Characterization of trehalose-6-phosphate synthase and Na/H antiporter genes in Vuralia turcica and expression analysis under salt and cadmium stresses

Vuralia turcica (Fabaceae; Papilionoideae) is a critically endangered endemic plant species in Turkey. This plant grows naturally in saline environments, although the photosynthesis and physiological functions of many plants are affected by salt stress. Molecular control mechanisms and identifi cation of genes involved in these mechanisms constitute the critical fi eld of study in plant science. Trehalose-6-phosphate synthase (TPS) is one of the essential enzyme genes involved in trehalose biosynthesis, which is protective against salt stress. Also, the vacuolar Na/H antiporter gene (NHX) is known to be useful in salt tolerance. In this study, the TPS and NHX–like genes in V. turcica were partially sequenced using degenerate primers for the fi rst time and submitted to the NCBI database (accession numbers MK120983 and MH757417, respectively). Also, the expression levels of the genes encoding TPS and NHX were investigated. The results indicate that the increase in both the level of applied salt and cadmium is coupled with the increase in the expression level of NHX and TPS genes. However, salt exposure signifi cantly affected the expression level of the NHX gene. The fi ndings suggest that the NHX gene might play a crucial role in the salt tolerance ability of V. turcica.


INTRODUCTION
Salt stress is an often encountered problem in agriculture that decreases yield by enabling healthy plant growth (Zhu 2001). Trehalose-6phosphate synthase (TPS) and vacuolar Na + /H + antiporter (NHX) genes are known to be useful against abiotic stress conditions such as salinity and drought.
Trehalose (α-D-glucopyranosyl-1,1-α-Dglucopyranoside) is one of the significant carbohydrate stores in not only plants also in a large variety of microorganisms such as yeast and animals. Various organisms, such as bacteria, fungi, plants, insects, and invertebrates, contain genes that enable them to produce trehalose endogenously (Elbein et al. 2003). Trehalose is a non-reducible disaccharide composed of two glucose units and provides rapid adaptations to an organism under various environmental conditions, and has a signifi cant role in glucose uptake. It also functions as an osmoprotectant (Elbein 1974, Crowe et al. 1984. Trehalose is catalyzed by two enzymes: trehalose-6phosphate synthase (TPS) and trehalose-6phosphate phosphatase (TPP). Currently, there has been a great interest in trehalose metabolism to improve stress-tolerant plants. TPS gene was successfully transferred to tomato, potato, and tobacco plants to obtain stress-tolerant species, especially against drought (Jang et al. 2003, Avonce et al. 2004, Zhang et al. 2005, Kissoudis et al. 2015. Also, there have been several studies conducted on the function of the TPS gene under salt and drought conditions and reported that overexpression of TPS was beneficial on the resistance mechanism (Kwon et al. 2004, Wu et al. 2006. Sodium ions can cause salt stress in plants. Plants remove excess Na + ions by exclusion or compartmentation in saline soils. Na + /H + antiporters, membrane proteins, play an essential role in Na + homeostasis and pH regulation in plant species. Na + /H + antiporter genes are also found in animals, yeast, and bacteria (Shi & Zhu 2002, Fukuda et al. 2004. It is known that NHX is responsible for cadmium stress from previous studies (Cong et al. 2013). Cadmium is a non-essential heavy metal and toxic for plants, animals, and also humans due to causing many adverse effects such as oxidative stress and disturbance in metabolism (Adabnejad et al. 2015).
Although the molecular identification and the expression pattern of TPS and NHX genes are very important in terms of salt stress mechanism in Vuralia turcica (Tan et al. 1983) Uysal et al. (2014, to date, these genes have not been investigated. This study aims to identify NHX and TPS genes in V. turcica and to analyze gene expressions of these genes in response to cadmium and salt stresses.

Plant materials
The leaves of salt (0, 1000, and 2000 ppm) and Cd (0, 5, 25, and 50 ppm) treated plants in the previous research (Tekdal & Cetiner 2018) were used for total RNA isolation. Leaf samples were obtained from the samples collected in the last experiment, treated with liquid nitrogen, and maintained at -80°C (Tekdal & Cetiner 2018).

Genomic DNA isolation
The MiniPrep DNA isolation method was used in gDNA isolation of V. turcica and was performed according to the protocol proposed by Edwards (1998). The purity of DNAs was achieved by spectrophotometric (NanoDrop ND 100, Wilmington, DE, USA) and electrophoresis (1% agarose) methods. Samples were adjusted with DNase and RNAse-free water at a concentration of 50 ng µl -1 for further analysis.

Detection of orthologs of TPS and NHX genes and degenerate primers design
Since the sequences of the V. turcica TPS and NHX genes are unknown, the degenerate primer design was first performed. To find the orthologs of TPS and NHX genes, the corresponding gene sequences of the legumes were first selected from the NCBI database (Table I). The known gene fragments (TPS and NHX) of the selected legumes were compared, and degenerate primers were designed for amplification of candidate orthologs from the genomic DNA of V. turcica. Degenerate primers for TPS and NHX were developed based on conserved sequence segments (Figures 1 and 2, respectively) identified using Clustal Omega (https://www.ebi.ac.uk/ Tools/msa/clustalo/) (Sievers et al. 2011). The primers designed for TPS and NHX sequences and selected for qRT-PCR analysis are described in Table II. Primers were designed manually using Primer3Plus (http://www.bioinformatics. nl/cgi-bin/primer3plus/primer3plus.cgi) and synthesized by Sentebiolab (https://sentebiolab. com.tr/), Ankara, Turkey.

PCR reaction and agarose gel electrophoresis
To obtain the amplification product using the designed primers, gradient PCR was performed to determine the optimal temperatures at which the primers were bound to DNA. The best DNA binding temperature of each primer applied with gradient PCR was determined according to the cleanest banding condition in 2% agarose gel. PCR amplifications were performed in a reaction volume of 25 µL reaction mixture containing 5 ng DNA, 0.8 µM of each primer (forward and reverse), 0.2 mM dNTP (Fermentas, #R0192), 0. 125 unit Taq DNA Polymerase (Fermentas, #EP0402), 1X Taq DNA Polymerase buffer (Fermentas, 00061586), including 2.5 mM MgCl 2 (Fermentas, 00061590). The cycling parameters were as follows: 10 min at 95°C for initial denaturation and 35 cycles of 30 seconds each at 95°C (melting) and 30 seconds at a temperature specific for every primer pair -48°C for TPS and 53°C for NHX primers -(annealing) and 1 min at 72°C (extension). These cycles were then followed by a final extension step at 72°C for 7 min. 10 µl of PCR products obtained from the designed degenerate primer analyzes were withdrawn, and 2 µl loading buffer (40% sucrose, 10 mM EDTA, 25% bromophenol blue) was added. The prepared mixture was studied in 2% agarose gel by adding 0.5x TBE (Trism Base, Boric Acid, EDTA) buffer. The gel was stained with 0.1% ethidium bromide solution. The presence of amplification products was determined by photographing under UV light (302 nm) on a Biorad Imager (Bio-Rad Laboratories, Segrate (Milan). The amplified and expected size PCR products were purified from the gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, USA) according to the protocol recommended by the manufacturer.

DNA sequencing and BLAST analysis
The sequencing service was commercially provided by Medsantek Ankara, Turkey (http:// www.medsantek.com.tr/). The raw sequence data transmitted from Medsantek were manually checked, and the forward and reverse complemented sequences were aligned using the Pairwise Sequence Alignment (Nucleotide) (http://www.ebi.ac.uk/Tools/psa/emboss_ needle/nucleotide.html) constructed by EMBL-EBI. The resulting consensus sequence was searched via the GenBank database at the National Center for Biotechnology Information (NCBI), Bethesda, USA, using the BLAST (Basic Local Alignment Search Tool; http://blast.ncbi. nlm.nih.gov/Blast.cgi) search program (Altschul et al. 1990) to determine similar sequences. The identified V. turcica TPS and NHX partial sequences were recorded in the NCBI GenBank using the Sequin program (https://www.ncbi.nlm. nih.gov/Sequin/). Target gene expression was analyzed with a mathematical method proposed by Pfaffl (2001).

RESULTS
The salt and Cd stress tests on V. turcica were established by applying different concentrations of NaCl (0, 1000, and 2000 ppm) and Cd (0, 5, 25, and 50 ppm) (Tekdal & Cetiner 2018). In this study, leaf samples of piyan plants, which were applied to salt and Cd stresses (target groups) and not applied to any stress (control group), were obtained from the study implemented by Tekdal & Cetiner (2018). Genomic DNAs that were needed for TPS and NHX gene identification were isolated from the leaves of NaCl-and Cd-treated V. turcica (target) and non-treated V. turcica (control) successfully. The quality of isolated gDNAs and RNAs was in good quality, with a 260/280 ratio of 1.8-2.0, and their integrity was determined using agarose gel electrophoresis ( Figure S1 -Supplementary Material).
To determine the effect of salt and Cd on the expression pattern of the TPS and NHX genes, total RNAs from leaves of NaCl-and Cdtreated plants were extracted and analyzed using agarose gel electrophoresis ( Figure S2).
In this study, gDNAs were used as the template for PCR-based amplification using degenerate primers designed to TPS and NHX genes using conserved regions of TPS and NHX mRNAs available in the GenBank database (Table  I). As a result of PCR analysis using degenerate primers, multiple bands were produced when used designed TPS primers, whereas single bands were obtained when used designed NHX primers (Figure 3). Multiple bands production is possibly related to amplifying unrelated sequences. The bands were expected sizes were extracted from the gel and purified for sequencing.
To identify the putative orthologues of TPS and NHX in V. turcica, isolated and purified PCR products were directly sequenced by the Sanger method. The sequences obtained through PCR were verified to be 230 bp in length for the NHX gene and 237 bp in length for the TPS gene and were deposited in the NCBI GenBank database with the following accession numbers: MH757417.1 and MK120983.1, respectively. The result of the BLAST search against the GenBank nucleotide database indicated that V. turcica and Arachis ipaensis Krapov. & W.C. Greg. shared the maximum identity (92%) at the nucleotide level in terms of the TPS gene, whereas and the identity between V. turcica and Cajanus cajan (L.) Millsp. was very high in terms of the NHX gene (Table III). Conceptual translation of the ORFs of the identified TPS sequence of V. turcica yielded an amino acid sequence of 79 aa ( Figure  4), and the NHX sequence produced an amino acid sequence of 69 aa ( Figure 5).
To understand whether Cd exposure influences salt tolerance ability, NHX and TPS expressions in Cd-treated plants were examined. In low Cd concentration (5 ppm), the transcript level of TPS gradually increased, and the expression level of TPS increased by >5-fold in medium Cd concentration (25 ppm). In a high concentration of NaCl (2000 ppm) and Cd (50 ppm), NHX expression level and salt tolerance ability increased.

DISCUSSION
Abiotic stress factors affect crop production negatively in Turkey as well as all over the world. Plants activate stress-related genes against such stress conditions. Therefore, elucidating the molecular control mechanisms developed by plants against stress conditions will contribute Table II. The list of the designed degenerate primers, control and housekeeping gene primers employed in this research (F, forward, R, reverse).

Target
Name Sequence (5´-3´) Definition Product size (  to plant development. Salt stress is one of the abiotic stress factors and has a signifi cant effect on product yield. Therefore, it is crucial to elucidate the molecular control steps of tolerance mechanisms in plants that can quickly develop in soils with high salt content. V. turcica (piyan) is an essential source of a gene that is in danger of extinction. As a result of the studies carried out with this plant, it was determined that the plant could develop in soils with high salt content. As a result of studying with plants exposed to high temperature and drought, trehalose, a nonreducible disaccharide, was found to be effective in salt tolerance (Garg et al. 2002). TPS is the most well-known enzyme gene that plays a role in the biosynthesis of trehalose, which is protective against salt stress (Carcia et al. 1997). Many studies indicate an increase in the expression of the TPS gene in stressed plants (Almeida et al. 2005, Zhang et al. 2005, Kosmas et al. 2006, Lunn 2007. The vacuolar Na + /H + antiporter gene has been reported to be effective in salt tolerance in many plants  )). In salt stress study with V. turcica, it was determined that the salt tolerance of the species was found as a result of phenological and physiological studies (Tekdal & Cetiner 2018). Within the scope of the present study, the partial sequences of the TPS and NHX genes were characterized. Salt tolerance ability of plants can be affected by increasing the osmolyte production or stress proteins (Zhu 2001). In a previous study, the NHX gene of A. thaliana enhanced salt tolerance ability of wheat and tomato , Xue et al. 2004. Expression levels of NHX and TPS in response to NaCl and Cd were evaluated by qRT-PCR ( Figure 6). Likewise, as a result of PCR study with primers designed for TPS and NHX genes and cDNAs of samples subjected to salt stress,   the expression level of the NHX gene was higher than that of the TPS gene. Under salt stress, NHX and TPS gene expressions were examined in V. turcica leaf tissues, and there was no significant change in TPS gene expression. It was thought that the difference in the expression of the TPS gene could be seen by prolonging the stress period applied or by examining the root tissues. According to the literature, it was found that the TPS gene was overexpressed in transgenic plants resistant to salt stress; Ginkgo biloba L. (Wu et al. 2006), rice (Garg et al. 2002, Jang et al. 2003), tobacco (Almeida et al. 2005, Zhang et al. 2005. The expression of NHX was high by applying 50 ppm Cd. It is, therefore, likely that the NHX gene was most active in V. turcica exposed to high Cd concentration (50 ppm).

CONCLUSION
Under salt and cadmium stress conditions, plants tend to change their developmental physiology mostly to overcome stress. NHX and TPS genes are essential for homeostasis in plants. Since there are no studies in the literature on the determination of V. turcica NHX and TPS genes and the elucidation of expression levels of these genes, it is thought that the present study findings will fill an essential gap in the literature. As a result of this study, partial sequences of TPS and NHX genes, which are thought to be useful in the mechanism of tolerance to salt stress in V. turcica, were determined for the first time and introduced into the literature.