Genome-Wide Identification of Genes Encoding for Rho-Related Proteins in ‘Duli’ Pear (Pyrus betulifolia Bunge) and Their Expression Analysis in Response to Abiotic Stress

Twelve Rho-related proteins (ROPs), namely PbROPs, were identified from the genome of the recently sequenced ‘Duli’ pear (Pyrus betulifolia Bunge), a wild-type pear variety routinely used for rootstocks in grafting in China. The length and molecular weight of these proteins are between 175 and 215 amino acids and 19.46 and 23.45 kDa, respectively. The 12 PbROPs are distributed on 8 of the 17 chromosomes, where chromosome 15 has the highest number of 3 PbROPs. Analysis of the deduced protein sequences showed that they are relatively conserved and all have the G domain, insertion sequence, and HVR motif. The expression profiles were monitored by quantitative RT-PCR, which showed that these 12 PbROP genes were ubiquitously expressed, indicating their involvement in growth and development throughout the life cycle of ‘Duli’ pear. However, they were altered upon treatments with abscisic acid (ABA, mimicking abiotic stress), polyethylene glycol (PEG, mimicking drought), and sodium chloride (NaCl, mimicking salt) to tissue-cultured seedlings. Further, transgenic Arabidopsis expressing PbROP1, PbROP2, and PbROP9 exhibited enhanced sensitivity to ABA, demonstrating that these 3 PbROPs may play important roles in the abiotic stress of ‘Duli’ pear. The combined results showed that the ‘Duli’ genome encodes 12 typical ROPs and they appeared to play important roles in growth, development, and abiotic stress. These preliminary data may guide future research into the molecular mechanisms of these 12 PbROPs and their utility in molecular breeding for abiotic stress-resistant ‘Duli’ pear rootstocks.


The 12 PbROPs of 'Duli' Pear Are Typical ROPs of Plants
Multiple sequence alignment was performed to identify the conserved domains of the 12 newly identified 'Duli' pear PbROPs. Similar to Arabidopsis and other plant ROPs, 3 typical conserved functional domains were identified [10,11] from the deduced protein sequences of all 12 PbROPs, i.e., the G domain, insertion sequence, and HVR. The G1-G5 regions of the G domains for GTP binding and hydrolysis were also present ( Figure 3). Therefore, the 12 PbROPs of 'Duli' pear are typical ROPs of plants based on their sequences.

The 12 PbROPs of 'Duli' Pear Are Typical ROPs of Plants
Multiple sequence alignment was performed to identify the conserved domains of the 12 newly identified 'Duli' pear PbROPs. Similar to Arabidopsis and other plant ROPs, 3 typical conserved functional domains were identified [10,11] from the deduced protein sequences of all 12 PbROPs, i.e., the G domain, insertion sequence, and HVR. The G1-G5 regions of the G domains for GTP binding and hydrolysis were also present ( Figure 3). Therefore, the 12 PbROPs of 'Duli' pear are typical ROPs of plants based on their sequences. Multiple sequence alignment was performed to identify the conserved domains of the 12 newly identified 'Duli' pear PbROPs. Similar to Arabidopsis and other plant ROPs, 3 typical conserved functional domains were identified [10,11] from the deduced protein sequences of all 12 PbROPs, i.e., the G domain, insertion sequence, and HVR. The G1-G5 regions of the G domains for GTP binding and hydrolysis were also present ( Figure 3). Therefore, the 12 PbROPs of 'Duli' pear are typical ROPs of plants based on their sequences.   PbROPs are predicted to have 7 to 8 exons (Figure 4b). Consistent with the number of exons of the type I and IV ROPs in Arabidopsis, PbROP1-6, PbROP8, and PbROP12 also have 7 exons, while PbROP7 and PbROP9-11 in type III have 8 exons, which are the same as those in Type III AtROPs of Arabidopsis. PbROPs are predicted to have 7 to 8 exons (Figure 4b). Consistent with the number of exons of the type I and IV ROPs in Arabidopsis, PbROP1-6, PbROP8, and PbROP12 also have 7 exons, while PbROP7 and PbROP9-11 in type III have 8 exons, which are the same as those in Type III AtROPs of Arabidopsis.
Seven conserved motifs were identified from the PbROPs (Figure 4c and Table S1). Motifs 1-6 were found in all the 12 PbROPs, except PbROP4, which did not have Motif 3, while motif 7 was only found in PbROP10 and PbROP11. The conserved GTP-binding domain fell in motifs 1, 2, 3, and 5.  Table S1 in Supplementary Materials. The number of amino acids in each motif can be estimated using the scale below the figure.

PbROPs Have Gone through Purifying Selection
The selection pressure of PbROPs was estimated (Table S2). This showed that the ratios of Ka (synonymous mutations) / Ks (not synonymous mutations) of all PbROPs were less than 1, indicating that the PbROP family genes have been undergoing purifying selection [26].
The collinearity analysis conducted for gene fragment replication relationships put 10 of the 12 PbROPs into 5 pairs, i.e., PbROP10/PbROP11, PbROP8/PbROP12, PbROP3/PbROP5, PbROP2/PbROP1, and PbROP2/ PbROP4 (Figure 5a). High sequence  Table S1 in Supplementary Materials. The number of amino acids in each motif can be estimated using the scale below the figure.
Seven conserved motifs were identified from the PbROPs (Figure 4c and Table S1). Motifs 1-6 were found in all the 12 PbROPs, except PbROP4, which did not have Motif 3, while motif 7 was only found in PbROP10 and PbROP11. The conserved GTP-binding domain fell in motifs 1, 2, 3, and 5.

PbROPs Have Gone through Purifying Selection
The selection pressure of PbROPs was estimated (Table S2). This showed that the ratios of Ka (synonymous mutations) / Ks (not synonymous mutations) of all PbROPs were less than 1, indicating that the PbROP family genes have been undergoing purifying selection [26].
The collinearity analysis conducted for gene fragment replication relationships put 10 of the 12 PbROPs into 5 pairs, i.e., PbROP10/PbROP11, PbROP8/PbROP12, PbROP3/ PbROP5, PbROP2/PbROP1, and PbROP2/ PbROP4 (Figure 5a). High sequence similarity was found between each pair of these PbROPs, indicating that the two PbROPs in a pair is conserved and may have similar functions during evolution.  To further understand the evolutionary relationship of the PbROP genes with those from Arabidopsis, syntenic and collinear regions of their ROP genes were analyzed by the Multiple Collinearity Scan toolkit. This resulted in the identification of many syntenic blocks (McScanX; Figure 5b). In total, 19 gene pairs were detected, indicating that the ROPs are evolutionarily conserved between 'Duli' pear and Arabidopsis (Table S4). To further understand the evolutionary relationship of the PbROP genes with those from Arabidopsis, syntenic and collinear regions of their ROP genes were analyzed by  Figure 5b). In total, 19 gene pairs were detected, indicating that the ROPs are evolutionarily conserved between 'Duli' pear and Arabidopsis (Table S4).

Cis-Acting Elements Related to Growth and Development, and Hormonal and Stress Response Are Present in the Promoter Regions of PbROPs
The cis-acting elements within the promoter region are vital for the transcription and expression of genes, hence their functions [27]. Therefore, the~2000 bp sequence upstream of transcriptional start codon ATG of all PbROPs was analyzed and the results are shown in Figure 6. In general, the cis-acting elements could be grouped into three types where type I is involved in growth and development, type II is involved in the hormonal response, and type III is involved in the stress response [28]. Within type I, there are 12 light response elements including 3 AF1 and 3 AF3 binding sites, MRE, G-box, lamp element, GT1-motif, GATA-motif, AE-box, BOX4, chs-CMA1a, I-box, and TCT-motif. Other type I cis-acting elements were also found, such as the GCN-motif (regulatory element required for endosperm expression), O 2 -site (cis-acting regulatory element involved in zein metabolism regulation), MBSI (MYB binding site involved in flavonoid biosynthetic genes regulation), HD-zip3 (leaf development correlated), MSA-like (involved in cell cycle regulation), CAT-box (involved in endosperm development), circadian (involved in meristem expression), and NON-box (meristem-specific activation). Within type II, the hormone response cis-acting elements, ABRE (ABA-responsive elements), CGTCAmotif (MEJA-responsive elements), ERE (ethylene-responsive elements), and TCA-element (salicylic acid-responsive elements), as well as TGA-elements, AuxRE related to the IAA response and gibberellin-responsive elements, TATC-box, P-box, and GARE-motif, were all present. Type III, the stress-responsive cis-acting elements, included ARE (involved in anaerobic induction), DRE (involved in dehydration, low-temperature, and high-salinity stresses), STRE (involved in stress), TC-rich repeats (involved in defense and stress), LTR (low temperature), WUN-motif (involved in wound), GC-motif (involved in anoxic specific inducibility), and MBS (involved in drought). ARE and STRE are shared by all PbROPs. Interestingly, PbROP1 has 7 ARE elements, indicating that it may play a significant role in anaerobic induction.

Tissue-Specific Expression Pattern of PbROP Genes
In order to understand the biological functions of PbROPs, qRT-PCR was carried out to determine the temporal and spatial expression patterns of PbROP genes. The results in Figure 7 show that the expression levels of the type I PbROPs, PbROP1-6, were much

Tissue-Specific Expression Pattern of PbROP Genes
In order to understand the biological functions of PbROPs, qRT-PCR was carried out to determine the temporal and spatial expression patterns of PbROP genes. The results in Figure 7 show that the expression levels of the type I PbROPs, PbROP1-6, were much higher in leaves and roots than other tissues, with the lowest levels found in fruits, indicating that these PbROPs play major roles in the growth and development of vegetative tissues. The expression levels of the PbROP8 and PbROP12, the two type III PbROPs, were similar where they were much higher in roots than other tissues, implying that they are mainly involved in the regulation of root growth and development. The type IV PbROPs have a more complicated expression pattern where the highest expression levels of PbROP10 and 11 were found in roots, and those of PbROP7 and PbROP9 were found in leaves and fruits, respectively.

Expression Levels of PbROPs were Elevated upon Stress
Tissue-cultured seedlings of 'Duli' pear were treated with ABA, PEG, and NaCl to mimic overall abiotic stress, drought, and salt stress, respectively. The transcript level of each PbROP was quantified by real-time RT-PCR using total RNAs isolated from the treated seedlings collected after various time points. This showed that all the 12 PbROPs were upregulated to different degrees when treated with 1 µ M ABA where the highest expression levels of 7 PbROPs (PbROP2, 3, 4, 7, 8, 9, and 12) were reached at 72 h, while those of PbROP1 and 10 were at 120 h ( Figure 8). It is noteworthy that the expression level of PbROP12 increased 14-fold after 72 h compared to 0 h. Upon treatment with 150 mM of NaCl, the transcript levels of PbROPs first increased and then decreased, where 7

Expression Levels of PbROPs Were Elevated upon Stress
Tissue-cultured seedlings of 'Duli' pear were treated with ABA, PEG, and NaCl to mimic overall abiotic stress, drought, and salt stress, respectively. The transcript level of each PbROP was quantified by real-time RT-PCR using total RNAs isolated from the treated seedlings collected after various time points. This showed that all the 12 PbROPs were upregulated to different degrees when treated with 1 µM ABA where the highest expression levels of 7 PbROPs (PbROP2, 3, 4, 7, 8, 9, and 12) were reached at 72 h, while those of PbROP1 and 10 were at 120 h ( Figure 8). It is noteworthy that the expression level of PbROP12 increased 14-fold after 72 h compared to 0 h. Upon treatment with 150 mM of NaCl, the transcript levels of PbROPs first increased and then decreased, where 7 PbROPs (PbROP1, 2, 3, 4, 6, 10, and 11) reached the highest at 24 h, whilst those of PbROP7, 8, 9, and 12 and PbROP5 were at 72 h and 12 h, respectively (Figure 8). Similarly, the expression levels of PbROPs in PEG-treated seedlings first increased and then decreased, where PbROP3 and PbROP6 showed the highest expression levels at 3 h; PbROP8, 10, and 11 at 6 h; PbROP1, 2, 4, 7, and 9 at 12 h; and PbROP5 and 12 at 24 h (Figure 8). Seeds of the four individual PbROPs overexpressing homozygous transgenic Arabidopsis plants were germinated on ½ MS with or without 0.5 μM ABA. After 10 days, the percentage of seedlings with green cotyledons were calculated. As shown in Figure 9,

PbROP1, 2, 3, and 9 Overexpressing Transgenic Arabidopsis Seedlings Have Different Sensitivity to ABA
Seeds of the four individual PbROPs overexpressing homozygous transgenic Arabidopsis plants were germinated on 1 /2 MS with or without 0.5 µM ABA. After 10 days, the percentage of seedlings with green cotyledons were calculated. As shown in Figure 9, while the number of seedlings with green cotyledons were 56.7% in WT, this was much reduced in the PbROP1, 2, and 9 transgenic Arabidopsis seedlings where the percentages of seedlings with green cotyledons of the 3 lines of PbROP1 over-expressers were 22.7%, 22.7%, and 22.0%, those of the three PbROP2 transgenic lines were 29.8%, 25.5%, and 19.2%, while only 16.3%, 15.6%, and 17.0% of seedlings with green cotyledons were found in the 3 lines of PbROP11 transgenic seedlings. However, 0.5 µM ABA had little effect on the percentage of seedlings with green cotyledons of the transgenic Arabidopsis seedlings expressing PbROP3, where 58.2%, 57.5%, and 56.7% of green seedlings were recovered, which were very similar to the ABA-treated WT seedlings. Therefore, the fact that PbROP1, 2, and 9 transgenic lines are more sensitive to ABA than PbROP3 transgenics suggests that PbROP1, 2, and 9 may play specific roles in ABA signal regulation. effect on the percentage of seedlings with green cotyledons of the transgenic Arabidopsis seedlings expressing PbROP3, where 58.2%, 57.5%, and 56.7% of green seedlings were recovered, which were very similar to the ABA-treated WT seedlings. Therefore, the fact that PbROP1, 2, and 9 transgenic lines are more sensitive to ABA than PbROP3 transgenics suggests that PbROP1, 2, and 9 may play specific roles in ABA signal regulation. software. ** indicate significant differences in comparison with WT at p < 0.01. ns, no significant difference.

Discussion
Because of its resistance to drought, flood, salinity, and diseases, 'Duli' pear (Pyrus betulifolia Bunge) is used as the main rootstock for pear planting in north China [29]. However, the 'Duli' pear plant is naturally tall with well-developed taproot roots, and fewer and weaker lateral roots in the seedling stage. It is also difficult in rooting from cutting. However, as desired rootstocks, dwarf and easy rooting are the required characteristics. Previous studies of ROPs found that they play important roles in root hair development, plant growth, and stress tolerance [3,7,30]. Therefore, research on ROPs of Figure 9. PbROP1, 2, 3, and 9 overexpressing transgenic Arabidopsis seedlings have different sensitivity to ABA. (a) The phenotype of the 35S::PbROPs Arabidopsis seedlings grown on plates without or with 0.5 µM ABA; (b) percentage of seedlings with green cotyledons of the 35S::PbROPs transgenic Arabidopsis seedlings grown on plates without or with 0.5 µM of ABA. Seeds of homozygous transgenic Arabidopsis were surface-sterilized and germinated on 1 /2 MS with or without 0.5 µM ABA. After 10 days, the phenotype was observed and the percentage of seedlings with green cotyledons was calculated. The values represent the means ± SE (n = 3). The data were analyzed via the Student's t-test using GraphPad Prism (8.0.2, Harvey Motulsky, California, USA) software. ** indicate significant differences in comparison with WT at p < 0.01. ns, no significant difference.

Discussion
Because of its resistance to drought, flood, salinity, and diseases, 'Duli' pear (Pyrus betulifolia Bunge) is used as the main rootstock for pear planting in north China [29]. However, the 'Duli' pear plant is naturally tall with well-developed taproot roots, and fewer and weaker lateral roots in the seedling stage. It is also difficult in rooting from cutting. However, as desired rootstocks, dwarf and easy rooting are the required characteristics. Previous studies of ROPs found that they play important roles in root hair development, plant growth, and stress tolerance [3,7,30]. Therefore, research on ROPs of 'Duli' pear is of great significance not only in revealing their roles in the development and stress resistance mechanism of woody plants, but also providing information on candidate genes to use in molecular breeding for improved rootstock for the pear industry.
The analysis of gene structure, phylogeny, and amino acid sequence similarity showed that the members of the ROP family were much conserved in structure and amino acid sequence (Figures 3 and 4A). According to the amino acid sequence similarity and the structure of the highly variable C-terminal regions, the PbROPs were divided into three types rather than four in Arabidopsis because type II, i.e., the AtROP7 homologous gene, is missing from 'Duli' pear ( Figure 1). A study in Arabidopsis showed that AtROP7 and AtROP2 are functionally redundant; together, they play negative roles in controlling lightinduced stomatal opening [31]. However, how and what the implication is for the 'Duli' pear genome to lack the type II ROP remains to be determined.
Many studies of plant ROPs showed that they are involved in plant growth and development as well as the stress response. For example, AtROP2 [32] and MaROP5 [7] participate in the response to salt stress, and EjROPs are related to cold stress [18]. To see if PbROPs play similar roles, we first analyzed the promoter regions of the 12 PbROPs. This revealed that the cis-acting elements related to growth and development, and the phytohormone and stress response are all present ( Figure 6). This indicates that the 12 PbROPs may also function in growth, development, and abiotic stress in 'Duli' pear. For example, the promoter regions of all 12 PbROPs contain the stress-responsive elements ARE and STRE. It is interesting that PbROP3 also contains six lower-temperature-responding (LTR) elements, indicating it may play a significant role in the regulation of low-temperature stress. We also identified cis-acting elements related to ABA, ethylene, IAA, GA, MeJA, and SA signaling, indicating that PbROPs may also be involved in many hormone signal regulation processes during growth and development of the 'Duli' pear plant.
Further analysis of the expression profiles of the PbROPs in different tissues of mature plants as well as in tissue-cultured seedlings treated with ABA, NaCl, and PEG provided more evidence to support the bioinformatic data in general. For example, we found that type I and IV PbROPs were expressed in roots to high levels, indicating that these two types of PbROPs may play an important role in root growth. Similar findings were also made with the AtROPs in Arabidopsis [33][34][35]. As a stress hormone, ABA plays crucial roles in plant responses to abiotic stress [17]. Transgenic Arabidopsis plants expressing AtROP2 and AtROP11 exhibited altered ABA responses [3,17]. Our study also showed that the expression levels of the majority of PbROPs were increased in 'Duli' pear seedlings treated with 1 µM of ABA ( Figure 8). Further, transgenic Arabidopsis expressing PbROP1, 2, or 9 became super sensitive to ABA because the survival rate of seedlings (with green cotyledons) was reduced from 56.7% for WT to 22.5%, 24.8%, and 16.3% for 35S:PbROP1, 35S:PbROP2, and 35S:PbROP9 transgenic seedlings, respectively, while that of PbROP3 expressing Arabidopsis was very similar to the WT (Figure 9). It is worth noting that one common feature shared by these four PbROPs is that they are all predicted to be S-acylated at Cys9. Given the important roles of S-acylation in membrane targeting, which is directly related to the functionality of ROPs, this warrants further investigation in order to understand if/how S-acylation of these four PbROPs occurs in ABA signaling and stress of 'Duli' pear [21].
When 'Duli' seedlings were treated with NaCl and PEG, the expression levels of PbROPs were increased and then decreased, indicating that these genes may participate in the initial stage of response to these stress conditions. In tobacco, the expression level of NtROP1 was increased upon treatment with NaCl. When NtROP1 was expressed in Arabidopsis, the transgenic plants showed increased salt sensitivity [36]. A study in Arabidopsis showed that the involvement of AtROP2 in the salt response is mediated by its involvement of the reassembly of microtubules where the constitutively active ROP2 promoted the reassembly of microtubules and the survival of seedlings, while the rop2-1 mutant showed a significantly low survival rate under salt stress [32].
The drought signal can excite the plant response to ABA, BR, ethylene, and ROP proteins [37,38]. Arabidopsis AtROP11 exerts its role in drought stress by playing a negative regulation role in the closure of stomata induced by ABA [17]. HDA15 (Histone modifier 15) loss-of-function mutant hap15 is sensitive to drought. This is because the MYB96-HDA15 complex binds to the promoters of a subset of ROPs, including ROP6, ROP10, and ROP11, to regulate their expression [39]. The fact that ABA can enhance the expression of PbROPs in 'Duli' pear seedlings and reduce the survival rates of transgenic Arabidopsis seedlings overexpressing three of the PbROPs indicated that the PbROPs may play important roles in the stress response via similar molecular mechanisms. Therefore, PbROPs may be crucial in the adaptation and survival of 'Duli' pear under abiotic stress conditions.

Data Acquisition
To build a local database, the reference sequence of the 'Duli' pear genome was downloaded from the database of the Chinese Academy of Sciences [26]. Using the sequences of AtROPs of Arabidopsis and Tbtools, the 'Duli' pear genome was blasted to identify homologous ROP gene sequences (E-value < 10 −10 ) [40]. This was followed by analyzing the conserved domains of the putative PbROP amino acid sequences using NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/ (accessed on 2 March 2022)) and ExPAsy (https://www.expasy.org/ (accessed on 5 March 2022)) [41].

Phylogenetic Tree, Gene Structure, and Sequence Analysis of PbROP Gene Family
Clustal (http://www.clustal.org/ (accessed on 8 March 2022)) was used to compare the amino acid sequences of the ROP family of 'Duli' pear. Sequence similarity analysis was calculated using Genedoc (http://www.psc.edu/biomed/genedoc/ (accessed on 12 March 2022)). MEGA6.0 software(version 6.0, Mega Limited, Auckland, New Zealand) was used to construct the phylogenetic tree by the Neighbor-Joining (NJ) method with bootstrap 1000 and other default parameters [42]. The gene structure was constructed using Tbtools [40].

Chromosome Location, Conserved Motifs, and the Cis-Elements in the PROMOTERS of PbROPs of 'Duli' Pear
The specific positions of each of the 12 PbROPs on the 17 chromosomes were mapped and drawn using Tbtools [40]. The online software MEME (http://meme-suite.org/meme/, accessed on 18 March 2022) was used to identify the conserved motifs of the PbROPs with the parameters set to 20 and the rest as default (https://meme-suite.org/meme/ doc/meme.html (accessed on 22 March 2022)) [43]. The important cis-elements 2000 bp upstream of the transcriptional initiation codon ATG of the promoter regions of each ROP were identified using the PlantCARE online software (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/ (accessed on 22 March 2022)) [44].

Selective Pressure and Collinearity Analysis of PbROPs
According to the gene clustering of the ROP families, the collinearity of the genes between Arabidopsis and 'Duli' pear were compared with Muscle built into MEGA6.0. The selection pressure between ROP genes was calculated by DNAsp6.12.01 (DNA Sequence Polymorphism). The collinearity of ROP genes of 'Duli' pear and Arabidopsis was analyzed by McScanX (http://chibba.pgml.uga.edu/mcscan2/ (accessed on 24 March 2022)), and the relatedness diagram was drawn by Tbtools [40].

Expression Patterns of PbROPs
Three 5-year-old 'Duli' pear plants with similar growth vigor were selected from the pear resources nursery of the Pear Engineering Technology Research Center of Hebei Agricultural University, China. Roots, shoots, leaves, flowers, and young fruits were collected for RNA isolation (see below).

Expression Profiles of PbROPs in Response to Different Stress Treatments
Tissue-cultured 'Duli' pear seedlings were grown in the plant tissue culture room under a long day of 16 h light and 8 h dark at a temperature of 24 ± 2 • C. The control seedlings were cultured in the base MS medium (MS 4.4 g/L + 6-BA 1.0 mg/L + IBA 0.1 mg/L + sucrose 30 g/L + Agar 8 g/L pH 5.8), while 1.0 µM ABA, 150 mM NaCl, or 20% PEG6000 were added to the base MS media for different stress treatments. Samples were collected at 0, 3, 6, 12, 24, 72, and 120 h after each treatment and immediately frozen in liquid nitrogen and stored at −80 • C until further analysis by RT-PCR (see below).

Total RNA Extraction and Synthesis of 1st-Strand cDNAs
Total RNA was extracted from the above collected tissues of 'Duli' pear plants using the TIANGEN RNA prep Pure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. The quality of the RNA was verified by gel electrophoresis and quantified using Nanodrop. One microgram of total RNA was reverse-transcribed to the first-stranded cDNA following the method provided by the TIANGEN FastKing cDNA first-strand synthesis kit (Tiangen Biotech, Beijing, China).

Real-Time PCR
The primers for quantitative real-time PCR were designed using Primer 5.0 and are listed in Table S3. A total of 20 µL of PCR mix containing 10 µL of SuperReal Premix Plus (TransGen Biotech, Beijing, China), 600 ng of cDNA, and 0.5 µL of each forward and reverse primer (10 µM) were set-up. The LightCycler 96 real-time PCR machine (Roche, Basel, Swiss Confederation) was used to run the PCR reaction with the following regime: pre-denaturation at 94 • C for 30 s, 45 cycles of 94 • C for 5 s, 55 • C for 10 s, and 72 • C for 10 s. The β-actin gene was used as the reference gene and the relative expression level of each PbROP was calculated. Three biological replicates and three technical replicates were included for each gene.

Over-Expression of PbROPs and ABA Sensitivity Assay of Transgenic Arabidopsis Seedlings
The full coding regions of PbROP1, PbROP2, PbROP3, and PbROP9 were individually cloned using the primers list in Table S3 into the plant expression vector pEarleyGate103 via the Gateway cloning strategy [45,46]. After transforming Agrobacterium strain GV3101, the floral dipping method was used to transform Arabidopsis [47]. The homozygous transgenic lines were selected in T 3 progenies. Forty seeds of each overexpression homozygous line were germinated on 1 /2 MS medium containing 0, 0.5, and 1 µM ABA, respectively. They were cultured under a long day with 16 h light/8 h dark at 25 ± 1 • C. After 10 days, the seedlings were scanned and the seedlings with green cotyledons were scored. Each treatment was repeated three times for statistical analysis.

Conclusions
In summary, we identified 12 PbROPs from the wild-type 'Duli' pear. They are typical ROPs because they share sequence similarity and have all the conserved domains and motifs similar to ROPs from Arabidopsis and other plant species. Expression analysis by qRT-PCR of all 12 PbROPs and transgenic studies of 4 PbROPs in Arabidopsis indicated that they play important roles in growth, development, and particularly in abiotic stress. Further research will focus on the molecular mechanisms, and importantly, the involvement of S-acylation in the regulation of biological function of PbROPs in 'Duli' pear.