Na Wang,Jun Chen,Yuan Gao,Yongin Zhou,Ming Chen,Zhaoshi Xu,Zhengwu Fang,Youzhi M

a Hubei Collaborative Innovation Center for Grain Industry/College of Agriculture,Yangtze University,Jingzhou 434000,Hubei,China

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

Keywords:TaIPT Phytohormone Abiotic stress Cytokinin biosynthesis Wheat

ABSTRACT Cytokinins(CKs)function in plant development and during stress responses,but their role in drought tolerance in wheat is unknown.In the present study,24 isopentenyltransferase(IPT)genes,encoding ratelimiting enzymes in CK biosynthesis were identified in the wheat genome.The chromosomal locations and structures of the genes,protein properties,and phylogenetic relationships were characterized.ATP/ADP TaIPT genes showed tissue-specific expression.TaIPT2,TaIPT7,and TaIPT8 expression was rapidly induced by 0.5–1 h drought treatments,which decreased to low levels after 2 h drought treatment,as did most other TaIPT genes.TaIPT8-5a/5b/5d triple mutants showed decreased levels of tZtype CK under normal and drought conditions and reduced drought tolerance,which,however,did not manifest as phenotype alterations.By contrast,transgenic wheat plants with drought-induced TaIPT8 showed increased drought tolerance.Our study provides a foundation for further investigation of TaIPT genes and novel insights into the role of CKs in the drought response of wheat.

1.Introduction

Wheat is a staple crop,particularly in arid and semi-arid areas where drought limits plant growth and productivity[1].Evolving in rapidly changing environments,plants have developed sensitive protection systems to respond and adapt to abiotic stressors.Plant hormones,even at minute concentrations,transmit local or longdistance signals in response to stress[2–4].

Although abscisic acid(ABA)has long been recognized as the primary stress-induced hormone,regulating biological activities including stomatal closure and induction of antioxidant defense.Increasing evidence[5–7]suggests that cytokinins(CK)too are involved in responses to abiotic stressors.CKs are a class of phytohormones first characterized as inducers of cell division[8,9],and they are synthesized primarily in growing root tips,from where they are transported to shoots[10,11].Along with auxins,CKs function in multiple biotic processes including inhibition of lateral root initiation,leaf expansion,shoot branching,photosynthesis,floral transition,seed germination,and leaf senescence[12].However,the levels of endogenous CKs and the molecular mechanisms of CK effects during stress are not well understood.Generally,production and transport of endogenous CKs decrease in response to extended periods of stress and result in a high ABA:CK ratio,which influences root-to-shoot signaling under abiotic stress[13–18].But CK levels can be rapidly elevated at the initial stage of stress and continue to increase in response to severe stress[19,20].In plants,CK biosynthesis and degradation are catalyzed by respectively phosphate-isopentenyl transferases(IPTs)and CK oxidase/dehydrogenases(CKXs),which are responsible for the fine-tuning of endogenous CK levels[21–23].Lonely Guy(LOG)converts inactive cytokinin nucleotides directly to the active free bases,while conjugation of CKs with glucose mediated by O-glucosyltransferases(ZOG)leads to rapid CK deactivation.The conjugation is reversible and O-glucosides can be converted back to free bases and ribosides by β-glucosidase(GLU),which is necessary for re-establishment or maintenance of CK homeostasis[21,24,25].The expression patterns of individual members of the IPT,CKX,LOG,ZOG,and GLU multigene families are tissue-and development-specific in wheat[25,26].IPTs act as rate-limiting enzymes in the biosynthesis of CKs,and were first identified in Agrobacterium tumefaciens[27,28].In plants,IPTs mediate two CK synthesis pathways:the tRNA and ATP/ADP pathways.The Arabidopsis genome encodes seven ATP/ADP IPT genes(AtIPT1,3,and 4–8)that control the biosynthesis of isopentenyladenine(iP)-and trans-zeatin(tZ)-type CKs and of two tRNA IPTs(AtIPT2 and 9),which are responsible for the synthesis of cis-zeatin(cZ)-type CKs[29–32].Their orthologs have been annotated or functionally verified in several crop species.Ten OsIPTs,ten ZmIPTs,and six TaIPTs have been isolated from rice,maize and wheat,respectively[26].Expression analyses of plant IPT genes under abiotic stress or during ABA treatment showed that the majority of abiotic responsive IPT genes in Arabidopsis were repressed;however,the contrasting pattern was observed in soybean,cabbage,and apple,in which they were up-regulated before returning to pretreatment levels,and a few were maintained at high expression levels under severe stress conditions[16,33–35].Upregulation of CKXs,which reduces CK levels,may be a common mechanism in plants continuously exposed to a stressor[33–37].

CK and ABA signaling are generally considered antagonistic in regulating seed germination and in response to stressors.In fact,both negative and positive effects of CKs on stress tolerance have been reported in numerous functional studies.In Arabidopsis and tobacco,CK downregulation via overexpression of the CKX gene led to higher drought and salt tolerance accompanied by lower leaf water loss,while under drought conditions,long-term CK elevation by overexpression of IPT or application of exogenous CK resulted in higher water loss,than observed in CKX transformants[16,36,38,39].Transgenic plants expressing an IPT gene through stress-or senescence-induced promoters such as PSARK and PSAG12 were generated[40–45]to examine the effects of increasing CK concentrations on stress tolerance,particularly drought tolerance,and these transgenic plants showed better protection of the photosynthetic system,delayed senescence,and increased redox potential,improving their drought resistance.

The concentrations of endogenous plant CKs are strongly regulated in response to abiotic stressors,and the roles of IPT genes in regulating CK levels have been identified in Arabidopsis and other plants.However,in wheat,the relationship between CKs and stress tolerance remains unclear.The objective of this study was to perform genome-wide identification of IPT genes in wheat and use phylogenetic and expression analyses to characterize the evolution of the wheat IPT gene family and the functions of its members in drought stress responses.Production of transgenic wheat plants with TaIPT8 homologs induced by CRISPR/Cas9 and droughtinducible expression of PNAC48::TaIPT8 was undertaken to investigate the regulatory roles of CK in wheat drought tolerance.

2.Materials and methods

2.1.In silico analysis of IPT genes in wheat

To identify all latent IPT genes in wheat,IPT protein sequences of Arabidopsis and rice were retrieved from the TAIR(https://www.arabidopsis.org)and RGAP(https://rice.uga.edu/)databases and were used as query sequences to search the wheat genome sequence database in Ensembl Plants(https://plants.ensembl.org/Triticum_aestivum/).A BLASTP search was performed,and all proteins with an E-value＜1e-5 were selected.The protein sequences of all putative wheat IPT genes were submitted to the Pfam(https://pfam.janelia.org)and SMART(https://smart.embl-heidelberg.de/)webtools to confirm the presence of a conserved IPPTbinding domain.Sequences lacking the IPPT domain were excluded.In total,24 wheat IPT gene family members were identified.Given that wheat is a hexaploid with three subgenomes(2n=6x=42;AABBDD),we further divided the 24 wheat IPT genes into nine homologous groups,which were designated TaIPTX-YA,TaIPTX-YB,or TaIPTX-YD,where X denotes the homologous group number and Y the wheat chromosome on which it was located.The molecular weights and isoelectric points of the TaIPT proteins were predicted using ExPASy(https://web.expasy.org/compute_pi).

2.2.Chromosomal locations and phylogenetic analyses

TBtools[46]was used to visualize the positions of IPT genes on chromosomes based on the physical location of each gene and the length of the respective chromosomes.The location information of each TaIPT gene was obtained from Ensembl Plants(https://plants.ensembl.org/).All TaIPT genes were mapped to 15 wheat chromosomes.For phylogenetic analysis,multiple sequence alignments were produced using ClustalX[47].The neighbor-joining method was used to construct a phylogenetic tree of 50 IPT proteins in wheat,Arabidopsis,rice and barley using MEGA 7.0[48].The confidence limits of the monophyletic groups in the phylogenetic tree were assessed using bootstrap analyses with 1000 replications.

2.3.Construction of a CRISPR/Cas9-based gene editing and overexpression vector

To generate TaIPT8-5A/5B/5D (TraesCS5A02G460000,TraesCS5B02G469600,and TraesCS5D02G471100)mutants,two sgRNA target sequences were designed according to the conserved exon sequences of TaIPT8-5A/5B/5D using CRISPR-P 2.0(https://crispr.hzau.edu.cn/CRISPR2/).The segment containing the wheat U3 promoter was amplified from the pCBC-MT1T2 vector using a pair of primers containing the two designed sgRNAs and was then cloned into the BsaI site of the CRISPR/Cas9 vector pBUE411[49]using T4 DNA ligase(TransGen Biotech,Beijing,China).

To generate overexpressing plants,the coding regions of TaIPT8-5A were amplified from wild-type(WT)cDNA using the DNA polymerase KOD FX(Toyobo,Osaka,Japan)and were cloned into a pMWB110 vector[50]using an In-Fusion Advantage PCR cloning kit(Clontech Laboratories,Takara,Tokyo,Japan).The recombinant plasmids were transformed into the spring wheat cultivar Fielder using Agrobacterium-mediated gene transformation[51].To generate transgenic plants with drought-inducible TaIPT8 overexpression,the ubiquitin promoter in the pMWB110 vector was replaced with a 1.4-Kb TaNAC48 promoter[52]using two restriction enzymes,HindIII and BamHI.All primer sequences are listed in Table S1.

2.4.Growth conditions and stress treatments

WT and transgenic plants were planted in pots containing peat soil and were grown in a greenhouse with a 16/8 h light/dark photoperiod,70% relative humidity,and 25/23 °C day/night temperature.Fifteen-day-old WT seedlings were dehydrated by placing on filter paper to induce rapid drought,and the seedlings were then sampled at 0,0.5,1,2,4,8,and 12 h.The samples were immediately frozen at-80 °C.

To allow WT and transgenic plants to grow under the same soil moisture conditions,seeds of both plant types were sown in the same pot.For drought-tolerance tests,seedlings were grown in the greenhouse under normal conditions for three weeks after which the drought treatment was applied by ceasing irrigation,and when marked differences in wilting were observed between transgenic and WT plants,all plants were rehydrated.All drought treatments were independently repeated three times.

2.5.RNA extraction and RT-PCR analyses

Total RNA was extracted from tissues using an RNeasy Plant Mini Kit(Qiagen,Valencia,CA,USA)according to the manufacturer’s instructions,and genomic DNA contamination was removed by digestion with DNase I(Takara).The first strand of cDNA was synthesized using the PrimeScript First Strand cDNA Synthesis Kit(Takara).For semi-quantitative RT-PCR,the wheat Actin gene was used as an internal control.Specific primers for TaIPT genes were designed using Premier 5.0(Premier Biosoft International,Palo Alto,CA,USA),based on cDNA sequences.All primer sequences are listed in Table S1.The PCR conditions included denaturation at 94 °C for 4 min,followed by predetermined number of cycles of denaturation at 94°C for 30 s,annealing at 58°C for 30 s,extension at 72 °C for 30 s,and a final extension at 72 °C for 5 min.The numbers of PCR cycles were chosen to ensure an exponential range of amplification.For the amplification of TaActin sequences,18 to 30 cycles were initially tested,and 24 cycles were used consistently thereafter.For TaIPT genes,the numbers of PCR cycles varied from 29 to 35,according to their expression levels.PCR products were separated by electrophoresis on a 1.2%agarose gel and stained with ethidium bromide.qRT-PCR was performed using gene-specific primers and SYBR Premix Ex Taq reagent(Takara)on an ABI 7500 Fast Real-Time PCR system(Applied Biosystems,Foster City,CA,USA).The TaActin gene was used as an internal control.PCR was performed in triplicate for each sample,using two independent biological replicates.

2.6.Detection of phytohormones

Fresh third-leaf samples of WT and transgenic seedlings were harvested 30 days after germination before and after drought treatment(0.5 h);they were immediately frozen in liquid nitrogen,ground to powder,and stored at-80°C until analysis.CK concentrations were detected using MetWare(https://www.metware.cn/)based on the AB Sciex QTRAP4500 LC-MS/MS platform.Three replicates were used per assay.

2.7.Physiological characteristics

To measure malondialdehyde(MDA),H2O2,O2-,and proline concentrations,wheat seedlings at the three-leaf stage were grown for approximately-two weeks under drought conditions,and 0.1 g third-leaf blade was sampled before and after drought treatment.Proline and MDA concentrations were measured using a commercial assay kit(Comin,Beijing,China)according to the manufacturer’s instructions.O2-and H2O2concentrations were measured using a superoxide anion content detection kit(BC1295;Solarbio Life Science,Beijing,China)and an H2O2content detection kit(BC3595;Solarbio Life Science,Beijing,China),respectively.Dry and fresh weights of WT and transgenic plants were recorded after one week of rehydration,and water loss was measured as previously described[53].Measurements were performed using three biological replicates,and ANOVA was fitted using SPSS 19.0(IBM Corp.,Armonk,NY,USA).

3.Results

3.1.In silico identification of IPT genes in wheat

To identify wheat IPT genes,a BLAST search using the amino acid sequences of Arabidopsis,rice,and barley IPT genes as queries was performed in the wheat DNA database.Twenty-four nonredundant putative wheat IPT sequences were identified and were designated according to their chromosomal order and genetic relationships(Figs.1A,S1).The 24 TaIPT genes were distributed widely and unevenly on all wheat chromosomes(Fig.S1)except 4A,4B,4D,6A,6B,and 6D.Sequence and phylogenetic analyses revealed that the 24 TaIPTs constituted nine sets,with each set including three homoeologous genes in the A,B,and D subgenomes,except for TaIPT1 and TaIPT2.TaIPT1-1A/1B had no homologs in the D subgenomes,and TaIPT2-1B had no homologs in either the A or D subgenomes(Figs.1A,S1).Each predicted gene was submitted to the Pfam database(https://pfam.janelia.org)to confirm the presence of domain signatures,and all 24 TaIPT proteins were predicted to contain one or two IPP transferase domains(PF01715;Fig.1B;Table 1).Amino acid sequence lengths of the 24 TaIPT proteins ranged from 280 to 466,the isoelectric points ranged from 5.10 to 10.32,and the molecular weights ranged from 30.64 to 52.18 kDa(Table 1).Among these 24 TaIPTs,TaIPT4-2A,TaIPT4-2B,and TaIPT4-2D contained many exons(11,11,and 10,respectively);TaIPT1-1A contained two exons,and the remaining 20 TaIPT members lacked introns(Fig.1C).

Table 1 IPT genes identified by in silico analysis in wheat.

Fig.1.Phylogenetic relationships,conserved domains,and gene structures of TaIPTs.(A)The unrooted phylogenetic tree was constructed using MEGA 7.0 by the neighborjoining method with 1000 bootstrap replications.(B)TaIPTs contain one or two IPPT domains.(C)Exon–intron structure of TaIPTs.Blue boxes indicate untranslated 5′-and 3′-regions;yellow boxes indicate exons;black lines indicate introns.

Fig.2.Phylogenetic relationship of IPTs.The phylogenetic tree was generated using MEGA 7.0 software based on multiple alignments of amino acid sequences from Arabidopsis,rice,barley,and wheat.

3.2.Phylogenetics of the IPT gene family in Arabidopsis,rice,and wheat

A phylogenetic analysis of 24 wheat TaIPTs,nine Arabidopsis AtIPTs[29],ten rice OsIPTs[54],and seven barley HvIPTs[55]was performed by generating a phylogenetic tree that classified all IPTs into four major groups.TaIPT4-2A/2B/2D and TaIPT9-7A7/B/7D grouped in groups I and group II with AtIPT2,OsIPT9 and AtIPT9,OsIPT10 respectively,suggesting their involvement in tRNA prenylation.The other 38 IPT genes presumably encoding ATP/ADP IPTs clustered in groups III and IV and were further divided into small subgroups(Fig.2).TaIPT2-1B and TaIPT8-5A/B/D showed the closest relationship with AtIPT3,5,and 7,which affect predominantly the formation of iP-and tZ-types in Arabidopsis.

3.3.Expression profiles of TaIPTs in tissues of wheat

Tissue-specific expression of the CK metabolic genes indicated their functional specification,which is useful for the genetic modification of specific traits.Semi-quantitative RT-PCR analysis revealed that TaIPT1 was expressed specifically in roots(Fig.3).By contrast,TaIPT6 and TaIPT9 were expressed mainly in aerial tissues.TaIPT2,TaIPT5,and TaIPT7 were expressed in relatively mature tissues,such as sheaths,stems,and spikes,whereas TaIPT8 was expressed in young roots,shoots,and leaves(Fig.3).Two TaIPTs,TaIPT3,and TaIPT4,were ubiquitously expressed in all examined tissues(Fig.3).

3.4.Expression analysis of TaIPTs in response to drought stress

To investigate the response of TaIPTs to the drought stress,the expression patterns of TaIPT genes under drought stress treatment were further studied.Transcription of TaIPT2 and TaIPT8 was induced by 0.5 h drought treatment,while TaIPT7 was highly expressed at 1 and 2 h drought treatments,after which they decreased to a low level.TaIPT3,TaIPT4,and TaIPT9 began to be suppressed at 1 h drought treatment and TaIPT6 at 2 h drought treatment(Fig.4).No significant changes were observed in TaIPT1 or TaIPT5(Fig.4).To further verify drought-induced transcription of TaIPT8,qRT-PCR analyses were performed using TaIPT8 universal primers and TaIPT8-5A,TaIPT8-5B,and TaIPT8-5D specific primers,and stronger upregulation of TaIPT8-5A and TaIPT8-5D was observed than TaIPT8-5B after 0.5 h drought treatment(Fig.S2).

3.5.Targeted mutagenesis of TaIPT8 induced by CRISPR/Cas9

To investigate the roles of CK in drought tolerance,CRISPR/Cas9 system was used to specifically disrupt the TaIPT8 gene which was closely related to the crucial ATP/ADP type Arabidopsis IPTs AtIPT3,5,7,and showed rapid responses to drought stress.

Fig.3.Tissue-specific expression patterns of TaIPT genes.The wheat actin gene was used as the control for equal cDNA concentrations.Roots and shoots were collected at seedling stage.Leaf blade,sheath,stem,and spike tissue was collected at heading stage.

Fig.5.Homozygous targeted mutagenesis of TaIPT8.(A)Schematic map of gRNA target site selection in the conserved regions of TaIPT8-5A,TaIPT8-5B,and TaIPT8-5D.(B)The sequences and sequencing chromatograms corresponding to the wild type and seven representative editing lines at target sites are shown;‘‘-”indicates deletion of nucleotides;PAM motifs are highlighted in red;Red arrow indicates the start of translation.

In order to simultaneously knock out TaIPT8 in all subgenomes(A,B,and D),we designed an sgRNA targeting the conserved region near the start codons of TaIPT8-5A,TaIPT8-5B,and TaIPT8-5D(Fig.5A),and a corresponding sgRNA/Cas9 vector was transformed into the wheat cultivar Fielder using Agrobacterium tumefaciens-mediated transformation.After DNA sequencing analysis of T2plants derived from five independent transgenic lines,we obtained four homozygous Taipt8-5a/5b/5d triple mutants designated Taipt8-M1 to Taipt8-M4 and Taipt8-M1;Taipt8-M2 plants were chosen for further functional analysis(Fig.5B).To avoid the influence of potential off-target sites on phenotype and functional analyses,six most likely off-target sites with 2–3 mismatches were selected for amplification and analysis using genome-specific PCR and Sanger sequencing.Sequencing revealed no mutations in the examined potential off-target loci of all homozygous Taipt8 mutants(Table S2).

3.6.Mutation of TaIPT8 results in reduced drought tolerance

There were no visible phenotypes in Taipt8-M1 and Taipt8-M2 under normal growth conditions.However,after drought treatment,the Taipt8 mutants showed significantly accelerated leaf rolling,compared to WT plants.After rehydration,the WT plants quickly recovered from water stress,whereas the Taipt8 mutants continuously withered and showed lower fresh and dry weights(Fig.6A–C).Although the water loss rate was comparable between mutant and WT plants(Fig.S3),the proline content of Taipt8 mutants was lower,while the concentrations of MDA,H2O2,and O2-were significantly higher than those of WT plants(Fig.6D–G).

Fig.6.Knockout of TaIPT8 leads to decreased drought tolerance in wheat.(A)Phenotypes of TaIPT8 mutants and control plants under normal growth and drought conditions.(B,C)Fresh weight(B)and dry weight(C)of TaIPT8 mutants and control plants under drought-stress conditions.(D–G)MDA content(D),proline content(E),O2-content(F),and H2O2 content(G)of TaIPT8 mutants and wild-type plants under normal and drought stress conditions.Shown are means±standard deviation(SD)of three independent biological replications.Differences were evaluated by Student’s t-test(*,P＜0.05;**,P＜0.01).

Fig.7.Cytokinin levels in Taipt8 mutant before and after drought treatment.(A)tZ,(B)IP,(C)IPA,and(D)cZ concentrations were measured in three-week-old seedlings before and after 0.5 h drought treatment.Shown are the means±SD of three independent biological replicates.

Fig.8.Quantitative RT-PCR analysis showing the relative expression levels of TaIPT8 in five drought-induced TaIPT8-overexpressing lines under drought stress.The x-axes shows treatment times of 0,0.5,1,2,4,and 8 h.The wheat actin gene was used as an internal reference.Shown are the means±SD of three independent biological replicates.Different letters in bar graphs indicate differences at P＜0.05.

3.7.Concentrations of CKs in WT and TaIPT8 mutant plants

As the expression of some TaIPT genes was upregulated after 0.5–1 h drought treatments,we first examined the effects of drought stress on CK metabolism in wheat plants by comparing CK concentrations in WT plants before and after the 0.5 h drought treatment.The tZ content was increased and iP and iPA content was decreased in WT leaves after drought treatment(Fig.7A–C).When the CK content was compared between WT and Taipt8-M1,the bioactive tZ content in the Taipt8 mutants was significantly reduced before and after drought treatment(Fig.7A).Levels of iP and iPA were not affected before treatment,whereas the iP content was decreased in Taipt8 mutants after drought treatment(Fig.7B,C).cZ levels were significantly increased in Taipt8 mutants with and without drought treatment(Fig.7D).

3.8.Drought-inducible expression of TaIPT8 improves wheat drought tolerance

To assess the effects of CK overproduction on wheat development and drought tolerance,we overexpressed TaIPT8 in transgenic wheat under the control of a maize ubiquitin promoter.Root formation was completely inhibited in all transgenic lines,which eventually withered without forming seeds(Fig.S4).We induced the ectopic expression of TaIPT8 with the previously identified drought-inducible promoter PNAC48[52],and based on the qRT-PCR results,two independent transgenic lines,DI-OE2 and DI-OE5,which maintained high expression levels of TaIPT8 under drought conditions,were selected from five positive transgenic lines for further analysis(Fig.8).No growth-restraining effect was observed in PNAC48::TaIPT8 transgenic wheat plants grown under well-watered conditions.After drought treatment,both PNAC48::TaIPT8 transgenic lines delayed wilting,and more transgenic plants were recovered with higher fresh weight and dry weight after seven days of rehydration(Fig.9).

Fig.9.Drought-induced overexpression of TaIPT8 confers improved drought tolerance in wheat.(A)Phenotypes of drought-induced overexpressing lines and wild-type plants under normal growth and drought conditions.(B,C)Fresh weight(B)and dry weight(C)of TaNAC48-overexpressing plants and wild-type plants under drought-stress conditions.Shown are the means±SD of three independent biological replications.Differences were evaluated by Student’s t-test(*,P＜0.05;**,P＜0.01).

4.Discussion

The IPT gene family has been characterized in many plant species because of its important biological functions in catalyzing the rate-limiting step of CK biosynthesis[32,54,56].In addition to the regulation of developmental processes,increasing evidence has shown that CK is essential for stress responses in plants.However,observations of endogenous CK levels under various stress conditions suggested[19]that CK metabolism is differentially regulated in response to abiotic stress in different plant species.To investigate the relationship between CK and drought tolerance in wheat,24 TaIPT genes were identified by in silico analysis.Not all TaIPT genes were represented by three homoeologous genes in the A,B,and D subgenomes,such as TaIPT2-1B,which had no homologs in the A and D subgenomes but is closely related to TaIPT8-5A/5B/5D.We predicted that TaIPT2-1B originated from gene duplication in the distant evolutionary history of the B genome donors.Phylogenetic analysis indicated that TaIPT4 and TaIPT9 would also be involved in the synthesis of cZ-type CKs,as they clustered with two Arabidopsis tRNA-dependent IPTs,whereas TaIPT1,2,3,5,6,7,and 8 may act as ATP/ADP-dependent TaIPTs that are responsible for bioactive CK biosynthesis.Previous study[26]have identified the six wheat IPT genes TaIPT2,3,5,6,7,and 8,which were named by their similarity to ZmIPT2,3,5,6,7,and 8 in maize.We found that TaIPT5 and TaIP6(previously identified)were two homoeologous alleles of TaIPT8(newly named)from 5A and 5B subgenomes and renamed the six previously identified TaIPT as TaIPT1-1D,TaIPT6-3D,TaIPT8-5A,TaIPT8-5B,TaIPT5-3D,and TaIPT3-1D according to the results of amino acid sequence alignment.

Although CKs have long been considered to be synthesized primarily in roots and transported acropetally to the shoots,recent study[57]confirmed that CKs synthesized locally rather than in roots are even more critical for axillary bud growth.The expression patterns of individual TaIPT genes,especially ATP/ADP-dependent TaIPTs,are differential.TaIPT1 was expressed specifically in roots,and TaIPT8 was expressed mainly in shoots,where it may be involved in the regulation of vegetative growth,whereas TaIPT2,TaIPT5,and TaIPT7 were expressed predominantly in sheath,stem,and spike tissue,where they may be involved in the regulation of reproductive growth.Similarly,considering their specificity of expression,15 wheat TaCKX genes were assigned to four groups specific to leaves,developing spikes,roots,and all the organs tested respectively.These assignments indicated that CK content is subtly controlled in diverse organs and development stages[23].The expression of IPTs has been shown[33–35]to be a response to abiotic and biotic stressors in many plant species.In maize,salt and osmotic stresses induce the expression of most CK biosynthetic genes in seedlings of maize,leading to a moderate increase of CKs lasting several days during acclimatization to stress[58].In the present study,expression of most TaIPTs was gradually reduced after 1 h drought treatment;however,TaIPT2,TaIPT7,and TaIPT8 were up-regulated within 0.5–1 h,which may have induced the elevation of tZ but not of iP or iPA in WT plants at the early stages of drought treatment.In TaIPT8-5a/5b/5d triple mutants,levels of tZ-type CK rather than iP-type CK were decreased under normal and drought conditions,indicating that TaIPT8 homologs are more critical for the formation of tZ-type CK in wheat.

In Arabidopsis,neither single-nor double-atipt mutants show visible changes in their phenotypes due to gene redundancy[29].Similarly,the decrease in CK content did not change the morphology of the TaIPT8-5a/5b/5d triple mutants in the present study;however,it reduced the drought tolerance of mutant plants.In contrast,downregulation of CK content via mutation of multiple IPT or overexpression of CKX genes has been reported[16,38]to increase drought tolerance in Arabidopsis and tobacco.These multi-gene mutants or overexpression plants show altered phenotypes,including slow growth rates,dwarf shoots,and small leaves,but enlarged root systems,which may contribute to lower transpiration rates and higher relative water content[29,38,39].The CKdeficient phenotype with enlarged root system and suppressed aerial part could also be observed in monocots.Constitutive overexpression of CKX in barley plants led to a substantially enlarged root system,but shoot growth was retarded and the plants died without reaching flowering[55].It thus remains uncertain whether the decreased CK levels achieved by overproduction of the CKX or mutation of IPT has a same effect on abiotic stress tolerance in other cereal crops.When the CK effect on drought tolerance in wheat was evaluated,the water loss rate was comparable between TaIPT8 mutant and WT plants,while the contents of MDA,H2O2,and O2-in transgenic plants were higher than those in WT plants under drought,owing to a decrease in active oxygen-scavenging ability of their antioxidant systems.Thus,although these TaIPTs may function redundantly during plant organ development,their spatially specific expression pattern probably influences plant stress response.

Given that employment of a constitutive promoter for IPT overexpression resulted in diminished root growth,genetic manipulation of crop plants using inducible promoters to increase stress tolerance is inevitable.Conditionally activated expression of IPT under stress-or senescence-induced promoters stimulated extreme drought tolerance without impairing plant morphology in cotton[40],maize[41],peanut[42],rice[44],and cassava[45].Most transgenic plants showed improved drought resistance by increased protection of the photosynthetic system,delayed senescence,and increased redox ability.A similar strategy was used in the present study,where TaIPT8 expression was induced by the wheat native drought-induced-promoter PNAC48.TaIPT8 maintained a high expression level in the resulting transgenic lines,increasing drought tolerance without change in plant development.Our findings of water loss rates in wheat Taipt8 mutants and WT plants also suggested that CKs exerted their beneficial effects on drought tolerance via regulation of cellular redox status.

In summary,we performed an in silico analysis of 24 TaIPTs in the wheat genome and characterized their chromosomal locations,phylogenetic relationships,gene structures,protein properties,and gene expression patterns.TaIPT8 mutants and drought-induced overexpression plants were produced,and functional analyses revealed beneficial effects of CKs on wheat drought tolerance.Further research may reveal the mechanisms and signaling pathways involved in the interactions of CK metabolism and stress tolerance in wheat.

CRediT authorship contribution statement

Na Wang:Data curation,Formal analysis,Visualization,Writing-review&editing.Jun Chen:Data curation,Formal analysis,Visualization,Writing-original draft.Yuan Gao:Investigation,Methodology,Validation.Yongbin Zhou:Resources,Software.Ming Chen:Resources,Software.Zhaoshi Xu:Supervision,Validation.Zhengwu Fang:Data curation,Project administration,Resources,Writing-review & editing.Youzhi Ma:Conceptualization,Data curation,Funding acquisition,Project administration,Resources,Writing-review & editing.

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 financially supported by the National Key Research and Development Program of China(2020YFE0202300)and the Agricultural Science and Technology Innovation Program(CAASZDRW202109 and CAAS-ZDRW202002).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.04.010.