Genome-Wide Identification, Expansion Mechanism and Expression Profiling Analysis of GLABROUS1 Enhancer-Binding Protein (GeBP) Gene Family in Gramineae Crops

The GLABROUS1 enhancer-binding protein (GeBP) gene family encodes a typical transcription factor containing a noncanonical Leucine (Leu-)-zipper motif that plays an essential role in regulating plant growth and development, as well as responding to various stresses. However, limited information on the GeBP gene family is available in the case of the Gramineae crops. Here, 125 GeBP genes from nine Gramineae crops species were phylogenetically classified into four clades using bioinformatics analysis. Evolutionary analyses showed that whole genome duplication (WGD) and segmental duplication play important roles in the expansion of the GeBP gene family. The various gene structures and protein motifs revealed that the GeBP genes play diverse functions in plants. In addition, the expression profile analysis of the GeBP genes showed that 13 genes expressed in all tested organs and stages of development in rice, with especially high levels of expression in the leaf, palea, and lemma. Furthermore, the hormone- and metal-induced expression patterns showed that the expression levels of most genes were affected by various biotic stresses, implying that the GeBP genes had an important function in response to various biotic stresses. Furthermore, we confirmed that OsGeBP11 and OsGeBP12 were localized to the nucleus through transient expression in the rice protoplast, indicating that GeBPs function as transcription factors to regulate the expression of downstream genes. This study provides a comprehensive understanding of the origin and evolutionary history of the GeBP genes family in Gramineae, and will be helpful in a further functional characterization of the GeBP genes.


Introduction
Transcription factors (TFs) are key regulators in plants, which play crucial roles in various growth and developmental processes, as well as in response to abiotic stresses [1][2][3][4]. Previous studies have systematically identified 129,288 TFs from 83 species, and more than 2000 TFs were identified in rice [5]. However, merely 60 TF families in plants had been reported and identified [6]. Thus, knowledge about some important yet unknown transcription factor families remains elusive. The GLABROUS1 enhancer-binding protein (GeBP) gene, first identified and isolated from Arabidopsis in 2003, was one of the new plant-specific transcription factor families whose members share a central DNA-binding defense responses and cell wall metabolism. These genes partially overlap with a subset of CPR5-regulated genes. The transcript levels of the pathogen response marker genes PR1 and PR5 are increased in the mutant, indicating that the GeBP/GPLs are repressors of PR genes. A recent study revealed that VirF and its plant functional homolog VBF of the Agrobacterium F-box effector interact with the Arabidopsis GeBP-like transcription factor VFP4 [27]. Loss-of-function mutation of VFP4 results in the differential expression of numerous biotic stress response genes, suggesting that one of the functions of VFP4 is to control a spectrum of plant defenses, including against Agrobacterium tumefaciens.
The Gramineae crops, such as rice, maize, and wheat, have high economic values and abundant nutritional values [28]. They are widely used in scientific research because of the availability of abundant diverse genetic resources and high-quality genome reference sequencing data [29]. Recently, many GeBP genes have been characterized in Arabidopsis. However, little is known about the evolutionary dynamics of the GeBP family in Gramineae crops. In this study, we have systematically and comprehensively characterized the GeBP gene family in nine Gramineae crops (Brachypodium distachyon, Hordeum vulgare, Oryza. sativa ssp. Indica, Oryza. sativa ssp. japonica, Oryza rufipogon, Sorghum bicolor, Setaria italica, Triticum aestivum, Zea mays) using bioinformatics analysis. We have analyzed the chromosomal distributions, phylogenetic relationships, duplication events, orthologous groups, selective forces, gene structure, and protein motifs of all the 125 GeBP genes. Additionally, expression analysis was performed in rice to characterize the functional differentiation of the GeBP gene family. Our study, therefore, lays a foundation for further functional characterization of the GeBP gene family in Gramineae crops.

Identification, Phylogenetic Analysis, and Classification in the Gramineae Crops
A total of 125 GeBP genes, including 13,13,19,10,18,9,17,15 Figure S1 and Table S2). We found that Bd, Sb, Si, and Zm had more GeBP gene members compared to Hv, Oi, Oj, Or, and Ta, implying a gene expansion of the GeBP family among different species. Furthermore, the phylogenetic tree was constructed based on the alignment of the GeBP protein sequences with the neighbor-joining (NJ) method. Results showed that the GeBP proteins were classified into four clades, including Clade I, Clade II, Clade III, and Clade IV ( Figure 1). All clades showed a clear expansion of gene numbers, while Clade II and Clade IV had more genes than Clade I and Clade III, and Clade III was absent in Oi and Ta. In addition, we calculated the values of Tajima'D for each clade and found that the Tajima'D values ranged from the lowest, −0.65172 (Clade II) to the highest, 0.18654 (Clade I) (Table S2). Those results indicated that two directional selective sweeps were observed for GeBP genes in nine crops. Taken together, these results suggested that the GeBP gene family showed different expansion mechanisms in the Gramineae crops during evolution.
To investigate the evolutionary pattern of GeBP genes in these species, the orthologous groups (OGs) were identified by OrthoFinder software 9 (Table S3). The results showed that 125 genes were divided into 9 OGs, and the gene number of each OGs was greatly varying. For example, the OG1, OG2, and OG3 contained 30, 25, and 19 genes, while the OG7, OG8, and OG9 included 7, 5, and 4 genes. In addition, among all the OGs, only three OGs (OG1, OG1, and OG5) were presented in all the Gramineae species, while other OGs were dispersed in the individual species. Interestingly, the 13 genes of Oj contained all OGs. These results suggested that unequal loss and expansion of OGs appeared during the evolutionary process. To characterize the selective pressure on the GeBP genes during the evolutionary process, the Tajima's D was calculated for the orthologous gene pairs among the tested Gramineae crops. We found most of the OG pairs with Tajima's D < 0, indicating that the GeBP gene family might have undergone purifying selection in the evolutionary process. To investigate the evolutionary pattern of GeBP genes in these species, the orthologous groups (OGs) were identified by OrthoFinder software 9 (Table S3). The results showed that 125 genes were divided into 9 OGs, and the gene number of each OGs was greatly varying. For example, the OG1, OG2, and OG3 contained 30, 25, and 19 genes, while the OG7, OG8, and OG9 included 7, 5, and 4 genes. In addition, among all the OGs, only three OGs (OG1, OG1, and OG5) were presented in all the Gramineae species, while other OGs were dispersed in the individual species. Interestingly, the 13 genes of Oj contained all OGs. These results suggested that unequal loss and expansion of OGs appeared during the evolutionary process. To characterize the selective pressure on the GeBP genes during the evolutionary process, the Tajima's D was calculated for the orthologous gene pairs among the tested Gramineae crops. We found most of the OG pairs with Tajima's D < 0, indicating that the GeBP gene family might have undergone purifying selection in the evolutionary process.

The Expansion and Evolutionary Pattern of the GeBP Genes
To better understand the expansion mechanism of the GeBPs paralogues in these species, the gene location and duplication pairs of each species were further analyzed. We mapped the GeBP gene sequences onto the genome and found that 125 GeBP genes of nine Gramineae species were unevenly distributed on the chromosomes ( Figure 2). For example, in O. sativa ssp. japonica (Oj), there were three GeBP genes, both on chromosome 2 and chromosome 9, and two GeBP genes on chromosome 1 and chromosome 3, and only one GeBP gene was detected on chromosome 6 to chromosome 8. Meanwhile, 29 duplication

The Expansion and Evolutionary Pattern of the GeBP Genes
To better understand the expansion mechanism of the GeBPs paralogues in these species, the gene location and duplication pairs of each species were further analyzed. We mapped the GeBP gene sequences onto the genome and found that 125 GeBP genes of nine Gramineae species were unevenly distributed on the chromosomes ( Figure 2). For example, in O. sativa ssp. japonica (Oj), there were three GeBP genes, both on chromosome 2 and chromosome 9, and two GeBP genes on chromosome 1 and chromosome 3, and only one GeBP gene was detected on chromosome 6 to chromosome 8. Meanwhile, 29 duplication gene pairs were identified in these Gramineae crops. All the duplication gene pairs were derived from the whole-genome duplication (WGD)/segmental duplication type (Table 1). Results showed that no duplication gene pair was found in Hv, but 4, 1, 3, 3, 4, 2, 5, and 7 duplication gene pairs were identified in Bd, Oi, Oj, Or, Sb, Si, Ta, and Zm, respectively. The numbers of duplication gene pairs varied greatly among those species, indicating that the gene expansion mechanism was different. Interestingly, the numbers and types of duplication gene pairs were the same between Oj and Or, which substantiated the previously reported that wild rice (Or) is an ancestor of cultivated rice [30]. derived from the whole-genome duplication (WGD)/segmental duplication type (Table  1). Results showed that no duplication gene pair was found in Hv, but 4, 1, 3, 3, 4, 2, 5, and 7 duplication gene pairs were identified in Bd, Oi, Oj, Or, Sb, Si, Ta, and Zm, respectively. The numbers of duplication gene pairs varied greatly among those species, indicating that the gene expansion mechanism was different. Interestingly, the numbers and types of duplication gene pairs were the same between Oj and Or, which substantiated the previously reported that wild rice (Or) is an ancestor of cultivated rice [30].   For selective force analysis of a duplication gene pair, the Ka/Ks rates were calculated using TBtools. The divergence time of each duplication gene pair was acquired using the formula T = Ks/ (2 × 9.1 × 10 −9 ) × 10 −6 . The MYA represents million years ago, the WGD represents whole genome duplication.
Further analysis showed that the divergence time of all duplication gene pairs greatly varied from 0.19 to 47.98 million years (MYA) among these tested species (Table 1) In addition to the BdGeBP12/BdGeBP10 (the ratio of Ka/Ks = 0.7902), the Ka/Ks ratios of GeBP gene pairs were far less than 1, suggesting that purifying selection was accompanied by the evolution of GeBP genes (Table 1). These results showed that multiple duplication events played a role in the long-term process of GeBP gene expansion in the nine Gramineae crops.

The Conserved Motif and Gene Structure Analysis
To clarify the functional relationships of the GeBP gene family members during the revolution, conserved protein motifs and gene structures of 125 GeBP protein sequences were analyzed using the MEME program. The results showed a total of 15 conserved motifs in GeBPs ( Figure 3). All the GeBP proteins contain motif1, motif2, and motif3, showing that these motifs might play crucial roles in the transcriptional regulation of their target genes. In addition, the motif distribution was similar among members of the same clade, but varied among different clades. These results suggested the diverse function of the GeBP proteins in Gramineae crops.
Meanwhile, we found that the structure of GeBPs had changed in the nine Gramineae crops, except for a fraction of genes. Most GeBP family members were found to be without intron, and few genes contained 2-8 exons. For example, OiGeBP6 contained 2 exons, and OrGeBP11 contained 8 exons. These results also suggested that the functions of the GeBP gene family were diverse and complicated in the plant kingdom.

Expression Profiles of the GeBP Genes across Different Rice Tissues and Developmental Stages
To better understand the possible function of GeBPs in rice, we investigated the expression pattern in different tissues and developmental stages, including the root, stem, leaf, and reproductive organs by qRT-PCR. The results showed that 13 genes were expressed in many tissues at different developmental stages, with especially high levels of expression in the leaf, palea, and lemma ( Figure 4). Through the expression profiles, 13 genes were mainly clustered into four groups based on hierarchical clustering analysis, revealing that four groups had a different function in rice growth and development. Interestingly, group I contains only one gene, OsGeBP1, which had the highest expression in the mature panicle, spikelet, and anther. Those results suggested that OsGeBP1 might play an important role in the development of mature spikelets and anther in rice. In addition, group II comprising OsGeBP3, OsGeBP7, OsGeBP4, and OsGeBP12 was also highly expressed in the young panicle and spikelet, apart from the palea and lemma, compared to that of group III and group IV. Meanwhile, we found that the structure of GeBPs had changed in the nine Gramineae crops, except for a fraction of genes. Most GeBP family members were found to be without intron, and few genes contained 2-8 exons. For example, OiGeBP6 contained 2 exons, and OrGeBP11 contained 8 exons. These results also suggested that the functions of the GeBP gene family were diverse and complicated in the plant kingdom.

Expression Profiles of the GeBP Genes Across Different Rice Tissues and Developmental Stages
To better understand the possible function of GeBPs in rice, we investigated the expression pattern in different tissues and developmental stages, including the root, stem, leaf, and reproductive organs by qRT-PCR. The results showed that 13 genes were expressed in many tissues at different developmental stages, with especially high levels of expression in the leaf, palea, and lemma ( Figure 4). Through the expression profiles, 13 genes were mainly clustered into four groups based on hierarchical clustering analysis, revealing that four groups had a different function in rice growth and development. Interestingly, group I contains only one gene, OsGeBP1, which had the highest expression in the mature panicle, spikelet, and anther. Those results suggested that OsGeBP1 might play an important role in the development of mature spikelets and anther in rice. In addition, group II comprising OsGeBP3, OsGeBP7, OsGeBP4, and OsGeBP12 was also highly expressed in the young panicle and spikelet, apart from the palea and lemma, compared to that of group III and group IV.

Expression Profiles of the GeBP Genes in Rice under Various Hormonal Stresses
Phytohormones are essential for plant growth and development and play important roles in stress response. Previous studies have reported that several GeBP genes function in response to hormone treatments. Here, to testify whether OsGeBPs respond to various hormones, the seedlings were treated with GA3, 6BA, and IAA. One gene (OsGeBP1) was significantly induced and four genes (OsGeBP2, OsGeBP5, OsGeBP6, and OsGeBP12) were obviously decreased by GA3 treatments (Figure 5a). Six genes (OsGeBP1, OsGeBP3, OsGeBP4, OsGeBP7, OsGeBP9, and OsGeBP13) were induced and three genes (OsGeBP2, OsGeBP6, and OsGeBP8) were decreased by 6BA (Figure 5b). Three genes (OsGeBP5, OsGeBP9, and OsGeBP10) were induced and four genes (OsGeBP1, OsGeBP2, OsGeBP11, and OsGeBP12) were extremely decreased by IAA (Figure 5c), respectively. The transcript levels significantly increased or decreased under different treatments, implying the diverse functions of OsGeBPs in rice. Moreover, the results demonstrated that OsGeBPs had extremely strong responses to the cytokinin stimuli.
OsGeBP4, OsGeBP7, OsGeBP9, and OsGeBP13) were induced and three genes (OsGeBP2, OsGeBP6, and OsGeBP8) were decreased by 6BA (Figure 5b). Three genes (OsGeBP5, OsGeBP9, and OsGeBP10) were induced and four genes (OsGeBP1, OsGeBP2, OsGeBP11, and OsGeBP12) were extremely decreased by IAA (Figure 5c), respectively. The transcript levels significantly increased or decreased under different treatments, implying the diverse functions of OsGeBPs in rice. Moreover, the results demonstrated that OsGeBPs had extremely strong responses to the cytokinin stimuli.

Expression Profiles of the GeBP Genes in Rice under Various Metal Ion Stresses
To assess the potential effects of metal ions on rice GeBPs expression during the development, the transcript levels were estimated under treatments with ZnCl2, CdCl2, and CuCl2. In our study, treatment with three metal ions significantly decreased the transcript levels of OsGeBP1, especially in response to CdCl2 and CuCl2 treatment (Figure 6a-c). In addition, OsGeBP2, OsGeBP4, OsGeBP11, and OsGeBP12 were also downregulated in response to CdCl2 treatment ( Figure 6c). Meanwhile, OsGeBP2 and OsGeBP11 were downregulated in response to CuCl2 treatment (Figure 6b). Only OsGeBP8 was gently induced by ZnCl2 treatment (Figure 6a). The above results demonstrated that GeBP genes may have played roles in response to various metal ion stresses in plant development.

Expression Profiles of the GeBP Genes in Rice under Various Metal Ion Stresses
To assess the potential effects of metal ions on rice GeBPs expression during the development, the transcript levels were estimated under treatments with ZnCl 2 , CdCl 2 , and CuCl 2 . In our study, treatment with three metal ions significantly decreased the transcript levels of OsGeBP1, especially in response to CdCl 2 and CuCl2 treatment (Figure 6a-c). In addition, OsGeBP2, OsGeBP4, OsGeBP11, and OsGeBP12 were also downregulated in response to CdCl 2 treatment (Figure 6c). Meanwhile, OsGeBP2 and OsGeBP11 were downregulated in response to CuCl 2 treatment (Figure 6b). Only OsGeBP8 was gently induced by ZnCl 2 treatment (Figure 6a). The above results demonstrated that GeBP genes may have played roles in response to various metal ion stresses in plant development.

The Subcellular Localization of OsGeBP11 and OsGeBP12 in Rice
The localization of proteins is closely related to their function. In general, transcription factors are localized to the nucleus. To confirm whether OsGeBPs are also localized to the nucleus, we selected two genes, OsGeBP11 and OsGeBP12, preferentially expressed in rice palea and lemma and constructed the 35s:OsGeBP11 and 35s:OsGeBP12 eGFP expression vectors. Both these vectors were transiently expressed in the rice protoplast, including 35S:eGFP were tested as the control (Figure 7). Results showed that the green fluorescence was characteristically observed in the nucleus of protoplast of the 35s:OsGeBP11-eGFP and 35s:OsGeBP12-eGFP, while the fluorescence was observed in the control. These results confirmed that the GeBP proteins were located in the nucleus, consistent with the previous studies on the other species [18]. Therefore, these data indirectly indicated that the OsGeBPs probably act as transcription factors to regulate the downstream genes during development and cope with various environments and stresses.

The Subcellular Localization of OsGeBP11 and OsGeBP12 in Rice
The localization of proteins is closely related to their function. In general, transcription factors are localized to the nucleus. To confirm whether OsGeBPs are also localized to the nucleus, we selected two genes, OsGeBP11 and OsGeBP12, preferentially expressed in rice palea and lemma and constructed the 35s:OsGeBP11 and 35s:OsGeBP12 eGFP expression vectors. Both these vectors were transiently expressed in the rice protoplast, including 35S:eGFP were tested as the control (Figure 7). Results showed that the green fluorescence was characteristically observed in the nucleus of protoplast of the 35s:OsGeBP11-eGFP and 35s:OsGeBP12-eGFP, while the fluorescence was observed in the control. These results confirmed that the GeBP proteins were located in the nucleus, consistent with the previous studies on the other species [18]. Therefore, these data indirectly indicated that the OsGeBPs probably act as transcription factors to regulate the downstream genes during development and cope with various environments and stresses.

Plant Materials and Growth Conditions
The Nipponbare rice seeds (O. sativa. ssp. japonica) were germinated for 3 days in water placed in an incubator at 37 • C. Then, the sprouting seeds were transferred into a greenhouse with environmental conditions similar to those during summer in Wuhan.

Hormone and Metal Ion Stress Treatments
For the hormone and metal ion stresses treatments, the seeds of Nipponbare were dehulled and sterilized with 0.15% HgCl 2 solution for 10 min, rinsed 4 times with sterile distilled water. Next, these seeds were sown on the 3.5% Phytagel-solidified half-strength Murashige and Skoog medium supplemented with 100 µM GA3, 100µM N 6 -benzyladenine (6BA), 100 µM indole-3-acetic acid (IAA), 70 µM CdCl 2 , 65 µM CuCl 2 , and 0.5 mM ZnCl 2 , respectively, and grown for two weeks, in a controlled environment with a 16/8-h light/dark photoperiod at 26 • C, and 60% relative humidity. The control groups were maintained on the normal nutrient solution or medium. The samples were quickly frozen in liquid nitrogen and stored at −80 • C until use. The experimental procedure was replicated three times.

Phylogenetic Relationship Analyses
To construct the phylogenetic tree of the GeBP genes of the nine representatives of Gramineae crops, all the protein sequences of GeBPs were aligned using the ClustalW program (http://www.clustal.org/clustal2/, accessed on 15 February 2021) and edited with Jalview (http://www.jalview.org, accessed on 15 February 2021). Then, the phylogenetic tree was constructed by the neighbor-joining (NJ) method of the MEGA 7.0 (https://www.megasoftware.net/, accessed on 1 March 2021) software based on the following parameters: p-distance, pairwise deletion option, and 1000 bootstrap replications [32]. The classified and annotated GeBP protein sequences were visualized using the iTOL software (https://itol.embl.de/itol.cgi, accessed on 3 March 2021).

Duplication Events, Orthologous Groups, Conserved Motifs, and Gene Structure Analyses
The duplicated gene pairs among the Gramineae crops were identified using the MC-ScanX with an E-value of 1 × e −5 in the BlastP search [33]. For the selective force analysis of a duplication gene pair, the nonsynonymous (Ka)/synonymous (Ks) substitution (Ka/Ks) rates were calculated using the TBtools [34]. The divergence time of each duplication gene pair was acquired using the formula T = Ks/ (2 × 9.1 × 10 −9 ) × 10 −6 (million years ago, MYA) [35].
To acknowledge the relationship of paralogous GeBPs in nine Gramineae, using the OrthoFinder v2.0 software, the phylogenetic tree of GeBPs was reconstructed depending on the result of orthologous groups using the STAG and STRIDE algorithms. The Tajima's D values were calculated using the DnaSP 5.0 [36].
The intron-exon organizations were analyzed with the TBtools software [34]. The conserved motifs were detected through the MEME server v5.0.4, with a maximum number of 20 motifs and motif length between 5 wide and 200 wide amino acids [37]. The phylogenetic tree, motifs and gene structures were visualized using the TBtools software.

RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR
The total RNA was extracted using the Trizol reagent (Invitrogen) (according to their instruction manuals) and RNase-free DNase I (Thermo Fermentas) treatment, to ensure that there is no contamination of DNA. The concentration of RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA). Approximately 5 µg of the digested total RNA was reverse-transcribed into the first-strand cDNA with the M-MLV reverse transcriptase (Invitrogen) and random primer (Thermo Fermentas, MA, USA). The quantitative real-time PCR (qRT-PCR) was carried out on a CFX96 Touch ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The specific qRT-PCR primers of 13 rice GeBP genes were designed using the Primer Premier 5 software and shown in Table S3. Actin was used as an internal control. The qRT-PCR was performed in three biological replicates and the technical replications were based on a previous study [38].

Transient Expression of the Enhanced Green Fluorescent Protein (eGFP) Constructs in the Rice Protoplast
To construct the transient expression vectors 35s:OsGeBP11-eGFP and 35s:OsGeBP11-eGFP, the whole cDNA fragments of OsGeBP11 and OsGeBP12 were amplified from the Nipponbare cDNA and cloned into the HBT95-sGFP vector with a CMV35s promoter [39]. These constructs were further transformed into rice protoplasts by the polyethylene glycol 4000 (PEG4000)-mediated transformation method [40]. After incubation in the dark for 16 h, the GFP fluorescence was observed with a confocal laser-scanning microscope, with excitation at 488 nm and emission at 498 nm. (Leica TCS SP5). Three independent experiments were carried out. In each experiment, more than ten cells with positive signals were analyzed.

Statistical Analysis
All the data were analyzed using the GraphPad Prism 7.00 statistics program (https: //www.graphpad.com/ accessed on 5 April 2021) and the means were compared by Student's t-test. Each assay was performed in three biological replicates and technical replications.

Characterization of the GeBP Genes in Gramineae Crops
The GeBP transcription factors have rarely been investigated in plants. In this study, a total of 125 GeBP genes were identified in nine Gramineae crops, including 18, 10, 9, 13, 13, 17, 15, 11, and 19 GeBP genes in B. distachyon  Figure S1). We found that Z. mays had the largest number of genes, containing 19 GeBP genes, consistent with the previous result that state that Z. mays underwent one specific WGDs compared to the other Gramineae plants [41]. Moreover, our results showed that the GeBP family genes could be classified into four clades by phylogenetic analysis and classification (Figure 1). The gene numbers varied greatly among each clade, indicating that the gene expansion mechanisms might be complicated.
Gene duplication and divergence events are the main processes that multiply genetic material during evolution and selection [42]. To further investigate the expansion mechanism in these species, the gene location and duplication pairs of each species were analyzed. In our study, we found that all of the GeBP genes were unevenly distributed on the chromosomes (Figure 2). Additionally, 29 duplication gene pairs were identified, all of which were derived from the WGD/segmental duplication type (Table 1). These results indicated that the WGD/segmental duplication type appears to have served as the most important driving force throughout the long period of Gramineae crops gene evolution. The numbers of duplication gene pairs varied greatly among these species, such as no duplication gene pairs in H. vulgare (Hv), and seven duplication gene pairs were found in Z. mays (Zm). The above results indicated that the gene expansion mechanism was different in these Gramineae crops. Further analysis showed that the divergence time of all duplication gene pairs varied greatly among these tested species, ranging from 0.1898 to 47.9755 Mya. The ratio of non-synonymous versus synonymous substitutions (Ka/Ks) is an indicator of the history of selection acting on a gene or gene region [43]. In our study, the Ka/Ks values of all the identified duplicate pairs were less than 1.0. These results suggested that multiple duplication events played essential roles in the gene expansion of Gramineae crop genomes during the long-term evolutionary process.
To better understand the functional relationships of the GeBP gene family, the protein conserved motif and gene structure were further analyzed using the MEME program. A total of 15 conserved motifs were identified among 125 GeBP proteins, and divided into different clusters (Figure 3). The result showed that different clusters had a great difference in members and distribution of the conserved motif. Interestingly, all GeBP proteins had motif1, motif2, and motif3, showing that these motifs may play crucial roles in transcript regulation of target genes expression during the evolutionary process. Moreover, the gene structure of GeBPs was quite different in the nine Gramineae crops, indicating that the function of the GeBP genes family was different in the plants.

Expression Profiles of the GeBP Genes in Gramineae Crops
As a sessile organism, Gramineae crops have to confront complicated environments. The transcription factors have been suggested as key regulators to modulate gene expression, responding to various environmental stress [44]. Previously, studies showed that the GeBP genes family play important roles in plant growth and development, as well as response to hormone and ion, implying that the GeBP genes are involved in various hormonal pathways [7,18,26]. We analyzed the expression profiles of the GeBP genes in rice at different developmental stages and in different tissues by quantitative real-time PCR. The results showed that 13 genes were expressed in many tissues at different development stages, and especially high expression in leaf, palea, and lemma ( Figure 5). This supported the previous report that GeBP genes can regulate the development of the plant epidermis cells [25].
Moreover, the GeBP genes were predicted to play a role in various hormonal pathways [16]. Here, we found that the rice GeBP genes respond differently to GA3, 6BA, and IAA, which suggest that they had functional differentiation ( Figure 5). For example, the transcription level of OsGeBP1 was upregulated and the transcription levels of OsGeBP2, OsGeBP5, OsGeBP6, and OsGeBP12 were downregulated by the application of exogenous GAs. These results were consistent with the previous report that the expression of the GeBP genes family was regulated by GA hormones. However, the mechanism by which the GAs regulates the expression of GeBP genes is still elusive. Additionally, the expressions of OsGeBP1, OsGeBP3, OsGeBP4, OsGeBP7, OsGeBP9, and OsGeBP13 were induced by cytokinin, which is also consistent with the report that GeBP/GPL play a redundant role in the cytokinin hormone pathway [26]. Meanwhile, we showed that auxin can also induce the expression of GeBP genes, indicating that auxin might promote the formation of trichomes by regulating the expression of GeBP family genes, but the mechanism still needs further study.
Previous studies have characterized a transcription factor, GeBP-LIKE 4 (GPL4), which was induced rapidly in the root tips in response to cadmium (Cd), and functioned as an inhibitor of root growth in Arabidopsis [21]. In our study, a small number of the GeBP genes were induced by heavy metal ions in the compounds, ZnCl 2 , CdCl 2 , and CuCl 2 , which implied that the GeBP genes played crucial roles in response to various metal ion stresses in plant development. Interestingly, we found that OsGeBP1 had significant responses to three hormones and three metal ions. The transcript levels of OsGeBP1 were significantly induced by gibberellin and cytokinin while decreased by auxin. Meanwhile, the transcript levels of OsGeBP1 were significantly decreased in response to three metal ion stresses. These results suggest that OsGeBP1 may play an important role in responding to environmental changes and stresses. However, the mechanism by which the GeBP genes respond to heavy metals needs further investigation. Subsequently, we confirmed that the subcellular localizations of the OsGeBP11 and OsGeBP12 were observed in the nucleus, suggesting that OsGeBPs functioned as transcription factors. Taken together, these results showed that OsGeBP genes can respond to various stresses or hormones to further regulate the downstream target genes in rice, suggesting that the GeBP family might also be involved in various regulatory networks to endure the complicated and unfavorable environments. Despite the biological functions of GeBP remaining elusive, our study has been able to present the fundamental data for the further exploration of GeBP in plants.

Conclusion
In our study, the comprehensive analysis of nine Gramineae crops of the GeBP family identified 125 genes that were classified into four clades, and divided into nine orthologous groups (OGs). The bioinformatic analyses and expression profiles indicated that there were different expansion mechanisms and there might be different functions of the GeBP gene family among these tested species, but further experimental work will be required to confirm this. Thus, these results provided a foundation for further understanding of the biological roles of the individual GeBP gene in Gramineae crops.