Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat（Triticum aestivum L.）

Xinguo Wang，Yanli Wang，Ruixia Xiao，Xin Chen，Jiangping Ren*National Wheat Engineering Research Center，Agricultural University of Henan，Zhengzhou 450002，China

?

Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat（Triticum aestivum L.）

Xinguo Wang，Yanli Wang，Ruixia Xiao，Xin Chen，Jiangping Ren*
National Wheat Engineering Research Center，Agricultural University of Henan，Zhengzhou 450002，China

A R T I C L E I N F OA B S T R A C T

Article history：

Received in revised form

3 March 2016

Accepted 15 March 2016

Available online 30 March 2016

The purpose of this study was to characterize Ta14S homoeologs and assess their functions inwheatseeddevelopment.ThegenomicandcDNAsequencesofthreeTa14S homoeologous genes encoding 14-3-3 proteins were isolated.Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure，whereas the cDNA sequences were variable in the 5′and 3′-UTR.The three genes，designated as Ta14S-2A，Ta14S-2B and Ta14S-2D，were located in homoeologous group 2 chromosomes.The polypeptide chains of the three Ta14S genes were highly similar.These genes were most homologous to Hv14A from barley.Real-time quantitative PCR indicated that the three Ta14S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues. Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated.The transcription of Ta14S-2B was clearly higher during seed germination，whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition（0-12 h），but declined thereafter.The results suggested that the three Ta14S homoeologous genes have regulatory roles in seed development and germination.

?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license

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

Common wheat

Gene expression

Homoeologous genes

Developmental regulation

1.Introduction

The 14-3-3 proteins are a family of highly conserved regulatory proteins found in virtually all eukaryotes.The N-and C-ends of these proteins are highly variable whereas the core structures are highly conserved［1］.These molecules are small acidic soluble proteins with a molecular mass of approximately 30 kDa.They usually form homo-and hetero-dimers to interact withdiversetargetproteinsbyspecificphosphoserine/ phosphothreonine-binding activity［2，3］.To date，there are over three hundred proteins identified as their interacting proteins ［4，5］，and the outcomes of binding are diverse，including alteration in conformation，subcellular localization and stabilization of the interacting proteins.They also mediate formationof protein complexes［6-9］.The physiological function of these proteins in plants has been the focus of considerable research. Various 14-3-3 isoforms have been isolated from diverse species including fifteen known protein isoforms in Arabidopsis［10］，eight in rice［11］，five in barley［12］and 7 potential isoforms in wheat［13-17］.Plant 14-3-3 proteins can be divided into two classes，the epsilon and the non-epsilon groups，based on sequence similarity and phylogenetic analyses of sequences obtained from Arabidopsis［3］.Functional analysis revealed that increased or decreased expression of 14-3-3 protein genes caused a number of phenotypic，developmental and stress tolerance changes.For example，in rice plants transformed with an over-expression construct of the maize 14-3-3 protein gene，GF14-6，tolerance to drought stress and response to pathogen infection were changed［18］.When overexpressed in Arabidopsis plants wheat 14-3-3 protein caused shorter primary roots，delayed flowering and retarded growth rates［16］.The overexpression of cotton 14-3-3 protein Gh14-3-3 L promoted fiber elongation，leading to an increase in mature fiber length.By contrast，the suppression of the expression of Gh14-3-3 L，Gh14-3-3 e and Gh14-3-3 h in cotton slowed down fiber initiation and elongation［19］.These results indicated that some 14-3-3 protein-coding geneshave roles in plant development and stress response.

Wheat is a globally important crop，accounting for 20%of the calories consumed by humans［20］.Research that focuses on mechanisms of developmental regulation at the molecular level has potential to accelerate progress of wheat improvement.Although 14-3-3 proteins are increasingly implicated as key factors in developmental regulation［21-24］，little is known about such genes in wheat seed development.Because hexaploidwheatcontainsthreesetsofchromosomes，Ta14-3-3 genes are likely to occur as homoeologous triplicates. Previously，we isolated a wheat cDNA designated as Ta14S with an open reading frame encoding a putative 14-3-3 protein［15］.To further clarify a regulatory function in wheat seed development，we isolated the genomic and cDNA sequences of Ta14S homoeologs in hexaploid wheat and located the genes to chromosomes using Chinese Spring nullisomic-tetrasomic lines.In addition，we analyzed their expression patterns in different tissues and at different seed developmental and germination stages.

2.Materials and methods

2.1.Plant sample preparation

Common wheat（Triticum aestivum L.）cultivar（cv.）Luohan 2 was used for gene cloning and expression analysis.After being sterilized，seeds were germinated on moist filter paper in growth chambers at 25°C under 12-h light/12-h dark conditions and transplanted into pots in a naturally lit glasshouse with normal irrigation and fertilization until mature.Dry seed embryos，primary roots and shoots at 1 day post-germination，roots and fully expanded leaves at 10 days post-germination，leaves at the tillering stage，stems at jointing，flag leaves，young panicles at heading，and developing seeds at 5，10，15，20，25，30 and 35 DAP （days after pollination）were sampled.For germination treatment，mature seeds were surface-sterilized and then imbibed water from moist filter paper in Petri dishes in a temperaturecontrolled cultivation chamber（16 h photoperiod at 25°C）. Seeds were collected at 0，6，12，24，36 and 48 h after initiation of imbibition.

All collected plant materials were frozen in liquid nitrogen immediately after collection and stored at-80°C until used. Three biologically independent replicates were assayed to ascertainreproducibility.AsetofChineseSpring（CS）nullisomic-tetrasomic lines，kindly provided by Dr.Xianchun Xia，Chinese Academy of Agricultural Sciences，was used to determine the chromosomal locations of Ta14S.

2.2.DNA extraction，primer design，PCR and sequencing

Genomic DNA was isolated from wheat seeds using a CTAB method［25］.Gene-specific primers were designed based on the sequence of Ta14S using Primer Premier 5.0 software （http://www.premierbiosoft.com/）and synthesized by Beijing LiuheHuadaGeneTechnologyCo.，Ltd.（http://www. bgitechsolutions.cn/）.PCR were performed in a Biametra-T3000 thermal cycler in total volumes of 20 μL，including 2 μL 10×PCR buffer，100 mmol L-1of each of dNTP，5 pmol of each primer，1 unit of Taq DNA polymerase（TIANGEN Biotech Co.，Ltd.，Beijing，http://www.tiangen.com/）and 100 ng of template DNA.Reaction conditions were 95°C for 3 min followed by 35 cycles of 94°C for 30 s，annealing at 55°C for 1 min and 72°C for 1-2 min，with a final extension at 72°C for 10 min. PCR products were separated by electrophoresis in 1.0% agarose gels.Targeted fragments of expected size were recovered and cloned into the pMD18-T vector and sequenced by Beijing Liuhe Huada Gene Technology Co.，Ltd.（http:// www.bgitechsolutions.cn/）.To ensure sequencing accuracy PCR and DNA sequencing were repeated at least three times.

2.3.RNA extraction and first-strand cDNA synthesis

Total RNA extractions from embryos and seeds were carried out using a hot-phenol method［26］and from leaves and roots using TRIzol Reagent（Invitrogen，Shanghai）according to the manufacturer's instructions.Quality and concentrations of total RNA were measured by spectrophotometer（NanoDrop ND-1000，Wilmington，USA）and agarose gel electrophoresis. Equal amounts（2 μg）of total RNA were transcribed into cDNA in a 20 μL reaction system containing 50 mmol L-1Tris-HCl （pH 8.3），75 mmol L-1MgCl2，10 mmol L-1DTT，50 mmol L-1dNTPs，200 U M-MLVreversetranscriptase（Promega，Madison，WI）and 50 pmol Oligo-dT15anchor primer.Reverse transcription was performed for 60 min at 42°C with a final denaturation step at 95°C for 5 min.

2.4.Rapid amplification of cDNA ends（RACE）

mRNAwaspurifiedthrougholigotexchromatography （Clontech，Beijing）from total RNA and 3′-RACE and 5′-RACE were performed using a SMART-RACE cDNA amplification kit （Clontech，Beijing）according to the manufacturer's protocol. Gene-specific primers used for PCR were 3′-GSP（3′-RACE）and 5′-GSP（5′-RACE），respectively（Table 1）.PCR was performed according to the manufacturer's protocol（Clontech）.PCR conditions were 94°C for 4 min，followed by 6 cycles of 94°Cfor 30 s，60°C/58°C for 30 s and 72°C for 3 min，followed by 20 cycles of 94°C for 30 s，56°C for 30 s，72°C for 3 min and finally 72°C for 10 min.PCR products were separated by 1% agarose gel electrophoresis，and subcloned into the pMD18-T vector（TaKaRa，Dalian）and sequenced.

Table1-Primersequencesforgenecloning，RACE，chromosome location and real-time PCR.

2.5.Real-time quantitative RT-PCR

Aliquots of 1 μg of total RNA were used for first-strand cDNA synthesis using a Primecript Reagent Kit with gDNA Eraser （Perfect real-time）according to the manufacturer's instructions（TaKaRa）.Real-time quantitative RT-PCR was performed using an Eppendorf Realplex4 Mastercycler EP Gradient S machine（Eppendorf，Hamburg，Germany）.Homoeologous Ta14S gene-specific primers were used for real-time quantitative PCR analysis.For normalization，the wheat β-actin gene （GenBank accession number:AB181991）was amplified as an endogenous control（Table 1）.

PCR was performed with the SYBR Green quantitative RT-PCR kit（TaKaRa）following the manufacturer's instructions.PCR cycling conditions comprised an initial cycle at 95°C for 30 s，followed by 40 cycles at 95°C for 15 s and at 60°C for 35 s and a final extension of 7 min at 72°C. No-template negative controls（H2O control）were set up using gene-specific primer pairs，and no-RT-PCR controls were set up in duplicate using actin gene primers.Reactions were conducted in triplicate to ensure reproducibility of results.

The cycle threshold（Ct），defined as the PCR cycle at which a statistically significant increase in reporter fluorescence is first detected，was used as a measure of the starting copy number of the target gene.Relative quantities of target gene expression levels were determined using the comparative Ct method.Relative quantification for each target gene was calculated by the 2-ΔΔCtmethod using β-actin as an internal reference gene for comparing data from different PCR runs or cDNA samples［27］.

3.Results

3.1.Isolation of cDNA sequences of wheat Ta14S homoeologs

To isolate the full-length cDNA sequence of the three wheat Ta14S homoeologs，the gene-specific primer pair Ta14S-F and Ta14S-R（Table 1）was designed according to the previously reported cDNA sequence of Ta14S（GB:JN650603）［15］in order to amplify the complete open reading frames（ORF）and cDNA from an immature seed mixture used as template.An approximate 1100 bp band was obtained；sequence alignment indicated that three different cDNA sequences were different，with lengths of 1052，1056 and 1058 bp，and we temporarily named them as Ta14S-1，Ta14S-2 and Ta14S-3，respectively. RACE was then performed to isolate the 5′and 3′-end fragments with specific primers（Table 1）.Agarose gel analysis showed three 5′-end fragments of about 598，562，484 bp and three 3′-end of fragments of about 387，392 and 389 bp. Accordingtotheoverlaps between the5′-and3′-end fragments and ORF sequences，three full-length cDNAs were obtained.As shown in Fig.1，the full-length cDNAs of Ta14S-1，Ta14S-2 and Ta14S-3 were 1269，1238 and 1156 bp，including the 189，153 and 75 bp of 5′UTR，287，292 and 289 bp of 3′UTR，respectively.The ORF of the three homoeologous genes were 792 bp.The sequence identity of Ta14S-1，Ta14S-2 and Ta14S-3 was 91%.Compared with Ta14S-1，Ta14S-2 has a 45 bp deletion and a 9 bp insertion in the 5′UTR，and Ta14S-3 has a 23 bp and 101 bp deletion in the 5′UTR.Ta14S-1，Ta14S-2 and Ta14S-3 have 3，4 and 5 TGG repeats，respectively（Fig.1）. Although there were 22 single-base differences in the coding regions，amino acid sequences of the three genes were almost completely conserved except for residues Asp and Asn being changed to Gly and His at position 241 and 257，respectively （Fig.2）.

3.2.Gene structure and chromosomal locations of the wheat Ta14S homoeologs

In order to better understand the genesis of the three Ta14S homoeologs，their genomic sequences were isolated with the specific primer pair Ta14S-F/Ta14S-R（Table 1）.To correctly identify exon and intron segments of the three genes，we compared the genomic and corresponding cDNA sequences using DNAMAN software.As shown in Fig.1，the three Ta14S homoeologous genes were composed of five exons and four introns.All exons and the first，second and fourth intron sequences of the three genes were relatively conserved，whereas the third introns had more frequent base substitutions and insertions/deletions.Compared with Ta14S-2 and Ta14S-3，Ta14S-1 had 9，207 and 3 bp deletions in the third intron，respectively.

Fig.1-Nucleotide sequence alignments of the Ta14S genes（Ta14S-1，Ta14S-2 and Ta14S-3）amplified from genomic DNA and cDNA from wheat cv.Luohan No.2.Exons and introns are shown in black and gray texts，respectively.Sequence identities and differences are shown in black and green shading，respectively.The positions of 5′and 3′-UTR primers used for gene cloning and RACE are indicated by arrows.Different colored boxes indicate the positions of primers used for chromosome mapping. The red rectangular frames show the locations of the start（ATG）and stop（TAA）codons，respectively.

Fig.1（continued）.

To determine the chromosome locations of the Ta14S homoeologs in the wheat genome，specific primer pairs Ta14S-F1/R1，Ta14S-F2/R2 and Ta14S-F3/R3（Table 1）were designed according to their 3′-end genomic sequences and tested on nulli-tetrasomic lines of Chinese Spring as templateto amplify the corresponding genes in the A，B and D genomes.Homoeologous Ta14S genes Ta14S-1，Ta14S-2 and Ta14S-3 were amplified in all nulli-tetrasomic lines except N2D-T2A，N2B-T2A and N2A-T2B（Fig.3）；Ta14S-1 amplified by primer pair Ta14S-F1/R1 was located on wheat chromosome 2D（Fig.3a）.Similarly，Ta14S-2 and Ta14S-3 amplified by primer pairsTa14S-F2/R2andTa14S-F3/R3，respectively，were located on chromosomes 2B and 2A（Fig.3b and c）. Accordingly，Ta14S-1，Ta14S-2 and Ta14S-3 were renamed as Ta14S-2D，Ta14S-2B and Ta14S-2A，respectively.

Fig.1（continued）.

Fig.2-Alignment of amino acid sequences of the three Ta14S homoeologous genes.Sequence similarities and differences are shown in black and green texts，respectively.

Fig.3-Three homoeologous Ta14S genes were located using Chinese Spring（C.S.）and its nulli-tetrasomics lines.M：DNA ladder；a，b and c indicate that PCR products were specifically amplified with primer pairs Ta14S-F1/Ta14S-R1，Ta14S-F2/ Ta14S-R2 and Ta14S-F3/Ta14S-R3.

3.3.Phylogenetic analyses of the Ta14S homoeologs

To determine the evolutionary relationship among the wheat 14-3-3 proteins and other plant species，32 amino acid sequences from wheat，rice，barley and Arabidopsis were aligned using Mega 6.0.An unrooted tree was constructed based on the alignment using the Neighbor-Joining method. As shown in Fig.4，the tree was clearly divided into two distinctbranches，non-epsilon（I）andepsilon（II）.The non-epsilon group contained most of the proteins，whereas theepsilongroupcomprisedonlyfourproteins.The non-epsilon group can be further divided into two subgroups，each including both monocotyledonous and dicotyledonous species.Ta14S-2A，Ta14S-2B and Ta14S-2D appear to belong to the non-epsilon group and all closely resemble Hv14A.In addition，four other 14-3-3 proteins of barley（Hv14B，Hv14C，Hv14D and Hv14E）cluster in small sub-groups with five reported 14-3-3 proteins of wheat（Ta14A，Ta14R1，Ta14R2，Ta14Win2 and Ta14Win1），indicating that wheat and barley have a close phylogenetic relationship.

3.4.Transcription profiling of the Ta14S homoeologs in different wheat tissues

To define possible functions of the Ta14S homoeologs，we performed tissue-specific expression analyses of the three homoeologous Ta14S genes at different growth and developmental stages，including dry seed embryos，primary roots and shoots at 1 day post-germination，roots and fully expanded leaves at 10 days poat-germination，leaves at tillering，stems at jointing，flag leaves，young panicles at heading and immature seeds（Fig.5a）.The expression of all three Ta14S homoeologs was detected in all tissues examined，and all shared high expression levels in 1-day-old primary roots after germination.Moreover，theexpressionsofthethree homoeologs changed with growth and developmental stages. They all showed high expression levels in early vegetative growth and decreased with seed maturity.Expression differences between the three homoeologs were also detected.For example，Ta14S-2Bhadrelativelyhigherexpressionin 1-day-old roots and shoots，10-day-old roots，leaves at tillering，and stems at jointing，whereas Ta14S-2D was highly expressed in 10-day-old fully expanded leaves.

3.5.Transcription profiling of the Ta14S homoeologs during seed development and seed germination

Total RNA was extracted from immature and mature seeds，and qRT-PCR was performed to define the expression profiles of the three Ta14S homoeologs.The expression of the three Ta14S homoeologs during seed development showed fluctuating trends（Fig.5b）.All three shared highest expression levels at early stages of development（5 DAP），followed by decreased levels in the middle stages（10-15 DAP）.Increases in transcription levels of all three genes were detected at 20 DAP，followed by decreases to minimum levels at 25 DAP. However，the three homoeologs showed different transcript abundancesduringseeddevelopment.Forinstance，Ta14S-2A was more highly expressed than Ta14S-2B and Ta14S-2D at 15，20 and 35 DAP，but the expression levels of Ta14S-2A and Ta14S-2D at 30 DAP were lower than that of Ta14S-2B.

The expression of the three Ta14S homoeologous genes during seed germination showed a gradually up-regulated trend at the beginning of seed imbibition（0-12 h）（Fig.5c）. Afterwards，significant differences in transcript were observed among the genes.The transcript levels of Ta14S-2A and Ta14S-2D showed a gradual downward trend at 24 and 36 h，and then increased at 48 h（Fig.5c）.By contrast，the transcription of Ta14S-2B was elevated in a time-dependent manner，and its transcript level was consistently higher than that of Ta14S-2A and Ta14S-2D during the entire seed germination process（Fig.5c）.These results indicate that all three homoeologs have a role in the seed germination process in wheat.

4.Discussion

The 14-3-3 proteins are phosphoserine-binding proteins that have vital roles in regulating a wide range of target proteins participatingindiversesignaltransductionandgeneregulation，such as primary metabolism，plant development，protein trafficking，signal transduction and biotic/abiotic stress response［18，19，28，29］.Therefore，isolation and functional analysis of 14-3-3 genes are critical steps in understanding their regulatory roles.In order to study the functions of 14S orthologs encoding 14-3-3 proteins in wheat seed development，genomic DNA and cDNA sequences of one set of Ta14S homoeologs from hexaploid wheat were isolated and characterized.Sequence analysis revealed that the gene structures and numbers of amino acids were similar，but the three genes were distinguished by their 5′-and 3′-UTR.The three genes were located on group 2 chromosomes and named as a homoeologous set（Ta14S-2A，Ta14S-2B and Ta14S-2D）.Phylogeneticanalysisrevealedthattheyare members of the non-epsilon group（I）of 14-3-3 proteins， sharing 50%to 100%identity with amino acid sequences in this group，which comprises more than 85%of plant 14-3-3 proteins.These results are in agreement with previous research［30］.

Fig.4-Phylogenetic analysis of Arabidopsis，rice，barley and wheat 14-3-3 proteins using the MEGA Neighbor-Joining program. GF14Chi，GF14Omega，GF14Psi，GF14Phi，GF14Upsilon，GF14Lambda，GF14Nu，GF14Kappa，GF14Mu，GF14Epsilon，GF14Omicron and GF14Iota are from Arabidopsis；Ta14A，Ta14B，TaR1，TaR2，TaWIN1，TaWIN2，Ta14S-2A，Ta14S-2B and Ta14S-2D are from wheat；Hv14A-E is from barley；and OsGF14A-F is from rice.

qRT-PCRassaysdemonstratedthatallthreeTa14S homoeologs were constitutively transcribed in various wheat organs at different developmental stages，including dry seed embryos，primary roots and shoots 1 day after germination，roots and fully expanded leaves 10 days after germination，leaves at tillering，stems at jointing，flag leaves and immature ears at heading，seeds at 10 and 25 days post-pollination. Moreover，mRNA abundances of the three genes also varied between tissues，and developmental stages.Interestingly，the three Ta14S homoeologous genes appeared to be highly expressed in primary roots 1 day post-germination，comparedto other plant organs.It was reported that a wheat Ta14-3-3 gene in transgenic Arabidopsis played an important role during root development［16］.Collectively，it can be concluded that the high expression of all three Ta14S homoeologs in young roots may regulate initial elongation of the radical and affect root development.

Fig.5-Expression patterns of the three Ta14S homoeologous genes in wheat.（a）In different tissues.1，dry seed embryos；2，roots at 1 day post-germination；3，shoots 1 day after germination；4，roots at 10 days post-germination；5，fully expanded leaves at 10 days post-germination；6，leaves at tillering；7，stems at jointing；8，flag leaves at heading；9，immature ears at heading；10，seeds at 10 DAP；11，seeds at 25 DAP.（b）During seed development stages.（c）During seed germination.

Earlier studies showed that 14-3-3 proteins are involved in signal transduction pathways with roles in late embryo development in seeds and germination.It was reported that 14-3-3 proteins are part of an abscisic acid viviparous-1（VP1）response complex in the Em gene promoter，one of the late embryogenesis-abundant（LEA）proteins which affect maturing embryos，and interact with VP1 and Em-binding protein 1 （EmBP1）to control abscisic acid-responsive gene expression ［6，31］.Three 14-3-3 isoforms，14-3-3A，14-3-3B and 14-3-3C，were induced in germinating barley embryos and their expression levels were all up-regulated by ABA［13，30］.In this study，we found that cDNA from the three Ta14S homoeologs share very high（95%）amino acid identities with a barley Hv14A amino acid sequence［32］.We hypothesized that the three homoeologs may be involved in regulation of seed development and germination processes.To test the hypothesis，we investigated their expression patterns in seed development and germination processes by qRT-PCR.All three homoeologs were expressed during the entire seed developmental process，and their mRNA abundances varied with the stage of seed development.Moreover，relatively high expression of all three genes was detected at 5 and 20 DAP，suggesting they may be involved in regulation of early seed development and later morphogenic processes.During germination all three homoeologous Ta14S genes were induced after seed imbibition but they were differentially expressed. Ta14S-2A and Ta14S-2D showed a similar expression pattern during the entire germination process whereas expression of Ta14S-2B was clearly different in that it increased after 12 h of imbibition.This differential expression suggested different functions of the three homoeologs in germination.

Expression divergence between homoeologs in wheat was reported previously.For example，three TaEXPA1 homoeologs had similar genomic structures，but TaEXPA1-B was silenced ［33］.Similarly，homoeologous sequences of methyl-binding domain proteins（TaMBD2）showed high conservation of nucleotide coding sequences and exon/intron structures，but the three TaMBD2 homoeologs were transcribed differentially in response to environmental stresses［34］.Epigenetic variation andpromotersequenceareconsideredtoaffecttheexpressions of homoeologous genes［33，35］.However，the mechanistic details are still unclear.Obviously，further studies are required todeterminewhetherTa14Shomoeologshavecriticalfunctions in regulating seed development in wheat.

Acknowledgments

This work was financially supported by the Key Transgenic Breeding Program of the Ministry of Agriculture of China（No. 2014ZX0800205B-003）and the National Natural Science Foundation of China（No.30771332）.

R E F E R E N C E S

［1］F.C.Denison，A.L.Paul，A.K.Zupanska，R.J.Ferl，14-3-3 proteins in plant physiology，Semin.Cell Dev.Biol.22（2011）720-727.

［2］D.H.Jones，S.Ley，A.Aitken，Isoforms of 14-3-3 protein can form homo-and heterodimers in vivo and in vitro: implications for function as adapter proteins，FEBS Lett.368 （1995）55-58.

［3］A.H.de Boer，P.J.van Kleeff，J.Gao，Plant 14-3-3 proteins as spiders in a web of phosphorylation，Protoplasma 250（2013）425-440.

［4］P.J.Schoonheim，H.Veiga，D.C.Pereira，G.Friso，K.J.van Wijk，A.H.de Boer，A comprehensive analysis of the 14-3-3 interactome in barley leaves using a complementary proteomics and two-hybrid approach，Plant Physiol.143 （2007）670-683.

［5］I.F.Chang，A.Curran，R.Woolsey，D.Quilici，J.C.Cushman，R. Mittler，A.Harmon，J.F.Harper，Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana，Proteomics 9（2009）2967-2985.

［6］T.F.Schultz，J.Medina，A.Hill，R.S.Quatrano，14-3-3 proteins are part of an abscisic acid VIVIPAROUS1（VP1）response complex in the Em promoter and interact with VP1 and EmBP1，Plant Cell 10（1998）837-848.

［7］D.Igarashi，S.Ishida，J.Fukazawa，Y.Takahashi，14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG，Plant Cell 13（2001）2483-2497.

［8］T.Gokirmak，A.L.Paul，R.J.Ferl，Plant phosphopeptide binding proteins as signaling mediators，Curr.Opin.Plant Biol.13（2010）527-532.

［9］J.C.Chi，J.Roeper，G.Schwarz，K.Fischer-Schrader，Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity，J.Biol.Inorg.Chem.20（2015）277-286.

［10］P.C.Sehnke，M.Rosenquist，M.Alsterfjord，J.Delille，M. Sommarin，C.Larsson，R.J.Ferl，Evolution and isoform specificity of plant 14-3-3 proteins，Plant Mol.Biol.50（2002）1011-1018.

［11］Y.Yao，Y.Du，L.Jiang，J.Y.Liu，Molecular analysis and expression patterns of the 14-3-3 gene family from Oryza sativa，J.Biochem.Mol.Biol.40（2007）349-357.

［12］P.J.Schoonheim，M.P.Sinnige，J.A.Casaretto，H.Veiga，T.D. Bunney，R.S.Quatrano，A.H.de Boer，14-3-3 adaptor proteins are intermediates in ABA signal transduction during barley seed germination，Plant J.49（2007）289-301.

［13］Y.Ikeda，N.Koizume，T.Kusano，H.Sano，Specific binding of a 14-3-3 protein to autophosphorylated WPK4，an SNF1-related wheat protein kinase，and to WPK4-phosphorylated nitrate reductase，J.Biol.Chem.275（2000）31695-31700.

［14］C.Wang，Q.H.Ma，Z.B.Lin，P.He，J.Y.Liu，Cloning and characterization of a cDNA encoding 14-3-3 protein with leaf and stem-specific expression from wheat，DNA Seq.19（2008）130-136.

［15］J.P.Ren，H.L.Liu，X.G.Wang，H.B.Niu，Y.C.Li，X.Wang，X. Chen，J.Yin，Cloning and expression analysis of a stress-related Ta14S gene from wheat，Sci.Agric. Sin.45（2012）1226-1234（in Chinese with English abstract）.

［16］J.Li，S.S.Song，Y.S.Zhao，W.W.Guo，G.H.Guo，H.R.Peng，Z.F. Ni，Q.X.Sun，Y.Y.Yao，Wheat 14-3-3 protein conferring growth retardation in Arabidopsis，J.Integr.Agric.12（2013）209-217.

［17］X.D.Meng，X.Chen，Y.Y.Wang，R.X.Xiao，H.L.Liu，X.G.Wang，J.P.Ren，Y.C.Li，H.B.Niu，X.Wang，J.Yin，Characterization and subcellular localization of two 14-3-3 genes and their response to abiotic stress in wheat，Chin.J.Biotechnol.30 （2014）232-246.

［18］S.Campo，C.P.Peris，L.Montesinos，G.Penas，J.Messeguer，B.S.Segundo，Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection，J.Exp.Bot.63（2012）983-999.

［19］Y.Zhou，Z.T.Zhang，M.Li，X.Z.Wei，X.J.Li，B.Y.Li，X.B.Li，Cotton（Gossypium hirsutum）14-3-3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signaling，Plant Biotechnol.J.13（2015）269-280.

［20］R.Brenchley，M.Spannagl，M.Pfeifer，G.L.Barker，R.Damore，A.M.Allen，N.McKenzie，M.Kramer，A.Kerhornou，D.Bolser，S.Kay，D.Waite，M.Trick，I.Bancroft，Y.Gu，N.Huo，M.C.Luo，S.Sehgal，B.Gill，S.Kianian，O.Anderson，P.Kersey，J.Dvorak，W.R.McCombie，A.Hall，K.F.Mayer，K.J.Edwards，M.W. Bevan，N.Hall，Analysis of the bread wheat genome using whole-genome shotgun sequencing，Nature 491（2012）705-710.

［21］J.D.Mayfield，A.L.Paul，R.J.Ferl，The 14-3-3 proteins of Arabidopsis regulate root growth and chloroplast development as components of the photosensory system，J. Exp.Bot.63（2012）3061-3070.

［22］P.J.van Kleeff，N.Jaspert，K.W.Li，S.Rauch，C.Oecking，A.H.de Boer，Higher order Arabidopsis 14-3-3 mutants show 14-3-3 involvement in primary root growth both under control and abiotic stress conditions，J.Exp.Bot.65（2014）5877-5888.

［23］H.Shi，Y.Zhang，Pear 14-3-3a gene（Pp14-3-3a）is regulated during fruit ripening and senescence，and involved in response to salicylic acid and ethylene signaling，J.Genet.93 （2014）747-753.

［24］Y.Dou，X.Liu，Y.Yin，S.Han，Y.Lu，Y.Liu，D.Hao，Affinity chromatography revealed insights into unique functionality of two 14-3-3 protein species in developing maize kernels，J. Proteomics 114（2015）274-286.

［25］G.L.Wang，H.J.Fang，Plant Genetic Engineering Principle and Technology，Science Press，Beijing，1998.

［26］T.C.Verwoerd，B.M.M.Dekker，A.Hoekema，A small-scale procedure for the rapid isolation of plant RNAs，Nucleic Acids Res.17（1989）2362.

［27］K.J.Livak，T.D.Schmittgen，Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCtmethod，Methods 25（2001）402-408.

［28］M.R.Roberts，J.Salinas，D.B.Collinge，14-3-3 proteins and the response to abiotic and biotic stress，Plant Mol.Biol.50（2002）1031-1039.

［29］D.Bridges，G.B.G.Moorhead，14-3-3 proteins:a number of functions for a numbered protein，Sci.STKE 242（2004）re10.

［30］C.Testerink，R.M.van der Meulen，B.J.Oppedijk，A.H.de Boer，S.Heimovaara-Dijkstra，J.W.Kijne，M.Wang，Differences in spatial expression between 14-3-3 isoforms in germinating barley embryos，Plant Physiol.121（1999）81-87.

［31］F.del Viso，J.A.Casaretto，R.S.Quatrano，14-3-3 proteins are components of the transcription complex of the ATEM 1 promoter in Arabidopsis，Planta 227（2007）167-175.

［32］J.Brandt，H.Thordal-Christensen，K.Vad，P.L.Gregersen，D.B. Collinge，A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator，Plant J.2（1992）815-820.

［33］Z.R.Hu，Z.F.Han，N.Song，L.L.Chai，Y.Y.Yao，H.R.Peng，Z.F. Ni，Q.X.Sun，Epigenetic modification contributes to the expression divergence of three TaEXPA1 homoeologs in hexaploid wheat（Triticum aestivum），New Phytol.197（2013）1344-1352.

［34］Z.R.Hu，Y.Yu，R.Wang，Y.Y.Yao，H.R.Peng，Z.F.Ni，Q.X.Sun，Expression divergence of TaMBD2 homoeologous genes encoding methyl CpG-binding domain proteins in wheat （Triticum aestivum L.），Gene 471（2011）13-18.

［35］O.Y.Shoeva，E.K.Khlestkina，H.Berges，E.A.Salina，The homoeologous genes encoding chalcone-flavanone isomerase in Triticum aestivum L.:structural characterization and expression in different parts of wheat plants，Gene 538 （2014）334-341.

27 November 2015

*Corresponding author.

E-mail address:xmzxrjp@126.com（J.Ren）.

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

http://dx.doi.org/10.1016/j.cj.2016.03.002

2214-5141/?2016 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http://creativecommons.org/licenses/by-nc-nd/4.0/）.