Genome-Wide Identification, Expression and Stress Analysis of the GRAS Gene Family in Phoebe bournei

GRAS genes are important transcriptional regulators in plants that govern plant growth and development through enhancing plant hormones, biosynthesis, and signaling pathways. Drought and other abiotic factors may influence the defenses and growth of Phoebe bournei, which is a superb timber source for the construction industry and building exquisite furniture. Although genome-wide identification of the GRAS gene family has been completed in many species, that of most woody plants, particularly P. bournei, has not yet begun. We performed a genome-wide investigation of 56 PbGRAS genes, which are unequally distributed across 12 chromosomes. They are divided into nine subclades. Furthermore, these 56 PbGRAS genes have a substantial number of components related to abiotic stress responses or phytohormone transmission. Analysis using qRT-PCR showed that the expression of four PbGRAS genes, namely PbGRAS7, PbGRAS10, PbGRAS14 and PbGRAS16, was differentially increased in response to drought, salt and temperature stresses, respectively. We hypothesize that they may help P. bournei to successfully resist harsh environmental disturbances. In this work, we conducted a comprehensive survey of the GRAS gene family in P. bournei plants, and the results provide an extensive and preliminary resource for further clarification of the molecular mechanisms of the GRAS gene family in P. bournei in response to abiotic stresses and forestry improvement.


Introduction
Throughout the life of a plant, temperature, water and salt stresses may recur over time and gradually affect its normal physiological activities. For example, salinity can cause oxidation, ionization, and osmosis, whereas heat and dryness can damage chloroplast membranes and promote chlorophyll breakdown, lowering photosynthetic efficiency [1]. These abiotic pressures may become more common in the natural environment as a result of climate change. Transcription factors (TFs) can precisely control the transcription of target genes via conjugation to deoxyribonucleic acid (DNA) sequences in response to unfavorable conditions [2][3][4]. TFs can help us understand the adaptive response networks that comprise many biological processes.
GRAS is an extremely significant TF that can regulate a variety of physiological processes in higher plants [5]. The GRAS gene family is named after the first three members to be discovered: Gibberellin Acid Insensitive (GAI), Repressor GA1 (RGA), and Scarecrow (SCR) [6,7]. The GRAS structural domain of a protein consists of an extremely volatile amino (N-) terminus region and a highly conserved carboxyl (C-) terminus region [8]. and beautiful chipping, it is widely considered to be one of the best timber species for building construction, furniture making, craft carving, and ornamental purposes [33,34]. As major players in the molecular mechanisms of plant stress resistance, GRAS family members are responsible for a wide diversity of physiological procedures, covering root formation, fruit development, hormone signaling, stem cell maintenance, initiation of the axillary meristem in response to light, generation of male gametophytes, and response to adversity [17,20,[35][36][37]. Despite the fact that genome-wide investigations of GRAS genes have been completed for a number of botanical species, no such analysis has been undertaken for P. bournei, and the role of GRAS proteins in abiotic strain responses remains unclear. In this work, we described 56 GRAS genes after conducting a genome-wide analysis of this gene family in P. bournei. Quantitative RT-PCR (qRT-PCR) was employed to examine the effects of exposure to drought, salt, and temperature stressors on a sub-set of PbGRASs genes.
In this research, we utilized the chromosome-level genomic information of P. bournei to systematically identify its GRAS family genes and analyzed the physicochemical nature of proteins and the structure and chromosomal distribution of the genes and established the evolutionary relationships, promoter cis-acting elements, and epistatic expression patterns of drought, salt and temperature stress genes in an attempt to offer a frame of reference for further research on the biological functions of P. bournei GRAS genes in the process of stress resistance. This research will be fundamental in elucidating the underlying molecular mechanisms of GRAS responsiveness to drought, salt and temperature stress, and will supply quality genes for genetically engineered breeding in P. bournei for improved resistance to either drought, salinity or high and cool temperatures.

Phylogenetic Analysis of PbGRAS Gene
On the basis of the updated genome assembly, we identified 150 presumptive GRAS genes: 56 in P. bournei, 34 in A. thaliana, and 60 in O. sativa. The evolutionary interrelationships of the proteins of PbGRAS were structured using phylogenetic trees ( Figure 1). Phylogenetic analysis revealed that these 150 GRAS proteins can be separated into nine groups (PAT1, SHR, LISCL, HAM, SCL4/7, LAS, SCL3, DELLA, and SCR). These results, as with those published for birch (Betula platyphylla), Dendrobium chrysotoxum, and soybean (Glycine max), demonstrated some variability in the number of GRAS genes in different subfamilies [15,38,39]. PbGRAS proteins are unevenly distributed in nine subfamilies, most of which belong to the DELLA subfamily (18 members), followed by the PAT1 subfamily (12 members). Only seven, six, five, three, two and one PbGRAS protein were detected in the HAM, SCR, SHR, SCL3, LISCL and LAS subfamilies, respectively. Several of the PbGRAS proteins were shown to have strong phylogenetic relationships with proteins from other species (bootstrap support ≥ 80), which could indicate that they are similar to GRAS proteins from A. thaliana or O. sativa serving the same purpose.

Chromosome Distribution and Synthesis Analysis of PbGRAS Gene
A total of 56 PbGRAS proteins were retrieved from the P. bournei genome databases and were characterized. Their physico-chemical properties were investigated further with the help of ExPasy (Table 1). The majority of these proteins have classical GRAS structural domains with around 400-700 amino acids (aa), reaching up to 777, while PbGRAS5 has a severely truncated GRAS domain with only 119 amino acids. The 56 PbGRASs had molecular weights ranging from 13.682 kDa to 84.836 kDa. Theoretical pI values were between 4.86 and 6.9 for the majority of PbGRAS proteins, with only a few pI values in the range of 7 to 9. This finding suggests that the majority of GRAS proteins are negatively charged, and only a minority are positively charged. The grand average of hydropathicity index for all PbGRAS proteins (from −0.555 to −0.014) indicates that all PbGRAS proteins are hydrophilic; these results are similar to those of GRAS proteins in Betula platyphylla [15]. The majority of the forecasted instability indices were above 40 (from

Chromosome Distribution and Synthesis Analysis of PbGRAS Gene
A total of 56 PbGRAS proteins were retrieved from the P. bournei genome databases and were characterized. Their physico-chemical properties were investigated further with the help of ExPasy (Table 1). The majority of these proteins have classical GRAS structural domains with around 400-700 amino acids (aa), reaching up to 777, while PbGRAS5 has a severely truncated GRAS domain with only 119 amino acids. The 56 PbGRASs had molecular weights ranging from 13.682 kDa to 84.836 kDa. Theoretical pI values were between 4.86 and 6.9 for the majority of PbGRAS proteins, with only a few pI values in the range of 7 to 9. This finding suggests that the majority of GRAS proteins are negatively charged, and only a minority are positively charged. The grand average of hydropathicity index for all PbGRAS proteins (from −0.555 to −0.014) indicates that all PbGRAS proteins are hydrophilic; these results are similar to those of GRAS proteins in Betula platyphylla [15]. The majority of the forecasted instability indices were above 40  The 56 PbGRASs identified were further localized on 12 Phoebe bournei chromosomes (Chr01 to Chr12) ( Figure 2). Overall, the distribution of these 56 PbGRASs on the 12 chromosomes was heterogeneous. The distribution of PbGRASs on P. bournei chromosomes was variable and heterogeneous among chromosomal regions. No PbGRASs were detected on Chr06. Among the above chromosomes, the highest distribution of PbGRASs was on Chr08, with 10 PbGRASs (17.9%), followed by Chr07 (9, 16.1%), then Chr01 and Chr02 (6, 10.7%). Five PbGRASs (8.9%) were located on Chr05, four PbGRASs (7.1%) were located on Chr03, 04, and 09, three PbGRASs (5.3%) were located on Chr10 and 12, and two PbGRASs (3.6%) were located on Chr11. Based on previous studies, we hypothesized that no significant association or correlation was found between the amount of GRAS genes and chromosome length [5,40]. tected on Chr06. Among the above chromosomes, the highest distribution of PbGRASs was on Chr08, with 10 PbGRASs (17.9%), followed by Chr07 (9, 16.1%), then Chr01 and Chr02 (6, 10.7%). Five PbGRASs (8.9%) were located on Chr05, four PbGRASs (7.1%) were located on Chr03, 04, and 09, three PbGRASs (5.3%) were located on Chr10 and 12, and two PbGRASs (3.6%) were located on Chr11. Based on previous studies, we hypothesized that no significant association or correlation was found between the amount of GRAS genes and chromosome length [5,40]. Tandem duplication events are defined as chromosomal regions that contain two or more genes within 200 kb and perform a critical task in the further generation of new functions in gene families [41]. Five tandem replication events were identified on Chr07 and Chr08, including PbGRAS1/ PbGRAS47, PbGRAS3/ PbGRAS17, PbGRAS5/ PbGRAS36, PbGRAS48/ PbGRAS3, and PbGRAS53/ PbGRAS37, involving a total of nine PbGRASs (Figure 2). Except for PbGRAS1 and PbGRAS47 from the SCR subfamily, and PbGRAS5 and PbGRAS36 from the PAT1 subfamily, all genes that participate in tandem replication events belong to the DELLA subfamily, implying that the DELLA cluster, as the largest Tandem duplication events are defined as chromosomal regions that contain two or more genes within 200 kb and perform a critical task in the further generation of new functions in gene families [41]. Five tandem replication events were identified on Chr07 and Chr08, including PbGRAS1/PbGRAS47, PbGRAS3/PbGRAS17, PbGRAS5/PbGRAS36, PbGRAS48/PbGRAS3, and PbGRAS53/PbGRAS37, involving a total of nine PbGRASs ( Figure 2). Except for PbGRAS1 and PbGRAS47 from the SCR subfamily, and PbGRAS5 and PbGRAS36 from the PAT1 subfamily, all genes that participate in tandem replication events belong to the DELLA subfamily, implying that the DELLA cluster, as the largest subfamily, plays a substantial role in the growing number of GRASs [7,42]. In addition, there are six fragment replicates situated at or near the terminal end of chromosomes 1, 2, 3, 4, 5, 7 and 10. To further investigate the gene duplications, we constructed a comparative tandem map of the three PbGRAS families ( Figure 3). Among them, the intensity of association with PbGRAS genes was ranked from highest to lowest as follows: O. sativa (22), A. thaliana (18). The linear homology plots suggest that tandem replication events are the main mechanism for gene family expansion in plants, and more importantly, for gene family diversity [43,44].
tive tandem map of the three PbGRAS families ( Figure 3). Among them, the intensity of association with PbGRAS genes was ranked from highest to lowest as follows: O. sativa (22), A. thaliana (18). The linear homology plots suggest that tandem replication events are the main mechanism for gene family expansion in plants, and more importantly, for gene family diversity [43,44].

Conserved Structural Domain Motif Analysis of PbGRASs
As a further exploration of the GRAS sequence characteristics and structure of P. bournei, we optimized the conserved motifs and intronic structures of P. bournei ( Figure  4). To examine the structural specifics of the PbGRAS protein, we estimated 10 conserved motifs in the MEME program. GRAS proteins from the same subfamily contain comparable motif combinations in general, implying that GRAS proteins of the same subfamily likely have identical functions. Nearly all GRAS proteins included Motifs 1, 2, 3, 4, 5, 6, 7 and 10, demonstrating that the motifs are highly conserved and might have significant effects within the GRAS family. Motif 8 is only distributed in the PAT1 subfamily; Motif 9 is missing in the HAM subfamily except for PbGRAS15. PbGRAS5 contains only Motif 3

Conserved Structural Domain Motif Analysis of PbGRASs
As a further exploration of the GRAS sequence characteristics and structure of P. bournei, we optimized the conserved motifs and intronic structures of P. bournei ( Figure 4). To examine the structural specifics of the PbGRAS protein, we estimated 10 conserved motifs in the MEME program. GRAS proteins from the same subfamily contain comparable motif combinations in general, implying that GRAS proteins of the same subfamily likely have identical functions. Nearly all GRAS proteins included Motifs 1, 2, 3, 4, 5, 6, 7 and 10, demonstrating that the motifs are highly conserved and might have significant effects within the GRAS family. Motif 8 is only distributed in the PAT1 subfamily; Motif 9 is missing in the HAM subfamily except for PbGRAS15. PbGRAS5 contains only Motif 3 and Motif 7, PbGRAS23 contains only Motifs 2, 4, 6 and 7, and PbGRAS43 contains only Motifs 1, 4, 5 and 7. Some motifs are distributed only in specific positions of the patterns. Motif 7, for instance, is consistently dispersed near the conclusion of the sequence, but Motif 5 is usually disseminated at the beginning. The intended uses of the majority of these preserved motifs are unknown. The variances in the order of appearance of these motifs across subfamilies imply that GRAS proteins from various subdivisions may have distinct purposes. Additionally, multiple genes within the same subfamily exhibit distinct motif distributions, implying that the tasks they perform may be unique as well. The tendency for similar motifs to cluster together in specific subgroups of GRAS proteins suggests the possibility of cross-functional similarities between these proteins. In addition, to determine the differences in gene structures, we compared the exon and intron structures of 56 P. bournei GRAS genes. Most of the GRAS genes have minimal or no introns, which was also evidenced in the present study with 67.86% of PbGRAS genes having no introns ( Figure 4 and Table 1). Five and three introns were detected in PbGRAS18 and PbGRAS29, respectively. This suggests that the family was relatively conservative in evolution.

Multiple Sequence Alignment and Cis-Acting Element Analysis of PbGRASs
Multiple sequence alignment of PbGRAS proteins showed a degree of conservation in the GRAS gene family. According to the output scores of multiple sequence alignment, there is a high similarity in the 320-360 and 460-510 regions, and it can be inferred that

Multiple Sequence Alignment and Cis-Acting Element Analysis of PbGRASs
Multiple sequence alignment of PbGRAS proteins showed a degree of conservation in the GRAS gene family. According to the output scores of multiple sequence alignment, there is a high similarity in the 320-360 and 460-510 regions, and it can be inferred that the function of these regions may be similar ( Figure 5). Cis-acting components can affect the transcribed state of their linked genes since they are non-coding DNA regions in gene promoters. The occurrence of cis-acting elements in promoters might be related to the variety of gene-related processes and expression trends. We submitted base pair sequences from the 2 kb region upstream of the PbGRAS gene promoter to the PlantCARE database and found 19 cis-acting parts linked to reactions to environmental stress or phytohormone transduction, including nine stress-responsive elements and five phytohormone-related elements ( Figure 6). As demonstrated in the picture, all PbGRAS genes have between seven and 11 cis-acting elements. Each GRAS gene has at a minimum one element associated with a strain response such as light responsiveness, essential for anaerobic induction, defense and stress responsiveness, low-temperature responsiveness, drought inducibility, anoxic specific inducibility, circadian control, and wound responsiveness, having a major effect in the generation of strain responses. Meanwhile, five phytohormone-related elements in the PbGRAS genes appear in most phytohormones; these include abscisic acid responsiveness, MeJA responsiveness, gibberellin responsiveness, auxin responsiveness, and salicylic acid responsiveness. Among them, we discovered that the photosensitive component is the richest cis-acting component, as it is extensively represented in the PbGRAS promoter region. These findings suggest that PbGRASs might be tolerant to non-biological stresses, such as light, salt, cold, and drought, and may be involved in plant evolutionary and developmental processes.

Heat Map of PbGRAS Gene Expression in Different Tissues
For analyzing the variation in PbGRAS exposure modes, we incorporated gene epistasis data from five tissues: leaf, root xylem, stem xylem, root bark, and stem bark (Figure 7). The outcomes displayed that PbGRAS7 and PbGRAS14 had the highest expression in the root xylem and stem xylem, and moderate expression in the root bark and stem bark; in particular, PbGRAS7 reached maximum expression in stem xylem, which indicated that the performance of PbGRA7 expression was closely related to the cultivation of the stem xylem. In addition, PbGRAS10, PbGRAS41, and PbGRAS16 genes were also highly expressed in these four tissues, but their expression was lower in the root xylem and stem xylem compared with PbGRAS7 and PbGRAS14. Meanwhile, the expression of PbGRAS10 was comparatively higher in the root and stem bark. This is similar to the results in pepper, where members of the PAT1 subfamily, such as CaGRAS30 and CaGRAS34, are expressed at higher concentrations in the stems than in other organs [3].

Abiotic Stress Experiments on the GRAS Gene Family of P. bournei
To investigate the responses of GRAS genes to abiotic stress, we tested the expression responsiveness of P. bournei genes to drought, salt, and temperature stress (Figure 8). The relatively high expression of PbGRAS7, PbGRAS10, PbGRAS14 and PbGRAS16 in different tissues can be seen more clearly in the gene expression heat map. In addition, cis-acting element analysis showed that PbGRAS7, PbGRAS10, PbGRAS14 and PbGRAS16 contain a large number of elements associated with drought and temperature stresses. Thus, we selected these four genes to validate the results further. The findings reveal that the levels of manifestation of GRAS genes were modulated in response to dryness, salt, and temperature. The expression of PbGRAS10, PbGRAS14, and PbGRAS16 did not change significantly after 4 h of immersion with a nutrient solution containing 10% PEG, and only the expression of PbGRAS7 was enhanced and was significantly higher at 6 h, about three times that of the control (Figure 8a). PbGRAS7 may have a non-significant effect on P. bournei's osmotic stress. When treated with 10% NaCl (Figure 8b), we discovered that PbGRAS7 and PbGRAS14 genes were repressed to different degrees. The expression of PbGRAS16 increased somewhat after 4 h, while PbGRAS10 increased significantly at 4 h after treatment and increased about 30-fold after 24 h compared with the expression at the beginning of treatment. One may presume that PbGRAS7 may have a significant action in the tolerance of P. bournei to drought strain. Under the 10 • C treatment (Figure 8c), the expression of all genes increased to different degrees after 12 h. The expression of PbGRAS10 and PbGRAS16 was already increased at 4 h, and PbGRAS7, PbGRAS14, and PbGRAS16 still maintained high expression levels after 24 h. The expression of PbGRAS10 and PbGRAS16 was increased at the beginning of the treatment. Among them, PbGRAS10 expression was remarkably enhanced after 12 h and was about 25-fold compared with the control group. Under 40 • C treatment (Figure 8d), the expression of every gene was increased to various extents and remained high after 24 h. In particular, PbGRAS10 and PbGRAS16 expression increased about 5-fold after 24 h of treatment compared with the CK group. It can be postulated that PbGRAS10 and PbGRAS16 have significant effects on the plant's reactions to temperature stress.
promoters. The occurrence of cis-acting elements in promoters might be related to the variety of gene-related processes and expression trends. We submitted base pair sequences from the 2 kb region upstream of the PbGRAS gene promoter to the PlantCARE database and found 19 cis-acting parts linked to reactions to environmental stress or phytohormone transduction, including nine stress-responsive elements and five phytohormone-related elements ( Figure 6). As demonstrated in the picture, all PbGRAS genes have between seven and 11 cis-acting elements. Each GRAS gene has at a minimum one element associated with a strain response such as light responsiveness, essential for anaerobic induction, defense and stress responsiveness, low-temperature responsiveness, drought inducibility, anoxic specific inducibility, circadian control, and wound responsiveness, having a major effect in the generation of strain responses. Meanwhile, five phytohormone-related elements in the PbGRAS genes appear in most phytohormones; these include abscisic acid responsiveness, MeJA responsiveness, gibberellin responsiveness, auxin responsiveness, and salicylic acid responsiveness. Among them, we discovered that the photosensitive component is the richest cis-acting component, as it is extensively represented in the PbGRAS promoter region. These findings suggest that PbGRASs might be tolerant to nonbiological stresses, such as light, salt, cold, and drought, and may be involved in plant evolutionary and developmental processes.  In sequence alignment, amino acid alignment of A, I, L, M, F, W, V, C is shown in blue, R, K in red, N, Q, S, T in green, E and D in magenta, G in orange, H, Y in cyan, and P in yellow.

Heat Map of PbGRAS Gene Expression in Different Tissues
For analyzing the variation in PbGRAS exposure modes, we incorporated gene epistasis data from five tissues: leaf, root xylem, stem xylem, root bark, and stem bark ( Figure  7). The outcomes displayed that PbGRAS7 and PbGRAS14 had the highest expression in the root xylem and stem xylem, and moderate expression in the root bark and stem bark; in particular, PbGRAS7 reached maximum expression in stem xylem, which indicated that the performance of PbGRA7 expression was closely related to the cultivation of the stem xylem. In addition, PbGRAS10, PbGRAS41, and PbGRAS16 genes were also highly expressed in these four tissues, but their expression was lower in the root xylem and stem xylem compared with PbGRAS7 and PbGRAS14. Meanwhile, the expression of PbGRAS10 was comparatively higher in the root and stem bark. This is similar to the results in pepper, where members of the PAT1 subfamily, such as CaGRAS30 and CaGRAS34, are expressed at higher concentrations in the stems than in other organs [3].

Abiotic Stress Experiments on the GRAS Gene Family of P. bournei
To investigate the responses of GRAS genes to abiotic stress, we tested the expression responsiveness of P. bournei genes to drought, salt, and temperature stress (Figure 8). The relatively high expression of PbGRAS7, PbGRAS10, PbGRAS14 and PbGRAS16 in different tissues can be seen more clearly in the gene expression heat map. In addition, cis-acting element analysis showed that PbGRAS7, PbGRAS10, PbGRAS14 and PbGRAS16 contain a large number of elements associated with drought and temperature stresses. Thus, we expression was remarkably enhanced after 12 h and was about 25-fold compared with the control group. Under 40°C treatment (Figure 8d), the expression of every gene was increased to various extents and remained high after 24 h. In particular, PbGRAS10 and PbGRAS16 expression increased about 5-fold after 24 h of treatment compared with the CK group. It can be postulated that PbGRAS10 and PbGRAS16 have significant effects on the plant's reactions to temperature stress.

Discussion
Plants primarily use gene regulation as an adaptive mechanism in response to environmental stressors [45]. The growth, differentiation, and stress responses of tissues and organs are tightly controlled by GRAS proteins [46]. The subtropical broadleaf evergreen tree P. bournei is extremely important, both economically and ecologically. Water scarcity and temperature variation severely affect P. bournei growth and wood accumulation. Jin and others found that P. bournei can regulate its own protective enzyme system and osmotic substances to adapt to drought under drought stress [47]. Wang showed that polyamines can increase soluble sugar and chlorophyll content under drought and improve the drought resistance of P. bournei [48]. However, there is a dearth of literature on the regulatory signaling pathways that help P. bournei to endure abiotic challenges such as drought. As GRAS genes have such a critical function in the signaling of P. bournei, it is crucial to comprehend their expression pattern. The correct identification of abiotic tolerance genes in P. bournei will be aided by a fundamental and thorough bioinformatic investigation of them.
Many plant species have been found to contain GRAS family genes, with 34 GRAS gene members in A. thaliana [17], 60 in O. sativa (Oryza sativa) [11], and 153 in wheat (Triticum aestivum) [49]. We undertook a genome-wide investigation of the GRAS gene family in P. bournei and identified 56 GRAS proteins, the majority of which have characteristic GRAS structural domains totaling roughly 360 aa, most of which share a conserved C-terminus (Table 1 and Figure 5), which is consistent with the study by Liu and Wang [50]. Most of the conserved sequences appeared in the 270-610 region. Given that intron loss in eukaryotes is massive due to selection pressure and that the GRAS group is a retrotransposon group that accelerates the generation of intron-less genes from bacterial to eukaryotic gene sets through both horizontal gene transfer and gene dimerization [51][52][53], it is possible that large and frequent gene duplication events have led to changes in the number of PbGRAS genes (38 out of 56, Table 1). This investigation found five simultaneous duplication events including a total of nine PbGRASs, and the majority of the genes implicated in these events belonged to the DELLA subfamily ( Figure 2). However, some PbGRASs evolved different exon-intron outcomes, which may indicate that they evolved adaptive functions in order to adapt to their environment. Moreover, most GRAS proteins in Table 1 have pI values located at around 5~7, with a smaller possibility of interacting nonspecifically with acidic proteins [54], and the GRAS set may be engaged in protein-protein interfaces [55]. The findings of chromosomal localization reveal that the 56 PbGRASs identified were unevenly located on 12 chromosomes of P. bournei (Figure 2), with the highest distribution being on Chr08, accounting for 17.9% of the PbGRASs, while no PbGRASs were detected on Chr06. These outcomes are analogous to other research, such as where no MdGRAS geneswere found on chromosome 16 of Malus domestica, while chromosome 11 contained the most, accounting for 15.75% of the MdGRAS genes [56]. It is possible to attribute this to evolutionary processes such as gene duplication and chromosomal transfer. Chr08 has the largest amount of GRAS genes and may be one of the critical chromosomes for plants to deal with abiotic pressures. The addition of GRAS genes from A. thaliana and O. sativa expanded the dataset to 150 GRAS proteins divided into nine subfamilies after phylogenetic analysis (Figure 1). However, the conserved motifs in different subfamilies are different, which may be due to the subfamilies of PbGRASs performing different biological functions in the growth of P. bournei, as the motif arrangement was mostly similar among PbGRAS proteins of the same subfamily, suggesting that PbGRAS proteins of the same subfamily may have similar functions. As shown by To and Wang et al., GRAS proteins are stochastically assigned in the phylogenetic tree [7,57], but individuals from the same subfamily tend to perform comparable tasks [50].
Characterization of cis-acting elements demonstrated that PbGRAS genes may participate in multiple biotic procedures by regulating a variety of target genes associated with hormone induction, evolution, metabolism, and abiotic stresses ( Figure 6). Among these cis-acting elements, those correlated to drought and temperature stress such as drought inducibility and low-temperature responsiveness are assigned to the promoter regions of PbGRAS genes, such as PbGRAS10 and PbGRAS14. There are many elements in PbGRAS14 that react to drought stress and salt stress, but fewer light response elements compared with other PbGRAS genes, which indicates that this gene may be less involved in light response and is the main gene used to cope with salt and drought stress. By comparing the other genes of the DELLA subfamily, there were relatively few cis-acting elements for light response, but more cis-acting elements for stress response, which indicated that the DELLA gene family may play a role in the stress response of P. bournei. Then, focusing on the stress experiment (Figure 8), it could be seen that the expression of PbGRAS14 changed significantly under NaCl and 40 • C stress, which is also consistent with our analysis results. A similar example is the case for CmGRAS genes, which are more closely related to the reference species in Chinese chestnut and were found to have more cis-elements and showed higher expression modes at different phases of buds and fertile or aborted ovules, suggesting their significant roles in the evolution and functioning of the CmGRAS gene family [14]. Additionally, the results can be seen more clearly in the expression heat map of the genes; PbGRAS7 and PbGRAS14 are highly expressed in the root xylem and stem xylem, while PbGRAS10 is relatively highly expressed in the root bark and stem bark. This likely relates to the fact that GRAS genes display a differential expression pattern across different tissues [13]. Consistent with some other observations, such as the expression of NnuGRAS-05, NnuGRAS-10, and NnuGRAS-25 in lotus root bark compared to their expression in the leaf and petiole, members of the PAT1 subfamily seem to be more highly expressed in the root than in the leaf [58]. The expression levels of CaGRAS30 and CaGRAS34 in pepper were higher in stems than in other organs [3]. Almost all PAT1 subfamily members were highly expressed in eggplant stems [25]. This may be due to the fact that PAT1 subfamily members help to maintain rootstock growth and resist environmental disturbances.
Combining these findings and analysis, we selected four genes (PbGRAS7, PbGRAS10, PbGRAS14, and PbGRAS16) for validation of the stress treatments. By subjecting abiotic stress-treated P. bournei seedlings to qRT-PCR analysis, it was found that drought, salt and temperature stress conditions significantly induced the expression of the four PbGRASs in P. bournei plants (Figure 8). In particular, the expression of PbGRAS10 significantly surged 30-fold and 25-fold after salt treatment and low-temperature treatment, respectively, PbGRAS7 was expressed at a three times higher level than in the control after PEG treatment, and PbGRAS10 and PbGRAS16 increased approximately five-fold after high-temperature treatment compared with the control, which was consistent with the outcomes of cisacting meta-analyses ( Figure 6). In contrast, a low-temperature-responsive cis-element was predicted at the PbGRAS14 promoter, but its expression level was comparable to the control condition. This, to some extent, reflects the fact that the accuracy of the cis-acting element prediction results needs to be further improved. Drought, salt, and temperature stress conditions significantly induced the expression of PbGRASs in P. bournei plants, suggesting that PbGRASs may have some degree of association with stress response. There have been other studies describing similar findings. In O. sativa (Oryza sativa), OsGRAS23 in the SCL subfamily is highly sensitive to salinity stress, and the expression of this gene is up-regulated under drought stress [26,59]. Similar results were found in Halostachys caspica, where the expression of HcSCL13 in the SCL subfamily was up-regulated under drought and salt treatments [30]. Salt stress in poplar (Populus euphratica) also regulates a stress response transcription factor, PeSCL7 [60]. This provides more evidence that the SCL subfamily is crucial for adapting to adverse conditions. The GRAS family of grapevine is also regulated by abiotic stresses, for example, in that drought treatment upregulates the expression of VviPAT3, VviPAT4, VviPAT6 and VViPAT7 in seedlings [61], while salt treatment has an effect on VviRGA5 in the DELLA subfamily [62]. Furthermore, the Lycopersicon esculentum gene SlGRAS2, which is homologous to VviPAT4, is highly sensitive to salt stress [51]. According to a recent report, GRAS genes play a key role in the regulation of salt stress [63], and thus, the role of GRAS in promoting plant growth and salt stress response has received widespread attention. However, further in-depth research is required to understand the function of GRAS genes in response to abiotic stressors in a wider range of plant species.
This study not only determined and examined the expression of PbGRASs using qRT-PCR, but it also systematically analyzed the GRAS gene family of P. bournei, suggesting that PbGRASs play an active part in the physiology of P. bournei's resistance to drought, salt, high, and low temperature stresses. This study provides a basis for elucidating the molecular mechanisms of the P. bournei GRAS response to drought, salt, and high and low temperature stresses and posits ideas for the future improvement of P. bournei using genetic engineering methods.

Genome Data and Plant Material Source
The genome sequence data and annotation information of P. bournei were downloaded from the Sequence Archive of China National GeneBank Database (CNSA) with accession number CNP0002030 [64]. Genome sequence files of A. thaliana (version: Ara- with accession number PRJNA628065. The plant materials were derived from 1-year-old P. bournei seedlings cultured in an artificial climate box under different treatments. P. bournei seedlings with the same growth potential for 1 year were selected for treatment, and the materials were divided into the control group and stress treatment group, with 30 strains in the treatment group and 3 strains in the control group. Every 2 strains in the treatment group were used as a biological replicate, and 3 groups of biological replicates were set in each time period. After various treatments, P. bournei leaf samples were collected and stored in liquid nitrogen at −80 • C for RNA extraction.

Identification and Physical and Chemical Property Analysis
The conserved domain of the A. thaliana GRAS gene family was downloaded from PlantTFDB (http://planttfdb.gao-lab.org/, accessed on 31 October 2022). A local BLASTp search was used to compare the conserved domain between P. bournei and A. thaliana to screen the candidate GRAS genes in P. bournei [39]. The repetitive results of the BLASTp search were removed. The identified protein sequences of GRAS genes from P. bournei were submitted to the NCBI for BLASTp to perform further searching. To further identify GRAS gene family members, the GRAS conserved domain HMM model (PF03514) was downloaded from the Pfam database (http://pfam.xfam.org/, accessed on 14 April 2023), using HMMER-3.2.1 (http://hmmer.org/download.html, accessed on 14 April 2023) with an e-value <10 -5 and other parameters by default. After the identification of GRAS genes in P. bournei, the online website ExPASy (https://web.expasy.org/prot-param/, accessed on 26 February 2023) was used to analyze the physical and chemical properties of the GRAS proteins that were identified [65].

Chromosomal Distribution and Gene Duplication of PbGRAS Genes
TBtools was used for grepping the location information of the PbGRAS genes from the genome (FASTA) file and the annotation (GFF) file of P. bournei [66]. The syntenic relationships of PbGRAS were determined using MCScanX with default parameters and plotted using Tbtools [67].

Collinearity Analysis of PbGRAS Genes
The syntenic relationships between PbGRAS genes and GRAS genes from A. thaliana and O. sativa were determined by using MCScanX software. Tbtools was used for visualization.

Phylogenetic Analysis
The sequences of GRAS proteins of P. bournei, A. thaliana and O. sativa were aligned using the Muscle program of MEGA11 with default settings for constructing maximum likelihood (bootstrap replications: 1000) phylogenetic tree [68,69]. iTOL (https://itol.embl. de/, accessed on 29 January 2023) was used to improve and beautify the phylogenetic tree.

Analysis of Conserved Motifs and Gene Structures
The protein sequence of P. bournei was identified using the online software MEME (http://meme-suite.org/, accessed on 8 March 2023), and the predicted value of the motif number was 10. The multiple GRAS protein sequences alignment was carried out using Jalview software (https://www.jalview.org/, accessed on 30 March 2023). We used the Batch CD-search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 8 March 2023) with default parameters to detect the conserved domains of the PbGRAS protein.

Multiple Sequence Alignment and Promoter Cis-Element Analysis of PbGRAS Genes
Multiple sequence alignment of PbGRAS was performed using Jalview software (version: 2.11.2.6). To explore the cis-acting elements in the sequence, we extracted the upstream 2000 bp sequences from the P. bournei genome. The online software PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 11 March 2023) was used to analyze the cis-acting regulatory elements in the promoter region of the PbGRAS genes. After selection and categorization, the data were visualized by means of TBtools software.

Treatment in Different Tissue
The fragments per kilobase of transcript per million fragments mapped (FPKM) transcriptomic data from five different tissues (leaf, root xylem, stem xylem, root bark and stem bark) were used to construct a expression profiling with TBtools.
Based on results of cis-element analysis and expression profiling of PbGRAS, we conducted different treatment. In the drought and salt stress experiment, 3 seedlings in the control group were soaked in distilled water after root washing, while those in the treatment group were soaked in a nutrient solution containing 10% PEG and 10% NaCl, respectively, and cultured in an artificial climate incubator with a temperature of 25 • C and humidity of 75%. The treatment group was sampled at 4 h, 6 h, 8 h, 12 h and 24 h, respectively, and the control group was sampled at 0h. In the temperature treatment (10 • C and 40 • C), the control group was incubated at room temperature, and the treatment group was incubated at 10 • C and 40 • C. The treatment group was sampled at 4 h, 6 h, 8 h, 12 h and 24 h, with 6 strains in each time period. The 40 • C and 10 • C control groups were sampled at 0 h.

qRT-PCR Analysis
An RNA extraction kit (HiPure Plant RNA Mini Kit from Magen, Shanghai, China) was used to extract RNA, while cDNA was synthesized using the Prime Script RT reagent Kit (Perfect Real Time from Takara, Dalian, China). Primer 3.0 software was used to design specific primers in the non-conserved region of the target gene, which were synthesized by Fuzhou Qingbaiwang Biotechnology Company. Real-time fluorescence quantitative analysis was used with cDNA template (1 µL), cDNA template SYBR Premix Ex TaqTM II (10 µL), specific primers (2 µL), and ddH 2 O reaction program (7 µL): 95 • C for 30 s; 95 • C for 5 s; 60 • C for 30 s; 95 • C for 5 s; 60 • C for 60 s; and 30 s at 50 • C, with 40 cycles in total. The internal reference gene was PbEF1α (GenBank No. KX682032) [70]. The expression level of the target gene was calculated using the 2−∆∆Ct method, and the quantitative data were analyzed via t test using SPSS26 software. Finally, GraphPad Prism 9.0 was used to construct the graphs.

Conclusions
In this study, a total of 56 PbGRAS proteins were identified from the P. bournei genome and phylogenetically classified into nine subfamilies. Four GRAS genes, PbGRAS7, Pb-GRAS10, PbGRAS14 and PbGRAS16, were induced by stress treatments such as drought, salt, and low and high temperature. The expression of each of the four genes varied under different treatments. These results suggest that PbGRASs may play a role in improving the tolerance of transgenic P. bournei to drought, salt, and high-and low-temperature stresses. This study lays the foundation for the further elucidation of the functions of PbGRAS family members, provides valuable information for further studies on the role of GRAS family genes in the development of abiotic stress resistance in P. bournei, and provides a basis for the improvement of P. bournei. Funding: This research was jointly funded by Fujian Agriculture and Forestry University Forestry peak discipline construction project (71201800739 to Shijiang Cao) and the 7th Project of Forest Seeding Breaking in Fujian Province (LZKG-202208 to Shipin Chen).
Data Availability Statement: Publicly available datasets were analyzed in this study. This data can be found here: The P. bournei genome data presented in this study are openly available in CNSA at https://db.cngb.org/search/project/CNP0002030/, accessed on 14