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Evolution and amplification of the trehalose-6-phosphate synthase gene family in Theaceae

BMC Genomics volume 26, Article number: 273 (2025) Cite this article

Abstract

Background

Trehalose-6-phosphate synthase (TPS) is an essential enzyme involved in the production of trehalose, and the genes associated with TPS are crucial for various processes such as growth, development, defense mechanisms, and resistance to stress. However, there has been no documentation regarding the evolution and functional roles of the TPS gene family within Theaceae.

Results

Here, we uncovered the lineage-specific evolution of TPS genes in Theaceae. A total of 102 TPS genes were discovered across ten Theaceae species with sequenced genomes. Consistent with the previous classification, our phylogenetic analysis indicated that the TPS genes in Theaceae can be categorized into two primary subfamilies and six distinct clades (I, II-1, II-2, II-3, II-4, II-5), with clade I containing a greater number of introns compared to those found in clade II. Segmental duplication served as the main catalyst for the evolution of TPS genes within Theaceae, and numerous TPS genes exhibited inter-species synteny among various Theaceae species. Most of the TPS genes were ubiquitously expressed, and expression divergence of TPS paralogous pairs was observed. The cis-acting elements found in TPS genes indicated their involvement in responses to phytohormones and stress.

Conclusion

This research enhanced our understanding of the lineage-specific evolution of the TPS gene family in Theaceae and offered important insights for future functional analyses.

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Introduction

Trehalose, a disaccharide that does not reduce, is found widely in nature and can be observed in a variety of organisms, including bacteria, plants, and mammals [1]. In plants, trehalose influences carbon distribution and is crucial for growth, development, and resilience to stress [2, 3]. In response to challenging environmental conditions like cold, drought, heat, and salinity, trehalose is produced in large quantities and serves as a protective agent to preserve cellular integrity and viability [3, 4].

Trehalose contains two glucose molecules connected by an α,α−1,1-glycosidic linkage [1]. Trehalose is produced from UDP-glucose (UDPG) and glucose 6-phosphate (Glc6P), involving two enzymatic reactions during its biosynthesis in plants [5, 6]. Initially, trehalose-6-phosphate synthase (TPS) facilitates the formation of trehalose-6-phosphate (T6P) from UDPG and Glc6P [6]. Subsequently, trehalose-6-phosphate phosphatase (TPP) removes the phosphate group from T6P to generate trehalose and inorganic phosphate [1]. Plant TPS proteins possess two crucial domains: Glyco_transf_20 (TPS domain, Pfam: PF00982) and Trehalose_PPase (TPP domain, Pfam: PF02358), whereas TPP proteins consist solely of the TPP domain [7]. Plant TPP proteins demonstrate TPP activities; however, the TPP domain in TPS proteins has lost its enzymatic function through the course of evolution [8].

TPS genes are widely distributed in the genomes of all major plant taxa [3, 9,10,11]. The TPS genes are highly abundant and diverse in higher plants [10]. So far, many TPS genes have been identified in plants, including 11 in A. thaliana [12], 11 in rice [8, 12], 12 in Populus [9], 13 in Quinoa [13], 13 in apple [14], 9 in peach [15], 53 in cotton [16], and 12 in wheat [17].

Based on sequence similarity, TPS genes are divided into two distinct classes [12]: class I and class II, which display different characteristics in gene numbers, gene structures, expression profiles, enzyme activities, and physiological functions [3]. Only AtTPS1 and OsTPS1 encode an enzyme with TPS activity, respectively [7, 18], whereas Class II TPS proteins lack the TPS activity [3, 19]. TPS genes are crucial for a range of biological processes, such as embryo development, flower induction, seed filling, and tolerance to both biotic and abiotic stresses. For instance, AtTPS1 plays a pivotal role in glucose, abscisic acid (ABA), and stress signaling. Mutation of AtTPS1 leads to embryonically lethal [20], while overexpressing AtTPS1 in A. thaliana and tobacco enhanced tolerance to drought, temperature and osmotic stresses, respectively [21]. Similarly, overexpression of OsTPS1 in rice increased tolerance to drought, salt, and cold stresses [22]. The attps9 mutants displayed salt sensitivity, while overexpressing AtTPS9 increased salt tolerance by enhanced suberin lamellae deposition in root endodermis [23]. AtTPS5 acts as a negative regulator of ABA signaling, and its mutation leads to increased sensitivity to ABA during seed germination [24]. Mutation of AtTPS6 reduced trichome branching and increased stomatal density [25]. Overexpression of OsTPS8 in rice improved salt tolerance [26] and heterogeneous expression of wheat TPS11 in A. thaliana enhanced cold tolerance [27].

Tea (Camellia sinensis (L.) Kuntze) and oil-tea (Camellia oleifera Abel) have great economic values, and are widespread in temperate, subtropical, and tropical regions [28]. They belong to the Theaceae, which contains more than 300 species [29]. During the past several years, the genomes of many tea and oil-tea were sequenced [30,31,32,33,34,35,36,37,38,39]. So far, TPSs have been identified in several species, such as A. thaliana, rice, Populus, apple, and cotton [7, 12, 14]. However, a comprehensive genome-wide identification or functional prediction of TPSs has not yet been conducted in tea and oil-tea plants. In this study, we examined the evolutionary relationships of TPSs in various tea species. This research identified 102 TPS genes from ten Theaceae species, and categorized them into two subfamilies. We analyzed its gene and protein structure, investigated the expression patterns in different tissues as well as the transcriptional responses under stress conditions. Our study offers a theoretical foundation for future research on the biological roles of TPS gene family members in tea and oil-tea plants.

Results

Identification and characteristics analyses of TPS genes in Theaceae plants

To identify all TPS homologs in Theaceae species, we conducted both BLAST and HMM searches using TPS sequences from A. thaliana and rice. All obtained sequences were filtered based on E values and subsequently analyzed with the SMART and Pfam databases to verify the existence of the conserved TPS domains PF00982 and PF02358. Finally, 15, 10, 11, 9, 9, 11, 11, 11, 8 and 7 TPS genes were identified from, Camellia sinensis var. sinensis cv. Shuchazao (Scz), Tieguanyin (Tgy), Biyun (By), Huangdan (Hd), Longjing-43 (Lj), Camellia sinensis var. assamica cv. Yunkang-10 (Yk), Camellia lanceoleosa (Cla), Camellia chekiangoleosa (CCH), Camellia oleifera (Col) and wild tea tree (DASZ), respectively (Table 1). The tea TPS genes that were identified were named based on their homologs in A. thaliana and the results of the phylogenetic analysis (Fig. 1, Table 1).

Table 1 TPS genes identified in ten Theaceae plants
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Fig. 1
figure 1

The phylogenetic tree of TPS genes from Arabidopsis thaliana (AtTPS), Oryza sativa (OsTPS), Shuchazao (Scz-TPS), Biyun (By-TPS), Longjing-43 (Lj-TPS), Huangdan (HdTPS), Yunkang-10 (Yk-TPS), Tieguanyin (Tgy-TPS), wild tea DASZ (DASZ-TPS), Camellia oleifera (Col-TPS), Camellia chekiangoleosa (Cch-TPS), and Camellia lanceoleosa (Cla-TPS). Colored circles represent different species. Gene subfamilies are indicated with different colors

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The physicochemical properties of tea TPS members were summarized in Table 1, including amino acid sequence length, isoelectric point (pI), and molecule weight (MW) (Table 1). The size of tea TPS proteins was highly variable from 282 (By-TPS7) to 1048 amino acids (Lj-TPS9), with an average length of 828 aa. The MW ranged from 31.18 (By-TPS7) KDa to 119.73 KDa (Lj-TPS9), and their pIs ranged from 4.87 (Lj-TPS9) to 9.04 (Yk-TPS10). These physicochemical parameters of tea TPS are comparable to those of TPS identified in A. thaliana, and rice.

Phylogenetic analysis of TPS gene families

To classify TPS family members within Theaceae, an unrooted phylogenetic tree was created using the complete protein sequence alignment of TPSs from A. thaliana (11 members), rice (11 members), and Theaceae (102 members) (Fig. 1). Based on the branches of the phylogenetic tree, the 124 TPS proteins could be distinctly categorized into two major subfamilies: Group I and Group II. The group II subfamily could be further assigned to 5 subclades (II-1, 2, 3, 4, and 5) with high bootstrap support (Fig. 1). This outcome is in agreement with prior studies conducted on A. thaliana, rice, and Populus [9, 12]. The number of TPS genes in A. thaliana, rice, and various Camellia species (Yk, Scz, By, Lj, Hd, Tgy, Col, Cch, Cla, and DASZ) across different groups are as follows: Group I (4, 1, 2, 3, 2, 2, 2, 2, 0, 2, 2, 1), Group II-1 (1, 2, 1, 1, 1, 1, 1, 1, 2, 1, 2, 0), Group II-2 (1, 0, 1, 1, 2, 2, 2, 1, 1, 1, 1, 1), Group II-3 (1, 3, 3, 5, 2, 2, 1, 2, 3, 3, 3, 2), Group II-4 (3, 2, 2, 3, 2, 1, 2, 2, 1, 2, 2, 2), and Group II-5 (1, 3, 2, 2, 2, 1, 1, 2, 1, 2, 1, 1) (Table 2). Rice had only one Group I TPS, whereas most Theaceae plants had at least two members, such as Yunkang-10 (two members), Shuchazao (three members), Biyun (two members), Longjing-43 (two members), Huangdan (two members), Tieguanyin (two members), Camellia chekiangoleosa (two members), Camellia lanceoleosa (two members). AtTPS1 and OsTPS1 were proved to have TPS activity [7, 18], suggesting that the Theaceae TPS genes in group I might have TPS activity. No Group I TPS was identified in Camellia oleifera, probably due to its incomplete genome annotation [38]. In most subgroups (II-1, 2, 3, 4, and 5), at least one number from all 12 species was present, with the exception of OsTPS and DASZ-TPS genes in group II-2, and II-1, respectively (Table 1, Fig. 1). The Theaceae TPS genes often clustered closer to A. thaliana TPS genes than to rice, that was expected since A. thaliana and Camellia are dicots whereas rice is a monocot. This result suggesting that A. thaliana and Camellia TPS genes share more similar functions.

Table 2 The distribution of the TPS genes in different subfamilies
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Gene structures and protein profiles of Theaceae TPSs

To gain a deeper insight into the molecular features of Theaceae TPS genes, we analyzed their gene structures, including exons, introns, and conserved motifs (Fig. 2). Most class I TPS genes possessed more than16 introns, whereas most class II TPS genes contained only two introns (Fig. 2A-B). Compared with group I TPSs, the average exon length was longer in group II TPSs, whereas the average gene length was shorter in group II TPSs. These differences in gene structures of Theaceae TPS are comparable to those of TPS existed in A. thaliana, and rice, indicating the evolutionary divergence between group I and II TPSs.

Fig. 2
figure 2

Illustrates the phylogenetic relationships (A), gene structure (B), and domain architecture (C, D) of TPS genes. In panel A, a protein IQ tree shows different TPS subfamilies represented in various colors. Panel B displays the exon/intron structures of TPS genes, where black lines indicate introns, green boxes represent exons, and blue boxes denote UTRs. The scale at the bottom provides an estimate of exon and intron sizes. Panel C highlights the conserved motifs found in TPS proteins, with ten putative motifs shown in differently colored boxes. Finally, panel D presents the domain architecture of full-length TPS proteins, featuring conserved domains such as glycosyltransferase family 20, trehalose-phosphatase, and haloacid dehalogenase-like hydrolase domains highlighted in distinct colors

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We next performed structural feature analysis of the conserved domains of Theaceae TPS proteins (Fig. 2C). All the TPS proteins consisted of two common conserved domains, one N-terminal glycosyltransferase 20 family (Glyco_transf_20) domin and one C-terminal trehalose phosphatase (Trehalose_PPase) domain. Additionally, the majority of Group II TPS proteins (80%, 68/84) contained a haloacid dehalogenase-like hydrolase (Hydrolase_3) domain in the C-terminal region. In addition, 10 distinct conserved motifs were identified using the MEME website (Fig. 2B), and these motifs were almost conserved in Theaceae TPS proteins. Motifs 1, 3, 4, 6, 7, 9, and 10 together composed the TPS domain (Glyco_transf_20), and motifs 2, 5, and 8 composed the TPP domain (Trehalose_PPase). The majority of the members of clades II-2 and II-4 harbored 10 motifs, while the majority of the members of clades II-1, II-3, and II-5 possessed two motif 7. Clade I TPSs display a different motif conservation, and all members of clade I lacked motif 8 (Fig. 2). In addition, 4 and 10 members of clade I contained two numbers of motif 7 and motif 3, respectively. The findings from the structural analysis validated the reliability of the phylogenetic tree, indicating functional distinctions between clades I and II.

Gene duplication analysis of TPS proteins

Previous analysis identified duplicate pairs of TPS genes created by segmental duplication in A. thaliana and rice [12], and segmental duplication contribute most to the expansion of TPS genes. To understand the expansion pattern of Theaceae TPS genes, a collinear relationship was examined (Fig. 3, Figure S1). 8 (TPS1/TPS2, TPS1/TPS3, TPS2/TPS3, TPS8/TPS9, TPS8/TPS10, TPS9/TPS10, TPS12/TPS13, TPS14/TPS15), 3 (TPS1/TPS2, TPS4/TPS5, TPS8/TPS9), 3 (TPS1/TPS2, TPS6/TPS7, TPS8/TPS9), 3 (TPS1/TPS2, TPS7/TPS8, TPS9/TPS10), 3 (TPS1/TPS2, TPS4/TPS5, TPS7/TPS8), 2 (TPS1/TPS2, TPS7/TPS8), 1 (TPS5/TPS6), and 1 (TPS3/TPS4) pairs of TPSs were identified in Shuchazao (SCZ), Biyun (By), Camellia chekiangoleosa (Cch), Camellia lanceoleosa (Cla), Huangdan (HD), Tieguanyin (Tgy), Camellia oleifera (Col), and wild tea (DASZ), respectively. In addition, 4 (TPS1/TPS2, TPS8/TPS9, TPS8/TPS10, TPS9/TPS10), and 6 (TPS2/TPS3, TPS2/TPS6, TPS5/TPS8, TPS5/TPS9, TPS8/TPS9, TPS1/TPS11) pairs of TPSs were identified in A. thaliana, and rice, respectively (Fig. 3). All these duplicated gene pairs were located in paralogous blocks, indicating that these duplicated gene pairs were formed by a segmental duplication event. The Ka/Ks ratios for all TPS pairs were found to be below 1 (Table S2), suggesting that these TPS genes have undergone purifying selection.

Fig. 3
figure 3

The intraspecies syntenic relationship pattern diagram of TPS genes in A. thaliana (A), DASZ (B), Shuchazao (C), rice (D), Camellia chekiangoleosa (E), and Biyun (F). The TPS genes were displayed with red colors. The collinearity genes were lined by lines and the tandem genes were displayed with black stars. Other colors indicate genes located around the TPS and on the collinear blocks

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To investigate the evolutionary mechanism of Theaceae TPS genes, the inter-species synteny was analyzed among Theaceae species, A. thaliana, and rice (Fig. 4). The results showed syntenic gene pairs were extensively present among these species. The number of gene pairs orthologous to A. thaliana TPS genes were 14 (Biyun), 8 (Shuchazao), 8 (Camellia chekiangoleosa), 11 (Camellia lanceoleosa), 10 (Huangdan), 10 (Tieguanyin), Camellia oleifera (Col), 7 (Longjing), and 7 (DASZ) (Fig. 4). One-to-many, or many-to-one homozygosity were identified among Theaceae species and A. thaliana. More orthologoue gene pairs were found between Theaceae species and A. thaliana than between Theaceae species and rice (Fig. 4), which is consistent with the result of phylogenetic analysis that the TPS genes of theaceae clustered closer to the TPS genes of Arabidopsi than to the TPS genes of rice (Fig. 1).

Fig. 4
figure 4

The interspecies syntenic relationships of TPS genes among Theaceae, rice, and A. thaliana are depicted. The collinearity of orthologous TPS genes is represented by black lines

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Analysis of cis-acting elements

To determine the possible regulatory mechanisms of TPS genes in Theaceae, we predicted the cis-acting elements of their promoters using Plant CARE (Fig. 5, Table S3). A total of 41 distinct types of cis-acting elements were identified and classified into four primary categories: light-responsive elements, plant growth-related elements, stress-responsive elements, and phytohormone-responsive elements. The light-responsive cis-acting elements were the most prevalent, followed by those related to stress response and phytohormones, while the plant growth-related elements were the least represented. 21 types of light responsive elements were identified, with Box4 and G-box account for 25% and 21%, respectively. In stress and phytohormone responsive elements, ARE (an anaerobic induction element), MBS (a drought-inducible element), ABRE (ABA-responsive element), ERE (ethylene-responsive element), and CGTCA-motif (methyl jasmonic acid [MeJA]-responsive element) were observed in most Theaceae TPS genes. The presence of these elements implied that Theaceae TPS genes could participate in the plant response to environmental stimuli and phytohormone signaling. Notably, 8 TPS genes (Cla-TPS8, Col-TPS6, Cch-TPS7, Yk-TPS7, DASZ-TPS4, Scz-TPS9, Scz-TPS10, and Lj-TPS7) belonging to clade II-3 contained the most elements of G-box and ABRE, indicating their possible function in light and phytohormone signal transduction (Fig. 5).

Fig. 5
figure 5

Predicted cis-elements in the promoters of TPS genes are shown. A The count of cis-acting elements. B The distribution of various types of cis-acting elements within each category

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Transcriptional profiling of TPS genes

To explore the expression pattern of Theaceae TPS genes, we acquired the public RNA-seq data and created a heat map with the TPM values of the TPS genes (Fig. 6, Table S4). In Shuchazao, eight different tissues were analyzed, including root, stem, young leaf, mature leaf, old leaf, apical bud, flower, and fruit (Fig. 6A). Most of the TPS genes were ubiquitously expressed, with the exception of TPS7/8/14 which were particularly expressed in the flower, albeit in very low level. Furthermore, both analogous and distinct expression patterns were observed among the TPS genes in each clade. TPS1, TPS2, and TPS3 (Clade I) were ubiquitously expressed, with TPS1 showing higher expression than TPS2 and TPS3. It is worth noting that TPS8 and TPS9 (Clade II-3), TPS14 and TPS15 (Clade II-5), as four duplication genes, displayed different expression patterns. TPS9 and TPS15 display a high expression in the tissues tested, whereas the transcripts of TPS8 and TPS14 were barely detected (Fig. 6A).

Fig. 6
figure 6

Expression profiles of TPS genes. A Shuchazao. B Camellia chekiangoleosa. C Camellia lanceoleosa. D DASZ. E Huangdan. F Longjing-43. G Biyun. H Tieguanyin. I Yunkang. Gene expression values were calculated and normalized with log2(TPM + 1)

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Similar expression patterns were also identified in TPS genes of other Theaceae species (Fig. 6B-I). As for the members of the Shuchazao clade I TPS genes, the clade I TPS genes of other species showed a similar expression pattern in the leaf, stem, flower and root tissues. For example, Cch-TPS1 and Cch-TPS2, Cla-TPS1 and Cla-TPS2, Hd-TPS1 and Hd-TPS2, Lj-TPS1 and Lj-TPS2, Tgy-TPS1 and Tgy-TPS2, By-TPS1 and By-TPS2, as twelve duplication genes, showed similar expression levels in the tissues tested. Cch-TPS7, Cla-TPS8, DASZ-TPS4, Hd-TPS8 were highly expressed in all the tissues, whereas the expression of their paralog genes Cch-TPS6, Cla-TPS7, DASZ-TPS3, Hd-TPS7 were barely detected (Fig. 6B-I). These findings suggest that Theaceae TPS genes have experienced significant functional divergence throughout their evolutionary history.

It has been shown that TPS proteins play a crucial role in regulating gene expression. To investigate the possible functions of tea TPS genes, we examined their expression patterns in Shuchazao under conditions of cold, drought, salt stress, and MeJA treatment (Fig. 7). During periods of cold, the expression of the majority of TPS genes were increased (Fig. 7A), whereas their expression levels were decreased under salt stress (Fig. 7C). TPS15 was upregulated under both drought and salt conditions, while TPS5 displayed an opposite trend to drought and salt stresses (Fig. 7B, D). In addition, distinct patterns of expression were observed between TPS paralogs. For example, TPS1 was upregulated under cold stress, while TPS2 was downregulated under drought and salt stresses. TPS15 was upregulated under cold, drought and salt stresses, while TPS14 was only downregulated under MeJA condition. TPS9 and TPS13 were differentially expressed under cold, salt and MeJA stresses, whereas the expressions of TPS8 and TPS12 were not changed.

Fig. 7
figure 7

Assessment of Shuchazao TPS genes expression under cold (A), drought (B), MeJA (C), and salt (D) conditions. Expression values were analyzed according to RNA-seq data. Under cold stress, all TPS genes were upregulated, while under MeJA treatment most TPS genes were downregulated

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We further examined the expression of TPS genes under both abiotic (heat, cold, drought, salt) and biotic stress (Ectropis obliqua) conditions in the local tea variety with quantitative RT-PCR (Fig. 8). The qPCR showed that TPS15 was significantly upregulated under heat, cold, drought, salt and Ectropis obliqua stresses, while TPS5 was significantly decreased under drought, salt and Ectropis obliqua stresses. TPS1 was upregulated under cold stress; TPS2, TPS3, TPS4, and TPS13 were upregulated under heat stress; TPS4 and TPS9 were upregulated under Ectropis obliqua stress. TPS10 and TPS11 were specially upregulated under salt and cold treatments, respectively. The findings reveal that different TPS genes exhibit distinct expression levels when subjected to various stresses, indicating their participation in stress response mechanisms.

Fig. 8
figure 8

The expression of TPS genes in response to heat, cold, drought, salt, and Ectropis obliqua Prout treatment was analyzed. Expression levels were normalized using PTB gene expression as a reference point, with the control group designated as '1'. Mean values were derived from three replicates, and error bars indicate the standard deviations of the biological replicates

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Discussion

Molecular evolution of TPS proteins in Theaceae

TPS genes play vital roles in plant growth, development, and stress responses [40, 41]. TPS genes have been identified and characterized in many plants, including A. thaliana [12], rice [12], wheat [17], cotton [16], and potato [42]. However, the TPS gene family in tea and oil tea has not been systematic studied. Herein, we present a first comparative evolutionary analysis of the TPS family in Theaceae species. 102 TPS genes were identified in ten Theaceae species, and the TPS gene numbers ranged from 7 to 15, indicating that TPS genes have experienced different gene duplication events in different Theaceae species (Fig. 1). Based on the phylogenetic analysis, the Theaceae TPS genes were divided into two main subfamilies (clade I and clade II), which was consistent with the classification in A. thaliana, rice, and populus [12]. More TPS genes were classified in clade II, and clade II TPSs was further classified into five clusters: II-1, II-2, II-3, II-4, and II-5.

The two TPS subfamilies displayed distinct characteristics in terms of conserved motifs and gene structures (Fig. 2). In comparison to clade II TPSs, clade I TPS genes had a higher number of introns and exhibited more complex structures. The TPS genes in clade I typically contained 16 introns, while those in clade II generally had two introns (Fig. 2), which was consistent with the gene structures of TPS genes in A. thaliana and rice [12]. Domain analysis showed that motif 8 and the hydrolase_3 domain were lost in clade I TPS proteins, whereas the majority of clade II TPSs contained the hydrolase_3 domain, in agreement with the results of other studies [7, 11, 12, 16, 43]. Structural diversification has been reported to play an important role in the evolution of diverse gene family [44]. Consequently, the distinctive features of clade I and clade II Theaceae TPSs imply that these two groups of TPS genes experienced different pathways of molecular evolution and functional divergence.

There are more TPS members identified in clade II than in clade I (Fig. 1). In addition, many lineage-specific duplication events were identified in Theaceae TPSs (Fig. 1). For example, in clade I, clade II-2, clade II-4 and clade II-5, Theaceae TPSs underwent one duplication forming two branches, in clade II-3 two duplication events were identified, while in clade II-1 no duplication event was occurred. In clade I, clade II-4, clade II-5, and clade II-3, the duplicated TPSs were maintained in most Theaceae species, while in clade II-1, most Theaceae species lost one copy of TPS duplicates. Numerous segmental duplication events were identified in Theaceae species, suggesting that the proliferation of TPS genes within this family primarily resulted from segmental duplication. This expansion of the TPS gene family indicates its significant biological functions in tea and oil tea.

Expression divergence of TPS genes

Expression divergence was reported in TPS genes of A. thaliana and rice [12] (Figure S2). Most TPS genes were expressed in all tissues examined, while AtTPS2, AtTPS2, AtTPS4, and OsTPS9 were selectively expressed at low levels in some specific tissues (Figure S2). It has been reported that clade I and clade II TPS genes displayed different characteristics in expression profiles, enzyme activities and physiological functions [3]. A. thaliana has four clade I TPS genes, with AtTPS1 was ubiquitously expressed while AtTPS2, AtTPS2, and AtTPS4 were low expressed. Only AtTPS1 was proved to have TPS activity [7, 8]. Most Theaceae species contained two clade I TPS genes (Fig. 1), and most of the duplicated paralogs displayed similar expression patterns (Fig. 6). Theaceae clade I TPS genes clustered closer and displayed higher sequence similarity to A. thaliana TPS1, indicating that Theaceae class I TPS proteins may have functions similar to AtTPS1. In addition, the low-copy numbers of TPS genes in clade I suggested a functional conservation during the long-term evolution.

The members of Class II TPS exhibit distinct expression patterns when exposed to different stress conditions (Fig. 7). Under cold stress, most of the TPS genes were upregulated, while under the MeJA condition, most of them were downregulated. Scz-TPS15 was observed to be induced by multiple stresses, such as drought, cold, and salt, whereas Scz-TPS5 was repressed by drought and salt (Figs. 7, 8). Expression divergence was also identified in the duplicate pairs of TPSs (Figs. 6, 7, 8). One copy of each seven duplicate gene (Cch-TPS7/Cch-TPS6, Cla-TPS7/Cla-TPS8, DASZ-TPS3/DASZ-TPS4, Hd-TPS7/Hd-TPS8, Scz-TPS8/Scz-TPS10, Scz-TPS14/Scz-TPS15, Tgy-TPS7/Tgy-TPS8) was highly expressed in all tissues examined, however, the transcript levels of the other paralog was low expressed only in a specific tissue (Fig. 6). Under stress conditions, expression divergence was observed in the duplicate gene pairs (Figs. 7, 8). Specifically, one copy of each of the four duplicate gene pairs (Scz-TPS1/Scz-TPS2, Scz-TPS9/Scz-TPS10, Scz-TPS12/Scz-TPS13, Scz-TPS14/Scz-TPS15) exhibited differential expression under stress conditions, while the other paralog did not respond to various treatments. These findings suggest that these duplicate genes have undergone subfunctionalization or functional divergence. Further research is required to gain deeper insights into their expression patterns and functional divergence in relation to tea plant growth and stress responses.

Materials and methods

Data sources and sequence acquisition

TPS genes from A. thaliana and rice were obtained from TAIR11 (https://www.arabidopsis.org/) and RiceFREND (https://ricefrend.dna.affrc.go.jp), respectively [45]. Genomic and protein sequences for tea plants and oil tea plants were sourced from TeaPGDB (https://eplant.njau.edu.cn/tea/index.html), GitHub (https://github.com/Hengfu-Yin/CON_genome_data), NGDC (https://ngdc.cncb.ac.cn/), and NCBI (BioProject: PRJNA780224) [46]. Although the tea plants have annotated genomes, the TPS gene candidates might be mis-annotated during the automated genome annotation process. Two approaches were employed to identify TPS homologs in tea and oil tea. The conserved domains of TPS proteins, specifically Glyco-transf-20 (PF00982) and TPP (Trehalose_PPase, PF02358), were retrieved from Pfam [47]. HMMER was utilized for candidate TPS protein identification with an E-value cutoff of 1e-10 [48]. Additionally, the Basic Local Alignment Search Tool algorithms (BLASTP) were applied using the amino acid sequences of A. thaliana and rice TPS members against a protein database with the following parameters: maximum target sequence: 100, expected threshold: 13, word size: 10, scoring matrix: BLOSUM62, gap cost: existence 11 and extension 1, compositional adjustments: conditional compositional score matrix adjustment. The intersection of genes acquired using these two methods was used as a screening criterion for candidate TPS genes. The identified TPS genes were input to Pfam (http://pfam.xfam.org/) to establish the existence of TPS domains. The integrity of the domain was confirmed using online tools such as CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/) [49], HMM (https://hmmer.org/), Geneious (http://www.geneious.com) and SMART (https://smart.embl-heidelberg.de/) [48, 50]. The basic information on TPS proteins, including molecular weight (MW), isoelectric point (pI), and subcellular locations were predicted using the Expasy (https://web.expasy.org/protparam/) and GenScript (https://www.genscript.com/wolf-psort.html) websites [51]. The secondary structures of TPS proteins were predicted using the PRABI website (https://npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl?page=npsa_sopma.html). The collinearity and selective evolutionary pressure of TPS genes were analyzed using the TBtools software [52].

Phylogenetic analyses, gene structure, motif analyses, and expression analysis

Multiple alignments of full-length TPS proteins were conducted using MAFFT software [53]. The maximum likelihood (ML) phylogenetic tree was generated using IQ-TREE2 with the parameters '-m MFP -B 1000' and included 1000 ultra-bootstrap replicates [54]. The gene structure was assessed and visualized through the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) [55]. Conserved motifs were detected using the online MEME program (http://meme-suite.org/tools/meme) [56]. Transcript levels of TPS genes were investigated by using the public available RNA-seq data as shown in previous studies [30, 31, 36, 37]. Gene expression values were calculated and normalized with log2(TPM + 1).

Synteny analysis

First, we utilized the BLAST tool to conduct sequence alignment. Specifically, we constructed a BLAST database using the protein sequence file of the target species as the reference. Subsequently, we employed the protein sequence file of the species to be compared as the query sequence and performed the alignment using blastp. The alignment parameters were set as follows: an E-value threshold of 1e-10, 12 threads, and output format 6 (standard table format). Upon completion of the sequence alignment, we merged the alignment result file with the genome annotation files of both species. We then executed the MCScanX tool, inputting the merged GFF file and the BLAST alignment result file, to generate collinearity analysis results. Following the completion of the MCScanX analysis, multiple output files were generated, including an HTML-formatted collinearity report, a collinearity alignment file, and a tandem repeat gene file. These files can be further utilized to construct collinearity dot plots, thereby providing a visual representation of the collinear regions between genomes.

RNA extraction and qRT‑PCR analysis

For the qRT-PCR analysis, a 2-year-old local tea variety (Camellia sinensis cv. Xinyanghong 10) cultivated at Xinyang Normal University (23 ± 3 °C, 65 ± 5% room humidity, with a day/night cycle of 16/8 h) was selected for the experiments. In the drought treatment, plants were subjected to water deprivation for seven days. For salt stress, leaves were treated with a solution of 150 mM NaCl for six hours. Heat and cold treatments involved exposing the plants to temperatures of 40 °C and 4 °C for six hours each. Additionally, twelve larvae from the third or fourth instar of E. obliqua were placed on three unfed tea plants for a duration of twenty-four hours to assess stress response. Plants that were not subjected to any stress served as the control (CK). Total RNA was extracted, and one microgram of this RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit. Quantitative RT-PCR (qRT-PCR) was conducted with SYBR Premix EX Taq on an ABI StepOnePlus instrument. The relative expression levels were determined using the 2 − ΔCt method, with CsPTB acting as the internal control gene. The experiments were conducted in triplicate. Specific primers were created using Primer 5 software (Table S5).

Data availability

The authors confirm that no new genes or proteins were generated in this study and all analyses were based on existing data in the databases. The data underlying the findings of this study are presented in the article and its supplementary materials. A. thaliana gene sequence data from this article can be found in the TAIR database (https://www.arabidopsis.org/). Oryza sativa gene sequence data from this article can be found in the NCBI database (https://www.ncbi.nlm.nih.gov/). Theaceae gene sequence data from this article can be found in the TPIA database (http://tpia.teaplants.cn/) and Github (https://github.com/Hengfu-Yin/CON_genome_data). The corresponding TPS accession numbers are in Table S1.

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Funding

This work was supported by the General Research Projects of Zhejiang Provincial Department of Education (Y202351039), Science and Technology Project of Huzhou (2023KT44), Huzhou Science and Technology Plan Project (2023GZ44), Research Program of Huzhou College (2023HXKM09), and National College Student Innovation and Entrepreneurship Training Program (202313287003).

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  1. School of Life and Health Science, Huzhou College, Huzhou, Zhejiang, China

    Zaibao Zhang, Kejia Li, Kexin Huang, Chunxia Liao & Guangqu Liu

  2. College of Life Science, Xinyang Normal University, Xinyang, Henan, China

    Tao Xiong

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Contributions

The study was conceived and directed by ZZB. ZZB and XT wrote the manuscript. XT, LKJ, HKX, LCX and LGQ performed the identification of TPS genes, protein structure, and evolution analysis. All the authors read and approved the final manuscript. All authors consent for publication.

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Supplementary Information

12864_2025_11475_MOESM1_ESM.pdf

Supplementary Material 1: Figure S1. The intraspecies syntenic relationship of TPS genes in Huangdan (A), Camellia lanceoleosa (B), Tieguanyin (C), Longjing (D), and Camellia oleifera (E),respectively.

Supplementary Material 2: Figure S2. The expression profiles of TPS genes in Arabidopsis thaliana and Oryza sativa.

12864_2025_11475_MOESM3_ESM.xlsx

Supplementary Material 3: Table S1. The identified TPS protein sequences. Table S2. The Ka/Ks analysis of TPS genes. Table S3. Cis-acting elements of TPS genes. Table S4. The expression of TPS genes. Table S5. Primers used to detect the expression of tea TPS genes in this study.

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Zhang, Z., Xiong, T., Li, K. et al. Evolution and amplification of the trehalose-6-phosphate synthase gene family in Theaceae. BMC Genomics 26, 273 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11475-3

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