TaNF-YB11,a gene of NF-Y transcription factor family in Triticum aestivum,confers drought tolerance on plants via modulating osmolyte accumulation and reactive oxygen species homeostasis

ZHAO Ying-jia,ZHANG Yan-yang,BAI Xin-yang,LIN Rui-ze,SHI Gui-qing,DU Ping-ping,XIAO Kai

State Key Laboratory of North China Crop Improvement and Regulation,Hebei Agricultural University/College of Agronomy,Hebei Agricultural University,Baoding 071001,P.R.China

Abstract Transcription factors (TFs) regulate diverse stress defensive-associated physiological processes and plant stress responses. We characterized TaNF-YB11,a gene of the NF-YB TF family in Triticum aestivum,in mediating plant drought tolerance. TaNF-YB11 harbors the conserved domains specified by its NF-YB partners and targets the nucleus after the endoplasmic reticulum (ER) assortment. Yeast two-hybrid assay indicated the interactions of TaNF-YB11 with TaNF-YA2 and TaNF-YC3,two proteins encoded by genes in the NF-YA and NF-YC families,respectively. These results suggested that the heterotrimer established among them further regulated downstream genes at the transcriptional level. The transcripts of TaNF-YB11 were promoted in roots and leaves under a 27-h drought regime. Moreover,its upregulated expression levels under drought were gradually restored following a recovery treatment,suggesting its involvement in plant drought response. TaNF-YB11 conferred improved drought tolerance on plants;the lines overexpressing target gene displayed improved phenotype and biomass compared with wild type (WT) under drought treatments due to enhancement of stomata closing,osmolyte accumulation,and cellular reactive oxygen species (ROS)homeostasis. Knockdown expression of TaP5CS2,a P5CS family gene modulating proline biosynthesis that showed upregulated expression in drought-challenged TaNF-YB11 lines,alleviated proline accumulation of plants treated by drought. Likewise,TaSOD2 and TaCAT3,two genes encoding superoxide dismutase (SOD) and catalase (CAT) that were upregulated underlying TaNF-YB11 regulation,played critical roles in ROS homeostasis via regulating SOD and CAT activities. RNA-seq analysis revealed that numerous genes associated with processes of ‘cellular processes’,‘environmental information processing’,‘genetic information processing’,‘metabolism’,and ‘organismal systems’modified transcription under drought underlying control of TaNF-YB11. These results suggested that the TaNF-YB11-mediated drought response is possibly accomplished through the target gene in modifying gene transcription at the global level,which modulates complicated biological processes related to drought response. TaNF-YB11 is essential in plant drought adaptation and a valuable target for molecular breeding of drought-tolerant cultivars in T.aestivum.

Keywords: wheat (Triticum aestivum L.),NF-YB transcription factor,drought stress,osmolyte accumulation,reactive oxygen species (ROS) scavenging

1.Introduction

Drought stress exerts negative impacts on plant growth,development,and the yield formation capacity for cereal crops by alleviating corresponding biochemical and physiological processes (Fahadet al.2017;Liet al.2021). Meanwhile,plants have evolved a suite of effective strategies to cope with the adverse effects initiated by drought stress (DaCostaet al.2004;Vanajaet al.2011;Reynoldset al.2015). Once challenged by water deprivation conditions,the root system firstly perceives the environmental stress signaling and transduces the drought signals across tissues through distinct signaling transduction mechanisms (Suet al.2013;Tianet al.2016;Zhanget al.2017;Monroeet al.2018). Thus far,various kinds of signaling molecules and regulators,including signal transductionassociated kinases,transcription factors (TFs),molecule chaperones,osmotic stress elicitors (Baldoniet al.2015;Tanet al.2018;Wanget al.2020;Zhanget al.2020),ion channels (Zhaoet al.2018),transporters (Gonget al.2013),and antioxidant enzymes (Koffleret al.2014;Liet al.2020),have been documented to be functional in plant responses to drought stress.

As one type of essential regulators,the TF proteins modulate plant osmotic stress responses due to modified transcription efficiencies upon stressors (Baldoniet al.2015;Heet al.2016;Tanet al.2018). Currently,a large set of genes categorized into various TF families,such as MYB,ZFP,NAC,AP2/ERF,and WRKY ones,have been recorded for their roles in improving the drought acclimation of plants (Baldoniet al.2015;Heet al.2016;Yinet al.2017;Zhanget al.2018). A set of genes has been used as valuable targets in the efforts for molecular breeding for drought-tolerant crop cultivars (Yinet al.2017).

The nuclear factor Y (NF-Y) proteins constitute a large class of TF families and are present in various species covering plants,animals,and other eukaryotes(Maheshwariet al.2019). The TF proteins in NF-Y class are also referred to asCCAATbinding factor (CBF) or Heme activator protein (HAP),regulating transcription efficiency of the downstream genesviaa heterotrimer form constituted by each of NF-YA (CBF-B/HAP2),NFYB (CBF-A/HAP3),and NF-YC (CBF-C/HAP5) subunits.Each subunit establishing a heterotrimer has potential to specifically bind the CCAAT motif,acis-acting regulatory element generally situated in promoters of the downstream genes (Nardiniet al.2013). Previous characterizations revealed that each NF-Y subunit fails to regulate the transcription of the downstream genes;instead,a heterotrimer covering all three kinds of different subunits (i.e.,NF-YA,NF-YB,and NF-YC) is necessary for transcriptionally modulating the downstream genes upon distinct internal or environmental cues (Maheshwariet al.2019). This kind of function mode for NF-Y TF proteins in regulating gene transcription is in contrast to most other TF family members,which regulates transcription of downstream genes with monomer form.

Each subunit constituting the heterotrimer is encoded by a set of genes in plant species,with different subfamilies defined based on the specific motifs/conserved domains situated in proteins (Petroniet al.2012). For example,in the model plantArabidopsis, a total of 36 genes were identified to encode the NF-Y proteins.Among them,10 code for NF-YA proteins,13 for NFYB ones,and 13 for NF-YC proteins (Sieferset al.2009). Functional characterizations of the NF-Y family genes have indicated that they are involved in the modulation of diverse physiological processes,which are associated with plant gametogenesis,embryogenesis,seed development,flowering time regulation,primary root elongation,abscisic acid (ABA) signaling,and the plant osmotic stress tolerance (Mantovani 1999;Gusmaroliet al.2002;Petroniet al.2012). These findings suggest the essential roles of the NF-Y proteins in regulating growth,development,and plant responses to various abiotic stressors.

Thus far,investigations on members of the NF-Y family have been extensively conducted in model plant species(Gusmaroliet al.2002;Zhaoet al.2016;Maheshwariet al.2019). However,the genes in the NF-Y families(i.e.,NF-YA,NF-YB,and NF-YC encoding ones) inTriticumaestivum,a major crop cultivated extensively worldwide,have been lacking functional documents and are needed for further characterization. In this study,we reportedTaNF-YB11,a gene of the NF-YB family in wheat,in mediating plant response to drought stress.TaNF-YB11was a response to drought at the transcriptional level and conferred improved drought adaptation on plants through regulating stomata movement,osmolyte accumulation,and cellular reactive oxygen species (ROS) homeostasis,due to the transcriptional regulation of genes at the global level. Our investigation provides insights into the plant drought response underlying the regulation of NF-YB TF family and benefits molecular breeding for elite droughttolerant cultivars inT.aestivum.

2.Materials and methods

2.1.Characterization on TaNF-YB11

TaNF-YB11(GenBank accession no.TraesCS1A02G411700)is located at chromosome 1A: 572 334 701-572 336 969 ofT.aestivum(https://www.ebi.ac.uk/Tools/hmmer/).The conserved domains of TaNF-YB11 were defined as described previously (Zhaoet al.2016). The homologous genes ofTaNF-YB11in various plant species released in GenBank database were identified based on BLAST search analysis. The phylogenetic relations amongTaNF-YB11and its plant counterparts were established using the MegAlign algorithm supplemented in DNAStar Software.

2.2.Determination of protein subcellular location

The open reading frame (ORF) ofTaNF-YB11was PCR amplified using gene-specific primers (Appendix A),which was inserted into a binary vector pCAMBIA3300 under the control of the CaMV35S promoter. Genetic transformation of the expression cassette harboringTaNFYB11intoAgrobacteriumtumefaciensstrain (EHA105)was conducted following the conventional approach.Further temporal transformation of the epidermal cells inNicotianatabacum(cv.Wisconsin 35) was performed based on anA.tumefaciens-mediated method. The signals in transgenic epidermal cells derived from green florescent protein (GFP) in control harboring empty vector and in those initiated by fusion TaNF-YB11-GFP were detected after 48 h of inoculation with theA.tumefaciensstrains,which were observed under fluorescent microscope as described previously (Guoet al.2013).

2.3.Yeast two-hybrid assays

The yeast two-hybrid assays covering TaNF-YB11 and the NF-YA and NF-YC family members inT.asetivumwere conducted as described previously (Guoet al.2013). The TaNF-YB11 protein and the proteins of NF-YA family (i.e.,TaNF-YA1 to TaNF-YA3) and NF-YC family (i.e.,TaNFYC1 to TaNF-YC6) were subjected to the yeast twohybrid experiments. Information together with the specific primers used forTaNF-YB11and the NF-YA and NF-YC family genes inT.aestivumis shown in Appendix A.

2.4.Expression analysis

Seedlings of Shimai 22,a drought-tolerant cultivar of wheat,were cultured hydroponically in a standard MS solution as described previously (Sunet al.2012).At the third leaf stage,they were subjected to the simulated drought treatment by growing in a modified MS solution supplemented with different concentrations of polyethylene glycol 6000 (PEG-6000) (i.e.,0,1,5,10,and 15% (w/v)). Tissues of the roots and leaves were harvested after 27 h of treatments. The transcripts of the target gene were evaluated based on qPCR performed as described previously (Guoet al.2013),using genespecific primers (Appendix A). In addition,to define the temporal pattern of gene expression,an aliquot of seedlings treated with 10% PEG for 27 h was subjected to the normal recovery condition. Tissues mentioned above were sampled at 0 h (prior to treatment),1,3,9,and 27 h during drought treatments,and 1,3,9,and 27 h following the normal recovery progression. Likewise,the transcripts ofTaNF-YB11in samples were evaluated based on qPCR using the gene-specific primers (Appendix A).Tatubulin,a constitutive gene inT.aestivum,was used as an internal standard to normalize the target transcripts.

2.5.Generation of transgenic lines

The ORF ofTaNF-YB11was PCR amplified in both sense and anti-sense orientations using gene-specific primers(Appendix A),then separately integrated into theNcoI/BstEII sites in the binary vector pCAMBIA3301 under the control of the CaMV35S promoter. The expression cassettes were separately subjected to transformation toA.tumefaciens(strain EHA105). Genetic transformation ofT.aestivum(cv.Shimai 22) and generation of transgenic lines were conducted as described previously(Guoet al.2013). The transcripts of the target gene in transgenic lines,including six overexpressing target genes (Sen 1 to Sen 6) and five with target knockdown expression (Anti 1 to Anti 5),were evaluated based on qRT-PCR performed similarly for evaluation of the target expression levels mentioned above.

2.6.Assay of growth traits

Four lines at T3generation,including Sen 1 and Sen 2 with strong target overexpression and Anti 1 and Anti 2 with the knockdown expression of the target gene(Appendices B and C) together with wild type (WT),were cultured under normal condition by growing the seedlings in standard MS solution and subjected to drought treatment by culturing them in the modified MS solution supplemented with 5% PEG (w/v). Five weeks after treatments,phenotypes of the transgenic lines,WT were recorded using a digital camera and biomass of the transgenic,and WT plants were obtained based on the oven-dried samples.

2.7.Assay of leaf water loss rate and photosynthetic parameters

The drought response-associated traits,including water loss rate of leaf (WLR) and photosynthetic parameters(i.e.,photosynthetic rate (Pn),stomatal conductance(gs),intercellular CO2concentration (Ci),photosystem II photochemical efficiency (ΨPSII),and non-photochemical quenching coefficient (NPQ)) following the drought progression,were measured using the representative leaves of transgenic and WT plants. Of which,WLR was determined based on the decreased weight of detached fresh leaves at indicated times (0.25,0.5,1,2,and 3 h)compared with time point 0 h as described previously(Imadiet al.2015);Pn,gs,andCiwere measured using a photosynthesis system (LiCOR-6200) following the manufactures’ suggestions;ΨPSII and NPQ shown in samples were assessed as reported previously (Guoet al.2013).

2.8.Assessment of osmolyte and ROS-associated traits

The osmolyte and ROS-associated traits of transgenic lines and WT were assessed under drought treatment.Of which,the contents of proline and soluble sugar were determined as reported previously (DuBoiset al.1956;ábrahámet al.2010);the ROS-associated traits,including antioxidant enzyme (AE) activities (e.g.,those of superoxide dismutase (SOD),catalase (CAT),and peroxidase (POD)) and the contents of malondialdehyde(MDA),were measured as previously described (Huanget al.2010).

2.9.Expression and function analyses of P5CS and AE family genes

Five genes in the P5CS family inT.aestivum,includingTaP5CS1toTaP5CS5,were evaluated for transcripts in the transgenic lines under drought treatments based on qRT-PCR using gene primer pairs (Appendix A) to define the genes in putatively modulating proline accumulation.Likewise,a subset of genes in the SOD,CAT,and POD families inT.aestivum,including five SOD genes (TaSOD1toTaSOD5),six CAT genes (TaCAT1toTaCAT6),and 11 POD genes (TaPOD1toTaPOD11),were subjected to transcripts evaluation in the transgenic and WT plants under drought treatments to understand the molecular processes underlying theTaNF-YB11-mediated ROS homeostasis. Information on the genes examined and the corresponding gene-specific primers for detecting the AE gene transcripts are shown in Appendix A.

The ORFs ofTaP5CS2,TaSOD2,andTaCAT3,the P5CS and AE genes significantly upregulated in expression in the drought-challenged transgenic lines,were PCR-amplified in anti-sense orientation and then integrated separately into theNcoI/BstEII sites of binary vector pCAMBIA3301 under the control of the CaMV35 promoter. Genetic transformation of the expression cassettes intoA.tumefaciensstrain EHA105 and further genetic transformation of the genes intoT.aestivum(cv.Shimai 22) were performed similarly to theNFYB11knockdown lines mentioned above. Typical transgenic lines AntiP5CS2-1 and AntiP5CS2-3 ofTaP5CS2,AntiSOD2-2 and AntiSOD2-3 ofTaSOD2,and AntiCAT3-1 and NtCAT3-2 ofTaCAT3at T3generation together with WT were subjected to drought treatments as mentioned previously. Four weeks after the treatment,the proline content and biomass in lines AntiP5CS2-1 and AntiP5CS2-3 were measured;the SOD activities and biomass in lines AntiSOD2-2 and AntiSOD2-3 were assessed;the CAT activities and biomass in lines AntiCAT3-1 and NtCAT3-2 were analyzed as mentioned above.

2.10.RNA-seq analysis

The Sen 2 (withTaNF-YB11overexpression) and WT plants after drought treatments were subjected to the RNA-seq analyses. Briefly,total RNA in root tissues of Sen 2 and WT was extracted using TRIzol reagent(Invitrogen,USA). The strand-specific RNA-seq libraries for the samples were constructed in triplicate following the procedures described previously (Zhonget al.2011) and sequenced using an Illumina HiSeq 2500 System. The raw reads identified in the libraries were processed using the Trimmomatic tool to remove the adaptor and lowquality sequences (Bolgeret al.2014). The reads with a sequence length less than 40 bp were discarded. The resulting reads were then subjected to alignment analysis against theT.aestivumtranscript database (Novogene Co.,Ltd.,Beijing). The genes with modified transcripts over two folds in the transgenic line and WT were defined as the differentially expressed (DE) ones,which were identified using the edgeR tool that calculated the raw count data (Robinsonet al.2010). Of which,the rawPvalues were corrected using a false discovery rate (FDR)less than 0.05 (Benjamini and Hochberg 1995). An online tool referred to as Plant MetGenMap (http://bioinfo.bti.cornell.edu/cgi-bin/MetGenMAP/home.cgi) was used to characterize the KEGG pathways for the DE genes based on the CPAN pearl module as described previously (Boyleet al.2004).

2.11.Statistical analysis

Averages of the gene transcripts,plant biomass,physiological traits,osmolyte contents,and ROSassociated traits were all derived from the triplicate results. Standard errors for the averages and statistical significance analyses for traits assessed were determined based on Statistical Analysis System Software (SAS Corporation,Cory,NC,USA).

3.Results

3.1.Characterization of TaNF-YB11

TaNF-YB11has a full-length cDNA of 1 018 bp,encoding a 136-aa polypeptide with a molecular mass of 14.93 kDa and an isoelectric point (pI) of 5.65. TaNF-YB11 harbors two conserved domains as its plant NF-YB counterparts,including one involving protein-protein interaction(aa 102-aa 126) and another functional protein-DNA interaction (aa 15-aa 88). At the nucleic acid level,TaNFYB11shares high similarities to a large set of the NF-YB family genes distributed in diverse plant species (Fig.1-A),which suggested that it acts as a member of the NFYB family inT.aestivum. The subcellular localization of TaNF-YB11 after endoplasmic reticulum (ER) assortment was defined based on the detected green florescent protein (GFP) signals in transgenic epidermal cells ofN.tabacum,which harbors the fusionTaNF-YB11-GFP.In contrast to that,the GFP signals freely distributed in the cells transformed with an empty vector,and the GFP signals derived from the transgenic cells integrated with the fusion were confined in nucleus (Fig.1-B). These results suggested that TaNF-YB11 targets nucleus after ER assortment. The subcellular localization of this NFYB protein is consistent with the nature of TF proteins functional in nucleus to regulate transcription of the downstream genes.

3.2.TaNF-YB11 interacts with NF-YA2 and NF-YC3 proteins

Yeast two-hybridization assays were performed to reveal the subunits in NF-YA and NF-YC families involving the establishment of a heterotrimer by interacting with TaNFYB11. As a result,it was found that TaNF-YB11 interacts specifically with distinct NF-YA and NF-YC proteins,including TaNF-YA2 and TaNF-YC3 (Fig.2). These results suggested that the heterotrimer constituted by TaNF-YB11/TaNF-YA2/TaNF-YC3 acts as a potential TF to putatively regulate transcription of the downstream genes.

3.3.Expression patterns of TaNF-YB11 under drought treatment

The expression patterns ofTaNF-YB11in roots and leaves under drought and the normal recovery treatments are shown in Fig.3-A and B. Under the drought treatment,the transcripts ofTaNF-YB11in the tissues examined were significantly upregulated along with the intensified drought extent within a 15% concentration range (i.e.,PEG-6000)(Fig.3-A). In addition,under a 27-h regime of treatment,the expression levels ofTaNF-YB11gradually elevated in the tissues and reached a peak at end of the treatment.Moreover,the induced expression levels ofTaNF-YB11under the drought treatment was gradually restored along with normal recovery condition,with reduced transcripts detected to be similar to that prior to drought treatment after a 27-h of recovery duration (Fig.3-B). These results suggested thatTaNF-YB11is sensitive in response to drought stressors at the transcriptional level.

3.4.Growth traits of TaNF-YB11 transgenic lines under drought treatment

Among the six lines withTaNF-YB11overexpression(i.e.,Sen 1 to Sen 6) and five lines with target anti-sense expression (Anti 1 to Anti 5),Sen 1 and Sen 2 displayed more target transcripts,whereas Anti 1 and Anti 2 decreased target expression with respect to wild type (WT)(Appendices B and C). Under the normal growth condition,all lines examined (i.e.,Sen 1,Sen 2,Anti 1,and Anti 2)were similar in phenotype and biomass compared with WT(Fig.4-A and B). Under drought treatments,however,the transgenic lines displayed modified growth traits mentioned.Among them,Sen 1 and Sen 2 were much better,whereas Anti 1 and Anti 2 more deteriorated in the phenotype behaviors and growth traits than the WT plants (Fig.4-A and B). These results together suggested thatTaNF-YB11plays a critical role in modulating plant drought tolerance.

3.5.Physiological parameters of the TaNF-YB11 transgenic lines

In agreement with the phenotypes and growth traits mentioned above,Sen 1,Sen 2,Anti 1,and Anti 2 showed comparable physiological traits,such as similar photosynthetic parameters (i.e.,Pn,gs,Ci,ψPSII,and NPQ) to the WT plants under the normal condition(Fig.5-B-F). Under the drought treatment,lower WLR and more improved photosynthetic function (i.e.,higherPn,gs,and ψPSII and lower WLR,Ci,and NPQ) were shown in Sen 1 and Sen 2,whereas higher WLR and more deteriorated photosynthetic traits (lowerPn,gs,and ψPSII and higher WLR,Ci,and NPQ) observed in Anti 1 and Anti 2 than those shown in WT plants (Fig.5-A-F).These results suggested that theTaNF-YB11-mediated drought tolerance is closely associated with the gene function in modulating the drought response-associated physiological processes.

3.6.Osmolytes and ROS-associated traits of transgenic lines

The osmolyte and ROS behaviors,including contents of proline and soluble sugar and activities of antioxidant enzymes (AE) (i.e.,SOD,CAT,and POD) and contents of MDA,were evaluated in the transgenic lines under drought treatment. Under the normal condition,all transgenic lines (Sen 1,Sen 2,Anti 1,and Anti 2)displayed similar osmolyte contents and ROS-associated traits to the WT plants (Fig.6-A-F). Under the drought treatment,Sen 1 and Sen 2 showed more proline and soluble sugar contents (Fig.6-A and B),higher activities of SOD,CAT,and POD,and less MDA amounts (Fig.6-CF) than the WT plants. In contrast,Anti 1 and Anti 2 were reverse on the osmolyte and ROS-associated traits to Sen 1 and Sen 2 under the drought treatment (Fig.6-AF). These results indicated thatTaNF-YB11exerts critical roles in modulating cellular osmotic-regulatory process and ROS homeostasis in the plants treated with drought stress.

3.7.Expression patterns of P5CS and AE encoding genes in TaNF-YB11 transgenic lines

Analysis of the expression patterns of genes in the delta-1-pyrroline-5-carboxylate synthetase (P5CS) family (i.e.,TaP5CS1-TaP5CS5) that modulate proline biosynthesis revealed thatTaP5CS2modified transcription efficiencies,with significantly upregulated transcripts in Sen 1 whereas downregulated expression in Anti 2 relative to WT (Fig.7-A).Likewise,the expression patterns ofTaSOD2andTaCAT3,the genes in SOD and CAT families,respectively,displayed more transcripts in Sen 1 and fewer in Anti 2 than WT plants (Fig.7-B-D). The two genes mentioned above with modified expression under the drought treatment were in contrast to other genes unchanged in expression in the transgenic lines and WT. These results suggested the potential roles of the differential genes in P5CS and AE families,which regulate proline biosynthesis and cellular ROS homeostasis underlyingTaNF-YB11regulation under the drought condition.

3.8.Proline and AE behaviors mediated by differential P5CS and AE genes

Compared with wild type,the proline contents and biomass in AntiP5CS2-1 and AntiP5CS2-3,two lines with significant knockdown expression ofTaP5CS2(Appendix D),were decreased under the drought treatment (Fig.8-A and B).Likewise,the SOD activities and biomass in AntiSOD2-2 and AntiSOD2-3,two lines with a knockdown expression ofTaSOD2(Appendix E),were reduced relative to WT (Fig.8-C and D);the CAT activities and biomass in AntiCAT3-1 and AntiCAT3-2 (two lines with a drastic knockdown expression ofTaCAT3(Appendix F)) were lowered (Fig.8-E and F).Therefore,the transgenic results on differential P5CS and AE genes validated their essential functions in promoting osmolyte accumulation and improving cellular ROS homeostasis under drought conditions.

3.9.RNA-seq analysis on TaNF-YB11 overexpression lines under drought treatment

High throughput RNA-seq analysis revealed that a total of 2 658 genes,including 1 371 upregulated and 1 287 downregulated,were differentially expressed in Sen 2(data sets S1-S2). Based on KEGG analysis,the DE genes were mainly enriched into the biological processes associated with ‘cellular processes’,‘environmental information processing’,‘genetic information processing’,‘metabolism’,and ‘organismal systems’ (Fig.9-A).These results suggested thatTaNF-YB11modifies the transcription efficiency of a number of genes at the global level,which is involved in the regulation of diverse physiological processes and biochemical pathways and impacts plant drought defensiveness.

4.Discussion

4.1.TaNF-YB11 is a NF-YB family member and its translated product interacts with TaNF-YA2 and TaNF-YC3

The genes encoding the NF-Y TF proteins comprise those to code for the NF-YA,NF-YB,and NF-YC class members(Petroniet al.2012;Nardiniet al.2013;Maheshwariet al.2019). The NF-Y family proteins are specified by the conserved domains involving binding for distinctcisregulatory elements,namely,the CCAAT box situated in the promoters of downstream genes (Nardiniet al.2013). In this study,two conserved domains like their plant homologous counterparts were identified in TaNFYB11. Additionally,TaNF-YB11shares high similarities to the NF-YB family genes at the nucleic acid level,and its translated products target nucleus after ER assortment.These findings suggested thatTaNF-YB11acts as a member of the NF-YB family inT.aestivum.

The NF-Y TFs transcriptionally regulate the downstream genesviaa heterotrimer constituted by each of three kinds of subunits,namely,NF-YA (CBF-B/HAP2),NF-YB (CBF-A/HAP3),and NF-YC (CBF-C/HAP5) (Nardiniet al.2013).To address the partners in NF-YA and NF-YC families that interact with TaNF-YB11,yeast two-hybrid assays were performed using TaNF-YB11 protein expressed in an expression system as a bait and the proteins of NFYA and NF-YC inT.aestivumexpressed in an expression system as preys,respectively. Our results indicated that TaNF-YB11 specifically interacts with TaNF-YA2 and NFYC3. These results suggested the putative heterotrimer established among TaNF-YB11 and the NF-YA member TaNF-YA2 as well as the NF-YC member TaNF-YC3,which exerts potential roles in regulating the expression of the downstream genes at the transcriptional level.

4.2.TaNF-YB11 confers improved drought tolerance on plant due to its role in mediating leaf water loss rate

The genes in the NF-Y family mediate plant responses to various abiotic stresses. For example,PdNF-YB7,a NF-YB family gene in popular,upregulated expression levels in plants upon the drought and ABA signaling. The transgenic lines withPdNF-YB7overexpression displayed enhanced root architecture establishment,photosynthetic function,water use efficiency (WUE),and improved drought tolerance of plants with respect to wild type under drought treatments (Hanet al.2013). Likewise,NF-YB2,a member of the NF-YB family inArabidopsis,exhibited induced transcripts when plants were treated with water deprivation. Overexpression ofNF-YB2conferred an elevated ability of drought adaptation on plants,whereas its knockout caused plants a typical phenotype to be drought stress sensitive (Satoet al.2019). These findings together confirm the conserved function of the NF-Y family members in mediating plant adaptation to drought stress conditions. In this study,the transgenic lines ofTaNF-YB11with overexpression and knockdown expression were generated to characterize the target gene function in regulating drought tolerance. The lines with modified expression ofTaNF-YB11displayed altered phenotypes,biomass,and photosynthetic parameters under drought treatments. Compared with wild type,the lines withTaNF-YB11overexpression (Sen 1 and Sen 2) significantly improved,whereas lines with target knockdown expression (Anti 1 and Anti 2) deteriorated the growth traits and photosynthetic function under drought conditions. These results suggested thatTaNFYB11,a gene in the NF-YB family,acts as an essential regulator in mediating plant drought acclimation. In this study,the results for defining theTaNF-YB11function in mediating drought adaptation were obtained based on the hydroponic culture approach,which was consistent with the growth traits of transgenic lines grown in soil treated with drought conditions (Appendix G). In this investigation,Sen 1 and Sen 2 reduced the leaf water loss rate (WLR),whereas Anti 1 and Anti 2 promoted leaf WLR under drought treatments. The mechanisms of the modified WLR underlying theTaNF-YB11regulation in response to drought stresses are needed to be further characterized.

4.3.Upregulated expression of TaP5CS2 underlying TaNF-YB11 enhances osmolytes accumulation to contribute to plant adaptation to drought stress

Proline and other types of osmolytes,such as soluble sugar,act as the crucial biochemical compounds in plant drought response,given their functions in the sustainment of cellular water potential and biochemical metabolism(Fuet al.2018). In this study,the contents of osmolytes,including proline and soluble sugar,were evaluated in the transgenic lines after the drought treatment. More or less accumulation of the above osmolytes was shown in lines with the overexpression or knockdown expression ofTaNF-YB11,respectively. These findings suggested the positive function ofTaNF-YB11in regulating osmolytes biosynthesis,which contributes to plant drought defensiveness. Previously,it was recorded that delta 1-pyrroline-5-carboxylate synthetase (P5CS)proteins act as the rate-limiting enzyme in regulating proline metabolism,whose increased activities benefit the improved plant responses to osmotic stress conditions(Savouréet al.1995;Yoshibaet al.1995;Székelyet al.2008). Furthermore,the P5CS family genes displayed modified transcription efficiencies upon external cues,such as signals initiated by osmotic stresses and H2O2,and variations of calcium ion,PLC (phospholipase C),PLD (phospholipase D),and ABA at the cellular level(Yoshibaet al.1995;Savouréet al.1997;Strizhovet al.1997;Parreet al.2007;Verslueset al.2007). These findings suggest that the genes in the P5CS family are involved in various physiological processes of the plants.In this study,to understand the molecular processes related to proline biosynthesis underlyingTaNF-YB11regulation,the transcripts of the P5CS family genes inT.aestivumwere analyzed in the drought-challenged transgenic lines. One of which referred to asTaP5CS2showed expression of upregulated in Sen 1 and Sen 2 and downregulated in Anti 1 and Anti 2,respectively.Moreover,the transgenic results ofTaP5CS2validated its positive role in enhancing proline accumulation.Therefore,TaNF-YB11andTaP5CS2constitute a major pathway contributing to plant drought adaptation through improving osmotic-adjustment ability at the cellular level.The transcriptional mechanisms ofTaP5CS2underlyingTaNF-YB11modulation are needed to be further defined.

4.4.Antioxidant enzyme genes TaSOD2 and Ta-CAT3 are transcriptionally regulated by TaNF-YB11 to improve ROS homeostasis of cells challenged with drought stressor

Antioxidant enzymes (AE),such as SOD,CAT,and POD,are essential in plant stress responses,given their functions in improving the cellular protection system under adverse stress conditions (Ajithkumar and Panneerselvam 2014;Heet al.2017). Thus far,it has been reported that enhancement of the AE activities positively impacts plant osmotic stress defensiveness. For example,several genes in the Cu/Zn-SOD family confer increased drought tolerance on plants (Wuet al.2016).CAT2,a gene in the catalase family,positively mediates plant adaptation to drought due to its role in scavenging hydrogen peroxide and sustaining cellular ROS homeostasis (Sofoet al.2015). Enhancement of the POD activities lowered the extent of cellular oxidative stress condition,improving photosynthetic organ function and productivity of the plants challenged with water deprivation (Wanget al.2010). In this study,assessment of the ROS-associated traits,such as activities of SOD,CAT,and POD,and contents of MDA were conducted using transgenic lines treated with drought as samples. Compared with wild type,Sen 1 and Sen 2,the lines withTaNF-YB11overexpression,displayed improved ROS homeostasisassociated traits,including increased AE activities and decreased MDA contents. In contrast,Anti 1 and Anti 2,two lines withTaNF-YB11knockdown expression,alleviated the ROS scavenging ability,showed decreased AE activities,and increased MDA accumulation under drought conditions. These results suggested the contribution of improved cellular ROS homeostasis to drought adaptation ability in transgenic lines. To understand the modified AE activities in transgenic lines,the expression patterns of genes in the SOD,CAT,and POD families were determined in transgenic lines after drought treatments. Results indicated thatTaSOD2andTaCAT3,the genes encoding the SOD and CAT proteins,respectively,modified expression in the transgenic lines with respect to wild type. Both of them showed upregulated transcripts in Sen 1 and Sen 2 and downregulated expression in Anti 1 and Anti 2 compared with those shown in WT. Previously,it was recorded that the modified transcription efficiencies of genes underlying NF-Y TF regulation are closely associated with a conserved CCAAT box,acis-regulatory element situated in gene promoter regions (Frontiniet al.2004;Liu and Howell 2010;Dolfiniet al.2012). Further characterization of CCAAT box as well as othercis-regulatory motifs in the AE gene promoters can deepen the understanding of the transcription mechanisms of AE genes upon the drought signaling. Moreover,transgene analyses of the differential AE genes validated their positive roles in modulating corresponding AE activities. Therefore,TaNF-YB11modulates cellular ROS homeostasisviatranscriptionally regulating distinct AE genes,which contributes to the drought tolerance of plants.

4.5.TaNF-YB11-mediated drought tolerance is accomplished via synergistically regulating diverse physiological processes

High throughput RNA-seq analysis provides useful genetic information and deepens understanding of the molecular processes underlying plant stress responses (Krepset al.2002;Metzker 2010;Haoet al.2020). In this study,the drought-challenged lines withTaNF-YB11overexpression were subjected to RNA-Seq analyses to characterize the differentially expressed (DE) genes underlyingTaNF-YB11regulation at the global level. Based on the DE genes identified in the transgenic lines with respect to wild type,it was shown that they were concentrated on five functional processes,including ‘cellular processes’,‘environmental information processing’,‘genetic information processing’,‘metabolism’,and ‘organismal systems’. Therefore,these findings suggested that theTaNF-YB11-mediated drought tolerance is a comprehensively integrated result covering huge amount of genes in modifying physiological processes,which associate with biomass production,photosynthetic function,osmolyte biosynthesis,and cellular ROS homeostasis of plants. The diagram that illustrates the physiological mechanisms contributing to plant drought adaptation underlyingTaNF-YB11regulation is shown in Fig.9-B. Further functional characterization of the essential genes involving signaling transduction and regulatory function can help elucidate the biochemical pathways associated with drought adaptation regulated by NF-Y TF genes.

5.Conclusion

TaNF-YB11is similar to its counterparts distributed in various plant species. The TaNF-YB11 protein targets nucleus after ER assortment,establishing a heterotrimer by interacting with TaNF-YA2 and TaNF-YC3.TaNFYB11is sensitive in response to drought stresses in root and leaf tissues,with a typical of dose and temporal dependent mode. Overexpression ofTaNF-YB11confers improved drought tolerance on plants,which is associated with positively regulating leaf water retention capacity,photosynthetic function,osmolyte accumulation,and cellular ROS homeostasis. Distinct genes in the P5CS family gene (i.e.,TaP5CS2) and those in AE families(i.e.,TaSOD2andTaCAT3) are regulated byTaNFYB11at the transcriptional level,playing critical roles in promoting proline accumulation and sustaining cellular ROS homeostasis in the drought-challenged plants.TaNF-YB11also regulates quantities of genes at the transcriptional level;its modified transcription leads to plant drought acclimationviamodulating various biological processes associated with plant growth and drought stress defensiveness.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31872869),the State Key Laboratory of North China Crop Improvement and Regulation (NCCIR2022ZZ-7),the National Key R&D Program of China (SQ2022YFD1200002),the Science and Technology Planning Project of Hebei Province,China(216Z6401G),and the Postgraduate Innovation Funding Project of Hebei Province,China (CXZZSS2021071).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm