Yang Liu*,Daxing Li,Qiping Song,Tianpeng Zhang,Dequan Li,Xinghong Yang*

State Key Laboratory of Crop Biology/Shandong Key Laboratory of Crop Biology/College of Life Sciences,Shandong Agricultural University,Tai'an 271018,Shandong,China

Keywords:Maize LEA proteins Dehydrins Zm DHN13 Copper stress

A B S T R A C T Late em bryogenesis abundant(LEA)proteins accum ulate in the late stage of plant seed development,and are upregulated in m ost plants during drought,cold,heat,or salinity stress.LEA proteins can be classified by amino-acid sequence into seven groups.Dehydrins belong to LEA protein group II.In previous studies,the m aize KStype dehydrin Zm DHN13 increased the tolerance of transgenic tobacco to oxidative stress.In the present study,Zm DHN13 w as identified under copper stress conditions,and the protein w as then characterized using transgenic yeast and tobacco plants to investigate its functions.Zm DHN13 bound Cu2+.Its overexpression in transgenic tobacco conferred tolerance to copper stress by binding m etals and reducing the accum ulation of reactive oxygen species(ROS).Three conserved dom ains displayed a cooperative effect under copper stress conditions.

1.Introduction

Plants produce diverse hydrophilic proteins to prevent damage caused by abiotic stresses.Late em bryogenesis abundant proteins(LEA proteins)accum ulate in the late stage of plant seed developm ent and w ere first found in cotton seeds[1].Ectopic expression of som e plant LEA proteins in plants and yeast confers tolerance to various abiotic stresses[2,3].According to their conserved m otifs,LEA proteins can be grouped into seven fam ilies[4].Dehydrins belong to group II,whose members accumulate in embryogenesis during seed m aturation and in vegetative tissue in response to salinity,drought,or cold.

Dehydrins contain four conserved segm ents:K,S,Y([V/T]D[E/Q]YGNP),andΦsegm ents.Based on the sequence and number of K,S,and Y segm ents,the dehydrins can be divided into five subgroups,SKn,Yn SKn,Kn S,Kn,and Yn Kn.As intrinsically disordered proteins,dehydrins lack a w elldefined three-dim ensional structure.How ever,K segm ents are predicted to form am phipathicα-helical structures,w hich can protect the enzym e and m em branes.The K segm ent present in all dehydrins is a lysine-rich m otif that consists of 15 am ino acid residues([E/H]KKGIMDKIKEKLPG).The K segment of w heat dehydrin WZY2 is important for its protective functions under tem perature stress[5].The K segm ent of m aize dehydrin DHN1 is essential for binding to anionic phospholipid vesicles,and adopts anα-helix conform ational change in DHNs upon binding to sodium dodecyl sulfate or anionic phospholipid vesicles[6].

Although the functional role of dehydrins rem ains speculative,m any studies have show n that dehydrins can increase plant resistance to m any abiotic stresses.Overexpression of the Prunus mume dehydrin and the Saussurea involucrata dehydrin SiDHN increased transgenic tobacco tolerance to drought and cold stresses[7,8].Overexpression of the Sorghum bicolor YSK2 dehydrin increased transgenic plant tolerance to high tem perature and osm otic stresses[9].

Toxic heavy m etals such as copper(Cu)are a grow ing threat to the environm ent.Elevated Cu induces toxicity in plants and severely reduces the quantity and quality of crop production[10].Study have show n that dehydrins can bind free m etals,an activity that m ay alleviate oxidative dam age by preventing the formation of toxic reactive oxygen species(ROS)and protect the activity of proteins from dam age caused by environmental stresses[11].The Arabidopsis thaliana dehydrin AtHIRD11 can bind m etals,and the histidine residues of the protein contribute to reducing the activity of the radicals[12,13].The Vigna radiata LEA protein VrDhn1 enters the nucleus.And then it interacts nonspecifically w ith DNA,w hich w as prom oted by the metals Ni2+and Zn2+during seed maturation.Zn2+prom oted the interaction betw een the citrus dehydrin Cu COR15 and DNA[14,15].

We describe here the isolation and functional characterization of Zm DHN13 and its m utant proteins under copper stress conditions.Our experim ents confirm ed that overexpression of Zm DHN13 confers tolerance to copper stress in transgenic tobacco,and the three m otifs displayed a cooperative effect in response to copper stress in vivo.

2.Material and m ethods

2.1.Plant materials and grow th conditions

Maize(Zea mays L.cv.Zhengdan 958)plants w ere grow n for 2 weeks in Hoagland's solution(p H 6.0)in a grow th chamber at 26°C/22°C(day/night)w ith a photoperiod of 14 h/10 h(day/night),and a photosynthetically active radiation of 600μmol m-2s-1.The solution was replaced daily.

Seeds of w ide type(WT)tobacco(Nicotiana benthamiana)and transgenic plants w ere treated w ith 70%ethanol for 30 s and w ith 2.6%hypochlorite for 10 m in,w ashed six tim es w ith sterile w ater,and plated on MSm edium under day/night cycle conditions of 16 h/8 h at 25°C.

2.2.Determining expression profiles of Zm DHN13 in different stress conditions by real-time PCR

Tw o-w eek-old m aize plants w ere treated w ith 100μm ol L-1abscisic acid(ABA),20μm ol L-1H2O2,10%(w/v)polyethylene glycol(PEG-6000),low tem perature(4°C),and 100μm ol L-1Cu Cl2or w ater(control),and q RT-PCR w as perform ed as previously described[16].ZmDHN13 w as am plified using SYBR Green qRT-PCR Super Mix(TransGene Biotech,China)and prim ers(forw ard 5′-CGCATAGCATTCTCTTCC-3′and reverse 5′-CGCTCCTGGATCTTGTC-3′).The m aize Zmactin and ZmUBCP(ubiquitin carrier protein)genes w ere am plified along w ith the ZmDHN13 gene to allow gene expression norm alization and subsequent quantification[2,17].

2.3.Protein expression and purification

To determ ine w hether the conserved segm ents are necessary for the function of Zm DHN13,segm ent deletion proteins w ere produced(Fig.1).The sequences of ZmDHN13,ZmDHN13ΔS,ZmDHN13ΔK,and ZmDHN13ΔNLS w ere cloned into the p ET30a vector and transform ed into an Escherichia coli(BL21 DE3)expression system.The proteins w ere purified w ith a Ni-NTA spin column(Tiangen,China).The 6×His tag was removed by HRV 3C protease(Takara)digestion follow ing the m anufacturer's instructions.The pure proteins w ere then exchanged into a low-m edium salt buffer(20 m m ol L-1Tris-HCl,100 m mol L-1NaCl,p H 8.0)using a HiPrep Desalting colum n(GEHealthcare).The purified proteins w ere separated by SDSPAGEand quantified by bicinchoninic acid assay[2].

2.4.Metal binding assay

Interactions betw een the m etal ions and polypeptides w ere detected by immobilized metal ion affinity chromatography(IMAC)on a HiTrap Chelating HPcolumn(Am ersham Pharm acia Biotech,Japan)follow ing previous reports[18,19].The colum n w as charged by application of 3 m L of 100 mm ol L-1Cu Cl2.Recom binant proteins were loaded onto the column.Unbound proteins were w ashed out w ith EQ buffer(50 mmol L-1Tris-HCl,p H 7.4,1.0 mol L-1NaCl),and bound proteins w ere eluted w ith 600 mmol L-1im idazole or 100 mmol L-1EDTA.A 5-μg aliquot of protein was separated by SDS-PAGE.

The metal binding of the recombinant protein w as analyzed by ultrafiltration[19,20].Mixtures containing 10μmol L-1protein,100 m mol L-1NaCl,Tris-HCl(10 mm ol L-1,p H 7.4),and appropriate concentrations of Cu Cl2w ere incubated at 4°Cfor 10 m in.Insoluble protein w as rem oved by centrifugation at 12,000×g for 10 min at 4°C.The concentration of Cu2+was determ ined using the BCA com petition assay[21].

Fig.1-Com parison of the sequences of Zm DHN13 and deletion m utants.The sequences of Zm DHN13 and deletion m utants(Zm DHN13ΔS,Zm DHN13ΔK,and Zm DHN13ΔNLS)are show n.Blue indicates the K segm ent,green the NLS segm ent,and red the Ssegm ent.

Table 1-Characteristics of d ehyd rin genes id entified in m aize.

Fig.2-Transcription of Zm DHN13 under stress treatments.Maize seedlings w ere treated w ith 100μmol L-1 ABA or 20 m m ol L-1 H2O2,10%(w/v)PEG-6000,low tem perature,100μm ol L-1 Cu Cl2,and control.The transcript level of Zm DHN13 w as m easured by q RT-PCR.Total RNA w as isolated from leaves at the indicated tim es after the treatm ents.Rrepresents rem oved treatm ents.Each d ata p oint represents an average of three replicates,and error bars represent standard d eviation.

2.5.Expression of Zm DHN13 and its mutant proteins in Pichia yeast GS115

The coding sequences of ZmDHN13,ZmDHN13ΔK,and ZmDHN13ΔNLS w ere amplified from cDNA using the follow ing primers,forward 5′GAGAAGTAGCCACAAGCATG-3′(Bam H I site underlined),and reverse,5′-ACAACAA TCTTGGCGAGT-3′(Eco R I site underlined).ZmDHN13ΔS w as am plified from c DNA using the follow ing prim ers:forw ard 5′-AGAGAAGTAGCCACAAGCATG-3′(Bam H I site underlined),and reverse,5′-CAGTGTCCGTCACCA TCAC-3′(Eco R I site underlined).The sequences w ere cloned into the plasmid p PIC3.5K(Invitrogen,USA)w ith the AOXI promoter.Yeast expression of the proteins was performed as previously described[3,22].

2.6.Copper stress tolerance assays of yeast transformants

Culture of recom binant colonies w as perform ed as previously described[3,22].For copper stress conditions,200 m m ol L-1Cu Cl2w as added to the medium.At each time point,3 m L of culture w as used for spectrophotom etric m easurem ent of OD600.Under the norm al condition,OD600w as recorded at 2-h intervals for 38 h.Under the copper stress condition,OD600w as recorded at 4-hs interval for 60 h.

2.7.Measurements of physiological parameters

Copper stress treatment w as perform ed as previously described w ith som e m odifications[23].Six-w eek-old plants of the control and transgenic lines w ere treated w ith 70μm ol L-1Cu Cl2for the indicated tim e.Relative electrolytic leakage,m alondialdehyde(MDA)and superoxide radical(O2-)concentrations w ere determ ined as previously described[16,24].

3.Results

3.1.Dehydrin-encoding genes in the maize genome

The availability of the complete maize genom e sequence has m ade it possible to identify dehydrin gene family members in maize(https://www.maizegdb.org/).Dehydrin genes from Arabidopsis thaliana w ere searched w ith blast against the maize genom e,identifying 11 putative maize dehydrin gene fam ily members(Table 1).Nine had two exons and ZmDHN13 and ZmDHN13-2 only one.The num bers of am ino acid residues ranged from 100 to 326 and the isoelectric points(p I)from 5.51 to 8.85(http://ww w.expasy.org/tools/pi_tool.html).Tw o dehydrins were found on chromosome 5 and four on chromosome 8,w hereas only one was found on each of chromosomes 1,3,4,6,and 9.In silico(ProtComp 9.0,http://linux1.softberry.com/berry.phtm l?topic=protcomppl&group=programs&subgroup=proloc)and subcellular localization analyses indicated that the LEA proteins w ere located in the nucleus or cytoplasm.

3.2.Transcription of Zm DHN13 under stress treatments

To investigate the transcription patterns of ZmDHN13,q RTPCR analysis w as perform ed using RNA from non-stressed and stressed Zea mays seedlings.The ZmDHN13 transcript level in the leaves peaked at 36 h under ABA treatm ent.The transcription of ZmDHN13 w as eight tim es greater under ABA treatment than under normal conditions.Treatment with H2O2and Cu Cl2m arkedly increased transcript levels in leaves at 12 h,follow ed by a gradual reduction to the untreated level.The transcription of ZmDHN13 w as upregulated fivefold.Under 10% PEG-6000 treatm ent,the transcript level of ZmDHN13 in the leaves peaked at 12 h and then returned to the norm al level.Transcription of ZmDHN13 w as increased tw ofold under 10%PEG-6000 treatm ent.Under cold treatm ent,transcription of ZmDHN13 in leaves peaked at 48 h and then fell slow ly to the preinduction level.It increased 17-fold under cold conditions.Under control conditions,there w ere no significant differences at the indicated time points.Thus,transcription of ZmDHN13 could be induced by drought,low tem perature,H2O2,Cu Cl2,and treatm ent w ith ABA(Fig.2).results of Cu2+,m olar protein:m etal ratios w ere m easured.According to the results,one m olecule of Zm DHN13 could bind up to 14 Cu(Fig.4-b).

Fig.3-SDS-PAGE of purified Zm DHN13 and d eletion p roteins.Purified proteins w ere sep arated by 15%SDS-PAGE.Protein m arkers are show n on the left p anel in k Da.

Fig.4-Investigation of m etal-Zm DHN13 binding using im mobilized metal ion affinity chromatography.(a)Columns w ere charged w ith Cu 2+.Protein w as loaded onto a colum n equilibrated w ith EQbuffer(50 m m ol L-1 Tris-HCl p H 7.4,1 m ol L-1 Na Cl).Unbound protein on the colum n w as w ashed out w ith EQ buffer.Bound p rotein w as eluted w ith 100 m m ol L-1 EDTA or 600 m m ol L-1 im idazole.A colum n not charged w ith m etal w as used as a positive control.Sam p les w ere collected,subjected to SDS-PAGE,and stained w ith Coom assie Brilliant Blue.(b)Cu2+and Zm DHN13 protein binding in vitro.

3.4.Overexpression of ZmDHN13 increased yeast GS115 tolerance to copper stress

3.3.Metal-binding property of Zm DHN13 proteins

Zm DHN13 and its m utant recom binant protein w ere purified on a Ni colum n(Fig.3).Zm DHN13 w as retained in colum n immobilizing Cu2+but not in columns immobilizing no m etal.The proteins that w ere retained in the colum n w ere eluted w ith EDTA and 600 m m ol L-1im idazole(an analogue of His),suggesting that Zm DHN13 m ight bind Cu2+.The three conserved segm ents show ed no significant influence on these binding roles(Fig.4-a).

IMAC analysis is prelim inary w hen used to estim ate the binding between copper and Zm DHN13.To confirm the

To determine the function of the Zm DHN13 protein under copper stress conditions,the ZmDHN13 gene w as cloned into vector p PI3.5K and then transform ed into yeast(GS115)cells.The growth curves of the transgenic yeast cell lines were measured under copper stress conditions.Under optimal conditions,there w as no significant grow th difference betw een the transgenic yeast and the control.How ever,under copper stress,the transgenic yeast displayed a higher growth rate than the control,and the lag phase of the transgenic yeast lines w as shorter than that of the control(Fig.5).Overexpression of the mutant genes(ZmDHN13ΔS,ZmDHN13ΔK,and ZmDHN13ΔNLS)increased the transgenic yeast tolerance compared to that of the control,but the effect the m utant genes w ere low er than that of ZmDHN13,and the three m utant genes did not display significant differences in copper stress tolerance.

Fig.5-Overexp ression enhances tolerance to cop per stress in Zm DHN13 transform ant yeast.(a)The transform ant yeast(GS115)w as grow n in non-stress BMGY m ed ium.(b)The transform ant yeast w as grow n in BMGY m edium supplem ented w ith 150 mm ol L-1 cop per.Each data p oint represents an average of three replicates,and error bars represent standard deviations.

3.5.Overexpression of Zm DHN13 increase transgenic tobacco tolerance to copper stress

Since Zm DHN13 can bind metals,it is reasonable to speculate that overexpression of ZmDHN13 w ould increase the tolerance of transgenic tobacco to copper stress.To test the hypothesis of Zm DHN13 action under copper stress,the copper stress tolerances of the control and transgenic tobacco lines w ere evaluated.Six-w eek-old tobacco plants w ere treated w ith 70 m m ol L-1Cu Cl2for 12 h.After treatm ent,the transgenic tobacco accum ulated less ROS than did control plants.To confirm the results,six-w eek-old tobacco plants w ere exposed to 70 m m ol L-1Cu Cl2for three days.After treatment,the leakage of the electrolytes MDA and O2-w as higher in control plants than in the transgenic lines under copper stress conditions.Although the conserved segm ents show ed negligible influence on the protein's ability to bind m etals,the m utant transgenic plants(Zm DHN13ΔS,Zm DHN13ΔK,and Zm DHN13ΔNLS)displayed low er tolerance to copper stress than did the Zm DHN13 transgenic plants(Fig.6).

Fig.6-Assay for copper tolerance in transgenic tobacco p lants.Transgenic and control tobacco plants w ere grow n at a norm al tem perature(25°C)for 6 w eeks,and then w ere treated w ith 70μm ol L-1 Cu Cl2.(a)In situ d etection of O2-by NBT staining of control and transgenic leaves w as perform ed after 12 h under 70μm ol L-1 Cu Cl2 treatment.Leakage of electrolytes(b)MDA and(c)O2-(d)in transgenic and control tobacco p lants w as m easured at the indicated tim es after treatm ent w ith Cu Cl2.Statistical analyses w ere perform ed w ith Sigm a Plot 11.0 and SPSS 13.Each data p oint rep resents an average of three replicates,and error bars rep resent standard deviation.Letters rep resent significant(P＜0.05)d ifferences am ong different plant lines.

Table 2-Com p arison of am ino acid num ber and content(%),m olecular w eight(Da),and p I of Zm DHN13 and segm entd eleted proteins.

4.Discussion

Dehydrins belong to LEA proteins group 2,w hich are also considered hydrophilins.They are highly hydrophilic,containing a high proportion of polar am ino acids[4].As low-molecular-w eight dehydrins,the KS-type dehydrin contains one K-segm ent,one NLS-segm ent(Lys-rich)and one S-segm ent.Although the functional m echanism s rem ain unclear,m any studies have dem onstrated that LEA proteins are involved in abiotic stresses during the past decades.The present study show ed that the expression of Zm DHN13 is constitutive but can also be induced by environm ental stresses,such as low tem perature,high osm olarity,oxidative stress,copper stress,and ABA application.

Metals such as copper and zinc are essential for gene expression and m etabolic processes in plant grow th;how ever,reactive transition m etals are released from enzym es and organelles under environmental stresses.Free m etals are a source of ROS generation via Fenton-type reactions[23,25].Many dehydrins stabilize transition m etal ions by binding them[26].The Ricinus KS-type dehydrin ITP is the first m em ber of the LEA protein fam ily found to be involved in the long-distance transport of m icronutrients[27].The Arabidopsis thaliana KS-type dehydrin AtHIRD11 can bind m etal ions and reduce ROS generation due to copper m etal.It has also been proposed[12]that histidine content and peptide length are fundam ental factors that influence the strength of ROSreduction by KS-type dehydrins.The proteins Zm DHN13,Zm DHN13ΔS,Zm DHN13ΔK,and Zm DHN13ΔNLS bound Ni2+,Fe3+,Cu2+and Zn2+in vitro,and binding w as inhibited by the chelator EDTA or im idazole.Zm DHN13 contains high levels of His.The finding that the content of this amino acid residue did not significantly change w hen the conserved segm ents w ere deleted from Zm DHN13 indicates that the three conserved segm ents did not influence the ability to bind m etal ions in vitro.

Dehydrins show mostly unordered structures in solution,but the K segm ent is predicted to form am phipathicα-helical structures and is thought to protect proteins and mem branes.K segm ents play im portant roles in response to abiotic stresses.Hara[28]reported that the cryoprotective activities of dehydrins depended on the hydrophobic residues of the K segm ents.Although the Ksegment is thought to be the core sequence,the S and NLS segments also play important roles in dehydrins.Phosphorylation of the Ssegment by a protein kinase has been suggested[25,29]to promote specific signal peptide interactions with dehydrins,leading to dehydrin transportation into the nucleus.In a previous study[16],the S and NLS segments of Zm DHN13 w ere the core sequences involved in phosphorylation and subcellular localization.In the present study,overexpression of ZmDHN13ΔS,ZmDHN13ΔK,and ZmDHN13ΔNLS in transgenic yeast and tobacco also induced a higher tolerance than that of control yeast and tobacco under copper stress,but these roles w ere w eaker in ZmDHN13ΔS,ZmDHN13ΔK,and ZmDHN13ΔNLS transgenic yeast and tobacco than in ZmDHN13 transgenic yeast and tobacco.Although the deleted proteins have similar abilities to bind metal ions,the K,S,and NLS segments influenced the roles of the proteins,including the amino acid content(Table 2),phosphorylation and protein subcellular localization,w hich also play important roles in response to oxidative stress in vivo[16].

5.Conclusions

The m aize dehydrin gene ZmDHN13,show ed stress-responsive expression.The Zm DHN13 protein bound Cu2+ions.Zm DHN13 positively regulates transgenic yeast and tobacco tolerance to copper stress by binding m etals and reducing the form ation of ROS.This activity depended partly on the presence of its three conserved segm ents.

Acknow ledgm ents

This w ork w as supported by the National Natural Science Foundation of China(31701334)and the Shandong Province Natural Science Foundation(ZR2016CQ34).