Genome-wide analysis of nuclear factor Y genes and functional investigation of watermelon ClNF-YB9 during seed development

Qin Feng,Ling Xio,Jif Wng,Jie Wng,Chenyng Chen,Jinyng Sun,Xixi Wu,Mn Liu,Xin Zhng,Shujun Tin,b,,Li Yun,b,

a State Key Laboratory of Crop Stress Biology for Arid Areas,College of Horticulture,Northwest A&F University,Yangling 712100,Shaanxi,China

b Shenzhen Research Institute,Northwest A&F University,Shenzhen 518000,Guangdong,China

Keywords: Watermelon NF-Y gene family Evolution relationship ClNF-YB9 gene function Seed development

ABSTRACT The nuclear factor Y (NF-Y) gene family is a class of transcription factors that are widely distributed in eukaryotes and are involved in various biological processes.However,the NF-Y gene family members in watermelon,a valued and nutritious fruit,remain largely unknown and their functions have not been characterized.In the present study,22 ClNF-Y genes in watermelon,29 CsNF-Y genes in cucumber,and 24 CmNF-Y genes in melon were identified based on the whole-genome investigation and their protein properties,gene location,gene structure,motif composition,conserved domain,and evolutionary relationship were investigated.ClNF-YB9 from watermelon and its homologs in cucumber and melon were expressed specifically in seeds.Its expression remained low in the early stages of watermelon seed development,increased at 20 days after pollination (DAP),and peaked at 45-50 DAP.Moreover,the knockout mutant Clnf-yb9 exhibited abnormal leafy cotyledon phenotype,implying its critical role during seed formation.Finally,protein interaction assays showed that ClNF-YB9 interacts with all ClNF-YCs and the ClNF-YB9-YC4 heterodimer was able to recruit a ClNF-YA7 subunit to assemble a complete NF-Y complex,which may function in seed development.This study revealed the structure and evolutionary relationships of the NF-Y gene family in Cucurbitaceae and the novel function of ClNF-YB9 in regulating seed development in watermelon.

1.Introduction

Nuclear factor Y(NF-Y),also known as CCAAT binding factor,is a transcription factor family widely distributed in eukaryotes.It is a heterotrimeric complex composed of NF-YA,NF-YB,and NF-YC subunits [1].Two alpha helices,A1 and A2,constitute the highly conserved domains of NF-YA,which are responsible for NF-YB/C interaction and CCAAT binding,respectively [2,3].NF-YB has four alpha helices,three of which are located in a conserved domain sharing a similar structure and amino acids with the histone fold motif(HFM)of the core histone H2B[4].NF-YC is commonly characterized by an HFM domain,but is more closely related to the core histone H2A[5].Generally,NF-YB and NF-YC dimerize in the cytoplasm,enter the nucleus and associate with NF-YA to form a mature trimer transcription complex that binds specifically to CCAAT in the promoter regions of target genes to regulate their expression [6].In yeast and mammals,each subunit is encoded by a single gene;but in plants these subunits have evolved as gene families.Arabidopsis thalianacontains 36 NF-Y members: ten NFYA,thirteen NF-YB,and thirteen NF-YC[7].The expansion of these subfamilies leads to the possible presence of diverse combinations of NF-Y subunits in plants,suggesting their diverse functional possibilities.

In recent years,the role ofNF-Ygene family members in plant abiotic stresses,including drought,salt,heat and cold,has attracted increasing attention.Under drought conditions,expression ofArabidopsis AtNF-YA5in micro-tubule organism and guard cells is induced to reduce water loss and increase plant drought tolerance[8],and homologousGmNF-YA5in soybean has a similar function during drought stress[9].Overexpression of wheatTaNFYA10inArabidopsisreduces salt tolerance by down-regulating the transcription of stress-responsive genes [10].LikeNF-YA,the rootspecific NF-Y family transcription factorPdNF-YB21has been reported[11]to be a positive regulator of root growth and drought resistance by abscisic acid (ABA)-mediated indoleacetic acid (IAA)transportation inPopulus.In maize,ZmNF-YB16increases plant photosynthesis and antioxidant capacity and also increases maize drought-stress resistance and grain yield [12].AtNF-YC10 forms a complex with NF-YA2/NF-YB3 and participates in regulation of heat shock stress response[13].AtNF-YC1(AtHAP5A)can increase freezing resistance by interacting with a CCAAT motif in the promoter region ofAtXTH21inArabidopsis[14].GmNF-YC14 has been shown [15] to form a heterotrimer with GmNF-YA16 and GmNFYB2 to activate theGmPYR1-mediated ABA signaling pathway to regulate stress tolerance in soybean.

There is much evidence thatNF-Ygenes function not only in resistance to abiotic stress but in plant growth and development,including photosynthesis,male gametogenesis,flowering,embryogenesis,and fruit and seed development[16-19].NF-YCsmodulate histone the deposition variant H2A.Z to regulate photomorphogenic growth[20]and mediate the inhibition of the brassinosteroid (BR) signaling pathway by regulating BR biosynthesis and signaling transduction to promote photomorphogenesis inArabidopsis[21].In a recent study [22],NF-YB1 interacted with OsMADS14 to promote the expression ofOsAGPL2andWaxyto regulate the synthesis of storage starch in rice endosperm.

LEAFY COTYLEDON 1(LEC1)is an atypical subunit of NF-Y,also named AtNF-YB9.In a previous study [23] a single amino acid change in the HFM domain of NF-YB,from a lysine (K) to aspartic acid (D) at position 55 allowed a non-LEC1-typeNF-YBto be converted to aLEC1-typeNF-YBgene inArabidopsis.Several studies ofLEC1have focused on embryo and seed development [24-26].Alec1mutant showed pleiotropic phenotypes compared to the wild type,such as trichomes on cotyledons,abnormal stalk development,and seeds with defects in starch,protein,and oil accumulation [27-29].LEC1also promotes the initial establishment of an active chromatin state atFLOWERING LOCUS C(FLC) and activates its expressionde novoin the pro-embryo for reversing the silenced state inherited from gametes [30].

In view of the roles ofNF-Ygenes in various biological processes and with the development of whole-genome sequencing technology,more and more members ofNF-Ygene family have been identified in different species [31-36].However,the basic characteristics of physical and chemical properties and functions ofNF-Ysin Cucurbitaceae family and their relationships among Cucurbitaceae species have been little studied.Watermelon,cucumber,and melon are Cucurbitaceae crops cultivated worldwide.In this study,22 watermelon ClNF-Ys,29 cucumber CsNFYs,and 24 melon CmNF-Ys were identified in the newly released genome sequences of watermelon,cucumber and melon.Their basic properties,gene structures,conserved domains,evolutionary relationships andcis-elements in promoter regions were characterized using bioinformatic methods.LEC1-typeClNF-YB9was expressed specifically in watermelon seeds.Loss-of-function mutants ofClNF-YB9generated with the CRISPR/Cas9 system showed thatClNF-YB9functioned in watermelon seed development.Yeast assays indicated that ClNF-YB9 interacts with ClNFYCs and ClNF-YB9-YC4-YA7 complex act an important role in seed formation.Our study lays a foundation for further functional mining and verification ofNF-Ygenes in watermelon,cucumber,and melon and provides new materials useful for revealing the mechanisms of watermelon seed development and its regulatory network.

2.Materials and methods

2.1.Genome-wide identification of NF-Y genes

Protein sequences of NF-Ys inArabidopsiswere retrieved from TAIR (https://www.arabidopsis.org/).These sequences were searched by BLAST(https://cucurbitgenomics.org/v2/blast)against watermelon,cucumber,and melon genomes from the Cucurbit Genomics Database [37] with an E-value cutoff of 1e-10.Hidden Markov model (HMM) profiles of NF-Ys (PF02045.17 and PF00808.25) from the Pfam database (https://pfam.xfam.org/)were constructed to search for potential NF-Ys with HMMER 3.0(https://www.hmmer.org/).Results of NCBI CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi),the Pfam database,and SMART (https://smart.embl.de/) to identify conserved protein domains were combined to identify allNF-Ygenes in the three cucurbit genomes.ProtParam [38] was used for physical and chemical information prediction,including theoretical isoelectric point(pI),instability index,aliphatic index,and grand average of hydropathicity (GRAVY).

2.2.Chromosome location,gene structure,motif,and conserveddomain analysis

The lengths of chromosomes andNF-Ygene chromosomal positions were retrieved from the Cucurbit Genome Database and visualized with MapGene2Chromosome (https://mg2c.iask.in/mg2c_v2.0/).Gene structures were drawn by Gene Structure Display Server [39].MEME [40] was used for prediction of NF-Ys conserved motifs.Multiple protein sequence alignments were constructed with DNAMAN V6 software.

2.3.Phylogenetic analysis of NF-YA,NF-YB and NF-YC in multiple species

ArabidopsisNF-Y homologs in were retrieved as described previously [7].NF-YA,NF-YB and NF-YC subunit family members were aligned separately with ClustalW.A phylogenetic tree was generated with MEGA7.0 software by the neighbor-joining method.

2.4.NF-Y gene duplication and collinearity detection in Cucurbitaceae species

Identification of duplicatedNF-Ygene pairs and collinearity in the three Cucurbitaceae species was performed with TBtools [41].

2.5.Prediction of cis-elements in potential promoter regions of NF-Y genes

The promoter regions(2000 bp upstream of ATG)ofNF-Ygenes from watermelon,cucumber and melon were submitted to Plant-CARE (PlantCis-Acting Regulatory Element,https://bioinformatics.psb.ugent.be/webtools/plantcare/html/)forcis-element prediction,and heat maps were generated with TBtools.

2.6.Plant materials and expression profiles analysis using RT-qPCR

Wild-type YL watermelon cultivars,9930 cucumber,and OT melon were grown under natural conditions at 28-35 °C/16-20 °C (day/night) in an experimental field in March.Samples from eight tissues (root,stem,leaf,male flower,female flower,fruit flesh,seed,and pollen) and multiple seed development stages(0-60 DAP) were collected,immediately frozen in liquid nitrogen,and stored at -80 °C for RNA extraction.RT-qPCR was performed as described previously [42].The specific primers forClNF-Ys,CsNF-YB4/5/6/7andCmNF-YB8genes are listed in Table S1,ClACTIN,CsUBQ,andCmACTINwere used as internal controls.

2.7.Subcellular localization of ClNF-YB9 proteins

The complete CDS sequence without stop codon was amplified and inserted into the pGREEN vector fused with marker gene GFP.The control plasmid pGREEN-35S::GFPand pGREEN-35S::ClNFYB9::GFPwere introduced intoAgrobacterium tumefaciensGV3101(pSoup).Four-to six-week-oldNicotiana benthamianaleaves were used for transient transformation.The primers used to clone theClNF-YB9are described in Table S2.

2.8.CRISPR/Cas9 mediated ClNF-YB9 knockout in watermelon

A knockout vector was constructed with two target sites ofClNF-YB9designed with CRISPR-P v2.0 (https://crispr.hzau.edu.cn/CRISPR2/) andA.tumefaciensEHA105 carrying theClNF-YB9-gRNA was used for watermelon transformation as previously described [43].Target gene mutagenesis results were performed as described [44].Primers for vector construction and specific target-site amplification are described in Table S2.

2.9.Yeast-two-and three-hybrid assays

For yeast two-hybrid assay,ClNF-YB9was amplified and inserted into pGBKT7 as a bait,andClNF-YCswere constructed with pGADT7 as prey and co-transformed to Y2H Gold strain as recommended by the manufacturer(Clontech,Mountain View,CA,USA).A yeast-three-hybrid system pBridge (Clontech) was constructed as described previously [45].Primers for vector construction in yeast assays are described in Table S2.

3.Results

3.1.Genome-wide identification,gene structure,and motif contribution in representative Cucurbitaceae species

ArabidopsisNF-Y proteins were used as query sequences for BLAST against watermelon,cucumber,and melon genomes.All candidate NF-Y proteins were submitted to NCBI,Pfam and SMART for conserved-domain identification.The conserved-domain search identified seven ClNF-YA,ten ClNF-YB,and five ClNF-YC subunits in watermelon inbred line 97103 (Table S3);seven CsNF-YA,fourteen CsNF-YB,and eight CsNF-YC in Chinese Long cucumber(Table S4);and seven CmNF-YA,eleven CmNF-YB,and six CmNFYC in the DHL92 melon genome (Table S5).They were unevenly distributed on most chromosomes in the three species (Fig.S1),and were renamed according to their chromosomal locations.

The shortest proteins were CmNF-YB5 and CmNF-YC4(117 AA)and the longest was ClNF-YB2(1016 AA).Their molecular weights ranged from 12,807.38 Da (CsNF-YB14) to 112,368.07 Da (ClNFYB2).Their theoretical pIs varied from 4.75 (CsNF-YB1) to 10.1(CsNF-YC1),instability indices ranged from 29.11 (CsNF-YB2) to 69.13 (CsNF-YA4) and aliphatic indices varied from 46.68 (ClNFYB1) to 94.62 (CsNF-YC8).Every NF-Y protein showed a negative GRAVY value (Tables S3-S5).As shown in Fig.S2,all but 18 members (ClNF-YB1/6/7/8,CsNF-YB3/5/12,CsNF-YC2/3/6,CmNFYB1/7/8/9/11andCmNF-YC1/2/4) contained one or more introns.Almost all members harbored similar conserved motifs belonging to NF-YA,NF-YB or NF-YC subfamily (Figs.S3-S5).

3.2.Phylogenetic relationships and conserved domains of NF-Ys

Although the functions ofNF-Ygenes indicotssuch as the model plantArabidopsishave been extensively studied,those ofNF-Ygenes in Cucurbitaceae crops were unknown.To characterize the evolutionary relationships among the NF-Y subunits and their potential functions,a phylogenetic tree of NF-Ys from watermelon,cucumber,melon,andArabidopsis(22 watermelon ClNF-Ys,29 cucumber CsNF-Ys,24 melon CmNF-Ys,and 36ArabidopsisAtNFYs) was generated.As shown in Fig.1,these proteins formed clear subfamilies:NF-YA,NF-YB,and NF-YC.Most of the NF-Ys clustered together.The subfamilies and clan of NF-Ys matched the branches well,supporting the reliability of the phylogenetic tree.In subgroup branches,ClNF-YB9,CsNF-YB4/5/6/7/8,and CmNF-YB8 were clustered with AtNF-YB9,a typicalLEC1-type NF-YB functioning in embryo development and seed maturation [46].ClNF-YC3,CsNFYC2 and CmNF-YC1 were tightly clustered in a branch with AtNF-YC1/2/3/4/9,suggesting that these genes share similar functions,possibly in early flowering associated with photooxidative stress [47].However,CsNF-YC1/8 showed a distant evolutionary relationship with other NF-YC members,possibly owing to the evolutionary diversity of these members.

Fig.1.Phylogenetic relationships and subfamily classification of NF-Ys from four species.Phylogenetic relationships among NF-YA proteins(pink)from watermelon (seven proteins),cucumber (seven proteins),melon (seven proteins) and Arabidopsis (ten proteins);NF-YB proteins (gray) from watermelon (ten proteins),cucumber (fourteen proteins),melon (eleven proteins) and Arabidopsis (thirteen proteins);NF-YC proteins (orange) from watermelon (five proteins),cucumber (eight proteins),melon (six proteins) and Arabidopsis (thirteen proteins).The tree was constructed by neighbor joining method.Watermelon (ClNF-Ys),cucumber (CsNF-Ys),melon (CmNF-Ys),and Arabidopsis (AtNF-Ys),are shown as respectively blue,yellow,green and red dots.

The conserved region NF-YA is consisting of two highly conserved A1 and A2 alpha helices in Fig.2A shown as purple blocks and responsible for NF-YB-YC subunit interaction and DNA binding(CCAAT binding),respectively.Among cucurbit species,all members of the NF-YA subfamily contained relatively conserved domains except for CsNF-YA3 in cucumber and CmNF-YA5 in melon.NF-YB is made up of four alpha helices,three of which are located in the conserved domain and share similar structure and amino acids with the histone fold motif(HFM)of the core histone H2B (Fig.2B),while NF-YC is closer to the core histone H2A(Fig.2C).Several members of NF-YB have conserved lysine (K) to aspartic acid (D) conversion,which is important for protein interactions and preserved in most NF-YB proteins,such as ClNF-YB9,CsNF-YB4/5/6/7 and CmNF-YB8 (Fig.2B).Although the phylogenetic tree showed that CsNF-YB8 clustered withLEC1-type members (Fig.1),amino-acid residue deletions at the K-D site suggested that CsNF-YB8 was not a typical LEC1 NF-YB.Compared to NF-YA and NF-YB,NF-YC was less conserved among the four species,particularly in the NF-YB interaction domain,which contained multiple variations and substitutions.ClNF-YC1 and CsNFYC1/5/8 were less conserved in NF-YB and NF-YA interaction regions with multiple amino acid changes,suggesting that new functions may have emerged during the evolution of Cucurbitaceae crops.

Fig.2.Multiple sequence alignment of NF-Y proteins with labeled domains.(A) The NF-YA conserved region is composed of two alpha helices: A1 mediates NF-YB-NF-YC interaction and A2 is responsible for CCAAT binding.These two domains were highly conserved in Cucurbitaceae and shown by purple blocks.(B)NF-YB has four alpha helices(shown by blue blocks),three of which lie in a conserved domain sharing similar structure and amino acids with the histone fold motif(HFM)of the core histone H2B(shown with gray lines) while NF-YC (C) is close to the core histone H2A.

3.3.Gene duplication and collinearity of NF-Ys in three Cucurbitaceae species

Gene duplications,such as tandem and segmental duplication,lead to expansion of gene families.To identify evolutionary relationships in theNF-Ygene family in duplication events in watermelon,cucumber,and melon,we performed gene duplication and collinearity analysis in each species respectively as well as synteny analysis in three Cucurbitaceae species using TBtools.No tandem duplication events ofNF-Ys were found(Fig.S6).Segmental duplications of theNF-YAandNF-YBgenes were detected(Table S6),such asClNF-YA2/ClNF-YA3,ClNF-YA2/ClNF-YA5,andClNF-YB2/ClNF-YB10,suggesting that theNF-YAandNF-YBsubfamilies are expanded only by segmental repeats.In contrast,no gene replication was found in anyNF-YC.Comparative collinearity analysis revealed 80 homologous gene pairs(Fig.3),of which 20ClNFYsfrom watermelon were detected in both cucumber and melon,whereasClNF-YA6andClNF-YA7were not (Table S7).ClNF-YA3was represented by the most homologous genes:two in cucumber(CsNF-YA1andCsNF-YA7) and three in melon (CmNF-YA1,CmNFYA2andCmNF-YA4).In conclusion,gene duplication and collinearity analyses indicate a close evolutionary interspecific and intraspecific relationship among the three Cucurbitaceae species.

Fig.3.Collinearity analysis of ClNF-Ys,CsNF-Ys and CmNF-Ys.Collinearity analysis of all NF-Y genes from watermelon(ClNF-Ys),cucumber(CsNF-Ys)and melon(CmNF-Ys)as shown by TBtools.Yellow color indicates cucumber;purple,watermelon;and green,melon.Gray lines show all interspecific gene pairs among the three Cucurbitaceae species and blue lines show collinearity of NF-Y gene pairs.

3.4.Cis-elements in NF-Y promoter sequences

Thirty-onecis-elements of four types: eight light-responsive(ACE,AE-box,Box 4,GATA motif,GT1 motif,I box,MRE,and TCT motif),seven development-associated elements (CATbox,CCGTCC motif,circadian,GCN4 motif,HD-Zip 1,O2 site and RY element),six stress-responsive elements (ARE,GCWUN motif,GT motif,LTR,MBS,and TC-rich repeats) and ten phytohormoneresponsive elements (ABRE,AuxRR core,CGTCA motif,ERE,GARE motif,Pbox,TATC box,TCA element,TGA element,and TGACG motif),were identified in the promoter regions of genes from the three Cucurbitaceae species (Figs.4,S7).The promoters of 22ClNF-Yscontained respectively 168,166,and 93 elements associated with light,phytohormone,and stress-response.Only a few members of the family harbored development-associated regulatory elements in their promoter regions.A RY element,a seedspecific developmental regulatory motif appearing in many seedspecific gene promoter regions,was identified in theClNF-YA4andClNF-YB85′region (Fig.4).The RY element was proposed[48] to be the recognition and binding site of plant-specific B3 DNA binding factors,which include the seed development regulatory genesABI3,VAL1,andVAL2.The presence of the RY motif in the gene promoter region ofClNF-YA4andClNF-YB8suggested that they were targeted by seed-specific transcription factors.The ABRE element,which is known [49] to be bound by ABI3 to activate downstream gene expression during seed maturation,was found in the promoter region of ClNF-YA1/2/5/7 and all ClNF-YBs but ClNF-YB2 and ClNF-YC2/3/4.Light morphogenesis,hormone signaling,and stress response participate in many essential developmental programs,including plant growth,cell fate differentiation,and seed development.The presence of multiple regulatory elements in the promoter regions of theNF-Ygenes indicates the roles ofNF-Ygenes in multiple biological processes in Cucurbitaceae crops.

Fig.4. Cis-element analysis of promoter regions of ClNF-Y genes.ACE,AE-box,Box 4,GATA motif,GT1 motif,I box,MRE,and TCT motif are light-responsive elements;CAT box and CCGTCC motif are involved in meristem expression;GCN4 motif,in endosperm expression;O2 site,in zein metabolism regulation;circadian,HD-Zip 1,and RY element,involved in circadian control,differentiation of the palisade mesophyll,and seed-specific regulation.ARE,involved in anaerobic induction;LTR,low-temperature-responsive element;GT motif,involved in anoxic specific inducibility;MBS,TC-rich repeats,involved in defense and stress responsiveness;GCWUN motif,wound responsiveness;ABRE,abscisic-acid responsive element;AuxRR core,auxin-responsive element;TGA element,auxin-responsive element;CGTCA motif,TGACG motif,MeJA-responsive elements;ERE,ethylene-responsive element;GARE motif,P box,and TATC box,gibberellin-responsive elements;TCA element,salicylic-acid-responsive element.

3.5.Expression profiles of ClNF-Ys in watermelon tissues

To investigate the roles and functions ofNF-Ygenes during plant growth and development in watermelon,we sampled eight different tissues (root,stem,leaf,male flower,pollen,female flower,fruit flesh,and seed) for expression profile analysis of 22ClNF-Ys(Fig.5A).A variety of gene expression patterns were observed.ClNF-YA3andClNF-YB10were expressed in all tissues,whereasClNF-YA1/4andClNF-YB3/4were hardly detectable in any tissue.ClNF-YA6andClNF-YC2were weakly expressed in roots and female flowers compared to other tissues.In fruit flesh,no members were expressed exceptClNF-YA3,ClNF-YB10,andClNFYC3/4.Seed-specific RYcis-elements were identified inClNF-YA4andClNF-YB8promoter regions,but onlyClNF-YB8showed expression in seeds,suggesting that many regulatory mechanisms may affect the expression of these genes.ClNF-YA2/5/7,ClNF-YB1/2/5/9andClNF-YC2/3/4/5were also expressed in seeds.ClNF-YB9was highly transcribed in seeds,suggesting that it functions during seed development.Because ClNF-YB9 in watermelon,CsNFYB4/5/6/7 in cucumber and CmNF-YB8 in melon were verified to beLEC1-type NF-YB factors,detailed expression analyses of these genes were performed in cucumber and melon.Similar toClNFYB9,CsNF-YB4/5/6/7andCmNF-YB8were highly expressed in seeds but less so in other tissues(Fig.S8).This highly specific expression pattern implied their conserved functionality in seed development in cucurbit crops.

Fig.5.Gene expression profiles of ClNF-Ys. (A) Gene expression patterns of ClNF-Ys in eight watermelon tissues.Red boxes indicate higher and green,lower expression.All experiments were performed with three independent replicates. ClACTIN was used as control.The heat map was represented by log2 (value of relative expression).(B)Relative expression of ClNF-YB9 at 12 stages during seed development in watermelon.(C) Gene subcellular location analysis of ClNF-YB9 protein.Relative expression of NF-Ys was calculated by the 2-ΔΔCT method.

3.6.Potential functions of ClNF-YB9 during seed development in watermelon

The development of seeds in dicots is a complex process that starts with gamete fusion,followed by embryo development and seed maturation.One of the best-investigated members of the NF-Y transcription factor family is LEAFY COTYLEDON1 (LEC1),functions in embryogenesis and seed maturation,but its function in watermelon has not been studied.Given that ClNF-YB9,CsNFYB4/5/6/7 and CmNF-YB8 shared a conserved domain ofLEC1-type proteins and were highly expressed in seeds (Fig.S8),we adopted watermelon as a model cucurbit for investigating the functions of these genes.The expression of ClNF-YB9 was analyzed at different development stages during seed formation.Transcription ofClNF-YB9remained low in early stages,but increased sharply at 20 DAP and peaked again at 45-50 DAP (Fig.5B).In the transient expression assay of ofClNF-YB9fused with GFP(Fig.5C),fluorescence signals were strong in the nucleus and weak in cytoplasm.

To further investigate the function ofClNF-YB9gene during watermelon seed development,CRIPSR/Cas9 system was employed to mutate theClNF-YB9(Fig.6A).In the T1generation,three homozygous gene-editing lines,Clnf-yb9_1,Clnf-yb9_2andClnf-yb9_3,were obtained.The presence of a 1-bp deletion inClnf-yb9_1,74 deletions plus 32 insertions inClnf-yb9_2,and a 69-bp deletion inClnf-yb9_3were confirmed at theClNF-YB9locus(Fig.6B).No vegetative growth defects were observed in the mutant plants (Fig.S9A),and fruits were able to expand normally(Fig.S9B).Although the 69-bp deletion inClnf-yb9_3resulted in a truncated protein with a 23-amino acid deletion,Clnf-yb9_3showed no abnormality during seed development.Thus,the truncated protein seems be function normally,probably because the missing amino acids are not located in an essential domain ofClNF-YB9.In contrast,Clnf-yb9_1andClnf-yb9_2,2 potential null mutants,showed similar abnormal seed phenotypes during development.Accordingly,further seed phenotype analysis was performed mainly withClnf-yb9_1.

TheClnf-yb9_1mutant set fewer seed on maturation than did WT plants (Fig.6C),some of which were shrunken,aborted seeds.About 43%ofClnf-yb9_1seed developed into mature seeds containing plump embryos,but the remainder aborted at early seed developmental stage and eventually became yellowish,hollow seed shells after desiccation (Fig.6D,E).Tracing their phenotypes back to earlier developmental stages showed that at 25 DAP aborted seeds enlarged normally like the wild type,indicating that seed coat development was not impaired.But these abnormal seeds showed lighter color than the wild type.At 60 DAP,the mature stage of the normal seeds,the abnormal seeds continued to show color differences from the wild type (Fig.6F).The abnormal seeds contained defective embryos that displayed leafy cotyledon leaves with curling leaf edges(Fig.6G),exactly resembling the phenotype observed in anArabidopsishomozygouslec1mutant[50].The seeds of homozygouslec1mutant were not viable during seed desiccation inArabidopsis[26].But the 43% normal-looking homozygous mutant seeds inClnf-yb9_1reached maturity and could germinate immediately after harvest,without showing any seed dormancy(Fig.6C and data not shown),inviting further analysis of the gene function ofClNF-YB9in seed maturation and germination.The phenotypes of loss-of-function mutants show that watermelonClNFYB9functions in embryo morphogenesis during early seed development,but its function during seed maturation awaits further study.

Previous studies have shown that the NF-Y trimer is the main functional form of these factors.The NF-YB subunit interacts with the NF-YC subunit to form the NF-YB-YC heterodimer and then recruits the NF-YA subunit to form a complete NF-Y complex that binds to CCAAT in the promoter regions of downstream genes via the NF-YA subunit to regulate their expression.The yeast twohybrid assay showed that ClNF-YB9 interacted with ClNF-YC1,ClNF-YC2,ClNF-YC3,ClNF-YC4,and ClNF-YC5 in yeast (Fig.7A).Given that all ClNF-YCs,except for ClNF-YC1,are highly expressed in seeds(Fig.5A),we used ClNF-YB9-YC4 as a representative of the NF-YB-YC dimer to investigate the potential NF-Y complex during seed formation by yeast three-hybrid assay.ClNF-YA1/6/7 showed interaction signals with ClNF-YB9-YC4,whereas ClNF-YA2/3/4/5 did not interact with ClNF-YB9-YC4 in yeast (Fig.7B),indicating that the ClNF-YB9-YC4 dimer could recruit ClNF-YA1/6/7 to form three different NF-Y complexes.ClNF-YA1andClNF-YA6transcripts were undetectable in seeds(Fig.5A),suggesting that the ClNF-YB9-YC4-YA7 trimer may be the unique NF-Y complex functions in seed development.These results suggested that ClNF-YB9 may interact with several ClNF-YC to form multiple dimers able to enter the nucleus,and that the ClNF-YB9-YC4 dimer recruits ClNF-YA7 to assemble the complete NF-Y complex.

Fig.7.ClNF-YB9 interacted with ClNF-YC and ClNF-YA to assemble mature NF-Y complexes in yeast.(A)A yeast two-hybrid assay showed that ClNF-YB9 interacted with all the ClNF-YC members in watermelon.ClNF-YB9 was fused with the pGBKT7 as bait,ClNF-YC1,ClNF-YC2,ClNF-YC3,ClNF-YC4,and ClNF-YC5 were fused to pGADT7 as preys.Empty pGADT7 vector was used as a negative control.Transformed yeast cells were grown on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade medium.(B)Yeast three-hybrid assays show interactions between NF-YAs and NF-YB9 in the presence of NF-YC4.Transformed yeast cells were grown on SD/-Trp-Leu-Met and SD/-Trp-Leu-His-Ade-Met medium.Empty pGADT7 vector was used as a negative control.

4.Discussion

4.1.Identification of NF-Y gene family

NF-Y transcription factor is a universal classification of transcription factors found in eukaryotes,also known as hemeactivator proteins (HAPs) or CCAAT-binding factor (CBF).The NFY complex,a heterotrimer consisting of NF-YA (CBF-B/HAP2),NFYB (CBF-A/HAP3) and NF-YC (CBF-C/HAP5) subunits,regulates the expression of multiple genes.In plants,theNF-Ygene was first discovered inBrassica napus[51].Increasing numbers of NF-Ys have been identified,but the number of members of NF-Ys varies among species.There are eight SbNF-YAs,11 SbNF-YBs and 14 SbNF-YCs,for a total of 33 in sorghum [52];8 VvNF-YAs,18 VvNF-YBs,8 VvNF-YCs,for a total of 34 in grape [53];6 PpNFYAs,12 PpNF-YBs and 6 PpNF-YCs for a total of 24 in peach [54];10 CsNF-YAs,15 CsNF-YBs and 10 CsNF-YCs for a total of 35 in tea plants [31];10 PhNF-YAs,13 PhNF-YBs,and 4 PhNF-YCs for a total of 27 in petunia [32];12 FtNF-YAs,17 FtNF-YBs,and 9 FtNF-YCs for a total of 38 in Tartary buckwheat [35];9 PtNF-YAs,9 PtNF-YBs and 10 PtNF-YCs for a total of 38 inPinus tabuliformis[33];13 PtNF-YAs,20 PtNF-YBs and 19 PtNF-YCs for a total of 52 inPopulus[34];11 MdNF-YAs,22 MdNF-YBsand 10 MdNF-YCsfor a total of 43 in apple [55];9 MsNF-YAs,26 MsNF-YBs,and 25 MsNF-YCs for a total of 60 in alfalfa [36].

There were 19 NF-Ys in watermelon genome V1.0,21 NF-Ys in cucumber genome V2.0,and 19 NF-Ys in melon CM_V3.5[56].But in the present study,the latest versions of watermelon(97103V2.5),cucumber (Chinese Long V3.0) and melon (DHL92 V4) were used to identify the family members of NF-Y genes.A total of 22 ClNF-Ys (seven ClNF-YAs,ten ClNF-YBs,and five ClNFYCs) in watermelon,29 CsNF-Ys (seven CsNF-YAs,fourteen CsNFYBs,and eight CsNF-YCs) in cucumber,and 24 CmNF-Ys (seven CmNF-YAs,eleven CmNF-YBs,and six CmNF-YCs) in melon were identified.Based on the information obtained from newly released genomes,after comparison and identification,we found that three previously reported genes,ClNF-Ys(Cla014640,Cla019279,andCla004399),sixCsNF-Ys(Csa1M569510,Csa3M644810,Csa3M048940,Csa3M049440,Csa3M055940,andCsa7M051420),and threeCmNF-Ys(MELO3C023161,MELO3C018689,andMELO3C026204),could not be found in the new version or did not contain the conserved domain of NF-Ys.Six new ClNF-Ys,four new CsNF-Ys,and eight new CmNF-Ys genes were newly discovered in this study.Many members identified in both old and new versions showed differing gene-length annotations.For example,the number of amino acids in ClNF-YA7 (868 AA) in the new version is much greater than that in Cla016849 (300 AA).

4.2.Conserved domains and evolutionary relationships among species

NF-YA contains a conserved domain consisting of two alpha helices [57],as confirmed in watermelon,cucumber and melon in the present study.The conserved domain of NF-YB contains four α helices,and the amino acid before the second α helix changes from lysine (K) to aspartic acid (D),resulting in conversion of the original non-LEC1 NF-YB to LEC1 NF-YB [23].ClNF-YB9 was the uniqueLEC1-type NF-YB in watermelon,as was CmNF-YB8 in melon.The phylogenetic tree showed CsNF-YB4/5/6/7/8 closely clustered with AtNF-YB9 in cucumber,while CsNF-YB8 was excluded owing to the lack of a conserved aspartic acid.Surprisingly,ClNF-YB5,CsNF-YB1 and CmNF-YB4 both carried a K-R amino acid transition at the site before the second α helix,differing from K-D in previous studies.Whether this change led to new gene functions is unknown.In comparison with NF-YA and NF-YB,NFYCs showed lower conservation,suggesting that new functions of these genes may have emerged during evolution.

Phylogenetic analysis of NF-Y subunits from watermelon,cucumber,melon,andArabidopsisclustered most NF-Y members in watermelon,cucumber and melon,showing a closer evolutionary relationship in cucurbit species and suggesting that these homologous genes have similar functions during plant development.All members of NF-YAs in Cucurbitaceae,including ClNFYA1/5,CsNF-YA6/7,and CmNF-YA1/6,were clustered with AtNFYA3/4/5/6/7/8.AtNF-YA3/8is functionally redundant during early embryogenesis [58],andAtNF-YA5is induced by drought stress at both transcriptional and posttranscriptional levels and influences drought resistance inArabidopsis[8],suggesting that they play multiple roles in plant stress resistance and embryonic development.Previous evidence [59] had shown that AtNF-YC3/4/9 is involved in the regulation of gibberellin (GA) and ABA-mediated functional redundancy during seed germination,and our results indicated that ClNF-YC3,CmNF-YC1,and CsNF-YC2 are more closely related to AtNF-YC3/4/9 than other NF-YCs,possibly indicating functional similarity between them (Fig.1).

Gene duplication,as one of the main drivers of gene family evolution and expansion,has been reported in the study of many gene families[60,61].Identification of collinear orthologs sheds light on gene evolution[62].In Cucurbitaceae,although the NF-Y family did not show tandem duplication,there were three segmental duplication events in watermelon and melon and four in cucumber.On three chromosomes,two pairs of tandem duplication genes(ClNF-YA2/ClNF-YA3,ClNF-YA2/ClNF-YA5) were identified,suggesting thatClNF-YA3andClNF-YA5may be derived from the same duplication event ofClNF-YA2in watermelon.Likewise,in cucumber and melon,(CmNF-YA1/CmNF-YA2,CmNF-YA1/CmNF-YA4;CsNF-YA1/CsNF-YA5,CsNF-YA1/CsNF-YA7)were also identified,suggesting that theNF-Ygene family expansion is driven by segmental duplication events in these three Cucurbitaceae crops.Synteny analysis revealed noClNF-YA6homolog in cucumber and noClNF-YA7in either cucumber or melon (Fig.3),suggesting thatClNF-YB6/7has novel functions in watermelon.The finding that watermelon,cucumber and melon showed high collinearity with one another indicates the high conservation of their ancestral genomes.

4.3.Analysis of cis elements,expression patterns of NF-Ys,and potential functions of ClNF-YB9 during seed development in watermelon

Functions of NF-Y transcription factors have been identified in many plant species.Root-specificPdNF-YB21was involved in abscisic acid-mediated indole acetic acid transport inPopulus[11],andAtNF-YB2andAtNF-YB3positively regulate plant stress resistance via drought and heat tolerance inArabidopsis[63].Becausecisacting elements are tightly coupled with gene function and regulatory mechanisms[64],with the aim of identifying functions of NFY in Cucurbitaceae crops,we identifiedcis-regulatory elements present in the promoter regions of allNF-Ys.Thecis-elements associated with stress response included TC-rich repeats and WUN motif,and phytohormones such as auxin,abscisic acid,gibberellin,ethylene,salicylic acid,and methyl jasmonate that have been detected in promoter regions,suggesting thatNF-Ysparticipate in multiple processes.

For Cucurbitaceae crops,the seed is the most important reproductive organ.Besides its essential function in sexual reproduction,the seed is the most economically important agricultural product,offering food for humans and wildlife and feed for livestock [65].Of genes showing tissue-specific expression,aClNF-YB9,was expressed mainly in seeds.ClNF-YB9was also identified as the onlyLEC1-typeNF-YBgene in watermelon.Functional studies ofLEC1have been performed in many species [25,26,66,67].LEC1is expressed specifically in embryo and endosperm and functions in many biological processes in embryo development and seed maturation [46,68-70].ClNF-YB9was highly expressed at 20 DAP and maintained high transcription levels during seed development.I was confirmed to be a nuclear factor with detectable amounts of protein in the nucleus and cytoplasm,perhaps because NF-YB itself is present in cytoplasm once it interacts with NF-YC or another transcription factor to form a heterodimer and enter the nucleus.Mutants ofClNF-YB9were defective in embryonic cotyledon morphogenesis during seed development without affecting seed coat development,suggesting thatClNF-YB9functioned during seed development in watermelon.

AbnormalClnf-yb9seeds showed phenotypes similar to those of the nullArabidopsis lec1mutant with leafy cotyledon,but the homozygouslec1mutant cannot survive during dehydration and no homozygous mutant could be obtained unless it was rescued at early embryo development stage[47].Normal-looking homozygous mutant seeds ofClnf-yb9_1were viable and eventually germinated (data not shown).The reason for this phenotype in watermelon could be that the thicker seed coat of watermelon seeds protects the embryo from desiccation at the later stage of seed maturity.Abnormal seeds with a chalky endosperm and elongated kernels can be harvested in the riceNF-YB9mutant,accompanied by deregulated expression of genes involved in starch synthesis [29].ForClnf-yb9,the specific reason for low embryonic lethality awaits further study,and these viable homozygous mutant seeds invite further study ofClNF-YB9gene function in seed maturation and germination.Yeast two-and three-hybrid experiments showed that ClNF-YB9 can interact with various ClNF-YCs to enter the nucleus and that the ClNF-YB9-YC4 dimer recruited ClNF-YA1/6/7 to assemble into three different NF-Y complexes.The finding thatClNF-YA1andClNF-YA6were not expressed in seeds(Fig.5A)suggests that ClNF-YB9-YC4-YA7 may be the NFY complex functioning in seed development.

5.Conclusions

Whole-genome investigation reveals that the watermelon,cucumber and melon separately encode 22,29 and 24 NF-Y subunits.We validated their close evolutionary relationship ofNF-Ygenes among three species based on phylogenetic analysis,gene duplication and collinearity detection.Cis-element prediction and expression patterns in diverse tissues indicated thatNF-Ygenes participate in multiple biological processes in watermelon.Further,we verified thatClNF-YB9gene is abundantly expressed in seed development,especially in the seeds 20-50 DAP and functions in seed/embryo development in watermelon.Eventually,ClNF-YB9 was validated to be able to interact with YC4 to form a heterodimer and recruit ClNF-YA7 to assemble a mature,complete NF-Y complex during seed development.This study deepens our understanding on watermelon seed development and provides valuable resources for further uncovering the molecular mechanism of regulating watermelon seed development.

CRediT authorship contribution statement

Qin Feng:Investigation,Data curation,Writing-original draft,Writing -review &editing.Ling Xiao:Data curation.Jiafa Wang:Data curation.Jie Wang:Data curation.Chenyang Chen:Investigation and Data curation.Jianyang Sun:Investigation and Data curation.Xixi Wu:Data curation.Man Liu:Funding acquisition.Xian Zhang:Data curation.Shujuan Tian:Funding acquisition,Data curation,Writing-review&editing.Li Yuan:Funding acquisition,Data curation,Writing -review &editing.All authors have read and agreed to the published version of the 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 the Horticulture Science Research Center at College of Horticulture,NWAFU for their technical support in this work.We thank Dr.Jing Zhang(Horticulture Science Research Center,Northwest A&F University,Yangling,Shaanxi,China) for her assistance with phenotype analysis.This work was supported by the National Youth Talent Program (A279021801),Key-Area R&D Program of Guangdong province (2022B0202060001),Key R&D Program of Shaanxi Province (2023-YBNY-008),the Science and Technology Innovation Team of Shaanxi (2021TD-32),the Natural Science Foundation of Shaanxi Province (2021JM-089,2022JM-112 and 2022JQ-162),the Key R&D Project from Yangling Seed Industry Innovation Center (K3031322016),and the Fundamental Research Fund from Northwest A&F University (2452022111).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2023.03.005.