Insights into the trihelix transcription factor responses to salt and other stresses in Osmanthus fragrans

Osmanthus fragrans is an evergreen plant with high ornamental and economic values. However, they are easily injured by salt stress, which severely limits their use in high salinity areas. The trihelix transcription factor (TF) family, as one of the earliest discovered TF families in plants, plays an essential part in responses to different abiotic stresses, and it has potential functions in improving the salt-tolerance capability of O. fragrans. In this study, 56 trihelix genes (OfGTs) were first identified in O. fragrans and then divided into five subfamilies in accordance with a phylogenetic tree analysis. The OfGTs were found to be located randomly on the 20 O. fragrans chromosomes, and an analysis of gene replication events indicated that the OfGT gene family underwent strong purification selection during the evolutionary process. The analysis of conserved motifs and gene structures implied that the OfGT members in the same subfamily have similar conserved motifs and gene structures. A promoter cis-elements analysis showed that all the OfGT genes contained multiple abiotic and hormonal stress-related cis-elements. The RNA-seq data suggested that the OfGTs have specific expression patterns in different tissues, and some were induced by salt stress. The qRT-PCR analysis of 12 selected OfGTs confirmed that OfGT1/3/21/33/42/45/46/52 were induced, with OfGT3/42/46 being the most highly expressed. In addition, OfGT42/OfGT46 had a co-expression pattern under salt-stress conditions. OfGT3/42/46 were mainly localized in the nuclei and exhibited no transcriptional activities based on the analysis of the subcellular localization and transcriptional activity assay. Furthermore, the expression levels of most of the selected OfGTs were induced by multiple abiotic and hormonal stresses, and the expression patterns of some OfGTs were also highly correlated with gibberellic acid and methyl jasmonate levels. Remarkably, the transient transformation results showed lower MDA content and increased expression of ROS-related genes NbAPX in transgenic plants, which implying OfGT3/42/46 may improve the salt tolerance of tobacco. The results implied that the OfGT genes were related to abiotic and hormonal stress responses in O. fragrans, and that the OfGT3/42/46 genes in particular might play crucial roles in responses to salt stress. This study made a comprehensive summary of the OfGT gene family, including functions and co-expression patterns in response to salt and other stresses, as well as an evolutionary perspective. Consequently, it lays a foundation for further functional characterizations of these genes.


Background
Transcription factors (TFs) are proteins that can regulate transcription and expression of target genes by binding to specific DNA sequences [1]. Presently, more than 60 TFs have been discovered in plants [2]. A trihelix TF was first discovered to specifically bind to a light-responsive element, namely the GT component; consequently, it is also known as the GT factor family [3]. The common feature of trihelix TFs in plants is that the DNA-binding domain contains three helical structures (helix-loophelix-loop-helix). The amino acid sequences of these functional domains are highly consistent and strongly conserved among the same subfamilies [4]. On the basis of the changes in the conserved domain, the trihelix TF is generally classified into five subfamilies: GT-1, SH4, GTγ, GT-2, and SIP1 [5]. Each subfamily contains an N-terminal conserved domain (apart from At5g47660 in Arabidopsis thaliana), but the C-terminal domains are different. In addition, the subfamilies have only one DNA-binding domain, except the GT-2 subfamily, which contains two DNA-binding domains [6]. Although all the members contain at least one domain, there are subtle differences in this domain among different subfamilies. In the GT-1 and SH4 subfamilies, the trihelix domains each have a tryptophan residue in the internal hydrophobic region of the tandem repeat. In the GTγ and GT-2 subfamilies, the third conserved tryptophan is substituted by phenylalanine, whereas in the SIP1 subfamily, it is replaced by isoleucine [6,7].
At present, the TF family of trihelix has been identified in A. thaliana, rice (Oryza sativa), Fagopyrum tataricum, Phyllostachys edulis, and Populus trichocarpa, and among them, Arabidopsis and rice have been studied in depth [2,[8][9][10][11]. The initial research on trihelix TFs showed that they help to regulate the light responses of plants [12,13]. Additionally, the genes universally regulate multiple processes of plant growth and development, such as, embryo sac development [14], seed separation [15], and floral organ development [16]. Furthermore, studies have shown that trihelix TFs are also related to the response of plants to biotic and abiotic stresses, such as, salinity, drought and methyl jasmonate (MeJA) [17][18][19]. Interestingly, some trihelix genes participate in the responses to multiple stresses. In Chrysanthemum morifolium, the genes are affected by high salt, drought, low and high temperature, abscisic acid (ABA), and methyl jasmoate MeJA [20]. The salt tolerances of many plants have been improved by isolating and cloning trihelix TF genes related to salt tolerance. The GTγ subgroup members OsGTγ-1, OsGTγ-2, and OsGTγ-3 in rice were induced by most of the abiotic stresses. Especially, overexpression of OsGTγ-1 in rice enhanced salt tolerance at the seedling stage [21]. In soybean (Glycine max),  GmGT-2B enhance the tolerance to salt stress [22]. In A. thaliana, the AtGT2L protein enhances plant tolerance to salt stress by up-regulating the expression levels of the marker genes RD29A and ERD10 [17]. In addition, AST1 which is a SIP1 subfamily TF member can combine with an AGAG-box or GT element to regulate downstream gene expression to enhance Arabidopsis salt-stress tolerance [23]. Interestingly, TFs also interact with other genes to enhance plant salt tolerance. For example, the AtGT4 interacts with the TEM2 gene to enhance the salt tolerance of Arabidopsis [24]. Some studies reported that MDA is closely related to cell membrane damage under abiotic stress [25]. Under cold stress, overexpressed Pub-HLH1 and NtbHLH123 have lower MDA content, which can reduce the oxidative damage of cell membrane by activating ROS-related genes [26,27].
Osmanthus fragrans is an evergreen plant with high ornamental and economic values. Research on O. fragrans has focused on floral fragrance and flower color [28,29], with research on abiotic stress tolerance being limited. High salinity and other abiotic stresses are critical adverse environmental factors that severely restrict plant growth and distribution [30]. However, the molecular regulatory mechanisms of O. fragrans involved in salt-tolerance and other abiotic stresses responses are still unclear. The publication of the whole-genome sequences of O. fragrans provides a resource for the screening of O. fragrans salt-tolerant genes and those involved in responding to other stresses [31].
In this research, 56 OfGT genes were screened from the O. fragrans genome data. They are located on 20 different chromosomes and were classified as five subfamilies. A thorough analysis of conserved motifs and gene structure was performed. In addition, the expression and co-expression patterns of 12 OfGT genes under three abiotic stress treatments (salt, waterlogging, and drought) and three hormonal stresses [MeJA, ABA, and gibberellic acid (GA 3 )] were examined. Furthermore, we analyzed the subcellular localizations and transcriptional activation activities of the potential salt tolerance genes OfGT3/42/46. Finally, the potential genes OfGT3/42/46 was transferred into tobacco for functional verification by transient transformation. The study will provide a beneficial genetic resource for improving the salt tolerance of O. fragrans.

Identification of OfGT gene family members in O. fragrans
We identified 56 trihelix genes from the database of O. fragrans genomes [31]. In accordance with their locations on the chromosomes, these genes were named OfGT1-56. The protein lengths encoded by the 56 OfGT genes range from 262 to 617 aa, with a mean length of 395 aa. The minimum isoelectric point value is 4.6, and the maximum is 9.58. The relative molecular masses range from 29.18 kDa to 69.75 kDa (Additional file 1: Table S1).

Phylogenetic analysis and subcellular localization predictions for OfGT genes
To analyses the evolutional relationships between the 56 OfGT proteins, a phylogenetic tree was constructed with the trihelix genes of rice (O. sativa) and Arabidopsis ( Fig. 1, Additional file 2: Table S2). In accordance with the classifications of trihelix TFs in rice and A. thaliana, the 56 trihelix genes in O. fragrans were classified into five subfamilies (GT-1, SH4, GTγ, GT-2, and SIP1). The largest subfamily is SIP1, which contains 20 members. The smallest subfamily is SH4, with only six members.
The subcellular localization predictions for the 56 OfGT protein sequences revealed that most (80.36%) of the proteins are localized in the nucleus, a small portion (14.29%) are localized in the chloroplasts, OfGT26 and OfGT27 (3.57%) are localized in the cytoplasm, and OfGT48 (1.79%) is localized in the mitochondria (Additional file 3: Table S3).

The analysis of genes structures, motif compositions, and a promoter for the OfGT gene family
According to the exon/intron structure analysis, 56 OfGT genes contain one to eight exons. In total, 23% of OfGT genes lack introns ( Fig. 2a-b). The majority of genes clustered in the identical subfamily showed similar exon/intron structures. For example, in the GT-2 subfamily, nine members contained two exons, whereas OfGT2/46/49 contained three exons. Using MEME, 15 motifs were identified among the 56 OfGT proteins (Fig. 2c). Specific amino acid sequences for each motif are provided (Additional file 4: Table S4). All the OfGT proteins contain motif 1, which is the most conserved motif among the subfamilies. The OfGT proteins, except  for the GTγ branch members, contain motif 3. The GT-2 branch genes have two trihelix domains. In addition, the conserved motifs of the most closely related members of the phylogenetic tree showed similar arrangements and positions, indicating that the functions of the trihelix proteins in each defined subfamily may be similar.
In the promoter analysis, four categories of cis-elements were discovered in the OfGT genes ( Fig. 3a-b). The first category is plant hormone-response elements, including the gibberellin-response element GAREmotif (1.79%), TATC-box (1.97%), and P-box (4.84%); MeJA (37.46%) homeopathic element CGTCA motif (19.18%) and TGACG motifs (18.28%), ABA homeopathic elements motifIIb (0.18%) and ABRE (21.96%); cis-regulatory elements involved in salicylic acidresponse TCA element (6.81%); and auxin cis-response regulatory elements AuxRR-core (3.76%) and TGA element (3.05%). The second category is abiotic and biotic stress-response elements, including drought (14.41%) and low temperature (0.42%) response elements, as well as defense and stress responses (TC-rich repeats, 10.59%), and anaerobic induction (ARE, 39.41%). The third category is plant growth and developmental factors, such as cis-elements involve in the specific activation of meristem (CCG TCC -box) and endosperm expression cis-regulating elements (Skin-1_motf and GCN4_motif ). The last category consists of lightresponsive elements, such as GT1 motif, G-box, and  Table S5). The above analysis of the hormonal and abiotic stress-response elements of the OfGT genes provides a basis for the subsequent hormonal and stress treatment of plants.

The gene duplication events of OfGT genes and distribution on the chromosomes
The MCScanX was used to analyze gene replication events, including tandem repeats and fragment replication. Three pairs of tandem repeat genes (OfGT1/OfGT2, OfGT33/OfGT34, and OfGT55/OfGT56) were identified. The 44 duplicated genes were also identified among the 56 OfGT genes (Fig. 4a). Furthermore, the OfGT gene pairs resulting from gene duplication events, nonsynonymous (Ka)/synonymous (Ks) substitution rate ratios were < 1, which indicated that the OfGT genes have undergone strong purification selection during the evolutionary process (Additional file 6: Table S6). In addition, the 56 OfGT genes are scattered across the 20 chromosomes (Fig. 4b). The OfGT genes numbers distributed on each chromosome ranges from one to seven. The most OfGT genes are located on Chr03, whereas Chr05, Chr20, and Chr23 do not contain any OfGT gene.

Expression pattern of OfGT genes in different tissues
The RPKM values of 56 OfGT genes were derived from the O. fragrans transcriptome databases of seven different tissue samples [31]. On the basis of the cluster analysis, the 56 OfGT genes were roughly divided into five different groups, and these genes in the same cluster having similar expression values. The overall expression revealed that 16 genes are expressed in all samples, 17 OfGT genes have different expression patterns in seven tissues, and 19 OfGT genes were not expressed in any tissue (Fig. 5). Some of the OfGTs genes showed tissue-specific expression. For example, OfGT47 is only expressed in roots, and OfGT19/22/43 are only expressed in stems. Most SIP1 subfamily members had higher expression levels in different tissues.

Expression analysis of OfGT genes in response to salt
The FPKM values of the 56 OfGT genes were derived from the transcriptome data of O. fragrans leaves treated with salt stress (Additional file 7: Table S7). Here, the OfGT genes that may be involved in the salt-stress response were initially screened. In total, the expression levels of 12 genes (OfGT1/3/12/13/15/21/23/33/42 /45/46/52) were higher (FPKM > 10) after the salt treatment and showed obvious changes (Fig. 6a). Then, qRT-PCR was used to determine whether these 12 genes were responded to salt stress. Under salt-stress conditions, the expression levels of OfGT1/3/42/45/46/52 were obviously upregulated. In particular, the FPKM values of the OfGT3/42/46 genes were highest after a 72 h salt treatment. In addition, the expression levels of OfGT21/23 obviously decreased, whereas the expression levels of the OfGT12/13/15/23 genes showed no changes under saltstress conditions (Fig. 6b). The expression patterns of these 12 genes showed that OfGT1/12/42/45/46/52 were strongly positively correlated, which indicates that these genes might enhance the tolerance to salt stress through cooperative effects (Fig. 6c). In general, the expression change trends of these 12 genes were basically the same as the change trends of their corresponding transcriptome FPKM values. A correlation analysis chart of the relative expression values and transcriptome data FPKM values showed a high correlation coefficient (r 2 = 0.76) (Fig. 6d), indicating the reliability of the transcriptome data.

Expression analysis of OfGT genes in response to other stresses
To further explore whether these 12 OfGT genes only respond to salt stress or respond to multiple stresses, the genes' expression profiles under drought and waterlogging stresses were analyzed. For drought stress, after 6 h of a PEG-6000 treatment, the expression levels of OfGT3/13/45/46/52 decreased and then gradually increased. OfGT12/15/42 were down-regulated after the drought treatment. In contrast, the OfGT1 gene showed up-regulated expression. The OfGT21/3/33 gene expression levels remained unchanged (Fig. 7a). The expression profiles of 12 genes under drought-stress conditions revealed that the OfGT3/13/46/52 gene group and the OfGT12/15 and OfGT33/45 gene pairs had strong positive correlations (Fig. 7b). For waterlogging treatment, the expression levels of OfGT12/13/15/42/45/52 showed increasing to decreasing trends. The levels of OfGT21/23 remained the same at the beginning and then declined after 24 h of treatment. The OfGT1/3//33/46 genes' expression levels showed no significant changes under waterlogging-stress conditions (Fig. 7a). The correlation analysis of 12 genes subjected to waterlogging stress revealed that OfGT1/12/13/45 gene group, and the OfGT3/21 and OfGT23/52 gene pairs, had strong positive correlations (Fig. 7b). To further investigate whether OfGT genes respond to hormonal stresses, we treated O. fragrans seedlings with MeJA, ABA, and GA 3 . The expression trends of the 12 OfGT genes, under different hormonal treatments were roughly the same, which indicated that they slowly dropped after 6 h of treatment and gradually rose after 12 h of treatment (Fig. 8a). The integration of expression data under multiple treatments have been used to perform the co-expression analysis (Additional file 8: Fig. S1). Under MeJA-stress conditions, the expression patterns of 12 genes showed that the OfGT33/42/46 and OfGT3/13/15/45/52 gene groups, as well as the OfGT1/23 and OfGT12/21 gene pairs have strong positive correlations (Fig. 8b) (Fig. 8b). Thus, some of OfGT genes had co-expression patterns under different stresses. We speculated that these OfGT genes improve tolerance levels to a specific stress through cooperative relationships in O. fragrans. In particular, the positive correlations of 12 OfGT genes was more obvious after GA 3 and MeJA treatments, which indicates that they may have closer cooperative relationships under these conditions. Furthermore, most of selected OfGTs were induced by multiple abiotic and hormonal stresses. For example, OfGT3/42/46 were induced by salt, drought, MeJA, ABA, and GA 3 stresses.

Subcellular localizations and transcriptional activation activities of OfGT3/42/46
On the basis of the FPKM values and expression level of OfGT3/42/46 genes were relatively high, they also have high degrees of homology with the salt-tolerant members of the trihelix TF family reported in soybean [22]. Therefore, we selected the OfGT3/42/46 genes for further study. The constructed GFP::pCAMBIA1300-3/42/46 fusion vectors and the 1300 empty vector were independently transiently transformed into tobacco (Nicotiana benthamiana) leaves. As shown in Fig. 9, the proteincoding nucleotide products of the three genes OfGT3, OfGT42 and OfGT46 in the trihelix TF family of O. fragrans were mainly expressed in the nuclei.
The transcriptional activation of OfGT3/42/46 was determined by constructing pGBKT7 vectors that were then transformed into yeast strain AH109. The positive control grew well on SD/−Trp, SD/−Trp-Ade, and SD/−Trp-Ade + X-α-gal media and produced a blue color. In contrast, yeast strains transformed with the negative control pGBKT7 vector and the OfGT3/42/46 pGBKT7 vectors were only able to grow well on the SD/− Trp culture medium. They did not grow on the SD/−Trp-Ade and SD/−Trp-Ade + X-α-gal media, and there were no blue color-producing reactions (Additional file 9: Fig.  S2). The results indicated that OfGT3/42/46 were not active in the yeast strain AH109.

The analysis of malondialdehyde content and the transient expression of OfGT3/42/46
Malondialdehyde (MDA) is a membrane lipid peroxidation product [32]. By determining the levels of MDA, the degree of damage to the cell membrane can be evaluated [33]. Under salt stress, the MDA content of tobacco infected with pCAMBIA1300-OfGT3/42/46 was lower than that of the control (pCAMBIA1300), and the difference reached a significant level (Fig. 10b). The results indicate that the cell membrane of tobacco infected by pCAMBIA1300-OfGT3/42/46 is less damaged than that in the control plants, indicating that the transient transformation of OfGT3/42/46 might enhance the salt tolerance of tobacco. In addition, the results of semiquantitative RT-PCR strongly proved that the pCAM-BIA1300-OfGT3/42/46 fusion protein was expressed in tobacco (Fig. 10a). The original uncropped gel (Fig. 10a) of qRT-PCR analysis was provided in the additional files (Additional file 10: Fig. S3).

Discussion
Many researchers have systematically identified the trihelix TF gene family in various plants, such as A. thaliana, P. edulis, Camellia sinensis, and P. trichocarpa [10,11,34,35]. However, no comprehensive research on the trihelix TF gene family of O. fragrans has been reported. In this study, 56 OfGT gene members were identified in O. fragrans (Additional file 2: Table S2). A phylogenetic analysis showed that the O. fragrans trihelix genes formed five clades (GT-1, SH4, GTγ, GT-2, and SIP1) (Fig. 1). According to previous studies, genes with few or no introns have high expression levels in plants [36], and 34% of trihelix genes in the woody plant P. edulis have no introns [10]. This proportion is greater in grasses (87% in Brachypodium distachyon and 90% in wheat) [34]. The level in O. fragrans, at 23%, was similar to that of other woody plants. Therefore, we speculated that the expression levels of trihelix genes may be higher in grasses than in woody plants. The specific sequence motifs present in each subfamily may confer specific functions for the trihelix protein members [37]. A MEME analysis showed that OfGT genes coming from the same subfamily usually have similar motif compositions (Fig. 2c); therefore, their functions may have similar. In addition, the similarities in the motif compositions and genetic structures of most OfGT genes in each subfamily indicated that the phylogenetic tree was reliable (Fig. 2).
The outspread of the gene family and the mechanisms of genome evolution mainly depend on gene duplication events, including tandem and fragment duplications [38]. Previous studies have shown that the O. fragrans has two whole-genome duplication (WGD) events [31], which could lead the expansion of the Trihelix family. In this study, most of the OfGT genes (78.6%) were fragment duplication genes (Fig. 4a), and there were more than in P. trichocarpa (50%) [11]. We suggest that the amplification of trihelix gene family members in O. fragrans mainly occurred through gene fragment replication events. Generally, positive selection pressure is conducive to gene amplification or functional differentiation, whereas purification selection pressure tends to increase gene conservation [39]. In addition, all the pairs of tandem repeats and fragment repeats in O. fragrans had Ka/ Ks < 1 (Additional file 6: Table S6), which indicates that most of the OfGT genes have undergone strong purification selection during the evolutionary process.
A gene expression profile is essential to analyze the functional roles of various trihelix genes [40]. In this study, the expression pattern of each OfGT gene in each different tissue varied (Fig. 5). The OfGT genes specifically expressed in tissues may have tissue-specific functions. For example, AtGT2, which plays a fundamental role in salt-stress responses, is highly expressed in rosette leaves [17]. In a comparison of three candidate genes (OfGT3/42/46), OfGT3 was highly expressed in roots and mature leaves, whereas OfGT42/46 were highly expressed in leaves. We speculate that the OfGT3/42/46 genes may be involved in responses to salt stress. In addition, OfGT47 is only expressed in roots, whereas three other genes (OfGT19/22/43) are only expressed in stems. These results implied that some OfGT genes have tissuespecific functions in O. fragrans, and the genes could play crucial roles in plant growth and development.
The RNA-seq data and qRT-PCR confirmed that OfGT3/42/46 were induced by salt stress (Fig. 6a-b). In addition, NP_001236630.1and NP_001236643.1 are salt tolerance genes reported in soybean, OfGT3/42 and OfGT46 have very high homology with NP_001236630.1 and NP_001236643.1, respectively [22]. (Additional file 11: Fig. S4). We speculated that the OfGT3/42/46 genes may play important roles in improving the salt-stress tolerance of O. fragrans. The subcellular localization results showed that OfGT3/42/46 localize in the nuclei (Fig. 9), which implies that the three TFs regulate the transcriptional processes of target genes in the nuclei. Furthermore, after transient expression of OfGT3/42/46 genes in tobacco, the MDA content was reduced compared with the control, which indicating that overexpression of OfGT3/42/46 increased the tolerance of plants to salt stress (Fig. 10). It is worth noting that the overexpressing of OfGT3/42/46 in the tobacco, the gene expression levels of NbAPX (ROSrelated genes) were significantly up-regulated (Fig. 11), implying that OfGT3/42/46 genes can reduce the oxidative damage of cell membrane by activating ROS-related genes. However, OfGT3/42/46 showed no transcriptional activities, as assessed by a transcriptional self-activation analysis (Additional file 9: Fig. S2). In A. thaliana, the AtGT4 TF interacts with the TEM2 gene to regulate the expression of the salt-responsive gene Cor15A to enhance the plant's salt tolerance [24]. Hence, we speculate that OfGT3/42/46 might regulate downstream genes by forming complexes with other TFs.
Promoter cis-elements play essential roles in responses to biotic and abiotic stresses in plants [41]. In this study, many important cis-acting elements relevant to plant abiotic and hormonal stresses were discovered in the 56 OfGT genes, including CGTCA-motif, P-box, ARE and ABRE (Fig. 3). Here, some OfGT genes were found to play roles in responding to stresses. For example, the expression levels of OfGT1/3/42/45/46/52 were up-regulated under salt-stress conditions (Fig. 6b), the expression levels of OfGT12/13/15/42/45/52 changed under waterlogging stress (Fig. 7a), the expression levels of OfGT3/13/45/46/52 were induced by drought (Fig. 7a), and the expression levels of OfGT3/12/15/33/42/45/46 were induced by MeJA, ABA, and GA 3 stresses (Fig. 8a). In addition, some OfGT genes responded to multiple abiotic and hormonal stresses in O. fragrans, such as, the OfGT3/42/46 genes were up-regulated under saltstress conditions, and they were also induced by drought, MeJA, ABA, and GA 3 stresses (Figs. 6b, 8a). In P. edulis, P. trichocarpa, and other species, there are reports of trihelix TFs responding to salt, drought, and hormonal stresses [10,35]. These analyses indicate that the trihelix TFs play important roles in plant adaptation to stressed environments, and some of the OfGT genes respond to multiple stresses in O. fragrans. Moreover, most OfGT genes' expression patterns are similar in different stresses, and there are many instances of co-expression (Figs. 6c,  7b, and 8b). For example, OfGT42/OfGT46 showed a coexpression pattern under salt-stress conditions (Fig. 6c), and this may play a role in improving the tolerance of O. fragrans to salt stress through cooperative relationships. In particular, the expression patterns of some OfGTs had high correlations after GA3 and MeJA treatments (Fig. 8b), which indicates that the OfGT genes might enhance the tolerance to GA3 and MeJA through cooperative effects. Remarkably, the integration of expression data under multiple treatments have been used to perform the co-expression analysis (Additional file 8: Fig.  S1). We found that OfGT13/OfGT46, OfGT3/OfGT13 and OfGT42/OfGT15 gene pairs had higher expression coefficients in most of stress conditions, indicating these gene pairs could have synergistic effect in response to different environmental signals.

Conclusion
In this study, 56 trihelix TF genes were identified in the O. fragrans genome, and they were divided into five subfamilies. The gene duplication events analysis showed that the fragment duplication events contributed to the expansion of the OfGT genes family in O. fragrans. Here, we determined that the OfGT genes have different expression patterns in specific tissues, and the OfGTs also contain a variety of cis-elements involved in responses to multiple abiotic and hormonal stresses. The qRT-PCR confirmed that these OfGT genes are induced by salt and other stresses. It is worth noting that the OfGT genes showed many coexpression patterns during different stress induction, which may mean that some OfGT gene members play cooperative roles in specific stresses. Furthermore, the decrease in MDA content and the expression levels of ROS-related genes up-regulated after the transient expression of nucleus located OfGT3/42/46 genes in tobacco indicated that these genes could enhance the salt tolerance of tobacco. In short, our research results strengthen our understanding of the OfGT gene family and the salt-tolerance mechanisms of O. fragrans.

Identification of the trihelix family in O. fragrans
The release of the genome-wide sequence data provided us an opportunity to research the members of the OfGT gene family [31]. The Hidden Markov Model (HMM) profiles of trihelix TFs (PF13837) were acquired from the Pfam database (http:// pfam. xfam. org/) to identify putative OfGT gene members. Then, the online software SMART (http:// smart. embl-heide lberg. de/), Search Pfam (http:// pfam. xfam. org/ search/), and CDD (https:// www. ncbi. nlm. nih. gov/ Struc ture/ bwrpsb/ bwrpsb. cgi/) were used to examine the OfGT domains that were conserved among these protein sequences. The isoelectric points and protein molecular weights of the OfGT genes were predicted using the online website ExPASy (https:// web. expasy. org/ compu te_ pi/).

Gene structure, motif compositions, and promoter analysis of the OfGTs gene family
The OfGT gene structures were visualized using TBtool tools [42]. The online website MEME (https:// meme-suite. org/ meme/) was utilized to analyze the conserved motifs of OfGT proteins, the parameters related to motif repeats were set to 'any' , the motif prediction number to 25, and the motif length to 6-200 aa [43]. The online tool PLACE [44] was used to identify the cis-acting elements of 2000 bp DNA sequences upstream of the OfGT genes.

Chromosomal distribution and gene duplication events of OfGT genes
Chromosomal information for OfGT genes were isolated from the genomic database for O. fragrans [31]. TBtools tools was used to visualize the distribution of the OfGT genes on the chromosomes [42]. A Multiple Collinearity Scan toolkit (MCScanX) was used to analyze duplication events involving OfGT genes [45]. The online tool DNAsp V6 (http:// www. ub. edu/ dnasp/) was utilized to calculate Ks and Ka substitution rates to further analyze the evolutionary selection pattern of OfGT genes in O. fragrans [46].

Expression profiles of OfGTs in salt-stressed leaves and different tissues of O. fragrans
We obtained the RPKM data for OfGT genes in the tissues roots, stems, leaves (young and mature), and flowers (initial, full-booming, and final fading flowering) from the O. fragrans transcriptome database [47]. The FPKM values for the OfGT genes in leaves were obtained from the O. fragrans transcriptome database under salt stress conditions (Additional file 7: Table S7). The TBtools software was used to construct heatmaps.

Plant materials and treatments
The material for this experiment was 2-year-old cutting seedlings of O. fragrans 'Rixianggui' [48], which was planted in the experimental field of Nanjing Forestry University, Nanjing, China (32°5′N,118°48E′). First, seedlings with the same growth trend were transplanted into a pot (10 cm pot height, 10 cm inner diameter), and placed in a growth chamber for 3 weeks under the following conditions: light/dark: 16/ 8 h, day/ night temperatures: 23 °C/21 °C, light intensity: 260 μmol m − 2 s − 1 , and relative humidity: 62% [49]. Then, the seedlings were exposed to abiotic stresses and hormones. Abiotic stress treatments included salt, waterlogging, and drought. The salt stress was achieved by treating with 250 mM NaCl solution containing 1/2 Hoagland's nutrient solution. Waterlogging stress was achieved by soaking the seedlings in a container with 1/2 Hoagland's solution. Drought stress was achieved by treating with 20% PEG6000. Hormone treatments involved spray plant leaves with MeJA (100 μM), GA 3 (50 μM), and ABA (100 μM) [11,34,50]. Finally, samples were collected at 0, 6, 24, and 72 h after the abiotic stress treatment, and hormone treatment samples were collected after 0, 6, and 24 h. Three biological replicates were collected for each sample. Identification of the plant variety was made by Qibai Xiang based on reliable sources available in the literature. A voucher specimen of 'Rixianggui' has been deposited in the National Germplasm Bank of Osmanthus fragrans in Nanjing, China (31°35′N,119°09E′).

RNA extraction and qPCR
The Plant RNA Extraction Kit V1.6 (Biofit, Chengdu, China) was utilized to extract total RNA from similarsized leaves from the top of O. fragrans. The cDNA Synthesis SuperMix kit (Transgen, Beijing, China) was used to transcribe the RNA into cDNA [51]. We used the Primer 5.0 tool to design specific primers (Additional file 12: Table S8). The internal reference gene was RAN in O. fragrans [47,49]. The selected gene expression levels were analyzed using the 2 −ΔΔCT method [52]. The comparative cycle threshold (Ct) values were adopted to calculate the relative expression levels of OfGT genes [53]. Each qRT-PCR assay provided three biological replicates and three technical replicates. SPSS and Origin2019 software were used to perform the statistical analyses.

Subcellular localization and transcriptional activation
The GFP::pCAMBIA1300-OfGT3/42/46 fusion expression vectors were constructed, and the fusion vectors were transformed independently into A. tumefaciens GV3101. The fusion vectors were injected into 40-dayold growing tobacco leaves, and the fluorescence signals of green fluorescent protein were observed using an LSM710 microscope (Zeiss, Germany). BD::PGBKT7-OFGT3/42/46 vectors were constructed, and both the constructed vectors carrying the target genes and the empty vector were transformed into the AH109 yeast strain. By observing the growth of the transformed yeast strains on SD/−Trp, SD/−Trp-Ade, and SD/−Trp-Ade + X-α-gal media, the transcriptional activation activity of each target gene was determined.

Transient transformation of OfGT3/42/46 and measuring the malondialdehyde content
Agrobacterium infection of N. benthamiana leaves is the commonly used transient expression system in plants [54][55][56]. The fusion vector pCAMBIA1300-OfGT3/42/46 and pCAMBIA1300 (control) were introduced into A. tumefaciens GV3101 and then injected into 35-day-old tobacco leaves. The transiently transformed tobacco was placed in a growth chamber for cultivation (the conditions were the same as previously described). After 2 days, the plants were irrigated with 500 mM NaCl solution and then samples were collected after 12 h, with three biological replicates for each sample. RNA was extracted from the collected samples and reverse transcribed into cDNA for semi-quantitative RT-PCR analysis. Tobacco NbL25 was used as an internal reference and the primers used in OfGT3/42/46 were the same as those used in the qRT-PCR analysis. The determination of MDA content was modified with reference to Zhou and Leul [57]. After grinding the fresh leaves without main veins with liquid nitrogen, a 0.2 g sample was weighed and homogenized in 5 mL 5% TCA. We mixed the extract with 2 ml of 0.67% TBA. The mixture was heated at 100 °C for 30 min and then placed in an ice bath to cool. After centrifugation at 8500 r/min for 20 min, the supernatant absorbance was measured at 450, 532, and 600 nm. The qRT-PCR analysis of the ROSrelated genes in the OfGTs and empty vector transient expression tobaccos were conducted.