Genome-wide identification of the trehalose-6-phosphate synthase gene family in sweet orange (Citrus sinensis) and expression analysis in response to phytohormones and abiotic stresses

Identification of TPS gene family in sweet orange

The candidate TPS protein sequences in sweet orange (C. sinensis) were downloaded from Phytozome v13 (https://phytozome-next.jgi.doe.gov) (Goodstein et al., 2012). Then, the TPS (Glyco-transf-20, PF00982) and TPP (Trehalose_PPase, PF02358) domains were predicted using the SMART website (http://smart.embl-heidelberg.de) and the National Center for Biotechnology Information Conserved Domain Database (NCBI-CDD; https://www.ncbi.nlm.nih.gov/cdd) (Lu et al., 2020; Letunic, Khedkar & Bork, 2021), and proteins lacking the TPS domain were removed.

The TPS cDNA sequences were used as queries to search the C. sinensis genome database at NCBI to confirm the chromosome localization of TPS genes. The TPS genes were named based on their location on C. sinensis chromosomes, and their physical locations were visualized using MG2C_v2.1 (http://mg2c.iask.in/mg2c_v2.1). The basic information on CisTPS proteins, including molecular weight (MW), isoelectric point (pI), grand average of hydropathicity (GRAVY), and subcellular locations were predicted using the Expasy (https://web.expasy.org/protparam/) and GenScript (https://www.genscript.com/wolf-psort.html) websites (Duvaud et al., 2021). The secondary structures of CisTPS proteins were predicted using the PRABI website (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). The collinearity and selective evolutionary pressure of TPS genes were analyzed using the TBtools software (Chen et al., 2020).

Phylogenetic analyses

Based on previous studies, 51 protein sequences were downloaded, including 10 PoTPS, 7 CsTPS, 12 PtTPS, 11 AtTPS and 11 OsTPS protein sequences (Blázquez et al., 1998; Dan et al., 2021; Sun, Chen & Tao, 2021; Vogel et al., 2001; Yang et al., 2012; Zang et al., 2011). Multiple alignments of CisTPS, PoTPS, CsTPS, PtTPS, AtTPS, and OsTPS protein sequences were performed using ClustalW, and the Neighbor-Joining (NJ) phylogenetic tree was constructed using MEGA-X with a 1000 bootstrap test.

Gene structure and motif analyses

The gene structure of CisTPS genes was analyzed and visualized using GSDS v2.0 (http://gsds.gao-lab.org/) (Hu et al., 2015). The conserved motifs in the CisTPS proteins were identified using MEME Suite v5.4.1 (https://meme-suite.org/meme/tools/meme) with the parameter settings: number of repetitions = any and maximum number of motifs = 20 (Timothy et al., 2009).

Prediction of cis-acting elements

Using Phytozome v13 (https://phytozome-next.jgi.doe.gov), upstream sequences (2000 bp) of CisTPS genes were extracted from the sweet orange genome as promoter sequences. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the cis-acting elements in the promoter sequences, and the results were illustrated with the TBtools software (Lescot et al., 2002; Chen et al., 2020).

Plant materials and treatment conditions

The outer and inner seed coats of the Ridge pineapple sweet orange seeds were removed, and sterilized seeds were cultured in Murashige and Skoog (MS) solid medium in a light incubator (27 °C, 16 h light/8 h dark) for 30 d. The culture seedlings were used as test materials. Roots, leaves, and stems of seedlings were collected and stored at −80 °C to calculate the CisTPS gene expression in different tissues.

Seedlings were transferred to an MS liquid medium and placed at 27 °C as control. For temperature stress treatments, the seedlings in the MS liquid medium were placed at a high temperature (40 °C) or a low temperature (4 °C) (Xie et al., 2018). For phytohormone and abiotic stress treatments, seedlings were transferred to an MS liquid medium containing 100 µM ABA, 50 µM indole-3-acetic acid (IAA), 10% (w/v) polyethylene glycol (PEG-6000), and 150 mM NaCl, and placed at 27 °C (Liu et al., 2020; Xie et al., 2015b; Xu et al., 2017). Leaves were immediately frozen in liquid nitrogen and stored at −80 °C after each treatment at 0, 6, 12, and 24 h. Three independent biological replicates were performed, and the leaves of each sample were collected from a single seedling.

Expression profile analysis

The specific primers for detecting CisTPS genes were designed by Primer Premier v6.0, and FBOX was the housekeeping gene used as an internal reference (Mafra et al., 2012). The primer sequences are listed in Table S1.

The total RNA was extracted with TRIzol Reagent (Invitrogen, Waltham, MA, USA), and first-strand cDNA was synthesized with 1 µg of total RNA using All-In-One 5 ×RT MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada). Total RNA extraction and cDNA synthesis were performed according to the manufacturers’ instructions. The synthesized cDNA solution was diluted 10 times with distilled water, and the diluted cDNA was used as a template for quantitative polymerase chain reaction (qPCR). qPCR was performed with TB Green^® Premix Ex Taq™ II kit (TaKaRa, Beijing, China). The qPCR reaction mixture consisted of 9 µL template cDNA, 0.5 µL each of 10 µM primers, and 10 µL SYBR Green Supermix. qPCR was performed for 3 min at 95 °C (one cycle), followed by 10 s at 95 °C, 60 s at 60 °C (40 cycles). Each reaction was performed in technical triplicates.

Relative gene expression was calculated by the 2^−ΔΔCt method (Livak & Schmittgen, 2001). Standard error bars represent standard error of the mean (SEM). The expression of CisTPS genes in different tissues was normalized by that in roots (Dan et al., 2021). Statistical differences were analyzed with Student’s t-test.

Genome-wide identification of TPS genes in sweet orange

Eight TPS genes were identified in the sweet orange genome by bioinformatics analysis. Based on the assessment of Pfam and CDD, these eight TPS proteins contained two conserved domains-an N-terminal TPS domain (Glyco_transf_20; Pfam: PF00982) and a C-terminal TPP domain (Trehalose_PPase; Pfam: PF02358) (Table 1). These results confirmed that the eight genes belonged to the TPS gene family. The TPS genes were named CisTPS1-CisTPS8 according to chromosome position (Fig. 1). Furthermore, CisTPS2-CisTPS5 proteins contained an extra Hydrolase_3 domain (Pfam: PF08282).

CisTPS genes were distributed on five chromosomes, three on chromosome 2, two on chromosome 5, and one on chromosomes 3, 4, and 7 (Fig. 1). The genes were mostly located at the proximal ends of chromosomes. No obvious correlation was observed between chromosome length and number of CisTPS genes based on their distribution on chromosomes.

Physicochemical properties analysis revealed that the size of CisTPS proteins was highly variable from 831 (CisTPS3) to 942 amino acids (CisTPS1), and MW was between 94.24 KDa and 106.78 KDa. pI ranged from 5.59 (CisTPS3) to 6.38 (CisTPS7). GRAVY was predicted from −0.392 (CisTPS7) to −0.132 (CisTPS4). Subcellular prediction of these CisTPS genes indicated their localization in the chloroplast and cytoplasm (Table 1).

Analysis of the secondary structure content in CisTPS proteins showed that these proteins consisted of alpha helix, beta turn, random coil, and extended strand (Table S2).

To better understand the evolutionary mechanism of sweet orange TPS family, a collinear relationship diagram of sweet orange, A. thaliana and rice was constructed. Ten pairs of orthologous genes were found between sweet orange and A. thaliana, and six between sweet orange and rice (Fig. 2). Furthermore, the orthologous genes of CisTPS2, CisTPS5 and CisTPS6 were detected in both dicotyledon (A. thaliana) and monocotyledon (rice) (Fig. 2), indicating the three genes may be highly conserved. The Ka /Ks ratios of all orthologous genes between sweet orange and A. thaliana were less than 1(Table S3), suggesting purifying selection had acted upon these orthologous genes.

Phylogenetic analysis of CisTPS gene family

To determine the evolutionary relationship of TPS genes among various species, a phylogenetic tree was constructed by the NJ method based on the TPS protein amino acid sequences from cucumber, tree peony, Populus, A. thaliana, rice, and sweet orange. CisTPS genes were classified into two subfamilies, Class I and Class II, as shown in the previous studies in cucumber, tree peony, Populus, A. thaliana and rice (Fig. 3) (Blázquez et al., 1998; Dan et al., 2021; Sun, Chen & Tao, 2021; Yang et al., 2012; Vogel et al., 2001; Zang et al., 2011). CisTPS1 and CisTPS7 belonged to Class I, and the other genes belonged to Class II (Fig. 3).

Most CisTPS and PtTPS genes clustered together, suggesting that CisTPS genes were closely related to PtTPS genes. For example, CisTPS1 clustered with PtTPS2, CisTPS6 clustered with PtTPS9 and PtTPS10, CisTPS2 clustered with PtTPS7 and PtTPS8, and CisTPS3 clustered with PtTPS4 and PtTPS6.

Structure analysis of CisTPS genes

To better understand the molecular characteristics of CisTPS genes, the gene structures such as exons, introns, and conserved motifs were analyzed. In Class I, CisTPS1 and CisTPS7 contained 18 and 17 exons, and 17 and 16 introns, respectively. However, in Class II, CisTPS3 and CisTPS6 possessed four exons and three introns, whereas all other genes contained three exons and two introns (Fig. 4B). The results indicated that functional diversity of closely related TPS genes might be caused by the gain and loss of exons in the course of evolution of the TPS gene family. In addition, 20 distinct conserved motifs were searched using the MEME website. The lengths of these conserved motifs ranged from 15 to 50 amino acids (Table S4). Members (CisTPS1 and CisTPS7) of Class I all harbored 15 motifs, which lacked motif 8, 10, 15, 19, and 20. Members of Class II contained all 20 motifs, except for CisTPS5, which contained 19 motifs but lacked motif 20 (Fig. 4C). The results of the structure analysis confirmed the reliability of the phylogenetic tree (Fig. 4A), suggesting functional differences between Class I and II.

Cis-acting elements of CisTPS genes

By analyzing the 2,000-bp region upstream of the transcription start site, 70 types of cis-acting elements were discovered in the promoter regions of CisTPS genes (Table S5). Among these, seven types of cis-acting elements were related to abiotic stress and nine types of cis-acting elements were related to phytohormone responses. In abiotic stress-related elements, ARE (an anaerobic induction element) and MBS (a drought-inducible element) were mainly found in the promoter regions of CisTPS genes. Phytohormone-responsive elements, including ABRE (ABA-responsive element), CGTCA-motif (methyl jasmonic acid [MeJA]-responsive element), TGACG-motif (MeJA-responsive element), and TGA-element (auxin-responsive element), were observed in most CisTPS genes. A total of 126 cis-elements related to light were recognized on the promoters, and each promoter harbored at least nine light-responsive elements (Fig. 5). In addition, the CAAT-box and TATA-box, which are considered the core and common promoter elements, were found in the promoter regions of all CisTPS genes. CisTPS genes may play essential roles in response to abiotic stresses, phytohormones, and light.

Expression analysis of CisTPS genes in different tissues

To determine the specificity of TPS gene expression in sweet orange, CisTPS gene expression in roots, stems, and leaves was quantified using qRT-PCR. All CisTPS genes were expressed in roots, stems, and leaves, and the expression levels of most CisTPS genes were lower in leaves than in other tissues. CisTPS2, CisTPS3, CisTPS4, CisTPS6, CisTPS7 and CisTPS8 were highly expressed in roots, whereas CisTPS1 and CisTPS5 were highly expressed in stems (Fig. 6).

Expression analysis of CisTPS genes under phytohormone treatment

TPS proteins are involved in the differential regulation of gene expression. To understand the potential roles of CisTPS genes, we measured their expression characteristics in sweet orange seedlings after phytohormone treatment. Under ABA treatment, the expression of CisTPS3 and CisTPS7 was slightly upregulated at 24 and 6 h, respectively. CisTPS4 was upregulated and peaked at 6 h, declined to its lowest expression level at 12 h, and recovered to an intermediate level at 24 h. CisTPS5 and CisTPS8 were slightly downregulated, with the lowest expression at 12 and 24 h, respectively. Under IAA treatment, CisTPS2 and CisTPS7 were upregulated at 6 and 12 h, respectively, CisTPS3 at 12 h, and CisTPS8 at 6 h, whereas CisTPS4 was slightly inhibited at 24 h (Fig. 7).

Expression analysis of CisTPS genes under abiotic treatment

We also examined the expression of CisTPS genes in response to various abiotic stresses, and qRT-PCR was used to calculate the expression patterns under different treatment conditions. Under NaCl treatment, CisTPS2 and CisTPS3 were significantly induced at 12 h. However, CisTPS7 was significantly upregulated at 12 h and slightly repressed at 24 h. Under PEG-6000, CisTPS1 showed strong expression at 6 h, and returned to normal levels after 6 h. At 24 h, CisTPS2 expression was slightly suppressed, whereas CisTPS3 and CisTPS7 expression was slightly increased. CisTPS2, CisTPS3, CisTPS4, and CisTPS7 were upregulated at low temperature; CisTPS2 and CisTPS7 were strongly expressed at both 12 and 24 h. CisTPS1 and CisTPS4 were significantly upregulated at 12 and 6 h at 40 °C treatment conditions, respectively. CisTPS7 was slightly induced by high temperature at 6 h (Fig. 8).