Molecular and Functional Analysis of Trehalose-6-Phosphate Synthase Genes Enhancing Salt Tolerance in Anoectochilus roxburghii (Wall.) Lindl.

Trehalose is a reducing disaccharide, acting as a protectant against various environmental stresses in numerous organisms. In plants, trehalose-6-phosphate synthase (TPS) plays a crucial role in trehalose biosynthesis. Anoectochilus roxburghii (Wall.) Lindl. is a prominent species of the Anoectochilus genus, widely utilized as a health food. However, the functional analysis of TPS in this species has been limited. In this study, TPS genes were cloned from A. roxburghii. The ArTPS gene, with an open reading frame spanning 2850 bp, encodes 950 amino acids. Comparative and bioinformatics analysis revealed that the homology was presented between the ArTPS protein and TPSs from other plant species. The ORF sequence was utilized to construct a prokaryotic expression vector, Pet28a-ArTPS, which was then transformed into Escherichia coli. The resulting transformants displayed a significant increase in salt tolerance under the stress conditions of 300 mmol/L NaCl. Quantitative RT-PCR analysis demonstrated that the expression of ArTPS genes responded to NaCl stress. The accumulation of G6P was upregulated, whereas the content of T6P exhibited an opposite expression trend. The glycometabolism products, including trehalose, exhibited notable changes under NaCl stress, although their variations may differ in response to stimulation. The content of kinsenoside, a characteristic product of A. roxburghii, was significantly upregulated under NaCl stress. These results suggest that the ArTPS genes function in response to NaCl stimulation and play a key role in polysaccharide and glycoside metabolism in Anoectochilus. This study provides new insights into the engineering modification of the health food A. roxburghii to enhance the medicinal activity of its ingredients.


Introduction
Anoectochilus roxburghii (Wall.) Lindl., an orchidaceae species, has a number of medicinal components that are essential for treating cancer and allergies [1][2][3][4]. Because to its protected status, the wild A. roxburghii is frequently exploited in artificial cultivation or tissue culture for quick multiplication [5][6][7]. However, unlike wild plants, cultivated A. roxburghii exhibits distinct accumulation patterns of medicinal components, mainly including polysaccharides and the glycoside derivative kinsenoside, primarily through secondary metabolites [5,8]. In the artificial cultivation or tissue culture of A. roxburghii, it is crucial to promote the accumulation of glycometabolites and glycoside derivatives.
Trehalose is a non-reducing disaccharide that is widely distributed in both plants and microbes [9]. While it is known to be present in specific drought-tolerant plants, it has also Molecules 2023, 28, 5139 2 of 12 been identified in common plants such as rice and Arabidopsis [10,11]. However, its content in these plants is typically very low [12,13]. In plants, trehalose synthesis is first catalyzed by trehalose-6-phosphate synthase (TPS), which transfers glucose from UDP-glucose to glucose-6-phosphate (G6P) to produce trehalose-6-phosphate (T6P). Then, T6P was dephosphorylated by trehalose-6-phosphate phosphatase (TPP) to produce trehalose [14,15]. Among these genes, the TPP gene can be substituted by the TPS gene, and TPS plays a crucial role in regulating the rate of biosynthesis for T6P and trehalose metabolites. These components are vital for a wide range of physiological and biochemical processes. [14,16].
In previous studies, the TPS gene was cloned and identified as AtTPS1 from cDNA libraries of Arabidopsis plants [17]. As research has progressed, TPS genes have been successfully cloned in various other plant species, such as Medicago truncatula, Dioscorea esculenta, and Tamarix hispida [15,18,19]. TPS has garnered significant attention, particularly due to its involvement in stress resistance mechanisms. The TPS gene has been observed to be upregulated and expressed in response to various stress conditions such as cold, salt, drought, and mannitol stresses in ginkgo plants [20]. The expression of the TPS gene was elevated under cold stress, and this gene may be related to the cold tolerance mechanism in tea trees [21]. The AtTPS5-dependent trehalose metabolism mediates basal defense responses [22]. In maize, the TPS gene was involved in the accumulation of trehalose and induced the expression of related genes in the early stages of salt and cold stress; in the late stages of stress, the TPS gene was upregulated and accumulated T6P, which opened other stress response pathways through the regulation of polysaccharide metabolism [14,23]. Thus, it was clear that TPS gene expression not only affected plant stress resistance but also regulated glycometabolism and glycoside through T6P.
In the present study, the TPS genes were amplified from A. roxburghii. After bioinformatics analysis, the expression of ArTPS genes in response to precursor substance NaCl stress was detected by real-time quantitative PCR (RT-qPCR). The transformation of the ArTPS genes into E. coli could increase tolerance to salt and cold. And the polysaccharides quantification and kinsenoside measurement of A.roxburghii were detected under the NaCl stress. These results demonstrate that ArTPS genes play an important role in glycometabolism and glycoside biosynthesis in Anoectochilus.

Open Reading Frame and Putative Proteins
The TPS gene sequences from A. roxburghii were obtained through RNA-seq analysis. To amplify segments longer than 2000 and 3000 base pairs, appropriate primers were designed based on the provided information ( Figure S1). The ArTPS gene consists of a single open reading frame spanning 2850 bp, encoding 950 amino acids. It has a molecular weight of 106.78 kDa, isoelectric point values of 6.81, and GRAVY values of −0.345. The molecular formula of ArTPS is C 4737 H 7424 N 1350 O 1410 S 29 . The secondary structure of ArTPS comprises 41.47% α-helices, 13.32% extended strands, and 38.11% random coils. These structural elements are present in the three-dimensional model of ArTPS ( Figure S2). Notably, the characteristics of ArTPS, PeTPS, and DcTPS were found to be comparable.

Multiple Sequence Alignment and Phylogenetic Analysis
A glycosyltransferase conserved domain and a HAD-like conserved domain were found in position 58-558 and 612-848, respectively. Meanwhile, the phosphorylation sites of T7, S8, S10, S113, S646, S852, and S868 were found in the ArTPS protein, respectively. Moreover, the conserved motif R160-S206 was found in multiple sequence alignment from TPS ( Figure 1). Multiple alignment and phylogenetic analysis showed that the putative ArTPS proteins were clustered into the same sub-group with the deposited functional TPS proteins of Dendrobium huoshanense and Phalaenopsis equestris (Figures 1 and 2), indicating that TPS from A. roxburghii is a member of the TPS family.
proteins of Dendrobium huoshanense and Phalaenopsis equestris (Figures 1 and 2), indicating that TPS from A. roxburghii is a member of the TPS family.

Overexpression of TPS Gene
A noticeable reduction in colony numbers on the LB agar plates was observed as the dilution fold increased. Furthermore, clear distinctions were observed between the lines transformed with the loaded vector pET−28a(+)−TPS and the empty vector pET−28a(+). Under the stress of 300 mmol L −1 NaCl, the E. coli colonies transformed with pET−28a(+)−TPS exhibited significantly higher tolerance to salt stress compared to those transformed with pET−28a(+) (Figure 3).

Relative Expression Level under Induction
The expressions of the ArTPS gene were significantly upregulated in A. roxburghii and there was a peak value at 0.5 h (3.81 times) in response to NaCl stress at the beginning. Then, the expressions plummeted and reached its valley at 4 h, only half or even less at 0.5 h. And the expressions were upregulated, and there was a peak value at 8 h (3.95 times). The ArTPS genes were continually downregulated at 12 h and 24 h (Figure 4). This result indicated the strong expression response of these ArTPS genes.

Overexpression of TPS Gene
A noticeable reduction in colony numbers on the LB agar plates was observed as the dilution fold increased. Furthermore, clear distinctions were observed between the lines transformed with the loaded vector pET−28a(+)−TPS and the empty vector pET−28a(+). Under the stress of 300 mmol L −1 NaCl, the E. coli colonies transformed with pET−28a(+)−TPS exhibited significantly higher tolerance to salt stress compared to those transformed with pET−28a(+) (Figure 3).

Overexpression of TPS Gene
A noticeable reduction in colony numbers on the LB agar plates was observed as the dilution fold increased. Furthermore, clear distinctions were observed between the lines transformed with the loaded vector pET−28a(+)−TPS and the empty vector pET−28a(+). Under the stress of 300 mmol L −1 NaCl, the E. coli colonies transformed with pET−28a(+)−TPS exhibited significantly higher tolerance to salt stress compared to those transformed with pET−28a(+) (Figure 3).

Relative Expression Level under Induction
The expressions of the ArTPS gene were significantly upregulated in A. roxburghii and there was a peak value at 0.5 h (3.81 times) in response to NaCl stress at the beginning. Then, the expressions plummeted and reached its valley at 4 h, only half or even less at 0.5 h. And the expressions were upregulated, and there was a peak value at 8 h (3.95 times). The ArTPS genes were continually downregulated at 12 h and 24 h ( Figure 4). This result indicated the strong expression response of these ArTPS genes.

Relative Expression Level under Induction
The expressions of the ArTPS gene were significantly upregulated in A. roxburghii and there was a peak value at 0.5 h (3.81 times) in response to NaCl stress at the beginning. Then, the expressions plummeted and reached its valley at 4 h, only half or even less at 0.5 h. And the expressions were upregulated, and there was a peak value at 8 h (3.95 times). The ArTPS genes were continually downregulated at 12 h and 24 h ( Figure 4). This result indicated the strong expression response of these ArTPS genes.

G6P and T6P Accumulation under Inducion
Under non-inducing conditions (the 0 h control), the accumulation of G6P and T6P of A. roxburghii was 17.78 µg/g and 15.77 µg/g from A. roxburghii, respectively ( Figure 5). In response to NaCl induction, the accumulation of G6P was both decreased by 41.28% and 46.24% after 4 d and 16 d, respectively ( Figure 5A), indicating its expression suppressed. On the contrary, the accumulation of T6P was 1.22 and 1.50 times (19.26 µg/g and 23.67 µg/g) of that after 4 d and 16 d by NaCl stress ( Figure 5B).

Glycometabolism and Kinsenoside Accumulation under Induction
The accumulation of monosaccharide from Glucose, D-Fructose, Methyl beta-D-galactopyranoside, D-Glucuronic acid, and D-Ribono-1,4-lactone was decreased during the first four days of the induction but rebounded after that with different ranges between different products ( Figure 6A-C). The content of monosaccharide from L-Fucose, Levoglucosan, Inositol, and D-Galactose showed a continuous decrease over the 16 days measured ( Figure 6C-D). But the accumulation of D-Mannose was increased continually ( Figure 6D).

G6P and T6P Accumulation under Inducion
Under non-inducing conditions (the 0 h control), the accumulation of G6P and T6P of A. roxburghii was 17.78 µg/g and 15.77 µg/g from A. roxburghii, respectively ( Figure 5). In response to NaCl induction, the accumulation of G6P was both decreased by 41.28% and 46.24% after 4 d and 16 d, respectively ( Figure 5A), indicating its expression suppressed. On the contrary, the accumulation of T6P was 1.22 and 1.50 times (19.26 µg/g and 23.67 µg/g) of that after 4 d and 16 d by NaCl stress ( Figure 5B).

G6P and T6P Accumulation under Inducion
Under non-inducing conditions (the 0 h control), the accumulation of G6P and T6P of A. roxburghii was 17.78 µg/g and 15.77 µg/g from A. roxburghii, respectively ( Figure 5). In response to NaCl induction, the accumulation of G6P was both decreased by 41.28% and 46.24% after 4 d and 16 d, respectively ( Figure 5A), indicating its expression suppressed. On the contrary, the accumulation of T6P was 1.22 and 1.50 times (19.26 µg/g and 23.67 µg/g) of that after 4 d and 16 d by NaCl stress ( Figure 5B).

Glycometabolism and Kinsenoside Accumulation under Induction
The accumulation of monosaccharide from Glucose, D-Fructose, Methyl beta-D-galactopyranoside, D-Glucuronic acid, and D-Ribono-1,4-lactone was decreased during the first four days of the induction but rebounded after that with different ranges between different products ( Figure 6A-C). The content of monosaccharide from L-Fucose, Levoglucosan, Inositol, and D-Galactose showed a continuous decrease over the 16 days measured ( Figure 6C-D). But the accumulation of D-Mannose was increased continually ( Figure 6D).

Glycometabolism and Kinsenoside Accumulation under Induction
The accumulation of monosaccharide from Glucose, D-Fructose, Methyl beta-Dgalactopyranoside, D-Glucuronic acid, and D-Ribono-1,4-lactone was decreased during the first four days of the induction but rebounded after that with different ranges between different products ( Figure 6A-C). The content of monosaccharide from L-Fucose, Levoglucosan, Inositol, and D-Galactose showed a continuous decrease over the 16 days measured ( Figure 6C-D). But the accumulation of D-Mannose was increased continually ( Figure 6D). Molecules 2023, 28, x FOR PEER REVIEW 6 of 12 The trehalose content was 67.32 µg/g under 0 h control from A. roxburghii. In NaCl induction, the accumulation of trehalose in A. roxburghii was increased and reached their peak values (1.15 times of the 0 d control) on the fourth day. The difference of trehalose content was not significant between the 0 h control and the 16th day ( Figure 7A). In NaCl induction, the expression of the phenylglucoside were continually downregulated from 47.14 µg/g to 31.75 µg/g ( Figure 7B). The accumulation of maltose and cellobiose was significantly lower in the untreated than the salt-treatment model on the 4th and 16th day ( Figure 7C,E). The sucrose content was continually upregulated and reached about 1.5 times (31.44 mg/g) of the 0 d control at the fourth day ( Figure 7D). In contrast, the downregulated expression reached the same level of the 0 d control at 16 d of the NaCl induction in A. roxburghii ( Figure 7D). The content of sucrose exhibited a similar trend of change as that of trehalose. (Figure 7D). Using the heat map, the glycometabolism content was clustered in A. roxburghii under non-inducing conditions and NaCl stress (Figure 8). The trehalose content was 67.32 µg/g under 0 h control from A. roxburghii. In NaCl induction, the accumulation of trehalose in A. roxburghii was increased and reached their peak values (1.15 times of the 0 d control) on the fourth day. The difference of trehalose content was not significant between the 0 h control and the 16th day ( Figure 7A). In NaCl induction, the expression of the phenylglucoside were continually downregulated from 47.14 µg/g to 31.75 µg/g ( Figure 7B). The accumulation of maltose and cellobiose was significantly lower in the untreated than the salt-treatment model on the 4th and 16th day ( Figure 7C,E). The sucrose content was continually upregulated and reached about 1.5 times (31.44 mg/g) of the 0 d control at the fourth day ( Figure 7D). In contrast, the downregulated expression reached the same level of the 0 d control at 16 d of the NaCl induction in A. roxburghii ( Figure 7D). The content of sucrose exhibited a similar trend of change as that of trehalose. (Figure 7D). Using the heat map, the glycometabolism content was clustered in A. roxburghii under non-inducing conditions and NaCl stress (Figure 8).
The accumulation of kinsenoside was 0.84 mg/g under 0 h control from A. roxburghii. The contents were increased sharply under salt induction, peaking at 32.94 mg/g on the 4th day, and then decreasing to 22.82 mg/g on the 16th day, while the upregulated expression     roxburghii under non-inducing condition and NaCl stress. CK stands for non-inducing, SS stands for NaCl stress after 4 d, SL stands for NaCl stress after 16 d; red stands for high relative expression, green stands for low relative expression, yellow stands for medium relative expression; the right side indicates the sorting of glycometabolism clusters.

Discussion
Trehalose metabolism is involved in the growth, development, and abiotic stress response in higher plants [24,25]. TPS is an essential enzyme in the trehalose biosynthetic pathway [26,27]. The TPS gene also plays a vital role in abiotic stress tolerance in plants [28][29][30][31][32]. In this study, the TPS gene was identified from A. roxburghii cDNA. The study presented the homology of the open reading frame (ORF) and putative protein sequences with the deposited functional TPS proteins of Dendrobium chrysotoxum as well as Phalaenopsis equestris. The TPS proteins contained both a glycosyltransferase conserved domain, a HAD-like conserved domain, and six phosphorylation sites, indicating that the TPS protein is highly conserved and the structures of the two domains might be essential for TPS functions in different plants. This characteristic was consistent with previous studies in Arabidopsis, rice, and Populus [19,33,34].
Expression level analyses of NaCl stress would help to better understand and predict gene function. The qRT-PCR results show that the expression of the ArTPS gene was significantly upregulated after NaCl treatments. Our results were consistent with research in Medicago truncatula, Dioscorea esculenta, and Tamarix hispida [15,19]. The TPS gene of A. roxburghii could be endowed with the same TPP and TPS catalytic activity as the maize. The TPS gene was upregulated in the early stage, and one of its functions could be to accumulate trehalose with the TPP gene, thus inducing the expression of downstream stress response related genes. The upregulation of the TPS gene expression in the later stage was regulated glycometabolism and glycoside through T6P, which was confirmed by the content of T6P and trehalose [14]. The accumulation of G6P and T6P, as the substrate and product of TPS catalysis, mainly showed opposite expression trends; however, the content of trehalose was short-changed after salt treatments, indicating that the ArTPS may be involved in increasing the T6P content in salt stress responses; while the accumulation of trehalose was increasedto counteract stress for the short-term, it could be maintained at a certain level for the long-term [23]. The other products from the glycometabolism were significantly changed under the NaCl stress, while its variation may differ in responses to stimulation. The content of kinsenoside was sharply upregulated, increasing T6P to regulate the polysaccharide and glycoside metabolism [15,23].
The overexpression of the ArTPS gene in E. coli demonstrated higher suitability compared to the control group when subjected to a stress condition of NaCl of 300 mmol L −1 stress. TPS genes play important roles in plant growth, development, and response to biotic and abiotic stresses [26,27]. AtTPS1 overexpression can enhance drought resistance in A. thaliana [35]. Overexpressing the gene encoding the bifunctional fusion of TPS and TPP genes from Escherichia coli in transgenic tomato plants improved drought and salt resistance and photosynthetic rates [36].

Sample Preparation
The A. roxburghii seedlings were surface-sterilized with 10% NaClO for 5 min. Then, they were plated onto MS media in a chamber with a 12 h light/12 h dark photoperiod at 28 • C and 60-80% relative humidity. The Hoagland nutrition solution was used to transplant the 4-month-old seedlings into an aquaculture plastic mesh grid. For stress conditions, 100 nm/L of NaCl was added to the nutritional solution on the sixth day.
Three biological replicates of the seedlings were collected at time points of 0 h (control), 0.

Cloning of the TPS Gene
The forward and reverse primer (5 -ATGCTGGGAAATAAGTACACGAG-3 /5 -TCAA ATACCATTGGAATAGCGGTGG-3 ) were designed to amplify the ArTPS gene open read-ing frame (ORF) from cDNA based on RNA-seq annotation. PrimeStar HS DNA Polymerase (TakaRa, Japan) was used to carry out the PCR amplification along with the proofreading process. The temperature cycle consisted of 38 cycles of 95 • C for 2 min, 98 • C for 30 s, 56 • C for 30 s, 72 • C for 150 s, and 72 • C for 5 min. The amplified product was purified using the Universal DNA Purification Kit (Tiangen, China), dATP was added to the sequence tails using the TaKaRa TaqTM (TakaRa, China), the amplified product was cloned into the pMD19-T vector (TakaRa, China), and Shanghai Sangon Biotech Co., Ltd. (China) sequenced the product.

Bioinformatic Analysis
The ArTPS gene sequencing was aligned for gene structure using NCBI blast (http://www.ncbi.nl-m.nih.gov accessed on 15 April 2023), and the same sequence was used for the analysis of physical and chemical properties, secondary structure, functional domains, and genetic structure of the putative proteins by using ProtParam

Vector Construction
A pair of specific primers (5 -cgggatccATGCTGGGAAATAAGTACACGAG -3 /5 -ccctcgagTCAAATACCATTGGAATAGCGGTGG -3 ) was designed with the introduction of the recognition sites (the lower-case bases in the bracket), and used to amplify the ORF of ArTPS without termination codon for homologous recombination by using Prime STAR HS DNA Polymerase (TaKaRa, Japan). The product was inserted into the expression vector pET-28a(+) using CloneExpress One Step Cloning Kit (Vazyme, China), to generate a set of expression vectors bearing fusion genes between the ArTPS genes ( Figure S3).

Protokaryotic Expression
pET-28a(+)-TPS was transformed into E.coli strain Rosetta (DE3) with empty prokaryotic pET-28a(+) as negative control and screened on a Luria-Bertani (LB) agar plate containing 50 mg L −1 ampicillin at 37 • C overnight. After confirmation by bacterial PCR and double digestion of the extracted plasmids with BamH I/Xho I, the positive single colonies were cultured in LB liquid medium containing 50 mg L −1 ampicillin at 37 • C. After 4 h, until OD600 = 0.6, the cultures were added with isopropyl β-D -thiogalactopyranoside (IPTG) to a final concentration of 1 mmol L −1 , and induced at 37 • C for 4 h. Then, each culture was adjusted to OD600 = 0.6 with the ampicillin LB liquid medium. Referring to He et al. (2012) and Udawat et al. (2014), each of the cultures was diluted by ten-fold serial to 1:104 with the ampicillin in LB liquid medium [37,38]. Two microliters of each dilution were spotted on the ampicillin LB agar plates containing NaCl of 300 mmol L −1 . After incubation for 16 h, the growth status of the colonies was observed.
So that a melting curve could be constructed and utilized to distinguish between specific and non-specific amplicons, the temperature was then raised by 0.5 • C/s to 95 • C. The internal reference and TPS gene expression were normalized using the 2 −∆∆CT method of the CFX Manger TM software version 2.0 (Bio-Rad, USA) [40]. The IBM-SPSS software (http://www-01.ibm.com/software/analytics/spss/ accessed on 15 April 2023) was used to evaluate the statistics.

G6P, T6P, and Glycometabolism Quantification
The seedling sample from 0 d, 4 d, and 16 d were dried at 55 • C, ground to fine powder, with 50 mg powder extracted in 500 mL 70% CH 3 OH. The extracts were through protein precipitation plate (MPPT9601A, Biocomma Limited). Two microliters of each of the filtrates were loaded into UPLC (Waters ACQUITY H-ClassD) equipped with ACQUITY UPLC BEH Amide column (1.7 µm, 2.1 × 100 nm) and MS/MS (QTRAP ® 6500+). Gradient elution was conducted with 5% mobile phase A (H 2 O with 10 mM CH 3 COONH 4 and 0.3% NH 3 ·H 2 O) and 95% mobile phase B (CH 3 CN) for 1.2 min, 30% mobile phase A and 70% mobile phase B for 8 min, 50% mobile phase A and 50% mobile phase B for 2 min, and 5% mobile phase A and 95% mobile phase B for 4 min at a flow rate of 1 mL/min and column temperature of 30 • C. And the MS/MS conditions included 550 • C of Elektrospray-Ionenquelle, 5500 V/−4500 V of the mass spectrometry voltage in positive/negative ion mode, and 35 psi Curtain Gas. Each ion pair was scanned through declustering potential and collision energy.

Kinsenoside Measurement
The seedling sample from 0 d, 4 dm and 16 d were dried at 55 • C, ground to fine powder, with 0.5 g powder extracted in 10 mL CH 3 OH in an ultrasonic at 25 • C for 30 min. The extracts were filtered through a 0.22 um filter membrane. Ten microliters of each filtrates were loaded into HPLC (Agilent 1260) equipped with Brownlee SPP C18 column (E-Merck, 2.7 µm, 4.6 × 150 nm). The elution was conducted with 10% CH 3 OH, 5% CH 3 CN, and 85% H 2 O as mobile phase at a flow rate of 1 mL/min and column temperature of 30 • C. The kinsenoside constituents were monitored at 215 nm and identified by comparison of retention time with their authentic samples. Their relative contents were quantified and expressed as percentage peak area.

Conclusions
The open reading frame sequences of the TPS genes were obtained from A. roxburghii. After bioinformatics analysis, the homology of the TPS proteins and conserved domains was found in their orthologs in related species. The overexpression of the ArTPS gene in E. coli showed higher tolerance to salt stress. Meanwhile, the relative expressions of the TPS genes were significantly higher under the NaCl stress. ArTPS gene expression not only affected plant stress resistance but also regulated the accumulation of glycometabolites and kinsenoside which is the characteristic substances of A. roxburghii through T6P.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28135139/s1, Figure S1: The fragments of the TPS gene from cDNA in A. roxburghii. Figure S2: Predicted three-dimensional models of the putative proteins of the TPS genes between (A) A.roxburghii, (B) D. chrysotoxum, and (C) P. equestris. Figure S3