Overexpression of the ThTPS gene enhanced salt and osmotic stress tolerance in Tamarix hispida

Peilong Wang · Xiaojin Lei · Jiaxin Lü · Caiqiu Gao

Abstract Trehalose is a non-reducing disaccharide with high stability and strong water absorption properties that can improve the resistance of organisms to various abiotic stresses.Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling.In this study, the full-length cDNA of ThTPS was cloned from Tamarix hispida Willd.A phylogenetic tree including ThTPS and 11 AtTPS genes from Arabidopsis indicated that the ThTPS protein had a close evolutionary relationship with AtTPS7.However, the function of AtTPS7 has not been determined.To analyze the abiotic stress tolerance function of ThTPS, the expression of ThTPS in T.hispida under salt and drought stress and JA, ABA and GA3 hormone stimulation was monitored by qRT-PCR.The results show that ThTPS expression was clearly induced by all five of these treatments at one or more times, and salt stress caused particularly strong induction of ThTPS in the roots of T.hispida.The ThTPS gene was transiently overexpressed in T.hispida.Both physiological indexes and staining results showed that ThTPS gene overexpression increased salt and osmotic stress tolerance in T.hispida.Overall, the ThTPS gene can respond to abiotic stresses such as salt and drought, and its overexpression can significantly improve salt and osmotic tolerance.These findings establish a foundation to better understand the responses of TPS genes to abiotic stress in plants.

Keywords Trehalose-6-phosphate synthase (TPS) · Tamarix hispida · Salt tolerance · Osmotic resistance

Introduction

Trehalose is a non-reducing disaccharide composed of two glucose molecules linked by α,α 1-1 glycosidic bonds (Elbein et al.2003; Bansal et al.2013).It was first discovered in bacteria by Wiggers in 1832, and the French chemist Berthelot subsequently discovered it in a sweet substance secreted by weevils in the Asia Minor Desert and named it trehalose (Drennan et al.1999).Trehalose is widely found in various organisms such as bacteria, yeasts, molds, edible fungi, insects, lower plants such as algae and mosses, as well as some higher plants (Drennan et al.1999; Goddijn and Dun 2006; Satoh-Nagasawa et al.2006; Schluepmann et al.2012; Jin et al.2018; Alicandri et al.2020).

Trehalose can increase the resistance of organisms to adverse environmental conditions and the resistance by many species is directly related to the concentrations of trehalose in their tissues (Crowe et al.1984; Hottiger et al.1994).Trehalose is a typical stress metabolite (Tang et al.2018).When an organism grows under suitable conditions, it does not accumulate trehalose but when it is under stress (starvation, drought, high temperatures and salinity), trehalose rapidly accumulates (Laere 1989; Wiemken 1992).Trehalose molecules become degraded when the adverse conditions are relieved.In addition, increased trehalose has clear protective effects on active substances such as proteins, enzymes and cell membranes (Reshkin et al.1988).In plants, trehalose is an important substance regulating diverse processes such as development (Chary et al.2008; Van Houtte et al.2013; Wahl et al.2013), response to biotic stresses (Reignault et al.2001; Brodmann et al.2002; Foster et al.2003; Govind et al.2016), and abiotic stresses (Zhang et al.2006; Li et al.2011; Henry et al.2015; Ritonga and Chen 2020).

The synthesis of trehalose in plants is based on the formation of trehalose-6-phosphate (Tre6P) from UDPglucose and glucose-6-phosphate catalyzed by trehalose-6-phosphate synthase (TPS), and Tre6P was then catalyzed by trehalose-6-phosphate phosphatase (TPP) to produce trehalose.However, when trehalose is synthesized in plants, there are many non-phosphatase enzymes that can catalyze Tre6P into trehalose, and plants can directly catalyze the dephosphorylation of Tre6P to produce trehalose without TPP (Goddijn and Dun 2006).Nevertheless, the TPS protein has an irreplaceable role in plant trehalose synthesis, and the successful transcription and expression of theTPSgene is required for the synthesis of trehalose.

Eleven TPS homologs have been found in theArabidopsis thaliana(L.) Heynh.genome (Kolbe et al.2005).These 11AtTPSgenes are classified into two classes (Avonce et al.2006).Class I includes theTPS1-4genes and is closely related to the yeastTPS1gene, whereas class II includes theTPS5-11genes and contains sequences corresponding to phosphatase and synthase domains (Paul et al.2008; Schluepmann and Paul 2009).In addition, eightTPSgenes were identified in potato, 53 in cotton, 20 in soybean and 9 inP.campanulatagenome (Xie et al.2014; Mu et al.2016; Xu et al.2017; Huang et al.2019).TPS genes have also been cloned in other plants such asSaccharum officinarumL.(Zhang et al.2006) and in fungi, includingPlasmodiophora brassicae(Brodmann et al.2002) andMagnaporthe grisea(Foster et al.2003).SomeTPStransgenic plants significantly improve abiotic stress tolerance.For example, theA.thaliana TPS1gene enhanced osmotic, drought, desiccation and temperature stress resistance of transgenic tobacco (Andre et al.2005).Transformation of the yeastTPS1gene into potato significantly improved the drought resistance of transgenic plants (Yeo et al.2000; Stiller et al.2008).Garg et al.(2002) transferred the trehalose synthesis genes (otsAandotsB) ofEscherichia coliinto rice and reported improved salt, drought and lowtemperature stress tolerance.Jang et al.(2003) showed that transformation of theE.colitrehalose synthase gene into rice can increase trehalose accumulation and tolerance to drought, high salt and cold.Overexpression of theTPS1gene in sorghum enhanced tolerance to salt stress (Yellisetty et al.2015), and transformation of theGrifola frondosa Fr.TPSgene into tobacco enhanced resistance to drought and salt (Zhang et al.2005).

Tamarix hispidaWilld., the Kashgar tamarisk, is a woody halophyte adapted to grow in saline conditions with well-developed roots and strong sprouting ability.The species has strong resistance to drought, cold, salt and alkaline conditions, making it a good species for sandy soils.It is an ideal species for resistant gene cloning and for the study of stress resistance mechanisms.In this study, theThTPSgene was cloned fromT.hispida, and its sequence characteristics and expression pattern after abiotic stress were analyzed.In addition,ThTPSwas transiently overexpressed inT.hispida.Physiological indexes were determined and staining analysis was carried out; the results were compared between plants overexpressingThTPSand controlT.hispidaunder salt and osmotic stress.This study will establish a theoretical foundation to further analyze the stress tolerance functions of theTPSgene and use genetic engineering to improve plant stress resistance.

Materials and methods

Plant material and stress treatment

Tamarix hispidaseeds (The Turpan Desert Botanical Garden, Xinjiang, China) were sown in a plastic basket with culture medium (vegetative flower soil: vermiculite=1:1).The greenhouse culture conditions were humidity 70-5%, average temperature 25 °C and photoperiod 14 h/10 h.The seedlings when approximately 5 cm, were moved to pots and cultured in the greenhouse.After two months, separate groups of seedlings were irrigated with 0.4 mol/L NaCl, 20% (w/v) PEG6000, 150 μmol/L ABA, 50 μmol/L GA3 and 100 μmol/L JA.Normally watered seedlings were used as a control.After 6, 12, 24, 48 and 72 h of treatment, the roots and leaves of each group were collected separately, immediately frozen in liquid nitrogen, and stored in at-80 °C for subsequent experiments.All treatments were replicated three times.

Bioinformatics analysis of the ThTPS gene in T.hispida

Through searching the transcriptome data ofT.hispidausing "trehalose-6-phosphate synthase" as a key word, a full-lengthThTPSgene sequence was obtained.Through BLASTx alignment (https://blast.ncbi.nlm.nih.gov/Blast .cgi) of theThTPSsequence and ORFfinder (https://www.ncbi.nlm.nih.gov/orffi nder/) analysis, the open reading frame (ORF) and amino acid sequence of theThTPSgene were identified.The relative molecular mass and theoretical isoelectric point of the ThTPS protein were identified through ProtParam software, and the domains of the ThTPS protein identified using the InterProScan online tool.The ThTPS amino acid sequences were aligned using the BLASTP program in NCBI to obtain nine other plant TPS proteins with higher homology, includingZiziphus jujuba,Coffea eugenioidesS.Moore,Quercus suberL.,Vitis viniferaL.,Juglans regiaL.,Gossypium raimondii,Actinidia chinensisvar.chinensis,Camellia fraternaHance andChenopodium quinoaWilld, and multiple sequence alignment analysis was carried out using ClustalX 1.83.In addition, the amino acid sequences of 11 TPS proteins in theArabidopsisfamily were selected and phylogenetic tree construction analysis performed using MEGA 5.0 software.Through MEME analysis, the conserved motifs of the ThTPS protein andArabidopsisTPS family members were analyzed.

RNA extraction and qRT-PCR analysis

The RNA of each sample was extracted using a plant RNA extraction kit (BioTeke, Beijing, China) and concentrations and masses were measured using a NanoVue microphotometer and 0.8% agarose gel electrophoresis.The total RNA of each sample was reverse transcribed into cDNA using TransScript One-step gDNA Removal and cDNA Synthesis SuperMix.qRT-PCR was carried out using theactin(FJ618517),α-tubulin(FJ618518) andβ-tubulin(FJ618519) genes as internal controls (reference genes).The internal controls andThTPSgene primer sequences are shown in Table 1.The qRT-PCR system consisted of 10 μl SYBR Green Mix, 1 μl each of the forward and reverse primers (10 μmol/L), cDNA 2.0 μl, and ddH2O supplemented to 20 μl.The reaction procedure was 95 °C for 3 min and 45 cycles of 95 °C for 20 s, 58 °C for 15 s, and 72 °C for 30 s.Each sample was repeated three times.The qRT-PCR experiments were performed using an Opticon Monitor 2 real-time PCR machine (Bio-Rad, USA), and data analyzed using the 2-ΔΔ(Ct)method (Livak and Schmittgen 2001).

Table 1 Primer sequences of real-time RT-PCR

ThTPS gene cloning and plant overexpression vector construction

To insert the gene sequence ofThTPSinto the multiple cloning site of the plant overexpression vector pROKII, Xba I and Kpn I restriction endonuclease sites were introduced at the 5′ and 3′ ends of theThTPSgene, respectively.The primers TPS-CF (5′CTA GTC TAG AATG ATG TC CAGA TC TTA TAC C3′) and TPS-CR (5′CGGG GT AC CCTA AG AGG GGC TGC CGC TAC3′) were used to obtain theThTPSgene by RT-PCR amplification.The digested gene and the vector fragment were then ligated and transformed into Top10E.colicompetent cells by the heat shock method.After culturing at 37 °C for 8-12 h, single colonies were picked for PCR verification using vector and gene primers.The strains with the correct fragment sizes were sent for sequencing analysis.The sequenced, correct overexpression vector strain was designated pROKII-ThTPS,and the plasmid was transformed intoAgrobacterium tumefaciensEHA105 to obtain an overexpression strain.

Transient transformation of the ThTPS overexpression vector into T.hispida and stress resistance analysis

According to the method of Zhang et al.(2018), the pROKIIThTPSoverexpression strain (OE) and the pROKII empty vector (Con) strain were transiently transformed intoT.hispida.After culturing in MS medium for 24 h, separate groups ofT.hispidaseedlings were again cultured in MS with 150 mM NaCl and 200 mM mannitol for 12, 24 and 36 h.Seedlings cultured on normal MS medium were used as controls.The RNA of each sample was extracted, and the expression of theThTPSgene analyzed by qRT-PCR.Furthermore, the physiological indexes of each sample, including MDA, chloroplast, trehalose (plant trehalose, trehalose determination by ELISA kit), and H2O2(hydrogen peroxide assay kit, NanJing Jiancheng, China) content were measured.Nitro blue tetrazolium chloride (NBT), diaminobenzidine (DAB) and Evans blue staining analyses were carried out after the seedlings had been treated for 12 h.Each experiment contained three replicates.

Results

Sequence and evolutionary analysis of the T.hispida ThTPS gene

A full-lengthThTPSgene sequence was obtained from theT.hispidatranscriptome.The GenBank number was MN615274.The ORF of theThTPSgene was 2577 bp and encoded 858 amino acids.ProtParam predicted that the molecular weight of the ThTPS protein was 96.81 kDa with a theoretical isoelectric point of 5.86, indicating an acidic protein.The ThTPS protein belongs to the HAD hydrolase IIB subfamily (IPR006379) and contains a glycosyltransferase domain (IPR001830, 61-544) and a HADlike domain (IPR023214, 591-842).The multiple sequence alignment indicated that ThTPS had high sequence homology (83.4-6.4%) to the selected amino acid sequences (Fig.1a).The phylogenetic tree, including the ThTPS protein sequence and 11 TPS members in theArabidopsisfamily, indicated that the ThTPS protein belongs to the class II subfamily and had a close evolutionary relationship with AtTPS7 (Fig.1b).Similar to other class II members, ThTPS contains three conserved motifs (motif 1, motif 2 and motif 3).

Fig.1 Multiple sequence alignment and evolution analysis of ThTPS gene.a Multiple sequence alignments of ThTPS protein sequence and other TPS proteins were performed using ClustalX 1.83.The conserved motifs are indicated by box.b Phylogenetic tree constructed with the neighbor-joining method using ThTPS protein and Arabidopsis TPS family proteins; MEME analysis of these sequences were performed

Expression analysis of the ThTPS gene under different treatments in T.hispida

To preliminarily analyze the function of theThTPSgene, we examined its expression levels in the roots and leaves ofT.hispidaunder treatments with 150 μmol/L ABA, 100 μmol/L JA, 50 μmol/L GA3, 0.4 mol/L NaCl and 20% (w/v) PEG6000by qRT-PCR.The results show thatThTPSexhibits different expression patterns under hormone, salt and drought treatments (Fig.2).

Under ABA treatment, the expression of theThTPSgene was upregulated at 6 h and 48 h, reaching 54.7-and 3.1-fold that of the control, respectively.At 12 h, 24 h and 72 h, it showed a downward trend in roots.In leaves,ThTPSshowed an opposite expression pattern from that in roots (Fig.2a).Under JA treatment, theThTPSgene showed an upregulated expression in roots, reaching the highest level, which was ten times that of the control at 6 h.While this gene mainly showed a downregulated expression in leaves, it reached its lowest point which was 5.9% of the control at 12 h (Fig.2b).Under GA3 treatment, the expression of theThTPSgene showed upregulation at 12 h and 48 h in roots, reaching almost 10 times and more then 30 times, respectively, that of controls.At 24 h, the gene expression changed little but was downregulated at 6 h and 72 h.In leaves, theThTPSgene showed the opposite trend (Fig.2c).

Under NaCl stress, in addition to a downward trend at 6 h, theThTPSgene showed a trend of upregulation at other times in roots and leaves, reaching the highest values of 361.33 and 7.55 times the controls at 24 h and 48 h, respectively (Fig.2d).After PEG6000stress, the expression ofThTPSgene showed upregulation at 6 h and 12 h, while down regulation after 12 h in roots and the expression ofThTPSgene in leaves showed the opposite expression trend from roots (Fig.2e) These results show that theThTPSgene can respond to the five stress treatments but the expression patterns were not exactly the same.

Fig.2 Expression analysis of ThTPS gene in T.hispida under a 150 μmol/L ABA, b 100 μmol/L JA, c 50 μmol/L GA3, d 0.4 mol/L NaCl and e 20% (w/v) PEG6000 treatment; each sample has three replicates; data were treated by 2-ΔΔ(Ct)

ThTPSoverexpression inT.hispidacan enhance salt and osmotic tolerance.

To further explore the stress resistance function of theThTPSgene, pROKII-ThTPS(OE) and pROKII (control) were transiently transformed intoT.hispida, and the OE and the control treated with 150 mM NaCl and 200 mM mannitol stress.The expression level of theThTPSgene in the OE plants was 100.3, 3.5, and 13.8 times that of the control after NaCl treatment for 12, 24 and 36 h, respectively.After mannitol treatment for 12, 24 and 36 h, the expression levels were 23.6, 10.1 and 13.9 times that of the controls, respectively.These results indicate that transient transgenicT.hispidahad been successfully obtained, and the expression level of theThTPSgene was higher after 12 h of treatment (Fig.3a).

Based on the results of qRT-PCR, DAB, NBT and Evans blue staining and physiological indexes were performed for the transiently transformedT.hispidatreated with NaCl and mannitol for 12 h.The results show that there were no color or physiological differences between the OE and control plants under normal conditions.However, the DAB and NBT staining colors of OE plants were lighter than those of the control under NaCl and osmotic stress (Fig.3b).Correspondingly, H2O2contents were measured and compared.In OE plants after NaCl and mannitol stress, the H2O2contents were 4.1-and 5.1-times those under normal conditions, while in control plants, it increased 6.5-and 6.96-times (Fig.3c).These results indicate that H2O2or O2-contents were lower in OE plants than in the controls under these two stress conditions.The Evans blue staining results show that the OE plants were a lighter color than the controls (Fig.3b).At the same time, the MDA contents in OE plants were 3.14-and 3.1-times those in normal conditions, while it was 4.7-and 3.55-times increased in the controls plants (Fig.3c), indicating that the cells of the OE plants were less damaged.In addition, the cell protection product trehalose increased 3.98-and 4.42-fold in OE plants, higher than that in the controls.The plant growth indicator, chlorophyll, decreased 33% and 26% in OE plants while it decreased 54% and 50% in the controls after NaCl and mannitol stress, respectively (Fig.3c).These results show that overexpression of theThTPSgene can enhance the ability of transgenicT.hispidacells to scavenge reactive oxygen species (ROS) and promote lower accumulations of O2-and H2O2, thereby reducing cell death and enhancing the salt and osmotic tolerance ofT.hispida.

Fig.3 Resistance tolerance analysis of the ThTPS gene in instantly overexpression T.hispida under NaCl and mannitol treatments; a qRT-PCR analysis and the control under NaCl and mannitol at different stress times; Each sample replicated three times and the data treated by 2-ΔΔ(Ct); b Chemical staining of T.hispida under 12 h stress; c related physiological analysis of T.hispida under 12 h stress

Discussion

Trehalose accumulates in plants and exerts a protective function to improve specific undesirable traits.Trehalose-6-phosphate synthase (TPS), a gene related to stress resistance, in addition to glutamate, proline and betaine synthases (Liang et al.1997), functions in catalyzing the conversion of substrate to trehalose.In this study, aThTPSgene was cloned fromT.hispida.Sequence analysis showed that the ThTPS protein contained a glycosyltransferase domain and a HAD-like domain.Multiple sequence alignment showed that the ThTPS protein and other TPS proteins have conserved motifs at the N-terminus and C-terminus, indicating that the TPS protein is highly conserved in different plants.Phylogenetic analysis including theA.thalianaTPS family showed that the ThTPS protein was closely related to theArabidopsisfamily member AtTPS7 and belonged to the class II subfamily.

Class II subfamily members may have a regulatory function transcriptionally by carbon status and stress (Schluepmann et al.2012).In recent years, several approaches have found that theA.thalianaTPS class II subfamily proteinAtTPS6regulates plant architecture, epidermal pavement cell shape, and trichome branching (Chary et al.2008).AtTPS5plays a role in thermotolerance through its interaction with the transcriptional coactivator MBF1c (Suzuki et al.2008).However, there have been no reports describingAtTPS7function.Other class II TPS proteins appear to lack significant enzymatic activity and many are extensively regulated by hormones, light and nutrient availability at the transcriptional level (Wang et al.2003; Contento et al.2004; Scheible et al.2004; Brenner et al.2005; Baena-Gonzalez et al.2007; Osuna et al.2007; Usadel et al.2008).

qRT-PCR results show that the expression of theThTPSgene inT.hispidawas significantly changed after high salt, drought, JA, ABA and GA3 treatments.After JA, ABA and GA3 treatments, the expression of theThTPSgene mainly showed opposite expression trends in roots and leaves, except at specific times.However, after NaCl and PEG6000treatments, the expression was mainly upregulated in roots and leaves, indicating that it may be involved in drought, salt stress and hormone stimulation responses, while its function may differ in root and leaf responses to hormone stimulation.It has been shown thatAtTPS1participates in the Glc and ABA signaling pathways controlling germination and vegetative development (Avonce et al.2006).Overexpression of theTSasegene from the mushroom,Grifola frondosa(Dicks.) Gray, reduced water loss, cell damage and photosynthesis and preserved superoxide dismutase (SOD) and peroxidase (POD) activities, thereby improving drought tolerance in sugarcane (Zhang et al.2006).OsTPS1-overexpressing rice seedlings showed improved tolerance to cold, high salinity and drought without other significant phenotypic changes (Li et al.2011).In this study, the physiological indicators and staining results of plants transiently overexpressingThTPSalso showed that O2-and H2O2levels were lower in OE plants and that the plant cells were less damaged.This finding indicates that overexpression of theThTPSgene can increase salt and osmotic tolerance byT.hispida.In future experiments, the specific resistance mechanism of theThTPSgene will be examined.

Conclusions

The TPS protein is an irreplaceable protein in plant trehalose synthesis and theTPSgene is successfully transcribed and expressed in plants.In this study, theThTPSgene was cloned fromT.hispida.The expression of theThTPSgene could be induced by drought, salt stress and hormone stimulation, revealing that it may play a vital role in plant responses to stresses and hormone stimulation.Overexpression of the gene inT.hispidapromoted the biosynthesis of trehalose, decreased the accumulation of O2-and H2O2, and therefore enhanced salt and osmotic tolerance.These results help to establish a foundation for the study of TPS functions and research onT.hispidastress resistance.

Author contributionsWPL carried out all the experiments and data analysis.WPL and GCQ conceived the project, designed the experiments and drafted the manuscript.GCQ supervised the analysis and critically revised the manuscript.LXJ and LJX provided help during the experimental process.All authors read and approved the final manuscript.

Compliance with ethical standards

Conflicts of interestThe authors declare no conflicts of interest.