SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet

16 Pages·2014·6.12 MB·English

Wangetal.BMCPlantBiology2014,14:290
http://www.biomedcentral.com/1471-2229/14/290
RESEARCH ARTICLE Open Access
SiLEA14, a novel atypical LEA protein, confers
abiotic stress resistance in foxtail millet
Meizhen Wang1,2†, Ping Li1†, Cong Li1, Yanlin Pan1, Xiyuan Jiang1, Dengyun Zhu1, Qian Zhao1 and Jingjuan Yu1*
Abstract
Background: Late embryogenesis abundant (LEA) proteins are involved in protecting higherplants from damage
caused by environmental stresses. Foxtailmillet (Setaria italica) is an important cereal crop for food and feed in
semi-arid areas.However,themolecular mechanismsunderlying tolerance to these conditions are not well defined.
Results: Here, we characterized a novel atypical LEA gene named SiLEA14 from foxtail millet. It contains two exons
separated by one intron. SiLEA14 was expressed in roots, stems, leaves, inflorescences and seeds at different levels
under normal growthconditions. Inaddition, SiLEA14 was dramatically induced by osmotic stress,NaCl and
exogenous abscisic acid. The SiLEA14protein was localized in thenucleusand the cytoplasm. Overexpression of
SiLEA14 improved Escherichia coligrowthperformance compared with the control under salt stress. Tofurther
assess thefunction of SiLEA14 in plants, transgenicArabidopsisand foxtail millet plantsthatoverexpressed SiLEA14
were obtained.The transgenicArabidopsis seedlings showed higher tolerance to salt and osmotic stressthan the
wild type(WT). Similarly, thetransgenicfoxtailmillet showed improved growth under salt and drought stresses
compared withthe WT. Takentogether, our results indicated that SiLEA14 is anovelatypical LEAprotein and plays
important roles in resistance to abiotic stresses in plants.
Conclusion: We characterized a novel atypical LEA geneSiLEA14 from foxtail millet, which plays important roles in
plant abiotic stress resistance. Modification of SiLEA14 expression may improve abiotic stressresistance in
agricultural crops.
Background the development of deep sequencing technology, an
Environmental stresses, such as drought and high salinity, increasing number of LEA proteins have been identified.
can cause severe damage to plants, leading to considerable On the basis of their amino acid sequence similarities and
reductionintheirproductivity.Tosurviveundersuchcon- conserved motifs, LEA proteins are categorized into differ-
ditions,plantshavedevelopedaseriesofdefenseresponsive entgroups[10-13].Inthiswork,weadopttheclassification
pathways. Among them, Ca2+-dependent signaling leads to introduced by Battaglia’s group, in which LEA proteins are
the activation of late embryogenesis abundant (LEA)-type categorizedintosevendistinctfamilies[12].Groups1,2,3,
genes,whichmayfunctioninprotectionanddamagerepair 4, 6 and 7, which share specific motifs within each group,
ofplants[1]. are considered to be hydrophilic or “typical” LEA proteins.
LEA proteins were first identified in cotton seeds [2]. Conversely, group 5 corresponds to atypical LEA proteins.
The proteins accumulated to high levels in the late This group includes all LEA proteins with higher content
stages of seed development [3,4]. Subsequently, they of hydrophobic residues than typical LEA proteins. Onthe
were found to be expressed in vegetative tissues [5,6] basisoftheirsequencesimilarity,group5LEAproteinsare
andcould beinducedbyabscisic acid (ABA) andvarious divided into the subgroups 5A, 5B, and 5C, corresponding
abiotic stresses, such as drought and cold [5-9]. With to the first described proteins D-34, D-73, and D-95 [3,14]
inthisgroup,respectively.Physicochemicalpropertiesshow
*Correspondence:[email protected] that group 5 LEA proteins are not soluble after boiling,
†Equalcontributors suggesting that they may adopt a globular conformation
1StateKeyLaboratoryofAgrobiotechnology,CollegeofBiologicalSciences,
and are not heat stable [3,15,16]. Subsequent reports show
ChinaAgriculturalUniversity,No.2YuanmingyuanWestRoad,Haidian
District,Beijing100193,China thatsubgroup5CLEAproteinsarenativelyfoldedandhave
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more β-sheets than α-helixes [5,17], which is different from Results
subgroups5Aand5BLEAproteinsthatareintrinsicallyun- SiLEA14isanatypicalLEAprotein
structured [17-19]. For example, Arabidopsis LEA14 has an The full-length sequence of SiLEA14 was determined by
αβ-fold consisting of one α-helix and seven β-strands that 5′ and 3′ rapid amplification of cDNA ends (RACE)
form two antiparallel β-sheets as determined by nuclear [GenBank: KJ767551]. The sequence is 821 bp in length,
magnetic resonance spectroscopy [16]. Moreover, subgroup witha 100bp5′ untranslatedregion(UTR) and a 208bp
5C LEA proteins have other outstanding characteristics, 3′ UTR (with polyA tail) (Additional file 1A). SiLEA14
such as lower instability index, narrower range of GRAVY harbors two exons separated by an intron (Additional file
values,andlowerproportionofpolar(hydrophilic)andsmall 1B), and encodes an open reading frame of 170 aa with a
residues, but higher proportion of non-polar residues than predictedmolecularmassof18.77kDandpIof5.56.Itis
subgroup 5A and 5B members [5]. All of these differences richinSer(10.6%),Lys(8.8%),andIle(8.2%),butcontains
indicatethatsubgroup5CLEAproteinsmayfunctiondiffer- low quantities of Trp (1.2%), Asn (1.8%), Cys (1.8%), and
entlyfromotherLEAproteinsinplants. Gln(1.8%).Threehydrophobicregions(I,II,andIII)were
At present, only a small number of subgroup 5C LEA identified in the SiLEA14 protein (Additional file 1C). A
genes have been characterized. Their transcripts can be motif search of the SiLEA14 protein in InterProScan
upregulatedinresponsetodiversestresses,asreportedfor revealed that it contains a “LEA_2” motif (PF03186, 3.4e-
cottonLEA14-A[15],CraterostigmaplantagineumPcC27- 20),whichwasclassifiedintosubgroup5C(D-95)accord-
45 [20], soybean D95-4 [21], tomato ER5 [22], hot pepper ing to Battaglia’s classification of LEA proteins [12].
CaLEA6[23],ArabidopsisLEA14[24]andAt2g44060[17], Further analysis showed that SiLEA14 contains a lower
andsweetpotatoIbLEA14[25].OverexpressionofCaLEA6 percentage of polar amino acids (47.2%) and higher
in tobacco improves tolerance to dehydration and NaCl percentage of non-polar amino acids (25.8%) than other
but not to low temperature [23]. Transgenic sweetpotato group LEA proteins (Additional file 1D) [5]. The GRAVY
non-embryogenic calli that overexpress IbLEA14 show value and the instability index of SiLEA14 is −0.155 and
increasedtolerancetodroughtandsaltstressbyenhancing 34.68, respectively (Additional file 1D). All these charac-
lignification[25].Recently,riceOsLEA5hasbeenreported teristics were in accordance with those of other members
to enhanceresistanceagainstdiverseabioticstressesinre- ofsubgroup5C[5].
combinant Escherichia coli cells. In vitro analysis showed Two additional SiLEA14 homologs (Si003774m and
that OsLEA5 was able to protect lactate dehydrogenase Si003233m, Additional file 2) existed in foxtail millet.
from aggregation under different abiotic stresses [5]. All They showed 57.7% and 48.24% identity to SiLEA14, re-
these results suggest that subgroup 5C LEA proteins are spectively. Homologous sequences of SiLEA14 were also
closely associated with resistance to multiple abiotic present in other plant species, such as rice OsLEA5
stresses. (77.06%), maize Lea14-A (50.88%) and Arabidopsis
Foxtail millet (Setaria italica (L.) Beauv.), a member of AtLEA14 (47.06%). The homologs shared different levels
the Poaceae family, has a long history in cultivation of of sequence similarities, indicating different levels of evo-
about 7000 years. It has been widely planted in northern lutionary relationship among these proteins (Figure 1A).
ChinaandotherAsiancountries.Recently,adraftgenome A phylogenetic tree for SiLEA14 and its homologs was
sequenceforfoxtailmillethasbeencompleted[26],which constructed from protein sequences (Additional file 2).
enables foxtail millet to be a tractable experimental grass The proteins weremainly separated into two clades com-
model [27]. As a diploid C panicoid crop species, foxtail prising dicot and monocot species, respectively. The
4
milletiswellknownforitsremarkabledroughtresistance. SiLEA14 protein shared the closest relationship with the
However,themolecularmechanismsunderlyingthistoler- pearl millet LEA-like protein, followed by OsLEA5
ancearenotwelldefined. (Figure 1B). Sequence logos were produced to examine the
In this study, we isolated and functionally characterized conservation level at each residue position. The sequences
a novel member of the atypical subgroup 5C LEA gene, lacked significant signature motifs or consensus sequences,
SiLEA14, from foxtail millet. The expression of SiLEA14 except for several conserved residues at specific positions,
wasinducedbyABA,polyethyleneglycol(PEG)andNaCl. such as proline at positions 41 and 83 (Additional file 3).
Overexpression of SiLEA14 resulted in enhanced resist- Thisisacommonfeatureofsubgroup5CLEAproteins[5].
ance to abiotic stresses in E. coli, Arabidopsis and foxtail
millet. The SiLEA14 promoter mediated remarkable in- SubcellularlocalizationofSiLEA14
duction of β-glucuronidase (GUS) expression in trans- To investigate the potential role of SiLEA14, we examined
genic Arabidopsis under various stresses. Cis-acting the subcellular localization of SiLEA14 fused to GFP and
regulatory elements in the SiLEA14 promoter were also GFP alone (as a control) in onion epidermal cells and
predicted. These data reveal the potential application of foxtail millet root protoplasts. The constructs used were
SiLEA14inthegeneticengineeringofothercrops. shown in Figure 2A. When observed by confocal
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Figure1MultiplesequencealignmentandphylogeneticrelationshipfortheSiLEA14proteinanditshomologs.(A)Multiplesequence
alignmentofSiLEA14withitshomologsfromvariousplantspecies.Theconserved“LEA_2”motif(PF03168)isboxed.(B)Phylogenetic
relationshipsofSiLEA14anditshomologs.Thedivergenceofthecladesbetweenthemonocotsanddicotsishighlighted.SiLEA14anditstwo
homologsinfoxtailmilletarehighlightedbysoliddarkcircleandtriangle,respectively.TheGenBankaccessionnumbersareasfollows:SiLEA14
(Setariaitalic,KJ767551),AtLEA14(Arabidopsisthaliana,NM_100029),Lea14-A(Zeamays,NM_001159174),D95-4(Glycinemax,U08108),IbLEA14
(Ipomoeabatatas,GU369820),ER5(Solanumlycopersicum,U77719),Lemmi9(Solanumlycopersicum,Z46654),CaLEA6(Capsicumannuum,
AF168168),OsLEA5(Oryzasativa,JF776156),pcC27-45(Craterostigmaplantagineum,M62990),pcLEA14(Pyruscommunis,AF386513),At1g01470
(Arabidopsisthaliana,BT015111),LEA14-A(Gossypiumhirsutum,M88322),Lea14homolog(Glycinemax,NM_001251780),At2g46140(Arabidopsis
thaliana,NM_130176),Os01g0225600(Oryzasativa,NM_001048996),LEA14-A-like(Brachypodiumdistachyon,XM_003567779),LOC100274480(Zea
mays,NM_001148839),SORBIDRAFT09g029860(Sorghumbicolour,XM_002441543),LEA-likeprotein(Cenchrusamericanus,AY823547),OsI21161
(Oryzasativa,CM000130),Os05g0526700(Oryzasativa,NM_001062639),Os05g0584300(Oryzasativa,NM_001062985),At2g44060(Arabidopsis
thaliana,BT024723),LOC100285131(Zeamays,EU970969)andumc2111(Zeamays,NM_001155750).Thesequencesusedforalignmentand
phylogenetictreeconstructionwereshowninAdditionalfile2.
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Figure3SiLEA14expressionpatternsinfoxtailmillet
determinedbyqRT-PCR.(A)Tissue-specificexpressionofSiLEA14.
TotalRNAwasextractedfromroots(R),stems(S),leaves(L),
inflorescences(I)andseedsat5(S1),15(S2)and25(S3)daysafter
pollination,respectively.FoxtailmilletActin7wasusedasaninternal
control.(B-D)SiLEA14transcriptsaccumulationinresponsetovarious
treatments.Two-week-oldfoxtailmilletseedlingsweretreatedwith
100μMABA(B),20%(v/v)PEG6000(C)and250mMNaCl(D)forthe
indicatedtimes,respectively.TotalRNAwasextractedfromtheabove-
groundpartsoftenplantsattheindicatedtimesafterthetreatments.
Datarepresentmeansandstandarderrorsforthreebiologicalreplicates.
Figure2SubcellularlocalizationofSiLEA14protein.(A)Diagram
oftheconstructsofpROK219-GFPandpROK219-SiLEA14-GFPusedfor
The expression level of SiLEA14 was also examined by
subcellularlocalization.35S-Pro,cauliflowermosaicvirus35Spromoter;
qRT-PCR under various stresses. Under ABA treatment,
SiLEA14,theSiLEA14codingregionwithoutthestopcodon;GFP,
greenfluorescentprotein;NOS,nopalinesynthaseterminator. SiLEA14 transcription abruptly reached its highest level
(B-C)FluorescentmicroscopicimagesofGFPandSiLEA14-GFPfusion (six-fold)after1htreatment,andthendecreasedgradually
proteinintheonionepidermalcells(Bar=200μm)(B)andinthe andrevertedalmosttothecontrollevelat24h(Figure3B).
foxtailmilletrootprotoplasts(Bar=10μm)(C).
Under PEG stress, SiLEA14 transcription was rapidly
inducedwithin0.5h andincreased to apeaklevel (8-fold)
microscopy, the green fluorescent protein (GFP) fluores- at 12 h (Figure 3C). Salt treatment resulted in increased
cence of SiLEA14-GFP was distributed throughout the accumulation of SiLEA14 to a maximum level (11-fold)
cell, consistent with the GFP control, both in the onion only after 3 h (Figure 3D). These results indicated that
cells (Figure 2B) and the foxtail millet root protoplasts SiLEA14 might play important roles in the responses to
(Figure 2C). These results indicated that SiLEA14 was saltanddroughtstresses.
localizedinthecytosol.
SiLEA14enhancessalttoleranceintransformedE.coli
SiLEA14expressionprofilesundernormalandstress In both prokaryotes and eukaryotes LEA proteins might
conditions have similar protective mechanisms [28]. Therefore, the
Quantitative reverse-transcription PCR (qRT-PCR) was up-regulationofSiLEA14inresponsetoNaClpromptedus
carried out to reveal the temporal and spatial expression to evaluate the salt stress tolerance of recombinant E. coli
of SiLEA14 in foxtail millet (Figure 3A). SiLEA14 was overexpressingSiLEA14(Figure4).Therearenosignificant
expressed at the highest level in roots, followed by stems differences in colony number between transformed E. coli
and leaves, and at the lowest level in inflorescences. harboring SiLEA14 and the control under normal condi-
After pollination, SiLEA14 transcription was gradually tions, indicating that overexpression of SiLEA14 did not
upregulated with seed maturation, which indicated a affect the growth of E. coli recombinants in non-stress
potential role for SiLEA14 in the maturation and desic- conditions. However, when grown on Luria–Bertani (LB)
cation phasesoffoxtailmillet seed development. plates supplemented with 600 mM NaCl, the number of
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Figure4AssayforsaltstresstoleranceofSiLEA14transformedE.coli.TransformantE.coli(BL21)withpET30a-SiLEA14weregrownonLB
plateswithorwithout600mMNaClandKClfor12and24h,respectively.TransformantwithemptyvectorpET30a(+)wasusedasacontrol.The
numberofcoloniesontheplatewascalculated.The200×,500×,1000×,2000×and4000×representthedilutionfold.Bar=1cm.Datarepresent
meansandstandarderrorsforthreebiologicalreplicates.StatisticalsignificancewasdeterminedbyStudent’st-test.**P<0.01.
transformed cells was much higher than that of the OverexpressionofSiLEA14increasesthesalttoleranceof
control.AsimilarresultwasobtainedonLBmediumsup- transgenicfoxtailmillet
plementedwith600mMKCl.Theseresultsdemonstrated To further analyze the function of SiLEA14, it was
that overexpression of SiLEA14 in E. coli significantly transformed into foxtail millet under the control of ubi-
enhancedtolerancetosaltstress. quitin promoter (Figure 6A). The SiLEA14 integration
was confirmed by genomic PCR using two pairs of
primer sets specific to Hpt and 35S promoter, respect-
SiLEA14improvestheabioticstressresistanceof ively (Additional file 4). QRT-PCR analysis showed
transgenicArabidopsis higher expression level of SiLEA14 in transgenic lines
ToassessthefunctionofSiLEA14inplants,itwasoverex- L68, L76, and L78, which showed a 3:1 segregation
pressed in Arabidopsis under the control of Super pro- ratio at T generation, than that of the WT (Figure 6B).
1
moter (Figure 5A). At least 30 transgenic plants were The T generation of these three lines were used for
2
obtained,andtwoindependenthomozygousT transgenic further analysis.
3
lines (L6 than L9) with high expression levels of SiLEA14 First, we examined the salt tolerance of transgenic fox-
(Figure5B)werechosenforfurtherinvestigation. tailmilletduringgermination,andtheresultswereshown
Under non-stress conditions, no significant differences in Figure 6. Compared with the WT plants, transgenic
in the fresh and dry weights were observed between the lines showed better growth performance (Figure 6C) with
WT and transgenic plants (Figure 5C and D). However, enhanced shoot and root growth(Figure6E and G) when
whengrownonMurashigeandSkoog(MS)mediumsup- germinatedinwaterfor4days.Undersaltstressfor4days,
plemented with 125 mM NaCl, the transgenic seedlings the transgeniclinesshowed significantly longershoot and
showed significantly larger cotyledons and longer roots root than the WT (Figure 6E and G), even though the
thanthoseoftheWT,eventhoughgrowthoftheseorgans growth of both WT and transgenic lines were seriously
of the transgenic and WT plants was inhibited compared suppressed (Figure 6C). Similar results were obtained for
with normal conditions (Figure 5C). Consistently, the 9dayssaltstresstreatment(Figure6D,FandH).
fresh and dry weights oftransgenicseedlings weresignifi- Further, we examined the salt tolerance of transgenic
cantly higher than that of the WT (Figure 5E). Similar foxtail millet seedlings in soil. No striking differences in
results were obtained when seedlings were treated with the height were observed between the transgenic and the
250 mM mannitol (Figure 5C and F). These results WT plants under non-stress conditions, but wider leaves
suggest that SiLEA14 may enhance the osmotic stress were observed in the transgenic foxtail millet compared
resistanceoftransgenicArabidopsis. with those of the WT (Figure 7A). This finding is in
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Figure5AssayforsalinityandmannitolstresstoleranceofSiLEA14overexpressionArabidopsis.(A)DiagramoftheT-DNAregionofthe
binaryvectorpS1300-SiLEA14.LB,leftborder;RB,rightborder;35S-Pro,cauliflowermosaicvirus35Spromoter;Hpt,hygromycinBphosphotransferase
gene;Super-pro,Superpromoter;SiLEA14,SiLEA14gene;NOS,nopalinesynthaseterminator.(B)RT-PCRanalysisofSiLEA14intransgenic
Arabidopsislines(L6andL9).WTwasusedasacontrol.Actin2wasaloadingcontrol.(C)ThephenotypeoftransgenicandWTArabidopsis
germinatedonMS,MS+125mMNaClandMS+250mMmannitol,respectively,for7days.Thisexperimenthadthreereplicates,andeach
experimentcomprised30–40plants.Bar=1cm.(D-F)Fresh/dryweightsofseedlingsgerminatedonMS,MS+125mMNaClandMS+
250mMmannitol,respectively.Datarepresentmeansandstandarderrorsforthreebiologicalreplications.Statisticalsignificancewas
determinedbyStudent’st-test.*P<0.05;**P<0.01.
accordance with the result that transgenic lines showed accumulate several metabolites, such as amino acids (e.g.,
longer shoot and root lengths when germinated in water proline), sugars, and sugar alcohols (e.g., mannitol and
compared with the WT (Figure 6C–H). Under 150 mM trehalose), to prevent detrimental changes caused by
NaCl stress for 6 days, the first and second leaves of WT severe osmotic stress [30,31]. Therefore, we measured
plantsbecamecurled,whereastherewasnoobviouseffect the electrolyte leakage and change in free proline and
onthetransgeniclines. TheWTplants wereshorterthan soluble sugar contents in control and SiLEA14 trans-
those of the transgenic lines. Under 250 mM NaCl stress genic lines with or without salinity stress, respectively.
for6days,thegrowthofWTplantswasseverelyinhibited Under optimal or salt-stress conditions, the amount of
andleaveswerecurled,bleached,andsenescent.However, electrolyte leakage in the transgenic lines was signifi-
the transgenic lines showed healthy growth except that cantly lower than that of the WT plants (Figure 7B).
onlyafewoldleavesbecamebleached(Figure7A). This finding is in agreement with the result that the
Electrolyte leakage always occurs following membrane SiLEA14 transgenic lines showed enhanced germination
damage under salinity stress [29]. Furthermore, plants and growth compared with the WT under both normal
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Figure6(Seelegendonnextpage.)
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(Seefigureonpreviouspage.)
Figure6AssayforNaCl,PEGandABAstressonSiLEA14overexpressionfoxtailmilletgermination.(A)DiagramoftheT-DNAregionofthe
binaryvectorpCOU-SiLEA14-flag.LB,leftborder;RB,rightborder;35S-Pro,cauliflowermosaicvirus35Spromoter;Hpt,hygromycinBphosphotransferase
gene;Ubi-pro,maizeUbiquitinpromoter;SiLEA14-flag,SiLEA14genefusedwithaflagtag;NOS,nopalinesynthaseterminator.(B)QRT-PCRanalysisof
SiLEA14intransgenicfoxtailmilletlines(L68,L76andL78).Actin7wasusedasaninternalcontrol.(C-D)ThephenotypeoftransgenicandWTfoxtail
milletundervariousabioticstresstreatmentduringthegerminationstage.TheT seedssoakedinwater(ascontrol)orinwatercontaining150mM
2
NaCl,250mMNaCl,10%PEG,20%PEGand10μMABAfor1dayat30°CandthenplacedonthefilterpaperinaPetridishwetwiththesame
solutionsmentionedabovefor4and9days,respectively.Thisexperimenthadthreereplicates,andeachexperimentcomprised30–40plants.
Bar=1cm.(E-F)TheshootlengthoftransgenicandWTfoxtailmilletgerminatedunderaboveconditionsfor4and9days,respectively.(G-H)Theroot
lengthoftransgenicandWTfoxtailmilletgerminatedunderaboveconditionsfor4and9days,respectively.DatainB,E-Hrepresentmeansand
standarderrorsforthreebiologicalreplicates.StatisticalsignificancewasdeterminedbyStudent’st-test.*P<0.05;**P<0.01.
and salt-stress conditions (Figures 6C–H, 7A). No sig- was significantly more pronounced in the transgenic
nificant differences in free proline and soluble sugar lines than in the WT plants (Figure 7C, D). All of these
contents were observed between the WTand the trans- increasedosmoticprotectantsarebeneficialforprotection
genic lines under normal conditions. Under 150 and of the plants against salt stress. Moreover, we noted that
250 mM NaCl stress, the free proline and soluble sugar thetransgenic line L68demonstrated bettersalt tolerance
contents increased in all plants. However, this increase than the other two transgenic lines (L76 and L78), which
Figure7SalttoleranceofSiLEA14overexpressingfoxtailmilletseedling.(A)ThephenotypeoftransgenicandWTfoxtailmilletunder
normalandsaltstresscondition.Two-week-oldseedlingsinsoilwereirrigatedwithwater,150and250mMNaClsolutionevery3days.After
6days,thephenotypesofthetransgeniclinesandWTwereinvestigated.Thisexperimenthadthreereplicates;andsixtoeightplantsgrownin
oneplotwereusedineachexperiment.Bar=10cm.(B)RelativeelectrolyteleakageinWTandtransgeniclinesaftersaltstress.(C)Proline
contentinWTandtransgenicfoxtailmilletaftersaltstress.(D)SolublesugarcontentinWTandtransgenicfoxtailmilletaftersaltstress.DatainB-D
representmeansandstandarderrorsforthreebiologicalreplicates.StatisticalsignificancewasdeterminedbyStudent’st-test.*P<0.05;**P<0.01.
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correlated withthe higheraccumulation of SiLEA14 tran- and root length of transgenic lines was longer than that of
scripts in L68. All of these results indicated that SiLEA14 the WT no matter germinated in water, 10% or 20% PEG
overexpressing foxtail milletis more tolerant tosalt stress (Figure 6E and G). When germinated in water or PEG for
comparedwiththeWT. 9days,similarresultswereobtained(Figure6D,FandH).
TheobservationthatSiLEA14improvedosmoticstress
OverexpressionofSiLEA14increasesdroughtresistanceof resistance during germination prompted us to investi-
transgenicfoxtailmillet gate the drought resistance of transgenic foxtail millet
Wealsoexaminedthedroughtresistanceoftransgenicfox- seedlings in soil. As it is shown in Figure 8A, no signifi-
tail millet during germination. When germinated in water cant differencesintheheightwere observedbetween the
for 4 days, the transgenic lines showed better growth transgenic lines and the WT when watered well, except
performance than WT as described above (Figure 6C, E that the leaves of the transgenic lines were wider. How-
and G). Compared with in water, no obvious suppression ever, under drought stress for 7 days, more than 90% of
under 10% PEG stress but serious suppression under 20% WT leaves, but less than 20% of transgenic plant lines
PEGstressfor4dayswereobserved(Figure6C).Theshoot leaves, became curled and wilted. After rewatering, 74–
Figure8DroughttoleranceofSiLEA14overexpressionfoxtailmilletinsoil.(A)ThephenotypesofWTandtransgenicfoxtailmilletunder
normalcondition,droughtstressfor7daysandafterrewateringfor3days.Two-week-oldseedlingswereused.Sixtotenplantsgrowninone
plotwereusedineachexperiment.Thisexperimenthadthreereplicates.Bar=10cm.(B)ThesurvivalrateofWTandtransgenicfoxtailmillet
plantsin(A)underdroughtstress.(C)FreeprolinecontentinWTandtransgenicplantsafterdroughtstress.(D)SolublesugarcontentinWTand
transgenicplantsafterdroughtstress.DatainB-Drepresentmeansandstandarderrorsforthreebiologicalreplicates.Statisticalsignificancewas
determinedbyStudent’st-test.*P<0.05;**P<0.01.
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100% plants of trangenic lines survived in contrast to was performed.No visibleGUS activity wasnoted for the
only14%ofWT(Figure 8B). control.However,inductionofGUSactivitywasobserved
The change in free proline and soluble sugar contents with ABA, PEG or NaCl application for 3 to 18 h,
in control and SiLEA14 transgenic lines with or without although the GUS signals were not particularly strong
drought stress was examined. No significant differences (Additional file 5). Nevertheless, collectively these results
in free proline and soluble sugar contents were observed suggest that the selected SiLEA14 promoter sequence is
between the WT and the transgenic lines under non- enough to regulate ABA, PEG and NaCl induction of the
stress conditions. Under drought stress for 3 days, the gene.
free proline and soluble sugar contents increased in all
WT and transgenic plants. However, a significantly SiLEA14promotercontainsstress-associatedcis-elements
higher increase in free proline content was observed in To characterize the mechanism of SiLEA14 function, a
the transgenic lines L68 and L78 when compared to the 1273 bp SiLEA14 promoter fragment (without the 96 bp
WT. In addition, compared with the WT, the soluble 5′UTR) was subjected to a putative cis-acting regulatory
sugar content increased significantly in the transgenic element search using the Plant Cis-acting Regulatory
lines L68 and L76 (Figure 8C, D). Taken together these DNA Elements (PLACE) database [32]. Cis-acting ele-
results indicate that SiLEA14 overexpressing foxtail ments reported to be involved in ABA- and dehydration-
millet showed improveddroughtresistance. mediatedgeneexpressionwereidentified(Table1).Absci-
sic acid-responsive elements (ABREs) are common motifs
GUSactivityisinducedbyvariousstressesinproSiLEA14:: for ABA- and dehydration-mediated regulation of tran-
GUStransgenicArabidopsis scription[33-35].IntheSiLEA14promoter,sixABRE-like
The putative SiLEA14 promoter region was isolated from sequences and four ABA- and dehydration-responsive
the foxtail millet genome using PCR. This fragment con- ACGT-box motifs [34,36] were identified. A dehydration-
sisted in nt −1273 to +96 (transcription start site is +1) responsive element (DRE)-like site, as a cis-acting element
upstreamofSiLEA14codingsequence.TheSiLEA14pro- involvedinABAinduction,drought,salinity,orcoldstresses
moter was fused to the GUS gene and transformed into [37,38],wasalsoidentifiedatposition−158.MYCandMYB
Arabidopsis. Then, qRT-PCR was carried out to examine binding sites have been identified as regulatory elements
the GUS expression level under various stresses. As it is associated with ABA, drought, or cold responsiveness in
showninFigure9,GUStranscriptionwasup-regulatedto plants [34,39-43]. In the SiLEA14 promoter, as many as 10
its highest level (about 3- and 8-fold increases) after 6 h andthreeMYC-andMYB-likesequences,respectively,were
treatment with ABA and NaCl, respectively. For PEG observed.TheseresultssuggestthatSiLEA14maybeunder
stress,GUSexpressiongraduallyincreasedandpeaked(an the regulation of DREB-, DBF-, CBF-, MYC- or MYB-like
almost 12-fold increase) after 18 h treatment. To further transcription factors as in Arabidopsis and rice. In addition,
confirm these results, a histochemical GUS-staining assay other regulatory cis-acting elements were identified in the
SiLEA14 promoter, such as low temperature-responsive,
pathogen-, defense- and wounding-associated elements.
However, whether SiLEA14 participates in these abiotic or
bioticstressesawaitsfurtherinvestigation.
Discussion
Typical LEA proteins can retain water molecules and
protect other proteins from aggregation or desiccation
because of their highly hydrophilic properties [44].
Conversely, atypical LEA proteins have higher content
of hydrophobic residues than typical LEAs. The latter
have been speculated to be involved in diverse stress
tolerances, although few studies have been carried out
Figure9ExpressionofGUSgeneinproSiLEA14::GUStransgenic tocharacterizetheirfunctions[5,23,25].Here,wereported
ArabidopsisuponvariousstressesdeterminedbyqRT-PCR.
theidentificationandcharacterizationofSiLEA14,anovel
One-week-oldseedlingsweretreatedwith100μMABA,250mMNaCl
atypicalLEAmember,infoxtailmillet.
and20%(m/v)PEG6000solutionfor0,3,6and18h,respectively.Total
RNAwasextractedfromatleast30wholeseedlingspertreatment.The As a key phytohormone, ABA plays an important role
expressionofGUSinproSiLEA14::GUStransgenicArabidopsiswas in plant stress responses. Osmotic stress-regulated genes
determinedbyqRT-PCRusingGUS-specificprimers(Additionalfile4). can be activated through both ABA-dependent and
Actin2wasusedtonormalizethegeneexpression.Datarepresent
ABA-independent pathways [45]. However, it is consid-
meansandstandarderrorsforthreebiologicalreplicates.
ered that stress-signalling pathways for the activation of