A MITE insertion into the 3′-UTR regulates the transcription of TaHSP16.9 in common wheat

*

aCollege of Resources and Environment Science,Pingdingshan University,Pingdingshan 467000,China

bState Key Laboratory for Agrobiotechnology/Department of Plant Genetics&Breeding,China Agricultural University,Beijing 100193,China

A MITE insertion into the 3′-UTR regulates the transcription of TaHSP16.9 in common wheat

JingtingLia,b,ZhenzhongWangb,HuiruPengb,ZhiyongLiub,*

aCollege of Resources and Environment Science,Pingdingshan University,Pingdingshan 467000,China

bState Key Laboratory for Agrobiotechnology/Department of Plant Genetics&Breeding,China Agricultural University,Beijing 100193,China

A R T I C L E I N F O

Article history:

Received 18 June 2014

Received in revised form 22 July 2014 Accepted 29 July 2014

Available online 22 August 2014

MITE

sHSP

3′-UTR

Gene transcription

Common wheat

Miniature inverted-repeat transposable elements(MITEs)are a type of DNA transposon frequently inserted into promoters,untranslated regions(UTR),introns,or coding sequences of genes.We found a 276-bp tourist-like MITE insertion in the 3′-UTR of a 16.9 kDa small heat shock protein gene(TaHSP16.9-3A)on chromosome 3A of common wheat.Haplotype analysis revealed two haplotypes,sHSP-W(wild type without MITE insertion)and sHSP-M(mutantwith MITE insertion),present in wheat germplasm.Both semiquantitative PCR and quantitative real-time PCR analyses showed increased transcription levels of TaHSP16.9-3A in sHSP-M compared with those of sHSP-W after heat treatment at 42°C.It appeared that the MITE insertion into the 3′-UTR enhances the transcription of TaHSP16.9-3A.

?2014 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.Allrights reserved.

1.Introduction

All living organisms can be induced to produce heat-shock proteins(HSPs)in response to heat stress.However,plant HSPs are more complex than HSPs in other organisms.Plant HSPs can be classified into five major classes:HSP60,HSP70, HSP90,HSP100,and small heat shock proteins(sHSPs)[1]. Plant sHSPs can also be divided into six classes according to their cellular localization and similarity.Three classes(CI,CII, and CIII)are present in the cytosol or in the nucleus,and the other three are present in the plastid,endoplasmic reticulum, and mitochondria,respectively[1–4].Ta HSP16.9 in wheat belongs to class Iand is the first eukaryotic s HSP for which a high-resolution structure has been described[5].In vitro analysis of the chaperone activity of Ta HSP16.9 has indicated substrate specificity for sHSP[6].In plants,abundant sHSPs are induced to be synthesized in response to environmental stresses and developmentalstimuli,whereas most sHSPs are restricted to low expression levels at certain development stages under normal environmental conditions,indicating that sHSPs play an important role in stress tolerance[2,3].It has been suggested that sHSPs act as molecular chaperones that selectively bind non-native proteins to prevent aggregation in an ATP-independent manner[3].

Transposable genetic elements(TEs)are repeated DNA sequences that are able to move from one site in the genome to another and replicate during mobilization.TEs are essential components of many eukaryotic genomes and may play important roles in size,structure,polymorphism,genome evolution,and regulation of gene expression[7–9].According to

their mode of transposition,TEs are divided into two classes, retrotransposons and DNA transposons.Retrotransposons transpose indirectly through an RNA intermediate and consist of two principal groups,long terminal repeat(LTR)and non-LTR retrotransposons.DNA transposons transpose directly as DNA that is excised from the original site in the genome and inserted into a new site.DNA transposons may also be divided into two classes.Autonomous DNA transposons contain genes encoding transposases that mediate DNA transposition,such as Ac transposons in maize.Although nonautonomous DNA transposons lack genes encoding transposases,they can transpose by mediation of transposases encoded by autonomous transposons such as the Ds transposons in maize[7,8,10].

Miniature inverted-repeat transposable elements(MITEs) are a particular class of DNA transposons that have typical structures of non-autonomous DNA transposons,containing target site duplications(TSD)and terminal inverted repeats (TIR)[10,11].In plant,MITEs are divided into tourist-like, stowaway-like and pogo-like groups,according to the similarity of their terminal inverted repeat and target site duplication sequences[11–13].However,MITEs differ from classic DNA transposons by their small size(usually less than 500 bp),large copy number(usually hundreds or thousands),and consistency of related elements,which are the features ofretrotransposons. The small size and large copy number of MITEs lead to their frequent insertion into promoters,untranslated regions,introns,or coding sequences ofplantgenes[14–18].The proximity between MITEs and adjacent genes promotes the hypothesis that MITEs play an important role in regulating gene expression.

Researchers continue to investigate the function of MITEs in gene regulation.Earlier studies discovered two types of rice ubiquitin2(rubq2)promoter in rice lines,with two nested MITEs(Kiddo and MDM1)inserted in IR24 and only MDM1 inserted in T309[15].The insertion of Kiddo increased the transcription rate of rubq2 in rice,but methylation of Kiddo neutralized this enhancement effect[19].MITE insertion into the coding region of an oleoyl-PC desaturase gene(ahFAD2B) resulted in a premature stop codon with a putatively truncated protein,leading to a reduced transcript level of ahFAD2B and high oleate content[18].A MITE-like insertion close to the start codon of the water-stress tolerance gene Hsdr4 in barley regulated the transcription of Hsdr4 by forming a hairpin-like secondary structure[20].The size of a repeated structure harboring a tourist-like MITE insertion in the upstream region of the SbMATE gene(multidrug and toxic compound extrusion)positively corresponded with aluminum tolerance in sorghum.The results suggested that the MITEs act as cis-acting elements to multiplicatively enhance the expression of SbMATE,explaining the positive correlation between the repeat structure and aluminumtolerance[21].Recent research in the Solanaceae showed that MITEs generated small RNA by a TE-derived siRNA pathway as described in Arabidopsis,and supported the hypothesis that a MITE-derived siRNA targeted the gene with MITE insertion in post-transcriptional silencing pathway[22].In rapeseed(Brassica napus L.),haplotype analysis revealed a tourist-like MITE insertion/deletion polymorphismin the upstream region of BnFLC.A10 distinguishing most winter types(insertion)from spring types (deletion).The polymorphism was positively associated with the difference in BnFLC.A10 expression between Tapidor(insertion) and Ningyou 7(deletion).Association analysis among two types of rapeseed showed that the MITE insertion was significantly associated with vernalization requirement[23].

Common wheat(Triticum aestivum L.)is one of the most important food crops.Owing to its large genome size,hexaploidy,and highly repetitive DNA sequence,regulation by MITEs of wheat gene expression has been poorly investigated.Here we report the identification and transcription regulation effects ofa 276-bp tourist-like MITE insertion into the 3′-UTR of TaHSP16.9, a 16.9-kDa smallheat shock protein in common wheat.

2.Materials and methods

2.1.Plant materials

Two hexaploid bread wheat genotypes,the heat-tolerantcultivar TAM107 and the heat-susceptible landrace Chinese Spring[24], were used as materials to clone the promoter,5′-UTR,coding region,and 3′-UTR of TaHSP16.9-3A.Also,17 common wheat cultivars and 23 Chinese landraces were selected for haplotype analysis of the MITE insertion(Table 1).Chinese Spring and its nullisomic-tetrasomics and ditelosomics were kindly providedby Drs.W.J.Raupp and B.S.Gill,Wheat Genetics Resource Center, Kansas State University,USA.

2.2.Hightemperaturestresstreatments

Seeds were surface-sterilized with 1%sodium hypochlorite for 30 min,rinsed with distilled water,and soaked in the dark overnight at roomtemperature.After germination,seeds were planted in flasks(10 seedlings per flask)containing 1%agar culture medium and grown in a climate-controlled incubator (16 h day/8 h night,22°C day/18°C night,60%humidity)[24]. Ten days later,seedlings were transferred to another incubator(16 h day/8 h night,42°C day/18°C night,80%humidity) for heat treatment at 42°C for 0.5 h,1 h,2 h,and 3 h.A total of30 seedlings in three flasks were subjected to each treatment, and three independentbiologicalreplications were prepared.At the end of each treatment,leaf samples from three flasks were immediately frozen in liquid nitrogen and stored at-80°C until total RNA extraction.

2.3.DNAextractionandDNAamplification

Genomic DNA was extracted from 10-day-old seedling leaves by the CTAB method[25].DNA amplification reactions were performed in a 20-μL volume containing 100 ng DNA,0.2 mmol L-1dNTPs,0.3μmol L-1of each primer,1 U Taq polymerase,and 1× assay buffer.The amplification parameters were as follows: 94°C for 5 min;40 cycles of 94°C for 45 s,53–60°C(depending on primers used)for 45 s,72°C for 1 min;and 72°C for 10 min. The PCR products were checked on 8%nondenaturing polyacrylamide gels or 2%agarose gels.

2.4.Genomewalking

Genomic DNA extracted from wheat genotypes TAM107 and CS was purified with phenol/chloroform after RNase (TaKaRa,Japan)treatment at 37°C for 1 h.DNA samples were evaluated by electrophoresis on a 1%agarose gel inTris-acetate ethylenediaminetetraacetic acid(TAE)buffer containing ethidiumbromide(0.5μg mL-1)for DNAintegrity and quantified by spectrophotometry.A genome walker library was constructed using the Genome Walker Universal Kit(Clontech, Japan)according to the manufacturer's protocol.The cycling conditions were as follows:94°Cfor 3 min;7 or 5 cycles of 94°C for 25 s,72°Cfor 3 min;32 or 25 cycles of94°Cfor 25 s,67°Cfor 3 min;with a final extension of 67°C for 7 min.

Table 1–Common wheat cultivars and landraces used for MITE haplotype analysis.

2.5.RNAextractionandreversetranscription

Three sHSP-W(without MITE insertion)and three sHSP-M (with MITE insertion)cultivars or landraces were randomly chosen and subjected to heat treatment and total RNA was extracted from 10-day-old seedlings using TRIzol reagent (Tiangen,Beijing)according to the manufacturer's instructions.Total RNA samples were treated with DNase I(Promega, USA)at 37°C for 1 h and subjected to phenol/chloroform extraction to eliminate residual DNA.RNA was precipitated with 0.3 mol L-1NaAc(pH 5.2)and two volumes ofethanol,collected by centrifugation(12,000×g,15 min at 4°C).RNA concentration was determined by measuring absorbance at 260 nm.Firststrand cDNA was synthesized from 2μg of total RNA using (dT)18primer with MMLV reverse transcriptase(Promega,USA) following the manufacturer's instructions.The reverse transcription reaction conditions were as follows:37°C for 60 min, 95°C for 5 min.The product of reverse transcription was examined by amplifying Tabeta-actin(300 bp),which was used as the endogenous control in semi-quantitative PCR.

2.6.Quantitativereal-timePCR

Quantitative real-time PCR was performed on a CFX96 realtime system(Bio-Rad,USA)using SYBR premix Ex Taq mixture (TaKaRa,Japan)with cycle conditions as follows:95°C for 5 min;38 cycles of 95°C for 10 s,56°C for 10 s,72°C for 20 s; melt curve from 65°C to 90°C,increment 0.3°C for 0.03 s, plate read.Allreactions were run in triplicate and included no template and no reverse transcription controls.Quantification results were expressed in terms of the cycle threshold(CT) value according to the baseline adjusted to 0.04.The comparative CT method(PE Applied Biosystems,USA)was used to quantify relative gene expression compared with Tabeta-actin (120 bp).Briefly,the CT values were averaged for each triplicate.Differences between the mean CTvalues of specific genes and those of Tabeta-actin were calculated as=In the final results,the relative expression levels were determined by the 2–ΔCtmethod.Statistical significance was tested using Student's t test(P＜0.05)[25].Each PCR product was evaluated in at least two independent experiments.The primers used for semiquantitative PCR and quantitative real-time PCR are listed in Table 2.

3.Results

3.1.IdentificationofaMITEinsertioninthe3′-UTRofTaHSP16.9incommonwheat

A 276-bp tourist-like MITE insertion was identified in the 3′-UTR of a small heat shock protein gene,TaHSP16.9,in wheat during annotation of the TaHSP16.9 sequence from a public database.Two haplotypes,sHSP-W(wild type without MITE insertion)and sHSP-M(mutant with MITE insertion), were found in public NCBI wheat EST data sets and then were confirmed in wheat germplasm by specific primers (CJD/MITER1 and GSP3/MIDE)flanking the MITE(Table 2; Fig.1).A set of common wheat cultivars and landraces werethen selected for MITE insertion polymorphism analysis.The sHSP-Mhaplotype was detected in 5 of 23 wheat landraces and 10 of17 wheatcultivars.Interestingly,the MITE insertion was present in a heat-tolerant wheat cultivar,TAM107,but absent from a heat-sensitive wheat landrace,Chinese Spring (Table 1).

Table 2–Primers used for semiquantitative PCR,quantitative real-time PCR,chromosome assignment,and haplotype analysis.

The chromosomal location of TaHSP16.9 was determined using nullitetrasomic and ditelosomic lines of Chinese Spring. TaHSP16.9 was assigned to chromosome 3AL(Fig.2).

3.2.GenomicstructureofTaHSP16.9-3AbetweenTAM107 andChineseSpring

The promoter,5′-UTR,coding sequences,and 3′-UTR of Ta HSP16.9-3A were amplified from genomic DNAof TAM107 and Chinese Spring by genome walking.TaHSP16.9-3A is a single-exon gene without introns.Two SNPs(G/C and A/G) in the coding region,27 SNPs in the promoter region,and a 276-bp MITE insertion and deletion(InDel)in the 3′-UTR were observed in the TaHSP16.9-3A between TAM107 and Chinese Spring.The MITE was located 142 bp downstream of the stop codon(TGA)and upstream of the polyadenylation termination sequence(AATAAA)signal site in the 3′-UTR with typical tourist-like structure of TAA as target site duplication (TSD)and 5′-GGCTGCTCATAGTGG-3′as terminal inverted repeat(TIR)(Fig.1).

The promoter regions of TaHSP16.9-3A between TAM107 and Chinese Spring were scanned in the PlantCARE web site(http://bioinformatics.psb.ugent.be/)to predict putative regulatory motifs,some of which were stress responsive.No significant differences in stress-response elements were identified between TAM107 and Chinese Spring(data not shown).The G/C and A/G SNPs in the coding regions resulted in two amino acid polymorphisms(N/K;E/K).The N/K amino acid polymorphisms were located in theβ-strand 5 within the conservedα-crystallin domain of Ta HSP16.9[5].

3.3.ExpressionpatternofTaHSP16.9-3Aunderhightemperaturestress

Semiquantitative PCR and quantitative real-time PCR analysis showed that TaHSP16.9-3A was expressed in a heat-inducible fashion and that the expression increased significantly in both TAM107 and Chinese Spring with increasing seedling exposure time to high temperature stress(42°C).However, the expression levels were higher in TAM107 than in Chinese Spring at each time point.No TaHSP16.9-3A expression was detected under the controlcondition(22°C day/18°Cnight)in either TAM107 or Chinese Spring(Fig.3).

To test whether the regulatory effects of MITE insertion in the 3′-UTR on TaHSP16.9-3A were universal,semiquantitative PCR and quantitative real-time PCR analysis were performed to evaluate the transcription of TaHSP16.9-3A in 6 Chinese wheat landraces under 42°C heat-treatment for 2 h.The results revealed an increased transcript level of TaHSP16.9-3A in the sHSP-M haplotype(Huangmaizi,Huangxiaomai,and Jiyumai)compared with the sHSP-W haplotype(Chinese Spring,Jianermai,Jianmai)(Fig.4).

Fig.1–Structure comparisons of the TaHSP16.9-3A gene between Chinese Spring and TAM107.

Fig.2–Chromosome arm assessment of TasHSP16.9 in Chinese Spring homoeologous group 3 nullisomic–tetrasomics and ditelosomics.M:marker;CS:Chinese Spring;1:N3A-T3B;2:N3B-T3A;3:N3B-T3D;4:N3D-T3A;5:N3D-T3B;6:Dt3AS;7:Dt3AL;8: Dt3BL;9:Dt3DS;10:DT3DL;11:ddH2O.

4.Discussion

Wheat is one of the most important food crops,and is grown under various environmental conditions worldwide.High temperature is one of its key limiting factors,causing wheat yield loss and reducing wheat quality.Plants are induced to synthesize abundant low molecular weight HSPs(sHSPs)in response to heat stress,suggesting that plant HSPs play a role in tolerance to heat shock.

In the present study,a miniature inverted-repeat transposable element(MITE)insertion in the 3′-UTR of TaHSP16.9-3A resulted in two haplotypes(sHSP-Wand sHSP-M)in wheat germplasm.An increased transcript level of TasHSP16.9-3A was detected in sHSP-M haplotypes compared with sHSP-W haplotypes under high-temperature stress.Comparative analysis of the promoter sequences revealed no recognizable cisacting stress-response elements differing between TAM107 and Chinese Spring.The nucleotide diversity in the promoter sequence may not contribute to the increased transcript level of TaHSP16.9-3A under heat stress treatment(Fig.S1).The G/C and A/G SNPs in the coding region resulted in two amino acid polymorphisms(Fig.S2).The N/K and E/K amino acid polymorphisms may influence the 3-D structure of the sHSP protein,but are unlikely to influence the transcription level.It appeared that the MITE insertion into the 3′-UTR could be a key factor enhancing the transcription of TaHSP16.9-3A.

Previous studies showed that increased HSP gene expression was positively correlated with genetic differences in cellular thermal tolerance in winter wheat.Earlier synthesized and higher levels of sHSP mRNAs were found in the heat-tolerant wheat cultivar Mustang than in the heatsusceptible wheat cultivar Sturdy under heat stress[26,27]. We propose that MITE insertion into the 3′-UTRof TaHSP16.9-3A improves the heat tolerance of wheat genotypes by increasing the transcription of TaHSP16.9-3A under heat stress.Association analysis of heat tolerance with the MITE insertion using a diversity panelof wheat germplasm and over-expression of the TaHSP16.9-3A in heat-sensitive wheat varieties should allow testing this hypothesis.

Fig.3–The TaHSP16.9-3A transcription pattern of TAM107 and Chinese Spring under 42°C heat treatment for different time intervals(0 h,0.5 h,1 h,2 h,3 h)detected by semiquantitative PCR(a)and quantitative real-time PCR(b).

Fig.4–The TaHSP16.9-3A transcription pattern in Chinese wheat landraces detected by semiquantitative PCR(a)and quantitative real-time PCR(b).1:Huangmaizi;2:Huangxiaomai;3:Jiyumai;4:Chinese Spring;5:Jianermai;6:Jianmai.Heat treatment was at 42°C for 2 h.MITE insertions were present in the 3′-UTRs of Huangmaizi,Huangxiaomai,and Jiyumai but absent from Chinese Spring(CS),Jianermai,and Jianmai.

The 3′-UTR could play an important role in gene regulation by controlling mRNA stability and translation efficiency.The 3′-UTR may act as a binding site of trans-acting factors such as proteins and miRNA.A recent study showed that the binding of miRNA23b to the 3′-UTR of mouseμ-opioid receptor(MOR1) suppressed MOR1 mRNA translation by inhibiting mRNA interaction with polysomes[28].Detailed analysis of SAURAC1 expression in Arabidopsis showed that the 3′-UTRacted as an mRNA instability determinant[29].The highly conserved DST(downstream element)sequence in the 3′-UTR may contribute to the mRNA instability and mediate mRNA decay[30].The mRNAs of heat shock genes have structures like the regions in the 3′-UTR that allow their selective translation in stressed cells by increasing mRNA stability[31].Studies have suggested that the abundance of gene expression is often regulated at the posttranscriptional levellike mRNA degradation,which is associated with mRNA stability[29,30].In this study,our results suggested that the MITE insertion enhanced the transcription of TaHSP16.9-3A mRNA in wheat lines containing MITE insertion in the 3′-UTR.Further analysis is needed to determine whether the MITE insertion improved the stability of TaHSP16.9-3A mRNA.

In plants,recent studies have suggested that MITEs contribute to genome structure and gene regulation[16,17,32–34].TEs are usually silenced by methylation,but they may affect the expression of adjacent genes under certain stresses.In other words,various environmental stresses are associated with the activation of TEs.The contribution of the MITE(Kiddo)insertion in the promoter to the enhancement of rubq2 promoter activity in rice was neutralized by methylation of Kiddo[19].The DNA transposon mPing acts as an enhancer to up-regulate the expression ofnearby genes under cold stress regardless of the site of TE insertion in rice[16].In sorghum,MITEs may act as cis-acting elements to multiplicatively enhance expression of SbMATE to induce aluminum tolerance[21].In this study,we speculate that either heat stress activates the MITE to enhance the expression of TaHSP16.9-3A,or the MITE acts as a cis-acting element interacting with trans-acting elements to enhance the expression of TaHSP16.9-3A in wheatlines(sHSP-M),resulting in heat tolerance.

5.Conclusions

Annotation of TaHSP16.9 sequence from a public database revealed the presence of a tourist-like MITE insertion in the 3′-UTR of TaHSP16.9 on chromosome 3A in wheat.Haplotype analysis of a set of common wheat cultivars and landraces revealed two haplotypes,sHSP-Wand sHSP-M.Transcription analysis suggested that MITE insertion is the key factor up-regulating the expression of TaHSP16.9-3A in the sHSP-M haplotype after heat treatment at 42°C,indicating a possible role of the MITE in gene regulation in wheat.

Acknowledgments

This work was supported by the China Agricultural University Graduate Research and Innovation Project(kycx09019).

Supplementary material

Supplementary material to this article can be found online at http://dx.doi.org/10.1016/j.cj.2014.07.001.

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*Corresponding author.

E-mail address:zhiyongliu@cau.edu.cn(Z.Liu).

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

http://dx.doi.org/10.1016/j.cj.2014.07.001

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