EbbHLH80 Enhances Salt Responses by Up-Regulating Flavonoid Accumulation and Modulating ROS Levels

bHLH transcription factors are involved in multiple aspects of plant biology, such as the response to abiotic stress. Erigeron breviscapus is a composite plant, and its rich flavonoids have strong preventive and therapeutic effects on cardio cerebral vascular disease. EbbHLH80, a gene from E. breviscapus that positively regulates flavonoid synthesis, was previously characterized. However, it is unclear whether EbbHLH80 increases flavonoid accumulation, which affects salt tolerance. The function of EbbHLH80 in transgenic tobacco seeds was identified by phylogenetic analysis and metabolome-transcriptome analysis. We investigated the role of EbbHLH80 in salt stress response. Our results showed that the expression of EbbHLH80 increased following salt treatment. Integrating the metabolome and transcriptome analysis of EbbHLH80-OE and Yunyan 87 (WT) seeds, we identified several genes and metabolites related to flavonoid biosynthesis and salt stress. Moreover, EbbHLH80-OE plants displayed higher salt tolerance than wild-type plants during seed germination and seedling growth. After salt treatment, transgenic tobacco had significantly lower levels of reactive oxygen species (ROS) than WT, with enhanced levels of antioxidant enzyme expression. Altogether, our results demonstrated that EbbHLH80 might be a positive regulator, promoting salt tolerance by modulating ROS scavenging and increasing stress-responsive genes.


Introduction
Salt stress induces both osmotic and toxicity stresses in plants, which lead to growth inhibition, developmental changes, metabolic adaptations, and ion sequestration [1]. Salt stress triggers rapid production of ROS molecules, such as hydrogen peroxide, singlet oxygen, superoxide, and hydroxyl radicals, leading to oxidative damage to plants [2][3][4][5]. Previous studies have shown that transcription factors play a critical role in plant adaptation to abiotic stresses [6].
Several eukaryotes, from yeast to plants and animals, possess basic helix-loop-helix (bHLH) proteins, which belong to a large transcription factor (TF) family. This protein contains a region of 60 amino acids, which is divided into two functional domains [7]. bHLH transcription factors regulate plant growth and development, metabolism, and stress responses. Therefore, transcription factors act as molecular switches in plants while they respond to abiotic stress [8,9]. For example, bHLH106 has been shown to be associated with salt tolerance in Arabidopsis [10]. AtbHLH112 increased the expression of the AtPOD and AtSOD genes, while simultaneously reducing the expression of the P5CDH and ProDH genes, thus enhancing salt resistance in Arabidopsis [11]. ThbHLH1 gene has been shown to increase peroxidase (POD) and superoxide dismutase (SOD) activities in response to high salt induction in Tamarix hispida [12]. Meanwhile, bHLHs may also be involved in the regulation of secondary metabolites that contribute to resistance to salinity. Salt stress activates AtMYC2 via a mitogen-activated protein kinase (MAPK) cascade. AtMYC2 can then bind to the promoter of P5CS1, thereby regulating proline synthesis and salt tolerance [13]. Additionally, AtbHLH122 inhibited CYP707A3 expression under NaCl stress [11]. Moreover, EcbHLH57 can enhance tobacco resistance to salt stress by increasing the expression of stress responsive genes [14]. It appears that OsbHLH068 has a similar effect on salt stress regulation as OsbHLH112 that heterogenic overexpression of OsbHLH068 can reduce salt-induced accumulation of H 2 O 2 in Arabidopsis [15].
Plants produce a wide range of secondary metabolites with diverse chemical and physical properties. Plant secondary metabolites play a crucial role in their growth, development, and immunity [16]. The flavonoid pathway is usually initiated by physiological and environmental fluctuations as a protection against oxidative stresses, resulting from pathogen infections, UV, drought, salt, and nutrient deficiency, etc. [17]. Furthermore, flavonoids exhibit strong biological activity and significant antioxidant properties [18,19] and are involved in the protection of plants against UV radiation, salt, heat, and drought. Plants can resist biotic and abiotic stresses with flavonoids, such as flavonols and anthocyanins. Several studies have shown that the overexpression of MYB12 confers resistance to insect attack in tobacco, and tolerance to salt and drought in Arabidopsis [20,21].
In a previous study, we characterized EbbHLH80 in Erigeron breviscapus, which positively regulates flavonoid biosynthesis [22]. However, it is still unclear whether the increased flavonoid content was responsible for improving stress tolerance. With integrative metabolome-transcriptome analysis, we identified metabolites and genes related to flavonoid biosynthesis and salt stress in seeds of EbbHLH80-OE and Yunyan87 (WT). Furthermore, a positive correlation was observed between flavonoid contents with expression level of EbbHLH80, while EbbHLH80-OE plants exhibited hypersensitive phenotypes to salt stress during seed germination and seedling growth, compared to wild-type plants.
EbbHLH80-OE modulated ROS scavenging, flavonoid biosynthesis, and stress-responsive gene expression in tobacco to enhance salt tolerance. Therefore, the above findings indicate that EbbHLH80 plays a crucial role in salt stress tolerance. Furthermore, it could act as a critical available gene for genetic engineering breeding for plant salt resistance.

Phylogenetic Analysis of EbbHLH80
Our previous studies reported that EbbHLH80 increases the flavonoid contents in tobacco leaves [22]. Additionally, the protein sequences of highly homologous Attt8 genes from various plants were conducted to further clarify the evolutionary relationship, the phylogenetic analysis showed that Brassica napus TT8 (BnTT8, NP_ 001302903.1), Brassica juncea TT8 (BjTT8, AIN41653.1), Arabidopsis thaliana TT8 (AtTT8, CAC14865.1), and EbbHLH80 were highly similar (Supplementary Figure S1). Previous studies showed that TT8 acted as a crucial component of a well-conserved complex controlling flavonoid accumulation [23,24]. While we know that EbbHLH80 plays a role in flavonoid biosynthesis, the role of EbbHLH80 in seeds involved in responding to various stresses has not been reported. Thus, we investigated whether EbbHLH80 is also involved in salt tolerance.

Differences in Metabolites with WT and EbbHLH80-OE Seeds
The phenotypes of EbbHLH80-OE (OE1, OE5, and OE9) and WT seeds showed no significant differences (Figure 1). To evaluate the effects of EbbHLH80 in tobacco seeds, the seeds of overexpression of EbbHLH80 in the tobacco were confirmed by RT-qPCR analysis ( Figure 2). The result confirmed that the transcript levels of EbbHLH80 were significantly higher in OE5 than in OE1 and OE9. Subsequently, to further explore the changes in the metabolic levels in seeds of EbbHLH80-OE tobaccos, OE5 was chosen and used for metabolite analysis. significant differences (Figure 1). To evaluate the effects of EbbHLH80 in tobac the seeds of overexpression of EbbHLH80 in the tobacco were confirmed by analysis ( Figure 2). The result confirmed that the transcript levels of EbbHLH significantly higher in OE5 than in OE1 and OE9. Subsequently, to further ex changes in the metabolic levels in seeds of EbbHLH80-OE tobaccos, OE5 was ch used for metabolite analysis.  The metabolites of samples were analyzed by principal component analys to illustrate significant differences in metabolites between WT and OE5 seeds a separated in the PCA score plots with 65.58% of PC1 and 9.93% of PC2 (Figure obtained a total of 1136 metabolites, of which 301 were differentially accumul tabolites (DAMs), including 301 upregulated-accumulated metabolites and 57 d ulated-accumulated metabolites (Supplementary Table S2). These metabolites w sified into 11 classes. The top 4 major classes of DAMs were phenolic acids (19 pids (16.48%), flavonoids (15.08%), and alkaloids (13.69%), respectively. To fur cidate the differences in metabolite accumulation in the seeds of WT and OE chical clustering analysis was performed on all the DAMs of the obtained com pairs, and the relative quantitative values of the DAMs were normalized and ( Figure 3B). The results showed that the metabolites changed significantly bet seeds of WT and OE5.
Annotation of the metabolites was performed using the KEGG pathway The results identified that the DAMs in KEGG terms were principally related analysis ( Figure 2). The result confirmed that the transcript levels of E significantly higher in OE5 than in OE1 and OE9. Subsequently, to furth changes in the metabolic levels in seeds of EbbHLH80-OE tobaccos, OE5 w used for metabolite analysis.  The metabolites of samples were analyzed by principal component to illustrate significant differences in metabolites between WT and OE5 s separated in the PCA score plots with 65.58% of PC1 and 9.93% of PC2 (F obtained a total of 1136 metabolites, of which 301 were differentially acc tabolites (DAMs), including 301 upregulated-accumulated metabolites an ulated-accumulated metabolites (Supplementary Table S2). These metabo sified into 11 classes. The top 4 major classes of DAMs were phenolic aci pids (16.48%), flavonoids (15.08%), and alkaloids (13.69%), respectively. cidate the differences in metabolite accumulation in the seeds of WT an chical clustering analysis was performed on all the DAMs of the obtain pairs, and the relative quantitative values of the DAMs were normalized ( Figure 3B). The results showed that the metabolites changed significant seeds of WT and OE5.
Annotation of the metabolites was performed using the KEGG path The results identified that the DAMs in KEGG terms were principally re The metabolites of samples were analyzed by principal component analysis (PCA) to illustrate significant differences in metabolites between WT and OE5 seeds and were separated in the PCA score plots with 65.58% of PC1 and 9.93% of PC2 ( Figure 3A). We obtained a total of 1136 metabolites, of which 301 were differentially accumulated metabolites (DAMs), including 301 upregulated-accumulated metabolites and 57 downregulatedaccumulated metabolites (Supplementary Table S2). These metabolites were classified into 11 classes. The top 4 major classes of DAMs were phenolic acids (19.27%), lipids (16.48%), flavonoids (15.08%), and alkaloids (13.69%), respectively. To further elucidate the differences in metabolite accumulation in the seeds of WT and OE5, hierarchical clustering analysis was performed on all the DAMs of the obtained comparison pairs, and the relative quantitative values of the DAMs were normalized and clustered ( Figure 3B). The results showed that the metabolites changed significantly between the seeds of WT and OE5.
Annotation of the metabolites was performed using the KEGG pathway database. The results identified that the DAMs in KEGG terms were principally related to flavonoid biosynthesis ( Figure 3C), indicating that flavonoid accumulation was much higher in seeds of OE5 than those in WT. These results suggested that the function of EbbHLH80 not only affect the flavonoid biosynthesis in leaves, but also in seeds. Thus, we speculated that DAMs in flavonoid biosynthesis might be initiated by overexpressing EbbHLH80. noid biosynthesis ( Figure 3C), indicating that flavonoid accumulation was much higher in seeds of OE5 than those in WT. These results suggested that the function of EbbHLH80 not only affect the flavonoid biosynthesis in leaves, but also in seeds. Thus, we speculated that DAMs in flavonoid biosynthesis might be initiated by overexpressing EbbHLH80.

Transcriptome Profiling of WT and EbbHLH80-OE
To understand the molecular basis of the metabolic differences between WT and EbbHLH80-OE, transcriptome sequencing was performed using the seeds of WT and OE5, with three biological replicates for each group. Data filtering produced an average of 6.69 Gb data for each sample. The Q20 value was in the range of 96.82-98.10%; the Q30 value was in the range of 91.55-94.14%; and the GC content was in the range of 43.69-44.45%. In general, the transcriptome data indicated that the sequencing quality was high, and the data obtained by sequencing are accurate and reliable.
A total of 6438 differentially expressed genes (DEGs; A false discovery rate (FDR) < 0.05 and log2fold change ≥|1|) were identified (Supplementary Table S3), of which 3326 were up-regulated and 3112, as down-regulated, as shown in the volcano plots ( Figure  4A). These DEGs were significantly associated with 55 GO functions and 136 KEGG pathways ( Figure 4B,C). KEGG analysis showed that several metabolic processes, including plant hormone signal transduction, alpha-linolenic acid metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and flavone and flavanol biosynthe-

Transcriptome Profiling of WT and EbbHLH80-OE
To understand the molecular basis of the metabolic differences between WT and EbbHLH80-OE, transcriptome sequencing was performed using the seeds of WT and OE5, with three biological replicates for each group. Data filtering produced an average of 6.69 Gb data for each sample. The Q20 value was in the range of 96.82-98.10%; the Q30 value was in the range of 91.55-94.14%; and the GC content was in the range of 43.69-44.45%. In general, the transcriptome data indicated that the sequencing quality was high, and the data obtained by sequencing are accurate and reliable.
A total of 6438 differentially expressed genes (DEGs; A false discovery rate (FDR) < 0.05 and log2fold change ≥|1|) were identified (Supplementary Table S3), of which 3326 were up-regulated and 3112, as down-regulated, as shown in the volcano plots ( Figure 4A). These DEGs were significantly associated with 55 GO functions and 136 KEGG pathways ( Figure 4B,C). KEGG analysis showed that several metabolic processes, including plant hormone signal transduction, alpha-linolenic acid metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and flavone and flavanol biosynthesis were significantly enriched in DEGs ( Figure 4C). This indicates that EbbHLH80 might affect more genes that can be related to secondary metabolites biosynthesis in seeds. sis were significantly enriched in DEGs ( Figure 4C). This indicates that EbbHLH80 might affect more genes that can be related to secondary metabolites biosynthesis in seeds. The abscissa represents the gene expression fold change, and the ordinate represents the significance level of the differential genes. (B) GO classification of differentially expressed genes in WT and EbbHLH80-OE seeds. The abscissa represents the GO item, and the ordinate represents the number of differential genes of the GO item. (C) KEGG classification histogram of differentially expressed genes in WT and EbbHLH80-OE seeds. The abscissa represents the ratio of the number of differential genes annotated to the pathway to the number of differential genes annotated, and the ordinate represents the name of the KEGG pathway.

EbbHLH80 Positively Regulates Tobacco Tolerance to Salt Stress
In plants, flavonoids are antioxidative agents that scavenge reactive oxygen species (ROS). Flavonoids inhibit ROS-generating enzymes, thereby preventing ROS production. Flavonoids are considered secondary antioxidant systems, since they complement other ROS scavenging systems that are reduced in activity. Our results showed that EbbHLH80 could increase the accumulation of flavonoids in transgenic tobacco seeds; for example, the content of kaempferol increased 12.94-fold compared with that in WT, suggesting that these flavonoids contribute to the improvement of antioxidant capacity, thereby exhibiting a greater increase in responding salt stress in transgenic tobacco plants. Additionally, transcriptome analysis showed that some salt resistance-related genes were up-regulated. Therefore, we speculate that the impact of overexpressing EbbHLH80 on salt tolerance is primarily from the increasing flavonoids accumulation, which improves the resistance of tobacco to salinity.
To evaluate whether EbbHLH80 could improve salt tolerance in transgenic plants, the response to salt stress was analyzed by measuring the germination rate of seeds from The abscissa represents the gene expression fold change, and the ordinate represents the significance level of the differential genes. (B) GO classification of differentially expressed genes in WT and EbbHLH80-OE seeds. The abscissa represents the GO item, and the ordinate represents the number of differential genes of the GO item. (C) KEGG classification histogram of differentially expressed genes in WT and EbbHLH80-OE seeds. The abscissa represents the ratio of the number of differential genes annotated to the pathway to the number of differential genes annotated, and the ordinate represents the name of the KEGG pathway.

EbbHLH80 Positively Regulates Tobacco Tolerance to Salt Stress
In plants, flavonoids are antioxidative agents that scavenge reactive oxygen species (ROS). Flavonoids inhibit ROS-generating enzymes, thereby preventing ROS production. Flavonoids are considered secondary antioxidant systems, since they complement other ROS scavenging systems that are reduced in activity. Our results showed that EbbHLH80 could increase the accumulation of flavonoids in transgenic tobacco seeds; for example, the content of kaempferol increased 12.94-fold compared with that in WT, suggesting that these flavonoids contribute to the improvement of antioxidant capacity, thereby exhibiting a greater increase in responding salt stress in transgenic tobacco plants. Additionally, transcriptome analysis showed that some salt resistance-related genes were up-regulated. Therefore, we speculate that the impact of overexpressing EbbHLH80 on salt tolerance is primarily from the increasing flavonoids accumulation, which improves the resistance of tobacco to salinity.
To evaluate whether EbbHLH80 could improve salt tolerance in transgenic plants, the response to salt stress was analyzed by measuring the germination rate of seeds from WT and EbbHLH80-OE after NaCl treatment (0, 50 mM) for 7 days. The results indicated that the germination rate of EbbHLH80-OE (OE1, OE5, and OE9) reached a substantially higher level than those in WT, while their response to salt stress, which was approximately 2.32-fold, 2.90-fold, and 2.32-fold higher, respectively ( Figure 5). To demonstrate that salt stress would have an effect on the tobacco seedling phenotype of EbbHLH80-OE, tobacco seedlings were treated using different concentration of NaCl (0, 50, 100, 150 mM) for four weeks, respectively ( Figure 6). It was observed that high concentrations of NaCl significantly inhibited the growth of tobacco and turned the leaves yellow. However, compared to wild-type tobacco, transgenic tobacco showed excellent growth conditions. We found that no significant difference was observed between the wild-type (WT) and EbbHLH80-OE lines (OE1, OE5, and OE9) when grown under normal conditions. WT and EbbHLH80-OE after NaCl treatment (0, 50 mM) for 7 days. The results indicated that the germination rate of EbbHLH80-OE (OE1, OE5, and OE9) reached a substantially higher level than those in WT, while their response to salt stress, which was approximately 2.32-fold, 2.90-fold, and 2.32-fold higher, respectively ( Figure 5). To demonstrate that salt stress would have an effect on the tobacco seedling phenotype of EbbHLH80-OE, tobacco seedlings were treated using different concentration of NaCl (0, 50, 100, 150 mM) for four weeks, respectively ( Figure 6). It was observed that high concentrations of NaCl significantly inhibited the growth of tobacco and turned the leaves yellow. However, compared to wild-type tobacco, transgenic tobacco showed excellent growth conditions. We found that no significant difference was observed between the wild-type (WT) and EbbHLH80-OE lines (OE1, OE5, and OE9) when grown under normal conditions.   WT and EbbHLH80-OE after NaCl treatment (0, 50 mM) for 7 days. The results indicated that the germination rate of EbbHLH80-OE (OE1, OE5, and OE9) reached a substantially higher level than those in WT, while their response to salt stress, which was approximately 2.32-fold, 2.90-fold, and 2.32-fold higher, respectively ( Figure 5). To demonstrate that salt stress would have an effect on the tobacco seedling phenotype of EbbHLH80-OE, tobacco seedlings were treated using different concentration of NaCl (0, 50, 100, 150 mM) for four weeks, respectively ( Figure 6). It was observed that high concentrations of NaCl significantly inhibited the growth of tobacco and turned the leaves yellow. However, compared to wild-type tobacco, transgenic tobacco showed excellent growth conditions. We found that no significant difference was observed between the wild-type (WT) and EbbHLH80-OE lines (OE1, OE5, and OE9) when grown under normal conditions.  To investigate whether salt stress could cause the root length to be affected, we examined the root length growth of WT and EbbHLH80-OE on 50 mM NaCl medium. Root length of WT seedlings was more severely inhibited than that of EbbHLH80-OE on MS plates supplemented with 50 mM NaCl (Figure 6), while EbbHLH80-OE tobacco showed longer roots ( Figure 7A,E). Salt treatment reduced tobacco seedling growth parameters regardless of EbbHLH80 expression changes. However, a less significant inhibition was observed in EbbHLH80-OE plants. Furthermore, after 3 weeks of salt treatment, EbbHLH80-OE plants showed greener leaves as compared to WT plants. To further explain the stress phenomenon, the fresh weight, dry weight, and root length were measured on tobacco plants. The results showed a higher level under 50 mM salt stress than those of the WT plants, indicating that EbbHLH80 suppresses NaCl-induced growth ( Figure 7B-D,F-H). Moreover, WT plants displayed a slower germination rate than EbbHLH80-OE plants in the presence of 50 mM NaCl. These results confirm that salt tolerance was significantly higher in EbbHLH80-OE (OE1, OE5, and OE9) than in WT. To investigate whether salt stress could cause the root length to be affected, we examined the root length growth of WT and EbbHLH80-OE on 50 mM NaCl medium. Root length of WT seedlings was more severely inhibited than that of EbbHLH80-OE on MS plates supplemented with 50 mM NaCl (Figure 6), while EbbHLH80-OE tobacco showed longer roots ( Figure 7A,E). Salt treatment reduced tobacco seedling growth parameters regardless of EbbHLH80 expression changes. However, a less significant inhibition was observed in EbbHLH80-OE plants. Furthermore, after 3 weeks of salt treatment, EbbHLH80-OE plants showed greener leaves as compared to WT plants. To further explain the stress phenomenon, the fresh weight, dry weight, and root length were measured on tobacco plants. The results showed a higher level under 50 mM salt stress than those of the WT plants, indicating that EbbHLH80 suppresses NaCl-induced growth ( Figure 7B-D,F-H). Moreover, WT plants displayed a slower germination rate than EbbHLH80-OE plants in the presence of 50 mM NaCl. These results confirm that salt tolerance was significantly higher in EbbHLH80-OE (OE1, OE5, and OE9) than in WT.

EbbHLH80 Reduced the Damage Caused by Salt Stress in Transgenic Tobacco Seedlings
Reactive oxygen species (ROS) are produced by plants under unfavorable conditions [25,26]. ROS hyperaccumulation causes endogenous stress that damages plant growth and development [27]. The DAB staining was used to assess the amount of hydrogen peroxide (H2O2) released under salt stress, showing darker leaves in the salt treatment group than in the control group. It has been shown that plants under salt stress produce a large amount of H2O2, which can cause serious damage to them. To further address the role of EbbHLH80 in salt stress response, all lines were transferred on 1/2 MS medium plates containing 300 mM NaCl and grown for 1 day. Salt stress directly reflects the degree of plant oxidation, exhibiting the extent of plant damage via H2O2. EbbHLH80-OE and WT seedlings accumulated H2O2 in different patterns after 300 mM NaCl treatment. The DAB staining showed a large accumulation of H2O2 in WT seedlings, whereas H2O2

EbbHLH80 Reduced the Damage Caused by Salt Stress in Transgenic Tobacco Seedlings
Reactive oxygen species (ROS) are produced by plants under unfavorable conditions [25,26]. ROS hyperaccumulation causes endogenous stress that damages plant growth and development [27]. The DAB staining was used to assess the amount of hydrogen peroxide (H 2 O 2 ) released under salt stress, showing darker leaves in the salt treatment group than in the control group. It has been shown that plants under salt stress produce a large amount of H 2 O 2 , which can cause serious damage to them. To further address the role of EbbHLH80 in salt stress response, all lines were transferred on 1/2 MS medium plates containing 300 mM NaCl and grown for 1 day. Salt stress directly reflects the degree of plant oxidation, exhibiting the extent of plant damage via H 2 O 2 . EbbHLH80-OE and WT seedlings accumulated H 2 O 2 in different patterns after 300 mM NaCl treatment. The DAB staining showed a large accumulation of H 2 O 2 in WT seedlings, whereas H 2 O 2 accumulated less in OE1, OE5, and OE9 seedlings (Figure 8). These results suggest that EbbHLH80 reduces the damage caused by salt stress in transgenic tobacco seedlings, indicating that EbbHLH80 negatively responds to NaCl-induced cell death. accumulated less in OE1, OE5, and OE9 seedlings (Figure 8). These results suggest that EbbHLH80 reduces the damage caused by salt stress in transgenic tobacco seedlings, indicating that EbbHLH80 negatively responds to NaCl-induced cell death.

Expression Analysis of Genes Involved in ROS and Flavonoid Biosynthesis
To illustrate that the expression of EbbHLH80 will be affected under salt stress, we examined the expression of EbbHLH80 by using quantitative real-time PCR (RT-qPCR). EbbHLH80 transcript level was induced in 2-week-old WT and EbbHLH80-OE line seedlings after the treatment of 50 mM NaCl (for 1 h, 3 h, 6 h, 9 h, and 12 h), and reached the highest peak after 6 h, decreasing thereafter ( Figure 9). The results showed that the expression level of EbbHLH80 in transgenic tobacco seedlings was the highest at 6 h under 50 mM NaCl treatment (Figure 9).

Expression Analysis of Genes Involved in ROS and Flavonoid Biosynthesis
To illustrate that the expression of EbbHLH80 will be affected under salt stress, we examined the expression of EbbHLH80 by using quantitative real-time PCR (RT-qPCR). EbbHLH80 transcript level was induced in 2-week-old WT and EbbHLH80-OE line seedlings after the treatment of 50 mM NaCl (for 1 h, 3 h, 6 h, 9 h, and 12 h), and reached the highest peak after 6 h, decreasing thereafter ( Figure 9). The results showed that the expression level of EbbHLH80 in transgenic tobacco seedlings was the highest at 6 h under 50 mM NaCl treatment ( Figure 9). accumulated less in OE1, OE5, and OE9 seedlings (Figure 8). These results suggest that EbbHLH80 reduces the damage caused by salt stress in transgenic tobacco seedlings, indicating that EbbHLH80 negatively responds to NaCl-induced cell death.

Expression Analysis of Genes Involved in ROS and Flavonoid Biosynthesis
To illustrate that the expression of EbbHLH80 will be affected under salt stress, we examined the expression of EbbHLH80 by using quantitative real-time PCR (RT-qPCR). EbbHLH80 transcript level was induced in 2-week-old WT and EbbHLH80-OE line seedlings after the treatment of 50 mM NaCl (for 1 h, 3 h, 6 h, 9 h, and 12 h), and reached the highest peak after 6 h, decreasing thereafter ( Figure 9). The results showed that the expression level of EbbHLH80 in transgenic tobacco seedlings was the highest at 6 h under 50 mM NaCl treatment (Figure 9). To further illustrate that EbbHLH80-OE can affect the changes of the expression amounts of some genes in transgenic tobacco seeds, we further analyzed the expression levels of genes related to salt stress, flavonoid biosynthesis, and ROS scavenging. Our results showed that after 300 mM NaCl treatment for 24 h, the expression levels of some genes involved in the flavonoid biosynthetic pathway were significantly higher in the tobacco seedlings of EbbHLH80-OE than those in WT, indicating that the expression patterns of these genes were associated with the regulation of EbbHLH80 under salt stress, which was consistent with our previous findings. In addition, the expressions of genes related to POD, SOD, and proline that we found in our transcriptome data were also significantly up-regulated under salt stress, and this result indicated that EbbHLH80 played a role in salt tolerance of transgenic tobacco. These results showed that the expression levels of genes involved in flavonoid biosynthesis and reactive oxygen species (ROS) system were significantly higher in transgenic tobacco compared with WT (Figure 10), and this finding was consistent with the transcriptome data, demonstrating the reliability of the transcriptome data. To further illustrate that EbbHLH80-OE can affect the changes of the expression amounts of some genes in transgenic tobacco seeds, we further analyzed the expression levels of genes related to salt stress, flavonoid biosynthesis, and ROS scavenging. Our results showed that after 300 mM NaCl treatment for 24 h, the expression levels of some genes involved in the flavonoid biosynthetic pathway were significantly higher in the tobacco seedlings of EbbHLH80-OE than those in WT, indicating that the expression patterns of these genes were associated with the regulation of EbbHLH80 under salt stress, which was consistent with our previous findings. In addition, the expressions of genes related to POD, SOD, and proline that we found in our transcriptome data were also significantly up-regulated under salt stress, and this result indicated that EbbHLH80 played a role in salt tolerance of transgenic tobacco. These results showed that the expression levels of genes involved in flavonoid biosynthesis and reactive oxygen species (ROS) system were significantly higher in transgenic tobacco compared with WT ( Figure  10), and this finding was consistent with the transcriptome data, demonstrating the reliability of the transcriptome data. Figure 10. Relative expression level of the EbbHLH80-OE related genes in the transgenic tobacco under salt stress. Transgenic tobacco and WT seedlings were placed in a cultivation chamber and Figure 10. Relative expression level of the EbbHLH80-OE related genes in the transgenic tobacco under salt stress. Transgenic tobacco and WT seedlings were placed in a cultivation chamber and treated with 300 mM NaCl for 1 week to analyze the genes expression. The Nicotiana tabacum actin gene was used as an internal control. Mean values and standard errors were obtained from three biological and six technical replicates. The one-way analysis of variance was used for statistical analysis (** p < 0.01; * p < 0.05).

Discussion
High salt stress is a major abiotic stress that inhibits plant growth, caused by ionic and osmotic stressors eventually and affecting crop production. Therefore, salt-tolerance in plants is a complex trait, acquired through the functions of multiple genes. Plant stress tolerance can be enhanced by utilizing stress-responsive transcription factors that regulate a wide range of downstream genes [28]. Several transcriptional factors have been identified in the response to salt stress in plants, including NAC, WRKY, MYB, AP2/ERF, and bHLH [29][30][31][32][33][34]. The NAC TF family, among the different TF families, has been receiving a lot of attention because of its functional diversity [35,36]. A previous study found that one of the NAC TFs, PgNAC21, is important for regulating salt tolerance in plants [37]. It has been found that MYB proteins contribute to plant salt tolerance. Salt tolerance is modulated by MYB49 through the formation of cuticles and antioxidant defenses [38]. Furthermore, MYB12 can also induce the expression of ABA biosynthesis genes, thereby enhancing salt tolerance in plants [23]. MYB12 overexpression upregulates gene expression associated with flavonoid biosynthesis and ROS scavenging, as well as genes involved in ABA and proline biosynthesis, thus conferring salt tolerance [23,39]. Rd29A and AP2/ERF family transcription factor DREB2A, are upregulated by salt treatment, both of which contain DRE in their promoters [40].
In previous studies, bHLHs have been found to regulate plant adaptation to stress across several pathways, including resistance to mechanical damage, drought, high salt, oxidative stress, low temperature stress, heavy metal stress, iron deficiency, and osmotic stress, according to previous research [15,41]. The main environmental factor limiting plant growth and productivity is salt stress. The effects of salt stress can include ionic stress, osmotic stress, and secondary stresses, especially oxidative stress [42,43]. These studies suggested that transcription factor regulation might affect plant salt tolerance. Herein, it was demonstrated that the over-expression of EbbHLH80 contributed to a considerable improvement of salt-stress tolerance. EbbHLH80 reduced cell death of transgenic tobacco under salt stress. Similarly, some bHLH TFs have a regulatory effect under salt stress. Plant cells simultaneously undergo osmotic stress, ion toxicity, and oxidative stress under salt stress [42]. BnTT8 and AtTT8 have been demonstrated to be involved in salt tolerance [24,44]. AtNIG1 is also a bHLH transcription factor that has been shown to play a role in salt stress signaling pathways in Arabidopsis [45]. Additionally, OsbHLH062 regulates ion transport genes, such as OsHAK21, and modulates Jasmonic acid (JA) signaling pathways in order to confer salt tolerance to plants [46].
The antioxidant properties of flavonoids are generally responsible for enhancing stressresistance. Abiotic stresses, such as low temperatures, drought, and salt stress, result in a series of physiological and biochemical changes in plant cells, primarily causing a decrease in photosynthesis efficiency and generating reactive oxygen radicals to make serious cell damage [47]. Oxidative stress activates flavonoid synthesis, protecting cells from oxidative damage and preventing reactive oxygen species [48]. Moreover, ZmSRO1e prevents anthocyanins from hyperaccumulating, helping to maintain ROS homeostasis and balance between growth and abiotic stress [49]. Moreover, AmDEL gene overexpression increased the expression of genes related to flavonoids, proline, and reactive oxygen scavenging under salt and drought stress [50]. Therefore, flavonoids are not only involved in removing reactive oxygen species in plants, but also act as signaling molecules [51]. We found that several flavonoid synthesis genes were upregulated in transgenic tobacco seeds, suggesting that these genes may be involved in salt tolerance mediated by EbbHLH80. To determine whether EbbHLH80 mediates salt tolerance by up-regulation of these genes, PAL, C4H, 4CL, CHS, CHI, FLS2, DFR, and ANS were randomly selected for salt tolerance study. The results showed that these genes were all positively correlated with salt stress tolerance ( Figure 9). Together, the results indicated that flavonoid biosynthesis-related genes played a role in salt stress, and EbbHLH80 improved salt tolerance by promoting their expression. ROS are produced by plants while they encounter adverse environments. In plants, ROS play a dual role, acting as signaling molecules at low levels and causing oxidative stress at high levels [52]. Therefore, the regulation of ROS at appropriate levels is vital for plants under abiotic stress [53].
Salt tolerance is mediated by bHLH TFs regulating ROS balance through direct regulation of peroxidase genes. AtbHLH92 enhanced Arabidopsis tolerance to osmotic and salt stresses by partially regulating ABA and SOS2 [54]. In addition, BvbHLH92 is expressed in both roots and leaves of beets when exposed to salt stress [54]. We found that EbbHLH80 could induce the expression of genes including SOD, POD, and Pro (Figure 9), accompanied by decreased ROS levels ( Figure 8). Furthermore, GO classification analysis showed that genes involved in antioxidant activity were highly expressed. Together, these findings suggest that EbbHLH80 may induce ROS scavenging-related genes, including SODs and PODs, resulting in increased POD and SOD activity and subsequently improved salt tolerance.

Plant Materials and Growth Conditions
Nicotiana tabacum cv. Yunyan87 (Y87, WT) and E. breviscapus obtained from the Yunnan Agricultural University (Kunming, China) were used in this study. Yunyan87 (Y87, WT) and EbbHLH80-OE lines were grown at greenhouse in Yunnan Agricultural University (Kunming, China). Fully mature seeds were collected from normally grown T2 generation EbbHLH80-OE tobacco for the following experiments. For exogenous addition of NaCl on 1/2 MS medium (Murashige and Skoog medium, with sugar, 0.7% agar, pH 5.7) experiments, tobacco seeds were disinfected with 75% ethanol for 6 min and rinsed six times with sterile water. After seed germination, 10-day-old seedlings were transferred to MS medium supplemented with NaCl to observe the growth phenotype. Surface-sterilized WT and EbbHLH80-OE seeds were grown in 1/2 MS medium at 4 • C for 2 days before placing them in tissue culture at 24 • C for 16-h-light/8-h-dark cycle. The subsequent addition of 300 mM NaCl was used in RT-qPCR experimental material.

Metabolite Extraction and Analysis
The WT and EbbHLH80-OE (OE5) tobacco seeds metabolites were detected by UPLC (SHIMADZU Nexera X2) and MS/MS (Applied Biosystems 4500 QTRAP). According to the method with modifications [55]. WT and OE line seeds are vacuum freeze-dried in a freeze dryer (Scientz-100F) and then ground into powder with a grinder (MM 400, Retsch, Haan, Germany). Dissolve 100 mg of sample in 1.2 mL 70% in the chromatographic sample bottle for UPLC-MS/MS analysis. For peak alignment, pickup, and quantification of each metabolite by UHPLC-MS/MS. HPLC: column, Agilent SB-C18, 1.8 µm, 2.1 mm × 100 mm; mobile phase: phase A is ultrapure water (add 0.1% formic acid), and phase B is acetonitrile (add 0.1% formic acid); elution gradient: 5% phase proportion at 0.00 min, linear increase of phase proportion to 95% in 9.00 min and maintenance at 95% for 1 min, 10.00-11.10 min, 5% phase proportion drop in B, and equilibration at 5% for 14 min; flow rate 0.35 mL/min; temperature, 40 • C; injection volume: 4 mL.
ESI source operating parameters were as follows: ion source, turbo spray; source temperature 550 • C; ion spray voltage (IS) 5500 V (positive ion mode)/−4500 V (negative ion mode); the ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25.0 psi, respectively, and the collision induced ionization parameter was set to high. A specific set of MRM ion pairs was monitored in each epoch based on the metabolites eluted within each epoch.

RNA Extraction and Sequencing
Frozen seeds (WT and EbbHLH80-OE) were ground into powder using liquid nitrogen. Total RNA was extracted from frozen seeds using the HiPure HP Plant RNA Mini Kit (Magen, Guangzhou, China). High quality RNA was used to synthesize cDNA libraries. After completion of cDNA library construction, the library quality was checked by initial quantification with Qubit2.0 and insert size of the library by Agilent 2100, followed by accurate quantification by Q-PCR (effective concentration of the library is >2 nM). The fragments were sequenced on an Illumina HiSeq 4000 platform.

RNA Sequencing (RNA-Seq) Differential Gene Expression Analysis
DESeq2 was used to examine gene expression differences between two groups samples. False discovery rate (FDR) values were then corrected for multiple hypothesis testing with the Benjamini-Hochberg method. Differential genes were filtered with a log2fold change ≥ |1| and FDR < 0.05.

Determination of H 2 O 2 Content in Seedlings after Salt Stress Treatment
Seedlings grown on 1/2 MS medium (0.7% agar) for 2 weeks were treated with 300 mM NaCl for 1 h, control is treated with water of the same volume. Seedlings of WT and EbbHLH80-OE lines were soaked in 1 mg/mL DAB staining solution, staining at room temperature in the dark for 6 h. After rinsing in pure water, the stained seedlings were decolorized by immersion in 95% ethanol at 40 • C for 12 h and were photographed after the decolorization ended by washing well and placing them in DAB sample preserving solution for 30 min (Solarbio, Beijing, China).

RT-qPCR Analysis of Gene Expression
RT-qPCR was used to verify the feasibility of the overexpression experiments and the credibility of the transcriptome data. Seeds for analyzing EbbHLH80 expression patterns and seedlings after salt stress were frozen with liquid nitrogen. WT and OE line of RNA was isolated following the instructions of manufacturer (Magen, Guangzhou, China) and the cDNA was synthesized using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Beijing, China). RT-qPCR was performed in the ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), with six biological replicates. NtActin (XM_016628756) was used as an internal reference gene to calculate the difference in specific expression of the target gene. Specific primer pairs for each gene (Supplementary Table S1) were designed by Primer3 (v. 0.4.0).

Statistical Analysis
All data presented in this manuscript were collected from more than three independent replicates and were statistically analyzed by one-way analysis of variance. Standard errors were calculated for the means and significant differences determined at the significance levels of p ≤ 0.05 and p ≤ 0.01.

Conclusions
Overall, a bHLH family gene, EbbHLH80, has been identified from E. breviscapus in a previous study. EbbHLH80 was induced by salt-stress treatment. The overexpression of EbbHLH80 resulted in enhanced resistance to salt stress in tobacco. Furthermore, transgenic plants displayed higher expression levels of three antioxidant enzymes (SOD, POD, and Pro), causing less ROS accumulation. Several flavonoid biosynthesis genes were also found to be up-regulated in the transgenic lines. In the future, EbbHLH80 can act as a candidate gene to enhance the stress tolerance of E. breviscapus with genetic engineering techniques.