Transcriptome-Wide Identification, Evolutionary Analysis, and GA Stress Response of the GRAS Gene Family in Panax ginseng C. A. Meyer

GRAS transcription factors are a kind of plant-specific transcription factor that have been found in a variety of plants. According to previous studies, GRAS proteins are widely involved in the physiological processes of plant signal transduction, stress, growth and development. The Jilin ginseng (Panax ginseng C.A. Meyer) is a heterogeneous tetraploid perennial herb of the Araliaceae family, ginseng genus. Important information regarding the GRAS transcription factors has not been reported in ginseng. In this study, 59 Panax ginseng GRAS (PgGRAS) genes were obtained from the Jilin ginseng transcriptome data and divided into 13 sub-families according to the classification of Arabidopsis thaliana. Through systematic evolution, structural variation, function and gene expression analysis, we further reveal GRAS’s potential function in plant growth processes and its stress response. The expression of PgGRAS genes responding to gibberellin acids (GAs) suggests that these genes could be activated after application concentration of GA. The qPCR analysis result shows that four PgGRAS genes belonging to the DELLA sub-family potentially have important roles in the GA stress response of ginseng hairy roots. This study provides not only a preliminary exploration of the potential functions of the GRAS genes in ginseng, but also valuable data for further exploration of the candidate PgGRAS genes of GA signaling in Jilin ginseng, especially their roles in ginseng hairy root development and GA stress response.


Introduction
Ginseng is one of the most valuable medicinal plants in the Araliaceae family. Ginseng ingredients include saponins, polysaccharides, volatile ingredients, proteins, vitamins, and other compounds. The main active ingredient is ginsenosides. Asian regions, particularly China, Korea and Japan, are the major growing areas of ginseng. The Chinese ginseng is mainly distributed in Jilin Province and is commonly named as Jilin Ginseng. Ginseng has anti-thrombosis and anti-aging effects, prevents tumor angiogenesis and has other functions [1,2]. In recent years, the study of ginseng has become a hotspot for research.

Gene Expression Pattern, GO Function Classification and Enrichment Analysis
In order to understand the expression pattern of genes, the expression patterns of genes were further analyzed. The heatmaps were constructed using the TBtools version 0.6673 software [36]. Blast2go (https://www.blast2go.com/free-b2g-trial) software was used to annotate PgGRAS transcription functions [37]. Then, Gene Ontology (GO) was used for further analysis of the annotation results. The enrichment of the number of PgGRAS transcriptions classified into each subcategory was tested by the Chi-square test method.

Network Analysis of PgGRAS Transcripts Genes
The R programming language and software (http://www.rproje ct.org/) was used to calculate the correlation coefficient of Spearman. A gene co-expression network was constructed using BioLayout Express 3D version 3.2 software [38].

Response of PgGRAS Genes to Different Concentrations of GA
Ginseng hairy roots were cultured in 1/2 MS liquid medium for 33 days with no hormones added. Then, we obtained the secondary generation to cultivate as 1.0 g ginseng hairy root in 1/2 MS liquid medium each bottle. The GA was added after 23 days of culture; that is, ginseng hairy roots were cultivated by six gradients of GA concentration, which included 0.0 µM as control, 2.5, 5.0, 7.5, 10.0 and 15.0 µM (four repetitions per concentration gradient) [39]. At each treatment, each group of treatments was repeated three times biologically. The result is three duplicate averages, and all processing experiments were carried out in separate periods of time. The fresh weight and dry weight of the hairy roots were weighed after 30 days with the untreated hairy root as the control. All these samples were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction [40].
Plants 2020, 9, 190 4 of 18 For qPCR analyses, the total RNA extraction of ginseng hairy roots used the improved TRIzol method based on the published manufacturer's instructions. The quality and concentration of each RNA sample was determined using gel electrophoresis and the ScanDrop 2000 spectrophotometer (AJ, Germany). First-strand cDNA synthesis was performed using 2.00 µg of total RNA in a 20 µL reaction volume, according to the manufacturer's instructions for RNA purification with HiFiScript gDNA Removal cDNA Synthesis Kit (ComWin, Beijing, China). The GADPH (GenBank Accession No. KF699323.1) [41] of Panax ginseng was used as a reference gene. The qPCR of relative fluorescence quantification was measured using the Applied Biosystems 7500 Real-Time System (ABI, USA) and the Ultra SYBR Mixture Kit (Low ROX) (ComWin, Beijing, China). The designed primers (Table 1) were used to detect the response of the PgGRAS genes under GA treatment using SYBR qPCR analysis. The relative expression of the 4 PgGRAS genes were determined by the 2 −∆∆Ct method [42] and all the samples of biological replicates and technical replicates were repeated three times. Table 1. Sequences of PgGRAS and GADPH gene primers used in this study.

Transcriptome-Wide Identification GRAS Gene Family in Ginseng
Although the GRAS gene family has been investigated in many studies, it has not been specifically studied in Panax ginseng. In order to ensure the accuracy of the extracted sequence, two methods were used to find the sequence in this experiment. Using the GRAS, the conserved domain (PF03514) has been investigated as the inquiry sequence to search PgGRAS genes by the Hmmer method [29]. In another way, the latest Hmm (PF03514.11) of the GRAS domain was used as a blast inquiry sequence to search directly in the Jilin ginseng Transcriptome Database [30,31]. Exercising these two methods, we searched the Jilin Ginseng Transcriptome Library and finally got 131 transcripts derivatives under 79 gene IDs (Tables S1 and S2). Only 59 of these transcripts' derivatives were intact, of which five PgGRAS genes pertained to the DELLA sub-family, namely PgGRAS44-04, PgGRAS48-01, PgGRAS50-01, PgGRAS68-01 and PgGRAS19-01 of the PgGRAS genes in ginseng [43,44].
The transcripts' sequence lengths of the PgGRAS genes are between 201 (PgGRAS15-01) to 3343 bp (PgGRAS65-02). Through the analysis of the physical and chemical properties of PgGRAS proteins, we found that the molecular weights ranged from 431.00 to 87,107.53 Daltons (Da). There are also significant differences in the theoretical PI values of predictions. The maximum theoretical PI value is 11.22 (PgGRAS75-01) and the minimum theoretical PI value is 4.37 (PgGRAS02-01). The hydrophilic range of PgGRAS proteins are from −0.652 (PgGRAS69-04) to 0.684 (PgGRAS66-06) ( Table 2). Only 12 PgGRAS proteins have hydrophilicity greater than 0, so it can be inferred that most PgGRAS proteins are hydrophilic. There were 101 PgGRAS proteins in which the number of positive amino acids was higher than that of negative amino acids, while in 25 PgGRAS proteins, the content of negative amino acids was higher than that of positive amino acids. Additionally, there were five PgGRAS proteins in which the number of positive amino acids was equal to the number of negative amino acids. It is speculated that the number of positive amino acids is greater than the number of negative amino acids in most of PgGRAS proteins. The data observation shows that most PgGRAS proteins are stable, while there are still 22 unstable PgGRAS proteins, for the reason that its instability index is less than 40. Based on these results, it could be concluded that there are great differences in physical and chemical properties among PgGRAS proteins (Table 2).

Conserved Motif Analysis and Systematic Analysis of PgGRAS Gene Family
By analyzing these results, we have obtained 131 transcripts' sequences of GRAS genes in ginseng. For the sequence-structure study, we used the NCBI ORF Finder to search for the ORFs of 131 transcripts. We found that 59 transcripts had a complete ORF. However, the GRAS gene family has a unique conserved domain, in addition to other motifs. Additionally, there are five main motifs in conserved domains. The GRAS gene family has 20 motifs found throughout the literature (Figure 1a) [45]. Applying MEME for the analysis of these conserved motifs, we found that most of the transcripts have similar conserved motifs. The stability of the conserved domain is proven in Figure 1b.

Conserved Motif Analysis and Systematic Analysis of PgGRAS Gene Family
By analyzing these results, we have obtained 131 transcripts' sequences of GRAS genes in ginseng. For the sequence-structure study, we used the NCBI ORF Finder to search for the ORFs of 131 transcripts. We found that 59 transcripts had a complete ORF. However, the GRAS gene family has a unique conserved domain, in addition to other motifs. Additionally, there are five main motifs in conserved domains. The GRAS gene family has 20 motifs found throughout the literature ( Figure  1a) [45]. Applying MEME for the analysis of these conserved motifs, we found that most of the transcripts have similar conserved motifs. The stability of the conserved domain is proven in Figure  1b.  Tables S2 and S3), respectively [46,47]. As shown in Figure 2, we identified a total of 15 sub-families of the GRAS genes. For ginseng, we identified nine PgGRAS genes in the PAT subfamilies, six in the LISCL sub-family, five in the DELLA sub-families, two in the HAM sub-family, two in the SHR sub-family, two in the NSP2 sub-families, one in the SCL3 sub-family, one in the DLT sub-family, one in the NSP1 sub-families, one in the SCL4/7 sub-families, one in the RAM1 subfamily, one in the SCR sub-family and one in the PG1 sub-family (Figure 2a). The LLSCL sub-family has 13 members, and six PgGRAS genes (PgGRAS08-1, PgGRAS24-03, PgGRAS60-01, PgGRAS63-01, PgGRAS65-01 and PgGRAS69-01) are highly homologous to AtSCL9 (AtGRAS27) and may be associated with the regulation of the occurrence of microspores in meiosis [48,49]. The PAT subfamily has 15 members, nine of which (PgGRAS12-01, PgGRAS22-01, PgGRAS34-02, PgGRAS47-01, PgGRAS55-01, PgGRAS56-02, PgGRAS57-01, PgGRAS61-01 and PgGRAS62-01) have a high degree of similarity to AtPAT (AtGRAS30) [50]. Based on previous studies, it was found that the Arabidopsis GRAS protein PAT was involved in the early stage of photosensitive pigment a signal transduction. These nine members may have similar functions. It has been reported that the HAM and LS families  Tables S2 and S3), respectively [46,47]. As shown in Figure 2, we identified a total of 15 sub-families of the GRAS genes. For ginseng, we identified nine PgGRAS genes in the PAT sub-families, six in the LISCL sub-family, five in the DELLA sub-families, two in the HAM sub-family, two in the SHR sub-family, two in the NSP2 sub-families, one in the SCL3 sub-family, one in the DLT sub-family, one in the NSP1 sub-families, one in the SCL4/7 sub-families, one in the RAM1 sub-family, one in the SCR sub-family and one in the PG1 sub-family (Figure 2a). The LLSCL sub-family has 13 members, and six PgGRAS genes (PgGRAS08-1, PgGRAS24-03, PgGRAS60-01, PgGRAS63-01, PgGRAS65-01 and PgGRAS69-01) are highly homologous to AtSCL9 (AtGRAS27) and may be associated with the regulation of the occurrence of microspores in meiosis [48,49]. The PAT sub-family has 15 members, nine of which (PgGRAS12-01, PgGRAS22-01, PgGRAS34-02, PgGRAS47-01, PgGRAS55-01, PgGRAS56-02, PgGRAS57-01, PgGRAS61-01 and PgGRAS62-01) have a high degree of similarity to AtPAT (AtGRAS30) [50]. Based on previous studies, it was found that the Arabidopsis GRAS protein PAT was involved in the early stage of photosensitive pigment a signal transduction. These nine members may have similar functions. It has been reported that the HAM and LS families are involved in the development and regulation of axillary birth tissue, the apex meristem and leaf axils meristem of tiller formation, respectively [49]. The HAM sub-family consists of six members (PgGRAS49-01, PgGRAS51-01, AtGRAS15, AtGRAS22, AtGRAS23 and AtGRAS26) and the LS sub-family consists of Plants 2020, 9, 190 8 of 18 one member (AtGRAS7). The SCR and SHR sub-families are critical to the growth of radial tissue during the development of roots and buds. A total of three members (PgGRAS01-01, PgGRAS30-01 and PgGRAS54-01) of the GRAS gene family belong to the SCR and SHR families and may be associated with the development of roots and buds [48].
are involved in the development and regulation of axillary birth tissue, the apex meristem and leaf axils meristem of tiller formation, respectively [49]. The HAM sub-family consists of six members (PgGRAS49-01, PgGRAS51-01, AtGRAS15, AtGRAS22, AtGRAS23 and AtGRAS26) and the LS subfamily consists of one member (AtGRAS7). The SCR and SHR sub-families are critical to the growth of radial tissue during the development of roots and buds. A total of three members (PgGRAS01-01, PgGRAS30-01 and PgGRAS54-01) of the GRAS gene family belong to the SCR and SHR families and may be associated with the development of roots and buds [48].
The RAM1 sub-family contains only one ginseng gene (PgGRAS33-01), and the RAM1 protein is involved in inducing many AM (Arbuscular mycorrhiza)-related genes. Through the above, we can boldly infer that the GRAS gene is likely to be involved in the related process of AM. The SCL3 subfamily has two members (AtGRAS5 and PgGRAS45-01) and one member in ginseng. The DLT genes were involved in the brassinosteroid (BR) signaling, but only one gene (PgGRAS36-01) belongs to the DLT sub-family in ginseng [51]. NSP1 and NSP2 regulate the expression of genes associated with strigolactone biosynthesis, so that the root can be stimulated to mycorrhization by affecting hormones and their derivatives [3]. After analysis, it was found that three ginseng genes (PgGRAS26-01, PgGRAS43-01 and PgGRAS52-01) belong to the NSP1 and NSP2 subfamilies. Three genes (AtGRAS20, AtGRAS33 and PgGRAS53-01) were observed in the evolutionary tree belonging to the SCL4/7 subfamily, which may be involved in the resistance system in the plant [2]. However, the SCL32 subfamily contains only two members (AtGRAS19 and AtGRAS34) of the Arabidopsis thaliana, and there are no members of the SCL32 sub-family in the Panax ginseng. There was a unique subfamily found, namely PG1 (PgGRAS67-12), in ginseng ( Figure 2a). As is known, the GRAS gene family has a subfamily DELLA that is involved in gibberellin signal responses and steadystate balance; the sub-family DELLA consists of 10 members (PgGRAS68-01, PgGRAS19-01,  PgGRAS50-01, PgGRAS44-04, PgGRAS48-01, AtGRAS10, AtGRAS3, AtGRAS28, AtGRAS16 and AtGRAS9), among which five members are from ginseng (Figure 2a). By understanding the DELLA sub-family, we can further study its influence on GA and speculate on its role in GA pathway [52]. The RAM1 sub-family contains only one ginseng gene (PgGRAS33-01), and the RAM1 protein is involved in inducing many AM (Arbuscular mycorrhiza)-related genes. Through the above, we can boldly infer that the GRAS gene is likely to be involved in the related process of AM. The SCL3 sub-family has two members (AtGRAS5 and PgGRAS45-01) and one member in ginseng.
The DLT genes were involved in the brassinosteroid (BR) signaling, but only one gene (PgGRAS36-01) belongs to the DLT sub-family in ginseng [51]. NSP1 and NSP2 regulate the expression of genes associated with strigolactone biosynthesis, so that the root can be stimulated to mycorrhization by affecting hormones and their derivatives [3]. After analysis, it was found that three ginseng genes (PgGRAS26-01, PgGRAS43-01 and PgGRAS52-01) belong to the NSP1 and NSP2 sub-families. Three genes (AtGRAS20, AtGRAS33 and PgGRAS53-01) were observed in the evolutionary tree belonging to the SCL4/7 subfamily, which may be involved in the resistance system in the plant [2]. However, the SCL32 subfamily contains only two members (AtGRAS19 and AtGRAS34) of the Arabidopsis thaliana, and there are no members of the SCL32 sub-family in the Panax ginseng. There was a unique subfamily found, namely PG1 (PgGRAS67-12), in ginseng ( Figure 2a). As is known, the GRAS gene family has a subfamily DELLA that is involved in gibberellin signal responses and steady-state balance; the sub-family DELLA consists of 10 members (PgGRAS68-01, PgGRAS19-01, PgGRAS50-01, PgGRAS44-04, PgGRAS48-01, AtGRAS10, AtGRAS3, AtGRAS28, AtGRAS16 and AtGRAS9), among which five members are from ginseng (Figure 2a). By understanding the DELLA sub-family, we can further study its influence on GA and speculate on its role in GA pathway [52].
According to the ML evolutionary tree, it can be seen that the genes of Panax ginseng, Arabidopsis thaliana, Solanum lycopersicum and Oryza sativa are mainly distributed in 13 sub-families (Figure 2b).
No genes of ginseng were found in the SCLB sub-family. These results suggest that the origin of the PgGRAS gene family was before the separation of monocotyledons and dicotyledons [9,53].

Network Analysis of Gene Expression in PgGRAS Gene Family
By means of the analysis of gene family genes, it can be said that there are great differences in expression and differentiation among functions of the family members of the gene. In order to observe the network of interactions among genes, we selected 131 transcripts expressed in 14 tissues from 42 farmers' ginseng cultivars. Figure 3 shows a network of expression interactions among 131 transcripts (Figure 3a). These results show that with the functional differentiation of the PgGRAS gene family, the expression of its gene members is also different [54]. This phenomenon is not only observed within the gene family, but also sub-families. For the sake of checking whether the interaction among genes is associated with expression activities to some extent, we randomly sampled as the negative control the transcripts of 14 tissues of 4-year-old ginseng. Then, 131 PgGRAS transcripts tend to have associated expression and form a co-expression network (Figure 3b). These results show that the function and expression of the gene members of the PgGRAS gene family have been significantly differentiated. However, they do maintain a slight link between relevant expression and functional cooperation [55].
Plants 2020, 9, 190 9 of 18 According to the ML evolutionary tree, it can be seen that the genes of Panax ginseng, Arabidopsis thaliana, Solanum lycopersicum and Oryza sativa are mainly distributed in 13 sub-families (Figure 2b). No genes of ginseng were found in the SCLB sub-family. These results suggest that the origin of the PgGRAS gene family was before the separation of monocotyledons and dicotyledons [9,53].

Network Analysis of Gene Expression in PgGRAS Gene Family
By means of the analysis of gene family genes, it can be said that there are great differences in expression and differentiation among functions of the family members of the gene. In order to observe the network of interactions among genes, we selected 131 transcripts expressed in 14 tissues from 42 farmers' ginseng cultivars. Figure 3 shows a network of expression interactions among 131 transcripts (Figure 3a). These results show that with the functional differentiation of the PgGRAS gene family, the expression of its gene members is also different [54]. This phenomenon is not only observed within the gene family, but also sub-families. For the sake of checking whether the interaction among genes is associated with expression activities to some extent, we randomly sampled as the negative control the transcripts of 14 tissues of 4-year-old ginseng. Then, 131 PgGRAS transcripts tend to have associated expression and form a co-expression network (Figure 3b). These results show that the function and expression of the gene members of the PgGRAS gene family have been significantly differentiated. However, they do maintain a slight link between relevant expression and functional cooperation [55].   Table S4). At least 85 transcription profiles were   Table S4). At least 85 transcription profiles were found in 131 transcripts. At least 85 of 131 transcripts were expressed, and a number of PgGRAS genes had similar expression patterns. Heatmaps indicated that lots of PgGRAS genes not only had similar expression patterns, but also interactions. It is further shown that there is a network of interactions among genes to control gene expression. In order to improve the understanding of the functional relationships among gene sub-families, we used the Gene Ontology (GO) terms to analyze gene functions. According to the results, we found that 131 transcripts are divided into three main GO function categories-Biological Processes (BP), Molecular Function (MF) and Cellular Components (CC) [56]. Most of these transcripts have three functions at the same time, with only 15 genes having a single function (Figure 5a). These three terms have eight subcategories in Level 2-BP includes cellular process, biological regulation, metabolic process, signaling, multicellular organismal process, regulation of biological process, response to stimulus, developmental process and single-organism process; MF includes transcription factor activity, protein binding, binding and nucleic acid binding transcription factor activity; CC includes cell parts, cells and organelles. For more information about this study, the enrichment analysis was carried out. An enrichment analysis revealed that 11 subcategories in 15 subcategories were significantly enriched (p ≤ 0.01) in comparison with total transcriptome transcripts ( Figure 6). Then, we performed a functional analysis of 131 PgGRAS genes that were found to have intact conserved domains (Figure 5b-d and Table S5). These results indicate that the gene function of each type of PgGRAS family had differentiated as they evolved.

Expression Analysis and Functional Evolution of PgGRAS Gene Family
Plants 2020, 9,190 10 of 18 found in 131 transcripts. At least 85 of 131 transcripts were expressed, and a number of PgGRAS genes had similar expression patterns. Heatmaps indicated that lots of PgGRAS genes not only had similar expression patterns, but also interactions. It is further shown that there is a network of interactions among genes to control gene expression. In order to improve the understanding of the functional relationships among gene sub-families, we used the Gene Ontology (GO) terms to analyze gene functions. According to the results, we found that 131 transcripts are divided into three main GO function categories-Biological Processes (BP), Molecular Function (MF) and Cellular Components (CC) [56]. Most of these transcripts have three functions at the same time, with only 15 genes having a single function (Figure 5a). These three terms have eight subcategories in Level 2-BP includes cellular process, biological regulation, metabolic process, signaling, multicellular organismal process, regulation of biological process, response to stimulus, developmental process and single-organism process; MF includes transcription factor activity, protein binding, binding and nucleic acid binding transcription factor activity; CC includes cell parts, cells and organelles. For more information about this study, the enrichment analysis was carried out. An enrichment analysis revealed that 11 subcategories in 15 subcategories were significantly enriched (p ≤ 0.01) in comparison with total transcriptome transcripts ( Figure 6). Then, we performed a functional analysis of 131 PgGRAS genes that were found to have intact conserved domains (Figure 5b-d and Table S5). These results indicate that the gene function of each type of PgGRAS family had differentiated as they evolved.

Analysis of PgGRAS Genes Expression under Different Concentrations of GA
Plant hormones play an important role in the growth and development of plants, and GRAS transcription factors play an important role in the pathogenesis of metabolism and biosynthesis in plant growth [57,58]. Firstly, the growth and development of ginseng hairy roots treated with different concentrations of GA were measured. The fresh weight and dry weight of all the treatments reached their peak at the GA concentration of 10 μM, but were still less than those of control group (Figure 7). GRAS transcription factors are also involved in plant growth metabolic and biosynthetic pathways. After previous analysis, it was identified that PgGRAS44-04, PgGRAS48-01, PgGRAS50-01, PgGRAS68-01 and PgGARS19-01 belong to the DELLA sub-family, thus, it is speculated that it has the function of responding to gibberellin. After detection, the expression levels of these five genes

Analysis of PgGRAS Genes Expression under Different Concentrations of GA
Plant hormones play an important role in the growth and development of plants, and GRAS transcription factors play an important role in the pathogenesis of metabolism and biosynthesis in plant growth [57,58]. Firstly, the growth and development of ginseng hairy roots treated with different concentrations of GA were measured. The fresh weight and dry weight of all the treatments reached their peak at the GA concentration of 10 μM, but were still less than those of control group (Figure 7). GRAS transcription factors are also involved in plant growth metabolic and biosynthetic pathways. After previous analysis, it was identified that PgGRAS44-04, PgGRAS48-01, PgGRAS50-01, PgGRAS68-01 and PgGARS19-01 belong to the DELLA sub-family, thus, it is speculated that it has the function of responding to gibberellin. After detection, the expression levels of these five genes Figure 6. The Chi-square test was used to calculate the prediction and actual situation of the same function (Level 2) of the PgGRAS genes, further embodying the differentiation diversity of the PgGRAS genes function. Gray, the control group. Yellow, the PgGRAS genes group. **, very significant (p ≤ 0.01).

Analysis of PgGRAS Genes Expression under Different Concentrations of GA
Plant hormones play an important role in the growth and development of plants, and GRAS transcription factors play an important role in the pathogenesis of metabolism and biosynthesis in plant growth [57,58]. Firstly, the growth and development of ginseng hairy roots treated with different concentrations of GA were measured. The fresh weight and dry weight of all the treatments reached their peak at the GA concentration of 10 µM, but were still less than those of control group (Figure 7). GRAS transcription factors are also involved in plant growth metabolic and biosynthetic pathways. After previous analysis, it was identified that PgGRAS44-04, PgGRAS48-01, PgGRAS50-01, PgGRAS68-01 and PgGARS19-01 belong to the DELLA sub-family, thus, it is speculated that it has the function of responding to gibberellin. After detection, the expression levels of these five genes were different under different concentrations of GA treatment. PgGARS19-01 was not detected in the qPCR assay. Hence, no further analysis was performed. Here, we found that three genes-PgGRAS44-04, PgGRAS48-01 and PgGRAS50-01-all reached their peaks of expression at 10.0 µM among treatments with different concentrations of GA (Figure 8a-c). The expressions of these three genes were gradually upregulated until GA reached the concentration of 10.0 µM, from which the expression levels decreased (Figure 8a-c). The expression level of PgGRAS68-01 began to gradually decline after the concentration of 7.5 µM (Figure 8d). The results show that PgGRAS44-04, PgGRAS48-01 and PgGRAS50-01 had similar expression patterns. PgGRAS44-04, PgGRAS48-01, PgGRAS50-01 and PgGRAS68-01 all had different responses to exogenous GA addition. It is further demonstrated that the PgGRAS gene family can be applied to GA stress response.
Plants 2020, 9, 190 13 of 18 were different under different concentrations of GA treatment. PgGARS19-01 was not detected in the qPCR assay. Hence, no further analysis was performed. Here, we found that three genes-PgGRAS44-04, PgGRAS48-01 and PgGRAS50-01-all reached their peaks of expression at 10.0 μM among treatments with different concentrations of GA (Figure 8a-c). The expressions of these three genes were gradually upregulated until GA reached the concentration of 10.0 μM, from which the expression levels decreased (Figure 8a-c). The expression level of PgGRAS68-01 began to gradually decline after the concentration of 7.5 μM (Figure 8d)

Function and Analysis of GRAS Transcription Factor Family
Since transcription factors play a very important role in plants, a large number of TFs are analyzed in many species [59]. However, the analyses of the GRAS gene family have been performed far less than those of other transcription factor families, and the study of the GRAS gene family in ginseng has not been reported until now. In this study, 131 transcripts of 79 genes from the Jilin ginseng transcriptome data of Panax ginseng were analyzed. The number of genes isolated from plants such as Solanum lycopersicum (53) and Oryza sativa (60) was similar, but higher than that of GRAS in Arabidopsis thaliana (34). The identification of these genes can further fill the gap of ginseng resources in GenBank. According to the gene tree, it can be observed that the PgGRAS gene family has been well clustered with the GRAS genes of Arabidopsis thaliana, Oryza sativa and Solanum lycopersicum. These results suggest that the origins of the PgGRAS gene family can be traced back to the division between monocotyledons (Oryza sativa) and dicotyledons (Arabidopsis thaliana, Solanum lycopersicum and Panax ginseng).
Through the functional analysis of PgGRAS, we found that the functions of the PgGRAS gene family have been largely differentiated. On the same level (Level 2), the PgGRAS gene family was categorized into at least eight GO functional subcategories and distributed across all three major GO functions, namely Biological Processes (BP), Molecular Function (MF) and Cellular Components (CC). There were great differences among the functions of the members of the PgGRAS gene family. However, according to the analysis, it was found that there were similar expression patterns among family members. Therefore, we can confirm that there is a network of co-expression among PgGRAS genes. Notwithstanding this, the function and expression activity of the PgGRAS genes have been greatly diversified. Most genes tend to have related expressions, and they also form a co-expression

Response of DELLA Sub-Family to Gibberellin and Its Effect on Ginsenosides
Expression analysis by qPCR revealed that four PgGRAS genes were involved in the GA regulatory pathway. According to previous reports, many families of transcription factors are involved in the regulation of hormones, and GA is involved in many aspects of plant growth and development, such as seed growth and development, stem elongation and flower development. After the comprehensive analysis of all the qPCR data (Figure 8), we found that four genes belonging to the DELLA sub-family (PgGRAS44-04, PgGRAS48-01, PgGRAS50-01 and PgGRAS68-01) had significant changes in response to GA expression levels, suggesting that these genes could be involved in GA signaling and stress response. GA as a plant hormone (gibberellin) is mainly synthesized in fruits or seeds, elongated stem ends and root organs of higher plants. Additionally, it promotes the germination of plant seeds, breaks dormancy and promotes stem and root growth. In addition, studies have found that GA and jasmonic acid (JA) interact with each other, which can play a synergistic role in plant development and an antagonistic role in plant defense. Predictions from other species indicate that GRAS proteins

Function and Analysis of GRAS Transcription Factor Family
Since transcription factors play a very important role in plants, a large number of TFs are analyzed in many species [59]. However, the analyses of the GRAS gene family have been performed far less than those of other transcription factor families, and the study of the GRAS gene family in ginseng has not been reported until now. In this study, 131 transcripts of 79 genes from the Jilin ginseng transcriptome data of Panax ginseng were analyzed. The number of genes isolated from plants such as Solanum lycopersicum (53) and Oryza sativa (60) was similar, but higher than that of GRAS in Arabidopsis thaliana (34). The identification of these genes can further fill the gap of ginseng resources in GenBank. According to the gene tree, it can be observed that the PgGRAS gene family has been well clustered with the GRAS genes of Arabidopsis thaliana, Oryza sativa and Solanum lycopersicum. These results suggest that the origins of the PgGRAS gene family can be traced back to the division between monocotyledons (Oryza sativa) and dicotyledons (Arabidopsis thaliana, Solanum lycopersicum and Panax ginseng).
Through the functional analysis of PgGRAS, we found that the functions of the PgGRAS gene family have been largely differentiated. On the same level (Level 2), the PgGRAS gene family was categorized into at least eight GO functional subcategories and distributed across all three major GO functions, namely Biological Processes (BP), Molecular Function (MF) and Cellular Components (CC). There were great differences among the functions of the members of the PgGRAS gene family. However, according to the analysis, it was found that there were similar expression patterns among family members. Therefore, we can confirm that there is a network of co-expression among PgGRAS genes. Notwithstanding this, the function and expression activity of the PgGRAS genes have been greatly diversified. Most genes tend to have related expressions, and they also form a co-expression network, even if the correlation is limited. This indicates that members of the gene family maintain functional coordination or interaction to a certain extent. The formation of numerous mutual networks within the gene family provides a series of evidence for this inference, and it further confirmed the functional differentiation among members of the PgGRAS gene family.

Response of DELLA Sub-Family to Gibberellin and Its Effect on Ginsenosides
Expression analysis by qPCR revealed that four PgGRAS genes were involved in the GA regulatory pathway. According to previous reports, many families of transcription factors are involved in the regulation of hormones, and GA is involved in many aspects of plant growth and development, such as seed growth and development, stem elongation and flower development. After the comprehensive analysis of all the qPCR data ( Figure 8), we found that four genes belonging to the DELLA sub-family (PgGRAS44-04, PgGRAS48-01, PgGRAS50-01 and PgGRAS68-01) had significant changes in response to GA expression levels, suggesting that these genes could be involved in GA signaling and stress response.
GA as a plant hormone (gibberellin) is mainly synthesized in fruits or seeds, elongated stem ends and root organs of higher plants. Additionally, it promotes the germination of plant seeds, breaks dormancy and promotes stem and root growth. In addition, studies have found that GA and jasmonic acid (JA) interact with each other, which can play a synergistic role in plant development and an antagonistic role in plant defense. Predictions from other species indicate that GRAS proteins are an important component of the GA signaling pathway. In Arabidopsis, the DELLA protein acts as a repressor for GA response to plant growth [60,61]. Since studies have shown that the exogenous addition of JA can promote the content of ginsenosides in ginseng, it is speculated that there is a relationship between the synthesis of GA and ginsenoside content. The analysis of bioinformatics provides a basic information for studying the molecular regulation of GRAS proteins during ginseng development. This study provides ideas for future studies for the interaction between GA synthesis and the signal pathway and GA and ginsenoside synthesis in ginseng, and further improves ginseng resources.

Conclusions
In this study, 59 GRAS genes (PgGRAS) are found in Jilin ginseng and divided into 13 sub-families. These 59 PgGRAS genes have a correlation of expression and form a co-expression network to function. We identified that four PgGRAS genes belonging to the DELLA sub-family (PgGRAS44-04, PgGRAS48-01, PgGRAS50-01 and PgGRAS68-01) had significant changes in expression levels in response to GA. The result of qPCR suggests that these genes can be involved in GA signaling and stress response. In summary, our results provide valuable data for further exploring the candidate PgGRAS genes of GA signaling in ginseng, especially their roles in ginseng hairy root development and stress response.
Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/2/190/s1, Table S1. The transcript sequences of the PgGRAS genes identified in this study. Table S2. The transcript protein sequences of the PgGRAS genes in this study. Table S3. The identified genes used as evolutionary tree for PgGRAS genes evolutionary analysis. Table S4