Genome-Wide Identi cation and Functional Analysis of U-Box E3 Ubiquitin Ligases Gene Family Related to Drought Stress Response in Chinese white pear (Pyrus bretschneideri)

Chunmeng Wang Nanjing Agricultural University Weigang Campus: Nanjing Agricultural University Bobo Song Nanjing Agricultural University Weigang Campus: Nanjing Agricultural University Yuqin Dai Nanjing Agricultural University Weigang Campus: Nanjing Agricultural University Shaoling Zhang Nanjing Agricultural University Weigang Campus: Nanjing Agricultural University Xiaosan Huang (  huangxs@njau.edu.cn ) Nanjing Agicultural University


Identi cation of PbrPUB gene family members
In our study, we used a strictly pipeline to identify PUB genes in pear genome. First, the hidden Markov model (HMM) of the U-box domain (PF04564) was downloaded from the Pfam30.0 database, and used as a query to identify the candidate PUB members in Chinese white pear genomic database (http://peargenome.njau.edu.cn/) usingHMMER3.0. As a result, a total of 91 candidate PUB genes were identi ed in pear genome. Second, SMART tools were performed to verify the accuracy of 91 candidate PUB gens, and 29 PUB genes were removed because of the incomplete of PUB domain. At last, 62 PUB genes with complete U-box domain were obtained for further analysis (Table 1). We named these PbrPUB genes from PbrPUB1 to PbrPUB62 according to their location information on the chromosome. The ExPASy ProtParam (https://www.expasy.org/), an online proteomics and sequence analysis tool, was used to estimate molecular weight and isoelectric point (pI). The molecular weight for the PbrPUB gene family range from 39.33 kDa to 151.30 kDa (Kilodalton) and the pI range from 4.99 to 8.83, with an average of 6.78. Subcellular localization were also predicted by Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/), and we found that most PUB gene were located in nucleus, except six pub located in cytoplasm and three located in cell membrane.

Phylogenetic analysis of PbrPUB gene family members
To investigate the evolutionary relationships of PbrPUB genes family, we constructed a neighbor joining(NJ) phylogenetic tree using the Mega-X program based on the full-length PUB proteins of pear (62 members), tomato (62 members) and Arabidopsis (61 members) (Fig. 1a). The protein sequences of PUB genes of tomato and Arabidopsis were obtained from previous study [10,12]. Based the result of phylogenetic tree, 185 members of PUB genes from these three species were clustered into ve groups, including Group I, Group II, Group III, Group IV and Group V. The member number of Group III was biggest in ve subgroups, and it harboured 64 PUB genes. However, Group IV harboured least PUB genes, with 10 PUB genes. In generally, the PUB genes of pear and tomato were clustered into one subclade, suggesting that pear and tomato exhibited relatively closer relationship compared to Arabidopsis.
It is interesting to note that the member number of PUB gene family in these three species is similar. This result indicated that the number of PUB genes in these three species is conserved. To explore which group of pears had occurred expansion or lost during evolution process, we measure the number of PUB genes of each species in each group. In pear, group I, II, III, IV and V contain 11, 21, 21, 3 and 6 PbrPUB gene family members, respectively. In tomato, groups I, II, III, IV and V contain 12, 21, 21, 3, and 5 SIU-box genes, respectively. In Arabidopsis, groups I, II, III, IV and V contain 20, 12, 22, 4 and 3 AtPUB genes, respectively (Fig. 1b). The member number of each group in pear and tomato is almost equal, suggesting that pear had not undergone expansion or lost compared to tomato. However, compared to pear and tomato, the group I of Arabidopsis had undergone rapid expansion, while the group II of Arabidopsis had undergone rapid lost.
Analysis of PbrPUB gene family conserved motifs and gene structures To further verify the classi cation results of phylogenetic tree, we investigated the conserved motif and gene structure of PbrPUB genes in pear. The MultipleEm for Motif Elicitation (MEME) motif search tool were used to predict the conserved motif of PbrPUB genes. A total of 20 motifs were estimated in our study, and we named as motif 1-20 ( Fig. 2a, 2b, Additional le 1: Fig. S1). Among them, motif 1, 3 and 5 were found in all groups, indicating that were highly conserved in all PbrPUB proteins. Based on the SMATR website, we determined that the U-box was comprised of Motif1, Motif3 and Motif5 (Additional le 2: Fig.S2). This result provided evidence to support the accuracy of PUB gene set identi ed in our study. Based on the SMATR website, we also found the other conserved domain: ARM and Pkinase domin. The ARM is comprised of motif 2, 4 and 7; the Pkinase is comprised of motif 11, 13 and 20.Generally, most PbrPUB members in the same groups had similar motifs. For example, most of the members of group II contained motif 6, 10 and 8. This result indicated that these three motifs might be key functional domain of group II PUB genes, suggesting that these proteins might have conservative functions.
To explore the gene structure of PbrPUB genes in pear, we extracted the exon-intron information of 62 PbrPUB genes from pear database using in-house scripts. Based on the information, TBtools software were preformed to show the gene structure of PbrPUB (Fig. 2c). The number of exon in PUB genes was greatly divergent, ranging from 1 to 20. Among 62 PUB genes in pear, PbrPUB24 contained the greatest number of exon (20), while 16 PUB genes only contained one exon. Furthermore, the lengths of the exon and intron were differential. There are 30 PUB genes have been found contain UTR structure. Similarly, to the result of motif analysis, the PUB genes with similar gene structure were cluster into same subclade. For example, most members of class II only housed one exon. This result indicated that the members of same groups exhibited similar gene structure and conserved motifs. These results from conserved motifs and gene structure analysis provided strong evidence to support the accuracy of the classi cation result of phylogenetic tree.

Chromosomal localization and homologous gene analysis of PbrPUB genes
To further explore the distribution pattern of PUB gene in pear genome, the PbrPUB chromosomal distribution map was plotted using TBtools (Fig. 3a). The location information of PUB genes in pear were extracted by our in-house scripts. As a result, a total of 50 PbrPUB genes (82.26%) were unevenly mapped on the 17 pear chromosomes, and no member of PbrPUB gene family was mapped on chromosome 8. Therefore, we didn't shown chromosome 8 in our Fig. 3. In addition, 12 genes were located on scaffold contigs, and we also didn't show them in our Fig. 3. Chromosome 15 had the largest number of PbrPUB genes, with eight PbrPUB genes, followed by chromosome 5 with 6 genes. Chromosome 1, 2 and 12 each contained 4 PbrPUB genes. Two or three PbrPUB genes were mapped on chromosomes 3, 6, 7, 9, 10, 11, 13, 14, and 16. Chromosome 4 and chromosome 17 contained only one gene. We also identi ed the homologous genes of PUB gene family using MCscanX software. As result, 16 homologous gene pairs were identi ed in pear PUB gene family, which contained 26 homologous genes. Three homologous gene pairs were detected between chromosomes 5 and chromosomes 10 (Fig. 3b).
Cis-acting elements analysis in the putative promoter of PbrPUB genes Cis-acting elements were important clues for the prediction of gene functions. To further investigate the function of PbrPUB genes, we predicted the cis-acting element of the putative promoter region of PbrPUB genes using PlantCARE databse. In this study, the 2000 bp upstream region of transcription initiation site of PbrPUB genes was de ned as the putative promoter region. As a result, a tot al of 41 cis-acting elements were identi ed (Fig. 4), and we selected 15 interesting cis-acting elements for further analysis. These 15 cis-acting were associated with stress, hormone, plant growth and development. As shown in Fig. 4a, some diverse distribution patterns of cis-acting elements were observed in the promoter region of PbrPUB genes, indicating that the PUB gene family of pear particular in various different biology process. Meanwhile, we found that all PbrPUB genes contained the cis-acting related to hormone regulation, such as, abscisic acid (ABA), methyl jasmonate (MeJA), gibberellin (GA), salicylic acid, and auxin responsiveness elements. Previous study had reported that DSG1, which encodes a U-box domain, could regulate cell division and elongation by responding to multiple hormones, such as auxin, salicylic acid and ethylene [33]. In our study, 55 genes were identi ed as the responsiveness elements of ABA, suggesting that PUB gene family might particular in resistance under ABA treatment (Fig. 4b). In Arabidopsis, AtPUB9, AtPUB18, AtPUB19, and AtPUB44 were identi ed to involve in ABA response [34]. It is notable that the element related to MYB binding site involved in drought was predicted in 44 PbrPUB genes, suggesting that these 44 PbrPUB genes might mediated by MYB genes response to drought stress. Moreover, there were 30 PbrPUB genes have cis-acting elements related to cold, suggesting that these 30 PbrPUB genes might particular in resistance under low temp treatment. As we all know, avonoid biosynthesis is one of important phenomenon during the process of response to stress in plant. In the study, we found PbrPUB10, PbrPUB24 and PbrPUB5 contained MYB binding site involved in avonoid biosynthetic.

Expression pro le of PbrPUB genes in different tissues of pear
To further explore the tissues-speci c expression of PbrPUB genes, we conducted RNA-seq analysis using transcriptomic data from seven different tissues of 'Dangshansuli' cultivar pear, including stem, ovary, petal, sepal, bud, fruit and leaf [35]. We used RPKM (reads per kilobase per million) values to estimate the expression level of PbrPUB genes. The RPKM value equal to 0 means no expression in one library, and the RPKM value greater than 10 means highly expression in one library. Then, we investigated the expression level of 62 PUB genes. Pheatmap, an R package, was used to show the expression patterns of 62 PbrPUB genes (Fig. 5a). Based on the expression pattern of 62 PUB genes, they were clustered into four main classes. Gene in Class IV exhibited highly expression level in all of seven examined tissues, while gene in Class II exhibited almost no expression in all of seven tissues. Class I was speci cally expressed in pear leaf, and a diversity of expression pattern were detected in Class III (Fig. 5a). Among the 62 PbrPUB genes, 52 genes (83.87%) were at least expressed in one tissue, even though the transcript abundance of several genes was relatively lower for certain tissues. Approximately 10 non-expressed PUB genes were identi ed in all of seven tissues, and they may be lost the function during the evolution process of PUB genes family in pear. 29 PbrPUB genes were expressed in all seven different tissues, indicating that they have various roles in the development of different tissues. Interesting, we found 28 PUB genes exhibited highest expression in leaf, suggesting that these 28 genes might involve the development of leaf. Due to leaf is an important plant organ involved resistance, we referred that these 28 PUB genes might particular in resistance in the process of pear growth and development.
To verify the transcriptome sequences analysis was reliable, 15 PbrPUB genes were randomly selected to conduct a quantitative real-time PCR (qRT-PCR) experiment to investigate the expression levels in seven different tissues of the 'Dangshansuli' pear, including stem, leaf, petal, fruit, sepal, bud, and ovary ( Fig. 5b). We found that all of 15 PbrPUB genes exhibited a diversity of expression patterns in the seven different tissues, suggesting that PbrPUB genes family may function in different tissues and participate in diverse metabolic processes. Seven genes (PbrPUB1, PbrPUB3, PbrPUB7, PbrPUB9, PbrPUB18, PbrPUB36 and PbrPUB38) exhibited a similar expression pattern with a high expression level in leaf tissues, suggesting that PbrPUB genes play critical functions during leaf development. All of these seven genes exhibited highly expression level in leaf in transcriptome data. These result provided further evidence to support our transcriptome sequences analysis was reliable. Interestingly, most of 15 PUB genes were highly expressed in reproductive organ, suggesting that PbrPUB genes might associate with the development of reproductive organ.

The expression pattern of PbrPUB genes under abiotic stresses
Previous study had extensively reported PbrPUB gene family involved in various abiotic stresses [36]. To explore the function of PUB gene family in pear, Pyrus betulaefolia from grown 45-day-old seedlings were subjected to four different stress treatments including dehydration, low temperature, ABA and salt. To identify whether PUB genes in pear could respond to abiotic stresses, 11 PbrPUB genes were randomly selected from each group to conduct a qRT-PCR experiment to detect their expression level. 11 genes are comprise of 2 from group I (PbrPUB1 and PbrPUB14), 4 from group II (PbrPUB12, PbrPUB18, PbrPUB36 and PbrPUB38), 2 from group III (PbrPUB3 and PbrPUB25), 2 from group IV (PbrPUB7 and PbrPUB48) and 1 from group V (PbrPUB34).
To identify candidate PbrPUB genes related to dehydration treatment, the shoots of seedlings of pear were placed on dry lter papers for 0, 1, 6, 9, 12 and 24 h at ambient environment (Fig. 6a). Among the eleven PUB genes, 9 PUB genes were up-regulated expressed and one PUB gene (PbrPUB7) was downregulated expressed under dehydration stress. However, PbrPUB14 was not signi cantly differential expressed under dehydration stress. Among the 9 up-regulated genes, PbrPUB18 exhibited highly increased expression level during the process of dehydration treatment, while PbrPUB12, PbrPUB3 and PbrPUB36 were up-regulated expressed during 12 h dehydration treatment and recovered to normal levels at 24 h. PbrPUB1, PbrPUB38 and PbrPUB25, exhibited highest expression level at 1 h, where PbrPUB12, PbrPUB14, PbrPUB3 and PbrPUB36 exhibited highest expression level at 12 h under dehydration treatment. These results suggested that PbrPUB1, PbrPUB38 and PbrPUB25 respond to dehydration treatment faster than that of PbrPUB12, PbrPUB14, PbrPUB3 and PbrPUB36. Therefore, PUB gene family in pear play vital role in the process of low temperature stress response.
To verify whether PUB gene in pear involve in low temperature stress, the seedlings were placed in the chamber set at 4°C for 0, 1, 6, 9, 12, 24, 48 and 96 h (Fig. 6b). We detected the expression level of those 11 PbrPUB genes by qRT-PCR experiment. 4 genes (PbrPUB12, PbrPUB3, PbrPUB36 and PbrPUB48) were up-regulated expressed under cold stress, suggesting that those PbrPUB genes might respond to low temperature. PbrPUB12, PbrPUB48 and PbrPUB36 were highly increased during the 48 h low temperature exposure. The expression level of PbrPUB3 was reached to double peak at 1 h and 48 h.
To explore the functions of PbrPUB genes under the salt stress, the seedlings were placed in solution containing 200 mM NaCl solution for 0, 2, 4, 6, 8, 12 and 36 h (Fig. 6c). We detected the expression level of those 11 PbrPUB genes by qRT-PCR experiment. On the whole, all of the selected 11 PbrPUB genes were signi cantly up-regulated expressed under the 200 mM salt stress treatment. The expression level of PbrPUB14, PbrPUB25, PbrPUB3, PbrPUB48 and PbrPUB7 were highly increased during the 12 h salt exposure. Moreover, PbrPUB1, PbrPUB12, PbrPUB18, PbrPUB34, PbrPUB36 and PbrPUB38 were highest expressed at 4 h under salt stress, suggesting that these 6 PbrPUB genes respond to salt treatment actively. We focus on the expression level of PbrPUB18. In the 4-8 h, the expression level of PbrPUB18 was signi cantly increased, and then it was down-regulated at 12 h, nally recovered normal level at 36 h.
Previous study had reported that PUB gene involved in ABA-mediated drought stress responses. To investigate the roles of PUB gene family in ABA stress, the seedlings were dipped in solution containing 100 µM ABA for 0, 1, 3, 6, 9, 12 and 36 h (Fig. 6d). As results, all of 11 PUB genes were respond to the ABA stress, and these gene were unregulated expressed at rst, and then were down-regulated at 36 h after ABA treatment. These results indicated that PUB genes play important roles in ABA-regulated pathway. The expression levels of three genes (PbrPUB1, PbrPUB25, and PbrPUB36) were reached to peak at 1 h, suggesting that these three genes were actively responded to ABA stress. Interestingly, we found that PbrPUB18 was expressed in 6 h and 12 h after ABA treatment.

Subcellular localization of PbrPUB18
To further explore the function of PUB gene in pear, we constructed subcellular localization experiment to identify where the PUB genes act function. To further verify the biologic function of PbrPUB genes in pear under drought stress, PbrPUB18 was selected from 9 up-regulated expressed genes for further study. The full-length ORF of PbrPUB18 was fused to the N-terminal of GFP (Green Fluorescent Protein), under the control of CaMV 35S promoter to form a fusion construct 35S-PbrPUB18-GFP. Then the 35S-PbrPUB18-GFP fusion protein and 35S-GFP (as a control) was transient transformed into tobacco leaves and the uorescence signal was observed with a confocal laser scanning microscope. The green uorescence of GFP control was found in the membrane and the nucleus (Fig. 7a). In contrast, 35S-PbrPUB18-GFPfused GFP protein was only existed in the nucleus and integrated perfectly with DAPI (4′, 6-diamidino-2phenylindole) regime (Fig. 7b), suggesting that PbrPUB18 was located in the nucleus, which was consistent with our prediction in Table 1.
Assessment of drought tolerance in transgenic lines of PbrPUB18 To further con rm the biologic function of PbrPUB18 gene under drought stress, Arabidopsis Col-0 plants (WT) were transformed by the oral dip method [37]. Two overexpression lines OE-4 and OE-5 were screened out by PCR identi cation at DNA level in T0 generation plants. The selected robust Arabidopsis thaliana seedlings were transplanted into the soil as T1 generation plants, and the T1 generation positive seedlings were identi ed again by semi-quantitative PCR at mRNA level. QRT-PCR also veri ed the expression of PbrPUB18 in OE-4 and OE-5 far above in WT (Additional le 3 Fig S3). To assess the function of overexpression PbrPUB18 in Arabidopsis on drought tolerance, 15-day-old WT and transgenic lines were used to expose to drought stress by withholding water for 12 days. There was no morphological difference between WTs and the transgenic lines in the normal condition. After 12 days without water, the two transgenic lines showed more tolerance to the drought stress, as manifested by lesser leaf-wilting symptoms compared with the WT plants (Fig. 8a). In addition, chlorophyll uorescence measurements were recorded to further verify drought tolerance of WTs and the transgenic lines (Fig. 8b).
The maximum quantum e ciency of the photochemistry (Fv/Fm) values was not affected by species and growth conditions, but under stress conditions, this parameter decreased signi cantly. After 12 days drought treatment, the Fv/Fm values of WT was signi cantly lower than of the two transgenic lines, suggesting WT showed more sensitivity to the drought stress (Fig. 8e). Electrolyte leakage (EL), an important indicator of cell injury, was measured after drought stress. The EL of two transgenic lines were only approximate 15 %-20 % compared to WT (37.3 %), suggesting that WT suffered more severe membrane damage than transgenic lines of Arabidopsis by overexpressing PbrPUB18 (Fig. 8c). The transgenic plants displayed signi cantly lower malondialdehyde (MDA) contents than WT exposure to drought condition (Fig. 8d).
In the drought tolerance stress assay, we found that two transgenic lines had lower values of EL and MDA, implying that they might be subjected to lighter extent oxidative stress than the wild type.
Histochemical staining with 3, 3′-diaminobenzidine (DAB) and nitro-blue tetrazolium chloride (NBT) was used to analyze the in situ accumulation of H 2 O 2 and O 2 -, two main reactive oxygen species (ROS), respectively. After drought stress the leaves of WT lines (Fig. 8f) were stained to a more serious extent compared with transgenic Arabidopsis, implying that more ROS was produced in the WT under the drought conditions. Similar to staining results, quantitative measurements further demonstrated that H 2 O 2 contents in the two transgenic lines were remarkably lower than those of WT (Fig. 8g). And anti-O 2 contents in the two transgenic lines were remarkably more than those of WT (Fig. 8h), implying that less ROS was produced in the two transgenic lines under the drought conditions.

Discussion
Genome-wide and phylogenetic analysis of PbrPUB genes in pear As a family of ubiquitin ligases, U-box genes encode a conserved U-box motif of about 70 amino acids and regulated the ubiquitination of the substrates [23]. U-box genes were widely distributed in the plants and reported to participate in many biological processes including plant hormone signaling regulations [6], self-incompatible or pseudo-self-compatibility regulations [18] as well as in biotic stress [19][20][21] and abiotic stress [5,22,23]. Due to PUB gene play important role during plant development, PUB genes have been identi ed in different plant species, such as Arabidopsis thaliana (61) [9,10], rice (77) [11], tomato (62) [12], cotton (93) [13], and banana (91) [14]. Pear, one of Rosaceae fruit trees, is widely cultivated all over the world. However, the analysis related to PUB genes in pear was poor until now. In the present study, 62 genes were identi ed as PUB gene family in pear using bioinformatics analysis, and the number of PUB gene in pear is similar to that of Arabidopsis thaliana (61) and tomato (62). We therefore speculate that the number of PUB genes in plant kingdom is relatively conserved.
Phylogenetic tree analysis indicated that a total of 185 PUB protein members in these three species (containing 62 pear, 62 tomato, and 61 Arabidopsis) were categorized into ve subgroups (I-V). Although there are some differences, this phylogenetic tree was largely consistent with the results from a previous study [12,14]. For example, 125 GmPUB genes in soybean proteins were classi ed into six groups using phylogenetic tree analysis [17]. Through the phylogenetic relationship analysis, it was showed that PbrPUBs exhibited closer relations with SIU-boxs compared with AtPUBs. This result was consistent with the fact that pear and tomato exhibited closely relationship than Arabidopsis. Although the number of PUB genes was similar in three species, we found that the genes of Group I in had undergone rapid expansion and Group II had undergone rapid lost. In addition to the U-box domain, 62 PbrPUB proteins are found to bind to different domains including armadillo (ARM) repeats, the tetratricopeptide (TPR) domain and WD40 repeats. The majority of PUB proteins that have been elucidated for biological functions are from the U-box proteins with ARM repeats [18]. The ARM repeats have been shown primarily mediating the interaction with substrates, suggesting that interaction make the substrates available for ubiquitination [23]. 25 member of PUB genes in pear only housed U-box domain, and 25 members housed both U-box and ARM domain. Moreover, TPR domain was found in PbrPUB14 gene and WD40 repeats was found in PbrPUB40 gene.

The function predication of PUB gene family in pear based on cis-acting and speci c-tissues expression analysis
The cis-acting analysis of putative promoter indicated the U-box gene family was involved in stressrelated mechanisms, hormonal regulation, growth and development. Previous study had reported that PUB proteins were responded with ABA. For instance, AtPUB44 could regulated the biosynthesis of ABA through ubiquitinating the AAO3 (abscisic aldehyde oxidase 3) via 26 proteasomes [38]. In additional, one transcription factor of ABI3 was regulated by AtPUB9 and increased the ABA sensitivity of Arabidopsis during seedling germination [39]. AtPUB18, AtPUB19 and AtPUB44 were found to directly interrupt the biosynthesis of ABA directly. In our study, 55 genes contained the ABA responsiveness elements on the putative promoter region. Especially, we found that eight ABA responsiveness elements were identi ed in the promoter region of PbrPUB43. This result indicted that PUB gene might play important role during ABA signal transduction in pear. In Arabidopsis and Nicotiana, the expression levels of PUB genes were regulated by abiotic and biotic stress [40]. In here, we found MYB binding site involved in drought induction responsive element, abscisic acid responsive element, defense and stress responsive element, low temperature responsive element, wound responsive element in the promoter region of PUB genes in pear. The present of these elements indicated that most of PUB genes in pear were involved in stress response.
Based on our previous RNA-Seq data in seven different tissues of 'Dangshansuli' cultivar pear, transcriptome sequencing analysis and qRT-PCR expression pro ling were conducted to investigate PUB gene expression patterns in seven different tissues. The members of the PUB gene family exhibited a variety of expression patterns in seven different tissues. These results indicated that PUB might particular in a diversity functions during plant growth and development. Among the 62 members of PUB gene family in pear, 29 PbrPUB genes were expressed in all seven different tissues. Additionally, 72.58 % of PbrPUBs were detected in transcriptional abundance in pear sepal. Whereas of PbrPUBs expressed in all tissues, 45.16 % were highest in leaves, suggesting these genes may have important roles in the development of pear leaves. Further, the expression levels of 15 randomly selected members of the PbrPUB gene family were validated using qRT-PCR. Based on qRT-PCR expression pro les, we found that the 15 PbrPUB genes were highly expressed in petal, sepal, ovary and leaf, suggesting that PbrPUB genes may function in the development of petal, sepal, ovary and leaf.

Roles of PbrPUB genes in response to different abiotic stresses
Previous studies have reported that PUB genes in plants involved in the process of stress responses [24,26,41,42]. A large of PUB genes were induced expressed during abiotic stress conditions [40]. In this study, the differential expression levels of 11 PbrPUB genes under various abiotic stresses were investigated by using qRT-PCR, including drought, low temperature and salt stress. From the result, PbrPUB12, PbrPUB3, PbrPUB36 and PbrPUB48 were signi cantly up-regulated expressed under four treatment, suggesting these three genes could response to dehydration, ABA, cold and salt stress. PbrPUB7 was down-regulated expressed under dehydration stress, suggesting that PbrPUB7 might negatively regulate the response process of dehydration. Drought is one of most critical stresses and could signi cantly affect the growth of plant. In our study, we found that the expression level of PbrPUB18 were signi cant up-regulated after dehydration treatment, suggesting that PbrPUB18 genes might respond to drought stress. Therefore, PbrPUB18 gene was selected for further functional identi cation. Subcellular localization experiment suggested that PbrPUB18 was located at the cell nucleus. This result indicated that PbrPUB18 might act biology function at the cell nucleus. Furthermore, different from previous studies, overexpression of PbrPUB18 gene in Arabidopsis thaliana resulted in resistance to drought tolerance. Summary, we systematically identi ed the PUB gene family in pear, and further function identi cation laid a foundation for the functional study of PUB genes of in pear in future.

Conclusions
In our study, a total of 62 PbrPUB members were identi ed in Chinese white pear genome, and were unevenly distributed on 17 pear chromosomes. According to phylogenetic tree analysis, the pear PUB gene family was divided into ve groups. The conserved motif and gene structure analysis provided strong evidence to support the result of classi cation phylogenetic tree. Cis-acting element analysis indicted PUB genes might participate in diverse biological processes, especially in the response to abiotic stresses and phytohormones. Transcription sequencing data from different seven tissues exhibited diverse of expression level of PbrPUB genes. Further qRT-PCR was used to identify candidate genes associated with abiotic stresses. In addition, PbrPUB18 was cloned and functionally identi ed. Subcellular localization revealed PbrPUB18 genes were located on cell nucleus. Heterologous overexpression of PbrPUB18 in Arabidopsis indicated that the over-expression of PbrPUB18 could enhance resistance in drought treatment. Our data and analysis lay a solid foundation for the future research on molecular mechanism of PbrPUB genes in responding to abiotic stresses.

Genome identi cation of PUB gene family members in Chinese white pear
To identify the potential members of the PUB protein family, the complete genome sequence of pear (Pyrus bretschneideri) was downloaded from the pear genome project (http://peargenome.njau.edu.cn/) [32]. The hidden Markov Model (HMM) pro le of the U-box domain (PF04564) was obtained from Pfam30.0 (http://pfam.xfam.org/), and was used as a query to identify the candidate PUBs from the pear genome protein database using HMMER3.0.
All candidate PUB genes were further veri ed by using SMART conserved domain search tools (http://smart.embl-heidelberg.de/) to ensure that each candidate protein contained a U-box domain. In addition, the ExPASy ProtParam (https://www.expasy.org/), an online proteomics and sequence analysis tool, was performed to predict the physical and chemical properties of PbrPUB proteins, such as the number of amino acids (AA), molecular weights, isoelectric points, and instability index [43]. The subcellular localization prediction was carried out in Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) [44].

PbrPUB protein phylogenetic analysis
Arabidopsis PUB protein sequences were downloaded from the Arabidopsis Information Resource (TAIR) (https://www.Arabidopsis.org/browse/genefamily/pub.jsp) [10] Tomato PUB protein sequences were obtained from previous study [12]. All PUB full-length protein sequences in Pyrus bretschneideri, Arabidopsis thaliana and Solanum lycopersicum were aligned using ClustalW withed fault parameters. Then, MEGA-X was used to construct an unrooted phylogenetictree based on the result of multiple sequence alignment using the NJ (Neighbor Joining) method with 1000 bootstrap replicates [45].
Gene structure, motif analysis and cis-acting elements analysis To identify and visualize the structural organization (introns, exons, and untranslated regions) of the pear PUB gene family, the information of gene structure was extracted from whole genome database of pear using in-house scripts. The novel conserved motifs of PbrPUB genes were identi ed by MEME suite (http://meme-suite.org/tools/meme). A total of 20 motifs and a width limit of 200 amino acids were used for the analysis with other default parameters. TBtools were used to visualize the results of gene structure and conserved motif analysis.
Bedtools software was used to extract the 2000 bp upstream (putative promoter region) sequences of the transcription start site of all PbrPUB genes [47]. Then, PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the cis-acting elements in the putative promoter region of PbrPUB genes [48]. TBtools were used to visualize the distribution of putative cis-acting elements.

Synteny analysis and chromosomal localization
Synteny analysis was performed by the method described in the Plant Duplicate Gene Database (PlantDGD) (http://pdgd.njau.edu.cn:8080/). The collinear block was identi ed by PbrPUB duplication events in the MCScanX [49]. The data were integrated and plotted by using Circos [50]. Based on the annotation information of PUB genes in pear, the corresponding location distributions of PbrPUB genes in chromosomes were displayed by TBtools [51].
Gene expression analysis of PbrPUB on the RNA-SEq. Data The RNA-seq data of the cultivar 'Dangshansuli' in seven different tissues were acquired from our previous study, including bud, stem, leaf, sepal, petal, fruit and ovary [52]. We downloaded them from the

Subcellular localization
The full-length cDNA of PbrPUB18 without a stop codon was ampli ed by RT-PCR with primer pair (GSP16, Additional le 4: Table S1) containing restriction sites of Xba I and BamH I, then inserted into the pCAMBIA1302 vector and fused in-frame to the N-terminal of GFP (Green Fluorescent Protein), under the control of CaMV 35S promoter to form a fusion construct 35S pro: PbrPUB18-GFP. After validation by sequencing, the fusion constructs 35S pro: PbrPUB18-GFP and 35S pro: GFP (as a control) were mobilized into Agrobacterium tumefaciens strain GV3101 by heat shock. Transient transformation of Nicotiana benthamiana was done as described earlier [54]. The uorescence signal was observed with a confocal laser scanning microscope (LSM410; Carl Zeiss) after 72 h post in ltration and the position of nucleus was revealed by staining with 4′, 6-diamidino-2-phenylindole (DAPI).

Arabidopsis transformation and characterization of transgenic plants
Arabidopsis thaliana ecotype Columbia Col-0 plants were transformed for heterologous over-expression PbrPUB18 by using the oral dip method [37]. And Agrobacterium tumefaciens suspension containing the vector 35S-PbrPUB18-GFP (OD 600 = 0.80) was used for transformation. T0 seeds were identi ed by Murashige and Skoog (MS) solid mediumwith 20 mg·L − 1 hygromycin and then veri ed by PCR analysis using speci c primers pair (GSP17, Additional le 4: Table S1). Semi-quantitative RT-PCR and qRT-PCR was used to further analyze the transcript levels of PbrPUB18 in T1 plants with primers pair (GSP18 and GSP5, Table S1). Two overexpressing lines (OE-4 and OE-5) of PbrPUB18 were selected to generate T3 homozygous plants for the subsequent stress tolerance assay.

Assessment of drought tolerance in transgenic lines
To test the drought tolerance, The WT and transgenic lines were subjected to drought. For drought treatments, 15-day-old soil-grown Arabidopsis seedlings were subjected to withholding water for 12 days. At the end of treatment, the leaves were collected for measurement of electrolyte leakage, MDA content and ROS level. Electrolyte leakage was measured as described [55]. The MDA content, H 2 O 2 and O 2 content were measured using speci c analytical kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) following the manufacturer's instructions. Histochemical staining with 3, 3′diaminobenzidine (DAB) and nitro-blue tetrazolium chloride (NBT) was used to analyse the in situ accumulation of H 2 O 2 and O 2 respectively, according to [56]. In addition, chlorophyll uorescence measurements were recorded using an IMAGING-PAM chlorophyll uorometer and ImagingWin software (Walz; Effeltrich, Germany) according to [57]. For chlorophyll uorescence imaging, the 20 mins-dark-