Jun Chen ,Yan Gong ,Yuan Gao ,Yongin Zhou ,Ming Chen ,Zhaoshi Xu ,Changhong Guo ,Youzhi Ma a,,*

a Key Laboratory of Molecular and Cytogenetics,College of Life Science and Technology,Harbin Normal University,Harbin 150025,Heilongjiang,China

b The National Key Facility for Crop Gene Resources and Genetic Improvement,Key Laboratory of Biology and Genetic Improvement of Triticeae Crops,Ministry of Agriculture,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

ABSTRACT NAC family transcription factors (TFs) are important regulators in plant development and stress responses.However,the biological functions of NAC TFs in wheat are rarely studied.In this study,43 putative drought-induced NAC genes were identified from de novo transcriptome sequencing data of wheat following drought treatment.Twelve wheat NACs along with ten known stress-related NACs from Arabidopsis and rice were clustered into Group II based on a phylogenetic analysis. TaNAC48,which showed a higher and constitutive expression level in Group II,was selected for further investigation.TaNAC48 transcript was up-regulated by drought,PEG,H2O2 and abscisic acid (ABA) treatment and encoded a nuclear localized protein.Overexpression of TaNAC48 significantly promoted drought tolerance with increased proline content,and decreased rates of water loss,malondialdehyde (MDA),H2O2 and O2- content.Root length and a stomatal aperture assay confirmed that TaNAC48-overexpression plants increased sensitivity to ABA.Electrophoretic mobility shift assay (EMSA) and luciferase reporter analysis indicated that TaAREB3 could bind to a cis-acting ABA-responsive element (ABRE) on TaNAC48 promoter and activate the expression of TaNAC48.These results suggest that TaNAC48 is essential in mediating crosstalk between the ABA signaling pathway and drought stress responses in wheat.

Keywords:TaNAC48 Drought tolerance NAC transcription factor Transgenic wheat ABA

1.Introduction

Over evolutionary time,plants developed a variety of physiological,biochemical and molecular response mechanisms to survive environmental changes.Drought is one of the most severe environmental factors restricting crop survival and yield by affecting root hydraulic conductance and aboveground biomass production[1].Wheat(Triticum aestivumL.)is one of the most important staple food across the world.It is mainly grown in arid and semiarid areas and drought stress is one of the main factors affecting wheat yield and growth [2].

In order to adapt to the harm caused by abiotic stress,plants modify the levels of multiple phytohormones.Abscisic acid (ABA)is one of the most studied plant hormones and it functions directly in plant adaptation to abiotic stresses by regulating stomatal closure and inducing antioxidant defense [3].In Arabidopsis,endogenous ABA level increases rapidly under abiotic stress such as drought and salinity.In the presence of ABA,the receptor protein pyrabactin resistance 1 (PYR1)/PPYR1-LIKE (PYL)/regulatory components of ABA receptor (RCAR) form a complex with clade A protein phosphatase 2C (PP2C) and inhibit its activity [3–6].The repression of PP2C activates downstream targets such assucrose non-fermenting 1-related protein kinase 2(SnRK2) [7,8],which further trigger ABA-dependent genes such asABA-responsive element-binding protein(AREB)/ABRE-binding factor(ABF)transcription factors(TFs)containing a basic leucine zipper structure[9,10].A recent study indicated that the promoter regions of many ABAinducible genes contain a conservedcis-acting element called the ABA-responsive element (ABRE),and ABREs are recognized by AREB/ABF [11].

Transcriptional regulation is a crucial mechanism in protecting crops from environmental stress.Several TFs previously identified as necessary for stress tolerance have been verified to participate in signal transduction and transcriptional regulation of some other stress-related genes [12].NAC(NAM,ATAF1/2,andCUC2) is one of the largest families of plant-specific TFs.Most NAC proteins have a highly conserved NAC domain at the N-terminus and a diverse transcriptional activation domain at the C-terminus [13].A genome-wide analysis of theNACTF family in Arabidopsis and rice had identified 151 non-redundantNACgenes in rice and 117 in Arabidopsis [14].According to the phylogenetic analysis of majorNACgenes of Arabidopsis,rice,lycophytes and moss,NAC proteins are classified into six categories [15],and they are involved in physiological and developmental processes including secondary wall synthesis[16,17],seed germination[18],cell division[19],lateral root development[20],flowering[21,22],leaf senescence[23]and abiotic/biotic stress responses [15].Multiple stress responses ofNACgenes are under the control of the central ABA perception and signaling module,such asANAC019,ANAC055,andANAC072/RD26,which play synergistic or antagonistic roles in ABA signaling and osmotic stress responses [24,25].OsNAC6showed high sequence similarity toANAC019,ANAC055,andANAC072and was induced by cold,high salinity,and drought [26].OsNAC6 along with the other four stress-responsive NAC proteins OsNAC3,OsNAC4,OsNAC5,and SNAC1,were clustered in the same SNACA subgroup [15].OsNAC5andOsNAC6were strongly induced by ABA and three ABA-responsive elements (ABREs) were identified in theOsNAC5andOsNAC6promoters [27].The overexpression ofOsNAC5,OsNAC6,andSNAC1could enhance drought resistance of rice.

Despite extensive research ofNACgenes in Arabidopsis and rice,their regulating effects under abiotic stress in wheat are still poorly understood.A few wheat stress-responsive NAC genes have been cloned,such asTaNAC2andTaNAC29,and their functions in stress tolerance were confirmed in Arabidopsis [28,29].Overexpression ofTaNAC2andTaNAC29resulted in enhanced tolerance to drought and salt in Arabidopsis.Recently,a total of 260 NAC-domain proteins were identified in the wheat genome and a drought responsive gene,TaSNAC8-6A,which is tightly associated with drought tolerance in wheat,was cloned and characterized via linkage disequilibrium (LD)-based association mapping [30].In this study,we identified 43 NAC genes from a comparison between transcriptome sequencing data of wheat with and without drought treatment.Further analysis indicated thatTaNAC48positively regulated drought tolerance in wheat seedlings via the ABA signaling pathway.

2.Materials and methods

2.1.Gene isolation and sequence analysis

The wheat droughtde novotranscriptome data was available in the Sequence Read Archive (SRA) under accession number SRP071191 (http://www.ncbi.nlm.nih.gov/sra) [31].Full-length cDNA ofTaNAC48was obtained using gene-specific primers and cloned into the pEASY vector (TransGene,China) for sequencing.Maximum likelihood was used to infer a phylogenetic tree of previously reported NACs and 43 drought-responsive NAC genes found in the wheat drought transcriptome using the MEGA5.1 program.The confidence levels of monophyletic groups were estimated using bootstrap analyses with 1000 replicates [32].

2.2.Plant materials and stress treatments

The wild-type (cv.Fielder) and transgenic wheat were planted in flowerpots containing peat soil and grown in controllable greenhouses with 70%relative humidity,at 25/23°C,with a 16 h light/8 h dark photoperiod.Fifteen-day-old WT seedlings were dehydrated.Seedlings were placed on filter paper to induce rapid drought and sampled at 0,0.5,1,2,4,8,12,and 24 h.The sampled seedlings were immediately frozen in liquid nitrogen and stored at -80 °C prior to RNA extraction.

In order to make WT and transgenic plants grow under the same soil moisture conditions,transgenic plants and wild-type(WT) plants were seeded in the same pot in a controllable greenhouse.For drought tolerance analysis,transgenic plants and WT wheat seedlings were grown normally in a controlled greenhouse for 3 weeks(until seedlings were at the 3-leaf stage)and were subjected to drought treatment until wilting and significant differences were observed in transgenic and WT plants,and then plants were rehydrated.All drought treatments were independently repeated three times.

2.3.RNA extraction and qRT-PCR analyses

Total RNA was extracted with Trizol reagent in accordance with the manufacturer’s instructions (TIANGEN,China),and total RNA was treated with DNase I (TaKaRa,Japan) to remove genomic DNA contamination.The first strand of cDNA was then synthesized using PrimeScript first strand cDNA synthesis kit,and qRT-PCR was performed using the PrimeScript RT reagent kit (TaKaRa)in accordance with the manufacturer’s protocol.Specific primers were designed according to NAC-related genes and stress response genes in wheat using Primer Premier 5.0.The wheat TaActin gene was used as a control (RT-TaActin).An ABI Prism 7500 real-time PCR system (Applied Biosystems,Foster City,CA) was used for qRT-PCR.Quantitative analyses of data were performed as previously described [33].

2.4.Generation of transgenic wheat

In order to generateTaNAC48transgenic wheat plants,the coding region (CDS) ofTaNAC48was inserted into pMWB110 vector[34],a plant transformation vector driven by the ubiquitin promoter ofZea mays,to produce wheat overexpression plants.Genetic transformation ofTaNAC48was carried out using anAgrobacterium-mediated transformation system.To isolate positive transgenic wheat lines,RNA was isolated from leaves of transgenic wheat seedlings at 15 days of age,and qRT-PCR analysis was conducted.All results were confirmed by sequencing,homozygous T3 seeds of transgenic lines were obtained,and phenotypic analysis was performed.Primers used in these studies are shown in Table S1.

2.5.Measurements of water loss rate,malondialdehyde (MDA),H2O2, and proline content

For the analysis of physiological characteristics,before measurements,wheat seedlings at the 3-leaf stage were treated for about 12 days under drought conditions,and about 0.1 g of wheat leaves were taken for the determination of physiological parameters.The water loss was measured according to the method described previously [35].The contents of MDA and proline were measured with an assay kit (Comin,Beijing,China) according to the manufacturer’s protocols.Thecontents were measured following the protocol of the superoxide anion content detection kit(BC1295;Solarbio Life Science).The H2O2contents were measured following the protocol of the H2O2content detection kit (BC3595;Solarbio Life Science).All measurements were from three biological replicates,and ANOVA was used for statistical analysis.

2.6.Stomatal aperture analysis

Under 20,000 lx high light intensity,seven-day-old wheat leaves were incubated in stomatal-opening solution for 4 h.The leaves were then transferred to a solution containing different concentrations of ABA for 2 h(0,15,or 20 μmol L-1).The epidermis of the blade was then removed and placed on a glass slide for observation with a laser confocal microscope [36].

2.7.Effects of ABA treatment on wheat growth

Seedlings at the germination stage for WT andTaNAC48-OE lines in the same hydroponic box were separately treated with exogenous ABA at 0,2.5,or 5 μmol L-1.The hydroponic boxes containing Hoagland nutrient solution were placed in an incubator for 6 days.The root length was measured in the longest primary seminal root of each plant.The shoot lengths of buds were measured in the primary bud of each plant.Each experiment was performed with at least three independent biological replicates.

2.8.Transcriptional activation activity of TaNAC48 and subcellular localization analysis

In the transcriptional activation experiment,the full-length CDS ofTaNAC48was amplified by PCR with specific primers and fused with the Gal4-DNA binding domain of pGBKT7 vector.pGBKT7-EHD1 was used as a positive control [37],and all fusion vectors and empty vectors were transformed into yeast strain Y2HGold.Yeast cells were then cultured on SD/-Trp/-His/-Ade medium containing X-α-Gal.

The CDS ofTaNAC48was inserted into the C-terminal GFP protein driven by the CaMV 35S promoter of the subcellular localization vector pAN580.The protoplast of wheat mesophyll cells was isolated,and pAN580-TaNAC48and nuclear marker EHD4-mCherry[38]were co-transformed into the protoplast for instantaneous expression via the PEG(polyethylene glycol)4000-mediated method.A Leica TCS-SP4 confocal microscope was used for fluorescence detection.

2.9.Preparation of fusion protein and EMSA assay

The CDS ofTaNAC48was inserted into the pGEX-4T-1 vector.The fusion proteins of GST and GST-TaNAC48 were expressed inEscherichia coliTransseta (DE3) and purified by glutathione-Sepharose TM 4B(GE Healthcare,Sweden)according to the manufacturer’s protocol).The biotin-labeled probes used in this experiment were synthesized (AuGCT,China),and their sequences are shown in Table S2.Oligonucleotides were heated at 95 °C for 10 min and then annealed at room temperature to obtain double-stranded DNA.EMSA was detected using The Light Shift Chemiluminescent EMSA Kit(Thermo,USA).In short,2 mg purified fusion protein GST-TaNAC48 or GST protein was added to the binding reaction at 25°C and left standing for 30 min.The DNA was isolated using a 6% polyacrylamide gel and transferred to nylon membranes (Millipore,USA).The signals were detected using an EasySee Western Blot kit.

2.10.Transient luciferase assay in tobacco

In the transcriptional activation experiment,the 1.5-kbTaNAC48promoter fragment was inserted into the LUC reporter plasmid pGreen II-0800,which contained theRenillaluciferase(REN) gene driven by the CaMV 35S promoter.The CDS of wheat TaAREB3 was cloned into pCAMBIA 1305.1 by replacing the GUS reporter gene driven by CaMV 35S.The constructed effectors and report plasmids were transfected into tobacco by injection.Transfected tobacco was cultured in a dark environment for 12 h at 23 °C,followed by a light environment for 24 h.The activity of LUC and REN was determined by a Dual-Luciferase Reporter Assay System (E1910;Promega).Data was collected as the ratio of LUC/REN.All transient expression experiments were repeated three times.

3.Results

3.1.Identification of drought-responsive NAC genes in wheat

To identify drought-inducible NAC genes in wheat,the transcriptome sequencing data of wheat treated with or without drought were analyzed and 43 NACs showing significant up-or down regulation in transcription level (more than a two-fold change)were chosen for further study(Table S1).To study the evolutionary relationships of drought-inducedNACsbetween wheat and other species,a phylogenetic analysis of 43 wheat droughtinduced NACs,six Arabidopsis NACs,and five rice NACs was performed by generating a maximum likelihood phylogenetic tree.The phylogenetic tree classified all NACs into four distinct groups.Twelve drought-induced wheat NACs along with ten known stressrelated NACs from Arabidopsis and rice were clustered into Group II,the other 31 drought-induced wheat NACs and AtNAC2 were clustered into Group I,Group III and Group IV respectively(Fig.1).Phylogenetic analysis also revealed that TaNAC2 and TaNAC5 had the closest relationship with OsNAC6 and OsNAC52,two rice NACs involved in the ABA-dependent stress-signaling pathway.

Among the 43 drought-induced wheatNACgenes,seven genes(TaNAC1-7) exhibited significantly up-regulated expression under drought stress,whileTaNAC11/13/23/26/32had relatively high endogenous expression levels among all drought-regulated NAC genes (Table S1).The expression patterns of these 12 wheatNACgenes under drought stress were further studied.As shown in Fig.2,the transcript levels of TaNAC1-7 were rapidly induced by 0.5 h drought treatment.SomeTaNACslikeTaNAC2,TaNAC5,andTaNAC6,maintained a high expression level while others were restored to their previous level after 12 h drought treatment.In addition,we also investigated the response of theseNACgenes under ABA and H2O2treatments for the essential role the reactive oxygen species(ROS)play in ABA-induced plant drought tolerance.Results showed that the expression ofTaNAC2,TaNAC5,TaNAC11,andTaNAC23were significantly increased after both treatments,and TaNAC2 showed highest levels at 4 h (>7-fold) and 12 h(>16-fold) after ABA and H2O2treatments respectively (Fig.S1).

3.2.TaNAC48 encodes a nuclear localized protein and has transcriptional activation activity

AsTaNAC2was closely related toOsNAC6andOsNAC52and maintained at a high transcript level under drought stress,ABA,and H2O2treatments,we selected it for further investigation.TaNAC2has an open reading frame (ORF) encoding a protein of 301 amino acid which shares a high level of sequence similarity with NAC domain-containing protein 48-like protein inAegilops tauschii(GenBank ID XP_020166636) andHordeum vulgare(Gen-Bank ID KAE8789022) (98% and 94% amino acid sequence identities respectively) and we renamed it asTaNAC48hereafter.To determine whetherTaNAC48has transcriptional activation activity,the full-length CDS ofTaNAC48was fused downstream of the pGBKT7 vector GAL4-binding domain (BD) and transformed into yeast strain Y2HGold.As shown in Fig.3A,similar to the positive control EHD1,all yeast cells carrying the BD-TaNAC48 could grow on a selective medium containing SD/-Trp/-His/-Ade/+X-α-gal,indicating thatTaNAC48could activate the transcription of the reporter genes in yeast (Fig.3A).

Fig.1.Maximum likelihood phylogenetic tree of 43 drought-responsive TaNAC proteins,six AtNAC proteins,and five OsNAC proteins.The phylogenetic tree was generated using MEGA 5.1 software based on amino acid sequence comparison.

To investigate the subcellular localization of TaNAC48,TaNAC48-GFP fusion protein driven byCaMV 35Spromoter was co-transformed with nuclear marker gene into wheat mesophyll protoplasts.The fluorescence of TaNAC48-GFP was detected specifically in the nucleus(Fig.3B–E),suggesting that it may function in the nucleus.

3.3.Overexpression of TaNAC48 significantly improved drought tolerance of transgenic wheat

In order to clarify potential functions,the responses ofTaNAC48under PGE and NaCl were analyzed by qRT-PCR (Fig.S2).TaNAC48was continuously induced by PGE but was not affected by NaCl.To assess the function ofTaNAC48in drought tolerance,we induced the ectopic expression ofTaNAC48with the maize ubiquitin promoter in spring wheat cultivar Fielder through theAgrobacteriummediated transformation method.Three independent transgenic lines with different degrees of high expression level ofTaNAC48were selected based on qRT-PCR results for further analysis(Fig.4A).Under normal growth conditions,TaNAC48-overexpression lines(OE-1,OE-2,and OE-5)were not significantly different from control plants(Figs.4B and S3).After drought treatment,theTaNAC48-OE plants had significantly delayed leaf rolling compared with the control plants.After re-watering,the transgenic plants started to recuperate,while the control plants continuously withered with no signs of recovery.In addition,the fresh weight and dry weight of transgenic plants were significantly higher than control plants and the water loss rate of control plants was higher than transgenic plants(Fig.4C–E).Moreover,the contents of MDA,H2O2,andin transgenic plants were significantly lower than those in control under drought,but the opposite change was shown for the proline content of transgenic and control plants(Fig.4F–I).Therefore,TaNAC48regulated the physiological process of improving drought resistance of transgenic wheat.

3.4.Overexpression of TaNAC48 significantly increased ABA sensitivity of transgenic wheat

To explore the role ofTaNAC48in ABA-mediated drought response,wheat seedlings were grown to the 3-leaf stage and treated with different concentrations of ABA(2.5 and 5 μmol L-1).The results showed that there was no significant difference betweenTaNAC48-OE plants and control plants under normal conditions,while the root length and shoot length ofTaNAC48-OE were significantly shorter than control plants under 5 μmol L-1ABA treatment (Fig.5A–C).These results indicated thatTaNAC48-OE plants increased sensitivity to ABA.

Fig.2.Expression patterns of 12 wheat NAC genes under drought stress.The y-axes are fold changes compared to non-stressed control,and the x-axes are treatment time of 0,0.5,1,2,4,8,and 12 h.The TaActin gene was used as an internal reference.The data represent the means±SDs of three independent biological replicates.

Fig.3.Transcriptional activation activity of TaNAC48 and subcellular localization of its protein.(A)TaNAC48 has transcriptional activation activity.Serial yeast dilutions were grown on SD medium lacking Trp and the same medium containing X-α-gal but lacking Trp,His,Ade.Rice EHD1 gene was used as positive control.(B–E)TaNAC48 was located in the nucleus;TaNAC48-GFP and nuclear marker OsEHD4-mCherry was co-transformed into wheat protoplasts.(B)Signal of TaNAC48-GFP.(C)Signal of OsEHD4-mCherry.(D)Bright field.(E)Merged.Scale bars=20μm.

Fig.4.Overexpression of TaNAC48 confers wheat drought tolerance.(A)The expression level of TaNAC48 in TaNAC48-OE and control plants;the TaActin gene was used as an internal reference gene,and three replicates were performed in each group.(B)Phenotypes of TaNAC48-OE plants(OE-5)and control plants under normal growth and drought conditions.(C) Water loss rate of TaNAC48-OE plants and control plants.(D,E) Fresh weight (E) and dry weight (E) of TaNAC48-OE plants and control plants under drought stress conditions.(F–I)MDA content(F),Proline content(G),H2O2 content(H)and O2-content(I)of TaNAC48-OE plants and control plants under normal growth and drought stress conditions.The data represent the means ± SDs of three independent biological replications.The different letters in the bar graphs indicate significant differences at P <0.05 between TaNAC48-OE and control plants.

3.5.Overexpression of TaNAC48 significantly increased ABA-mediated stomatal response.

According to the water loss rate assay,we found that the water loss rate of transgenic plants was lower than control plants.To investigate the role ofTaNAC48in ABA-mediated stomatal response,we selected 7-day-old detached leaves of control plants and transgenic plants and added different concentrations of ABA(15 and 20 μmol L-1) into the stomatal opening solution for 2 h to observe stomatal closure.Mean stomatal apertures of both control and transgenic wheat leaves decreased after 2 h of ABA treatment.However,with the increase of ABA concentration,guard cells of transgenic plants showed greater sensitivity to ABA-induced stomatal closure than control plants(Fig.5D,E).These results indicated thatTaNAC48could positively regulate ABA-mediated stomatal closure in transgenic wheat leaves.

3.6.TaNAC48 is a potential target of TaAREB3 and positively regulates drought-tolerance relative genes

To further explore howTaNAC48was regulated by ABA signaling,we analyzed the promoter sequences ofTaNAC48using the online tool plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and found three ABA-responsive elements (ABRE).To test whetherAREBTF physically bound to the ABRE motif ofTaNAC48 in vitro,an electrophoretic mobility shift assay (EMSA) was carried out with three biotin-labeled probes(P1–P3)and TaAREB3-GST fusion protein.As shown in Fig.6,wheat TaAREB3 protein caused a mobility shift in the labeled probes,especially probe P1,located 467-bp upstream of ATG whereas no such shift was observed with the GST control protein (Fig.6A,B).

We further used the tobacco transient expression system to evaluate whether this combination could drive the expression ofTaNAC48gene expressionin vivo.The pGreenII-0800 vector carrying the LUC reporter gene driven by theTaNAC48promoter was cotransformed into tobacco with the pCAMBIA1305-TaAREB3overexpression vector.Compared with the control samples with empty vector,LUC/REN ratio ofTaNAC48pro-pGreenII co-transformed with 1305-TaAREB3was significantly increased(Fig.6C).Moreover,the transcript level of five wheat homologs of Arabidopsis drought marker genesAVP,RZF1,NCED3,DREB1AandCOR47containing theNACrecognition core sequence (CACG) in their promoters was investigated (Fig.7).Increased expression ofTaNCED3,TaDREB1AandTaCOR47was observed in overexpression lines indicating thatTaNAC48might enhance wheat drought tolerance by activating the expression of these downstream genes.

Fig.5.Plants overexpressing TaNAC48 are sensitive to ABA.(A)Phenotypes under different concentrations of ABA treatment and measurements of root and shoot length(B,C).(D)TaNAC48-OE and control plants were used to analyze stomatal closure under different concentrations of ABA(0,15,and 20μmol L-1).Bright-field images were taken with a confocal microscope at 20×magnification.Scale bar=20μm.(E)Stomatal apertures of TaNAC48-overexpression plants and control plants under different concentrations of ABA were measured.The data represent the means±SDs of three independent biological replications.The data represent the means±SDs of three independent biological replications.The different letters in the bar graphs indicate significant differences at P<0.05 between TaNAC48-OE and control plants.The asterisks indicate significant differences between TaNAC48-OE5 and control plants(ANOVA,*,P<0.05 and**,P<0.01).

Fig.6.TaAREB3 directly regulates the expression of TaNAC48.(A)The graph shows the structure of the TaNAC48 promoter and P1,P2 and P3 are the binding sites of ABRE ciselements.(B)TaAREB3 binding to the promoter of TaNAC48.Biotin-labeled TaAREB3 probes were incubated with GST or GST-TaNAC48 proteins.(C)TaAREB3 increased TaNAC48 promoter activity in tobacco.TaABRE3 was co-transfected with the TaNAC48 promoter.The LUC/REN ratio indicates the activity of transcription factors on promoter expression levels.The data represent the means±SDs of three independent biological replications.The asterisks indicate significant differences between experimental group and control group(ANOVA,*,P<0.05 and**,P<0.01).

Fig.7.Expression levels of stress response genes regulated by TaNAC48.The y-axes are fold changes,and the x-axes are different lines.The data represent the means±SDs of three independent biological replications.The asterisks indicate significant differences between TaNAC48-OE and control plants (ANOVA,*, P <0.05 and **, P <0.01).

4.Discussion

TheNACTF superfamily has been studied by many researchers using biochemistry and bioinformatics approaches due to its diverse biological functions.Increasing evidence shows thatNACTFs are essential in both biotic and abiotic stress responses in plants [15].However,knowledge about the role of wheatNACsin regulating abiotic responses was still limited.In this study,43 drought-responsiveNACgenes were identified from transcriptome sequencing data from wheat with and without drought treatment.Phylogenetic results showed that 12 wheatNACsalong with ten stress-relatedNACsfrom Arabidopsis and rice were clustered into Group II.Most of these genes exhibited a response to drought stress and acted as positive regulators of drought resistance [39,40].TraesCS5B02G480900 (TaNAC23)and TraesCS5D02G481200 (TaNAC29),two homologous alleles from previously reportedTaNAC2were clustered into Group II on the same branch as riceSNAC1.Overexpression ofTaNAC2andSNAC1both improved plant drought resistance and salt tolerance by enhancing root development and expression of abiotic stressresponse genes [29,41].

Recently,39 wheat stress-responsiveNACTFs were isolated from a total of 260NACdomain containing proteins based on Arabidopsis and rice NAC superfamily phylogenies [30].Consistent with this result,TaNAC48and eight other drought induced wheatNACgenes (TaNAC10,TaNAC15,TaNAC21,TaNAC27,TaNAC30,TaNAC34,TaNAC38 and TaNAC40) belonging to Group II are also members of those 39 TFs.Therefore,it was supposed thatNACsin Group II might be involved in drought response and functions of those eight genes are probably worth determining.

Several stress responsiveNACstended to have pleiotropism and overexpression of theseNACsconferred pleiotropic effects to plant defense and stress tolerance,affecting growth,flowering and senescence.Transgenic rice plants over-expressingOsNAC6showed an improved tolerance to dehydration,high-salt stresses and blast disease at the expense of growth retardation and low reproductive yields [4].Rice plants overexpressingOsNAPhad enhanced tolerance to high salinity,drought and low temperature at the vegetative stage,however,plant senescence was promoted,whereas knockdown ofOsNAPmarkedly delayed senescence [23].In this study,TaNAC48transgenic plants showed no clear differences in plant architecture and grain yield under well-watered conditions.qRT-PCR analysis confirmed thatTaNAC48was highly induced by drought and PEG6000 treatments but not NaCl treatment and no effects ofTaNAC48on salt tolerance were observed in wheat seedlings (data not shown).

In addition,the expression pattern ofTaNAC48under ABA treatment was like the expression pattern under drought and PEG treatments,and the root length,shoot length and stomatal aperture of transgenic plants were more sensitive to ABA.This indicated thatTaNAC48may participate in regulating drought tolerance through the ABA-mediated signaling pathway.ABA is regarded as the primary plant hormone in the plant abiotic stress response.ABAresponsive element binding (AREB) proteins are reported to target a core ABRE motif present in downstream gene promoters [42].Plants overexpressing NACs with ABRE motif-containing promoters such asANAC002/ATAF1[33],ANAC019,ANAC055[39,43] andANAC072/RD26[39,40] show increased drought tolerance based on an ABA-dependent pathway.There are also severalABRE cisacting elements on the promoter ofTaNAC48,and the EMSA and LUC experiments indicated that TaAREB3 could bind directly to the ABRE motifs in promoters ofTaNAC48and promote the expression ofTaNAC48.Relative expression analysis further showed that ABA biosynthesis geneTaNCED3inTaNAC48overexpression lines was transcriptionally elevated,indicating thatTaNAC48might also control ABA synthesis via a positive feedback mechanism.

Overall,we characterizedTaNAC48as a newNACtranscription factor in the ABA-mediated transcriptional regulation in response to drought stress in wheat.Overexpression ofTaNAC48in wheat enhanced sensitivity of the plant to ABA and drought tolerances,but the more detailed mechanism need to be further studied.Also,generation ofTaNAC48and its homologous genes deficient plants using RNA interference or gene editing strategy would be useful to investigate whetherTaNAC48could mediate the ABA-signaling pathway in other aspects of plant growth and stress tolerance.

CRediT authorship contribution statement

Youzhi Ma and Changhong Guo coordinated the project,conceived and designed experiments,and edited the manuscript;Jun Chen performed experiments and wrote the first draft of the manuscript;Yan Gong conducted the bioinformatic work and performed experiments;Jun Chen and Yan Gong generated and analyzed data;Yuan Gao and Zhaoshi Xu contributed with editing the manuscript;Yongbin Zhou and Ming Chen provided analytical tools and managed reagents.All authors have read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Dr.Genying Li from the Shandong Academy of Agricultural Sciences for help with the wheat genetic transformation.This work was supported by the National Natural Science Foundation of China (31701414) and the National Key Research and Development Program of China (2016YFD0101004).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.09.010.