Whole-genome identification and expression profiling of growth-regulating factor (GRF) and GRF-interacting factor (GIF) gene families in Panax ginseng
BMC Genomics volume 24, Article number: 334 (2023)
Panax ginseng is a perennial herb and one of the most widely used traditional medicines in China. During its long growth period, it is affected by various environmental factors. Past studies have shown that growth-regulating factors (GRFs) and GRF-interacting factors (GIFs) are involved in regulating plant growth and development, responding to environmental stress, and responding to the induction of exogenous hormones. However, GRF and GIF transcription factors in ginseng have not been reported.
In this study, 20 GRF gene members of ginseng were systematically identified and found to be distributed on 13 chromosomes. The ginseng GIF gene family has only ten members, which are distributed on ten chromosomes. Phylogenetic analysis divided these PgGRFs into six clades and PgGIFs into two clades. In total, 18 of the 20 PgGRFs and eight of the ten PgGIFs are segmental duplications. Most PgGRF and PgGIF gene promoters contain some hormone- and stress- related cis-regulatory elements. Based on the available public RNA-Seq data, the expression patterns of PgGRF and PgGIF genes were analysed from 14 different tissues. The responses of the PgGRF gene to different hormones (6-BA, ABA, GA3, IAA) and abiotic stresses (cold, heat, drought, and salt) were studied. The expression of the PgGRF gene was significantly upregulated under GA3 induction and three weeks of heat treatment. The expression level of the PgGIF gene changed only slightly after one week of heat treatment.
The results of this study may be helpful for further study of the function of PgGRF and PgGIF genes and lay a foundation for further study of their role in the growth and development of Panax ginseng.
Growth-regulating factors (GRFs) are unique plant transcription factors that play vital roles in regulating plant growth and development, as well as abiotic stress response [1, 2]. The first GRF gene was found in rice (Oryza sativa) and was named OsGRF1; it was induced by GA3 (gibberellin)  and played an essential role in regulating stem elongation. After that, GRFs have been reported in various plants, such as Arabidopsis thaliana, Zea mays, Medicago truncatula, and Brassica rapa [4,5,6,7]. GRFs are a large gene family with highly conserved proteins. Most GRFs contain unique QLQ (Glu-Leu-Glu, glutamine, leucine, glutamine) and WRC (Trp-Arg-Cys, tryptophan, arginine, cysteine) domains in the N-terminal region [1, 3, 6, 8]. The WRC domain can be combined with the cis-acting regions of downstream genes to regulate their expression. The QLQ domain can interact with the SNH domain in the GIF protein to form a transcription activator [3, 6, 8, 9].
Initially, GRF was only shown to play a regulatory role in stem and leaf development [10, 11]. Subsequent studies found that GRF can also regulate the growth and development of other plant tissues, including flower organ development , root development [13, 14], leaf lifespan , and plant stress response [16, 17]. Studies have also shown that most members of the GRF family have higher expression levels in the meristem. GRF genes are usually expressed at higher levels in young tissues but at lower levels in mature tissues . In recent years, studies have shown that GRF transcription factors play essential roles in plant growth development and defence responses to biological and abiotic stresses. Further functional classification of the hypothetical downstream targets of AtGRF1 and AtGRF3 shows that most of them are involved in defence responses and disease resistance processes [6, 10, 11, 17].
GIF proteins are SSXT superfamily genes, a class of plant transcriptional coactivators that are functionally homologous to human SYT transcriptional coactivators . AtGIF1, the first member of the GIF family, has been used as bait in yeast two-hybrid assays . GIF protein has strong transcriptional activity and strong cell division ability . AtGIF1 is also involved in the control of leaf growth and morphology [14, 19]. AtGIF2 and AtGIF3 have similar biological functions to AtGIF1, and they play an important role in regulating the cell division ability of plants .
Panax ginseng is a perennial herbaceous plant with a long growth period. Ginseng must cope with complex environmental changes during its growth and development time, such as extreme temperature and drought, as well as biotic stresses such as pests and diseases . As essential transcription factors in plant growth and development, response to exogenous hormones, and stress resistance, GRF and GIF transcription factor families have been found in many plant species. For example, in Chinese cabbage (Brassica rapa L. spp. pekinensis), most of the BrGRF genes were induced by GA3 treatment. Moreover, overexpression of BrGRF8 in Arabidopsis (A. thaliana) could increase the sizes of leaves and other organs by regulating cell proliferation . Overexpression of ZmGRF11-ZmGIF2 and ZmGRF2-ZmGIF3 accelerated inflorescence stem growth compared with the wild type . In Prunus persica, PpGRFs responded to UVB and GA3 treatment and participated in the growth process of new shoot elongation . In Fragaria vesca, FvGRFs play a potential role in the growth and development of vegetative organs . In callus cells of Beta vulgaris, ectopic expression of Arabidopsis GRF5 accelerated shoot formation and improved transformation efficiency . The molecular characterization of ginseng GRF and GIF genes has not been studied. Therefore, identifying and analysing the ginseng GRF gene is of great significance.
GRF and GIF may also be involved in controlling the growth and development of ginseng tissues or organs as a class of important transcription factors. This study identified 20 GRF and ten GIF genes in ginseng. The structural characteristics, phylogenetic relationships, gene duplication events, collinearity, and expression patterns of the PgGRF and PgGIF genes were also analysed at the genome level, laying the foundation for further study of PgGRF and PgGIF.
Identification and phylogenetic analysis of the PgGRF and PgGIF gene families
Based on the hidden Markov model (HMM) of the WRC (PF08879) and QLQ (PF08880) domains, a total of 20 GRF genes were identified from the ginseng genome, ranging from 346 aa (PgGRF14 and PgGRF16) to 1240 aa (PgGRF17) amino acids in length, and the coding sequences (CDSs) of PgGRFs ranged from 1041 bp (PgGRF14 and PgGRF16)—3723 bp (PgGRF17) in length. In addition, the molecular weights ranged from 38.44 kDa (PgGRF14) to 139.81 kDa (PgGRF17), and the isoelectric points were between 5.96 (PgGRF10) and 9.31 (PgGRF9). The pI values of 16 PgGRF members were greater than 7, while only PgGRF3, PgGRF10, PgGRF17 and PgGRF20 had pI values less than 7. This may be related to the different effects of PgGRF on the growth and development of ginseng (Table S1).
We obtained ten PgGIF genes by validating GIF’s conserved domain SSXT (PF05030). The amino acid lengths of the GIF proteins in ginseng ranged from 176 aa (PgGIF7) to 214 aa (PgGIF5), and the coding sequences (CDSs) of PgGRFs ranged from 531 bp (PgGIF7) to 645 bp (PgGIF5) in length. In addition, the molecular weights ranged from 18.49 kDa (PgGIF7) to 22.97 kDa (PgGIF5), and the isoelectric points varied from 5.73 (PgGIF8) to 7.94 (PgGIF1), thereby indicating that these GIF proteins are rich in acidic amino acids (Table S2).
IQ-TREE was used to construct a maximum likelihood phylogenetic tree (Fig. 1A and Table S3) of P. ginseng (20), O. sativa (12), and A. thaliana (9). All 41 GRFs from different species are divided into six clades (A-F). Among the six clades, Clade C is relatively small and contains only four members. In addition, the other three clades (B, D and F) have six members each. In contrast, Clade E contains the most significant number of GRFs (twelve), followed by Clade A (seven). Clade B contains only the GRFs of rice and ginseng, while Clades C and D, contain only the GRFs of Arabidopsis and ginseng. The phylogenetic tree indicated that PgGRFs are more closely related to AtGRFs than OsGRFs, which may be partly because ginseng and Arabidopsis are both dicotyledonous plants .
A phylogenetic tree of the GIF genes from six species (A. thaliana, O. sativa, Z. mays, Solanum tuberosum and Solanum lycopersicum) was constructed (Fig. 1B and Table S3). PgGIFs and their counterparts were used for the phylogenetic analysis. The ten PgGIF proteins were clustered into two clades (I and II): four ginseng GIFs (PgGIF2, PgGIF3, PgGIF8 and PgGIF9) are in Clade I, while Clade II contains PgGIF1, PgGIF4, PgGIF5, PgGIF6, PgGIF7 and PgGIF10.
Gene structure and conserved domain analysis of PgGRFs and PgGIFs
All PgGRFs contained motif 1 and motif 2, annotated as the GRF-specific domains WRC and QLQ, respectively (Fig. 1C and Figure S1). Ginseng GRFs are divided into six clades, and each PgGRF contains three to six conserved motifs. The PgGRFs belonging to the same clade have a similar motif composition. Additionally, some motifs only appear in specific clades. For example, motif 9 is unique to Clade A, motif 5 is unique to Clade B, motif 6 is unique to Clade D, and motif 4 and 8 are specific to Clade E. Overall, the gene structure and motif features support the phylogenetic relationship of PgGRFs. Similarly, we identified conserved motifs in the ginseng GIF gene, as shown in Figure S2 and Figure S3.
The PgGRF gene structure shows that the ginseng GRF family member genes contain two to 23 introns, most of which contain two to six introns. Most PgGRFs have three to seven exons. However, two genes (PgGRF10 and PgGRF17) in Clade A have 21 to 24 exons. Members of the PgGIF gene family have three to five exons. The numbers of exons and introns within the same subfamily have high degrees of similarity.
Promoter cis-element analysis of PgGRF genes
The abundant hormone response elements show that PgGRF plays an important role in ginseng hormone signal transduction (Fig. 1D and Table S4). These cis-elements, which included ABRE elements (related to abscisic acid); P-box, GARE-motif, and TATC-box (gibberellin response elements); TGA element (auxin response element); CGTCA motif (involved in MeJA reactions) and TGACG motif; TCA element (participates in the salicylic acid reaction), and AuxRR core (participates in the abscisic acid reaction). In addition, defence and stress response elements (TC-rich repeats), light response elements (GT1-motif, Sp1, MRE, ACE), drought-inducing elements (MBS), and low-temperature response elements (LTR) were identified.
Overall, 16 PgGRFs (80.0%) had more than one GT1 motif, suggesting that they may respond to light. Thirteen PgGRFs (65.0%) had more than one ABRE motif, which suggested that they may respond to abscisic acid. Additionally, 12 PgGRFs (60.0%) possessed at least one CGTCA and TGACG motif, which showed the potential of PgGRFs to respond to MeJA. In addition, cis-acting elements related to gibberellin were found in 11 PgGRFs. We also found five LTRs and six MBSs in PgGRF promoter regions, indicating that these genes might play a role in cold and drought.
Duplication, synteny and evolution analyses of PgGRF and PgGIF gene members
We visualized and analysed the distribution of PgGRF gene family members in the chromosome (Fig. 2). The 20 PgGRF family genes were distributed on 13 chromosomes of ginseng. Among them, chr2, 3, 4, 8, 11 and 18 belong to subgenome A, and chr10, 14, 15, 17, 19, 20 and 24 belong to subgenome B. Chr11 and 14 contain three PgGRF genes, and chr18 and chr20 have two PgGRF genes. Other chromosomes contain one PgGRF gene. The ten ginseng PgGIF genes were distributed on ten chromosomes. Most genes were mainly distributed at the both ends of chromosomes. It can be seen from the above results that during the genetic evolution of ginseng, PgGRF genes were distributed on different chromosomes of ginseng, and the number of genes distributed on each chromosome was different. Gene replication usually mutates genes to derive new functions or divide the functions of ancestral genes crucial to plant adaptation. The expansion of known gene families and the development of new functions contribute to gene replication (tandem and segmental) and differentiation. An intraspecific collinearity analysis showed that eight pairs of PgGRFs originated from segmental replication (whole-genome duplication, WGD), accounting for 90% of all ginseng GRF family members. In the PgGIF family, two pairs of PgGIFs originated from segmental replication. Based on the above results, we can infer that WGD events lead to the derivation of new PgGRF gene members.
To study the collinear relationship between ginseng genes and members of the same family and genus, we analysed the genomic collinearity of PgGRF and PgGIF in ginseng, Panax quinquefolium and Panax notoginseng (Fig. 3). The results showed that 19 PgGRF genes were collinear with the P. quinquefolium GRF gene, and 14 PgGRF genes were collinear with the P. notoginseng GRF gene. This shows that the PgGRF gene family is more closely related to P. quinquefolium than P. notoginseng. Similarly, the results of interspecific collinearity of ginseng PgGIF are shown in Fig. 3. The relationship between PgGIF and P. quinquefolium was closer.
Repeated genes showed that the Ka/Ks ratios of the PgGRF gene family were between 0.276151 and 1.3834. Among them, the Ka/Ks ratios of the PgGRF1-PgGRF5 gene pair were > 1. High Ka/Ks ratios may have been retained to adapt to the environment, indicating that this family may have a complex evolutionary history. The Ka/Ks values of all PgGIF gene pairs were < 1. This shows that the PgGRF and PgGIF gene families may evolve under the action of negative selection (Table S5).
Expression profiles of PgGRF and PgGIF genes in different tissues
The expression trends of 20 PgGRFs in different tissues are shown in Fig. 4A and Table S6. The 14 tissues included fibre root, leg root, main root epiderm, main root cortex, rhizome, arm root, stem, leaf peduncle, leaflet pedicel, leaf lade, fruit peduncle, fruit pedicel, fruit flesh and seed, and expression profiling based on existing transcriptome data. The results showed that only one PgGRF gene (PgGRF15, FPKM < 1) was not expressed in any tissue. Three PgGRF genes, PgGRF8, PgGRF10 and PgGRF17, were expressed in 14 tissues (FPKM > 1). The expression patterns of PgGRFs are low-level, tissue-distinct and constitutive [25, 26]. Eleven PgGRF genes showed low-level expression patterns in all tissues. Only one PgGRF gene (PgGRF11) was expressed in the rhizome, and three PgGRFs (PgGRF12, PgGRF14 and PgGRF16) were expressed in seeds. The two GRF genes (PgGRF8 and PgGRF9) were only expressed in rhizomes and seeds. In addition, PgGRF6 was expressed in the root of ginseng, while PgGRF10 was expressed in the aboveground parts of ginseng (FPKM > 5). The different expression trends of PgGRFs indicated that these genes might be involved in various biological processes in different ginseng tissues.
Because the GIF protein is involved in the process of plant growth, we preliminarily understood the relationship between PgGIF and ginseng growth and development by analysing the expression profiles of the PgGIF gene in different tissues (Fig. 5A and Table S6). Among PgGIF genes, PgGIF4, PgGIF5, PgGIF6 and PgGIF7 were very prominent. These four genes were highly expressed in 14 ginseng tissues, and the FPKM values were greater than five. Only the PgGIF3 gene was not expressed in 14 tissues (FPKM < 1). In addition, eight PgGIF genes were expressed in rhizomes (FPKM > 1), and seven PgGIFs were expressed in seeds (FPKM > 1), which was similar to the PgGRF gene expression previously described.
Transcriptional responses of PgGRFs and PgGIFs to exogenous hormone treatments
To elucidate the hormone responses of PgGRF and PgGIF genes, we exposed five-week-old ginseng seedlings to exogenous GA3, 6-BA, IAA, and ABA. The comprehensive expression profiles of genes under hormone treatment are shown in Fig. 4B and Table S7. A small number of PgRGFs showed significantly altered transcriptional levels after hormone treatment. Ten PgGRFs in the GA3 treatment, five PgGRFs in the IAA treatment, seven PgGRFs in the 6-BA treatment, and seven PgGRFs in the ABA treatment were upregulated by 1.5-fold or more. The highest fold (fold > 10) inductions in the transcriptional responses to hormones were exhibited by PgGRF16 (12.4-fold to GA3 and p < 0.05), PgGRF7 (11.8-fold to GA3 and p < 0.05), PgGRF2 (39.6-fold to IAA), and PgGRF15 (58.4-fold to GA3, 35.7-fold to ABA and 7.7-fold to 6-BA). Notably, PgGRF6 and PgGRF18 accumulated higher transcription levels in response to GA3 treatment but responded only slightly to other hormones. Across all hormone treatments, PgGRF7 was elevated more than fivefold. These results suggest that PgGRF genes may function in a manner responsive to hormonal signalling. According to Fig. 5B and Table S7, we found that the expression levels of all PgGIF genes did not change significantly under hormone treatment.
Expression analysis of PgGRF and PgGIF genes under different abiotic stresses
The published data on different abiotic treatments for ginseng can provide more information for further study of the PgGRF and PgGIF genes in response to abiotic stress (Figs. 4C and 5C and Table S9). To further explore the response of ginseng GRF and GIF to low temperature, salt, drought and heat treatments, we analysed the public transcriptome data of ginseng under abiotic stress. Compared with the control group, the expression levels of three ginseng GRF genes (PgGRF4, PgGRF6 and PgGRF11) increased under cold stress, the expression of one GRF gene (PgGRF16) increased under drought stress, and the expression levels of two GRF genes (PgGRF7 and PgGRF16) increased under salt stress (fold > 2). Similarly, compared with CK, there was no significant change in ginseng GRF gene expression after one week of heat treatment, and the expression levels of two GRF genes (PgGRF7 and PgGRF16) increased (fold > 2). However, after three weeks of heat treatment, the expression levels of six ginseng GRF genes were significantly increased (fold > 2), of which PgGRF7 (12.7-fold) and PgGRF15 (5.8-fold) were significantly changed. Similar to the results described in the previous section, ginseng GIF genes did not change significantly under cold, drought and salt stress (fold < 2). Compared with CK, the expression levels of PgGIF4, PgGIF6 and PgGIF7 were increased after one week of heat treatment, and only PgGIF8 was increased after three weeks of heat treatment (fold > 2), which was different from the change trend of the PgGRF gene. The functions of these genes in ginseng need further study.
Coexpression analysis between PgGIF and PgGRF
The GIF1 protein acts as a transcription coactivator to interact with GRF proteins in Arabidopsis and rice [27,28,29,30]. To further understand whether there is a regulatory relationship between ginseng GIF and GRF genes, the correlation between PgGIFs and PgGRFs was analysed (Fig. 6 and Table S10). A total of 27 pairs of PgGIFs and PgGRFs had correlation r-values greater than 0.6, and we considered them to be strongly correlated. According to the phylogenetic tree analysis results, we focused on five PgGRFs (PgGRF1, PgGRF4, PgGRF5, PgGRF11 and PgGRF17) because of their high homology with AtGRF1 and AtGRF5. The expression levels of PgGIF and PgGRF genes were highly correlated, suggesting a regulatory relationship between these genes.
The evolution and characterization of PgGRFs and PgGIFs in ginseng
Studies have suggested that the GRF gene family expanded significantly during evolution from lower plants to higher plants, and the number of GRF transcription factor genes in land plants ranged from eight to 20 . Only two GRF genes have been found in mosses. Genes with regulatory functions are preferentially retained after mass duplications . In previous studies, nine GRF genes were found in Arabidopsis, with a genome size of 0.12 Gb; 12 in O. sativa, with a genome size of 0.46 Gb; and 14 in Z. may, with a genome size of 2.3 Gb. In some recent studies, 30 GRF genes were found in wheat (Triticum aestivum L.), with a genome size 17 Gb, 8 in H. vulgare, with a genome size of 4.5 Gb; and 20 in Populus. trichocarpa, with a genome size of 0.48 Gb. We identified 20 GRF transcription factors in the ginseng genome in the present study, with a genome size of 2.98 Gb . This result indicated that the number of GRF genes was not associated with genome size.
GIF acts as a transcriptional coactivator and can form a complex with GRF. In our study, we identified ten GIF genes in ginseng. GIF exists in most eukaryotic organisms, such as embryophytes and metazoans, but is not present in fungi and protists, suggesting that GIF genes possess ancient origins . For embryophytes, M. polymorpha has only one GIF gene, and both P. patens and S. moellendorffii have four. The basal angiosperm Amborella trichopoda has two GIF genes. Monocots such as rice and maize have three GIF genes, and wheat has four. Dicotyledons, such as tomato (S. lycopersicum), have four, and Chinese cabbage has five. Although research on the GIF gene family is deepening, no rule has been identified regarding the numbers of GIF genes in eudicots or monocots. Similarly, the number of GIF genes between annuals and perennials has no obvious rule. For example, the annual plant G. max has 11, while Z. mays has three. Among the biennial plants, B. rapa has five and Beta vulgaris has three, while among the perennial plants, P. trichocarpa has six, and Theobroma cacao has three .
Previous studies have reported that the expansion of the GRF and GIF families mainly occurs through gene duplication, especially large-scale duplication (i.e., whole-genome duplication or fragment duplication), to enhance plant adaptation to environmental changes [27, 33, 34]. This phenomenon exists in soybeans and many other plants. For example, soybean has 22 GRF genes. There were also two WGD events (58 million and 13 million years ago) during the evolution of the soybean (Glycine max) genome . However, there are only ten GRF genes in Medicago, and the genome experienced only one WGD event 58 million years ago . Ginseng underwent two whole-genome duplication (WGD) events between 2.2 million and 28 million years ago [20, 36]. In fact, TFs are usually preserved after WGD events . In our study, both ginseng GRF and GIF were mainly families expanded by WGD events [24, 27]. Therefore, ginseng has more GRF and GIF genes, which are closely related to the WGD event of ginseng.
According to the phylogenetic results (Fig. 1), 20 GRF members in ginseng were clustered into six clades; this evolutionary relationship is similar to previous GRF taxonomic studies , and the homology of ginseng GRF and Arabidopsis GRF is higher than that of rice GRF, which may be related to the fact that ginseng and Arabidopsis are dicotyledonous plants. Gain or loss events of exons or introns provide structural and functional differences . From the gene structure of each group of responses, most of the PgGRF genes have similar structures, and most of the PgGRF genes have two to four introns/exons, which are similar to those of rice and Arabidopsis [6, 8]. Previous reports showed that suppressed OsGRF3, OsGRF4 and OsGRF5 in clade B could cause plant dwarfing, delayed growth and inflorescence formation . The AtGRF7 gene in clade D is involved in osmotic stress . Overexpression of AtGRF9 can produce smaller leaves and petals . We speculate that the PgGRF genes in these three clades also have the same function.
In Arabidopsis, mutants of AtGIF1 can change leaf shape . AtGIF1 interacts with AtGRF1, AtGRF2, AtGRF4, AtGRF5 and AtGRF9 through its conserved QLQ domain [11, 41]. Three ginseng GRF genes (PgGRF10, PgGRF15, and PgGRF17) in branch A are highly homologous to AtGRF5, so we believe they may have similar functions, such as regulating cell proliferation, improving plant tolerance or participating in stem growth and delaying leaf senescence . Studies of AtGRF proteins have shown that AtGRF5 only tightly interacts with AtGIF1 [11, 43]. Therefore, we speculate that PgGRF10, PgGRF15 and PgGRF17 can also bind to the SNH conserved domain of PgGIF proteins to obtain a better gene expression effect.
PgGRFs and PgGIFs are involved in the growth and development of ginseng
Previous studies have confirmed that GRFs are expressed in different tissues, usually in growth areas where cell proliferation occurs, such as germinating seeds, calli and shoots [1, 6, 11,12,13, 30]. The expression levels of PgGRFs in 14 tissues were calculated based on FPKM. As shown in Fig. 4A, PgGRFs had different expression patterns in 14 tissues. These tissue-specific expression patterns suggest that these PgGRFs may be involved in tissue-specific developmental and signalling processes. Some PgGRFs were highly expressed in other tissues, such as leaf blade, fruit peduncle, stem and fruit pedicel (FPKM > 10). This means that the vast majority of PgGRFs are involved in the growth and development of ginseng. The expression levels of GRF genes were significantly higher in actively growing tissues than in mature tissues, and GRF transcription levels decreased with plant senescence [6, 8, 41]. The expression level of AtGRF decreased with increasing plant age [6, 44]. GRF genes in rice are strongly expressed in shoots and immature leaves . The median FPKM values of fibre roots, leg roots, fruit pedicels, rhizomes and seeds were higher than those in other tissues, indicating that PgGRF may be involved in more physiological processes in these tissues. PgGIFs may be transcriptional coactivators of PgGRFs because the expression profiles of PgGIFs showed a similar trend to PgGRFs. GRF and GIF proteins positively regulate leaf size by promoting cell expansion and proliferation [11, 15, 41, 45]. The specific functions of PgGRF and PgGIF in different parts of ginseng need further research and discovery.
PgGRFs and PgGIFs are involved in abiotic stresses responses of ginseng
Previous studies have shown that plant hormones regulate many physiological processes, such as growth, differentiation, and development. The first GRF was OsGRF1, found in gibberellin-treated rice . GRF acts as an upstream repressor of the KNOX gene that inhibits GA3 biosynthesis, and GA3 treatment leads to the upregulation of GRF . GA3 treatment increased the expression of some PgGRF genes in tobacco and some AhGRF genes in peanuts [33, 47]. However, GRFs in Arabidopsis were not significantly affected by GA3 [6, 8]. In our study, we tested the responses of the PgGRF genes to various hormones. Eight of the 20 PgGRFs were upregulated by twofold or more in GA3 treatment. In addition, the expression levels of most PgGRF genes in this study were maintained or enhanced under 6-BA and ABA treatment, while the expression levels of most genes decreased under IAA treatment. PgGRF genes showed significant differential expression, suggesting that these PgGRFs may play different roles in hormonal feedback regulation. Various hormone-related cis-elements are found in the PgGRF promoters. The results showed that six of the ten PgGRF genes upregulated in the GA3 treatment group contained P-box, GARE-motif, and TATA-box elements, and six of the seven PgGRF genes upregulated in the ABA treatment group contained ABREs. We therefore believe that cis-acting element analysis can predict the responses of certain transcription factors to hormonal treatment. PgGRF and PgGIF genes may regulate physiological processes through interactions with molecular and hormonal signals. Further qRT-PCR analysis was carried out for genes with significantly increased expression under different hormone treatments. The results showed that the expression patterns of 12 genes (PgGRF2, PgGRF4, PgGRF6, PgGRF7, PgGRF14, PgGRF16, PgGRF18, PgGIF1, PgGIF3, PgGIF7, PgGIF9, PgGIF10) were basically consistent with RNA-seq data.
During the long evolutionary process, plants have acquired a series of signalling pathways and defence systems to resist environmental stresses, and transcription factors play crucial roles in plant responses to various environmental stresses. GRF transcription factors play important roles in plant growth by coordinating stress responses and defence signals [16, 17, 48]. For example, under stress conditions, overexpression of Arabidopsis AtGRF7 increases resistance to drought stress . AtGRF1 and AtGRF3 coordinate plant growth, defence signals, and stress responses [14, 49]. Transcriptome data showed that 13 PgGRFs were upregulated under cold treatment, five PgGRFs were upregulated under drought stress, six PgGRFs were upregulated under salt stress, and ten PgGRFs were upregulated under a one-week heat treatment. The expression levels of ten PgGRFs increased under a three-week heat treatment. Three PgGIF genes (PgGIF4, PgGIF6, and PgGIF7) responded to all treatments, indicating that they may play important roles in the response of ginseng to abiotic stress. This study found that the expression levels of the PgGRF and PgGIF genes had similar trends under cold, drought and salt stresses. Their gene changes were not very significant under these three abiotic stresses, and the genes were relatively more responsive to cold stress. Although there were significant changes in the two family members in the heat treatment group, the PgGRF gene was more responsive to a three-week heat treatment, while PgGIF gene was more responsive to a one-week heat treatment. These findings suggest that both GRF and GIF genes may be involved in biological processes related to abiotic stress responses, especially in plant responses to changes in temperature conditions. The responses and functions of PgGRF and PgGIF in ginseng under environmental stress need further verification.
Regulatory relationship between PgGRFs and PgGIFs
Functional studies have shown that AtGIF1 interacts with six GRF proteins in Arabidopsis [11, 15, 41], while OsGIF1 interacts with three GRF proteins [29, 48, 50]. GIF genes may also mediate different plant growth and development pathways by interacting with different GRF genes . In the above results, we focus on five PgGRFs (PgGRF1, PgGRF4, PgGRF5, PgGRF11 and PgGRF17), which have a strong correlation with at least one PgGIF (r > 0.6), which also means that they may have a synergistic effect with PgGIF in ginseng. Phylogenetic tree analysis showed that PgGIF8 and PgGIF9 had homology with AtGIF1/AN3 among the eight PgGIF genes with a strong correlation with PgGRF, and they interacted with different PgGRF genes, which indicated the difference in PgGIF genes in the evolution process . Coexpression analysis showed that 27 pairs of PgGIFs and PgGRFs had high correlations (r > 0.6), suggesting that they may be regulated by the same TF . In addition, it has been reported that several GRF proteins in rice  and maize  are located downstream of the GIF gene, and increasing the expression of the GIF gene can increase the transcription level of the GRF gene. The specific functions and modes of action of the different GRFs and GIFs in ginseng may require further research.
In our study, the genome-wide identification and analysis of GRF and GIF TFs in ginseng and their induction in different tissues, upon exposure to different hormones, and in response to different abiotic stresses were performed. The results of coexpression studies indicated potential interactions between PgGRFs and PgGIFs. Our results lay a foundation for further research on the roles of PgGRFs and PgGIFs in the growth and development of ginseng, provide valuable information for the functional study of transcription factors in ginseng, and provide a theoretical basis for ginseng variety selection.
Materials and methods
Plant cultivation and treatment
Hormone treatment: sown ginseng seeds in a culture bowl cultivate at room temperature at 25 °C, with a relative humidity of about 60%, and 16 h light/8 h dark. When the seedlings (JIMEI Ginseng) grow for five weeks and have three real leaves, spray with different hormones, including ABA: 50 mM, IAA: 10 mM, 6-BA: 75 mM, GA3: 100 mM and the control group is treated with distilled water. The treatment time is five hours, the biological repetition is three times, and the seedlings are collected and stored at -80 °C. The environmental stress analysis uses public data .
GRF and GIF sequence retrieval and identification
The candidate GRF and GIF genes were firstly obtained from the Ginseng Genome Data resource . Hidden Markov Models (HMMs) for GRF and GIF conserved domains WRC (PF08879), QLQ (PF08880) and SSXT (PF05030) were extracted from the Pfam database (http://pfam.xfam.org). The GRF and GIF genes retrieved from the ginseng genome were detected by HMMER 3.2.1 software, and the E-value threshold was 10−2. All candidate PgGRFs and PgGIFs were further validated using the SMART data resource (http://smart.embl.de/), NCBI-Conserved Domain Database (CDD) and PlantTFDB (Plant Transcription Factor Database) (http:// planttfdb.cbi.pku.edu.cn) to ensure that they contain both GRF or GIF domains.
Phylogenetic analysis and gene structure analysis
Mafft (https://mafft.cbrc.jp/alignment/software/) with default parameters was used for multiple alignments of ginseng GRF sequences as well as for multiple alignments of GRFs among other species. The ginseng GRF phylogenetic tree was established by the maximum likelihood method IQ-TREE based on the JTTDCMut + F + R4 model , and the nodes were tested 1000 times by bootstrap analysis. Further annotation of the phylogenetic tree results was handled by Evolview (https://evolgenius.info/).
TBtools 1.053 was employed to demonstrate the gene structure . Conserved motifs of PgGRFs were identified using MEME native software (version 4.12.0) in Linux with a maximum of 10 mismatches and an optimal motif width of 6–100 amino acid residues. In addition, theoretical isoelectric point (pI) along with the molecular weight (MW) of PgGRF proteins were predicted by the online Sequence Manipulation Suite (http://www.detaibio.com/sms2/reference.html) . Likewise, the GIF gene was also analyzed using the method described above.
Cis-acting elements analysis
The sequence of 2000 bp upstream of the start codon of PgGRFs and PgGIFs was obtained for promoter analysis. Use PlantCARE (http://bionformatics.psb.ugent.be/webtools/plantcare/html) to predict cis-acting elements in the promoter region and use PlantTFDB software (http://planttfdb.cbi.pku.edu.cn/) online Predict the distribution of promoter transcription factor binding sites (p-value ≤ 1e−6).
To analyze gene expression among different tissues and responses to different abiotic treatments. We retrieved RNA-Seq datasets from 14 different tissues from NCBI (accession number PRJNA302556)  and 15 RNA-Seq datasets for abiotic treatment (No.24–38 in ginseng transcriptome data resource, http://ginsengdb.snu.ac.kr/transcriptome.php) from Ginseng Genome Data Resource (http://ginsengdb.snu.ac.kr/) were retrieved. The clean reads were aligned to the ginseng genome using Hisat2 software. Hisat2, StringTie and ballgown were used to assemble and calculate the expression value for each transcript.
The hormone treatment cDNA libraries were established in a previous study . These 15 cDNA libraries were finally sequenced on HiSeq 2500 (Illumina) with the PE125 strategy. The FRKM was calculated using the same protocol for the other 16 RNA-Seq datasets. The heatmap was generated by the R package “Heatmap”.
Quantitative-real time PCR analysis
Total RNA was prepared from samples using a EasyPure Plant RNA Kit (TransGen Biotech). RNase-free DNase I (TransGen Biotech) was used in the extraction process to remove DNA contamination. Both the concentration and the quality of the RNA samples were evaluated with a NanoPhotometer N50 (Implen, GER). Use the PerfectStart Uni RT&qPCR Kit (TransGen Biotech) to reverse transcribe RNA into cDNA and perform two-step Quantitative Real-time PCR. qRT-PCR was performed using a Stratagene Mx3000P SYBR-GREEN I Master (Agilent, USA). β-Actin gene is used as internal control . For the data analyses, the 2−ΔΔCT method was used for calculating the relative expression of PgGRF and PgGIF genes . Primers for qRT-PCR were synthesized by Sangon Biotech (ShangHai, China), and sequences are listed in Supplementary Table S8.
Chromosomal location, duplication, synteny and evolution analyses
The MCScanX program was used for inter- and intra-species collinearity analysis of proteins with an E value of 1e−5, and the Duplicate Gene Classifier script in the MCScanX program was used to quantify various forms of duplication (WGD or segmental, tandem, dispersed and proximal duplication) and visualized by the Circos [60, 61].
Using KaKs-Calculator-2.0 calculates the non-synonymous replacement rate (Ka) and synonymous replacement rate (Ks) of replicated gene pairs and analyzes the environmental selection pressure through the Ka / Ks ratio .
Correlation coefficient analysis between PgGRFs and PgGIFs
The FPKM value of PgGRF and PgGIF under hormone-induced and abiotic environmental stress conditions was used to calculate the Pearson’s correlation between the two gene families by the R package “Hmisc”.
Availability of data and materials
The raw RNA-Seq data of 14 P. ginseng tissues were downloaded from the NCBI Sequence Read Archive ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA302556). The raw RNA-Seq datasets of drought, salt and cold treatment were downloaded from Ginseng Genome Database (http://ginsengdb.snu.ac.kr/download.php?filename=DSC.tar.gz), and the heat stress was downloaded from Ginseng Genome Database ( http://ginsengdb.snu.ac.kr/download.php?filename=Heat.tar.gz). All other data generated or analysed in this study are included in this article and its additional files.
Multiple expectation maximization for motif elicitation
Fragments per kilobase of transcript per million mapped fragments
Hidden Markov model
Molecular evolutionary genetics analysis
Million years ago
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This research was funded by National Natural Science Foundation of China (81703635), Jilin Province Science and Technology Development Project (20210101190JC), National Key Research and Development Program of China (2021YFD1600900) and Natural Science Foundation of China (U21A20405).
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GRF conserved motif structure of ginseng.
Gene structure of ginseng GIF family.
GIF conserved motif structure of ginseng.
expression analyses of PgGRF and PgGIF genes under different hormone treatments conditions analyzed by qRT-PCR. CK: control sample. ABA: 50 mM, IAA: 10mM, GA3: 100 mM. Data were normalized to β-actin gene and vertical bars indicated standard deviation.
Identification and characterization of PgGRF genes in P. ginsneg. Table S2. Identification and characterization of PgGIF genes in P. ginsneg. Table S3. GRF genes found in Arabidopsis thaliana and Oryza sativa. And protein sequences of GIFs from A. thaliana, O. sativa, Z. mays, S. tuberosum and S. lycopersicum. Table S4. The detailed information of cis-elements in the promoter regions of PgGRF genes in P. ginseng. Table S5. The ka/ks values of PgGRF and PgGIF genes in P. ginseng. Table S6. The FPKM values of PgGRF and PgGIF genes in different P. ginseng tissues. Table S7. The FPKM value of PgGRF and PgGIF genes transcriptional response to exogenous hormones treatments in P. ginxeng. Table S8. Primers for qRT-PCR of candidate genes in P. ginseng GRF and GIF gene families. Table S9. The FPKM value of PgGRF and PgGIF genes transcriptional response to abiotic stresses in P. ginseng. Table S10. The FPKM value is the correlation between PgGRF and PgGIF genes in P. ginseng.
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Wang, P., Xiao, Y., Yan, M. et al. Whole-genome identification and expression profiling of growth-regulating factor (GRF) and GRF-interacting factor (GIF) gene families in Panax ginseng. BMC Genomics 24, 334 (2023). https://doi.org/10.1186/s12864-023-09435-w