Genome-Wide Identi ﬁ cation of the F-box Gene Family and Expression Analysis under Drought and Salt Stress in Barley

: The F-box protein-encoding gene family plays an essential role in plant stress resistance. In present study, 126 non-redundant F-box genes were identi ﬁ ed in barley ( Hordeum vulgare L., Hv ). The corresponding proteins contained 165 – 887 amino acid residues and all were amphiphilic, except 5 proteins. Phylogenetic analysis of F-box protein sequences in barley and stress-related F-box protein sequences in wheat and Arabidopsis thaliana ( At ) was used to classify barley F-box genes are divided into 9 subfamilies (A – I). A structure-based sequence alignment demonstrated that F-box proteins were highly conserved with a total of 10 conserved motifs. In total, 124 F-box genes were unevenly distributed on 7 chromosomes; another 2 genes have not been anchored yet. The gene structure analysis revealed high variability in the number of exons and introns in F-box genes. Comprehensive analysis of expression pro ﬁ les and phylogenetic tree analysis, a total of 12 F-box genes that may be related to stress tolerance in barley were screened. Of the 12 detected F-box genes, 8 and 10 were upregulated after drought and salt stress treatments, respectively, using quantitative real-time polymerase chain reaction (qRT-PCR). This study is the ﬁ rst systematic analysis conducted on the F-box gene family in barley, which is of great importance for clarifying this family ’ s bioinformatic characteristics and elucidating its function in barley stress resistance. These results will serve as a theoretical reference for subsequent research on


Introduction
The F-box gene family is one of the most abundant and functionally diverse families found in plants [1,2], which mainly degrades most alienated proteins through the ubiquitin proteasome pathway (UPP) in order to cope with adverse external stimuli, such as drought, saline-alkali, low temperature, and heavy metal stress. The F-box protein was first recognized by Kumar et al. [3] when they studied the WD (Trp-Asp) domain but was named after the F-box protein after the F-box domain was discovered by Bai et al. [4]. The N-terminus of the F-box protein usually contains a polypeptide region of 40-50 amino acids, which is the binding site of Skp1 or Skp1 similar proteins in the SCF complex and mediates protein-protein interactions. However, these conserved amino acids are less in quantity [5]. The C-terminal domain specifically recognizes substrates and is used for classification of the F-box protein family. However, F-box genes with high structural similarity can identify the same type of substrate, and, thus, some F-box proteins serve similar functions.
Drought and salinity are the main environmental stressors that severely restrict plant growth and sustainable agricultural development. Previous studies found that some F-box genes in M. truncatula contain drought-responsive cis-acting elements and respond to drought stress [14]. Additionally, the TaFBA1 gene identified in wheat was upregulated in response to drought, high salt, and abscisic acid (ABA) stress, proving that TaFBA1 is strongly resistant to stress [15]. Overexpression of TaFBA1 in tobacco under salt stress promoted seed germination and root growth, and as exposure to stress increased over time, photosynthesis in transgenic plants became more stable than the wild type, which is conducive to mass accumulation [16]. The overexpression of Triticum aestivum SKP1-like 1 (TSK1) in A. thaliana resulted in delayed seed germination and hypersensitivity to ABA [17]. Through a bioinformatics analysis and identification of the promoter and related drought-resistant elements of the F-box gene in A. thaliana, an F-box gene, AtPP 2 -B 11 , was downregulated under drought stress. Further research found that the AtPP 2 -B 11 protein interacted with a downstream drought response factor, LEA14, which affects plant drought resistance [18]. Jia et al. [19] found a total of 51 F-box genes that were differentially expressed under salt stress. Among them, 34 were upregulated and 17 were downregulated, indicating that F-box genes are involved salt stress response in soybeans [19]. OsMsr9, which contains an F-box domain, was overexpressed in rice and A. thaliana. These plants exhibited strong salt tolerance, and the expression of other genes related to salt tolerance was also enhanced, indicating that OsMsr9 has a positive effect on enhancing salt tolerance [20]. Chen et al. [21] found that GmSK 1 , an SKP 1 homolog in soybeans, was constitutively expressed in all tested tissues, especially the roots. GmSK 1 -overexpressing lines exhibited a significant increase in root number and an obvious decrease in water loss [21]. These results indicated that the F-box gene family plays a different regulatory role in plant stress resistance.
Barley belongs to the Poaceae family and is a widely planted cereal crop [22], which is highly tolerant to drought and salt stress and is of great research importance. Completion of barley genome sequencing provided favorable conditions for barley genome identification. Since then, several gene families have been identified in barley by comparing homologous sequences from other plants. Research on the SQUAMOSA PROMOTER BINDING PROTEIN box (SBP-box) gene family revealed that there were 13 SBP genes unevenly distributed on 5 chromosomes in barley, which were divided into 5 evolutionary subfamilies according to their diverse structures [23]. Based on a genome-wide analysis of Mitogenactivated protein kinase (MAPK), a total of 20 MAPKs, 6 MAPKK, and 156 MAPKKK genes have been discovered in barley. A gene duplication analysis revealed that the amplification of MAPK cascade genes originated from tandem repeats [24]. Moreover, 45 WRKY proteins were identified in barley by using the protein sequences of different WRKY domains in A. thaliana. Further analysis revealed that HvWRKY proteins had high sequence similarity and expression pattern correlations with A. thaliana [25]. F-box proteins greatly affect plant growth, endogenous hormone signal transduction, and cell cycle regulation. Therefore, many studies have investigated the structure and function of F-box proteins in important plants, including rice, wheat, and A. thaliana, but only a few reports have explored the F-box gene family in barley. In this study, 126 F-box genes were identified by utilizing different bioinformatic methods, including gene structure analysis, chromosomal location, physicochemical properties, phylogenetic relationships, and tissue expression. Additionally, qRT-PCR was used to study the expression patterns of some family members under drought and salt stress. The combined analyses on the biological characteristics and expression patterns of the F-box gene family in barley will provide a theoretical reference in future applications for improving barley drought-and salt-tolerant varieties.

Plant Materials
The barley variety Morex was planted in the experimental field of the Hangzhou Normal University (Xiasha Campus) and selected as the materials. Morex seeds were cultured in a light culture incubator at 26°C under a 14/10 h light/dark photoperiod and photosynthetically activated radiation at 18,000 lx. Drought and salt stress experiments were performed at the two-leaf stage. A total of 3 groups were tested: (1) control (CK) (0); (2) salt stress (200 mmol·L -1 NaCl); (3) drought stress (20% PEG6000). Leaves were collected for total RNA extraction after 24 h of treatment. Duplicate samples were stored at -80°C in an ultra-low temperature refrigerator.

Total RNA Extraction and cDNA Synthesis
Total RNA was extracted from the barley leaves with the reference RNA plant extraction kit (AxyPrep). DNA contaminants were removed through DNase I treatment (RNase free). Total RNA was reversetranscribed to generate cDNA using the Hifair TM II 1st Strand cDNA Synthesis Kit. cDNA products were stored at -20°C prior to qRT-PCR analysis.

Phylogenetic Analysis of the F-box Gene Family
The identified full-length sequences of F-box proteins in barley and F-box proteins from A. thaliana and wheat with well-defined stress resistance from NCBI were aligned with the default settings of ClustalW.

F-box Gene Expression Analysis
At the two-leaf stage, hydroponic barley seedlings were exposed to drought and salt stress treatments for 24 h and subsequently used for RNA extraction. Then, the expression levels of 12 F-box genes were quantitatively analyzed. All primers selected for qRT-PCR were designed by Primer Premier v5.0 (PREMIER Biosoft, San Francisco, CA, USA) (Tab. 1). Relative expression was calculated using the 2 -ΔΔCT Livak method [35]. Each sample was repeated 3 times. qRT-PCR data were analyzed using SigmaPlot v10.0 (SYSTAT, San Jose, CA, USA) and IBM SPSS Statistics v20 (IBM, Armonk, NY, USA) statistical software. ANOVA was used to test significance (p < 0.05 indicates significant difference, indicated by *; p < 0.01 indicates high significance, indicated by **; p < 0.001 indicates high significance, indicated by ***).

Molecular Characteristics of F-box Genes
In this study, 126 F-box protein sequences were obtained after removing redundant sequences and secondary verification of the structure using CD-HIT, SMART, and NCBI Conserved Domains. These sequences were identified as F-box gene family members in barley and named HvFBXs. The physicochemical analysis revealed that the average number of amino acids was 420. The theoretical isoelectric point (pIs), relative molecular mass, and subcellular localization of F-box genes in barley were summarized in Appendix A. HvFBX61 contained the largest number of amino acids, encoded 819 amino acid residues, had a relative molecular mass of 91.29346 kDa, and a theoretical isoelectric point of 5.73. HvFBX21 contained the smallest number of amino acids and encoded 165 amino acid residues. The predicted molecular weight of HvFBX21 was 18.77672 kDa, with a theoretical isoelectric point 9.00. The isoelectric point of HvFBXs ranged from 4.54 to 11.62. The isoelectric point and relative molecular weight information of 6 F-box proteins were not provided as they contained unidentified amino acid residues. Of the 120 F-box proteins, 53 were acidic amino acids and 67 were basic amino acids. According to the principle of hydrophilicity index, amphoteric proteins ranged from -0.5 to 0.5 (GRAVY is a negative value for hydrophilicity and a positive value for hydrophobicity); only HvFBX17, HvFBX135, HvFBX149, HvFBX131, and HvFBX152 were hydrophilic proteins, and all others were amphoteric proteins. The subcellular localization of the 126 HvFBX proteins obtained in this study revealed that 39.68% of the total proteins were located in the Chloroplast, 24.60% were located in the Cytoplasm, and 18.25% were located in the Nucleus; a few proteins were localized in Mitochondria and Peroxisomes.

Identification and Phylogenetic Analysis of F-box Family Genes
NCBI database was used to search F-box genes related to plant stress resistance, and finally 1 wheat stress-resistant F-box protein sequence and 10 A. thaliana stress-resistant F-box protein sequences were selected according species affinities. In order to further screen the F-box genes associated with stress resistance in barley, 126 HvFBX protein sequences and 11 protein sequences mentioned above were used to construct a phylogenetic tree for subsequent analysis. The phylogenetic tree revealed that the F-box genes in barley, wheat and A. thaliana were divided into 9 subfamilies based on sequence similarity and named subfamilies A-I. Among them, subfamily D and I contained the largest number of F-box family members, accounting for 18.25% and 19.84%, respectively. However, subfamily B contained the smallest number of F-box family members, accounting for only 5.56% (Fig. 1). The anti-stress functional proteins from A. thaliana in subfamilies A and B were regarded as orthologs of barley [36][37][38][39][40]. Therefore, it is speculated that the genes in subfamilies A and B may serve anti-stress functions. Members of subfamily E were highly evolutionarily similar to FBP7 (GenBank: BAE99994.1) from A. thaliana [41] and unnamed proteins (GenBank: AEL33721.1) from wheat [42]. Thus, it is speculated that genes in subfamily E possess high homology with F-box genes in A. thaliana and wheat.

Chromosomal Mapping of F-box Family Genes
Based on the genome annotations, 124 of the 126 HvFBXs genes were anchored to 7 chromosomes with the largest number of genes detected on Chr5 and Chr6 (26), followed by 20 genes on Chr7, 19 on Chr3, and 9 on Chr1 and Chr4 (Fig. 3). Additionally, HvFBX103 and HvFBX139 were not anchored to the chromosome due to many unknown amino acid residues, and no clear localization information was obtained. These results suggested that F-box genes were non-randomly distributed on barley chromosomes, and most were concentrated on or near the end of the chromosomes (Fig. 3).

Conserved Domain Analysis of F-box Gene Family Members
Conservative analysis of HvFBX protein sequences revealed a total of 10 conserved motifs. However, HvFBX family members contained 1-6 motifs and all members with > 4 motifs gathered in subfamilies D and I, which confirmed the evolutionary tree results and indicated that the results had high reliability  Fig. 4). Additionally, all members contained Motif1 and -2 individually or together, but only a few proteins contained Motif5, -7, and -10, indicating that the occurrence frequency of different motifs greatly differed. With the exception of HvFBX65 located in subfamily E, Motif5-containing proteins were all distributed in subfamily D, Motif7-containing proteins were distributed in subfamily D, and Motif10-containing proteins were concentrated in subfamilies C and D. Compared to other subfamilies, the conserved sequences of subfamily D were considerably different; thus, it is speculated that stress resistance may be functionally diverse.  from the same branch; thus, the genetic structure of F-box genes in barley possesses evolutionary similarity. HvFBX125 in subfamily B contained 10 introns and 11 exons, and HvFBX69 in subfamily C contained 6 introns and 7 exons, indicating that some genes were located on the same branch, but there were still some differences in the number of introns and exons. Members of subfamily A also contained 0-5 introns and were closer to the subfamily B-D branch, indicating that the genetic structure of its members to the closer family was similar. Additionally, members of subfamilies E and G contained 0-2 introns, while members of subfamily H contained 1-4 introns.

Expression Analysis of F-box Genes under Drought and Salt Stress
The F-box proteins serve various functions, such as delaying plant senescence [47], regulating flowering [48], and responding to various abiotic stressors. In order to verify the diversity of screened genes, qRT-PCR The qRT-PCR results of the 12 HvFBX gene expression patterns were highly consistent with the RNA-Seq results (Fig. 5). Under salt stress, 10 genes were upregulated and 2 were downregulated. Under drought stress, 8 genes were upregulated and 4 were downregulated. Some target genes were intensely induced by salt treatment; specifically, the expression of HvFBX75 increased by 96.72%, that of HvFBX20 and HvFBX158 increased by~70%, that of HvFBX6, HvFBX10, and HvFBX56 increased by~60%, and that of HvFBX152 decreased by 33.82%. Additionally, the expression of HvFBX10, HvFBX20, and HvFBX75 increased by 161.62%, 157.77%, and 140.00%, respectively, after drought stress, while that of HvFBX152 decreased by 39.66%. These 4 genes were clearly sensitive to drought, but the underlying molecular mechanism remains unknown. Combined with the phylogenetic analysis, these 12 genes were found to be distributed in subfamilies A-E and I, thus, it was speculated that genes in these 8 subfamilies were related to barley drought and salt tolerance.
In summary, 10 upregulated genes under salt stress and 8 upregulated genes under drought stress were identified (Fig. 5). The expression of HvFBX158 significantly increased after salt (ANOVA, p = 0.042, df = 2) and drought (ANOVA, p = 0.025, df = 2) stress, and that of HvFBX152 significantly decreased after salt (ANOVA, p = 3.6 × 10 -5 , df = 2) and drought (ANOVA, p = 1.1 × 10 -5 , df = 2) stress. Thus, these 2 genes may play influential roles in barley drought and salt tolerance. Based on the above results, members of subfamilies A-E and I are speculated to exert an enormous effect on barley drought and salt stress response, thereby providing a basis for further research.

Discussion
The F-box gene family possesses a large number of members that exist in various eukaryotes from nematodes to humans. Previous studies found that model organisms, Drosophila and Caenorhabditis elegans, contained 22 and 326 F-box genes [5], respectively. Humans possess 68 F-box genes [49], which is close to the 74 F-box genes identified in mice [49], which indicates the similarity between these 2 species at the genomic level. The identification of F-box gene families in multiple species has been completed, including 359 in corn [7] and 687 in rice [50], reflecting the large Gramineae F-box gene family.
In this study, 126 non-redundant HvFBX genes were identified in barley. The phylogenetic analysis revealed that the genes divided into 9 subfamilies. Kelch repeats were found in HvFBX95, HvFBX30, and HvFBX43 of subfamily A and HvFBX67, HvFBX104, and HvFBX145 of subfamily G; the remaining family members contained FBOX or FBD domains. Kelch possesses a specific domain in plant F-box proteins. A previous study indicated that a few characterized wheat FBKs were involved in heat stress tolerance [42]. HvFBX101 of subfamily B and HvFBX137 of subfamily C contained LRR domains, which are Leu repeat units. These LRR domains play an important role in protein-ligand and protein-protein interactions and are involved in plant immune responses [51]. COI1, an F-box protein with an LRR domain in A. thaliana, plays a fundamental role in response to jasmonates (JA), which regulate plant root growth, pollen fertility, wounding and healing, and defense against pathogens and insects [52]. Wang et al. [53] isolated an F-box protein, GmCOI1, with an LRR domain from soybeans, which also mediates JA-regulated plant defense and fertility. Thus, it is speculated that HvFBX101 and HvFBX137 are related to plant signaling and resistance in biological stress [54]. In this study, 38 family members were found in subfamilies D-F. The phylogenetic tree results revealed that they only contained NaCl and columns in green represent 20% PEG6000. ANOVA was used to test significance. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Error bars represent standard deviation an FBOX domain. HvFBX134 contained an FBA domain, while the other 8 members of subfamily H only contained an FBOX domain. FBA specifically recognizes the target protein and plays an important role in plant 26S proteasome-mediated ubiquitination of proteins. In subfamily I, only HvFBX131 contained an FBA domain, and the remaining members only contained an FBOX domain. Therefore, compared to other subfamilies, members of subfamilies A-C and G-I with special domains were more likely to play important roles in plant signal transduction and coping with various stressors.
Gene replication events provide more choice sites for evolution, leading to several gene family members, which primarily contribute to gene family expansion and genetic novelty. Some plausible explanations may account for the large number of F-box genes members. First, whole genome duplication (WGD) is universal in plant genomes [55], which has doubled the entire nuclear gene pool and produced a large number of supergene families, such as the F-box gene family. Second, in addition to several WGD events that occurred in the common ancestor of angiosperms, some additional lineage-specific (LS)-WGDs were found in plants, including A. thaliana, corn, soybeans, poplar, and pears. LS-WGDs make new repeat genes in F-box gene families of different species, which differentiate in size [10]. Paterson et al. [56,57] found that Poaceae had a genome-wide doubling event before differentiation. In this study, analysis of the phylogenetic tree results revealed that HvFBX140, HvFBX135, and HvFBX75 of subfamily A, HvFBX31 and HvFBX78 of subfamily B, HvFBX108 of subfamily E, and HvFBX33 of subfamily I were also found in A. thaliana and have orthologs. However, HvFBX76, HvFBX152, and HvFBX20 of subfamily E all have paralogs in A. thaliana. Therefore, it can be assumed that the genes corresponding to the above 10 F-box proteins differentiated relatively early. Additionally, HvFBX95 of subfamily A was likely to have branch-specific repetitive events after differentiation from barley, which in turn produced HvFBX30 and HvFBX43 that are highly similar in protein sequence, gene structure, and expression. Such events are common in the F-box gene family in barley; thus, it can be speculated that among the 126 HvFBXs identified, gene duplication had an effect on the amplification and evolution of this gene family, which eventually lead to F-box gene diversity in terms of quantity, structure, and function in barley.
F-box proteins are widely involved in plant stress resistance. The homology analysis of F-box proteins revealed that some of these proteins served unclear functions and were more conducive for exploring the potential function of barley F-box proteins. In this study, protein sequences corresponding to the 11 F-box genes and 126 F-box protein sequences were used to construct a phylogenetic tree (Fig. 1). By comprehensively analyzing the results of the evolutionary tree and the expression profile, 12 genes were screened that exhibited high expression levels and may be related to stress responses (Tab. 1). The qRT-PCR results revealed that the expression of HvFBX152 decreased significantly, while the expression of HvFBX20 and HvFBX158 increased after salt and drought stress treatments. HvFBX152 and HvFBX20 of subfamily E are homologous to the drought-resistant and salt-tolerant-related gene, AtPP2-B11, which encodes an SCF E3 ligase that responds to drought stress as a negative regulator and to salt stress by regulating gene expression [46]. HvFBX158 of subfamily B is homologous to the salt stress-related gene, TIR1 [43], and the drought and salt tolerance-related gene, AFBA1 [40]. HvFBX6 is homologous to TIR1 and AFBA1 and was upregulated by salt stress and downregulated by drought stress in this study. The above results indicate that the changes in expression after adversity control were in line with theoretical expectations and that the evolutionary tree results have high credibility.
This study revealed that HvFBXs could respond to two different stress, which suggested that HvFBXs might not only improve salt tolerance but also drought tolerance. But the diverse expression level also been observed. For example, HvFBX6, HvFBX10, HvFBX20, HvFBX56, HvFBX75, HvFBX80 and HvFBX158 were more sensitive to salt than HvFBX30, HvFBX43 and HvFBX67 among 10 upregulated genes after salt stress. Meanwhile, HvFBXs in leaf tissues were also sensitive to drought. For example, HvFBX10, HvFBX20, HvFBX75 and HvFBX158 were more sensitive to drought than HvFBX43, HvFBX56, HvFBX67 and HvFBX137 among 8 upregulated genes after salt stress. Salt stress causes barley cell membrane damage, weakens photosynthesis, and leads to nutrient and water deficiency. It often manifests as physiological drought, and drought stress causes similar damage to barley. Therefore, the changes in gene expression caused by physiological and environmental drought may have certain similarities. According to the results of this study, HvFBX10, HvFBX20, HvFBX43, HvFBX56, HvFBX67, HvFBX75, HvFBX158 and HvFBX152 changed in the same direction after subjugation to drought and salt stress; therefore, it can be speculated that there is a certain relationship between the internal response mechanisms of these 8 genes to stress.

Conclusions
This study conducted bioinformatics research in various aspects on the F-box gene family in barley. Results revealed its origin, amplification, evolution, and possible functional differentiation, as well as screened out 12 highly expressed genes and their expression levels after stress. Among the 12 target genes, 10 genes were upregulated and 2 genes were downregulated after salt stress. The genes whose expression was upregulated were HvFBX6, HvFBX10, HvFBX20, HvFBX30, HvFBX43, HvFBX56, HvFBX67, HvFBX75, HvFBX80, and HvFBX158. However, among the 12 target genes, 8 genes were upregulated and 4 genes were downregulated after drought stress. The genes whose expression was downregulated were HvFBX10, HvFBX20, HvFBX43, HvFBX56, HvFBX67, HvFBX75, HvFBX158, and HvFBX137. This study is the first to confirm that HvHBX152 and HvHBX158 play important roles in drought resistance and salt tolerance, providing a theoretical basis for future in-depth research on related genes and applications aimed at improving barley varieties.