Genome-wide identification and expression analysis of the trehalose-6-phosphate synthase (TPS) gene family in cucumber (Cucumis sativus L.)

Genome-wide identification and bioinformatics analysis

We downloaded the whole genome in the gff, cds, pep and FASTA file format from the EnsemblPlants- Cucumber genome database (ASM407v2) (http://plants.ensembl.org/index.html) (Li et al., 2019b). The TPS (Glyco-transf-20, PF00982) and TPP (Trehalose_PPase, PF02358) domains’ hidden Markov Models (HMM) were downloaded from the Pfam database (http://pfam.xfam.org) (Liu et al., 2019a). HMMsearch software and the TPS and TPP’s HMMs were used to search all possible TPS candidate sequences containing typical TPS and TPP domains under a Linux system (Chen et al., 2019). Pfam (Liu et al., 2019a) and NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) (Yan, Li & Zhao, 2019) databases were used to manually confirm that the candidate sequences had complete TPS and TPP domains. The remaining genes were subsequently identified as members of the cucumber TPS family and were named according to their location on the cucumber’s chromosome.

The length of open reading frames (ORFs) of cucumber TPS genes was predicted using the NCBI-ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) website (Xie et al., 2015). The physicochemical properties and subcellular locations of the cucumber TPS proteins were forecast using Protparam (https://web.expasy.org/protparam/) (Zhang et al., 2019) and the Cell-PLoc2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) website, respectively.

The cucumber TPS proteins’ secondary structures were determined using the PRABI (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) website (Xu et al., 2016).

Phylogenetic analysis

The phylogenetic tree, containing seven cucumber, 11 rice (Zang et al., 2011), 11 Arabidopsis (Yang et al., 2012) and 20 soybean (Xie, Wang & Huang, 2014) TPS protein sequences, was constructed based on multiple sequence alignments using Fasttree software and the maximum likelihood method. The bootstrap replication value was set as 1,000 and the other parameters remained constant. We used the evolview (https://evolgenius.info//evolview-v2/#login) website to improve the appearance of the evolutionary tree. The TPS protein sequences of Arabidopsis, rice, and soybean were downloaded from TAIR (https://www.arabidopsis.org/) (Song et al., 2019), the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) (Zang et al., 2011), and the Phytozome (https://phytozome.jgi.doe.gov) (Yue et al., 2019) database, respectively (Supplemental File 1).

Gene structure, chromosomal location and cis-acting element analysis

The GSDS2.0 (http://gsds.gao-lab.org/index.php) (Li et al., 2019a) website was used to analyze the gene structure of cucumber TPS genes and to plot the exon-intron diagram. We used the TBtools software to combine the evolutionary tree with the gene structure diagram and Mapchart software was applied to visualize the location of the genes on the chromosomes. The 2kb sequences in the cucumber TPS genes’ upstream region were screened as promoter sequences using Tbtools software. We used plantCARE (Song et al., 2019) (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to investigate the cis-acting elements in promoter regions to study the roles of genes in stress and hormone responses.

Conserved motifs analysis

We searched the MEME (http://meme-suite.org/) (Liu et al., 2019c) website for conserved motifs of cucumber TPS proteins. The maximum retrieval value for the motif was set to 10 and the other parameters were set to default. InterProScan software was used to annotate the retrieved motifs.

GO annotation

We used the EggNOG mapper software (http://eggnog-mapper.embl.de/) to perform the gene ontology analysis. Cucumber TPS protein sequences were uploaded and Arabidopsis TPSs were used as the reference. GO analysis was categorized as: molecular function (MF), biological process (BP), and cellular component (CC).

Plant materials, cultivation conditions and treatments

Cucumber seeds (C. sativus L. “Xin Chun 4”) were germinated and grown in culture dishes with wet filter paper. Seedlings were transferred into hydroponic boxes once the cotyledon fully unfolded. The boxes contained Yamazaki cucumber nutrient solution and were placed in plant incubators at 25 °C, a light intensity of 200 μmol.m⁻²s⁻¹, and a photoperiod of 14 h light/10 h dark (Niu et al., 2019). The nutrient solution was replaced every two days in order to maintain an adequate level of nutrients. Stress treatments were carried out when seedlings were at the two-leaf stage. Seedlings were grown in a 1/2 nutrient solution containing 8% (w/v) PEG, 50 mM NaCl, 1 μM IAA, 8% (w/v) H₂O₂, 50 mM sucrose and 50 mM mannitol, respectively, for drought, salt, IAA, H₂O₂, sucrose and mannitol treatments. The concentrations of these reagents were determined by a preliminary experiment. Whole seedlings were frozen with liquid nitrogen after treatment at 0, 6, 12 and 24 h, and were stored at −80 °C (Zhu et al., 2019). The roots, stems, and leaves of untreated seedlings were collected at the two-leaf stage and were stored at −80 °C to investigate the expression of cucumber TPS genes in the different tissues. Each treatment was performed with three biological replicates and each sample was collected from five cucumber seedlings.

RNA extraction, reverse transcription and quantitative real-time PCR

The total RNA from different tissues and whole seedlings under different treatments were extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China). The RNA concentration and purity were determined using the NaNo drop 1,000 spectrophotometer and agarose gel electrophoresis (Xie et al., 2018). The FastQuant first strand cDNA synthesis kit (TIANGEN, Beijing, China) was used for the synthesis of cDNA following the manufacturer’s protocol. The SuperReal PreMix Plus kit (TIANGEN, Beijing, China) and a Roche LightCycler instrument were used for qRT-PCR. The reaction system of qRT-PCR was as follows: 10 μL 2×SuperReal PreMix Plus, 0.6 μL 10 μM forward primers, 0.6 μL 10 μM reverse primers, 2 μL cDNA and 6.8 μL RNase-free ddH₂O. The qRT-PCR procedure was as follow: 95 °C for 15 min and 40 cycles of 95 °C for 10 s and 60 °C for 20 s. CsActin was used as an internal reference gene (Zhou et al., 2017). The primers of the cucumber TPS genes and CsActin for qRT-PCR were designed and synthesized using Sangon Biotech online software (Table 1). Three technical replicates were performed for each reaction.

Statistical analysis

The relative expression of the genes was calculated using the 2^−ΔΔCt method (Han et al., 2020). The expression of the CsTPS genes in the roots was used as a calibration sample to calculate their relative expressions in the stem and leaf (Zhang et al., 2014). The expression of CsTPS genes in untreated seedlings (0 h) was used as a calibration sample to calculate their relative expression at 6, 12 and 24 h. We adopted Duncan’s (p < 0.05) method for significance analysis.

Genome-wide identification of TPS family in cucumber

Seven TPS sequences were identified in the cucumber genome using bioinformatics methods. The Pfam and NCBI-CDD databases were used for domain analysis to further prove the reliability of these candidate sequences. Our results indicated that all seven sequences had a typical TPS domain and belonged to the cucumber TPS family. The cucumber TPS genes identified were named CsTPS1-CsTPS7 according to their location in the cucumber chromosome (Table 2).

Domain analysis revealed that all cucumber TPS proteins contained a typical TPS (Glyco_transf_20; Pfam: PF00982) domain in the N-terminal and a TPP (Trehalose_PPase; Pfam: PF02358) domain in the C-terminal (Table 2). CsTPS3, CsTPS5, and CsTPS6 proteins all contained an additional Hydrolase-3 (Pfam: PF08282) domain (Supplemental File 2).

Physical and chemical property analysis revealed that the length of the open reading frame of the CsTPS genes was between 2,553 bp (CsTPS6) and 2,916 bp (CsTPS7); the length of cucumber TPS proteins was between 850 (CsTPS6) and 971 (CsTPS7) amino acids; and their molecular weight was between 96.31 KDa (CsTPS6) and 110.33 KDa (CsTPS7). The isoelectric point (pI) ranged from 5.50 (CsTPS6) to 7.02 (CsTPS2). CsTPS2 was the only alkalescence (pI > 7) in the cucumber TPS proteins and the rest were acidic (pI < 7) (Table 2).

The subcellular localization prediction indicated that cucumber TPS genes were mainly distributed in the vacuole, chloroplast, and cytoplasm (Table 2).

The analysis of the secondary structure showed that cucumber TPS proteins were composed of an α-helix, random coil, extended strand and a β-turn (Table 3).

Phylogeny and gene structure analysis of CsTPSs

Seven cucumber TPS proteins were divided into two subfamilies, Class I and Class II, based on the results of previous studies (Lunn, 2007; Mu et al., 2016). Among the seven CsTPS proteins, CsTPS2 and CsTPS4 were classified as Class I and the remaining five proteins were classified as Class II (Fig. 1A). In addition, the exon-intron diagram revealed that two members (CsTPS2 and CsTPS4) of Class I possessed 17 and 16 introns, respectively. In Class II, only CsTPS7 had six introns, while the other members all contained two introns (Fig. 1B). We inferred that two subfamilies went through functional differentiation in the course of evolution (Du et al., 2017).

We aligned the full-length protein sequences of seven cucumber TPS proteins, 11 Arabidopsis TPS proteins (Yang et al., 2012), 11 rice TPS proteins (Zang et al., 2011) and 20 soybean TPS proteins (Xie, Wang & Huang, 2014) to establish a maximum likelihood phylogenetic tree (Fig. 2) to further investigate the evolutionary relationships of TPS family in various species. As previously described, the TPSs in cucumber, Arabidopsis, rice, and soybean may be differentiated into two subfamilies: Class I and Class II. The two subfamilies were further divided into seven subgroups: I-1, I-2, II-1, II-2, II-3, II-4 and II-5, based on the phylogenetic relationship with high bootstrap support (Xie et al., 2015). Subgroup I-1 contained nine members originating from Arabidopsis (1), rice (1), cucumber (2) and soybean (5), respectively. I-2 contained three members which all originated from Arabidopsis. II-1 contained nine members which were derived from Arabidopsis (2), rice (2), cucumber (1), and soybean (4), respectively. II-2 contained four members which derived from cucumber (1) and soybean (3), respectively. II-3 contained ten members which originated from Arabidopsis (3), rice (2), cucumber (1), and soybean (4), respectively. II-4 contained six members which originated from Arabidopsis (1), rice (3), and soybean (2), respectively. II-5 contained eight members which derived from Arabidopsis (1), rice (3), cucumber (2), and soybean (2), respectively. With the exception of I-2, II-2 and II-4, the other subgroups contained at least one member of four species. Our results showed that some of the TPS proteins of cucumber and soybean were divided into the same subgroup, indicating that they were closely related. In addition, previous studies have shown that most of TPS genes in the Class I had 16 introns, while TPS genes in the Class II possessed 2 introns (Yang et al., 2012). When combined with the analysis of the cucumber TPS gene structure, we can speculate that the genes of the same group were close to each other during the evolution process, while the genes of different groups were far away from each other during the evolution process (Xie et al., 2015).

Conserved motifs of cucumber TPS proteins

We used the MEME online website to study the characteristic regions of cucumber TPS proteins. We searched 10 conserved motifs in cucumber TPS proteins (Fig. 3). The lengths of these conserved motifs were between 29 and 50 amino acids and the sequence information of these 10 conserved motifs is listed in Table 4. Cucumber TPS proteins classified into the same subfamily in the evolutionary tree possessed a similar or identical motif composition (Ou et al., 2018). For instance, members of Class II possessed all 10 conserved motifs, while members (CsTPS2 and CsTPS4) of Class I lacked motifs 8 and 9. Motifs 4, 5, 1, 9, 6, 7 and 3 together constituted the TPS domain and motifs 10, 2 and 8 constituted the TPP domain, according to annotation. These results further supported the reliability of the phylogenetic classification of cucumber TPS proteins and indicated that the two subfamilies may have produced a functional difference during evolution (Xie et al., 2018).

Chromosomal location of cucumber TPS genes

We used Mapchart software to analyze the location of CsTPS genes on cucumber chromosomes. The results showed that seven CsTPS genes were randomly located on six cucumber chromosomes (Fig. 4). Two genes (CsTPS1 and CsTPS2) were located on chromosome 1, while only single genes existed on the other chromosomes. The majority of the CsTPS genes were located on the proximate or distal ends of the cucumber chromosomes (Zhao et al., 2018).

Cis-acting element analysis of cucumber TPS genes

A total of 72 types of elements were found in the promoter regions of the CsTPS genes. All CsTPS genes contained CAAT and TATA boxes in the promoter regions, which were core and common promoter elements (Zhou et al., 2017) (Supplemental File 3). The cis-acting elements were identified and categorized as stress-related elements and plant hormone-responsive elements (Zhu et al., 2019). Stress-related elements, including MYB (stress response element), MYC (the recognition site of cold-resistant element), and ARE (anaerobic induction element) were mainly found in promoter regions of most CsTPS genes (Zhou et al., 2017). Some plant hormone-responsive elements, including ERE (ethylene-responsive element), ABRE (abscisic acid responsive element), TCA-element (salicylic acid-responsive element), and TGA-element (auxin-responsive element) (Han et al., 2020) were also found in most CsTPS genes (Figs. 5 and 6). Light responsive elements including Box4, AE-box, G-Box and GATA-motif were widely found in promoter regions of cucumber TPS genes (Fig. 5 and Supplemental File 3). Our results indicated that TPS genes played a significant role in stress, hormone and light response (Zhao et al., 2018).

GO annotation of cucumber TPS proteins

We found that a majority of cucumber TPS proteins participated in UDP-glycosyltransferase activity, hydrolase activity, phosphatase activity, catalytic activity, and alpha-trehalose-phosphate synthase activity (Supplemental File 4). CsTPS2 and CsTPS4 were involved in transferase activity and phosphoric ester hydrolase activity. Cellular component analysis showed that most CsTPS proteins were located on the cytoplasm and cytosol. However, CsTPS2 and CsTPS4 were also located intracellularly. Biological process analysis indicated that most CsTPS proteins were involved in various biological processes, including metabolism, biosynthesis, cellular processes, and development. Some CsTPS proteins also participated in the stress response, signal transduction, post-embryonic development, and seed, fruit and reproductive system development.

Expression analysis of cucumber TPS genes in different tissues

We measured the expression of CsTPS genes in root, stem and leaf using qRT-PCR to determine the expression specificity of cucumber TPS genes in different tissues. The expression of CsTPS genes was detected in the root, stem and leaf (Fig. 7) and their expressions were high, moderate, and low in the root, leaf and stem, respectively. Our results indicated that CsTPS genes may play a specific role in the growth of cucumber seedlings.

Expression analysis of cucumber TPS genes under different treatments

We conducted qRT-PCR experiments and drew a cluster heatmap to determine the expression patterns of CsTPS genes under various treatments (Fig. 8). Our results indicated different patterns for the expression of CsTPS genes with various treatments. Under PEG, the expression of CsTPS3 was up-regulated significantly and peaked at 24 h and CsTPS2 and CsTPS5 were up-regulated slightly. However, CsTPS1, CsTPS4, CsTPS6 and CsTPS7 were down-regulated and reached their lowest expression levels at 24 h. The expression of CsTPS3 and CsTPS4 was up-regulated significantly with NaCl treatment and reached the highest expression level at 24 h; the expression of CsTPS2 and CsTPS7 also increased. However, CsTPS1 and CsTPS6 were down-regulated with the NaCl treatment and reached the lowest expression level at 24 h. Under H₂O₂ treatment, CsTPS1 and CsTPS5 were activated while others genes were inhibited (CsTPS2, CsTPS3, CsTPS4 and CsTPS6) or did not show obvious trends (CsTPS7). Under mannitol treatment, CsTPS3 and CsTPS7 were up-regulated with the highest expression level at 24 h and CsTPS1 and CsTPS4 were slightly induced. Conversely, the mannitol treatment caused a large decline in the expression of CsTPS2, CsTPS5 and CsTPS6. Under IAA treatment, CsTPS3 and CsTPS4 were up-regulated significantly, whereas others genes showed no clear trends. CsTPS3 and CsTPS4 showed a strong expression under sucrose treatment, whereas CsTPS1 and CsTPS7 were induced slightly by sucrose. CsTPS2, CsTPS5 and CsTPS6 were inhibited by sucrose.

These results showed that CsTPS genes may be involved in stress, hormone, and sugar responses.