The Identification and Expression Analysis of the Liriodendron chinense F-Box Gene Family

The F-box gene family is one of the largest gene families in plants, and it plays a crucial role in regulating plant development, reproduction, cellular protein degradation, and response to biotic and abiotic stresses. Despite their significance, a comprehensive analysis of the F-box gene family in Liriodendron chinense and other magnoliaceae species has not been reported. In this study, we report for the first time the identification of 144 full-length F-box genes in L. chinense. Based on specific domains and phylogenetic analyses, these genes were divided into 10 distinct subfamilies. We further analyzed their gene structure, conserved domain and chromosome distribution, genome-wide replication events, and collinearity. Additionally, based on GO analysis, we found that F-box genes exhibit functional specificity, with a significant proportion of them being involved in protein binding (GO:0005515), suggesting that F-box genes may play an important role in gene regulation in L. chinense. Transcriptome data and q-PCR results also showed that F-box genes are involved in the development of multiple tissues in L. chinense, regulate the somatic embryogenesis of Liriodendron hybrids, and play a pivotal role in abiotic stress. Altogether, these findings provide a foundation for understanding the biological function of F-box genes in L. chinense and other plant species.


Introduction
Ubiquitin is a small protein molecule that is found widely within eukaryotic cells.Its fundamental role is to promote protein degradation and DNA damage repair, and it achieves this by catalyzing a series of enzymatic reactions to attach to target proteins.The ubiquitin-mediated protein degradation pathway involves three enzymes: ubiquitinactivating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3.Among these enzymes, E3 is the most intricate and varied in structure and number [1].One of the most extensively studied E3 ubiquitin ligases is the SCF complex, which is comprised of Rbx1, Skp1, Cullin, and F-BOX.Rbx1, Skp1, and Cullin form the framework of the complex, while different F-BOX are capable of recognizing specific substrates, which are subsequently degraded via targeted degradation [2,3].
The F-BOX, which contains a conserved F-BOX motif of approximately 40 amino acids, is a member of one of the largest protein families present in eukaryotes [4].Since the discovery of the first F-BOX (Cyclin F) in humans [5], numerous F-BOX have been identified based on the conserved motif of its N-terminal [6].The number of F-box genes differs across species, and plants typically have a greater number of F-box genes than animals.For instance, while Caenorhabditis elegans has the highest number of F-box genes in animals with a count of 326, there are only 69 and 22 F-box genes in humans and Drosophila, respectively [6,7].On the other hand, plants have a greater number of F-box genes, such as Plants 2024, 13, 171 3 of 20 genome-wide analyses of gene families that shape the growth, development, and response to environmental stressors of this tree species.Several gene families have been studied, including CBF [30], WRKY [31], MYB [32], TPS [33], PIN [34], etc.Here, we conducted a genome-wide gene family analysis of F-box genes in L. chinense, exploring various aspects such as conserved domains, gene structure, phylogenetic tree, chromosome locations, gene replication, and collinearity.Additionally, we analyzed gene ontology and predicted the subcellular localization of the F-box gene family in L. chinense.Importantly, to further examine the function of the F-box gene family in L. chinense, we combined transcriptome and qRT-PCR to analyze the expression of F-box genes in different tissues during somatic embryonic development and under stress treatment.These findings contribute to our understanding of the mechanisms underlying plant growth, development, and response to environmental stress.Moreover, they provide potential avenues for the cultivation of desirable seedlings.

Genome-Wide Identification, Characteristics, and Classification of F-Box Genes in L. chinense
Based on our analysis using HMMER (v2.41.2), we identified 163 F-box genes in L. chinense.We then screened these genes using the SMART (2020), Pfam, and CDD databases and identified 144 F-box genes with conserved domains.We used TBtools software (v2.034) to extract the F-BOX and F-BOX-LIKE conserved domain sequences of these 144 F-BOX in L. chinense, and the two conserved motifs were visualized using the Weblogo online software (v2.8.2).The F-BOX motif is typically located at the N-terminal of F-BOX and has a length of approximately 40 to 50 amino acids.The conservative F-BOX sequence of L. chinense F-BOX contains approximately 50 amino acid residues, including an extremely conservative tryptophan residue (W) at the 43rd position, as well as other conservative amino acid residues such as leucine (L) at the 6th position, proline (P) at the 7th and 27th positions, tryptophan (W) at the 26th position, arginine (R) at the 29th position, valine (V) at the 38th position, and more (Figure 1A).These conserved amino acid residues may interact with tryptophan residues to maintain the α helix structure of the F-BOX motif of L. chinense [5] and enable the proteins to perform their specific functions.We also analyzed the F-BOX-LIKE conserved motif, which is also located at the N-terminal of the F-BOX and contains approximately 60 amino acid residues.In addition to an extremely conserved tryptophan residue (W) at the 41st position, other conserved amino acid residues include leucine (L) at the 4th and 21st positions, proline (P) at the 5th position, isoleucine (I) at the 17th position, valine (V) at the 40th position, and more (Figure 1B).Our analysis shows that the conserved amino acid residues of these two conserved motifs are largely consistent.
We also analyzed some basic characteristics of the 144 F-box genes.We found that the shortest protein length was 95 amino acids with a molecular weight (MW) of 10.71 kDa, while the longest protein length was 1582 amino acids with a MW of 181.88 kDa.The differences were quite large, with an average length of 461 amino acids and an average MW of 51.97 kDa.Additionally, we observed that among the 144 F-box genes, the isoelectric points (pI) of the corresponding proteins of 65 genes were acidic, while the remaining 79 genes had alkaline protein products (Table S1).
We classified the 144 F-box genes based on the C-terminal conserved domain types of each F-box gene, and they were divided into 10 subfamilies, including: FBX (only F-BOX), FBXL (F-BOX-LIKE), FBA (associated domain), FBD (FBD domain), FBDUF (domain of unknown functions), FBK (Kelch repeats), FBL (Leucine-rich repeats), FBP (PP2), FBT (Tubby), and FBO (other domain) (Figure 2A,B).The FBO subfamily members include Actin, Arm, DnaJ, EamA, LysM, Pkinase, Sel1, SnowaL_3, zf-cw, and others (Figure 2C).To verify our classification, we further constructed a phylogenetic tree using the full-length amino acid sequences of the F-box genes, which showed that members of the same subfamily tended to cluster on the same branch.For example, the FBD, FBA, FBK, and FBT genes were found to cluster closely together (Figure 3).This indicates that our classification based on the C-terminal conservative domain has some credibility.
the number of motifs tended to be consistent within the same gene family.For instance, the FBD and FBL subfamilies had a relatively large number of motifs, while the FBA and FBK subfamilies had a relatively small number of motifs.In contrast, the number of introns on the 144 F-box genes was highly variable, ranging from 0 to 16, while the number of exons ranged from 1 to 17.The numbers of UTRs ranged from 0 to 2 (Figure 4, Table S2).We also analyzed the gene structure and motif of the 144 F-box genes.We found that the number of motifs tended to be consistent within the same gene family.For instance, the FBD and FBL subfamilies had a relatively large number of motifs, while the FBA and FBK subfamilies had a relatively small number of motifs.In contrast, the number of introns on the 144 F-box genes was highly variable, ranging from 0 to 16, while the number of exons ranged from 1 to 17.The numbers of UTRs ranged from 0 to 2 (Figure 4, Table S2).

Chromosomal Locations, Gene Duplication Events, and Synteny Analysis in the L. chinense F-Box Gene Family
Based on the genomic annotation document, we found that F-box genes were distributed on all chromosomes of L. chinense.Among them, 18 F-box genes were distributed on several different contigs, while the rest were distributed on different chromosomes of L. chinense.Chromosome 1 had the most F-box genes, with a total of 14, followed by Chromosome 8.In contrast, Chromosome 14 had the least number of F-box genes, with only one (Figure 5A).Correlation analysis of 126 F-box genes that were distributed on chromosomes and chromosome length revealed that chromosome length showed no significant correlation with the distribution number of F-box genes (Figure 5B).We also found that among these 126 F-box genes, there were eight tandem repeating pairs.These pairs included Lchi00483/Lchi00484, Lchi08342/Lchi08343, Lchi17374/Lchi17375, Lchi21894/Lchi21895, Lchi22847/Lchi22848, Lchi23958/Lchi23959, Lchi27116/Lchi27117, and Lchi27131/Lchi27132 (Figure 5A).
We also analyzed the collinearity of these 144 F-box genes and found a total of four pairs of linear relationships.Two of these pairs belonged to the FBX subfamily, one belonged to the FBT subfamily, and one belonged to the FBK subfamily (Figure 5C).    ) was used to perform multiple sequence alignments on the full-length amino acid sequences of 144 F-box genes in L. chinense.The phylogenetic tree was constructed using the maximum likelihood (ML) method.The Jones-Thornton-Taylor (JTT) model algorithm was used to estimate 1000 bootstrap repeats.

Subcellular Localization, GO, KEGG Enrichment Analysis, and Protein Interaction Prediction of the F-Box Gene in L. chinense
To analyze the potential functions of these 144 F-box genes in L. chinense, we first predicted their subcellular localization (Table S3).We found that the 144 F-box genes were unevenly distributed across different cellular compartments, including the nucleus, cytoplasm, extracellular space, plasma membrane, mitochondria, Golgi, peroxisomes, and vacuoles.Specifically, the proportion of F-box genes distributed in these structures is 29:16:8:19:22:2:3:1 (Figure 6B).Moreover, we found that some subfamily genes were localized in various cellular structures.For instance, the FBL subfamily was distributed in cytoplasmic, nuclear, and peroxisomal cellular structures, while the FBX subfamily was distributed across all eight cellular structures to some extent (Figure 6A).
To further understand the biological functions of the F-box gene-encoded proteins in L. chinense, we conducted gene ontology analysis to predict the function of homologous genes based on studies of F-box genes in A. thaliana.We found that the majority of L. chinense F-BOX was identified as being involved in protein-binding molecular functions (Table S4), which was consistent with previous studies.Additionally, we found that 12 genes had multiple GO numbers, indicating that these F-box genes were involved in specific biological processes (Table 1).

Chromosomal Locations, Gene Duplication Events, and Synteny Analysis in the L. chinense F-Box Gene Family
Based on the genomic annotation document, we found that F-box genes were distributed on all chromosomes of L. chinense.Among them, 18 F-box genes were distributed on several different contigs, while the rest were distributed on different chromosomes of L. chinense.Chromosome 1 had the most F-box genes, with a total of 14, followed by Chromosome 8.In contrast, Chromosome 14 had the least number of F-box genes, with only one (Figure 5A).Correlation analysis of 126 F-box genes that were distributed on chromosomes and chromosome length revealed that chromosome length showed no significant correlation with the distribution number of F-box genes (Figure 5B).We also found that among these 126 F-box genes, there were eight tandem repeating pairs.These pairs included Lchi00483/Lchi00484, Lchi08342/Lchi08343, Lchi17374/Lchi17375, Lchi21894/Lchi21895, Lchi22847/Lchi22848, Lchi23958/Lchi23959, Lchi27116/Lchi27117, and Lchi27131/Lchi27132 (Figure 5A).We also conducted KEGG pathway enrichment analysis, which revealed that the majority of L. chinense F-box genes (35) were involved in the biological process of ubiquitination, consistent with their function as components of the SCF complex.Additionally, 13 genes were predicted to be involved in starch and sucrose metabolism, 12 in ascorbate and aldarate metabolism pathways, 9 in signaling protein pathways, and 7 in the transcription machinery pathway, among others (Figure 6C).The interaction between 144 F-BOXs was predicted by the STRING online website (Figure 7).The results showed that 35 F-BOX genes may interact, and 15 genes form the key nodes of the interaction network.They are Lchi10916, Lchi27083, Lchi10475, Lchi28264, Lchi01081 of the FBXL subfamily; FBX subfamily Lchi05172, Lchi17321, Lchi14963, Lchi00327; Lchi21170, Lchi23958, Lchi13056 of the FBD subfamily; Lchi22848, Lchi33849 of the FBA subfamily; and Lchi30203 of the FBL subfamily.

F-Box Gene Expression Levels across Different Tissue Types in Liriodendron Hybrids
To investigate the expression patterns of the F-box gene family, we obtained transcriptome data for different tissues of Liriodendron hybrids from the NCBI website.We extracted the F-box family genes and used them to generate a heatmap of their expression patterns (Figure S1, Table S5).Relatively speaking, most genes tend to be highly expressed in buds and pistils and lowly expressed in leaves and petals.A small number of genes are highly expressed in stamens, most of which belong to the FBA subfamily, indicating that the FBA subfamily genes may be related to some functions of stamens (Figure S1).The cluster analysis of gene expression showed that the tissue-specific expression patterns of

F-Box Gene Expression Levels across Different Tissue Types in Liriodendron Hybrids
To investigate the expression patterns of the F-box gene family, we obtained transcriptome data for different tissues of Liriodendron hybrids from the NCBI website.We extracted the F-box family genes and used them to generate a heatmap of their expression patterns (Figure S1, Table S5).Relatively speaking, most genes tend to be highly expressed in buds and pistils and lowly expressed in leaves and petals.A small number of genes are highly expressed in stamens, most of which belong to the FBA subfamily, indicating that the FBA subfamily genes may be related to some functions of stamens (Figure S1).The cluster analysis of gene expression showed that the tissue-specific expression patterns of F-box genes were concentrated in 14, 9, and 7 patterns, and the number of genes contained in the three patterns was 26, 22, and 11, respectively (Figure S2).To verify the reliability of the transcriptome data, we selected 1-2 genes (selection criteria: 1. the expression level of the gene cannot be too low to prevent the gene from being detected by qRT-PCR; 2. the designed primers were amplified by semi-quantitative PCR, and the product had a target band) from each subfamily for qRT-PCR analysis.We found that different subfamily genes showed different expression patterns in different tissues.For instance, Lchi33849 of the FBA subfamily had relatively high expression in stamens and relatively low expression in petals.We also observed that Lchi34216 of the FBDUF subfamily had relatively low expression in buds, Lchi02628 of the FBK subfamily had high expression in sepals and relatively low expression in leaves, and Lchi04963 of the FBT subfamily had high expression in roots and stems but relatively low expression in other tissues, among others.Additionally, different genes within the same subfamily showed different expression patterns in different tissues, such as Lchi21894 and Lchi27116 of the FBD subfamily and Lchi00327 and Lchi22556 of the FBX subfamily (Figure 8).In summary, we speculate that the specific expression of F-box genes in different tissues means that these genes may be related to the developmental regulation of specific tissues.
F-box genes were concentrated in 14, 9, and 7 patterns, and the number of genes contained in the three patterns was 26, 22, and 11, respectively (Figure S2).To verify the reliability of the transcriptome data, we selected 1-2 genes (selection criteria: 1. the expression level of the gene cannot be too low to prevent the gene from being detected by qRT-PCR; 2. the designed primers were amplified by semi-quantitative PCR, and the product had a target band) from each subfamily for qRT-PCR analysis.We found that different subfamily genes showed different expression patterns in different tissues.For instance, Lchi33849 of the FBA subfamily had relatively high expression in stamens and relatively low expression in petals.We also observed that Lchi34216 of the FBDUF subfamily had relatively low expression in buds, Lchi02628 of the FBK subfamily had high expression in sepals and relatively low expression in leaves, and Lchi04963 of the FBT subfamily had high expression in roots and stems but relatively low expression in other tissues, among others.Additionally, different genes within the same subfamily showed different expression patterns in different tissues, such as Lchi21894 and Lchi27116 of the FBD subfamily and Lchi00327 and Lchi22556 of the FBX subfamily (Figure 8).In summary, we speculate that the specific expression of F-box genes in different tissues means that these genes may be related to the developmental regulation of specific tissues.

F-Box Genes Are Universally Expressed at Different Stages of Somatic Embryogenesis in Liriodendron Hybrids
To further understand the role of F-box family genes in affecting plant development, we obtained expression data for the 144 F-box genes from the transcriptome data of somatic embryogenesis in Liriodendron hybrids (Figure S3, Table S6).During the whole process of somatic embryogenesis, the expression of the F-box gene is differentially regulated.Some are up-regulated in the early stages, while others are up-regulated in both the early and late stages.In addition, a small number of genes are highly expressed in the middle stage (Figure S3).Cluster analysis of these expression patterns showed that with the

F-Box Genes Are Universally Expressed at Different Stages of Somatic Embryogenesis in Liriodendron Hybrids
To further understand the role of F-box family genes in affecting plant development, we obtained expression data for the 144 F-box genes from the transcriptome data of somatic embryogenesis in Liriodendron hybrids (Figure S3, Table S6).During the whole process of somatic embryogenesis, the expression of the F-box gene is differentially regulated.Some are up-regulated in the early stages, while others are up-regulated in both the early and late stages.In addition, a small number of genes are highly expressed in the middle stage (Figure S3).Cluster analysis of these expression patterns showed that with the development of somatic embryos, these F-box genes were concentrated in five expression patterns, and 28 genes were clustered into two expression patterns, which showed that the expression decreased first, then increased, and finally decreased.Furthermore, 29 genes were clustered into 19 expression patterns, which showed that their expression levels continued to increase.Eighteen genes were clustered into one type of expression pattern, and their expression levels decreased first and then increased.The 16 genes were clustered into zero expression patterns, and their expression levels showed a continuous decrease.The 16 genes were Plants 2024, 13, 171 13 of 20 clustered into nine expression patterns, and their expression levels were similar to those of the two expression patterns (Figure S4).We conducted qRT-PCR verification and found that, during the somatic embryogenesis of Liriodendron hybrids, Lchi20923 and Lchi15582 showed a decreasing trend, and the expression of Lchi04963 decreased first, then increased, and then increased.In addition, other genes showed a trend of increasing first, then decreasing, and then increasing (Figure 9).In summary, the change in F-box gene expression during somatic embryogenesis indicates that it may be involved in some biological processes during somatic embryogenesis and thus plays a regulatory role in somatic embryo development.
patterns, and 28 genes were clustered into two expression patterns, which showed that the expression decreased first, then increased, and finally decreased.Furthermore, 29 genes were clustered into 19 expression patterns, which showed that their expression levels continued to increase.Eighteen genes were clustered into one type of expression pattern, and their expression levels decreased first and then increased.The 16 genes were clustered into zero expression patterns, and their expression levels showed a continuous decrease.The 16 genes were clustered into nine expression patterns, and their expression levels were similar to those of the two expression patterns (Figure S4).We conducted qRT-PCR verification and found that, during the somatic embryogenesis of Liriodendron hybrids, Lchi20923 and Lchi15582 showed a decreasing trend, and the expression of Lchi04963 decreased first, then increased, and then increased.In addition, other genes showed a trend of increasing first, then decreasing, and then increasing (Figure 9).In summary, the change in F-box gene expression during somatic embryogenesis indicates that it may be involved in some biological processes during somatic embryogenesis and thus plays a regulatory role in somatic embryo development.

F-Box Responds Strongly to Cold, Heat, and Drought Stress Treatments in Liriodendron Hybrids
Numerous studies have shown that F-box family genes play significant roles in stress responses.Therefore, we extracted transcription data for these 144 genes under drought, high temperature, and cold stress (Figures S5, S7, and S9; Table S7).In cold stress, the peak expression of some genes appeared at 0 h, while the peak expression of other genes appeared at 3 day (Figure S5).Its expression patterns are basically clustered between 0 and 19.The expression pattern 0 contained 45 genes, and the expression level continued to increase.Expression pattern 19 included 11 genes, and the expression level showed a continuous decrease (Figure S6).In heat stress, the peak of F-box gene expression appeared at 0 day, 1 day, and 3 day (Figure S7).Its expression patterns are clustered into 1 and 19 patterns.The expression pattern 1 contained 25 genes, and the expression level decreased first and then increased.The expression pattern 19 contains 19 genes, and the expression level is continuously reduced (Figure S8).Under drought stress, the peak of F-box gene expression appeared at multiple time points (Figure S9).Its expression patterns are clustered into zero and six patterns.The expression pattern 0 contained 17 genes, and the expression level showed a continuous decrease.The expression level 6 contained 12 genes, and the expression level showed a repeated fluctuation of decreasing first and then increasing (Figure S10).Then we conducted qRT-PCR verification of gene expression under drought stress.Interestingly, we found that during the process of drought stress, the gene expression levels of almost all subfamilies first decreased and then increased, reaching a peak expression level at 72 h of drought treatment (Figure 10).In summary, the differential

F-Box Responds Strongly to Cold, Heat, and Drought Stress Treatments in Liriodendron Hybrids
Numerous studies have shown that F-box family genes play significant roles in stress responses.Therefore, we extracted transcription data for these 144 genes under drought, high temperature, and cold stress (Figures S5, S7 and S9; Table S7).In cold stress, the peak expression of some genes appeared at 0 h, while the peak expression of other genes appeared at 3 day (Figure S5).Its expression patterns are basically clustered between 0 and 19.The expression pattern 0 contained 45 genes, and the expression level continued to increase.Expression pattern 19 included 11 genes, and the expression level showed a continuous decrease (Figure S6).In heat stress, the peak of F-box gene expression appeared at 0 day, 1 day, and 3 day (Figure S7).Its expression patterns are clustered into 1 and 19 patterns.The expression pattern 1 contained 25 genes, and the expression level decreased first and then increased.The expression pattern 19 contains 19 genes, and the expression level is continuously reduced (Figure S8).Under drought stress, the peak of F-box gene expression appeared at multiple time points (Figure S9).Its expression patterns are clustered into zero and six patterns.The expression pattern 0 contained 17 genes, and the expression level showed a continuous decrease.The expression level 6 contained 12 genes, and the expression level showed a repeated fluctuation of decreasing first and then increasing (Figure S10).Then we conducted qRT-PCR verification of gene expression under drought stress.Interestingly, we found that during the process of drought stress, the gene expression levels of almost all subfamilies first decreased and then increased, reaching a peak expression level at 72 h of drought treatment (Figure 10).In summary, the differential expression of F-box genes in these stress responses indicates that they may be regulated by certain factors in the process of stress response, thus responding to these abiotic stresses.

Discussion
The F-box gene family, which is involved in many biological pathways, is one of the largest gene families in eukaryotes and plays a critical role in the complete life cycle of plants, making its study essential for understanding plant growth and development.
The F-box gene family has been extensively analyzed and studied in various species, with the number of its members varying widely across species (Figure 11).This variability may be attributed to the different identification thresholds and criteria adopted by different researchers [12].For instance, the number of F-box gene family members in rice was reported to be 680, 858, 764, while in A. thaliana it was 660,698, and in aspen, it was 320, 337, 387 [8,35,36].Furthermore, differences in the number of F-box gene family members may be a result of gene duplication during species evolution, which could lead to both gene loss and expansion.Gene replication is considered an essential contributor to gene family expansion and functional diversity during evolution, and it may occur through chromosome segmental replication or tandem replication [37].In chickpeas, for instance, repeats made up 43.7% of the 192 F-box genes, of which 38 (13.3%) resulted from segmentary repeats and 62 (21.8%) arose from tandem repeats.These findings suggest that tandem repeats, rather than segmentary repeats, played a more significant role in the expansion of the F-box gene family in chickpeas [11].In L. chinense, there are also eight pairs of tandem repeats in the F-box gene family.Among the eight gene pairs, five pairs belong to the FBD subfamily, while previous studies have shown that the F-box gene subfamily shows bias in its amplified repeat pattern, and most genes involved in tandem and segmental duplications belong to the FBD, FBX, and FBL subfamilies [11].
The functional diversity of F-box genes is closely linked to the conserved C-terminal domain of the F-box genes.In addition to the conserved F-BOX domain, the C-terminal of the F-box gene usually contains a Kelch repeat, a leucine-rich repeat, the FBD domain, the FBA domain, Tubby, PP2, and other conserved domains.These domains have distinct

Discussion
The F-box gene family, which is involved in many biological pathways, is one of the largest gene families in eukaryotes and plays a critical role in the complete life cycle of plants, making its study essential for understanding plant growth and development.
The F-box gene family has been extensively analyzed and studied in various species, with the number of its members varying widely across species (Figure 11).This variability may be attributed to the different identification thresholds and criteria adopted by different researchers [12].For instance, the number of F-box gene family members in rice was reported to be 680, 858, 764, while in A. thaliana it was 660,698, and in aspen, it was 320, 337, 387 [8,35,36].Furthermore, differences in the number of F-box gene family members may be a result of gene duplication during species evolution, which could lead to both gene loss and expansion.Gene replication is considered an essential contributor to gene family expansion and functional diversity during evolution, and it may occur through chromosome segmental replication or tandem replication [37].In chickpeas, for instance, repeats made up 43.7% of the 192 F-box genes, of which 38 (13.3%) resulted from segmentary repeats and 62 (21.8%) arose from tandem repeats.These findings suggest that tandem repeats, rather than segmentary repeats, played a more significant role in the expansion of the F-box gene family in chickpeas [11].In L. chinense, there are also eight pairs of tandem repeats in the F-box gene family.Among the eight gene pairs, five pairs belong to the FBD subfamily, while previous studies have shown that the F-box gene subfamily shows bias in its amplified repeat pattern, and most genes involved in tandem and segmental duplications belong to the FBD, FBX, and FBL subfamilies [11].
The functional diversity of F-box genes is closely linked to the conserved C-terminal domain of the F-box genes.In addition to the conserved F-BOX domain, the C-terminal of the F-box gene usually contains a Kelch repeat, a leucine-rich repeat, the FBD domain, the FBA domain, Tubby, PP2, and other conserved domains.These domains have distinct biological functions.For example, the Kelch repeat domain is involved in circadian clock regulation, recognizes substrates, and mediates their ubiquitination [38].LRR is associated with plant resistance, and in A. thaliana, LRR is able to recognize flagellin (flg22) in the defense response, thus initiating plant resistance [39,40].The WD40 domain promotes flavonoid biosynthesis in protein-interacting plants [41][42][43].The FBA domain is involved in carbohydrate metabolism, signal transduction [44][45][46][47], and so on.Here, we divided F-box family genes into 10 subfamilies to conserved domains.However, in L. chinense, we did not find the WD40 domain, which we speculated may be due to the ancient evolutionary status of L. chinense, and the WD40 domain may have evolved later.
sponses to these stresses.Moreover, to investigate the role of F-box genes in plant gro and development, the transcriptome data of Liriodendron hybrid embryogenesis was lyzed.The q-RCR results revealed that during somatic embryogenesis, most F-box g exhibited a pattern of initial increase, then decrease, and finally increase again.This i cates that these genes may be involved in the initiation of embryonic development postembryonic growth.Furthermore, during drought stress treatment, the expression els of these F-box genes decreased initially and then increased, reaching their highest l after 72 h of stress treatment.This suggests that F-box genes may be upregulated du this period to resist the effects of drought stress.To delve further into the gene function of the F-box gene family in L. chinense, we predicted the subcellular locations of these genes and found that most are located in the nucleus, followed by the mitochondria, plasma membrane, and cytoplasm.Interestingly, different subfamilies show a certain bias in their predicted subcellular locations; for instance, the FBL subfamily is not predicted to be located in other cell structures aside from the nucleus, cytoplasm, and peroxisomic structures.The annotation structure of the gene body showed that most of the F-box family genes were annotated to the protein-binding (GO:0005515) module, indicating that F-box genes may play a key role in gene transcription regulation of pear, which is consistent with previous studies [9,12].KEGG enrichment results showed that most F-box genes (35) were involved in the biological process of ubiquitination, which is related to the fact that F-BOX is one of the most typical ubiquitin ligases in the SCF complex [2,3].
Gene expression analysis is an insightful tool for studying the functionality of genes, and RNA-seq is a powerful technique for studying the transcription patterns of specific genes through high-throughput sequencing.To explore the tissue expression specificity of F-box genes, we employed published RNA-seq data and studied F-box gene expression profiles under cold, heat, and drought stress in order to understand the F-box gene responses to these stresses.Moreover, to investigate the role of F-box genes in plant growth and development, the transcriptome data of Liriodendron hybrid embryogenesis was analyzed.The q-RCR results revealed that during somatic embryogenesis, most F-box genes exhibited a pattern of initial increase, then decrease, and finally increase again.This indicates that these genes may be involved in the initiation of embryonic development and postembryonic growth.Furthermore, during drought stress treatment, the expression levels of these F-box genes decreased initially and then increased, reaching their highest level after 72 h of stress treatment.This suggests that F-box genes may be upregulated during this period to resist the effects of drought stress.

Database Search for F-Box Proteins in L. chinense
To identify all the protein sequences in the L. chinense genome containing F-BOX and F-BOX-LIKE domains, we downloaded the L. chinense genomic dataset from the L. chinense genomic assembly data (https://db.cngb.org/search/project/CNP0000815/)(accessed on 23 May 2020) [29].We then obtained the Hidden Markov Model (HMM) files of F-BOX (PF00646) and F-BOX-LIKE (PF12937) from the Pfam database (http://pfam.xfam.org)(accessed on 5 December 2020) [48].HMMER v3.2.1 (https://www.ebi.ac.uk/Tools/hmmer/) (accessed on 5 December 2020) [49] with an E-value cutoff of 1.0 was used as a query tool to search for all protein sequences containing F-BOX and F-BOX-LIKE in the L. chinense genome.Additionally,

Figure 1 .
Figure 1.WebLogo based on the alignments of the F-BOX motifs and F-BOX-LIKE motifs from 144 F-BOX proteins in L. chinense.(A) represents F-BOX motifs, and (B) represents F-BOX-LIKE motifs.Figure 1. WebLogo based on the alignments of the F-BOX motifs and F-BOX-LIKE motifs from 144 F-BOX proteins in L. chinense.(A) represents F-BOX motifs, and (B) represents F-BOX-LIKE motifs.

Figure 1 .
Figure 1.WebLogo based on the alignments of the F-BOX motifs and F-BOX-LIKE motifs from 144 F-BOX proteins in L. chinense.(A) represents F-BOX motifs, and (B) represents F-BOX-LIKE motifs.Figure 1. WebLogo based on the alignments of the F-BOX motifs and F-BOX-LIKE motifs from 144 F-BOX proteins in L. chinense.(A) represents F-BOX motifs, and (B) represents F-BOX-LIKE motifs.

Figure 2 .
Figure 2. Conserved domain of the L. chinense F-box gene family.(A) represents the conserved domain of 144 F-box genes; (B) represents the number of members of different subfamilies; and (C) represents the conserved domain and the number of members contained in the FBO family, the number in the fan represents the number of members.

Figure 2 .
Figure 2. Conserved domain of the L. chinense F-box gene family.(A) represents the conserved domain of 144 F-box genes; (B) represents the number of members of different subfamilies; and (C) represents the conserved domain and the number of members contained in the FBO family, the number in the fan represents the number of members.

Figure 3 .
Figure 3. Phylogenetic tree of the L. chinense F-box family gene.The ClustalW program in MEGA-X software (v10.1.8)was used to perform multiple sequence alignments on the full-length amino acid sequences of 144 F-box genes in L. chinense.The phylogenetic tree was constructed using the maximum likelihood (ML) method.The Jones-Thornton-Taylor (JTT) model algorithm was used to estimate 1000 bootstrap repeats.

Figure 3 .
Figure 3. Phylogenetic tree of the L. chinense F-box family gene.The ClustalW program in MEGA-X software (v10.1.8)was used to perform multiple sequence alignments on the full-length amino acid sequences of 144 F-box genes in L. chinense.The phylogenetic tree was constructed using the maximum likelihood (ML) method.The Jones-Thornton-Taylor (JTT) model algorithm was used to estimate 1000 bootstrap repeats.

Figure 4 .
Figure 4. Gene structure and conserved motif analysis of the L. chinense F-box family.(A) represents the motifs of F-box genes in different F-box subfamilies, in which different color frames represent different motifs; (B) represents the gene structure of the F-box, where the green box represents the UTR, the yellow box represents the CDS, and black lines represent introns.

Figure 4 .
Figure 4. Gene structure and conserved motif analysis of the L. chinense F-box family.(A) represents the motifs of F-box genes in different F-box subfamilies, in which different color frames represent different motifs; (B) represents the gene structure of the F-box, where the green box represents the UTR, the yellow box represents the CDS, and black lines represent introns.

Figure 5 .
Figure 5. Chromosome distribution, gene tandem duplication, and collinearity analysis of the F-box gene in L. chinense.(A) represents the distribution of F-box genes on chromosomes, and the red arc represents tandem repeat genes.The blue line represents the gene, and its thickness represents the length of the gene on the chromosome.(B) represents the correlation analysis between F-box gene number and chromosome length; (C) represents the collinearity of the F-box gene within L. chinense, Lines of different colors represent different subfamilies.

Figure 6 .
Figure 6.Subcellular prediction and KEGG enrichment analysis of L. chinense F-box family genes.(A) represents the prediction of the subcellular structure of F-box genes in different subfamilies, and the number on the column represents the number of F-box genes.(B) represents the proportion of F-box genes predicted by different cell structures.(C) represents the KEGG enrichment pathway analysis of F-box family genes.

Figure 6 .
Figure 6.Subcellular prediction and KEGG enrichment analysis of L. chinense F-box family genes.(A) represents the prediction of the subcellular structure of F-box genes in different subfamilies, and the number on the column represents the number of F-box genes.(B) represents the proportion of F-box genes predicted by different cell structures.(C) represents the KEGG enrichment pathway analysis of F-box family genes.

Figure 7 .
Figure 7. Predicted protein-protein interaction network for the F-box gene in L. chinense.Different line colors represent different types of protein-protein interactions.Colored nodes: query proteins and the first shell of interactors; filled nodes: some 3D structure is known or predicted.

Figure 7 .
Figure 7. Predicted protein-protein interaction network for the F-box gene in L. chinense.Different line colors represent different types of protein-protein interactions.Colored nodes: query proteins and the first shell of interactors; filled nodes: some 3D structure is known or predicted.

Figure 8 .
Figure 8. Expression patterns of L. chinense F-box family genes in different tissues were analyzed by qRT-PCR.

Figure 8 .
Figure 8. Expression patterns of L. chinense F-box family genes in different tissues were analyzed by qRT-PCR.

Figure 9 .
Figure 9. Expression patterns of L. chinense F-box family genes during somatic embryogenesis of liriodendron hybrids were analyzed by qRT-PCR.

Figure 9 .
Figure 9. Expression patterns of L. chinense F-box family genes during somatic embryogenesis of liriodendron hybrids were analyzed by qRT-PCR.
of F-box genes in these stress responses indicates that they may be regulated by certain factors in the process of stress response, thus responding to these abiotic stresses.

Figure 10 .
Figure 10.The expression pattern of the F-box gene under drought stress was analyzed by qRT-PCR.

Figure 10 .
Figure 10.The expression pattern of the F-box gene under drought stress was analyzed by qRT-PCR.

Figure 11 .Figure 11 .
Figure 11.The number of F-box genes in different species-the number after the Latin name species-indicates the number of F-box genes in that species.

Table 1 .
The 12 F-box genes involved in biological processes (BPs) based on the known functions of orthologous Arabidopsis genes.