Genome-Wide Identification and Characterization of Wheat 14-3-3 Genes Unravels the Role of TaGRF6-A in Salt Stress Tolerance by Binding MYB Transcription Factor

14-3-3 proteins are a large multigenic family of general regulatory factors (GRF) ubiquitously found in eukaryotes and play vital roles in the regulation of plant growth, development, and response to stress stimuli. However, so far, no comprehensive investigation has been performed in the hexaploid wheat. In the present study, A total of 17 potential 14-3-3 gene family members were identified from the Chinese Spring whole-genome sequencing database. The phylogenetic comparison with six 14-3-3 families revealed that the majority of wheat 14-3-3 genes might have evolved as an independent branch and grouped into ε and non-ε group using the phylogenetic comparison. Analysis of gene structure and motif indicated that 14-3-3 protein family members have relatively conserved exon/intron arrangement and motif composition. Physical mapping showed that wheat 14-3-3 genes are mainly distributed on chromosomes 2, 3, 4, and 7. Moreover, most 14-3-3 members in wheat exhibited significantly down-regulated expression in response to alkaline stress. VIGS assay and protein-protein interaction analysis further confirmed that TaGRF6-A positively regulated slat stress tolerance by interacting with a MYB transcription factor, TaMYB64. Taken together, our findings provide fundamental information on the involvement of the wheat 14-3-3 family in salt stress and further investigating their molecular mechanism.


Introduction
General regulatory factor (GRF) proteins, also known as 14-3-3 proteins, are found among all eukaryotic organisms [1]. The 14-3-3 protein, first discovered in the bovine brain as a soluble acidic protein, acquired its name based on the fraction number in diethylaminoethyl (DEAE) cellulose chromatography and migration position on starch-gel electrophoresis [2]. Initially, 14-3-3 proteins were believed to be unique to animals. However, further research showed that these proteins are found in almost all eukaryotes [1,3,4]. The 14-3-3 proteins are a class of highly conserved regulatory proteins that can form homoor heterodimers, and each monomer in the dimer can interact with a separate target protein. This dimeric property of 14-3-3s allows them to serve as scaffolds for bringing together different regions of a protein or two different proteins in close proximity [5,6]. Therefore, 14-3-3 proteins regulate activities of numerous target proteins via protein-protein interactions, specifically by binding to the phosphoserine/phosphothreonine residues of target proteins. Evidence suggests that 14-3-3s are the most important phosphopeptide-binding proteins that play a regulatory role in various biological processes including the regulation of cell cycle, metabolism, apoptosis, development, protein trafficking, stress response, and gene transcription [7][8][9][10][11][12][13][14][15].
Wheat (Triticum aestivum L.) is the most widely cultivated crop in the world and one of the primary grains consumed by humans [19]. Drought, extreme temperatures, and salinity are the major abiotic stresses that reduce wheat production throughout the growing season [20]. The 14-3-3 proteins act as key regulators of signaling networks and abiotic stress responses [21]. For example, in rice, OsGF14e interacts with OsCPK21 to promote the salt stress response [22]. Additionally, studies show that OsGF14f has a negative effect on grain development and filling, while OsGF14e negatively impacts cell death and disease resistance [23,24]. In tomato, four 14-3-3 genes (TFT1, TFT4, TFT7, and TFT10) are significantly up-regulated under salt stress [25]. Ectopic expression of the wheat TaGF14b gene in tobacco (Nicotiana tabacum L.) enhanced the tolerance of mature tobacco plants to drought and salt stresses, which are related to the abscisic acid (ABA) signaling pathway, and improved their growth and survival compared with the control [26]. Overexpression of the soybean GsGF14o gene in Arabidopsis showed that GsGF14o regulates stomata size, root hair development, and drought tolerance [27].
Recently, Guo et al. [28] analyzed wheat TaGF14 genes based on the TGACv1 reference genome and found that five of these genes were up-regulated under drought stress, and all of the analyzed TaGF14s were down-regulated during heat stress. These results suggest that some of the TaGF14 genes play a vital role in combating drought stress, and all TaGF14s are negatively associated with heat stress. Additionally, the study showed that TaGF14s might be involved in starch biosynthesis. However, no information is available on the structure of 14-3-3 genes and their role in response to salt tolerance in wheat.
In this study, we conducted an in-depth analysis of the wheat 14-3-3 gene family members based on the newly released Chinese Spring reference genome (IWGSC RefSeq v1.1, https://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Annotations/v1.1/ (accessed on 13 February 2021)). We performed a comprehensive analysis of the structure, evolutionary relationship, cis-acting elements, Gene Ontology (GO) annotations, and expression profiles of the 14-3-3 genes, and deciphered the biological roles of these genes in salt stress tolerance using the virus-induced gene silencing (VIGS) assay and proteinprotein interaction analysis. It provided an updated view of the wheat 14-3-3 family and lays the foundation for studying the molecular mechanism of TaGRF6-A in regulating salt tolerance in wheat.

Genome-Wide Identification and Characterization of 14-3-3 Family Members in Wheat
A total of 32 14-3-3 protein sequences of wheat were identified using the basic local alignment search tool, BLASTp, and validated using the Hidden Markov Model (HMM) search tool, HMMER. These sequences were further confirmed using SMART and NCBI-CDD online tools, which revealed that all sequences contained conserved 14-3-3 protein domains. These protein sequences were encoded by 17 genes, including 12 genes showing alternative splicing, and splice variants with complete domains were chosen as representatives (Table 1). Multiple sequence alignment and secondary structure analysis revealed that the 14-3-3 family members contained nine typical α-helices. But the TraesCS4A02G167100 gene lacked the first α-helix, making it the first 14-3-3 family member that does not contain nine α-helices ( Figure S1). The 14-3-3 amino acid sequences were highly conserved, indicating that these proteins may perform similar functions to the 14-3-3 proteins of other plant species. The C-terminal end of 14-3-3 proteins was relatively divergent, which explains the functional diversity observed among these proteins. Genes with a 1:1:1 correspondence in all three sub-genomes (A, B, and D) of wheat are called triads [29]. We identified five triads based on the results of Ramírez-González et al. (Table S1). Because of the peculiarity of TraesCS4A02G167100, this gene was named TaGRF-like1. The remaining 16 genes were named according to their order on various homologous chromosomes (Table 1).
A detailed description of TaGRFs is summarized in Table 1. The deduced wheat 14-3-3 proteins contained 244-282 amino acid residues, and their molecular weight (MW) ranged from 27.25 to 31.93 kDa. The predicted isoelectric point (pI) of these proteins ranged from 4.67 to 6.32, implying that these proteins were acidic in nature. Three-dimensional modeling clearly showed the presence of α-helices and indicated that higher structures of these proteins were very similar ( Figure S2).

Gene Structure and Conserved Motif Analysis
Phylogenetic analysis revealed that TaGRFs were divided into two groups (ε and non-ε) and three corresponding members of each triad were closely clustered together ( Figure 1A). To examine the structural diversity of TaGRFs, we manually extracted the exonintron structure from the GFF3 annotation file (Table S2). All TaGRF genes, especially those within the same phylogenetic group, shared a similar exon-intron structure ( Figure 1B). The number of exons varied from 4 to 6 and the genes within each phylogenetic group exhibited nearly identical exon lengths. To understand the motif composition, a search was implemented using the MEME website, with the maximum number of motifs set at 10 ( Figure 1C). Functional analysis using SMART revealed that 3 of these 10 motifs (motifs 1-3) were annotated as 14-3-3, which were included in all members (Table S3). This result suggests that motifs 1-3 represent the main domains that determine the function of 14-3-3 proteins. At the same time, this result also indicates the functional conservation of the 14-3-3 genes during evolution. In addition, motif composition was highly similar within each phylogenetic group but was relatively divergent between groups. All members of the non-ε group contained six main motifs (1)(2)(3)(4)(5)(6), whereas members of the ε group contained two unique motifs (9 and 10), which indicates the potentially different roles during plant growth and response to various stresses.

Chromosomal Distribution and Evolutionary Analysis of TaGRF Genes
To clarify the chromosome position of TaGRFs, a schematic diagram was constructed. The TaGRF genes were mainly distributed on homoeologous chromosomes 4A/4B/4D, and only one gene was located on each of the remaining chromosomes ( Figure 2A).
To trace the evolutionary origin of wheat TaGRFs, we performed a synteny analysis of family members in common wheat (AABBDD) and its progenitor species Aegilops tauschii L. (DD), Triticum urartu L. (AA), and T. dicoccoides L. (AABB) ( Figure 2B). Given that the B sub-genome donor has not been sequenced, wild emmer wheat (T. dicoccoides L.) was used as the source of the B sub-genome. The results showed that TaGRFs had seven, twelve, and five orthologs in T. urartu, T. dicoccoides, and Ae. tauschii, respectively (Table S4). Interestingly, the origin of TaGRF genes was traceable. Our results indicated that the TaGRF genes in common wheat were entirely derived from the sub-genome donors and have the same number on the corresponding chromosomes. Wheat chromosomes 4A, 4B, and 4D harbored three, three, and two TaGRF genes, respectively. The same number of GRF homologs were detected on chromosomes 4A and 4B of T. dicoccoides and on chromosome 4D of Ae. tauschii. However, the corresponding gene was not detected on chromosome 2A of T. urartu. We speculated that the genes on chromosome 2A of common wheat evolved from T. dicoccoides or the corresponding gene on T. urartu used to exist but was later lost. This implied that members of the TaGRF gene family did not experience large fluctuations. The only polyploidization doubles the number of genes twice. In addition, the non-synonymous-to-synonymous substitution ratio (Ka/Ks) of TaGRF gene duplicates was <1 ( Figure 2C, Table S3), indicating that these genes are under negative selection pressure to maintain the protein sequence.
To understand the evolutionary relationships of wheat 14-3-3 proteins with those of other plant species, a phylogenetic tree of full-length 14-3-3 amino acid sequences from seven plant species, including T. aestivum, A. thaliana, O. sativa, G. max, Populus trichocarpa, Medicago truncatula, and V. vinifera, was constructed using the maximum likelihood (ML) method ( Figure 2D, Table S5). Consistent with the previous classification [30], all 14-3-3

Chromosomal Distribution and Evolutionary Analysis of TaGRF Genes
To clarify the chromosome position of TaGRFs, a schematic diagram was constructed. The TaGRF genes were mainly distributed on homoeologous chromosomes 4A/4B/4D, and only one gene was located on each of the remaining chromosomes ( Figure 2A).
To trace the evolutionary origin of wheat TaGRFs, we performed a synteny analysis of family members in common wheat (AABBDD) and its progenitor species Aegilops tauschii L. (DD), Triticum urartu L. (AA), and T. dicoccoides L. (AABB) ( Figure 2B). Given that the B sub-genome donor has not been sequenced, wild emmer wheat (T. dicoccoides L.) was used as the source of the B sub-genome. The results showed that TaGRFs had seven, twelve, and five orthologs in T. urartu, T. dicoccoides, and Ae. tauschii, respectively (Table S4). Interestingly, the origin of TaGRF genes was traceable. Our results indicated that the TaGRF genes in common wheat were entirely derived from the sub-genome donors and have the same number on the corresponding chromosomes. Wheat chromosomes 4A, 4B, and 4D harbored three, three, and two TaGRF genes, respectively. The same number of GRF homologs were detected on chromosomes 4A and 4B of T. dicoccoides and on chromosome 4D of Ae. tauschii. However, the corresponding gene was not detected on chromosome 2A of T. urartu. We speculated that the genes on chromosome 2A of common wheat evolved from T. dicoccoides or the corresponding gene on T. urartu used to exist but was later lost. This implied that members of the TaGRF gene family did not experience large fluctuations. The only polyploidization doubles the number of genes twice. In addition, the non-synonymous-to-synonymous substitution ratio (Ka/Ks) of TaGRF gene duplicates was <1 ( Figure 2C, Table S3), indicating that these genes are under negative selection pressure to maintain the protein sequence.
proteins were divided into two main evolutionary branches, namely, ε and non-ε. Only TaGRF-like1 and TaGRF5-B were grouped into the ε group, and the remaining TaGRFs clustered in the non-ε group. The proteins from wheat, Arabidopsis, and rice were closely related, suggesting that these proteins may perform similar functions, which provides clues for functional analysis.

Cis-Element Analysis and Functional Annotation of TaGRF Genes
To examine cis-acting elements in TaGRF promoter sequences, we searched 1.5 kb sequence upstream of the start codon (ATG) of all TaGRF genes. A total of 1789 cis-acting elements were identified (Table S6). These included elements involved in biotic and abiotic stress responses, plant growth and development, and phytohormone response processes. The number of elements involved in the first two processes was significantly more ( Figure 3A, Figure 3B). Motifs such as TGACG and CGTCA (involved in responding to To understand the evolutionary relationships of wheat 14-3-3 proteins with those of other plant species, a phylogenetic tree of full-length 14-3-3 amino acid sequences from seven plant species, including T. aestivum, A. thaliana, O. sativa, G. max, Populus trichocarpa, Medicago truncatula, and V. vinifera, was constructed using the maximum likelihood (ML) method ( Figure 2D, Table S5). Consistent with the previous classification [30], all 14-3-3 proteins were divided into two main evolutionary branches, namely, ε and non-ε. Only TaGRF-like1 and TaGRF5-B were grouped into the ε group, and the remaining TaGRFs clustered in the non-ε group. The proteins from wheat, Arabidopsis, and rice were closely related, suggesting that these proteins may perform similar functions, which provides clues for functional analysis.

Cis-Element Analysis and Functional Annotation of TaGRF Genes
To examine cis-acting elements in TaGRF promoter sequences, we searched 1.5 kb sequence upstream of the start codon (ATG) of all TaGRF genes. A total of 1789 cis-acting elements were identified (Table S6). These included elements involved in biotic and abiotic stress responses, plant growth and development, and phytohormone response processes. The number of elements involved in the first two processes was significantly more ( Figure 3A,B). Motifs such as TGACG and CGTCA (involved in responding to methyl jasmonate [MeJA]) and ABA-responsive element (ABRE; involved in the response to ABA) were commonly found in TaGRF gene promoters, suggesting that these genes are involved in hormone regulation. In addition, stress-related cis-elements, such as Myb, STRE, Sp1, G-box, Myc, as-1, LTR, and GC-motif, were also identified, implying that TaGRFs are important players in response to adverse conditions. ) and ABA-responsive element (ABRE; involved in the response to ABA) were commonly found in TaGRF gene promoters, suggesting that these genes are involved in hormone regulation. In addition, stress-related cis-elements, such as Myb, STRE, Sp1, G-box, Myc, as-1, LTR, and GC-motif, were also identified, implying that Ta-GRFs are important players in response to adverse conditions. To further predict the function of TaGRFs, we performed GO annotation analysis. The results showed that TaGRFs were divided into three categories (molecular function, cell composition, and biological process) ( Figure S3). In the molecular function category, GO terms such as "ATP binding" and "protein domain specific binding" were highly enriched, indicating that TaGRF proteins may bind to other proteins to perform various cellular functions. In the cell component category, GO terms such as nucleus, cytosol, plasma membrane, and organelles, such as mitochondrion, were highly enriched, suggesting that TaGRFs perform a wide range of functions. In the biological process category, TaGRFs play a prominent role in the response to cadmium ions. Both TaGRF5-B and TaGRF-like1 were annotated only under the GO term "protein domain specific binding", suggesting that these proteins play a limited role in wheat.

Expression Profiling of TaGRF Genes
The results of cis-element and GO annotation analyses suggested that TaGRFs are potentially involved in abiotic stress responses. To better understand the function of Ta-GRFs, we examined their expression patterns using RNA-seq data. The expression profiles of 16 TaGRFs in different wheat varieties under salt stresses are shown in Figure 4A. In general, the expression of TaGRFs decreased after the salt treatment. Furthermore, the expression levels of TaGRF4-A, TaGRF4-B, and TaGRF5-B were extremely low. In the wheat variety Kharchia Local, TaGRFs showed different expression profiles in different tissues; for example, TaGRF1-B, TaGRF1-D, and TaGRF2-A showed low expression in roots, while TaGRF6-A, TaGRF6-B, and TaGRF6-D showed high expression in leaves, which indicated that these genes may perform a function mainly in leaves. Compared with Kharchia Local, To further predict the function of TaGRFs, we performed GO annotation analysis. The results showed that TaGRFs were divided into three categories (molecular function, cell composition, and biological process) ( Figure S3). In the molecular function category, GO terms such as "ATP binding" and "protein domain specific binding" were highly enriched, indicating that TaGRF proteins may bind to other proteins to perform various cellular functions. In the cell component category, GO terms such as nucleus, cytosol, plasma membrane, and organelles, such as mitochondrion, were highly enriched, suggesting that TaGRFs perform a wide range of functions. In the biological process category, TaGRFs play a prominent role in the response to cadmium ions. Both TaGRF5-B and TaGRF-like1 were annotated only under the GO term "protein domain specific binding", suggesting that these proteins play a limited role in wheat.

Expression Profiling of TaGRF Genes
The results of cis-element and GO annotation analyses suggested that TaGRFs are potentially involved in abiotic stress responses. To better understand the function of TaGRFs, we examined their expression patterns using RNA-seq data. The expression profiles of 16 TaGRFs in different wheat varieties under salt stresses are shown in Figure 4A. In general, the expression of TaGRFs decreased after the salt treatment. Furthermore, the expression levels of TaGRF4-A, TaGRF4-B, and TaGRF5-B were extremely low. In the wheat variety Kharchia Local, TaGRFs showed different expression profiles in different tissues; for example, TaGRF1-B, TaGRF1-D, and TaGRF2-A showed low expression in roots, while TaGRF6-A, TaGRF6-B, and TaGRF6-D showed high expression in leaves, which indicated that these genes may perform a function mainly in leaves. Compared with Kharchia Local, QM6 and Chinese Spring showed greater differences in gene expression after the salt treatment, suggesting that genotype is an important factor affecting the differences in abiotic stress responses. QM6 and Chinese Spring showed greater differences in gene expression after the salt treatment, suggesting that genotype is an important factor affecting the differences in abiotic stress responses.
To verify the reliability of the transcriptome data, we analyzed the expression of two genes (TaGRF4-D and TaGRF6-A) in 1-week-old salt-treated wheat seedlings by quantitative real-time PCR. Both the TaGRFs responded to salt stress, but with different rates and intensities ( Figure 4B). Compared with the control, the expression of TaGRF4-D did not change significantly, while the expression of TaGRF6-A was significantly up-regulated at 12h post salt treatment, and then decreased sharply at 24h post salt treatment. This trend suggests that TaGRF6-A can be induced by salt stress at a certain stage, and may play a role during this period.

TaGRF6-A Positively Regulates Salt Tolerance in Wheat
The VIGS assay is used to silence specific genes in plants, thus enabling rapid characterization of gene function [31]. To further understand the role of TaGRF6-A in salt stress, we performed a virus-induced gene silencing assay. Two fragments of the TaGRF6-A gene were chosen for silencing ( Figure S3). At 10 days post-inoculation (dpi), wheat leaves inoculated with BSMV showed mild chlorotic mosaic symptoms while leaves inoculated with BSMV: TaPDS showed bleaching symptoms, indicating that the BSMV-induced gene silencing system was working ( Figure 5A). When treated with 300 mM NaCl for 12 days, both fragments of TaGRF6-A were well silenced by qRT-PCR ( Figure 5C), and at this time, the leaf curling degree of plants carrying BSMV: TaGRF6-A-1/2 was stronger than that of the control group carrying the empty vector ( Figure 5B). According to the statistical results, the number of leaf curls of plants carrying BSMV: TaGRF6-A-1/2 was significantly more than that of the empty carrier group (Figure 5D), indicating that Ta-GRF6-A contributes to salt tolerance in wheat. To verify the reliability of the transcriptome data, we analyzed the expression of two genes (TaGRF4-D and TaGRF6-A) in 1-week-old salt-treated wheat seedlings by quantitative real-time PCR. Both the TaGRFs responded to salt stress, but with different rates and intensities ( Figure 4B). Compared with the control, the expression of TaGRF4-D did not change significantly, while the expression of TaGRF6-A was significantly up-regulated at 12 h post salt treatment, and then decreased sharply at 24 h post salt treatment. This trend suggests that TaGRF6-A can be induced by salt stress at a certain stage, and may play a role during this period.

TaGRF6-A Positively Regulates Salt Tolerance in Wheat
The VIGS assay is used to silence specific genes in plants, thus enabling rapid characterization of gene function [31]. To further understand the role of TaGRF6-A in salt stress, we performed a virus-induced gene silencing assay. Two fragments of the TaGRF6-A gene were chosen for silencing ( Figure S3). At 10 days post-inoculation (dpi), wheat leaves inoculated with BSMV showed mild chlorotic mosaic symptoms while leaves inoculated with BSMV: TaPDS showed bleaching symptoms, indicating that the BSMV-induced gene silencing system was working ( Figure 5A). When treated with 300 mM NaCl for 12 days, both fragments of TaGRF6-A were well silenced by qRT-PCR ( Figure 5C), and at this time, the leaf curling degree of plants carrying BSMV: TaGRF6-A-1/2 was stronger than that of the control group carrying the empty vector ( Figure 5B). According to the statistical results, the number of leaf curls of plants carrying BSMV: TaGRF6-A-1/2 was significantly more than that of the empty carrier group (Figure 5D), indicating that TaGRF6-A contributes to salt tolerance in wheat.

TaGRF6-A Interacts with TaMYB64 In Vitro and In Vivo
Since TaGRF6-A expressed salt tolerance, we started to look for its internal mechanism. We focused on the expression levels of stress-related genes and their potential targets ( Figure 6A). It is worth noting that TaMYB64 is significantly down-regulated, suggesting that there may be a synergistic effect to enhance abiotic stress tolerance together with TaGRF6-A. Previous studies in soybean and rice showed that interactions between MYB transcription factors and 14-3-3 proteins enhance plant growth and stress tolerance [32,33]. These findings, combined with our qRT-PCR results, prompt us to investigate the association between TaMYB64 and TaGRF6-A. Before that, we analyzed the subcellular localization of TaGRF6

TaGRF6-A Interacts with TaMYB64 In Vitro and In Vivo
Since TaGRF6-A expressed salt tolerance, we started to look for its internal mechanism. We focused on the expression levels of stress-related genes and their potential targets ( Figure 6A). It is worth noting that TaMYB64 is significantly down-regulated, suggesting that there may be a synergistic effect to enhance abiotic stress tolerance together with TaGRF6-A. Previous studies in soybean and rice showed that interactions between MYB transcription factors and 14-3-3 proteins enhance plant growth and stress toler-ance [32,33]. These findings, combined with our qRT-PCR results, prompt us to investigate the association between TaMYB64 and TaGRF6-A. Before that, we analyzed the subcellular localization of TaGRF6-A and TaMYB64 by generating a C-terminal fusion of gene and yellow fluorescent protein (YFP) gene. After transient expression in N. benthamiana leaves using Agrobacterium-mediated transformation, compared with the YFP empty vector, confocal microscopy showed that TaGRF6-A has a similar subcellular localization and was detected in both the nucleus and the cytoplasm (Figure 6B), and TaMYB64 was detected in the nucleus, indicating the place where it functions. When exploring the interaction of TaMYB64 and TaGRF6-A, we first performed yeast two-hybrid (Y2H) assays. Although yeast transformants expressing both TaMYB64 and TaGRF6-A could grow on high stringency media, the number of yeast cells was less ( Figure 6C). To further verify the interaction between TaMYB64 and TaGRF6-A, we performed bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assay. After transiently co-expressing TaGRF6-A labeled with the N-terminal half of YFP (nYFP), together with TaMYB64 labeled with the C-terminal half of YFP (cYFP) in N. benthamiana leaves, the YFP signal was detected in the nucleus and cytoplasm of N. benthamiana leaf epidermal cells. While no signal was detected in the control combinations ( Figure 6D). The TaMYB64-YFP-3×FLAG fusion proteins were co-expressed with TaGRF6-A-YFP-HA or YFP-HA in N. benthamiana. After immunoprecipitation using the anti-HA antibody, Western blot was performed using the anti-FLAG antibody to detect TaMYB64 proteins. The TaMYB64-YFP-3×FLAG protein could be coimmunoprecipitated by TaGRF6-A-YFP-HA, but not the YFP-HA control, revealing a specific interaction between TaMYB64 and TaGRF6-A in vivo ( Figure 6D). These results proved that TaGRF6-A could physically interact with TaMYB64.

TaGRF6-A and TaMYB64 Work Together to Cope with Salt Stress
To clarify whether TaGRF6-A interacts with TaMYB64 to respond to salt stress, we chose to silence the TaMYB64 gene. Ten days post-inoculation (dpi), wheat leaves inoculated with BSMV showed mild symptoms of chlorosis, while leaves inoculated with BSMV: TaPDS showed symptoms of bleaching stripes, indicating that BSMV-induced gene silencing was effective ( Figure 7A). qRT-PCR results revealed that both fragments of TaMYB64 were successfully silenced ( Figure 7C). After treated with 300 mM NaCl for 12 days, the plants carrying BSMV: MYB64-1/2 had stronger curls and a significantly higher number of rolled leaves compared with the control group carrying the empty vector ( Figure 7B,D). These results indicated that TaMYB64 was also involved in response to salt stress.
3×FLAG fusion proteins were co-expressed with TaGRF6-A-YFP-HA or YFP-HA in N. benthamiana. After immunoprecipitation using the anti-HA antibody, Western blot was performed using the anti-FLAG antibody to detect TaMYB64 proteins. The TaMYB64-YFP-3×FLAG protein could be coimmunoprecipitated by TaGRF6-A-YFP-HA, but not the YFP-HA control, revealing a specific interaction between TaMYB64 and TaGRF6-A in vivo ( Figure 6D). These results proved that TaGRF6-A could physically interact with TaMYB64.  BSMV:TaPDS showed symptoms of bleaching stripes, indicating that BSMV-induced gene silencing was effective ( Figure 7A). qRT-PCR results revealed that both fragments of TaMYB64 were successfully silenced ( Figure 7C). After treated with 300 mM NaCl for 12 days, the plants carrying BSMV:MYB64-1/2 had stronger curls and a significantly higher number of rolled leaves compared with the control group carrying the empty vector (Figure 7B,D). These results indicated that TaMYB64 was also involved in response to salt stress.

Discussion
14-3-3 genes are ubiquitous in many species, and the study of its gene function is very necessary. Recent studies have shown that the four 14-3-3 proteins (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) of apples interact with the flower integrator TFL1/FT and participate in flowering regulation [34]. Under the condition of nitrate deficiency, the interaction of MdGRF11 and MdBT2 in apples can induce the accumulation of anthocyanins and provide a new mechanism for anthocyanin biosynthesis [35]. Under sugar starvation, MYBS2 interacted with 14-3-3 protein to inhibit the expression of α-amylase, thereby to improve rice plant growth, stress resistance, and grain weight [32]. As the hub of different signal pathways, 14-3-3 protein can transmit and integrate different hormone signals in plant signal transduction [36]. 14-3-3 interacts with AtWRI1, and the biosynthesis of oil increased in transgenic tobacco leaves co-expressing these two genes [37]. 14-3-3λ and k are important salt tolerance regulators. When there is no salt stress, 14-3-3λ and k interact with SOS2 and inhibit the kinase activity of SOS2; while in the presence of salt stress, the interaction of them weakens and activates SOS2 kinase activity [38].
In the present study, we performed a genome-wide characterization and in silico analysis of the 14-3-3 gene family in wheat. A total of 17 14-3-3 genes were identified in wheat based on the genome sequence of Chinese Spring. However, this result is inconsistent with previous studies, which identified 20 14-3-3 genes based on the TGACv1 wheat genome assembly [28]. We found that previous results contained duplicate genes and several other genes that do not exist in our newly released genome sequencing data. For example, Traes_2DL_1D912517E and Traes_2DL_21639EB55, which were previously identified as two distinct genes, represent the same gene in the new genome. In addition, TraesCS2D02G325600 and Traes_3B_3BF024700350CFD_c1 are not identified in the present study. We speculate that this difference between studies is caused by differences in genome sequencing depth, genome analysis, and gene annotations. We also cannot rule out the possibility of wheat germplasm resource differences. The 14-3-3 proteins are highly conserved and usually contain nine α-helices, which constitute the conserved core region of each monomer in the dimer [13,39]. In the current study, we showed that TaGRF-like1 lacks the first α-helix, which may affect the binding of phosphorylated target proteins. while the secondary structure of TaGRF proteins was conserved except in the C-terminal region ( Figure S1). Previous studies have shown that the variable C-terminus is the key to dimer formation, and it can interact with different ligands to show the target specificity of 14-3-3 protein [40,41].
Phylogenetic analysis of 89 14-3-3 proteins belonging to wheat and six other plant species showed that these proteins were clustered into two major groups (ε and non-ε), which is consistent with previous reports [18,34]. The result indicates that the formation of these two isoforms is a basic and ancient difference ( Figure 2D). As TaGRF6-A was adjacent to OsGF14d, which is expressed under salt, heat, and cold stress [42], the function of TaGRF6-A may be similar to that of OsGF14d. According to previous studies, the ε group genes usually contain a greater number of exons and motifs than the non-ε genes. However, this finding was not in agreement with our results; although the ε group genes contained more motifs, not all ε group genes contained more exons than non-ε group genes ( Figure 1B,C). For instance, the ε group gene TaGRF-like1 contained four exons, whereas the non-ε group gene TaGRF2-B contained six exons. This difference may be due to the insertion and deletion of introns over the long-term evolution of wheat. Nevertheless, gene structure and motif distribution were diverse between the two groups but similar within a group, which supports the results of the phylogenetic analysis ( Figure 1B,C).
Common wheat is an allohexaploid with a large and complex genome, which makes its genome research difficult. Genome sequencing of common wheat and its sub-genome donors has been completed, which provides an important reference for the evolutionary analysis of wheat. The chromosome map of common wheat shows that the distance between genes is much greater than 200 kb (Figure 2A), which means that these genes were not generated by tandem duplication [43]. Collinearity analysis of common wheat and its ancestors revealed the direct source of the wheat 14-3-3 sub-genomic donor ( Figure 2B), indicating that the entire family of wheat 14-3-3 proteins originated by polyploidization. The Ka/Ks ratio of 14-3-3 genes was <1 ( Figure 2C), implying that wheat 14-3-3 proteins may remain unchanged during the process of long-term domestication. Analysis of cis-acting elements in the promoter region of TaGRFs indicated that these genes may be involved in biotic and abiotic stress responses, growth and development, and phytohormone response ( Figure 3A). Previously, there have been some reports showing the involvement of 14-3-3 proteins in abiotic stress tolerance [44,45]. Considering the potential function of 14-3-3 proteins in abiotic stress, we performed transcriptome analysis of wheat plants under salt stress. Different TaGRF genes showed different expression profiles under salt stress. Most TaGRF genes were down-regulated after the salt treatment, suggesting that these genes may negatively regulate the response to salt stress ( Figure 4A). However, in rice, tomato, Brachypodium distachyon, and other plant species, members of the 14-3-3 family are involved in salt stress tolerance [22,25,46]. In addition, wheat TaGF14b (named as TaGRF1-B in this article) also enhanced salt stress tolerance when ectopically expressed in tobacco [26]. In the current study, we selected two genes for the salt treatment and found that TaGRF6-A was changed greatly responding to salt stress ( Figure 4B). To further verify the role of 14-3-3 family genes in salt stress, we used VIGS technology to silence the TaGRF6-A gene. The results showed that the level of leaf curling and number of BMSV: TaGRF6-A were more than the wild type ( Figure 5B-D), indicating that TaGRF6-A plays a positive role in response to salt stress.
To elucidate the possible mechanism of action of TaGRF6-A under salt stress, we examined the expression of seven stress-related genes or potential interacting partners of TaGRF6-A. Interestingly, the expression level of TaMYB64 was significantly down-regulated after TaGRF6-A silencing ( Figure 6A). Since the interaction between MYBS2 and 14-3-3 protein has been reported in rice and soybean [32,33,47], we cloned the homolog of OsMYBS2 in wheat and performed Y2H, BiFC, and CoIP assays. The results showed that TaMYB64 and TaGRF6-A did interact in wheat ( Figure 6C-E). Thus, we have the question of whether they use this interaction to improve salt tolerance. Next, we used the same method to silence TaMYB64 in wheat, and the results proved our deduction. Compared with the control, the silenced plants also have a higher degree and amount of curling ( Figure 7B,D). In summary, the results in the present study revealed the function of TaGRF6-A in salt stress, and initially elucidated its salt tolerance mechanism. Moreover, our study improved our understanding of the biological functions of the wheat 14-3-3 gene family.

Plant Material and Salt Treatment
Wheat (T. aestivum L.) cultivar Emai 170 was used in this study. Seeds of uniform size were selected and soaked in clear water for 24 h. Then, the seeds were sown in a small pot filled with nutrient-rich soil, and the pots were placed in a greenhouse maintained at 23 ± 2 • C, 16 h light/8 h dark photoperiod, and 200 lux light intensity. At the 2-3-leaf stage, wheat plants were treated with either double-distilled water (control) or 300 mM NaCl solution. Leaves were collected at 2, 4, 6, 12, 24, and 96 h after the treatment, frozen in liquid nitrogen and stored at −80 • C.

Chromosome Distribution, Synteny, Ka/Ks, and Phylogenetic Analysis of TaGRFs
Information about the start and end of TaGRFs was extracted from the GFF3 file. A physical map of TaGRFs was constructed using MapInspect software Version 1.0 ( http://www.softsea.com/review/MapInspect.html (accessed on 13 February 2021)) [57]. The TBtools software (https://github.com/CJChen/TBtools/ (accessed on 13 February 2021)) was used to determine the Ka and Ks values of TaGRFs, based on their coding sequences (CDSs) [58]. Reference genome sequences of wheat sub-genome donors were downloaded from NCBI, and 14-3-3 genes of each species were identified using the same methods as those used for determining the TaGRFs. To determine the paralogous or orthologous relationship between wheat TaGRFs and 14-3-3 genes of its sub-genome donors, the general tool "all against all BLAST searches" was used, with an E-value of 1 × 10 −10 and sequence similarity > 75% [59]. The "circlize" package of the R program was used to draw the relationship between wheat TaGRFs and 14-3-3 genes of its sub-genome donors [60]. Phylogenetic analysis was conducted using the ML method of MEGA7, based on the aligned 14-3-3 sequences of T. aestivum, A. thaliana, O. sativa, G. max, P. trichocarpa, M. truncatula, and V. vinifera, with 1000 bootstrap replications [61]. The phylogenetic tree file was then uploaded to the Interactive Tree of Life (https://itol.embl.de/ (accessed on 13 February 2021)) for adjustment and modified [62].

Cis-Acting Element Analyses
The 1.5 kb genomic DNA sequences upstream of the start codon (ATG) of each TaGRFs genes were extracted from the wheat genome sequence. Cis-regulatory elements in the promoters were identified using the PlantCARE database (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/ (accessed on 13 February 2021)) [63].

Expression Analysis of TaGRF Genes under Abiotic Stress Conditions
To determine the expression patterns of 14-3-3 genes in wheat under salt stress, wheat transcriptome data were downloaded from the NCBI Short Read Archive (SRA) database and mapped onto the reference genome of wheat using hisat2. The FPKM (fragments per kilobase of transcript per million) values obtained after "cufflinks" assembly were log-transformed, and a heatmap was drawn using the RStudio software "pheatmap" to display the expression profiles of TaGRFs [64,65].

RNA Isolation and qRT-PCR Analysis
Total RNA was extracted from leaf and root tissues of wheat plants treated with salt or water (control) using the RNAprep Pure Plant Kit (Invitrogen). The isolated total RNA was reverse transcribed to synthesize cDNA using the HiScript ® II 1st Strand cDNA Synthesis Kit (Vazyme) for qRT-PCR analysis. The cDNA was diluted to 100 ng/µL with RNase-free water, and qRT-PCR was performed in a 10 µL reaction volume containing 5 µL ChamQ Universal SYBR qPCR Master Mix, 0.5 µL each forward and reverse primer (10 µM), and 4 µL cDNA template. The following conditions were used for PCR: initial denaturation at 95 • C for 3 min, followed by 40 cycles of denaturation at 95 • C for 10 s, and annealing at 60 • C for 30 s. Fluorescence signals were collected after each cycle, and the temperature was increased from 60 • C to 95 • C after each cycle for melting curve analysis. The EF-1α gene (GeneBank accession: BT009129.1) was used as a reference gene. The relative expression level of genes was calculated using the 2 −∆∆CT method [66]. Three technical repeats were performed for each sample, and three independent replicates were carried out. Primers used for qRT-PCR are listed in Table S7.

Virus-Induced Gene Silencing (VIGS) Assay in Wheat
There are four kinds of vectors involved in the VIGS test: α, β, γ, and γ-PDS. The gene fragments were cloned into the γ vector to obtain a recombinant vector. The vector (α, β, γ, γ-PDS, and recombinant vector) were linearized, and the linearized plasmid was treated with RiboMAX ™ Large Scale RNA Production System-T7 and the Ribom7G Cap Analog (Promega) to obtain capped in vitro transcription products. VIGS inoculation was carried out at the 3-leaf stage [67]. Steps are as follows: Mix equal volumes of in vitro transcription products α, β, γ (or γ-PDS /recombinant γ), dilute with DEPC water, add 1 × FES buffer (0.1 M glycine, 0.06 M K2HPO4, 1% w/v tetrasodium pyrophosphate, 1% w/v bentonite, and 1% w/v celite, pH 8.5), and then rubbed onto wheat leaves. BSMV: γ-PDS (PDS: wheat phytoene desaturase gene) and BSMV: γ were used as controls for BSMV infection. After inoculation, when BSMV: γ-PDS showed bleaching and yellowing phenomenon (about 10 days later), ddH 2 O and 300 mmol NaCl solution were used for irrigation. After 12 days of irrigation, the curl phenotype of the fourth leaf was recorded and the curl rate was counted. Curl rate is the percentage of curled leaves in all leaves.

Subcellular Localization of the TaGRF6-A Protein
The CDS of TaGRF6-A was cloned into the pQBV3 Gateway entry vector and then cloned into the pEarlyGate101 destination expression vector [68]. The resulting TaGRF6-A-YFP fusion construct was transformed into Agrobacterium tumefaciens strain GV3101, which was grown on LB (add antibiotics: kanamycin, rifampicin, gentamicin) solid medium for 2 days. The positive colonies were verified by PCR and transferred to LB (add antibiotics: kanamycin, rifampicin, gentamicin) liquid medium. The culture was grown for 16 h at 28 • C on a shaker until the optical density of the culture (measured at 600 nm absorbance; OD600) reached 1.5-1.8. The cells were harvested by centrifugation at 4000× g for 15 min and resuspended in acetosyringone (AS) culture solution. Then, 1 mL culture (OD600 = 0.8) was injected into the abaxial surface of the leaves of 3-4-week-old N. benthamiana plants using a needleless syringe, followed by incubation in the dark for 4 h. At 48 h post-inoculation, the distribution of the YFP signal in leaf epidermal cells was observed under a confocal laser scanning microscope (Zeiss LSM710) [69].

Y2H, BiFC, and CoIP Assays
The CDSs of TaGRF6-A and TaMYB64 were cloned into pGBKT7 (BD) and pGADT7 (AD) vectors, respectively. According to the Yeast Protocols Handbook (Clontech, Mountain View, CA, USA), the recombinant plasmids were transformed into the yeast AH109 strain (Saccharomyces cerevisiae) and plated on an SD/-LW selection medium. The plates were incubated at 30 • C for 3-5 days until the appearance of colonies. Single colonies were picked using an inoculation ring and streaked onto SD/-LW and SD/-LWHA solid media. Plates were incubated at 30 • C for 3-5 days, and photographs were taken to record the growth of yeast colonies. T + P53 and T + lam served as positive and negative controls, respectively. Full-length cDNA sequences of TaGRF6-A and TaMYB64 minus the stop codon were PCR amplified using the Pfu polymerase (NEB). The PCR products were ligated into the pQBV3 vector and then cloned into pEarleyGate201-YN and pEarleyGate202-YC vectors using the LR enzyme (Gateway LR Clonase II Enzyme mix, Invitrogen). The resulting plasmids were transformed into A. tumefaciens strain GV3101 and then transiently expressed in N. benthamiana leaves using the method described above. For Co-IP analysis, the PCR products were constructed on pEarleyGate100 and pEarleyGate104 vectors. Using the method of Qiao et al. [69], the protein was transiently expressed in N. benthamiana and the total protein was extracted, and then incubated with HA magnetic beads (MBL, Tokyo, Japan) at 4 • C and enriched with magnetic beads on ice. The precipitated protein was then separated by