Analysis of the UDP-Glucosyltransferase (UGT) Gene Family and Its Functional Involvement in Drought and Salt Stress Tolerance in Phoebe bournei

Uridine diphosphate glycosyltransferases (UDP-GTs, UGTs), which are regulated by UGT genes, play a crucial role in glycosylation. In vivo, the activity of UGT genes can affect the availability of metabolites and the rate at which they can be eliminated from the body. UGT genes can exert their regulatory effects through mechanisms such as post-transcriptional modification, substrate subtype specificity, and drug interactions. Phoebe bournei is an economically significant tree species that is endemic to southern China. Despite extensive studies on the UGT gene family in various species, a comprehensive investigation of the UGT family in P. bournei has not been reported. Therefore, we conducted a systematic analysis to identify 156 UGT genes within the entire P. bournei genome, all of which contained the PSPG box. The PbUGT family consists of 14 subfamilies, consistent with Arabidopsis thaliana. We observed varying expression levels of PbUGT genes across different tissues in P. bournei, with the following average expression hierarchy: leaf > stem xylem > stem bark > root xylem > root bark. Covariance analysis revealed stronger covariance between P. bournei and closely related species. In addition, we stressed the seedlings with 10% NaCl and 10% PEG-6000. The PbUGT genes exhibited differential expression under drought and salt stresses, with specific expression patterns observed under each stress condition. Our findings shed light on the transcriptional response of PbUGT factors to drought and salt stresses, thereby establishing a foundation for future investigations into the role of PbUGT transcription factors.


Introduction
Glycosylation, a widespread compound modification pathway in plant secondary metabolism, is a crucial transformation process in organisms.It serves as the final stage for many biosynthetic products, altering the activity, stability, and solubility of receptors [1].Glycosylation plays a vital role in plant hormone balance, detoxification processes, and secondary metabolite production.Glycosyltransferases (GTs) are responsible for plant glycosylation and constitute a highly diverse multi-gene family present in various organisms.These enzymes catalyze the specific transfer of glycosyl groups from donors to receptors, forming αand β-glycosidic bonds and generating oligosaccharides, polysaccharides, complex sugars (such as glycoproteins and glycolipids), and glycoside compounds [2].The most prevalent glycosyl donor in plants is UDP-glucose, while receptors include glycolipids, proteins, and nucleic acids.The carbohydrate active enzyme database (CAZy) classifies glycosyltransferases (GTs) from different species into 114 gene families based on amino acid sequence similarity, substrate specificity, catalytic mechanism, signal membrane, and conformation of the glycosidic chain.The GT1 branch represents the largest division, indicating its close functional association with plants.Within the GT1 branch, there exists a subclass known as UDP-glucosyltransferases (UGTs) due to their utilization of UDPG as the glycosyl donor [3].One prominent characteristic of the plant UGT family is the presence of a highly conserved segment, known as the PSPG box, comprising 44 amino acids located at the protein's Cterminus [4].This conserved motif facilitates the recognition of and interaction with uridine diphosphate glycosyl donors.Conversely, the N-terminal sequence, which is considerably variable and less conserved, governs the recognition and binding of various receptors, contributing to the vast substrate diversity and complex structures of plant UGT genes, such as their involvement in the glycosylation of diverse compounds including hormones, terpenoids, and sterols [5].
The UGT gene family has been extensively identified and classified in various plants, including Arabidopsis thaliana [6], Linum usitatissimum [7], Triticum aestivum [8], Zea mays [9], Gossypium hirsutum [10], and Prunus persica [11].Arabidopsis thaliana, a model plant, possesses 122 UGT genes, among which 8 are pseudogenes.These genes are categorized into 14 distinct groups (A-N) based on their amino acid sequences [6].Subsequent studies have revealed the presence of O and P groups in higher plants [12].Oryza sativa has a total of 609 UGT genes, divided into 40 groups along with an unknown class [13].Citrus grandis exhibits 145 UGT genes distributed among 16 groups [14], while Zea mays contains 147 UGT genes classified into 17 groups, with 18 genes shared with Arabidopsis thaliana and 2 genes shared with Oryza sativa [9].The functional roles of the UGT gene family have been validated in various plants, including hormone balance regulation, localization control, secondary metabolite synthesis, detoxification, and defense responses [15].
Phoebe bournei, a species of the Lauraceae family, is characterized by a straight trunk and limited branches.It serves as an excellent timber species and is endemic to China [16].Noteworthy for its unique fragrance, P. bournei holds significant value as a timber tree.Its wood exhibits strong resistance to corrosion, making it resistant to cracking and bending during the drying process.Consequently, it possesses high economic worth and finds applications in shipbuilding and furniture production [17].However, due to excessive logging driven by its economic value and the adverse effects of environmental factors such as soil fertility degradation, P. bournei has experienced a decline in its wild population and is now listed as a secondary nationally protected plant.
While several gene families in other plants have been extensively studied, limited research has been conducted on the gene family of P. bournei.This dearth of studies can be attributed to the relatively recent sequencing of the P. bournei genome.The complete genome data of P. bournei was initially obtained by Liu Zhongjian and Chen Shiping from Fujian Agriculture and Forestry University in 2020 [18].However, this study did not unveil the chromosomal-level information.Subsequently, in 2022, the genome of P. bournei was deciphered by the Tong Zaikang team from Zhejiang Agriculture and Forestry University, shedding light on the synthesis mechanism of P. bournei sesquiterpenes [19].
The gene families in P. bournei primarily include commonly found families like WRKY [20], NF-Y [21], and PLR [22], while the UGT gene family has received limited attention.Previous studies have primarily focused on whole-gene family analyses, with few investigations on stress responses.Furthermore, existing research primarily addresses nutrient deficiency stress, neglecting drought and salt stresses.Therefore, our study aims to elucidate the response mechanisms of PbUGT genes under drought and salt stress conditions.
Previously, we studied transcriptional analysis of the GATA gene family under abiotic stress in Phoebe bournei [23].We know that under drought stress, the photosynthesis of P. fortunei is inhibited, the net photosynthetic rate, transpiration rate, and stomatal conductance of its leaves decrease, while the activities of oxidoreductase (POD and SOD) increase, and the ABA content in its roots increases.In terms of morphological characteristics, the Plants 2024, 13, 722 3 of 19 root color of the seedlings becomes darker with longer drought treatment [24].Moreover, in our previous experiments, we found that excessive stress time and excessive solution concentration may lead to the death of the seedlings.Therefore, we will use the method used in these previous experiments to stress Phoebe bournei, using 10% NaCl to apply salt stress to annual seedlings and 10% PEG-6000 to apply drought stress.Finally, qRT-PCR was used to detect the expression profile of the genes under drought and salt stress.This study provides a new idea for studying the evolution of UGT genes and helps to provide a better understanding of the abiotic stress response of Phoebe bournei.

Identification and Physicochemical Analysis of PbUGT Gene Members
Through rigorous verification using various methods, a comprehensive set of 156 PbUGT genes was successfully identified within the entirety of the genome of P. bournei.These UGT genes were subsequently designated PbUGT1-156 based on their chromosomal distribution and are presented in Table 1.To gain further insights into the physicochemical properties of the UGT proteins, ProtParam, MEME, and other online tools were employed for analysis.The results revealed that the amino acid count of the 156 PbUGT proteins ranged from 132 aa (PbUGT66) to 1019 aa (PbUGT89).The molecular weight of the proteins varied between 14948.04Da (PbUGT66) and 113398.82Da (PbUGT89).The isoelectric points spanned a range from 4.62 to 9.80.Specifically, there were 136 acidic proteins (PI < 6.5), 12 neutral proteins (6.5 < PI < 7.5), and 8 basic proteins (PI > 7.5).Subcellular localization analysis of these PbUGT proteins indicated that 88 proteins were localized within the chloroplast, 43 were in the cytoplasm, and 15 were in the nucleus, while the remaining proteins were distributed across various organelles such as vacuoles, peroxisomes, and mitochondria.Furthermore, instability coefficient analysis unveiled that 46 proteins were classified as stable, whereas the remaining 110 proteins exhibited instability.Notably, the maximum instability coefficient reached a significant value of 73.59, suggesting that the majority of the PbUGT proteins were characterized as unstable acidic proteins.

Chromosome Localization Analysis of the PbUGT Gene Family
To determine the precise chromosomal positions of the UGT genes, we utilized MapChart 2.32 software to construct a visual representation of their distribution.Our analysis revealed that the 156 UGT genes were dispersed across all 12 chromosomes.Notably, chromosome 10 harbored the highest number of PbUGTs, with a total of 32 genes, followed by chromosome 2, with 28 PbUGTs.Furthermore, the distribution pattern of UGT gene members within different subfamilies of P. bournei displayed irregularities, characterized by the presence of gene clusters.These clusters varied in size, ranging from 2 to 13 genes, with the largest cluster residing on chromosome 10.We further examined the chromosomal distribution within different gene groups.Chromosome 2 contained 28 genes belonging to eight distinct groups.Among these, group A exhibited the greatest number of members in the P. bournei UGT family, totaling 36 genes.Specifically, thirteen members from this group formed a cluster on chromosome 7, while four members clustered on chromosomes 2 and 4. The remaining six members were dispersed across chromosomes 1, 2, 5, and other regions encompassing chromosomes 8 to 12 (Figure 1).This uneven distribution of PbUGT genes across chromosomes indicates the presence of genetic variations during the evolution of P. bournei.
genes, with the largest cluster residing on chromosome 10.We further examined the chromosomal distribution within different gene groups.Chromosome 2 contained 28 genes belonging to eight distinct groups.Among these, group A exhibited the greatest number of members in the P. bournei UGT family, totaling 36 genes.Specifically, thirteen members from this group formed a cluster on chromosome 7, while four members clustered on chromosomes 2 and 4. The remaining six members were dispersed across chromosomes 1, 2, 5, and other regions encompassing chromosomes 8 to 12 (Figure 1).This uneven distribution of PbUGT genes across chromosomes indicates the presence of genetic variations during the evolution of P. bournei.

PbUGT Protein Conserved Motif Analysis
To gain further insights into the conserved domain characteristics of the UGT family in Phoebe bournei, we employed the online tool MEME to identify and assign 10 distinct motifs, designated as motifs 1 to 10 (Figure 2).The motif analysis revealed that the number of motifs present in each PbUGT family member ranged from three to thirteen.Notably, 114 out of the 156 members exhibited a combination of motifs 4 to 9.This unique motif

PbUGT Protein Conserved Motif Analysis
To gain further insights into the conserved domain characteristics of the UGT family in Phoebe bournei, we employed the online tool MEME to identify and assign 10 distinct motifs, designated as motifs 1 to 10 (Figure 2).The motif analysis revealed that the number of motifs present in each PbUGT family member ranged from three to thirteen.Notably, 114 out of the 156 members exhibited a combination of motifs 4 to 9.This unique motif combination serves as a distinctive feature of the family, setting it apart from other gene families and implying its close association with specific functional and performance sequences.Additionally, a notable observation was that members belonging to the same evolutionary group exhibited a high degree of similarity in the distribution of conserved motifs.This suggests that members classified within the same group are likely to share similar functions due to the similarity in motif distribution.Domain analysis unveiled the presence of four conserved domains, namely glycosyltransferase, GTB-type superfamily, GT1-Gtf-like, and PLN02448 domains, in all 156 UGT members.This observation suggests that the composition of these three conserved motifs is intricately linked to the synthesis and regulation of glycosyltransferases.
combination serves as a distinctive feature of the family, setting it apart from other gene families and implying its close association with specific functional and performance sequences.Additionally, a notable observation was that members belonging to the same evolutionary group exhibited a high degree of similarity in the distribution of conserved motifs.This suggests that members classified within the same group are likely to share similar functions due to the similarity in motif distribution.Domain analysis unveiled the presence of four conserved domains, namely glycosyltransferase, GTB-type superfamily, GT1-Gtf-like, and PLN02448 domains, in all 156 UGT members.This observation suggests that the composition of these three conserved motifs is intricately linked to the synthesis and regulation of glycosyltransferases.

Phylogenetic Analysis of the PbUGT Gene Family
Previous studies have classified UGT genes in Arabidopsis into 14 distinct groups, labeled A to N, with the identification of three additional groups, O, P, and Q, in higher plants.To elucidate the functional attributes of each PbUGT-family gene and determine

Phylogenetic Analysis of the PbUGT Gene Family
Previous studies have classified UGT genes in Arabidopsis into 14 distinct groups, labeled A to N, with the identification of three additional groups, O, P, and Q, in higher plants.To elucidate the functional attributes of each PbUGT-family gene and determine Plants 2024, 13, 722 7 of 19 its phylogenetic group, we obtained 122 sequences from A. thaliana via the Arabidopsis resource website.Subsequently, we constructed a phylogenetic tree by incorporating these sequences with the 156 identified P. bournei sequences.The phylogenetic tree revealed the presence of 14 A. thaliana groups within P. bournei, with an uneven distribution across these groups (Figure 3).Notably, the majority of PbUGT genes were found in groups A, G, E, and L, with the largest representation in group A, consisting of 36 members.Conversely, groups K, M, and N each contained only one member.The differences observed among various subfamilies may be linked to the evolutionary trajectory and functional diversity within P. bournei.
Plants 2024, 13, x FOR PEER REVIEW 7 of 19 its phylogenetic group, we obtained 122 sequences from A. thaliana via the Arabidopsis resource website.Subsequently, we constructed a phylogenetic tree by incorporating these sequences with the 156 identified P. bournei sequences.The phylogenetic tree revealed the presence of 14 A. thaliana groups within P. bournei, with an uneven distribution across these groups (Figure 3).Notably, the majority of PbUGT genes were found in groups A, G, E, and L, with the largest representation in group A, consisting of 36 members.Conversely, groups K, M, and N each contained only one member.The differences observed among various subfamilies may be linked to the evolutionary trajectory and functional diversity within P. bournei.

Organ and Tissue Expression Profile Analysis of the PbUGT Gene Family
By leveraging the original transcriptome sequencing data obtained from five distinct tissues in P. bournei (PRJNA628065), we constructed an expression heatmap to examine the tissue-specific expression patterns of the 156 PbUGT genes (Figure 4).The heatmap displayed expression levels ranging from low (blue) to high (dark red), highlighting considerable variations in the expression patterns across different tissues.Among the genes analyzed, PbUGT1, PbUGT21, PbUGT26, PbUGT27, PbUGT35, PbUGT55, PbUGT84, PbUGT104, PbUGT117, PbUGT121, PbUGT145, and PbUGT154 lacked corresponding expression data in the reported transcriptome, suggesting their limited involvement in the growth of P. bournei.In contrast, PbUGT101 and PbUGT136 exhibited significantly higher expression levels (FPKM > 15) in all tissues, indicating their crucial roles in the growth and development of P. bournei.On average, the expression levels ranked as follows: leaf > stem xylem > stem epidermis > root xylem > root epidermis.Among the fourteen subfamilies, the B subfamily displayed the highest average expression level, surpassing other subfamilies by a substantial margin.Conversely, the C, D, and K subfamilies exhibited notably low overall expression levels (FPKM < 1).The differential expression of PbUGT genes across various tissues signifies functional disparities among genes within distinct subfamilies.

Organ and Tissue Expression Profile Analysis of the PbUGT Gene Family
By leveraging the original transcriptome sequencing data obtained from five distinct tissues in P. bournei (PRJNA628065), we constructed an expression heatmap to examine the tissue-specific expression patterns of the 156 PbUGT genes (Figure 4).The heatmap displayed expression levels ranging from low (blue) to high (dark red), highlighting considerable variations in the expression patterns across different tissues.Among the genes analyzed, PbUGT1, PbUGT21, PbUGT26, PbUGT27, PbUGT35, PbUGT55, PbUGT84, PbUGT104, PbUGT117, PbUGT121, PbUGT145, and PbUGT154 lacked corresponding expression data in the reported transcriptome, suggesting their limited involvement in the growth of P. bournei.In contrast, PbUGT101 and PbUGT136 exhibited significantly higher expression levels (FPKM > 15) in all tissues, indicating their crucial roles in the growth and development of P. bournei.On average, the expression levels ranked as follows: leaf > stem xylem > stem epidermis > root xylem > root epidermis.Among the fourteen subfamilies, the B subfamily displayed the highest average expression level, surpassing other subfamilies by a substantial margin.Conversely, the C, D, and K subfamilies exhibited notably low overall expression levels (FPKM < 1).The differential expression of PbUGT genes across various tissues signifies functional disparities among genes within distinct subfamilies.

Synteny Analysis of Homologous UGT Genes among Phoebe bournei, Arabidopsis thaliana, Populus trichocarpa, and Cinnamomum camphora
To investigate the collinearity relationships within the UGT gene family among Phoebe bournei, Arabidopsis thaliana, Populus trichocarpa, and Cinnamomum camphora, a comprehensive genome-wide collinearity analysis was conducted.The aim was to unveil the interconnections within the UGT family.As depicted in Figure 5, a total of 13 PbUGT genes on 8 chromosomes displayed collinearity with UGT genes from A. thaliana, while 39 UGT genes on 10 chromosomes exhibited collinearity with UGT genes from P. trichocarpa.Additionally, 60 UGT genes on all 12 chromosomes displayed collinearity with UGT genes from Cinnamomum camphora.The collinearity analysis revealed that species with closer

Synteny Analysis of Homologous UGT Genes among Phoebe bournei, Arabidopsis thaliana, Populus trichocarpa, and Cinnamomum camphora
To investigate the collinearity relationships within the UGT gene family among Phoebe bournei, Arabidopsis thaliana, Populus trichocarpa, and Cinnamomum camphora, a comprehensive genome-wide collinearity analysis was conducted.The aim was to unveil the interconnections within the UGT family.As depicted in Figure 5, a total of 13 PbUGT genes on 8 chromosomes displayed collinearity with UGT genes from A. thaliana, while 39 UGT genes on 10 chromosomes exhibited collinearity with UGT genes from P. trichocarpa.Additionally, 60 UGT genes on all 12 chromosomes displayed collinearity with UGT genes from Cinnamomum camphora.The collinearity analysis revealed that species with closer genetic relationships exhibited stronger collinearity within the same gene family.This observation suggests a plausible association with species evolution, where these genes might have arisen earlier, with each gene family demonstrating homology prior to species differentiation.Over time, as the species diverged, these homologous gene relationships gradually diminished.
Plants 2024, 13, x FOR PEER REVIEW 9 of 19 genetic relationships exhibited stronger collinearity within the same gene family.This observation suggests a plausible association with species evolution, where these genes might have arisen earlier, with each gene family demonstrating homology prior to species differentiation.Over time, as the species diverged, these homologous gene relationships gradually diminished.

Duplication Analysis of PbUGT Genes in Chromosomal Replication
To elucidate the expansion and evolutionary mechanisms underlying the PbUGT gene family, potential gene duplication events in the P. bournei genome were investigated.The entire P. bournei genome was compared using MCScanX software, leveraging amino acid sequence homology.The analysis revealed the presence of 14 tandem repeat gene clusters within the UGT gene family members (Figure 6), indicating that tandem repeats played a pivotal role in the gene family's expansion.Additionally, this study examined the ratio of nonsynonymous substitutions (Ka) to synonymous substitutions (Ks) between the duplicated genes.The results demonstrated that the ratio was significantly below one, implying a negative selection effect on the PbUGT gene during the evolutionary process, indicative of purifying selection.

Duplication Analysis of PbUGT Genes in Chromosomal Replication
To elucidate the expansion and evolutionary mechanisms underlying the PbUGT gene family, potential gene duplication events in the P. bournei genome were investigated.The entire P. bournei genome was compared using MCScanX software, leveraging amino acid sequence homology.The analysis revealed the presence of 14 tandem repeat gene clusters within the UGT gene family members (Figure 6), indicating that tandem repeats played a pivotal role in the gene family's expansion.Additionally, this study examined the ratio of nonsynonymous substitutions (Ka) to synonymous substitutions (Ks) between the duplicated genes.The results demonstrated that the ratio was significantly below one, implying a negative selection effect on the PbUGT gene during the evolutionary process, indicative of purifying selection.

Enrichment Analysis of Stress-, Hormone-, and Light-Responsive Cis-Elements in the Promoter Region of Phoebe bournei UGT Genes
Cis-acting elements serve as indicators of gene function and predictors of transcriptional regulation patterns.The promoter regions of PbUGT genes encompass numerous cis-acting elements with distinct functions, which can be categorized into the stress response, hormone response, and light response.In total, 7, 35, and 11 cis-elements associated with stress responsiveness, hormone responsiveness, and light responsiveness, respectively, were identified across all PbUGT promoter sequences (Figure 7).Among them, the G-box (a light-response cis-element) exhibited the highest frequency, occurring 366 times in the promoter sequences of 131 genes.It was followed by ABRE (a hormone-response cis-element), which appeared 326 times in the promoter sequences of 124 genes.Previous studies have indicated that cis-element responses in various plants are involved in diverse stresses and hormone regulation.Hence, PbUGT genes may play a role in stress, hormone, and light regulation during the growth and development of P. bournei.This finding provides valuable insights for subsequent stress treatments in this experimental analysis.

Enrichment Analysis of Stress-, Hormone-, and Light-Responsive Cis-Elements in the Promoter Region of Phoebe bournei UGT Genes
Cis-acting elements serve as indicators of gene function and predictors of transcriptional regulation patterns.The promoter regions of PbUGT genes encompass numerous cis-acting elements with distinct functions, which can be categorized into the stress response, hormone response, and light response.In total, 7, 35, and 11 cis-elements associated with stress responsiveness, hormone responsiveness, and light responsiveness, respectively, were identified across all PbUGT promoter sequences (Figure 7).Among them, the G-box (a light-response cis-element) exhibited the highest frequency, occurring 366 times in the promoter sequences of 131 genes.It was followed by ABRE (a hormone-response cis-element), which appeared 326 times in the promoter sequences of 124 genes.Previous studies have indicated that cis-element responses in various plants are involved in diverse stresses and hormone regulation.Hence, PbUGT genes may play a role in stress, hormone, and light regulation during the growth and development of P. bournei.This finding provides valuable insights for subsequent stress treatments in this experimental analysis.

Screening and Verification of Stress Resistance Genes in PbUGT Genes
Based on the cis-acting elements, five genes-PbUGT14, PbUGT15, PbUGT56, PbUGT60, and PbUGT154-displaying the highest number of elements corresponding to adversity were identified.Subsequently, their transient expression levels were verified in response to various adversity stresses using RT-qPCR.The expression level analysis of PbUGT genes under 10% NaCl salt stress revealed that PbUGT14, PbUGT15, PbUGT56, PbUGT60, and PbUGT154 were all affected by salt stress (Figure 8A).Generally, their expression levels exhibited a "rise-fall-rise" trend over time.PbUGT14, PbUGT56, PbUGT60, and PbUGT154 reached their peak expression levels at 4 h, followed by an upward trend after a sudden decline.In contrast, the expression level of PbUGT15 continuously decreased with time.

Screening and Verification of Stress Resistance Genes in PbUGT Genes
Based on the cis-acting elements, five genes-PbUGT14, PbUGT15, PbUGT56, PbUGT60, and PbUGT154-displaying the highest number of elements corresponding to adversity were identified.Subsequently, their transient expression levels were verified in response to various adversity stresses using RT-qPCR.The expression level analysis of PbUGT genes under 10% NaCl salt stress revealed that PbUGT14, PbUGT15, PbUGT56, PbUGT60, and PbUGT154 were all affected by salt stress (Figure 8A).Generally, their expression levels exhibited a "rise-fall-rise" trend over time.PbUGT14, PbUGT56, PbUGT60, and PbUGT154 reached their peak expression levels at 4 h, followed by an upward trend after a sudden decline.In contrast, the expression level of PbUGT15 continuously decreased with time.
a sharp decrease and subsequent upward trend.PbUGT15, on the other hand, peaked at 4 h, exhibiting an overall "rise-decline-rise" pattern.The expression levels of the five genes demonstrated significant variations under 10% NaCl salt stress and 10% PEG-6000 drought stress, displaying both up-regulation and down-regulation.This indicates that these members of the gene family play crucial roles in the response of P. bournei to drought and salt stresses.

Discussion
In the present investigation, a total of 156 PbUGT genes were identified in the genome of Phoebe bournei.The number of PbUGT genes in P. bournei exceeded that of Cucumis sativus L. (94), Dendrobium officinale (44), and pomegranate (145), but was lower than Arachis hypogaea L. (267) and Glycine max (182).In comparison to other gene families, the UGT gene family demonstrated a higher abundance across all plant species.The initial stage of Under 10% PEG-6000 drought stress (Figure 8B), except for PbUGT15, PbUGT14, PbUGT56, PbUGT60, and PbUGT154 displayed peak expression levels at 6 h, followed by a sharp decrease and subsequent upward trend.PbUGT15, on the other hand, peaked at 4 h, exhibiting an overall "rise-decline-rise" pattern.The expression levels of the five genes demonstrated significant variations under 10% NaCl salt stress and 10% PEG-6000 drought stress, displaying both up-regulation and down-regulation.This indicates that these members of the gene family play crucial roles in the response of P. bournei to drought and salt stresses.

Discussion
In the present investigation, a total of 156 PbUGT genes were identified in the genome of Phoebe bournei.The number of PbUGT genes in P. bournei exceeded that of Cucumis sativus L. (94), Dendrobium officinale (44), and pomegranate (145), but was lower than Arachis hypogaea L. (267) and Glycine max (182).In comparison to other gene families, the UGT gene family demonstrated a higher abundance across all plant species.The initial stage of species differentiation showed a certain covariance, and the analysis of species covariance showed that UGT genes may have differentiated earlier in the evolutionary process, showing higher homology and closer linkage relationship with related plants.As species continue to differentiate, covariance gradually decreases.
Analysis of the physicochemical properties of P. bournei identified that 145 genes (93%) had an average molecular mass greater than 30 KDa, with an average of 50 KDa, and contained more than 100 amino acids.Regarding the proteins, 136 (87%) were found to be acidic, and 130 (83%) were hydrophilic.The instability coefficients of 110 proteins exceeded 40, indicating that most of the proteins regulated by PbUGT genes were both unstable and acidic.Subcellular localization analysis revealed an uneven distribution of PbUGT genes across organelles, with the majority located in the mitochondria (88), followed by 43 in the cytoplasm, and 15 in the nucleus, with the remaining in organelles such as vesicles and peroxisomes.The abundance of PbUGT genes in mitochondria may be related to the substantial energy requirements of glycosyltransferase, a process regulated by UGT.This finding provides a significant foundation and insight for further UGT gene research.
During natural growth, forest trees frequently encounter various abiotic stresses, including drought, low temperature, and salinity stresses, which impede their growth.These stresses can cause damage to plants, necessitating optimal water and fertilizer conditions for forest trees to achieve expected growth rates.However, this also results in greater nutrient consumption and longer recovery periods for the forest land.Genes play a vital role in enhancing the tolerance of forest trees to abiotic stresses [25].Forest trees respond to stress signals by modulating the expression of various genes, which, in turn, regulate metabolic responses and control tree growth under stressful conditions.
The current experiment, in conjunction with previous studies and cis-element analysis, has demonstrated that UGT genes across species respond to diverse abiotic stresses.For instance, maize mutants lacking the ZmUGT2 gene failed to complete flavonol glycosylation in vivo, resulting in inhibited germination rates and suppressed stem and root growth under abiotic stress conditions, particularly salt and drought stresses [26,27].In tea trees, abiotic stress suppressed the gene regulation of CsUGT91Q2 responsible for nerolidol glycol production, leading to impaired reactive oxygen species scavenging and reduced cold resistance, thereby increasing sensitivity to cold stress [28,29].
As transcription factors, UGT genes can activate the expression of stress-related genes, enhancing plant stress resistance and participating in the regulatory network governing adversity stress in forest trees.Phylogenetic analysis, transcriptome data, and RT-qPCR data confirmed that the five PbUGT genes can be rapidly induced and activated in response to abiotic stresses such as drought and salt stress, playing a significant role in the plant's response to these stresses.
Furthermore, transcriptome data analysis of P. bournei seedlings subjected to drought and salt stress revealed the involvement of all five UGT genes in abiotic stress responses.Their expression varied significantly with the duration of stress.The majority of genes exhibited up-regulated expression under drought and salt stress, displaying a "risingdeclining-rising" expression pattern under both 10% NaCl salt stress and 10% PEG-6000 drought stress.Particularly, under the 10% NaCl salt stress, all five UGT genes exhibited a high expression pattern after only 4 h.These findings suggest that PbUGT genes play a positive regulatory role in the abiotic stress response.This provides new insights for P. bournei species selection and stress regulation studies, aiming to expand the planting area of P. bournei and maximize its growth potential under stressful conditions.
Based on the whole-genome data and annotation files of P. bournei, specific location information for UGT genes on each chromosome was obtained.Subsequently, Mapchart 2.32 software [32] was employed to map the location information of each PbUGT gene onto the 12 P. bournei chromosomes.The ExPASy online (https://web.expasy.org/protparam(accessed on 15 July 2023)) [33] was utilized to predict and calculate the molecular weight, number of amino acids, theoretical PI (isoelectric point), number of positive (negative) residues, instability coefficient, lipolysis coefficient, and total average hydrophilicity of each UGT protein in P. bournei.The subcellular localization of each member of the P. bournei UGT gene family was predicted using the WoLFPSORT website (https://wolfpsort.hgc.jp(accessed on 17 July 2023)) [34].

Gene Structure, Protein Conserved Motifs, and Exon-Intron Analysis
The annotation file of the P. bournei genome and the amino acid sequences of the 156 UGT genes were used to construct a phylogenetic evolutionary tree for PbUGT genes using the One Step Build an ML Tree function in TBtools v1.120 [35].The MEME 5.5.2 online software (http://meme-suite.org/tools/meme(accessed on 19 July 2023)) [36] was employed to analyze the conserved motifs of PbUGT genes with a motif set of 10.The GSDS2.0 online tool (http://gsds.cbi.pku.edu.cn/(accessed on 21 July 2023)) [37] was utilized to map the gene structure, including the UTRs, exons, and introns of each UGT gene.The Batch CD Search function of the NCBI website (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi(accessed on 23 July 2023)) was used to query the structural domains by submitting the gene sequences.The Gene Structure View function in TBtools [35] facilitated the visualization and mapping of phylogenetic relationships, conserved motif composition, structural domains, and exon-intron gene structures for each PbUGT gene.

Phylogenetic Analysis and Construction of the Phylogenetic Tree
Nucleotide and amino acid sequences of 122 Arabidopsis thaliana UGT genes were obtained from the A. thaliana glycosyltransferase family 1 information website (http: //www.p450.kvl.dk/UGT.shtml(accessed on 27 July 2023)) [38][39][40] as a reference.Multiprotein sequence alignment was performed using MUSCLE [41] on the combined sequences of 156 UGT genes from P. bournei.Phylogenetic trees of related proteins were constructed using the maximum likelihood method in the MEGA11 program with a bootstrap value set to 1000 and default parameters [42].The constructed phylogenetic trees were further organized and embellished using the iTol 6.8.2 online website (https://itol.embl.de/(accessed on 30 July 2023)) [43].

Analysis of Tissue Expression Profiles
The sequence number PRJNA628065 of P. bournei was searched on the European Bioinformatics Centre repository EBI (https://www.ebi.ac.uk (accessed on 1 October 2023)).Transcriptome expression data of P. bournei in five different tissues were downloaded [19].RNA-seq analysis was conducted on the data to investigate the differential expression of the 156 PbUGT genes.Tbtools v1.120 software was used to algebraically transform the FPKM values from the analyzed data into log2 values, which were then used to generate heat maps.

Covariance Analysis
To elucidate the covariance patterns among PbUGT family homologous genes in Arabidopsis thaliana, Populus hirsutus, and Cinnamomum camphora, a genome-wide covariance analysis was conducted.This analysis integrated the chromosome-level genome data of P. bournei with the genome-wide data of A. thaliana [44], P. hirsutus [45], and C. camphora [46].The Synteny Visualization function in Tbtools was employed to map the covariance across species.The whole-genome data of A. thaliana and P. trichocarpa from the Ensembl Plants database (https://plants.ensembl.org/index.html(accessed on 3 October 2023)) and C. camphora genome-wide data from the National Genomics Data Center (https://ngdc.cncb.ac.cn/ (accessed on 3 October 2023)) (login number: GWH-BGXC00000000) were utilized.

Chromosomes Location, Gene Duplication, and Synteny Analysis
Circos [47] was used to identify each PbUGT gene on individual chromosomes.The results were then visualized using the Multiple Covariance Scanning Toolkit MCScan [48] with default settings.A comparison within the entire P. bournei genome was performed based on amino acid sequence homology.The duplication events in each PbUGT gene were analyzed, and the results were visualized using Circos and Dual Synteny Plot in Tbtools.

Enrichment Analysis of Stress-, Hormone-, and Light-Responsive Cis-Elements in the Promoter Region
The GTF/GFF3 Sequences Extract function of Tbtools was employed to obtain a 2000 bp segment upstream of the promoter region of each PbUGT gene based on the wholegenome sequencing sequence of Phoebe bournei.PlantCare (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/(accessed on 5 October 2023)) [49], an online software, was used for the prediction and screening of cis-acting elements associated with PbUGT genes.Subsequently, the data were categorized, organized, and analyzed, and the Heatmap function in Tbtools was utilized to classify and visualize the number of cis-acting elements for each stress-responsive PbUGT gene.

Material and Stress Treatments
One-year-old P. bournei seedlings from the Fujian Academy of Forestry were selected as the plant material and incubated in artificial climate chambers under different treatments.Before stress, the soil for planting the seedlings was prepared using peat soil, humus soil, sandy soil, perlite, etc., in a ratio of 5:2:2:1, and was cultivated in pots with a diameter of 150 mm.During stress treatment, the setting parameters of the artificial climate chambers were as follows: light cycle 12 h/d, temperature 25 • C, LED lights used for lighting, photosynthetically active radiation set to 1200 µmol•mol −1 •s −1 .The capacity of each pot was about 3.75 L. The seedlings with consistent growth potential were divided into two groups: the control group and the stress treatment group.The treatment group comprised thirty plants, while the control group consisted of three plants.For each biological replicate, two plants were included in the treatment group.Three sets of biological replicates were established for each time point.During stress treatment, we removed the P. bournei seedlings from the basin and cleaned their roots with pure water.The PEG treatment was transplanted into a beaker containing 10% PEG 6000 of ¼-strength Hogland solution.The salt treatment was transplanted into a beaker containing 10% NaCl of ¼-strength Hogland solution.The control group was maintained under normal moisture conditions.All stress treatments were conducted for 4, 6, 8, and 12 h.After the designated treatment periods, leaf samples from the P. bournei seedlings were collected, immediately frozen in liquid nitrogen, and stored for subsequent RNA extraction.

Figure 2 .
Figure 2. Conserved motif of the PbUGT gene family.Different colors correspond to different types of motifs with the numbers 1-10.

Figure 2 .
Figure 2. Conserved motif of the PbUGT gene family.Different colors correspond to different types of motifs with the numbers 1-10.

Figure 3 .
Figure 3. Phylogenetic tree of two plants' UGT proteins.The different colored arcs represent the UGT protein subfamilies.The tree was built using 156 PbUGTs from Phoebe bournei, 122 AtUGTs from Arabidopsis thaliana.

Figure 3 .
Figure 3. Phylogenetic tree of two plants' UGT proteins.The different colored arcs represent the UGT protein subfamilies.The tree was built using 156 PbUGTs from Phoebe bournei, 122 AtUGTs from Arabidopsis thaliana.

Figure 4 .
Figure 4. Expression heatmap of the PbUGT family in different organ and tissues.Differences in gene expression changes are shown by color, with warmer colors indicating higher expression.

Figure 4 .
Figure 4. Expression heatmap of the PbUGT family in different organ and tissues.Differences in gene expression changes are shown by color, with warmer colors indicating higher expression.

Figure 5 .
Figure 5. Synteny analysis of UGT genes among Phoebe bournei, Arabidopsis thaliana, Populus trichocarpa, and Cinnamomum camphora.The two rings in the middle represent the gene density of each chromosome, the gray lines represent the collinearity, and the red lines represent the highlighted collinearity of UGT genes.

Figure 5 .
Figure 5. Synteny analysis of UGT genes among Phoebe bournei, Arabidopsis thaliana, Populus trichocarpa, and Cinnamomum camphora.The two rings in the middle represent the gene density of each chromosome, the gray lines represent the collinearity, and the red lines represent the highlighted collinearity of UGT genes.

Plants 2024 , 19 Figure 6 .
Figure 6.Distribution and collinearity analysis of PbUGT genes.The two rings in the middle represent the gene density of each chromosome, the gray lines represent the collinearity, and the red lines represent the highlighted collinearity of UGT genes.

Figure 6 .
Figure 6.Distribution and collinearity analysis of PbUGT genes.The two rings in the middle represent the gene density of each chromosome, the gray lines represent the collinearity, and the red lines represent the highlighted collinearity of UGT genes.

Figure 7 .
Figure 7. Enrichment analysis of stress-, hormone-, and light-responsive cis-elements in the promoter regions of the PbUGT gene family.Differences in the number of cis-elements are indicated by color, with darker colors indicating more cis-elements.

Figure 7 .
Figure 7. Enrichment analysis of stress-, hormone-, and light-responsive cis-elements in the promoter regions of the PbUGT gene family.Differences in the number of cis-elements are indicated by color, with darker colors indicating more cis-elements.

Table 1 .
Physical and chemical properties of PbUGT family members.