In recent years, studies on the GRAS gene family have shown that it plays an important role in plant growth and development and particularly in the response to abiotic stress (Jaiswal et al., 2022). The GRAS gene family name is derived from the abbreviations of the earliest three members: gibberellic acid insensitive (GAI) (Peng et al., 1997), repressor of GAI (RGA) (Silverstone et al., 1998), and scarecrow (SCR) (Laurenzio et al., 1996). Genes of the GRAS gene family generally consist of 400–770 amino acids, with a changeable N-terminal sequence and a more conservative C-terminal sequence (Niu et al., 2016a). The C-terminal sequence has five conserved motifs: LHR I, LHR II, VHIID, PFYRE and SAW. Among them, VHIID, PFYRE, and SAW are more conserved (Pysh et al., 1999). GRAS gene family has been identified and analysed in many plants. According to the difference in N-terminal length and sequence, members of GRAS gene family can be divided into 17 subfamilies at most (DELLA, PAT1, LISCL, HAM, SCR, SHR, LAS, SCL3 in model plants, and NSP1, NSP2, RAD1, SCLA, SCLB, RAM1, DL, SCL4/7, SCL32 in other plants.) (Tian et al., 2004; Liu and Widmer, 2014; Cenci and Rouard, 2017).

First, in the growth and development of plants and morphological regulation, through the regulation of the LAS subfamily of the GRAS protein family, the formation of mutants prevents the development of axillary meristems, which is more conducive to the growth of meristems (Schumacher et al., 1999). OsMOC1 and LAS are homologous genes that are key factors in regulating tillering in rice (Liang et al., 2014). SHR induces the activity of SCR promoters in specific tissues (Helariutta et al., 2000), which play an important role in radial growth of roots and stems (Laurenzio et al., 1996). NSP1 (Smit et al., 2005) and NSP2 (Kalo et al., 2005) also play important roles in nodulation signalling in legumes. Second, in terms of plant hormone signal transduction, the introduction of an early stop codon in the PAT1 1 mutant indicated that the PAT1 protein inhibits phyA signalling (Bolle et al., 2000); AtSCL13 plays a positive regulatory role in phyB signalling (Torres-Galea et al., 2006); and the DELLA subfamily and SCL3 subfamily antagonize each other and regulate GA (gibberellin) signal transduction in plants, thereby regulating plant seed germination, stem elongation, and flower development (Heoa et al., 2011). In addition, GRAS proteins also play an important role in biotic and abiotic stresses. OsCIGR1 and OsCIGR2, members of the PAT1 subfamily, can inhibit the infection of Oryza sativa L. pathogens (Day et al., 2004), and their expression in Arabidopsis thaliana can improve the drought, cold, and salt resistance of plants (Yuan et al., 2016). The DELLA protein interacts with the cellular gibberellin (GA) receptor GID1, activates stress adaptation mechanisms by altering cellular reprogramming, and regulates plant growth under different adversities by combining plant intrinsic signal transduction. AtSCL9, AtSCL13, AtSCL14, and other genes can all respond to abiotic stress (Torres-Galea et al., 2006; Fode et al., 2008; Lee et al., 2008).

Orchidaceae is one of the largest angiosperm families, accounting for 10% of the world’s angiosperms and representing an ideal taxa for studying biodiversity and evolution. Orchids have abundant flower morphology and pollination mechanisms (Ramirez et al., 2011; Li et al., 2020; Lu et al., 2022), unique epiphytic phenomena, xerophytic physiology (Zhang et al., 2016), and extremely complex mycorrhizal relationships (Li et al., 2022). Dendrobium is the second largest genus of Orchidaceae and has high medicinal and ornamental value and strong stress resistance (Zhang et al., 2016; Zhang et al., 2021a). D. chrysotoxum is a valuable medicinal plant resource in Orchidaceae, and its functional components, such as erianin (Zhang et al., 2021b) and chrysotoxene (Dou et al., 2018), have been reported. The stout pseudobulb of D. chrysotoxum is not only rich in medicinal components but also closely related to its resistance to stress (Zhang et al., 2021a). Although GRAS genes have been extensively studied in model plants as well as in some crops, little is known about the characterization of GRAS genes in orchids. The advent of high-quality, chromosomal-level orchid genomes has made it possible to explore the role of the GRAS gene family in orchids. Exploring the regulatory mechanism of the GRAS gene family in the growth and development of D. chrysotoxum and the expression of functional genes under abiotic stress is of great significance for the development and utilization of D. chrysotoxum.

In this study, GRAS gene family members of D. chrysotoxum were examined by gene structure analysis, phylogenetic tree construction, selection pressure analysis, cis-acting element analysis, and analysis of expression patterns under abiotic stress. This study provides a reference for further research on the evolutionary relationship and functional characteristics of the GRAS family in orchids.

The ecotypes of orchids are diverse, with 70% being epiphytic (Zhang et al., 2016; Zhang et al., 2022). D. chrysotoxum, as a perennial epiphytic herb, is widely cultivated and used (Xiao et al., 2014). It has high ornamental, medicinal, and health value. In recent years, the molecular mechanism of flower development regulation of D. chrysotoxum and the formation mechanism of medicinal components have been extensively studied (Zhang et al., 2021a). However, there are few studies on the molecular mechanism of D. chrysotoxum under adverse conditions. The GRAS gene family has been widely identified and studied in a variety of plants, which proves that it is closely related to plant growth, development, abiotic stress, and hormone regulation (Jaiswal et al., 2022). In this study, 46 DchGRAS genes were obtained through comparison and screening based on GRAS-specific domains ( Table 1 ). The identification results were similar to those of Cicer arietinum (46) (Yadav et al., 2022), Prunus mume (47) (Lu et al., 2015), Dendrobium catenatum (47) (Zeng et al., 2019); more than those of A. thaliana (32) (Tian et al., 2004) and Pinus (32) (Abarca et al., 2014); and less than those of Rosa chinensis (59) (Kumari et al., 2022), O. sativa (63) (Tian et al., 2004), Manihot esculenta (77) (Shan et al., 2020), and Zea mays (86) (Guo et al., 2017). These differences may be due to gene duplication events or the different frequencies of retained copies after duplication events.

In general, the evolutionary relationship of species is inferred or evaluated through phylogenetic analysis and the construction of an evolutionary tree. The phylogenetic tree constructed in the study shows that the phylogenetic relationships of the GRAS gene family in D. chrysotoxum are more closely related to O. sativa rather than A. thaliana. All 140 GRAS genes of three species are divided into ten subfamilies ( Figure 1 ). HAM protein was found to be related to the maintenance of apical or lateral meristem in Petunia hybrida plants (Jaiswal et al., 2022). The HAM subfamily has the most members (10/46) in our study. The SCR and SHR subfamilies can form a tissue specific network with BIRD/IDD proteins (Moreno-Risueno et al., 2015), which plays an important role in the regulation of root and leaf radiation morphology. In this study, the members of SCR (6/46) and SHR (7/46) subfamilies may help D. chrysotoxum form a divergent root system, which can better adhere to trees or rocks. The DELLA and PAT1 subfamilies are widely studied in many plants, especially the DELLA subfamily. DELLA protein can stimulate GA signal transduction, which is crucial in seed germination, stem and root elongation and flower development (Niu et al., 2016a). DELLA (6/46), PAT1 (5/46), and LISCL (5/46) subfamilies also had more members in this study, and these subfamilies play a crucial part in other plants in coping with abiotic stresses such as drought, salt and photooxidative stress (Khan et al., 2021). GRAS proteins are fully involved in all stages of plant growth and development, and some specific subfamilies are differentiated during the process of contraction or expansion. Such as the Os19 subfamily is a specific subfamily of O. sativa, and the phylogenetic tree constructed in this study shows that the Os19 subfamily contains only two members, DchGRAS20 and OsGRAS21 (Liu and Widmer, 2014). Most plant GRAS gene families contain members of the Os19 subfamily, but A. thaliana (Liu and Widmer, 2014), Z. mays (Guo et al., 2017), Phaseolus vulgaris (Laskar et al., 2021) and other plants do not contain members of this subfamily. This suggests that DchGRAS20 may be formed after differentiation of the common ancestor with A. thaliana.

The structure of genes may also affect the phylogenetic relationship. A gene family may be structured differently in different species. The N-terminal intrinsically disordered region (IDR) sequence of GRAS proteins binds to different proteins when expressed, resulting in specific functions (Sun et al., 2012); VHIID is a core structure among five conserved domains at the C-terminus of GRAS proteins (Pysh et al., 1999). The results of the sequence alignment of the DchGRAS gene in this study showed that, except for DchGRAS2, all 45 members have a VHIID domain, in which aspartic acid (D) is extremely stable, and almost all subfamilies have different degrees of amino acid substitution ( Figure 6 ). For example, all members of the SHR subfamily have valine (V) replaced by leucine (L), while all members of the DELLA family with a complete motif backbone have isoleucine (I) replaced by valine (V), and the PAT1 subfamily also has a few amino acid substitutions. The PAT1 and DELLA subfamilies play an important role in regulating plant photosensitive signal transduction, GA signal transduction, and stress resistance (Torres-Galea et al., 2006; Wang et al., 2014; Zhang et al., 2021c). In this study, they have a complete motif skeleton structure without deletion. Therefore, the large number of amino acid substitutions in these two relatively stable subgroups may be the embodiment of the slow evolution of the D. chrysotoxum GRAS gene family to adapt to environmental changes. In addition, SAW is also a relatively conserved domain at the C-terminus of the GRAS protein. It is composed of three consecutive parts: WX7G, LW and SAW (Hirsch et al., 2009). In this study, most DchGRAS genes had this domain, and there was less substitution than seen in the prior study. Although the function of the SAW domain is unclear, these conserved elements play a vital role in maintaining the integrity of the domain and playing a role in stabilizing the protein (Sun et al., 2011).

During evolution, introns are considered to be one of the important reasons for the formation of new genes. 69.56% of DchGRAS genes in this study lacked introns ( Figure 2C ) and more than 70% of GRAS genes in other plants such as Z. mays (Guo et al., 2017), Glycine max (Wang et al., 2020) and Melilotus albus (Wang et al., 2022) lacked introns. This phenomenon is parallel to the early reports on the conserved characteristics of GRAS genes. Some studies have found that intron-free genes also belong to large families that regulate physiological and biochemical processes and participate in growth and development processes, such as signal transduction, protein synthesis and conversion, and metabolism (Jain et al., 2008). Although intron-free genes have no advantage in species evolution or recombination, they often respond quickly to stress (Sang et al., 2016). Therefore, many DchGRAS members may be able to respond quickly to environmental changes, and these intron-free genes are the main driving force of plant tissue-specific evolution. Orchids have the characteristics of high heterozygosity, high repetition, and long introns (Cai et al., 2015; Lu et al., 2019). Three genes in this study (DchGRAS17, DchGRAS26, and DchGRAS32) have introns over 10,000 bp in length, probably because they contain a large number of transposable elements ( Figure 7 ). Genes containing introns promote the evolution of plant species by participating in translation and energy metabolism, and these genes with long introns are important for the abundance of orchid species (Jain et al., 2008).

Cis-acting elements are involved in the dynamic network of gene regulation. This study identified many anti-stress responsive cis-acting elements in the upstream 2000 bp promoter region of 46 DchGRAS genes ( Figure 4 ). At the same time, some hormone-responsive elements, such as ABRE (ABA response), P-box (ABA and ethylene response), and TATC-box (GA response) (Cao et al., 2021), are also widely present in DchGRAS genes. Plants need to adapt to changes in the external environment through gene regulation. In recent years, an increasing number of studies have shown that GA anabolism and signal transduction pathways are involved in abiotic stress in plants. When GA content decreases, the corresponding cis-acting elements begin to respond, and DELLA proteins bind to transcription factors, inhibiting downstream gene expression and thereby inhibiting plant growth (Achard et al., 2008; Shan et al., 2020). This may be a key pathway for GRAS proteins to regulate plant growth and development under stress conditions.

We selected 12 DchGRAS genes from different subfamilies for RT−qPCR validation analysis. The results showed that after high temperature, drought and salt treatment, the expression levels of 12 genes were significantly different under different stresses, and there were also differences in expression among members of the same subfamily ( Figure 5 ). For example, DchGRAS13 and DchGRAS15 all belong to the LISCL subfamily, but the RT−qPCR results showed that DchGRAS13 is significantly induced under high temperature stress and increases up to 25-fold, while DchGRAS15 does not change significantly under high temperature stress but is upregulated ten-fold under salt stress and drought stress. This phenomenon also exists in GRAS genes from different subgroups of other plants after stress treatment, such as Solanum lycopersicum (Niu et al., 2016b), Populus (Liu and Widmer, 2014), and Camellia sinensis (Wang et al., 2018). This suggests that the genes of the same subfamily may have different roles in the signalling pathways of abiotic stress responses. A large number of studies have shown that the DELLA and PAT1 subfamily are fully involved in the growth and development processes of multispecies abiotic stress, light signal transduction, seed germination, and other processes and control plant height by influencing the growth of inflorescence stems (Khan et al., 2021; Jaiswal et al., 2022). In this study, members of the DELLA subfamily (DchGRAS25 and DchGRAS28) were induced under all three stress conditions, and their expression was significantly upregulated under drought stress. The expression of DchGRAS32 of the PAT1 subfamily was upregulated under all three stress conditions, but especially under drought stress, under which its expression was upregulated by more than five-fold. LAS subfamily is closely related to the development of plant lateral buds (Schumacher et al., 1999). OsMOC1 in rice belongs to LAS subfamily, and its overexpression can increase the number of rice tillers, thereby reducing plant height (Li et al., 2003). LAS homologous genes with similar functions have been found in S. lycopersicum, Nicotiana tabacum, Cucumis sativus, Zoysia tenuifolia and other crops (Chang et al., 2013). In this study, the expression of DchGRAS23, a member of the LAS subfamily, was also significantly upregulated under stress conditions, which indicated that LAS subfamily members likely function in stress tolerance and provided a reference for exploring more functions of LAS subfamily members. In addition, studies have shown that AtGRAS14 of the LISCL subfamily is also a key gene regulating plant development and stress and is related to the occurrence of anther microspores in Lilium longiflorum (Morohashi et al., 2003). DchGRAS15 and AthGRAS14 are homologous genes, which are also up-regulated under abiotic stress. To sum up, this study speculates that the normal growth and development of D. chrysotoxum and the maintenance of the swollen pseudobulb under abiotic stress may be regulated by members of the GRAS gene family.

In this study, 46 DchGRAS genes were identified from the D. chrysotoxum genome and divided into ten subfamilies according to their phylogenetic relationships, and members of the same subfamily had similar gene structures and conserved domains. The results of real-time reverse transcription quantitative PCR (RT−qPCR) experiments showed significant differences in the expression levels of 12 genes under abiotic stress, among which LISCL, LAS, and DELLA subfamily members were the most sensitive to stress. This result will provide valuable information for further studying of D. chrysotoxum and other orchids under abiotic stress.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

SL, Z-JL, and DZ contributed to conceptualization and validation. XZ, D-KL, and CZ prepared the original draft. SK, YL, Q-QW, and QZ analysed the data. All authors contributed to the article and approved the submitted version.

This work was supported by the Forestry Peak Discipline Construction Project of Fujian Agriculture and Forestry University (72202200205), and the National Key Research and Development Program of China (2019YFD1000400).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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