levels of the 12ThLEA genes wereplotted relative to expression of the actin gene Transcript levels ofthe actin gene in roots stems leaves and seeds were all assigned as100

Gene Relative abundanceRoots Stems Leaves Seeds

ThLEA-1 68 116 362 10ThLEA-2 53 264 121 230ThLEA-3 82 232 648 4ThLEA-4 1881 3656 3555 161ThLEA-5 15 02 03 20ThLEA-6 01 14 07 3ThLEA-7 08 195 279 10ThLEA-8 85 08 15 770ThLEA-9 177 617 262 180ThLEA-10 1983 20 51 1240ThLEA-11 1618 41 237 6050ThLEA-12 99 01 01 1220

only ThLEA-10 had greater expression while the transcriptlevels of ThLEA-5 and -6 were very low in all four tissues(roots stems leaves and seeds) The expressions ofThLEA-8and -12 were very high in seeds while those were very lowin the other three tissues In the roots the transcript levelof ThLEA-6 was lowest and only 01 (equivalent to 11000of actin) Meanwhile in stems and leaves the transcriptlevel of ThLEA-12 was lowest and less than 11000 of actinMoreover the transcript level of the ThLEA genes highlydiffered among each other and also strongly differed amongthe four tissues For instance ThLEA-10 -11 and -12 wereexpressed mainly in the roots and seeds and their transcriptlevels were all exceeded 1220-fold in the seedsThLEA-2 and -9were expressedmainly in the stems and seeds whileThLEA-1 -3 and -7 showed relatively high transcript level in theleaves

33 The Expression Profiles of ThLEAs in Response to NaClPEG and ABA Treatments

331 NaCl Stress The transcript patterns of all of theThLEAgenes were quite similar among the roots stems and leaves

4 The Scientific World Journal

ThLEA-7ThLEA-6At1g01470At2g46140At1g54410At2g44060ThLEA-1ThLEA-3At3g53770At4g15910At1g02820At4g02380At1g03120At5g27980At3g22490At3g22500At5g53260At5g53270ThLEA-4At1g72100ThLEA-2At4g38410At1g76180At1g20440At1g20450At2g21490At4g39130At3g50970At3g50980At5g66400At2g33690At2g23110At2g23120ThLEA-9At3g17520At3g02480At4g13560At1g52690At3g15670ThLEA-11At2g18340At4g36600ThLEA-12At4g21020At5g44310At4g13230ThLEA-10At2g36640At3g53040At2g42560At2g03740At2g03850At2g42530At2g42540At1g32560At2g35300At5g06760ThLEA-8At2g41260At2g41280ThLEA-5At2g40170At3g51810

LEA-2

SMP

LEA-3

LEA-4

Dehydrin

PvLEA18

LEA-4

LEA-1

AtM

LEA-5

Figure 1 Phylogenetic tree of the 12 ThLEAs and 51 Arabidopsis LEAs based on sequence alignments of the predicted proteins All the 12ThLEA proteins from T hispida and 51 Arabidopsis LEA proteins were subjected to phylogenetic analysis by constructing a phylogenetic treeby the neighbor-joining (NJ) method in ClustalX According to the classification of Hunault and Jaspard [9] the LEA proteins were classifiedinto nine groups

under NaCl stress Furthermore ThLEA-2 -3 -4 and -11were induced (gt2-fold) during NaCl stress period ThLEA-7was also upregulated during most treatment period and sixThLEA genes reached their peak expression level in the leavesat 24 h of stress The most highly induced gene wasThLEA-1which was induced 1596-fold HoweverThLEA-5 -6 -8 -10

or -12 were highly downregulated at almost each stress timepoint Except for ThLEA-5 the other four downregulatedgenes reached lowest transcript levels at 9 h in stems andleaves Interestingly distinct from these genes the transcriptpattern of ThLEA-9 was different among the three tissuesIn the roots it was induced at 3 h but downregulated at the

The Scientific World Journal 5

minus8minus6minus4minus2

02468

10Th

LEA-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2Re

lativ

e exp

ress

ion

leve

l

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10

minus5

0

5

10

15

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes

Relat

ive e

xpre

ssio

n le

vel

3h6h9h

12h24h

(c)

Figure 2 Expression analysis of the 12ThLEAs in response to 04M NaCl stress Relative expression level = transcription level under stresstreatmenttranscription level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

other time points while in stems and leaves it was mainlyupregulated Similar with the other upregulated ThLEAsThLEA-9 reached greatest expression at 24 h in the leaves(Figure 2)

332 PEG Stress Consistent with NaCl stress the transcriptpatterns of theseThLEA genes under PEG stress were dividedinto two distinct groups (Figure 3) One groupwas composedof ThLEA-1 -2 -3 -4 and -11 and these genes were mainlyupregulated The second group includedThLEA-5 -6 -8 -9-10 and -12 that were largely downregulated during the PEGtreatment period

333 ABA Treatment After application of exogenous ABAThLEA-1 -2 and -3 were only slightly differentially regulatedin the roots stems and leaves indicating that they may beregulated via the ABA-independent signaling pathway How-ever the genes that were downregulated by PEG andor NaClstress were also highly downregulated by ABA treatmentIn contrast ThLEA-4 -7 and -11 were mainly upregulatedespecially ThLEA-4 whose transcript levels in the stem at6 h were increased by 38-fold compared with the control(Figure 4)

4 Discussion

In the present study 12 ThLEA genes were identified fromTamarix RNA-seq data base and the transcript abundanceof each gene was assessed in normal growth conditions byreal-time RT-PCR Furthermore NaCl and PEG were usedto simulate salinity and drought conditions respectively andthe expression levels of theThLEA genes were investigated inroots stems and leaves in response to these stress environ-ments

Many studies have shown that members of the plant LEAfamily displayed tissue-specific expression and are involvedin various processes in plant growth and development Forexample in Arabidopsis 22 of the 51 LEA genes (43)showed high expression levels (relative expression gt10) inthe nonseed organs in the absence of a stress or hormonetreatment [27] The OsLEA3-2 gene is not expressed invegetative tissues under normal conditions and it is thoughtto play an important role in the maturation of the embryo[10] Moreover the transcripts of MsLEA3-1 were stronglyenriched in leaves compared with roots and stems of maturealfalfa plants [20] Expression profiles of the 12ThLEA genesin different tissues of T hispida were determined undernormal growth conditions and this showed that most of

6 The Scientific World Journal

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10minus8minus6minus4minus2

02468

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 3 Expression analysis of the 12ThLEAs in response to 20 PEG6000 stress Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

the ThLEAs were expressed in various organs and tissuesHowever they displayed different expression levels betweenthe different tissue types and this may reflect the complexityof functions performed by this gene family [28] Among the12 ThLEAs ThLEA-10 -11 and -12 were mainly expressed inthe roots suggesting that these genes may play roles in rootstress responseThLEA-2 and -9 were highly expressed in thestem while ThLEA-1 -3 and -7 may play important roles inthe leaves as these genes are highly and specifically expressedin leaves Moreover ThLEA-4 has very high expression levelin the roots stems and leaves suggesting that it might playimportant roles in all of these tissues The expression levelsof ThLEA-5 -6 -8 and -12 were very low in each of thetissues examined indicating that these genes may play arelatively unimportant role in these tissues under normalgrowth conditions

There is increasing evidence suggesting that LEA genesare associated with abiotic stress tolerance particularly inplant responses to dehydration salt and cold stresses [18 29]In Arabidopsis of the 22 genes highly expressed in nonseedtissues 12 were induced by more than 3-fold in response tocold drought and salt stresses [27] In sweet potato plantsIbLEA14 expressionwas strongly induced by dehydration andNaCl [30] In the present study half of the 12 ThLEA genes

(ThLEA-1 -2 -3 -4 -7 and -11) were induced under saltand drought stress indicating that these genes might playimportant roles in response to salt and drought stresses in Thispida Bies-Etheve et al [31] reported that LEA genes fromthe same group do not show identical expression profilesand regulation of LEA genes that show apparently similarexpression patterns does not systematically involve the sameregulatory pathway Consistent with this the six upregulatedThLEAs in T hispida belong to two LEAs groups (LEA-2and LEA-4) while LEAs in the same group (such as LEA-4)showed completely different expression patterns

There are ABA-dependent and ABA-independent regula-tory networks controlling plant responses to abiotic stressesLEA genes are often considered as being ABA-regulated andprevious studies have shown that some ThLEA genes canbe induced by exogenous ABA [10 30] However our studyshows that someThLEA genes includingThLEA-1 -2 and -3were highly induced by abiotic stresses but were not regulatedby ABA Consistent with this Bies-Etheve et al [31] reportedthat most of the LEA genes in Arabidopsis seedlings showno response at all following exogenous ABA treatment Hun-dertmark and Hincha [27] deduced the presence of differentsignal transduction pathways in different Arabidopsis tissues(vegetative plants and seeds) by comparing the expression of

The Scientific World Journal 7

minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus15

minus10

minus5

0

5

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus15minus12minus9minus6minus3

036

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 4 Expression analysis of the 12ThLEAs in response to 100 120583MABA treatment Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

LEA genes after ABA treatment In contrast we found thatexpression patterns were highly similar in the three tissuesexamined after ABA treatment Expression analyses showedthat nine ThLEA genes in three tissues of T hispida wereupregulated or downregulated by ABA which suggests thatthese ThLEA genes are regulated by ABA-dependent stressresistance pathways These findings demonstrate that theThLEA genes involved in salt and drought stress resistancein T hispida are probably regulated by two different pathways(ABA-dependent andABA-independent) Further studies arerequired to elucidate the functions of theThLEAs in responseto the abiotic stress and to fully characterize the signalingpathways that regulate their expression

5 Conclusion

In summary we identified 12 ThLEA genes from T hispidawhich belong to three groups The LEA-1 group includesThLEA-2 -4 -9 -10 -11 and -12 proteins the LEA-2 groupcontains ThLEA-1 ThLEA-3 ThLEA-6 and ThLEA-7 andThLEA-8 and -5 are in the Atm and LEA-5 groups TheThLEA genes displayed tissue-specific expression patternsunder normal growth conditionsThLEA-10 -11 and -12were

expressed mainly in the roots and seeds ThLEA-2 and -9were preferentially expressed in the stems and seeds whileThLEA-1 -3 and -7 showed high transcript level in the leavesFurthermore real-time RT-PCR analysis showed that theThLEA genes were regulated by salt and drought stressesThLEA-1 -2 -3 -4 -7 and -11 were mainly induced by saltand drought stress ButThLEA-5 -6 -8 -9 -10 and -12 werelargely downregulated during the NaCl and PEG treatmentAfter the application of exogenous ABA the expression levelsof all ThLEA genes (except for the ThLEA-1 -2 and -3) wereobviously different under the ABA treatment Our studieswill contribute to a better understanding of the functions ofThLEA genes involved in stress response in T hispida

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Caiqiu Gao and Yali Liu have the same contribution to thispaper

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 The Scientific World Journal

ThLEA-7ThLEA-6At1g01470At2g46140At1g54410At2g44060ThLEA-1ThLEA-3At3g53770At4g15910At1g02820At4g02380At1g03120At5g27980At3g22490At3g22500At5g53260At5g53270ThLEA-4At1g72100ThLEA-2At4g38410At1g76180At1g20440At1g20450At2g21490At4g39130At3g50970At3g50980At5g66400At2g33690At2g23110At2g23120ThLEA-9At3g17520At3g02480At4g13560At1g52690At3g15670ThLEA-11At2g18340At4g36600ThLEA-12At4g21020At5g44310At4g13230ThLEA-10At2g36640At3g53040At2g42560At2g03740At2g03850At2g42530At2g42540At1g32560At2g35300At5g06760ThLEA-8At2g41260At2g41280ThLEA-5At2g40170At3g51810

LEA-2

SMP

LEA-3

LEA-4

Dehydrin

PvLEA18

LEA-4

LEA-1

AtM

LEA-5

Figure 1 Phylogenetic tree of the 12 ThLEAs and 51 Arabidopsis LEAs based on sequence alignments of the predicted proteins All the 12ThLEA proteins from T hispida and 51 Arabidopsis LEA proteins were subjected to phylogenetic analysis by constructing a phylogenetic treeby the neighbor-joining (NJ) method in ClustalX According to the classification of Hunault and Jaspard [9] the LEA proteins were classifiedinto nine groups

under NaCl stress Furthermore ThLEA-2 -3 -4 and -11were induced (gt2-fold) during NaCl stress period ThLEA-7was also upregulated during most treatment period and sixThLEA genes reached their peak expression level in the leavesat 24 h of stress The most highly induced gene wasThLEA-1which was induced 1596-fold HoweverThLEA-5 -6 -8 -10

or -12 were highly downregulated at almost each stress timepoint Except for ThLEA-5 the other four downregulatedgenes reached lowest transcript levels at 9 h in stems andleaves Interestingly distinct from these genes the transcriptpattern of ThLEA-9 was different among the three tissuesIn the roots it was induced at 3 h but downregulated at the

The Scientific World Journal 5

minus8minus6minus4minus2

02468

10Th

LEA-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2Re

lativ

e exp

ress

ion

leve

l

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10

minus5

0

5

10

15

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes

Relat

ive e

xpre

ssio

n le

vel

3h6h9h

12h24h

(c)

Figure 2 Expression analysis of the 12ThLEAs in response to 04M NaCl stress Relative expression level = transcription level under stresstreatmenttranscription level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

other time points while in stems and leaves it was mainlyupregulated Similar with the other upregulated ThLEAsThLEA-9 reached greatest expression at 24 h in the leaves(Figure 2)

332 PEG Stress Consistent with NaCl stress the transcriptpatterns of theseThLEA genes under PEG stress were dividedinto two distinct groups (Figure 3) One groupwas composedof ThLEA-1 -2 -3 -4 and -11 and these genes were mainlyupregulated The second group includedThLEA-5 -6 -8 -9-10 and -12 that were largely downregulated during the PEGtreatment period

333 ABA Treatment After application of exogenous ABAThLEA-1 -2 and -3 were only slightly differentially regulatedin the roots stems and leaves indicating that they may beregulated via the ABA-independent signaling pathway How-ever the genes that were downregulated by PEG andor NaClstress were also highly downregulated by ABA treatmentIn contrast ThLEA-4 -7 and -11 were mainly upregulatedespecially ThLEA-4 whose transcript levels in the stem at6 h were increased by 38-fold compared with the control(Figure 4)

4 Discussion

In the present study 12 ThLEA genes were identified fromTamarix RNA-seq data base and the transcript abundanceof each gene was assessed in normal growth conditions byreal-time RT-PCR Furthermore NaCl and PEG were usedto simulate salinity and drought conditions respectively andthe expression levels of theThLEA genes were investigated inroots stems and leaves in response to these stress environ-ments

Many studies have shown that members of the plant LEAfamily displayed tissue-specific expression and are involvedin various processes in plant growth and development Forexample in Arabidopsis 22 of the 51 LEA genes (43)showed high expression levels (relative expression gt10) inthe nonseed organs in the absence of a stress or hormonetreatment [27] The OsLEA3-2 gene is not expressed invegetative tissues under normal conditions and it is thoughtto play an important role in the maturation of the embryo[10] Moreover the transcripts of MsLEA3-1 were stronglyenriched in leaves compared with roots and stems of maturealfalfa plants [20] Expression profiles of the 12ThLEA genesin different tissues of T hispida were determined undernormal growth conditions and this showed that most of

6 The Scientific World Journal

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10minus8minus6minus4minus2

02468

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 3 Expression analysis of the 12ThLEAs in response to 20 PEG6000 stress Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

the ThLEAs were expressed in various organs and tissuesHowever they displayed different expression levels betweenthe different tissue types and this may reflect the complexityof functions performed by this gene family [28] Among the12 ThLEAs ThLEA-10 -11 and -12 were mainly expressed inthe roots suggesting that these genes may play roles in rootstress responseThLEA-2 and -9 were highly expressed in thestem while ThLEA-1 -3 and -7 may play important roles inthe leaves as these genes are highly and specifically expressedin leaves Moreover ThLEA-4 has very high expression levelin the roots stems and leaves suggesting that it might playimportant roles in all of these tissues The expression levelsof ThLEA-5 -6 -8 and -12 were very low in each of thetissues examined indicating that these genes may play arelatively unimportant role in these tissues under normalgrowth conditions

There is increasing evidence suggesting that LEA genesare associated with abiotic stress tolerance particularly inplant responses to dehydration salt and cold stresses [18 29]In Arabidopsis of the 22 genes highly expressed in nonseedtissues 12 were induced by more than 3-fold in response tocold drought and salt stresses [27] In sweet potato plantsIbLEA14 expressionwas strongly induced by dehydration andNaCl [30] In the present study half of the 12 ThLEA genes

(ThLEA-1 -2 -3 -4 -7 and -11) were induced under saltand drought stress indicating that these genes might playimportant roles in response to salt and drought stresses in Thispida Bies-Etheve et al [31] reported that LEA genes fromthe same group do not show identical expression profilesand regulation of LEA genes that show apparently similarexpression patterns does not systematically involve the sameregulatory pathway Consistent with this the six upregulatedThLEAs in T hispida belong to two LEAs groups (LEA-2and LEA-4) while LEAs in the same group (such as LEA-4)showed completely different expression patterns

There are ABA-dependent and ABA-independent regula-tory networks controlling plant responses to abiotic stressesLEA genes are often considered as being ABA-regulated andprevious studies have shown that some ThLEA genes canbe induced by exogenous ABA [10 30] However our studyshows that someThLEA genes includingThLEA-1 -2 and -3were highly induced by abiotic stresses but were not regulatedby ABA Consistent with this Bies-Etheve et al [31] reportedthat most of the LEA genes in Arabidopsis seedlings showno response at all following exogenous ABA treatment Hun-dertmark and Hincha [27] deduced the presence of differentsignal transduction pathways in different Arabidopsis tissues(vegetative plants and seeds) by comparing the expression of

The Scientific World Journal 7

minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus15

minus10

minus5

0

5

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus15minus12minus9minus6minus3

036

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 4 Expression analysis of the 12ThLEAs in response to 100 120583MABA treatment Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

LEA genes after ABA treatment In contrast we found thatexpression patterns were highly similar in the three tissuesexamined after ABA treatment Expression analyses showedthat nine ThLEA genes in three tissues of T hispida wereupregulated or downregulated by ABA which suggests thatthese ThLEA genes are regulated by ABA-dependent stressresistance pathways These findings demonstrate that theThLEA genes involved in salt and drought stress resistancein T hispida are probably regulated by two different pathways(ABA-dependent andABA-independent) Further studies arerequired to elucidate the functions of theThLEAs in responseto the abiotic stress and to fully characterize the signalingpathways that regulate their expression

5 Conclusion

In summary we identified 12 ThLEA genes from T hispidawhich belong to three groups The LEA-1 group includesThLEA-2 -4 -9 -10 -11 and -12 proteins the LEA-2 groupcontains ThLEA-1 ThLEA-3 ThLEA-6 and ThLEA-7 andThLEA-8 and -5 are in the Atm and LEA-5 groups TheThLEA genes displayed tissue-specific expression patternsunder normal growth conditionsThLEA-10 -11 and -12were

expressed mainly in the roots and seeds ThLEA-2 and -9were preferentially expressed in the stems and seeds whileThLEA-1 -3 and -7 showed high transcript level in the leavesFurthermore real-time RT-PCR analysis showed that theThLEA genes were regulated by salt and drought stressesThLEA-1 -2 -3 -4 -7 and -11 were mainly induced by saltand drought stress ButThLEA-5 -6 -8 -9 -10 and -12 werelargely downregulated during the NaCl and PEG treatmentAfter the application of exogenous ABA the expression levelsof all ThLEA genes (except for the ThLEA-1 -2 and -3) wereobviously different under the ABA treatment Our studieswill contribute to a better understanding of the functions ofThLEA genes involved in stress response in T hispida

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Caiqiu Gao and Yali Liu have the same contribution to thispaper

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 5

minus8minus6minus4minus2

02468

10Th

LEA-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2Re

lativ

e exp

ress

ion

leve

l

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10

minus5

0

5

10

15

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes

Relat

ive e

xpre

ssio

n le

vel

3h6h9h

12h24h

(c)

Figure 2 Expression analysis of the 12ThLEAs in response to 04M NaCl stress Relative expression level = transcription level under stresstreatmenttranscription level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

other time points while in stems and leaves it was mainlyupregulated Similar with the other upregulated ThLEAsThLEA-9 reached greatest expression at 24 h in the leaves(Figure 2)

332 PEG Stress Consistent with NaCl stress the transcriptpatterns of theseThLEA genes under PEG stress were dividedinto two distinct groups (Figure 3) One groupwas composedof ThLEA-1 -2 -3 -4 and -11 and these genes were mainlyupregulated The second group includedThLEA-5 -6 -8 -9-10 and -12 that were largely downregulated during the PEGtreatment period

333 ABA Treatment After application of exogenous ABAThLEA-1 -2 and -3 were only slightly differentially regulatedin the roots stems and leaves indicating that they may beregulated via the ABA-independent signaling pathway How-ever the genes that were downregulated by PEG andor NaClstress were also highly downregulated by ABA treatmentIn contrast ThLEA-4 -7 and -11 were mainly upregulatedespecially ThLEA-4 whose transcript levels in the stem at6 h were increased by 38-fold compared with the control(Figure 4)

4 Discussion

In the present study 12 ThLEA genes were identified fromTamarix RNA-seq data base and the transcript abundanceof each gene was assessed in normal growth conditions byreal-time RT-PCR Furthermore NaCl and PEG were usedto simulate salinity and drought conditions respectively andthe expression levels of theThLEA genes were investigated inroots stems and leaves in response to these stress environ-ments

Many studies have shown that members of the plant LEAfamily displayed tissue-specific expression and are involvedin various processes in plant growth and development Forexample in Arabidopsis 22 of the 51 LEA genes (43)showed high expression levels (relative expression gt10) inthe nonseed organs in the absence of a stress or hormonetreatment [27] The OsLEA3-2 gene is not expressed invegetative tissues under normal conditions and it is thoughtto play an important role in the maturation of the embryo[10] Moreover the transcripts of MsLEA3-1 were stronglyenriched in leaves compared with roots and stems of maturealfalfa plants [20] Expression profiles of the 12ThLEA genesin different tissues of T hispida were determined undernormal growth conditions and this showed that most of

6 The Scientific World Journal

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10minus8minus6minus4minus2

02468

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 3 Expression analysis of the 12ThLEAs in response to 20 PEG6000 stress Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

the ThLEAs were expressed in various organs and tissuesHowever they displayed different expression levels betweenthe different tissue types and this may reflect the complexityof functions performed by this gene family [28] Among the12 ThLEAs ThLEA-10 -11 and -12 were mainly expressed inthe roots suggesting that these genes may play roles in rootstress responseThLEA-2 and -9 were highly expressed in thestem while ThLEA-1 -3 and -7 may play important roles inthe leaves as these genes are highly and specifically expressedin leaves Moreover ThLEA-4 has very high expression levelin the roots stems and leaves suggesting that it might playimportant roles in all of these tissues The expression levelsof ThLEA-5 -6 -8 and -12 were very low in each of thetissues examined indicating that these genes may play arelatively unimportant role in these tissues under normalgrowth conditions

There is increasing evidence suggesting that LEA genesare associated with abiotic stress tolerance particularly inplant responses to dehydration salt and cold stresses [18 29]In Arabidopsis of the 22 genes highly expressed in nonseedtissues 12 were induced by more than 3-fold in response tocold drought and salt stresses [27] In sweet potato plantsIbLEA14 expressionwas strongly induced by dehydration andNaCl [30] In the present study half of the 12 ThLEA genes

(ThLEA-1 -2 -3 -4 -7 and -11) were induced under saltand drought stress indicating that these genes might playimportant roles in response to salt and drought stresses in Thispida Bies-Etheve et al [31] reported that LEA genes fromthe same group do not show identical expression profilesand regulation of LEA genes that show apparently similarexpression patterns does not systematically involve the sameregulatory pathway Consistent with this the six upregulatedThLEAs in T hispida belong to two LEAs groups (LEA-2and LEA-4) while LEAs in the same group (such as LEA-4)showed completely different expression patterns

There are ABA-dependent and ABA-independent regula-tory networks controlling plant responses to abiotic stressesLEA genes are often considered as being ABA-regulated andprevious studies have shown that some ThLEA genes canbe induced by exogenous ABA [10 30] However our studyshows that someThLEA genes includingThLEA-1 -2 and -3were highly induced by abiotic stresses but were not regulatedby ABA Consistent with this Bies-Etheve et al [31] reportedthat most of the LEA genes in Arabidopsis seedlings showno response at all following exogenous ABA treatment Hun-dertmark and Hincha [27] deduced the presence of differentsignal transduction pathways in different Arabidopsis tissues(vegetative plants and seeds) by comparing the expression of

The Scientific World Journal 7

minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus15

minus10

minus5

0

5

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus15minus12minus9minus6minus3

036

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 4 Expression analysis of the 12ThLEAs in response to 100 120583MABA treatment Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

LEA genes after ABA treatment In contrast we found thatexpression patterns were highly similar in the three tissuesexamined after ABA treatment Expression analyses showedthat nine ThLEA genes in three tissues of T hispida wereupregulated or downregulated by ABA which suggests thatthese ThLEA genes are regulated by ABA-dependent stressresistance pathways These findings demonstrate that theThLEA genes involved in salt and drought stress resistancein T hispida are probably regulated by two different pathways(ABA-dependent andABA-independent) Further studies arerequired to elucidate the functions of theThLEAs in responseto the abiotic stress and to fully characterize the signalingpathways that regulate their expression

5 Conclusion

In summary we identified 12 ThLEA genes from T hispidawhich belong to three groups The LEA-1 group includesThLEA-2 -4 -9 -10 -11 and -12 proteins the LEA-2 groupcontains ThLEA-1 ThLEA-3 ThLEA-6 and ThLEA-7 andThLEA-8 and -5 are in the Atm and LEA-5 groups TheThLEA genes displayed tissue-specific expression patternsunder normal growth conditionsThLEA-10 -11 and -12were

expressed mainly in the roots and seeds ThLEA-2 and -9were preferentially expressed in the stems and seeds whileThLEA-1 -3 and -7 showed high transcript level in the leavesFurthermore real-time RT-PCR analysis showed that theThLEA genes were regulated by salt and drought stressesThLEA-1 -2 -3 -4 -7 and -11 were mainly induced by saltand drought stress ButThLEA-5 -6 -8 -9 -10 and -12 werelargely downregulated during the NaCl and PEG treatmentAfter the application of exogenous ABA the expression levelsof all ThLEA genes (except for the ThLEA-1 -2 and -3) wereobviously different under the ABA treatment Our studieswill contribute to a better understanding of the functions ofThLEA genes involved in stress response in T hispida

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Caiqiu Gao and Yali Liu have the same contribution to thispaper

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 The Scientific World Journal

minus10minus8minus6minus4minus2

02468

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus10minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus10minus8minus6minus4minus2

02468

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 3 Expression analysis of the 12ThLEAs in response to 20 PEG6000 stress Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

the ThLEAs were expressed in various organs and tissuesHowever they displayed different expression levels betweenthe different tissue types and this may reflect the complexityof functions performed by this gene family [28] Among the12 ThLEAs ThLEA-10 -11 and -12 were mainly expressed inthe roots suggesting that these genes may play roles in rootstress responseThLEA-2 and -9 were highly expressed in thestem while ThLEA-1 -3 and -7 may play important roles inthe leaves as these genes are highly and specifically expressedin leaves Moreover ThLEA-4 has very high expression levelin the roots stems and leaves suggesting that it might playimportant roles in all of these tissues The expression levelsof ThLEA-5 -6 -8 and -12 were very low in each of thetissues examined indicating that these genes may play arelatively unimportant role in these tissues under normalgrowth conditions

There is increasing evidence suggesting that LEA genesare associated with abiotic stress tolerance particularly inplant responses to dehydration salt and cold stresses [18 29]In Arabidopsis of the 22 genes highly expressed in nonseedtissues 12 were induced by more than 3-fold in response tocold drought and salt stresses [27] In sweet potato plantsIbLEA14 expressionwas strongly induced by dehydration andNaCl [30] In the present study half of the 12 ThLEA genes

(ThLEA-1 -2 -3 -4 -7 and -11) were induced under saltand drought stress indicating that these genes might playimportant roles in response to salt and drought stresses in Thispida Bies-Etheve et al [31] reported that LEA genes fromthe same group do not show identical expression profilesand regulation of LEA genes that show apparently similarexpression patterns does not systematically involve the sameregulatory pathway Consistent with this the six upregulatedThLEAs in T hispida belong to two LEAs groups (LEA-2and LEA-4) while LEAs in the same group (such as LEA-4)showed completely different expression patterns

There are ABA-dependent and ABA-independent regula-tory networks controlling plant responses to abiotic stressesLEA genes are often considered as being ABA-regulated andprevious studies have shown that some ThLEA genes canbe induced by exogenous ABA [10 30] However our studyshows that someThLEA genes includingThLEA-1 -2 and -3were highly induced by abiotic stresses but were not regulatedby ABA Consistent with this Bies-Etheve et al [31] reportedthat most of the LEA genes in Arabidopsis seedlings showno response at all following exogenous ABA treatment Hun-dertmark and Hincha [27] deduced the presence of differentsignal transduction pathways in different Arabidopsis tissues(vegetative plants and seeds) by comparing the expression of

The Scientific World Journal 7

minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus15

minus10

minus5

0

5

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus15minus12minus9minus6minus3

036

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 4 Expression analysis of the 12ThLEAs in response to 100 120583MABA treatment Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

LEA genes after ABA treatment In contrast we found thatexpression patterns were highly similar in the three tissuesexamined after ABA treatment Expression analyses showedthat nine ThLEA genes in three tissues of T hispida wereupregulated or downregulated by ABA which suggests thatthese ThLEA genes are regulated by ABA-dependent stressresistance pathways These findings demonstrate that theThLEA genes involved in salt and drought stress resistancein T hispida are probably regulated by two different pathways(ABA-dependent andABA-independent) Further studies arerequired to elucidate the functions of theThLEAs in responseto the abiotic stress and to fully characterize the signalingpathways that regulate their expression

5 Conclusion

In summary we identified 12 ThLEA genes from T hispidawhich belong to three groups The LEA-1 group includesThLEA-2 -4 -9 -10 -11 and -12 proteins the LEA-2 groupcontains ThLEA-1 ThLEA-3 ThLEA-6 and ThLEA-7 andThLEA-8 and -5 are in the Atm and LEA-5 groups TheThLEA genes displayed tissue-specific expression patternsunder normal growth conditionsThLEA-10 -11 and -12were

expressed mainly in the roots and seeds ThLEA-2 and -9were preferentially expressed in the stems and seeds whileThLEA-1 -3 and -7 showed high transcript level in the leavesFurthermore real-time RT-PCR analysis showed that theThLEA genes were regulated by salt and drought stressesThLEA-1 -2 -3 -4 -7 and -11 were mainly induced by saltand drought stress ButThLEA-5 -6 -8 -9 -10 and -12 werelargely downregulated during the NaCl and PEG treatmentAfter the application of exogenous ABA the expression levelsof all ThLEA genes (except for the ThLEA-1 -2 and -3) wereobviously different under the ABA treatment Our studieswill contribute to a better understanding of the functions ofThLEA genes involved in stress response in T hispida

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Caiqiu Gao and Yali Liu have the same contribution to thispaper

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 7

minus8minus6minus4minus2

0246

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(a)

minus15

minus10

minus5

0

5

10

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(b)

minus15minus12minus9minus6minus3

036

Relat

ive e

xpre

ssio

n le

vel

ThLE

A-1

ThLE

A-12

ThLE

A-11

ThLE

A-10

ThLE

A-9

ThLE

A-8

ThLE

A-7

ThLE

A-6

ThLE

A-5

ThLE

A-4

ThLE

A-3

ThLE

A-2

Genes3h6h9h

12h24h

(c)

Figure 4 Expression analysis of the 12ThLEAs in response to 100 120583MABA treatment Relative expression level = transcript level under stresstreatmenttranscript level under control conditions All relative expression levels were log

2transformed and error bars (SD) were obtained

from multiple replicates of the real-time RT-PCR (a) (b) (c) expression ofThLEAs in roots stems and leaves respectively

LEA genes after ABA treatment In contrast we found thatexpression patterns were highly similar in the three tissuesexamined after ABA treatment Expression analyses showedthat nine ThLEA genes in three tissues of T hispida wereupregulated or downregulated by ABA which suggests thatthese ThLEA genes are regulated by ABA-dependent stressresistance pathways These findings demonstrate that theThLEA genes involved in salt and drought stress resistancein T hispida are probably regulated by two different pathways(ABA-dependent andABA-independent) Further studies arerequired to elucidate the functions of theThLEAs in responseto the abiotic stress and to fully characterize the signalingpathways that regulate their expression

5 Conclusion

In summary we identified 12 ThLEA genes from T hispidawhich belong to three groups The LEA-1 group includesThLEA-2 -4 -9 -10 -11 and -12 proteins the LEA-2 groupcontains ThLEA-1 ThLEA-3 ThLEA-6 and ThLEA-7 andThLEA-8 and -5 are in the Atm and LEA-5 groups TheThLEA genes displayed tissue-specific expression patternsunder normal growth conditionsThLEA-10 -11 and -12were

expressed mainly in the roots and seeds ThLEA-2 and -9were preferentially expressed in the stems and seeds whileThLEA-1 -3 and -7 showed high transcript level in the leavesFurthermore real-time RT-PCR analysis showed that theThLEA genes were regulated by salt and drought stressesThLEA-1 -2 -3 -4 -7 and -11 were mainly induced by saltand drought stress ButThLEA-5 -6 -8 -9 -10 and -12 werelargely downregulated during the NaCl and PEG treatmentAfter the application of exogenous ABA the expression levelsof all ThLEA genes (except for the ThLEA-1 -2 and -3) wereobviously different under the ABA treatment Our studieswill contribute to a better understanding of the functions ofThLEA genes involved in stress response in T hispida

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Caiqiu Gao and Yali Liu have the same contribution to thispaper

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 The Scientific World Journal

Acknowledgments

This work was supported by Key Laboratory of Forest Genet-ics amp Biotechnology (Nanjing Forestry University) Ministryof Education (FGB200902) andThe Program for Young Top-Notch Talents of Northeast Forestry University (PYTT-1213-09)

References

[1] H B Shao L Y Chu M A Shao C A Jaleel and M Hong-mei ldquoHigher plant antioxidants and redox signaling underenvironmental stressesrdquo Comptes RendusmdashBiologies vol 331no 6 pp 433ndash441 2008

[2] H Shao L Chu C A Jaleel and C Zhao ldquoWater-deficitstress-induced anatomical changes in higher plantsrdquo ComptesRendusmdashBiologies vol 331 no 3 pp 215ndash225 2008

[3] F T Ni L Y Chu H B Shao and Z H Liu ldquoGene expressionand regulation of higher plants under soil water stressrdquo CurrentGenomics vol 10 no 4 pp 269ndash280 2009

[4] H Shao L Chu C A Jaleel P Manivannan R Panneer-selvam and M Shao ldquoUnderstanding water deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and sustainably improving agriculture andthe ecoenvironment in arid regions of the globerdquo CriticalReviews in Biotechnology vol 29 no 2 pp 131ndash151 2009

[5] H Cao Z Zhang P Xu et al ldquoMutual physiological geneticmechanism of plant high water use efficiency and nutrition useefficiencyrdquo Colloids and Surfaces B Biointerfaces vol 57 no 1pp 1ndash7 2007

[6] E Mazzucotelli A M Mastrangelo C Crosatti D Guerra AM Stanca and L Cattivelli ldquoAbiotic stress response in plantsWhen post-transcriptional and post-translational regulationscontrol transcriptionrdquo Plant Science vol 174 no 4 pp 420ndash4312008

[7] A Nezhadahmadi Z H Prodhan and G Faruq ldquoDroughttolerance in wheatrdquo The Scientific World Journal vol 2013Article ID 610721 12 pages 2013

[8] L Dure III S C Greenway and G A Galau ldquoDevelopmentalbiochemistry of cottonseed embryogenesis and germinationchanging messenger ribonucleic acid populations as shown byin vitro and in vivo protein synthesisrdquo Biochemistry vol 20 no14 pp 4162ndash4168 1981

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

[10] J Duan and W Cai ldquoOsLEA3-2 an abiotic stress induced geneof rice plays a key role in salt and drought tolerancerdquo PLoSONEvol 7 no 9 Article ID e45117 2012

[11] Y Zhang Y Li J Lai et al ldquoEctopic expression of a LEAprotein gene TsLEA1 fromThellungiella salsuginea confers salt-tolerance in yeast and ArabidopsisrdquoMolecular Biology Reportsvol 39 no 4 pp 4627ndash4633 2012

[12] Y Olvera-Carrillo F Campos J L Reyes A Garciarrubio andA A Covarrubias ldquoFunctional analysis of the group 4 lateembryogenesis abundant proteins reveals their relevance in theadaptive response during water deficit in arabidopsisrdquo PlantPhysiology vol 154 no 1 pp 373ndash390 2010

[13] S Cui J Hu S Guo et al ldquoProteome analysis of Physcomitrellapatens exposed to progressive dehydration and rehydrationrdquoJournal of Experimental Botany vol 63 no 2 pp 711ndash726 2012

[14] D Naot G Ben-Hayyim Y Eshdat and D Holland ldquoDroughtheat and salt stress induce the expression of a citrus homologueof an atypical late-embryogenesis of Lea5 generdquo PlantMolecularBiology vol 27 no 3 pp 619ndash622 1995

[15] N Ukaji C Kuwabara D Takezawa K Arakawa and SFujikawa ldquoCold acclimation-induced WAP27 localized inendoplasmic reticulum in cortical parenchyma cells of mul-berry tree was homologous to group 3 late-embryogenesisabundant proteinsrdquo Plant Physiology vol 126 no 4 pp 1588ndash1597 2001

[16] C NDong J Danyluk K E Wilson T Pocock N P A Hunerand F Sarhan ldquoCold-regulated cereal chloroplast late embryo-genesis abundant-like proteins Molecular characterization andfunctional analysesrdquo Plant Physiology vol 129 no 3 pp 1368ndash1381 2002

[17] H Zegzouti B Jones C Marty et al ldquoER5 a tomato cDNAencoding an ethylene-responsive LEA-like protein charac-terization and expression in response to drought ABA andwoundingrdquo Plant Molecular Biology vol 35 no 6 pp 847ndash8541997

[18] M Dalal D Tayal V Chinnusamy and K C Bansal ldquoAbioticstress and ABA-inducible Group 4 LEA from Brassica napusplays a key role in salt and drought tolerancerdquo Journal ofBiotechnology vol 139 no 2 pp 137ndash145 2009

[19] A RoyChoudhury C Roy and D N Sengupta ldquoTransgenictobacco plants overexpressing the heterologous lea geneRab16Afrom rice during high salt and water deficit display enhancedtolerance to salinity stressrdquo Plant Cell Reports vol 26 no 10pp 1839ndash1859 2007

[20] Y Bai Q Yang J Kang Y Sun M Gruber and Y ChaoldquoIsolation and functional characterization of a Medicago sativaL geneMsLEA3-1rdquoMolecular Biology Reports vol 39 no 3 pp2883ndash2892 2012

[21] B J Park Z Liu A Kanno andT Kameya ldquoIncreased toleranceto salt- and water-deficit stress in transgenic lettuce (Lactucasativa L) by constitutive expression of LEArdquo Plant GrowthRegulation vol 45 no 2 pp 165ndash171 2005

[22] X Zhao L Zhan and X Zou ldquoImprovement of cold toleranceof the half-high bush Northland blueberry by transformationwith the LEA gene from Tamarix androssowiirdquo Plant GrowthRegulation vol 63 no 1 pp 13ndash22 2011

[23] P Zhao F Liu M Ma et al ldquoOverexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli andprovides enhanced osmotic stress tolerance andABA sensitivityin Arabidopsis thalianardquo Molekuliarnaia Biologiia vol 45 no5 pp 851ndash862 2011

[24] C Wang C Gao L Wang L Zheng C Yang and Y WangldquoComprehensive transcriptional profiling of NaHCO

3-stressed

Tamarix hispida roots reveals networks of responsive genesrdquoPlant Molecular Biology vol 84 no 1-2 pp 145ndash157 2014

[25] L Wang C Wang D Wang and Y Wang ldquoMolecular charac-terization and transcript profiling of NAC genes in response toabiotic stress in Tamarix hispidardquo Tree Genetics amp Genomes vol10 pp 157ndash171 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

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

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

The Scientific World Journal 9

[28] I A Paponov M Paponov W Teale et al ldquoComprehensivetranscriptome analysis of auxin responses in ArabidopsisrdquoMolecular Plant vol 1 no 2 pp 321ndash337 2008

[29] J Boudet J Buitink F A Hoekstra et al ldquoComparative analysisof the heat stable proteome of radicles of Medicago trun-catula seeds during germination identifies late embryogenesisabundant proteins associated with desiccation tolerancerdquo PlantPhysiology vol 140 no 4 pp 1418ndash1436 2006

[30] S Park Y Kim J C Jeong et al ldquoSweetpotato late embryoge-nesis abundant 14 (IbLEA14) gene influences lignification andincreases osmotic- and salt stress-tolerance of transgenic callirdquoPlanta vol 233 no 3 pp 621ndash634 2011

[31] 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

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology