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Article

Genome-Wide Identification of UGT Genes and Analysis of Their Expression Profiles During Fruit Development in Walnut (Juglans regia L.)

by
Danhua Shi
1,
Jinyu Yang
1,
Gengyang Li
1,
Yuanting Zhou
1,
Pei Yao
1,
Yanyu Shi
1,2,
Jieyun Tian
1,2,
Xiaojun Zhang
1,2,* and
Qunlong Liu
1,2,*
1
College of Horticulture, Shanxi Agricultural University, Jinzhong 030801, China
2
Shanxi Provincial Key Laboratory of Fruit Tree Germplasm Creation and Utilization, Taiyuan 030031, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(11), 1130; https://doi.org/10.3390/horticulturae10111130
Submission received: 4 October 2024 / Revised: 19 October 2024 / Accepted: 21 October 2024 / Published: 23 October 2024
Browse Figures
Versions Notes

Abstract

:
Walnut (Juglans regia L.) possesses the ability to prevent coronary heart disease and promote cardiovascular health. This ability can be attributed to their rich content of polyphenols, particularly flavonoids. The biosynthesis of flavonoids is reliant on the catalytic activity of uridine diphosphate glycosyltransferase (UGT). However, the identification of UGTs in walnut has not been reported. In the current study, a total of 124 UGT genes containing the PSPG box were identified from the walnut genome. Based on phylogenetic analysis, the 124 UGTs could be classified into 16 distinct groups, which exhibited an uneven distribution across the 16 chromosomes. Subcellular localization prediction analysis revealed that approximately 78.23% of walnut UGT proteins were predominantly localized in the cytoplasmic compartment. Furthermore, motif annotation confirmed that motifs 1, 2, and 3 represented conserved structural features within UGT proteins, while interestingly, around 56.5% of walnut UGT members lacked introns. Through the analysis of promoter cis-regulatory elements, it was revealed that JrUGTs are involved in photoresponse, hormonal regulation, and other physiological responses. In conjunction with transcriptome analysis and quantitative expression, approximately 39% of UGT genes in walnut exhibited high expression levels during early fruit development. Correlation analysis between UGT genes’ expression and phenolic content in walnut indicated that JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 might be involved in phenolic biosynthesis in walnut. This comprehensive study provides an overview of the UGT genes in walnut, serving as a valuable reference and theoretical foundation for further investigations into the biological functions of JrUGTs in flavonoid biosynthesis.

1. Introduction

Walnut (Juglans regia L.), a member of the Juglans family, is extensively cultivated worldwide. Substantial evidence suggests that walnuts possess the capacity to prevent coronary heart disease and promote cardiovascular health owing to their abundant polyphenol content, particularly of flavonoids [1,2]. Flavonoids, as secondary metabolites in plants, represent a class of polyphenolic compounds that exhibit potent biological activity and are widely distributed throughout the plant kingdom. The biosynthesis of flavonoids relies on the catalytic function of the uridine diphosphate glycosyltransferase (UGT) family, especially Uridinediphosphate Glycosyltransferase [3]. Glycosylation, an important modification reaction in plants, plays a crucial role in growth and response to biotic and abiotic stresses, and it also enhances solubility, stability, transferability, and diversity of various plant secondary metabolites such as flavonoids [4,5,6,7].
Glycosylation modification is catalyzed by glycosyltransferases (GTs), which are highly diverse, multifunctional enzymes belonging to a polymorphic gene family [8]. According to the latest update on CAZy (http://www.cazy.org/GlycosylTransferases.html (accessed on 1 May 2023)), GTs can be classified into 115 distinct species based on amino acid sequence similarity, catalytic mechanism, and the presence of conserved sequence motifs [9]. Among these, the GT1 family, commonly referred to as uridine diphosphate glycosyltransferases (UGTs), represents the predominant group in plants and exerts significant influence on plant growth and development [10]. UGT catalyzes the transfer of glycogroups from glyconucleotide substrates to diverse receptor molecules while simultaneously engaging in the binding of hydroxyl groups along the growing chain of polysaccharides, thereby actively participating in polysaccharide synthesis. [11]. The UGT protein exhibits a highly conserved sequence of 44 amino acids (aa) near the C-terminal region and is referred to as the plant secondary product glycosyl transferase (PSPG) [12]. Moreover, UGT demonstrates broad substrate specificity in plants by recognizing multiple receptor molecules selectively [13,14].
A growing number of putative UGT-coding genes have been discovered in plants, with 107 members identified in Arabidopsis thaliana, which were categorized into 14 groups based on their amino acid sequences and designated as groups A-N [15]. The results showed that there were 147 UGT genes in Zea mays, 181 in Vitis vinifera, 241 in Malus × domestica, 145 in Citrus grandis, and 178 in Camellia sinensis [16,17,18,19]. Polygenic UGT family members collaborate synergistically to orchestrate intricate biochemical processes in plant cells, thereby exerting a profound influence on diverse biological activities and functions. Currently, the functionality of UGT genes has been validated in multiple species [20,21,22,23]. Yang et al. [24] demonstrated the functions of 276 glycosyltransferases in Nicotiana tabacum and observed a significant increase in the levels of quercetin-3-O-glucoside, quercetin-3-O-rutin, and kaempferol-3-O-rutin in transgenic tobacco leaves by overexpressing NtUGT217. In a study by Lim et al. [25], 91 UGT genes were identified in Arabidopsis thaliana using quercetin as a substrate, with 29 of them exhibiting catalytic activity towards related glycosylation reactions. Yao et al. [26] conducted a comprehensive genome-wide analysis of Epimedium pubescens, revealing that only the recombinant EpGT60 protein exhibited activity against 8-enyl kaempferol and icariin, leading to the biosynthesis of key bioactive compounds pyridoside II and pyridoside I. However, despite the large number of UGT genes in the plant genome, the functional validation of signature proteins remains limited [11].
The present study employed whole genome sequencing to identify the UGT gene family in walnut (JrUGT). Subsequently, sequence alignment and phylogenetic tree analysis were conducted to elucidate the genetic relationships among these UGTs in walnut. Furthermore, predictions regarding the physical and chemical properties of proteins, subcellular localization, chromosome distribution, collinearity, and gene structure were made for JrUGT. Based on this premise, the expression patterns of UGT genes in various developmental stages of walnut kernel pellicle were analyzed and subsequently validated using qRT-PCR. The correlation analysis between JrUGT and phenolic content establishes a fundamental for future investigations into the biosynthesis mechanism and accumulation pattern of walnut phenolics.

2. Materials and Methods

2.1. Identification of UGT Gene Family in Walnut

The genome sequence information of walnut was primarily obtained from the Juglans genome database (http://www.juglandaceae.net/ (accessed on 1 June 2023)). Arabidopsis thaliana UGT members were predominantly obtained from the TAIR database (https://www.arabidopsis.org/ (accessed on 1 June 2023)). The hidden Markov model HMM file for the conserved domain of the UGT gene family (PF00201) was accessible through Pfam (http://pfam.xfam.org/ (accessed on 1 June 2023)). By employing the Arabidopsis thaliana UGT amino acid sequence as a query, BLASTP was utilized to search for proteins in the walnut genome with an e-value threshold set at 1× 10−15, thereby constructing UGT candidate dataset 1. The hmmsearch function in TBtools was employed to retrieve the UGT candidate dataset 2 by utilizing the HMM file of the UGT family as a probe. Subsequently, two sets of candidate data were combined and subjected to verification using the NCBI CDD program, confirming the presence of conserved domain PSPG box in all walnut UGT members.

2.2. Physical and Chemical Properties, as Well as Cis-Acting Element Analysis

The Expasy online database (http://www.expasy.org/ (accessed on 1 June 2023)) was utilized for the prediction of physicochemical properties of the amino acid sequence, encompassing relative molecular weight, theoretical isoelectric point, and amino acid length. Additionally, subcellular localization analysis of walnut UGT protein was performed using the PSORT II Prediction online software (https://psort.hgc.jp/form2.html (accessed on 1 June 2023)).
JrUGTs’ gene upstream 2000 bp sequence was obtained from the Juglans genome database (http://www.juglandaceae.net/ (accessed on 1 June 2023)), and the PlantCARE database was used to identify cis-regulatory elements (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 1 June 2023)).

2.3. Phylogenetic and Chromosomal Localization Analysis

The UGT protein sequences of walnut, Arabidopsis thaliana, Zea mays, and Pilosella officinarum were aligned with MUSCLE 11 software. Subsequently, phylogenetic trees based on 1000 bootstrap replicates using the neighbor-joining (NJ) method were constructed in MEGA 11 software [27]. The genomic location information of JrUGT members was visually analyzed by TBtools 7.0 software, and the results were derived from walnut genome database.

2.4. Gene Replication and Collinearity Analysis

The gene replication events were analyzed using the One Step MCScanX program and visualized with the TBtools 7.0 program [28,29]. Collinearity analysis of JrUGTs and AtUGTs was conducted using TBtools software, as reported by the walnut genome database [29].

2.5. Conserved Sequence and Gene Structure Analysis

The conserved motifs of walnut UGT members were identified using the MEME program (http://meme-suite.org/tools/meme (accessed on 1 June 2023)). The conserved motifs recognized by MEME programs were further annotated using the NCBI CDD program, with a total of 10 motifs identified. Additionally, the walnut gene annotation files and coding sequences from its corresponding genome sequence comparison were inputted into the NCBI GSDS online tools (v2.0) at http://gsds.cbi.pku.edu.cn/ (accessed on 1 June 2023) to display information on exons and introns of walnut UGT.

2.6. Expression Pattern Analysis of 13 JrUGTs

The experimental materials used in this study were “NongHe 1” (“NH1”) and “FenHe 4” (“FH4”). Three plants of the same age and growth stage were carefully selected for sample collection. The collection periods included the hard core stage, fatty stage, and mature stage. These samples were obtained from the walnut planting resource nursery of the horticultural experimental station of Shanxi Agricultural University. Subsequently, all samples were rapidly frozen in liquid nitrogen and stored at −80 °C in an ultra-low temperature refrigerator for future use. Three biological replicates were performed on each sample. The kernel pellicle of two walnut varieties, “NH1” and “FH4”, at various developmental stages was submitted to Baimaike Biotechnology Co., Ltd. (Beijing, China). for transcriptome sequencing analysis. Three independent biological replicates were conducted for each sample. Transcript abundance was quantified using fragments per kilobase transcript per million fragment mapping (FPKM) values derived from RNA-Seq reads. Each gene exhibited distinct expression levels. The tissue-level data were averaged and presented as log2 values. Then, a heat map was generated using TBtools 7.0 software [30].
The total RNA was extracted using the improved CTAB method [31], and first-strand cDNA synthesis was performed using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper)(Novizan Biotechnology Co., Ltd., Nanjing, China). Specific primers for 13 selected JrUGTs were designed using Primer Premier 5.0 software, and their sequences are provided in Table S1. The reference gene employed was JrActin, with “NH1-6” serving as the control group. The relative expression of JrUGTs was determined by qRT-PCR reaction system with the 2−ΔΔCT method [32].

2.7. Determination of Phenolics

The kernel pellicle was freeze-dried and ground into a fine powder. An amount of 0.1 g of the kernel pellicle powder was measured and added to 5 mL of a 50% methanol solution. Three biological replicates were performed for each sample. Ultrasound-assisted extraction was conducted for 30 min at 50 °C, followed by centrifugation at 13,400× g for 10 min to obtain the supernatant. This process was repeated twice, and the resulting supernatants were combined and adjusted to a constant volume before being analyzed using HPLC. The content of phenolic compounds was determined following the methods described by Lu et al. [33] and Yang et al. [34]. The regression equation for the standard curve is presented (Table S2).

2.8. Data Processing

Data sorting and mapping were performed using Microsoft Excel 2020 and Prism 9. Significance difference analysis and correlation analysis were conducted using SPSS 26.0 software, while heat mapping was generated using TBtools 7.0 software.

3. Results

3.1. Identification of UGT Gene Family Members in Walnut

In order to identify JrUGT and UGT proteins, the walnut genome database was screened using an HMM file (PF00201) as a query. The screening criterion was set at an e-value < 1. We employed two screening methods, namely BLASTP and hmmsearch, resulting in the identification of 174 potential UGT genes in walnut. To verify the presence of conserved domains, TBtools was employed, and any UGT proteins lacking a PSPG box were manually excluded. Ultimately, 124 JrUGT genes were obtained and designated as JrUGT1-124.
The 124 JrUGT genes encoded proteins with diverse physicochemical properties (Table S3). Each member of the JrUGT family had a protein length ranging from 216 to 611 aa, with an average length of 474 aa. The theoretical molecular weight of the protein ranges from 29.29 to 61.50 KDa, with an average value of 52.90 KDa. The theoretical isoelectric point (pI) ranges from 5.17 to 8.1, with an average pI of 5.85. According to the PSORT II Prediction online tool for subcellular localization prediction, a majority of the UGT proteins in walnut (78.23%) were localized in the cytoplasm while the second-largest amount were found in mitochondria (12.10%). Additionally, eleven UGT proteins were found in the endoplasmic reticulum and one protein was located extracellularly within the cell wall.

3.2. Phylogenetic and Chromosomal Localization Analysis

The classification and phylogenetic relationship of UGT proteins in walnut were investigated by constructing a phylogenetic tree based on UGT protein sequences from walnut, Arabidopsis thaliana, Zea mays, and Pilosella officinarum. The main clusters of walnut UGT family members consisted of 18 subgroups, with a total of 124 JrUGTs being classified into these previously identified subgroups (Figure 1). Notably, JrUGTs were absent from groups F and Q, while the majority of them clustered within groups E (25), G (16), D (19), L (15), and A (15). It has been reported that AtUGT71B1, AtUGT72B1, and AtUGT88A1 of Arabidopsis thaliana were involved in flavonoid biosynthesis. Sequence analysis found JrUGT4, JrUGT13, JrUGT31, JrUGT32, JrUGT43, JrUGT44, JrUGT45, JrUGT57, JrUGT58, JrUGT59 JrUGT60, JrUGT61, JrUGT62, JrUGT67, JrUGT68, JrUGT69, JrUGT80, JrUGT81, JrUGT93, JrUGT108, JrUGT111, JrUGT114, JrUGT117, JrUGT118, and JrUGT121 clustered with AtUGT71B1, AtUGT72B1, and AtUGT88A1, suggesting that these JrUGTs may be involved in flavonoid biosynthesis in walnut.
Among the 124 JrUGTs identified, 124 JrUGTs were located on 16 chromosomes of walnut (Figure 2). Among the 16 walnut chromosomes, chromosome 9 contained only one UGT family member, while chromosome 1 contained 17 UGT genes. Chromosome 7 contained 15 UGT genes, chromosome 3 contained 13 UGT genes, and chromosomes 2 and 15 each contained only 2 UGT genes. This unbalanced distribution of UGT genes in walnut chromosomes indicates that there was genetic variation in walnut during evolution.

3.3. Gene Replication and Collinearity Analysis

Gene replication events play a pivotal role in the formation of gene families. In order to elucidate the expansion and evolution mechanism of the UGT gene family in walnut, the potential gene replication events in walnut genome were further investigated. The detection of UGT gene replication events in walnut was performed using TBtools software. A total of five genes located on chromosomes 1, 2, 4, 9, 10, and 14 were identified to undergo four gene replication events (Figure 3). Notably, chromosomes 1 and 10 exhibited the highest frequency of tandem repeat events (three occurrences each), implying that UGT genes might have originated from such replication events; these events could be considered as key drivers for UGT evolution. Furthermore, by comparing the DNA sequence similarity between the UGT gene of walnut and the homologous genes of Arabidopsis thaliana (Figure 4), a collinear relationship between 39 walnut genes and 36 Arabidopsis thaliana genes was found. These conserved genes are likely to possess crucial functions across different species.

3.4. Conserved Sequence and Gene Structure Analysis

To further elucidate the conserved domain characteristics of the walnut UGT family, 10 motifs were generated using the online tool MEME and then numbered from 1 to 10. Notably, motif 1 and motif 3 corresponded to the highly conserved PSPG box within the UGT family. The distribution pattern of these motifs among different types of walnut UGT members was depicted (Figure 5). Remarkably, our findings revealed that members belonging to the same group exhibited either identical or similar conserved motifs. The characteristic sequence motif 1-3, present in all walnut UGT proteins, was considered to be the glycosyltransferase recognition site for the glycosyl donor. With a few exceptions, most walnut UGT proteins exhibited the following characteristics: motif 6 was located proximal to motif 2, and motif 5 was also positioned near motif 8. In the majority of sequences, motif 4 appeared at the beginning while motif 7 occurred at the end. The protein’s sequence typically follows this pattern: motif 4-5-8-6-2-10-1-3-9-7; however, variations existed among certain proteins.
The diversity in intron-exon structure often plays a pivotal role in the evolutionary dynamics of gene families and provides supplementary evidence to support phylogenetic classifications. In order to further understand the gene structure, the intron-exon structure of UGT gene in walnut was analyzed. Out of the 124 UGT genes identified in this study, 47 contained a single intron, 70 were devoid of any introns, and 7 harbored two introns.

3.5. Promoter Cis-Acting Element Analysis

In order to identify the cis-acting elements within the promoter region of the JrUGT genes, we obtained a 2000 bp fragment upstream of the JrUGT genes and subjected it to analysis using PlantCARE online software. JrUGT gene promoter mainly contains a light responsive element, MYB binding site involved in flavonoid biosynthetic genes regulation, cis-acting element involved in defense and stress responsiveness, cis-acting element involved in low-temperature responsiveness, MYB binding site involved in drought inducibility, cis-acting element involved in abscisic acid responsiveness, auxin-responsive element, gibberellin-responsive element, cis-acting regulatory element involved in the MeJA-responsiveness, cis-acting element involved in salicylic acid responsiveness, and a cis-acting regulatory element related to meristem expression (Figure 6).

3.6. Expression Patterns of UGT Genes

In this study, the transcriptomic sequencing results of the kernel pellicle of two walnut varieties, “NH1” and “FH4”, were analyzed at different developmental stages to gain further insights into the expression pattern of UGT genes in walnut. The findings revealed that out of 118 UGT genes analyzed in walnut, JrUGT2, JrUGT11, JrUGT68, JrUGT113, JrUGT119, and JrUGT121 were not detected in the transcriptome data. A total of 19 JrUGTs (FPKM > 10) exhibited high transcription levels in “NH1” and “FH4”, respectively (Figure S1). The expression profiles of JrUGT39, JrUGT58, JrUGT75, and JrUGT118 were consistently elevated across all three developmental stages in both varieties, suggesting their potential involvement in diverse biological processes throughout growth and development. Group E, which represents the largest subset of the UGT gene family in walnut, exhibited predominant expression of UGT during the hard core stage in “NH1” and “FH4”. Additionally, some genes showed higher expression levels in the mature stage compared to those observed in the hard core stage and the fatty stage. Similarly, Group G also displayed peak expression during the hard core stage, with greater abundance detected in “FH4” than “NH1”. Most UGT genes demonstrated elevated transcript levels during early fruit development stages, but these decreased as fruit matured.
In this study, we performed a comprehensive analysis of the walnut UGT gene family through phylogenetic tree analysis and transcriptome differential gene screening. Subsequently, 13 differentially expressed walnut UGT genes were randomly selected for validation using real-time fluorescence quantitative PCR. The qRT-PCR results are presented (Figure 7). The gene expression levels of JrUGT39, JrUGT58, JrUGT88, and JrUGT95 were found to be higher in August, while JrUGT118 exhibited the lowest gene expression levels during this period. These findings were consistent with the transcriptome data analysis.

3.7. Analysis of Phenolic Substances in Walnut Kernel Pellicle at Different Periods

HPLC was used to determine the content of “NH1” and “FH4” phenolics in walnut kernel pellicle at different periods. With the growth and development of walnut fruit, the content of phenolics also changed, and the results are shown (Table 1). The content of gallic acid was the highest among the 11 substances, and the content of GCG was the lowest. The content of C was the highest among catechins, and the content of C in “FH4” was significantly higher than that of “NH1”. The contents of EC and GC showed a trend of continuous increase on the whole, the contents of EGC and EGCG in the two varieties showed a trend of first increasing and then decreasing, and the content of EGC in the fatty stage of “NH1” was significantly higher than that in other periods. The content of “FH4” in syringate was higher than that of “NH1” in different periods.

3.8. Correlation Analysis of UGT Gene Expression and Phenolic Content in Walnut

The Pearson coefficient was employed to investigate the correlation between UGT gene expression and phenolic substance content in walnut (Figure 8). No significant correlation was observed between JrUGT36 and phenolic substances. Only JrUGT118 showed a positive correlation with EGCG and chlorogenic acid (p < 0.05). JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 exhibited positive correlations with vanillic acid (p < 0.01). Additionally, JrUGT38, JrUGT39, JrUGT67, JrUGT69, JrUGT75, and JrUGT82 were positively correlated with C, GC, and EC. These findings suggest that these identified UGTs may play a role in the biosynthesis of phenolic substances in walnut, thus providing valuable insights for further study on this topic.

4. Discussion

Glycosylation is modified by GTs, which can be divided into at least 111 families, of which the UGT gene family is the largest family [35]. Currently, the UGT gene family has been found and analyzed in plants such as Arabidopsis thaliana, Triticum aestivum, Zea mays, and Morella rubra [15,17,36,37]. However, there was a lack of information regarding the UGT gene family in walnut. Through BLASTP and hmmsearch screenings along with conserved domain verification, 124 JrUGTs were identified. Compared to other species, the number of UGT genes in walnut showed lower numbers: 152 UGT genes were identified in Morella rubra and 168 UGT genes in Prunus persica [38]. This observation suggests that the amplification of the walnut UGT gene family was not significant, possibly due to the absence of genome-wide duplication events in walnut. The protein encoded by the UGT gene of walnut exhibits a wide range of amino acid lengths and molecular weights. The intron content in walnut UGT family members was 43.5%, which was lower compared to Arabidopsis thaliana (58%) [15], Linum usitatissimum (55%) [39], and Zea mays (60%) [17]. The UGT gene family was initially employed for evolutionary studies in Arabidopsis thaliana and is classified into 14 distinct clades (A-N) [15,40]. Subsequently, its application expanded to Malus × domestica [16] and grape [41]. Notably, Group O and Group P have been identified in other plant species. In the evolutionary process of higher plants, groups A, L, D, G, and E of the UGT gene family were considered as rapidly evolving groups [11], and these five groups also contain the highest number of UGT gene family members in walnut, consistent with the aforementioned results. To functionally identify UGT genes in walnut, a phylogenetic tree was constructed to classify the 124 identified UGT genes into 16 groups. The phylogenetic tree showed that group E contained the most UGT genes (25), accounting for 20.2% of the total UGT genes in walnut. There were 17, 22, and 35 UGT genes in group E in Arabidopsis thaliana, Linum usitatissimum, and Zea mays, respectively, indicating the expansion of group E in different plant species [11,39]. JrUGTs were not distributed in group F and group Q. Group F was first identified in Arabidopsis Thaliana [15], and group Q was first identified in Zea mays [17]; this group of genes was thought to be related to cytokinin glycosylation.
JrUGTs were found to be distributed across 16 chromosomes. In Dendrobium nobile and Gossypium hirsutum, the genes exhibited clustering with high homology on specific chromosomes, a pattern also observed in the distribution of UGT gene family on walnut chromosomes [42,43]. Qiao et al. [44] argued that gene replication events include five modes, namely WGD, TD, DSD, PD, and TRD. Tandem repeat events play an important role in the expansion of gene families. In Broussonetia papyrifera, TD was the main driver of expansion of the BpUGT gene family [45]. In the UGT family of walnut, a total of five tandem replicators were obtained, and four tandem replication events occurred. Therefore, tandem repetition events continue to occur in UGT gene families, which is a continuous process throughout the evolutionary history of UGT gene families. By analyzing the conserved motifs of UGT proteins in walnut, 10 different motifs were found, and all UGT proteins contained motif 1-3. The UGT protein of walnut in group E did not contain motif 9. These specific motifs may lead to differentiation of UGTs’ function in walnut. The widespread presence of JrUGT promoter photoresponse elements suggests that light signals may have a general impact on the regulation of UGT genes, similar to previous studies conducted in Petunia hybrida and Gossypium hirsutum by Dong et al. [46] and Sun et al. [47]. Furthermore, it has been observed that abiotic stresses, such as low temperature and drought induction, can induce the expression of UGTs, indicating their potential role in stress response mechanisms. However, further investigation is required to determine the specific types of adversities involved and elucidate their underlying mechanisms.
Understanding the spatiotemporal expression patterns of genes is crucial for predicting their functional roles. In “NH1”, a total of 62 UGT genes exhibited significant expression (FPKM > 1) during kernel pellicle development, while “FH4” showed the expression of 52 UGT genes with FPKM > 1. Notably, both varieties displayed higher expression levels of JrUGT58 and JrUGT118 in group E throughout fruit development. Additionally, JrUGT75 demonstrated the highest level of expression within group G. Interestingly, “FH4” exhibited a tenfold increase in FPKM value for JrUGT67 compared to “NH1”; conversely, JrUGT69 displayed an opposite pattern of expression between these two varieties. These specific gene expressions may potentially impact the metabolite composition in walnut fruits. Correlation analysis between UGT gene expression and phenolics content in walnut showed that JrUGT36 had no significant correlation with phenolics. JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 were significant correlated with C, GC, EC, and vanillic acid. This discovery improved the basis for subsequent research on phenolic biosynthesis of walnut.

5. Conclusions

In this study, a total of 124 JrUGTs’ protein sequences containing PSPG boxes were identified in the walnut genome, and they were categorized into 16 functional groups. The UGT protein motifs and gene structures in walnut exhibited similarities within the same group, while notable differences were observed between different groups. Significantly distinct expression patterns of UGT gene family members during various developmental stages were detected in the kernel pellicle of “Nonghe 1” and “Fenhe 4”. Correlation analysis between UGT gene expression and phenolic content in walnut indicated that JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 might be involved in phenolic biosynthesis in walnut. The results provided a basis for further study on the biosynthesis of phenolic substances in walnut.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae10111130/s1, Figure S1: Transcriptome expression patterns of UGT family in walnut. Table S1: Primers sequence. Table S2: Regression equation of standard curve for phenolic substances. Table S3: Genetic physicochemical properties of JrUGTs.

Author Contributions

Software, D.S.; validation, D.S. and J.Y.; investigation, G.L. and Y.Z.; data curation, P.Y. and Y.S.; writing—original draft preparation, D.S.; writing—review and editing, J.T., X.Z. and Q.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Major Special Program of Science and Technology of Shanxi province (202201140601027); the Key Research and Development Project of Shanxi province (202302140601014); the Basic Research Program of Shanxi Province (No. 20210302123396); and the Key Research and Development Plan (Agriculture) of Jinzhong City (No. Y212013).

Data Availability Statement

The data presented in this study are available upon request from the corresponding author due to privacy concerns.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic trees of walnut, Arabidopsis thaliana, Zea mays, and Pilosella officinarum were constructed using UGT protein.
Figure 1. Phylogenetic trees of walnut, Arabidopsis thaliana, Zea mays, and Pilosella officinarum were constructed using UGT protein.
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Figure 2. Localization of the UGT gene family on chromosomes in walnut.
Figure 2. Localization of the UGT gene family on chromosomes in walnut.
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Figure 3. JrUGT replication analysis. The yellow rectangle illustrates chromosomes 1–16 of the walnut, while the line, heat map, and histogram within the rectangle represent gene density across each chromosome. The gray lines indicate all identified homogenous blocks in the walnut genome, whereas the red lines denote duplicate gene pairs between chromosomes.
Figure 3. JrUGT replication analysis. The yellow rectangle illustrates chromosomes 1–16 of the walnut, while the line, heat map, and histogram within the rectangle represent gene density across each chromosome. The gray lines indicate all identified homogenous blocks in the walnut genome, whereas the red lines denote duplicate gene pairs between chromosomes.
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Figure 4. Collinearity analysis of UGT genes between walnut and Arabidopsis thaliana. The gray lines illustrate the presence of syntenic blocks within the genomes of walnut and Arabidopsis thaliana, while the red lines indicate pairs of UGT genes.
Figure 4. Collinearity analysis of UGT genes between walnut and Arabidopsis thaliana. The gray lines illustrate the presence of syntenic blocks within the genomes of walnut and Arabidopsis thaliana, while the red lines indicate pairs of UGT genes.
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Figure 5. Conservation of sequence and gene structure analysis in the UGT gene family of walnut.
Figure 5. Conservation of sequence and gene structure analysis in the UGT gene family of walnut.
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Figure 6. Cis-acting element of the JrUGT genes promoter.
Figure 6. Cis-acting element of the JrUGT genes promoter.
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Figure 7. The expression patterns of 13 different JrUGTs. “NH1-6” indicates the hard core stage of “NongHe 1”; “NH1-7” indicates the fatty stage of “NongHe 1”; “NH1-8” indicates the mature stage of “NongHe 1”; “FH4-6” indicates the hard core stage of “FenHe 4”; “FH4-7” indicates the fatty stage of “FenHe 4”; “FH4-8” indicates the mature stage of “FenHe 4”.
Figure 7. The expression patterns of 13 different JrUGTs. “NH1-6” indicates the hard core stage of “NongHe 1”; “NH1-7” indicates the fatty stage of “NongHe 1”; “NH1-8” indicates the mature stage of “NongHe 1”; “FH4-6” indicates the hard core stage of “FenHe 4”; “FH4-7” indicates the fatty stage of “FenHe 4”; “FH4-8” indicates the mature stage of “FenHe 4”.
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Figure 8. Analysis of the correlation between UGT gene expression and phenolic content in walnut. * indicates significance at the 0.05 level, and ** indicates significance at the 0.01 level.
Figure 8. Analysis of the correlation between UGT gene expression and phenolic content in walnut. * indicates significance at the 0.05 level, and ** indicates significance at the 0.01 level.
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Table 1. Content analysis of phenolics in different stages (mg/100 g). Different lowercase letters in the same line indicate significant difference in kernel pellicle at 0.05 level in different periods.
Table 1. Content analysis of phenolics in different stages (mg/100 g). Different lowercase letters in the same line indicate significant difference in kernel pellicle at 0.05 level in different periods.
Developmental StageNH1-6NH1-7NH1-8FH4-6FH4-7FH4-8
Substance
Name
C866.71 ± 2.71 b822.45 ± 16.30 c746.36 ± 12.40 e733.57 ± 5.98 e785.20 ± 11.82 d1797.77 ± 13.64 a
GC168.22 ± 2.31 e203.57 ± 2.49 c355.09 ± 2.61 b144.95 ± 4.70 f185.72 ± 4.39 d591.43 ± 4.24 a
EC56.97 ± 4.37 e230.58 ± 2.91 c213.56 ± 2.17 d57.44 ± 2.91 e243.31 ± 6.38 b315.93 ± 2.13 a
EGCG209.25 ± 6.12 bc254.38 ± 28.44 a152.46 ± 10.50 d231.61 ± 1.67 ab232.77 ± 4.42 ab191.65 ± 2.40 c
EGC318.98 ± 7.78 d646.48 ± 13.98 a186.41 ± 12.01 f282.21 ± 15.76 e369.06 ± 7.51 b344.71 ± 4.98 c
GCG39.93 ± 4.05 b8.18 ± 0.40 e15.21 ± 0.30 cd13.07 ± 1.69 d17.52 ± 0.58 c45.02 ± 2.73 a
ECG359.08 ± 25.51 a296.73 ± 24.67 b177.76 ± 4.80 c151.43 ± 0.80 cd154.68 ± 7.26 cd145.33 ± 1.43 d
Gallic acid1390.92 ± 46.78 b1535.90 ± 11.41 a1223.70 ± 4.08 c1086.18 ± 11.21 d1243.39 ± 19.68 c1218.43 ± 15.23 c
Chlorogenic acid366.99 ± 9.65 c263.89 ± 7.50 d142.40 ± 4.66 e547.09 ± 32.01 a256.41 ± 6.06 d449.65 ± 11.78 b
vanillic acid185.66 ± 5.70 f333.59 ± 6.35 c321.29 ± 2.72 d230.55 ± 3.22 e451.43 ± 11.37 b645.98 ± 2.52 a
Syringate55.07 ± 3.76 e97.62 ± 7.02 c63.65 ± 3.50 e76.84 ± 6.60 d186.88 ± 9.21 a139.95 ± 1.75 b
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Shi, D.; Yang, J.; Li, G.; Zhou, Y.; Yao, P.; Shi, Y.; Tian, J.; Zhang, X.; Liu, Q. Genome-Wide Identification of UGT Genes and Analysis of Their Expression Profiles During Fruit Development in Walnut (Juglans regia L.). Horticulturae 2024, 10, 1130. https://doi.org/10.3390/horticulturae10111130

AMA Style

Shi D, Yang J, Li G, Zhou Y, Yao P, Shi Y, Tian J, Zhang X, Liu Q. Genome-Wide Identification of UGT Genes and Analysis of Their Expression Profiles During Fruit Development in Walnut (Juglans regia L.). Horticulturae. 2024; 10(11):1130. https://doi.org/10.3390/horticulturae10111130

Chicago/Turabian Style

Shi, Danhua, Jinyu Yang, Gengyang Li, Yuanting Zhou, Pei Yao, Yanyu Shi, Jieyun Tian, Xiaojun Zhang, and Qunlong Liu. 2024. "Genome-Wide Identification of UGT Genes and Analysis of Their Expression Profiles During Fruit Development in Walnut (Juglans regia L.)" Horticulturae 10, no. 11: 1130. https://doi.org/10.3390/horticulturae10111130

APA Style

Shi, D., Yang, J., Li, G., Zhou, Y., Yao, P., Shi, Y., Tian, J., Zhang, X., & Liu, Q. (2024). Genome-Wide Identification of UGT Genes and Analysis of Their Expression Profiles During Fruit Development in Walnut (Juglans regia L.). Horticulturae, 10(11), 1130. https://doi.org/10.3390/horticulturae10111130

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