Overexpression of chalcone synthase gene improves avonoid accumulation and drought tolerance in tobacco

Background: Flavonoids are important secondary metabolites in plants that play important roles in maintaining the cellular redox balance of cells. Chalcone synthase (CHS) is the key enzyme in the avonoid biosynthesis pathway and has been found to monitor changes due to drought stress tolerance. Results: In this study, a CHS gene in tobacco (Nicotiana tabacum) was overexpressed. Results revealed that transgenic tobacco plants were more tolerant than control plants to drought stress. Transcription levels of the key genes involved in the avonoid pathway and the contents of seven avonoids signicantly increased in transgenic tobacco plants (p < 0.01). Overexpression of the CHS gene led to lower concentrations of the oxidative stress product, malondialdehyde (MDA). Additionally, 11 CHS family genes were mined from the tobacco genome. Based on the phylogenetic tree, these genes split into two groups with eight genes clustered together with the bona de Arabidopsis CHS gene, suggesting that those tobacco genes are CHS genes. Further phylogenetic analyses indicated that the tobacco CHS genes grouped further into three independent clades with the cloned tobacco CHS gene located within Clade iii. The tobacco CHS family genes exhibited a highly conserved CDS length, pI, and molecular weight of the encoded peptides. All CHS peptides contained two conserved domains, and the genes harbored two or three exons. Conclusions: Based on the results of this study, the NtCHS gene is considered a possible candidate gene for genetically engineering enhanced drought tolerance and improved responses to oxidative stress in plants.

template and two primers, npt II-F 5´-TGTCACTGAAGCGGGAAG-3´ and npt II-R 5´-CTTCCATCCGAGTAC GTG-3´. The PCR conditions were as follows: 1 min at 94°C; 35 cycles for 30 s at 94°C, 30 s at 50°C, and 1 min at 72°C. PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide staining and UV illumination. Genomic DNA was isolated from the pH7WG2D-NtCHS transformants and control plants using a DNeasy plant mini kit (Qiagen, Duesseldorf, Germany) following the manufacturer's instructions.
To select positive transgenic tobaccos, NtCHS gene expression was determined by uorescent real-time quantitative PCR (RT-qPCR). For each plant, 100 mg sample was ground in liquid nitrogen. Then, total RNA was extracted from the pH7WG2D-NtCHS transformants and control plants separately using an RNeasy plant mini kit (Qiagen, Duesseldorf, Germany) following the manufacturer's instructions. After treatment with DNase I (GeneAnswer, Zhengzhou, China), rststrand cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Sangon, Shaanghai, China) following the manufacturer's instructions. RT-qPCR was performed on a CFX96 instrument (BIO-RAD, CA, USA). Each reaction mixture (total volume = 20 μL) contained 10 μL SYBR Premix Ex Taq (2 × concentration) (TaKaRa, Dalian, China), 1 μL gene-speci c primers CHS-F/CHS-R (10 mM), and diluted cDNA (100 ng). Leaf samples of both CHSoverexpressing tobacco and control plants were tested in triplicate. Ampli cation conditions were as follows: 30 s at 95°C; 40 cycles for 5 s at 95°C, 40 s at 60°C; one cycle for 15 s at 95°C, 1 min at 60°C, 15 s at 95°C, 15 s at 60°C. The internal standard gene was the 26S rRNA-based gene primer. [16] No morphological differences were observed between the transgenic and control plants.

Plant growth conditions and drought treatment
Seeds were grown on soil or one-half-strength Murashige and Skoog (MS) agar medium in a growth chamber maintained at 22-24°C and 60% relative humidity under a16/8 h light/dark photoperiod. After germination with 50 mg mL −1 kanamycin, seedlings of transgenic tobacco seeds were transferred to culture pots in the greenhouse where the drought treatment was applied for 14 d. Fresh weight (FW) of leaves was measured after removal from plants. Turgid weight (TW) was determined after rinsing the leaves in water at 4°C for 12 h. Then, the dry weight (DW) of leaves was determined after drying at 80°C for 48 h.
The optimized MS parameters were as follows: drying gas at 350°C; capillary voltage 4.0 kV; drying gas ow rate 11 L min −1 ; nebulizer pressure 45 psi. The Mining of CHS family genes and bioinformatics analyses CDS and peptide sequence datasets were downloaded for Arabidopsis, tomato, coffee, tobacco (N. tabacum), and N. attenuata. Datasets were preprocessed to clean sequences and accession lines. [21][22][23][24][25] The cloned Arabidopsis and tobacco CHS genes were used for retrieving peptides with the same conserved domains using HMMER against a library of Pfam-A families with default parameters. [26] CDS sequences were retrieved from CDS datasets based on the locus IDs of peptide sequences. Peptide sequences were aligned using Probcons v1.12. [27] If required, the le formats of aligned sequences were converted to phy. Maximum-likelihood phylogenetic trees were reconstructed using PhyML using a bootstrap method with 1,000 replicates. [28] Unless otherwise indicated, default parameters of phylogeny inference were used. Phylogenetic trees were visualized with FigTree v1.3.1.

Results
Generation of NtCHS overexpressing tobacco plants An overexpression construct, pH7WG2D-NtCHS, with NtCHS cDNA (AF311783.1) under the control of the CaMV35S promoter was transformed into N. tabacum K326 (Fig. 1a). Three independent putative positive lines were selected on the MS medium with kanamycin. Positive lines were further con rmed with genomic PCR using the primer pair, npt II-F and npt II-R. The PCR results revealed that the npt II gene was expressed in the three selected transgenic lines (i.e., F1, F2, and F3), but not in the control plants (i.e., C1, C2, and C3) (Fig. 1b). Since the empty vector, pH7WG2D, contained a suicide gene, ccdB, between the attR1 and attR2 sites, the plasmid pH7WG2D-NtCHS was successfully transferred into tobacco plants. Transgenic lines were further selected by kanamycin resistance for subsequent analyses.

Expression patterns of avonoid genes in transgenic tobacco
In order to investigate the expression patterns of the avonoid genes, leaves at the vigorous growing stage were harvested from transgenic and control plants. The three transgenic tobacco lines exhibited signi cantly higher NtCHS transcription levels compared to the control plants (p < 0.01). Therefore, they were selected for subsequent experimentation (Fig. 2a). Moreover, the expression levels of the CHI, F3H, F3'H, FLS, and DFR genes in transgenic tobacco plants were signi cantly higher compared to the control tobacco plants (p < 0.01).

Flavonoid accumulation in tobacco leaves
In order to investigate the effect of the NtCHS gene on avonoid biosynthesis in tobacco, avonoid content in transgenic and control leaves was detected. The content of all avonoids was much higher in transgenic plants compared to control plants ( Table 1, Fig. 3).

Drought tolerance of tobacco plants
In order to evaluate whether NtCHS overexpression affects tobacco drought tolerance, a water-de cit treatment was performed. The growth of transgenic plants was notable better compared to control plants after 14 d (Fig. 4a). To further characterize the performance of transgenic tobacco plants under drought stress, changes in the concentrations of RWC, MDA, and H 2 O 2 were monitored before and after drought treatment. After 14 d of water shortage, the RWCs of the three transgenic lines were signi cantly higher compared to the control plants (p < 0.01) (Fig. 4b). Leaf MDA concentrations also increased in transgenic and control tobacco plants following drought stress (Fig. 4c). The concentrations of ROS were not signi cantly different under drought treatment conditions. However, control tobacco plants had higher concentrations of H 2 O 2 compared to transgenic plants under drought treatment conditions (Fig. 4d).

Mining of CHS family genes in the tobacco genome
Because cDNA we cloned and identi ed had a role in avonoid accumulation and drought tolerance, how this gene evolved was investigated and whether there were other CHS genes that possess potential relevant functions was explored. Using HMMER with the known tobacco CHS gene (accession No.: AF311783.1) as a query, 11 peptides were mined from the tobacco genome, all of which contained two conserved domains (i.e., Pfam ID: PF02797.14 and PF00195.18), which are signatures of CHS peptides. One of the mined peptides (accession No. XP_016480648.1) exhibited 100% identity with the gene cloned in this study (AF311783.1), suggesting that the two genes are virtually the same. Additionally, peptides were mined from the genomes of N. attenuate (n = 7), tomato (n = 7), coffee (n = 10), and Arabidopsis (n = 4). In total, 35 peptides were mined from four Solanaceae genomes.

Phylogenetic relationships of CHS peptides
In order to determine the phylogenetic relationships among the mined genes, 39 peptide sequences were aligned and used as input to infer the phylogenetic tree. The resulting tree revealed that the peptides were split into two distinct groups (Fig. 5a). Identity values among the genes within their respective groups were generally considerably higher than those between the two groups ( Table 2). Seven of the tobacco peptides clustered together with a bona de Arabidopsis CHS gene (AT5G13930.1) (Fig. 5a, I), while four peptides grouped with three other Arabidopsis peptides, one of which was previously reported to be hydroxyalkyl α-pyrone synthase. [29,30] Therefore, genes in Class I of the mined sequences putatively encode CHSs (Fig. 5a).
To better resolve CHS phylogeny, the sequences in Class I were retrieved after globally aligned results were used for phylogenetic inference, which revealed that the Solanaceae CHS genes further split into three independent clades with high branch bootstrap support (i.e., Clades i, ii, and iii) (Fig. 5b). In each clade, no less than two tobacco genes were present, while only one tomato copy was present, which was in agreement with the haploid nature of the tomato genome compared to the tetraploid nature of the tobacco genome. The cloned gene clustered in Clade iii (XP_016480648.1), which was phylogenetically close to another tobacco gene (XP_016494384.1) and one tomato gene (Solyc09g091510.2.1) (Fig. 5b). The two tobacco CHS peptide sequences in Clade iii exhibited a high identity value (98.7%). High identity values were also observed in tobacco CHSs within the other two clades ( Table 2).

Characteristics of the tobacco CHS family genes and encoded peptides
The 11 tobacco genes were distributed in different scaffolds of the tobacco genome, which may be due to low assembly quality. However, the CDS length (1167-1293 bp), pI values (5.57-8.02), and molecular weights (42.5-47.4 kDa) were highly similar among the genes and their encoded peptides, which represent conserved sequence characteristics among the 11 tobacco genes ( Table 3). All peptide sequences contained two conserved domains, with PF00195.18 located at the N-terminus and PF02797.14 at the C-terminus (Fig. 6a). All CDS sequences of the CHS family genes contained one or two introns with highly variable lengths, and the rst exon of the genes tended to be considerably shorter than the second and third exons (Fig. 6b).

Discussion
The avonoid biosynthesis pathway is an important secondary metabolite pathway, and CHS is the rst committed enzyme that catalyzes the synthesis of the avonoid branch. Previous work suggested a role for CHS in the production of avonoids and improving plant tolerance to drought stress. [13,31,32] In this study, overexpression of NtCHS in tobacco plants enhanced plant tolerance to drought stress and increased RWC. Additionally, transcripts of the avonoid biosynthesis genes and avonoid contents were considerably upregulated in transgenic plants. Four of the seven measured avonoids (i.e., rutin, quercetin, kaempferol-3-rutinoside, and kaempferol-glucopyranoside) were higher in content, [33,34] while naringin, naringenin, and isoliquiritigenin occupied key positions in the metabolic pathway. Overall, these results suggest that avonoids play a role in plant drought tolerance. [5,7] Exposure to drought conditions can result in the increased production of ROS, oxidative stress, and cell membrane damage in plants. [35] Flavonoids affect plant physiology in response to external stressors and play an important role in the maintenance of cellular redox balance. [36] It has been demonstrated that avonoids prevent the formation of ROS and improve the scavenging of ROS in drought-stressed plants. [37][38][39] Before exposure to drought conditions, the concentrations of MDA and H 2 O 2 in transgenic plants were similar to control plants. However, transgenic tobacco plants possessed lower concentrations of ROS and MDA than control plants when exposed to drought stress conditions . MDA is a lipid peroxidation product that is produced due to drought stress in plants. [40] Therefore, the results of this study suggest that the measured avonoids (i.e., rutin, quercetin, kaempferol-3-rutinoside, kaempferol-glucopyranoside, naringin, naringenin, and isoliquiritigenin) play a role in maintaining the redox balance of transgenic tobacco in response to drought stress, as indicated by the lower concentrations of MDA.
By using the 14 d drought tolerance treatment, it was demonstrated that the overexpression of the NtCHS gene in tobacco resulted in improved plant drought tolerance. Additionally, the transcripts of key avonoid pathway genes and contents of seven avonoids signi cantly increased in transgenic plants.
Furthermore, concentrations of ROS were lower in transgenic tobacco plants than control plants, suggesting a role for avonoids in maintaining cellular redox balance. The results of this study suggest that NtCHS is a potential candidate gene that could be targeted in the genetic engineering of tobacco plants in order to enhance drought stress tolerance.
In addition to the cloned tobacco CHS gene, CHS genes were systematically mined from the tobacco genome, leading to the identi cation of 11 CHS family genes. Phylogenetic analyses indicated that there are 8 CHS genes in the tobacco genome, which were derived from two rounds of duplications in Solanaceae. The genes exhibited a conservation of sequence characteristics, indicating that these genes are conserved in biological and enzymatic roles. Therefore, these genes may serve as targets in future studies on CHSs in tobacco and their relationship to avonoid biosynthesis and drought tolerance.

Conclusions
A CHS gene in tobacco (Nicotiana tabacum) was overexpressed. Results revealed transcription levels of the key genes involved in the avonoid pathway and the contents of seven avonoids signi cantly increased in transgenic tobacco plants (p < 0.01). Overexpression of the CHS gene led to lower concentrations of the oxidative stress product, malondialdehyde (MDA). Further phylogenetic analyses indicated that the tobacco CHS genes grouped further into three independent clades with the cloned tobacco CHS gene located within Clade iii. The tobacco CHS family genes exhibited a highly conserved CDS length, pI, and molecular weight of the encoded peptides. All CHS peptides contained two conserved domains, and the genes harbored two or three exons. Based on the results of this study, the NtCHS gene is considered a possible candidate gene for genetically engineering enhanced drought tolerance and improved responses to oxidative stress in plants. Availability of data and material Additional data are available in Additional les.

Competing interests
The authors declare that they have no competing interests. HB,JLF coordinated the project, conceived and designed experiments, and edited the manuscript; HY and YLG conducted bioinformatics analysis, performed experiments and wrote the rst draft; MJ and YL conducted bioinformatics analysis; RW contributed valuable discussion and substantively revised it; FL and JGG provided analytical tools and analyzed the data; KL and MYZ coordinated the project and edited the manuscript. All authors have read and approved the nal manuscript.

Supplementary Files
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