Comparative analysis of the P-type ATPase gene family in seven Rosaceae species and an expression analysis in pear (Pyrus bretschneideri Rehd.)

Journal Pre-proof Comparative analysis of the P-type ATPase gene family in seven Rosaceae species and an expression analysis in pear (Pyrus bretschneideri Rehd.)

Yuxin Zhang, Qionghou Li, Linlin Xu, Xin Qiao, Chunxin Liu, Shaoling Zhang PII:

S0888-7543(19)30509-9

DOI:

https://doi.org/10.1016/j.ygeno.2020.02.008

Reference:

YGENO 9470

To appear in:

Genomics

Received date:

1 August 2019

Revised date:

3 February 2020

Accepted date:

7 February 2020

Please cite this article as: Y. Zhang, Q. Li, L. Xu, et al., Comparative analysis of the P-type ATPase gene family in seven Rosaceae species and an expression analysis in pear (Pyrus bretschneideri Rehd.), Genomics (2019), https://doi.org/10.1016/j.ygeno.2020.02.008

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© 2019 Published by Elsevier.

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Comparative analysis of the P-type ATPase gene family in seven Rosaceae species and an expression analysis in pear (Pyrus bretschneideri Rehd.)

Yuxin Zhang1 † Qionghou Li1 † , Linlin Xu1 , Xin Qiao1 , Chunxin Liu1 , Shaoling Zhang1

These authors contributed equally: Yuxin Zhang, Qionghou Li

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Centre of Pear

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1

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†

Engineering Technology Research, Nanjing Agricultural University, Nanjing 210095,

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China

Corresponding author:

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Shaoling Zhang

Tel: +86-25-84396580

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E-mail: [email protected]

E-mail:

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Fax: +86-25-84396485

Yuxin Zhang: [email protected] Qionghou Li: [email protected] Linlin Xu: [email protected] Xin Qiao: [email protected] Chunxin Liu: [email protected] Shaoling Zhang: [email protected]

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Abstract P-type ATPases are integral membrane transporters that play important roles in transmembrane transport in plants. However, a comprehensive analysis of the P-type ATPase gene family has not been conducted in Chinese white pear (Pyrus

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bretschneideri) or other Rosaceae species. Here, we identified 419 P-type ATPase

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genes from seven Rosaceae species (Pyrus bretschneideri, Malus domestica, Prunus

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persica, Fragaria vesca, Prunus mume, Pyrus communis and Pyrus betulifolia).

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Structural and phylogenetic analyses revealed that P-type ATPase genes can be

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divided into five subfamilies. Different subfamilies have different conserved motifs and cis-acting elements, which may lead to functional divergence within one gene

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family. Dispersed duplication and whole-genome duplication may play critical roles

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in the expansion of the P-type ATPase family. Purifying selection was the primary

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force driving the evolution of P-type ATPase family genes. Based on the dynamic transcriptome analysis and transient transformation of Chinese white pear fruit, Pbr029767.1 in the P3A subfamily were found to be associated with malate accumulation during pear fruit development. Using a co-expression network, we identified several transcription factors that may have regulatory relationships with the P-type ATPase gene family. Overall, this study lays a solid foundation for understanding the evolution and functions of P-type ATPase genes in Chinese white pear and six other Rosaceae species. Keywords : P-type ATPase, Malate accumulation, Evolution, pear, Dynamic 2

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transcriptome, Rosaceae

1. Introduction In plants, there are primarily three kinds of proton pumps, the V-type ATPase, P-type ATPase and F-type ATPase [1-3]. P-type ATPase was named based on the presence of an intermediate phosphorylated process occurring during catalyzing, and

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first described as a pump that was thought to be associated with the transport of sodium ions in 1957 [4]. The structure of P-type ATPases in plants includes several

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transmembrane segments, four protein domains with highly conserved features, a

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single subunit N-terminus and a C-terminus exposed to the cytoplasm [5, 6]. This

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shared structure in plants indicates that P-type ATPases may have the same basic mechanism.

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Even though they have a shared basic mechanism, P-type ATPases from different

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subfamilies can translocate diverse type of cations, such as H+, Na+/K+, Ca2+, Zn2+,

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Cu2+ and some lipids. Based on sequence identities and functions of P-type ATPase genes, the P-type ATPase superfamily can be divided into five major subfamilies [7]. In subfamily P1, the P1A branch is the main branch, and it has more members than P 1B. Subfamily P2 has four branches (P 2A, P2B, P2C and P2D) [7]. P2A and P2B are reported to transport Ca2+, P2C may be involved in Na+/K+ and H+/K + transport, and P2D represents a small proportion of the proteins in the P2 subfamily [8]. P3A is a plasma membrane H+-ATPase, and P3B is related to Mg2+-ATPases in two bacterial species [7]. The P4 subfamily is involved in lipid transport. The P5 subfamily has not yet been

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functionally characterized [7–9]. P-type ATPases are vital membrane transporters in all kingdoms of life. Recent research has started to reveal the biological functions of P-type ATPase members. For example, in petunia, the PH5 gene, a member of the P-type ATPases, is involved in vacuolar acidification, which affects flower color [10]. In Arabidopsis and cotton,

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P-type ATPase genes that can affect the transport of anthocyanins and

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proanthocyanidins have been identified [11, 12]. Extensive research has been

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conducted on the P-type ATPase superfamily, especially P1B subfamily genes, and the

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functions of P1B ATPase heavy metal ATPase 4 (HMA4) in Arabidopsis thaliana and

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Anemone. halleri [13] have been revealed. In rice, a HAM gene, HAM4, can transport copper, zinc, and lead in cells [14]. In fruit trees, like apple and citrus [15, 16],

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researchers have often focused on the P-type ATPase genes that participate in the

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accumulation of organic acids, such as malic acid and citric acid. A series of Ma genes

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are reported to affect the acidity of apple fruit. For example, Ma10 plays an essential role in fruit vacuolar acidification in apple [15, 17]. Moreover, pH genes, which are also members of the P-type ATPase gene family, have commonly been studied in citrus. The molecular functions of PH1, PH5, PH8 and PH5-like genes have been elucidated in previous studies [18, 19]. Although the P-type ATPase gene family has been reported in Arabidopsis [6, 20, 21], cotton [11, 22], citrus [19, 23], soybean [24], rice [14], maize [25] and petunia [26], it has still not been systematically identified and investigated in Rosaceae, which is an important family that includes many commercial fruit species, such as apple, pear, 4

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peach, strawberry and apricot. Furthermore, the level of organic acids is a major factor that influences fruit flavor. P-type ATPase genes may regulate fruit acidity [15– 18, 27]. For example, in apple, the Ma10 gene has roles in proton pumping and affects fruit vacuolar acidification in apple [15]. Moreover, in Citrus limon, two vacuolar P-ATPases genes, CitPH1 and CitPH5, are highly expressed in all low pH species,

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while they are down-regulated in high PH species [18]. In apple, many important

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organic acid transport genes, including P-ATPases, can be found at the Ma locus [27].

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Therefore, studying the P-type ATPase gene family may aid in improving important

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traits in Chinese white pear and other Rosaceae fruits. In this study, we identified 419

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P-type ATPase genes in seven Rosaceae species. And the detection of duplication events uncovered the evolutionary driving force in the gene family. Finally, the

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dynamic fruit transcriptome and qRT-PCR validation aided in the identification of

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potential candidate genes that regulate acidity during fruit development. The results of

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this study will be useful in further investigations of the biological functions and molecular mechanism of the P-type ATPase family in Chinese white pear and other Rosaceae species that bear economically important fruits.

2. Materials and methods 2.1 Plant materials ‘Dangshansuli’ (P. bretschneideri, white pear group) fruit samples were collected from the same trees at 30, 60, 90 and 120DAF in an orchard (Gaoyou County, Jiangsu Province, China). The pear fruit of transient transformation were collected at 150 DAF from the same orchard. Pear fruit at 150 DAF were used to perform transient 5

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transformation experiment. 2.2 Collection and identification of P-type ATPase genes The genome sequences from seven Rosaceae species were used in this study. The genome sequence of Chinese white pear (Pyrus bretschneideri) was downloaded from the pear genome project (http://peargenome.njau.edu.cn/) [28]. The genome sequences of apple (Malus domestica), peach (Prunus persica), strawberry (Fragaria

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vesca), European pear (Pyrus communis) and Du Pear (Pyrus betulifolia) were downloaded from the Genome Database for Rosaceae (http://www.rosaceae.org/). The

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Japanese apricot (Prunus mume) genome sequence was collected from the Prunus

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mume Genome Project (http://prunusmumegenome.bjfu.edu.cn/index.jsp). The

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alignment file for the P-type ATPase domain (PF00690, PF00122 and PF13246) obtained from the Pfam database [29] was used to build a Hidden Markov Model

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(HMM) file. Then, an HMM search was conducted against the pear protein database

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by using HMMER3 software [30]. Redundant sequences were removed based on their

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identification numbers and chromosomal locations. Furthermore, all P-type ATPase protein sequences were verified by the SMART program (http://smart.embl-heidelberg.de/), and the protein sequences lacking the P-type ATPase domain were removed. 2.3 Conserved motifs, cis-elements and basic data of the Chinese white pear P-type ATPase genes The conserved motifs of P-type ATPase genes were searched by using MEME (http://meme-suite.org/tools/meme) [31]. Specifically, we searched for 30 conserved motifs, and the parameters were set as the default. We identified the promoter 6

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sequences (~1,500 bp) of P-type ATPase genes from the Pear Project database [28] and used PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict cis elements. Some basic information on the P-type ATPase proteins, including numbers of amino acids, molecular weights (MW) and isoelectric points (pI), were calculated by using the ProtParam tool (http://web.expasy.org/compute_pi/). 2.4 Phylogenetic analysis of the P-type ATPase genes

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First, all the P-type ATPase full- length protein sequences of seven Rosaceae species and Arabidopsis were aligned by using MAFFT [32]. Then, IQ-TREE [33]

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was used to construct a maximum- likelihood (ML) phylogenetic tree with all 465

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full- length protein sequences. The best-fit substitution model LG+I+G4 was

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determined by ModelFinder [34], which was incorporated in the IQ-TREE. The bootstrap values were 1,000. To verify the ML phylogenetic tree, a neighbor-joining

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phylogenetic tree was constructed use MEGA 7.0, with bootstrap values 1,000 [35].

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2.5 Identification of gene duplications and chromosomal locations, and Ka/Ks analysis

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Different duplication modes of gene pairs among P. bretschneideri, M. domestica, P. persica, F. vesca, P. mume, P. communis and P. betulifolia were identified by using the DupGen_finder pipeline [36]. In brief, Nelumbo nucifera was selected as the outgroup to identify duplicated gene pairs. Then, all the species were used in a self-BLASTN search and a BLASTN search against N. nucifera. The simplified gff files were generated by using an in-house perl script. Finally, the DupGen_finder pipeline was started with the following parameters: DupGen_finder.pl -i data -c Nnu -t species -o results -k 50 -g -1 -s 5 -e 1e-05 -m 25 -w 5. 7

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Following the method described in a previous study [37], a synteny analysis was performed. All-versus-All BLASTP was used to search for orthologous and paralogous gene pairs among these species (E_value < 1e-05, m8 format). Then, based on the BLASTP results, the MCScanX toolkit, was used to identify synteny blocks among these species [37, 38].

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Yn00, which was included in the PAML package, was used to calculate Ka and

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Ks substitution rates with the Yang and Nielsen method (YN method) [39]. ParaAT

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and MAFFT [32, 40] were also used to perform the multiple sequence alignment and

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convert the format. Then, the yn00.ctl file was uploaded into the yn00 program. To

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batch process these steps, a pipeline that was developed can be found at https://github.com/LQHHHHH/paml-yn00-run-pipeline. 2.6 Expression analysis

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The raw RNA-seq reads were downloaded from NCBI SRA

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(https://www.ncbi.nlm.nih.gov/sra) [41]. The adapter sequences, low quality reads

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(quality score < 15) and poly(A/T) tails were removed from raw reads to obtain clean reads. Hisat2 [42] was used to align clean reads to the reference genome, and featureCounts [43] was used to estimate transcript abundance levels. The fragments per kilobase million (FPKM) values were used to measure the expression levels of the P-type ATPase genes. The expression level of each P-type ATPase gene was displayed in a heatmap by using TBtools [44]. 2.7 Quantification of the malic acid content in Chinese white pear The soluble organic acid content in Chinese white pear ‘Dangshansuli’ was determined by high-performance liquid chromatography (HPLC). The liquid 8

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chromatograph used was the Waters1525 system, and the chromatographic column was a Zorbar SB - Aq column (4.6 mm × 250 mm, 5 μm), with a mobile phase of 2% methanol and 98% 20 mmol·L−1 sodium dihydrogen phosphate buffer (pH 2.4, mixed with phosphoric acid). The HPLC had the following settings: flow rate, 0.7 mL·min−1 ; column temperature, 35°C; detector type, Waters 2487 UV detector; wavelength, 210

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nm; and sample quantity, 5 µL. The soluble organic acid content was calculated

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according to the peak area of the sample and the standard curve of the organic acid. 2.8 Quantitative real-time PCR (qRT-PCR) analysis

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Total RNA was extracted from the four developmental stages of pears by using a

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Plant RNA Extraction Kit (AutoLab) for qRT-PCR analysis with three biological

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replicates. Genomic DNA contamination was removed by using DNase I. Genomic DNA was then reverse transcribed with 1 μg of RNA to obtain cDNA. The

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SYBR®Green Premix kit (TaKaRa Biotechnology, Dalian, China) was used for the

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qRT-PCR. The composition of the PCR mixture was as follows: 1 μL of each primer

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(20 μM), 5 μL of 2× SYBR Premix ExTaq TM, 1 μL of cDNA template and 3 μL of RNase-free water). The qRT-PCR was run as follows: 10 min at 95°C, then 45 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30s. The final expression level was calculated by using the 2–ΔΔCt method. The primer sequences were designed by using Primer 5.0 (PREMIER Biosoft International, USA) (Table S1a). 2.9 Transient transformation of ‘Dangshansuli’ pear fruit About 400 bp fragment at the C-terminal of Pbr029767.1 was amplified then inserted into the pTRV2 vector following Zhang et al. [45]’s method (Table S1b).

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pTRV2-Pbr029767.1 together with pTRV1 were individually introduced into A. tumefaciens strain GV3101. After incubation, the suspension was centrifuged and then re-suspended with the infiltration buffer (10 mM MgCl2 , 10 mM MES, pH 5.5, and 150 μM acetosyringone). The A. tumefaciens containing pTRV2-Pbr029767.1 and pTRV1 were mixed together with a 1:1 ratio and then were injected into the

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‘Dangshansuli’ flesh tissue [46]. pTRV1 and pTRV2 (empty plasmid) were also used

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for injection as control.

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We prepared a pCambia 1301 construct to overexpress Pbr029767.1(35S-

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Pbr029767.1; Table S1b), and the over-expression vector was introduced into A.

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tumefaciens strain GV3101 [47]. Then incubated at 28 °C until OD600 reached a value of 1. After centrifugation and resuspension of the bacterial strain in infiltration

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buffer,the A. tumefaciens containing the over-expression vector was injected into the

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‘Dangshansuli’ flesh tissue with sterile syringe. All samples were taken after

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preservation at 21 °C for 5 d.

2.10 Construction of gene co-expression network

To investigate the regulatory network between P-type ATPase genes and transcription factors (TFs), we constructed a co-expression network based on RNA-seq data and Pearson’s correlation coefficients (PCCs). First, we calculated PCCs between every P-type ATPase gene and TF, which were identified in previous reports [48]. Then, PCC values lower than 0.995 were removed. Then, networks were visualized by using Cytoscape software [49]. 2.11 Statistical analysis 10

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The data were statistically processed using the SAS software package (SAS Institute, North Carolina, USA); analysis of variance was used to compare the statistical difference based on t-test at the significance levels of P < 0.05 (*), P < 0.01 (**).

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3. Results

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3.1 Identification and classification of P-type ATPase genes in seven Rosaceae species

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First, three accession numbers (PF00122, PF00690 and PF13246) were identified

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from the protein sequences of the P-type ATPase family members reported in the

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Arabidopsis protein sequences. To identify the family members in seven Rosaceae species, we built three HMM files using Pfam seed files corresponding to the three

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accession numbers, and performed an HMM search against the protein databases of

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the seven species. In total, 419 P-ATPase genes were identified as having specific

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P-type ATPase domains in seven Rosaceae species. There were 76 P-type ATPase genes found in Chinese white pear, 80 in apple, 58 in European pear, 45 in Japanese apricot, 45 in peach, 41 in strawberry and 74 in Du Pear. The numbers of P-type ATPase genes in pear (Chinese white pear and Du Pear) and apple were nearly twice those in strawberry, peach and Japanese apricot, which were consistent with lineage-specific whole-genome duplication events in pear and apple. A phylogenetic tree was constructed by using the ML method with the protein sequences of seven Rosaceae species and Arabidopsis. Based on the classification of

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subfamilies in Arabidopsis, all the P-type ATPase genes in Rosaceae species could be divided into five major subfamilies: P 1B, P2B, P2A, P3A, P4 and P5. P2B contained the most P-type ATPase genes (141 of 419), followed by P3A (80 of 419), P1B (75 of 419) and P4 (72 of 419), whereas the P 2A (41 of 419) and P5 (10 of 419) subfamilies had the fewest members of the seven species (Figure 1). For the P3A subfamily, 9, 11, 16,

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14, 14, 9 and 7 genes were assigned to Japanese apricot, European pear, Du pear,

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Chinese white pear, apple, peach and strawberry, respectively (Table S2). In addition,

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the classification of the neighbor-joining phylogenetic tree was consistent with the ML phylogenetic tree, which indicated the reliability of the classification (Figure S1).

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3.2 Conserved motif and promoter analyses of P-type ATPase genes in Chinese white pear The conserved motif analysis of P-type ATPase genes supported the phylogenetic

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relationships and classifications of Chinese white pear P-type ATPase family genes. In

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total, 30 corresponding consensus motifs were identified from 419 P-ATPase family

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members. The P-type ATPase proteins of each subfamily contained similar motifs. The members of P2B had the greatest number of conserved motifs, with 19 motifs detected in Pbr041968.1 and Pbr024083.1, while P1B members had the fewest motifs. Different subfamilies included specific motifs that may contribute to the functional divergence of each subfamily. For example, motifs 20 and 6 were specific to subfamily P4, whereas motif 13 appeared in all the members of subfamily P 3A, indicating that this motif may have been added to the subfamilies via the evolutionary process and may have resulted in a new function (Figure 2, Table S3). As specific binding sites of TFs, cis-acting elements are important. We identified 12

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cis-acting elements in the 1,500-bp regulatory sequences upstream of the 76 pear P-type ATPase genes. Specifically, 54 P-type ATPase genes included abscisic acid (ABA)-responsive elements (ABREs), 44 P-type ATPase genes had ethylene responsive elements (EREs), and 55 P-type ATPase genes had methyl jasmonate -responsive elements (CGTCA motif). Plant hormones, such as ABA, ethylene and

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salicylic acid, were involved in fruit ripening and senescence, suggesting that P-type

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ATPase family members are likely associated with hormonal changes and may be

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involved in pear maturation and senescence. In addition, we also identified abiotic

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stress-related cis-acting elements of the P-type ATPases, including 41 members

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containing LTRs, which are related to low-temperature responsiveness, 60 members containing G-BOX, which are involved in light responsiveness, and 62 members

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containing AREs, which are light stress-related. These cis-acting elements were

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related to stress responses, suggesting that P-type ATPase family members are likely

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to be associated with stress responses in Chinese white pear (Figure S2). Moreover, the genes of the P3A subfamily shared a cis-acting MYC (drought stress) element, suggesting that they are associated with responses to drought stress (Table S4). 3.3 Analysis of gene duplication events and chromosomal distributions of the P-type ATPase gene family Duplicated genes usually evolve to obtain new functions or to partition existing functions of ancestral genes, which is important for plant adaptability [50, 51]. To investigate the origin of P-type ATPase family genes, five modes of gene duplication (whole-genome duplication [WGD], tandem duplication [TD], proximal duplication [PD], transposed duplication [TRD] and dispersed duplication [DSD]) were analyzed 13

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in seven Rosaceae species. We investigated different types of gene duplication events and identified their contributions to the expansion of the P-type ATPase gene family. A total of 832 duplicated gene pairs were found in seven Rosaceae species, with the maximum number of gene pairs derived from DSDs (454 gene pairs), followed by WGDs (174

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gene pairs) and TRDs (146 gene pairs), suggesting that the expansion of the P-type

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ATPase gene family was mainly associated with DSD, WGD and TRD events. In

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contrast, only 28 TD-pairs and 30 PD-pairs were identified in the P-type ATPase gene

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family. The numbers of WGD-pairs in Chinese white pear (33), apple (43), European

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pear (35) and Du Pear (42), which shared a recent lineage-specific WGD event, are greater than those in Japanese apricot (6), peach (7) and strawberry (8), indicating the

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important roles of WGDs in the P-type ATPase family expansion in pear and apple.

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DSD and TRD events occurred more frequently in strawberry, peach and Japanese

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apricot, which have not experienced more recent WGDs, suggesting the important roles of single-gene duplications in the expansion of the P-type ATPase family during the long-term evolution of these genomes (Figure 3, Table S5). The distribution of the P-type ATPase genes on the chromosomes of the seven species was analyzed. For Chinese white pear, the P-type ATPase genes were distributed on 16 of the 17 chromosomes. A total of 62 P-type ATPase genes were located on the pear chromosomes, and 14 genes were found on scaffolds. Among these 76 genes, there were 10 genes distributed on chromosome 15, which contained the most genes compared with the other chromosomes. Chromosomes 4, 5, and 7 each 14

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only contain one P-type ATPase gene (Table 1). Like Chinese white pear, the distributions of the P-type ATPase genes in the other six Rosaceae genomes were random (Table S6). Further, the collinearity relationships of the P-type ATPase genes between Chinese white pear and the other six Rosaceae species were analyzed. All seven species belong to the Rosaceae family

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and share a common ancient hexaploid ancestor. Therefore, we analyzed the

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collinearity relationships among the P-type ATPase family genes of these seven

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species. For the P-type ATPase family, 441 collinear gene pairs were identified among

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the seven species (Figure 4, Table S7), and these included 64 collinear gene pairs

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between strawberry and apple, 93 pairs between apple and European pear, 74 pairs between European pear and Chinese white pear, 103 pairs between Chinese white

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pear and Du pear, 65 pairs between Du pear and peach, and 42 pairs between peach

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and Japanese apricot. Intra-genomic collinearity was also investigated in each species,

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and 27 pairs were found in Chinese white pear, 33 pairs were found in apple, 32 pairs were found in Du pear, 24 pairs were found in European pear, 5 pairs were found in Japanese apricot, 3 pairs were found in strawberry, and 6 pairs were found in peach (Figure 5, Table S8). The results show a good collinearity relationship between Chinese white pear and the other six species, and illustrates a potential evolutionary mechanism between them. 3.4 Ka and Ks substitutions per site and a Ka/Ks analysis of the P-type ATPase family genes The Ks value is used to estimate the evolutionary history of WGD events [52]. The mean Ks values of WGD-derived gene pairs in Chinese white pear, apple, Du 15

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Pear, European pear, peach, strawberry and Japanese apricot were 0.4, 1.31, 0.56, 0.91, 2.20, 2.23 and 1.96, respectively. The lower Ks values of WGD-derived gene pairs in pear and apple suggested that they were duplicated and retained from recent WGD events, while peach, strawberry and Japanese apricot were derived from more ancient WGD events (Figure S3). Here, we calculated the Ka/Ks of orthologous gene

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pairs among seven Rosaceae species. The Ka/Ks ratios of duplicated gene pairs in

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apple, strawberry, peach, European pear and Japanese apricot were < 1, indicating that

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P-type ATPase genes evolved under strong purifying selection. However, in Chinese

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white pear and Du Pear, three gene pairs Pbr002730.1 and Pbr037298.2 (Ka/Ks

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~1.24), GWHGAAYT032823 and GWHGAAYT008503 (Ka/Ks ~1.96), and GWHGAAYT017397 and GWHGAAYT017489 (Ka/Ks ~1.90), had higher Ka/Ks

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ratios, suggesting that this family may have a complicated evolutionary history. For

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Chinese white pear, we calculated the mean Ka/Ks values for the DSD, PD, TD, TRD

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and WGD gene pairs, which were 0.21, 0.26, 0.52, 0.30 and 0.22, respectively (Figure 6, Table S9). Compared with other types of duplicated gene pairs, the TD gene pairs had a higher Ka/Ks ratio, indicating that they evolved at a higher rate than the other gene pairs (Figure 6). The mean Ka/Ks values of gene pairs in the P3A subfamily were lower than those of mean P-type ATPase genes, suggesting that these P 3A duplicates evolved at a slower rate (Figure 6, Table S9). 3.5 Expression patterns of P-type ATPase genes during the fruit developmental stages of Chinese white pear Previously published dynamic RNA-seq data were used to analyze P-type ATPase gene expression profiles in Chinese white pear [41], including the expression 16

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analyses of P-type ATPase genes in fruits at four developmental stages (15 d after full bloom [DAFB], 45 DAFB, 90 DAFB and 120 DAFB). We found that 70 of the 76 P-type ATPase genes were expressed in fruit developmental stages. Seven genes (Pbr036737.1, Pbr037631.1, Pbr021970.1, Pbr032213.1, Pbr021950.2, Pbr011498.1 and Pbr036112.1) have higher expression levels in all the developmental stages than

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that of other genes, indicating that they may be associated with fruit development and

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ripening. Notably, seven genes (Pbr024083.1, Pbr002476.1, Pbr031825.2,

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Pbr029767.1, Pbr007143.1, Pbr026279.1 and Pbr024958.1) exhibited preferential

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expression levels in one or two stages and low expression levels in other stages, suggesting stage-specific roles (Figure 7A, Table S10).

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3.6 qRT-PCR validation

Some P3A-ATPase genes exist in apple and citrus, such as Ma10 and PH1 [15,

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16], and they are involved in the accumulation and transport of H+. Therefore, we

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investigated whether such genes could be identified in Chinese white pear. The

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content of malic acid in white pear is higher than that of other acids [53]. First, the malate contents at different developmental stages of Chinese white pear fruit were measured (Table S11). As shown in Figure 7B, the malate content first decreased, gradually increased, peaking at 90 DAFB and then gradually decreased. The malate accumulation trend decreased initially, then increased gradually and finally decreased. To investigate genes in the P3A subfamily related to the accumulation and transport of organic acids in Chinese white pear, primers were designed and quantitative real-time PCR (qRT-PCR) was performed on ‘Dangshansuli’ fruits at 15, 45, 90 and 120 DAFB. 17

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The relative expression levels of some P3A-ATPase genes (Pbr023904.1, Pbr033845.1, Pbr033851.1, Pbr002140.1, Pbr008201.1 and Pbr007323.1) were zero. The expression levels of most P3A-ATPase genes were consistent with the RNA-seq data. Thus, the reliability of the transcriptome data was verified. In combination with RNA-seq data, we found that some P3A-ATPase genes, such as Pbr029767.1,

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Pbr041824.1 and Pbr036112.1 showed relative expression levels consistent with the

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accumulation of malic acid (Figure 7C). In addition, the positive correlation (90.5)

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between the expression of Pbr029767.1 and the content of malic acid was supported

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by PCC values. Therefore, Pbr029767.1 was chosen as a candidate gene for further study.

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3.7 Candidate gene Pbr029767.1 regulating the transport malic acid in Chinese white pear

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To test the role of Pbr029767.1 in malic acid accumulation, transient

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over-expression and VIGS-mediated gene silencing of Pbr029767.1 were conducted

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in pear fruit. The transient transformed samples were collected and the malic acid was measured using HPLC. As seen in Figure 8B, the malic acid level of silencing Pbr029767.1 sample were significantly lower than infiltrating with empty vectors (both pTRV1 and pTRV2) one. Meanwhile, malic acid was evidently increased in content in the sample over-expressing Pbr029767.1, compared to the sample infiltrating with empty vector (Figure. 8D, Table S12). Thus, Pbr029767.1 was positive correlated with accumulation of malic acid in pear fruit, and should be associated with transport of acid. 3.8 Gene co-expression network analysis 18

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To further investigate the regulation of the P-type ATPase genes in Chinese white pear, a co-expression network of the P-type ATPase genes was constructed. First, we obtained all the TFs in Chinese white pear from PlantTFDB (http://planttfdb.cbi.pku.edu.cn/). Then, 24 key genes were selected from 76 P-type ATPase genes of each subfamily, and the PCCs between these genes and TFs were

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calculated. After gene pairs with PCCs < 0.995 were removed, the remaining gene

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pairs were used to construct the co-expression network. We identified 16 P-type

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ATPase genes and 78 TFs, and 96 pairs of P-type genes and TFs have co-expression

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relationships (Figure 9A).

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Here, we predicted that 61 of 78 TFs regulated only one P-type gene, but 17 of 78 TFs regulated two P-type ATPase genes, such as Pbr006809.1 regulating

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Pbr011498.1 and Pbr017990.2. For P3A-ATPase genes, we found that Pbr036112.1

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was associated with 12 TFs, while only 3 TFs may regulate Pbr011498.1.

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Pbr026279.1 and Pbr024958.1 shared 10 common TFs. Additionally, Pbr029767.1 was only associated with one TF (Pbr007180.1) (Figure 9B, Table S13).

4. Discussion P-type ATPases form a large membrane protein family whose members combine the hydrolysis of ATP with the active transport of cations or other compounds, such as phospholipids, across the membrane [54, 55]. Previous studies have identified and analyzed P-type ATPase gene family members in different plants [10, 12, 26, 56]. The number and composition of P-type ATPase family members differ in different plants 19

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[8, 9, 11]. In this study, we identified 419 P-type ATPase gene family members in seven Rosaceae species. Surprisingly, Du pear and Chinese white pear have greater gene numbers than European pear, suggesting that gene losses have occurred more frequently in the European pear genome than in the Asian pear genome. Additionally, pear and apple have undergone a recent lineage-specific WGD. Thus, the numbers of

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P-type ATPase genes in Chinese white pear, Du Pear and apple were almost double

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the numbers in Japanese apricot, peach and strawberry. A phylogenetic analysis of the

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P-type ATPase family in seven Rosaceae species and Arabidopsis revealed that this

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super family can be divided into five major subfamilies, and P-type ATPase family

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genes in seven Rosaceae species were presented in each phylogenetic group. The differences among clades might be associated with different biological functions. For

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example. motif 13 is unique to the P3A subfamily; consequently, it may be associated

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with the subfunctionalization of P3A, which may be related to H+ transport. Motifs 20

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and 6 are specific to subfamily P4, suggesting that this subfamily has a special function that is different from other subfamilies and may be related to lipid transport. Gene duplications are important for the proliferation of P-type ATPase genes in plants [57]. Duplicated genes usually evolved to obtain new functions or to partition existing functions, which could improve the adaptability of plants [50, 51]. Different types of gene duplication events, including WGD, TD, PD, TRD and DSD, contribute differently to the expansion of plant gene families. The expansions of AP2/ERF and WRKY gene families mainly resulted from TD events [58, 59], whereas the expansions of SWEET and F-box gene families mainly resulted from WGD or 20

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segment duplications [60, 61]. In this study, we found that WGD, TRD and DSD made large contributions in the expansion of the P-type ATPase gene family. In addition, based on the Ka, Ks and Ka/Ks analyses, we discovered that in pear and apple, the mean Ks values of WGD-derived gene pairs were much lower than in peach, strawberry and Japanese apricot, supporting a linkage-specific WGD event (~30 Mya)

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shared by pear and apple. Additionally, in Chinese white pear, TD-pairs had a higher

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Ka/Ks ratio, indicating that TD-derived P-type ATPase genes experienced a rapid

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functional divergence.

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For the P-type ATPase gene family, we were more interested in P3A-ATPase subfamily. The functional diversification of H+-ATPase genes has been observed in

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different plant species. H+-ATPase plays an important role in salt tolerance [62, 63].

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In addition to ion transport, the transport of organic compounds, such as sugars, is

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also dependent on H+-ATPase activity [64, 65]. In this study, we investigated the

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relationship between the P3A-ATPase genes and the transport and accumulation of organic acids in Chinese white pear. From previous reports and our expression analysis, P3A-ATPases subfamilies were identified as having strong positive correlations with the malic acid content in fruit. Here, using HPLC and RNA-seq data, we identified a candidate gene Pbr029767.1 in the P3A-ATPases subfamily that was highly associated with the accumulation of malic acid in pear fruit and changed the malate concentration during pear development. Silence of Pbr029767.1 expression suppressed malic acid when compared with that of the control, while the content of malic acid significantly increased when Pbr029767.1 was overexpressed. In citrus, 21

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PH8 was identified as located in the tonoplast and shown to affect fruit acidity [16]. In apple, Ma10 proton-motive force is able to enhance the influx of protons, not only reduces the vacuolar pH, but also provides a driving force for additional organic acid uptake [15]. In summary, Pbr029767.1 was highly reliable gene, which displayed close relationships with the transport of acid and accumulation of malate and affect

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the growth and development of Chinese white pear. Through the analysis of the gene

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co-expression network, we predicted regulatory relationships between key gene

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family members in Chinese white pear and TFs, and successfully excavated 78 TFs,

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which belonged to 33 TF families. The 41 TFs are potentially related to the P 3A genes,

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which can be used to further understand the gene regulatory network of Chinese white

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5. Conclusions

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pear’s acid transport.

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In total, 419 homologous P-type ATPase genes were identified from seven Rosaceae species. P-type ATPase genes were classified into five subfamilies according to the conserved domain and phylogenetic analysis. WGD, TRD and DSD were identified as the primary forces driving the P-type ATPase gene family expansion. Integrated with bioinformatic analysis and experimental verification, Pbr029767.1 plays an important role in fruit acid transport and accumulation. Regulatory relationships between key gene family members in Chinese white pear and TFs were further predicted. The study of the P-type ATPase gene family provides

22

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insights into the evolutionary history and has laid a foundation for molecular mechanism-related studies.

Funding This work was funded by the National Key Research and Development Program of

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China (2018YFD1000107), Key Program of National Natural Science Foundation of

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China (31830081),"Taishan Scholar" project from Shandong Province of China, the

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Earmarked Fund for China Agriculture Research System (CARS-28) and Jiangsu

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Province Science and Technology Support Program (BE2018389)

Acknowledgments

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Y.Z., Q.L. and L.X. conceived and designed the experiments. Y.Z. and Q.L. carried

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out the experimental design. Y.Z. analyzed data and drafted the manuscript. L.X. and

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Y.Z. performed the qRT-PCR experiment. C.L. provided experimental methods. L.X. and X.Q. contributed to proofreading and critical review of this manuscript. S.Z. managed the research and experiments. All authors have read and approved the final manuscript. Bioinformatic analysis supported by the Bioinformatics Center of Nanjing Agricultural University

Author details 1

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Centre of

Pear Engineering Technology Research, Nanjing Agricultural University, Nanjing 23

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210095, China

Conflict of interest The authors declare that the research was conducted in the absence of any commercial

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or financial relationships that could be construed as a potential conflict of interest.

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References 1.

+

Beyenbach KW, Wieczorek H: The V-type H -ATPase: molecular structure and function, physiological roles and regulation. Journal of Experimental Biology 2006, 209(4):577.

2.

Futai M, Iwamoto A, Omote H, Maeda M: A glycine-rich sequence in the catalytic site of F-type ATPase. Journal of Bioenergetics & Biomembranes 1992, 24(5):463-467.

3.

Pedersen PL, Carafoli E: ion motive ATPases .1. ubiquity, properties, and significance to cell-function. Trends in Biochemical Sciences 1987, 12(4):146-150.

4.

Skou JC: The Influence of Some Cations on an Adenosine Triphosphatase from Peripheral Nerves. Biochim Biophys Acta 1957, 23(2):394-401.

5.

Kuhlbrandt W: Biology, structure and mechanism of P-type ATPases. Nat Rev Mol Cell Bio 2004, 5(4):282-295. Axelsen KB, Palmgren MG: Inventory of the superfamily of P-type ion pumps in Arabidopsis.

f

6. 7.

oo

Plant Physiology 2001, 126(2):696-706.

Axelsen KB, Palmgren MG: Evolution of substrate specificities in the P-type ATPase

8.

pr

superfamily. J Mol Evol 1998, 46(1):84-101.

Axelsen KB, Palmgren MGJPP: Inventory of the superfamily of P-type ion pumps in Arabidopsis. 2001, 126(2):696-706.

Baxter I, Tchieu J, Sussman MR, Boutry M, Palmgren MG, Gribskov M, Harper JF, Axelsen KB:

e-

9.

Genomic comparison of P-type ATPase ion pumps in Arabidopsis and rice. Plant Physiol 10.

Pr

2003, 132(2):618-628.

Li Y, Provenzano S, Bliek M, Spelt C, Appelhagen I, Machado dFL, Verweij W, Schubert A, Sagasser M, Seidel TJNP: Evolution of tonoplast P-ATPase transporters involved in vacuolar

11.

al

acidification. 2016, 211(3):1092-1107.

Chen W, Si GY, Zhao G, Abdullah M, Guo N, Li DH, Sun X, Cai YP, Lin Y, Gao JS: Genomic

rn

Comparison of the P-ATPase Gene Family in Four Cotton Species and Their Expression Patterns in Gossypium hirsutum. Molecules 2018, 23(5). Appelhagen I, Nordholt N, Seidel T, Spelt K, Koes R, Quattroc hio F, Sagasser M, Weisshaar B:

Jo u

12.

TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds. Plant J 2015, 82(5):840-849. 13.

Lekeux G, Crowet JM, Nouet C, Joris M, Jadoul A, Bosman B, Carnol M, Motte P, Lins L, Galleni M et al: Homology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase. Journal of Experimental Botany 2019, 70(1):329-341.

14.

Sichul L, Yu-Young K, Youngsook L, Gynheung A: Rice P1B-type heavy-metal ATPase , OsHMA9, is a metal efflux protein. Plant Physiology 2007, 145(3):831-842.

15.

Ma B, Liao L, Fang T, Peng Q, Ogutu C, Zhou H, Ma F, Han Y: A Ma10 gene encoding P-type ATPase is involved in fruit organic acid accumulation in apple. Plant Biotechnol J 2019, 17(3):674-686.

16.

Shi CY, Song RQ, Hu XM, Liu X, Jin LF, Liu YZ: Citrus PH5-like H(+)-ATPase genes: identification and transcript analysis to investigate their possible relationship with citrate accumulation in fruits. Front Plant Sci 2015, 6:135.

17.

Bai Y, Dougherty L, Cheng LL, Xu KN: A co-expression gene network associated with developmental regulation of apple fruit acidity. Mol Genet Genomics 2015, 25

Journal Pre-proof 290(4):1247-1263. 18.

Strazzer P, Spelt CE, Li S, Bliek M, Federici CT, Roose ML, Koes R, Quattrocchio FM: Hyperacidification of Citrus fruits by a vacuolar proton-pumping P-ATPase complex. Nat Commun 2019, 10(1):744.

19.

Guo L-X, Shi C-Y, Liu X, Ning D-Y, Jing L-F, Yang H, Liu Y-Z: Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant. Scientific Reports 2016, 6.

20.

Mayerhofer H, Sautron E, Rolland N, Catty P, Seigneurin-Berny D, Pebay-Peyroula E, Ravaud S: Structural Insights into the Nucleotide-Binding Domains of the P-1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana. Plos One 2016, 11(11).

21.

Yu HY, Yan JP, Du XG, Hua J: Overlapping and differential roles of plasma membrane calcium ATPases in Arabidopsis growth and environmental responses. Journal of Experimental Li TC, Fan HH, Li ZP, Wei J, Lin Y, Cai YP: The accumulation of pigment in fiber related to

oo

22.

f

Botany 2018, 69(10):2693-2703.

proanthocyanidins synthesis for brown cotton. Acta Physiol Plant 2012, 34(2):813-818. Shi CY, Hussain SB, Guo LX, Yang H, Ning DY, Liu YZ: Genome-wide identification and

pr

23.

transcript analysis of vacuolar-ATPase genes in citrus reveal their possible involvement in citrate accumulation. Phytochemistry 2018, 155:147-154.

Fang XL, Wang L, Deng XJ, Wang P, Ma QB, Nian H, Wang YX, Yang CY: Genome-wide

e-

24.

characterization of soybean P-1B-ATPases gene family provides functional implications in 25.

Pr

cadmium responses. Bmc Genomics 2016, 17.

Wang XZ, Qian XQ, Stumpf B, Fatima A, Feng K, Schubert S, Hanstein S: Modulatory ATP binding to the E2 state of maize plasma membrane H+-ATPase indicated by the kinetics of

26.

al

vanadate inhibition. Febs J 2013, 280(19):4793-4806. Verweij W, Spelt C, Di Sansebastiano GP, Vermeer J, Reale L, Ferranti F, Koes R, Quattrocchio F:

rn

An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour. Nat Cell Biol 2008, 10(12):1456-1462.

Verma S, Evans K, Guan Y, Luby JJ, Rosyara UR, Howard NP, Bassil N, Bink MCAM, van de Weg

Jo u

27.

WE, Peace CP: Two large-effect QTLs, Ma and Ma3, determine genetic potential for acidity in apple fruit: breeding insights from a multi-family study. Tree Genet Genomes 2019, 15(2). 28.

Wu J, Wang Z, Khan MA, Shu Z, Korban SS, Ming R, Zhang S: The Genome of Pear (Pyrus bretschneideri Rehd.). In: International Plant & Animal Genome Conference XXI: 2012; 2012.

29.

Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K et al: The Pfam protein families database. Nucleic Acids Res 2010, 38(Database issue):D211-222.

30.

Eddy SR: Accelerated Profile HMM Searches. PLoS computational biology 2011, 7(10):e1002195.

31.

Fang L, Xu Y, Jiang H, Jiang C, Du Y, Cheng G, Wei W, Zhu S, Han G, Cheng BJIJoMS: Systematic Identification, Evolution and Expression Analysis of theZea mays PHT1Gene Family Reveals Several New Members Involved in Root Colonization by Arbuscular Mycorrhizal Fungi . 2016, 17(6):930-.

32.

Nakamura T, Yamada KD, Tomii K, Katoh K: Parallelization of MAFFT for large-scale multiple sequence alignments. Bioinformatics 2018, 34(14):2490-2492.

33.

Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ: W-IQ-TREE: a fast online phylogenetic 26

Journal Pre-proof tool for maximum likelihood analysis. Nucleic Acids Res 2016, 44(W1):W232-W235. 34.

Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS: ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 2017, 14(6):587-+.

35.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011, 28(10):2731-2739.

36.

Qiao X, Li Q, Yin H, Qi K, Li L, Wang R, Zhang S, Paterson AH: Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol 2019, 20(1):38.

37.

Li Q, Qiao X, Yin H, Zhou Y, Dong H, Qi K, Li L, Zhang S: Unbiased subgenome evolution following a recent whole-genome duplication in pear (Pyrus bretschneideri Rehd.). Hortic Res 2019, 6:34.

38.

Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee TH, Jin H, Marler B, Guo H et al: MCScanX:

f

a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic 39.

oo

Acids Res 2012, 40(7):e49.

Yang Z: PAML 4: Phylogenetic Analysis by Maximum Likelihood. Molecular Biology and

40.

pr

Evolution 2007, 24(8):1586-1591.

Zhang Z, Xiao J, Wu J, Zhang H, Liu G, Wang X, Dai L: ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochemical and biophysical research

e-

communications 2012, 419(4):779-781. 41.

Qiao X, Yin H, Li LT, Wang RZ, Wu JY, Wu J, Zhang SL: Different Modes of Gene Duplication

Pr

Show Divergent Evolutionary Patterns and Contribute Differently to the Expansion of Gene Families Involved in Important Fruit Traits in Pear (Pyrus bretschneideri). Frontiers in Plant Science 2018, 9.

Kim D, Landmead B, Salzberg SL: HISAT: a fast spliced aligner with low memory

al

42.

requirements. Nat Methods 2015, 12(4):357-U121. Liao Y, Smyth GK, Shi W: featureCounts: an efficient general purpose program for assigning

rn

43.

sequence reads to genomic features. Bioinformatics 2014, 30(7):923-930. Chen C, Xia R, Chen H, He Y: TBtools, a Toolkit for Biologists integrating various HTS-data

45.

Zhang SL, Ma M, Zhang HP, Zhang SL, Qian M, Zhang Z, Luo WQ, Fan JB, Liu ZQ, Wang LB:

Jo u

44.

handling tools with a user-friendly interface. bioRxiv 2018:289660. Genome-wide analysis of polygalacturonase gene fam ily from pear genome and identification of the member involved in pear softening. Bmc Plant Biology 2019, 19(1). 46.

Hao PP, Wang GM, Cheng HY, Ke YQ, Qi KJ, Gu C, Zhang SL: Transcriptome analysis unr avels an ethylene response factor involved in regulating fruit ripening in pear. Physiol Plantarum 2018, 163(1):124-135.

47.

Wang LB, Wang L, Zhang Z, Ma M, Wang RZ, Qian M, Zhang SL Genome-wide identification and comparative analysis of the superoxide dismutase gene family in pear and their functions during fruit ripening. 2018, 143:68-77.

48.

Jin JP, Tian F, Yang DC, Meng YQ, Kong L, Luo JC, Gao G: PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res 2017, 45(D1):D1040-D1045.

49.

Maere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 2005, 21(16):3448-3449.

50.

Airoldi CA, Davies BJJoG, Genomics: Gene Duplication and the Evolution of Plant MADS-box 27

Journal Pre-proof Transcription Factors. 2012, 39(4):157-165. 51.

Wei B, Zhang RZ, Guo JJ, Liu DM, Li AL, Fan RC, Mao L, Zhang XQ: Genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. Plos One 2014, 9(1):e84781.

52.

Xu LL, Qiao X, Zhang MY, Zhang SL: Genome-Wide analysis of aluminum-activated malate transporter family genes in six rosaceae species, and expression analysis and functional characterization on malate accumulation in Chinese white pear. Plant Sci 2018, 274:451-465.

53.

Sha SF, Li JC, Wu J, Zhang SL: Characteristics of organic acids in the fruit of different pear species. Afr J Agr Res 2011, 6(10):2403-2410.

54.

Lutsenko S, ., Kaplan JH, %J Biochemistry: Organization of P-type ATPases: significance of structural diversity. 1995, 34(48):15607-15613. Møller JV, Juul B, Maire MLJBBA: Structural organization, ion transport, and energy transduction of P-type ATPases. 1996, 1286(1):1-51.

Faraco M, Spelt C, Bliek M, Verweij W, Hoshino A, Espen L, Prinsi B, Jaarsma R, Tarhan E, de

oo

56.

f

55.

Boer AHJCR: Hyperacidification of vacuoles by the combined action of two different 57.

pr

P-ATPases in the tonoplast determines flower color. 2014, 6(1):32-43. Alvarez-Buylla ER, Pelaz S, ., Liljegren SJ, Gold SE, Burgeff C, ., Ditta GS, Pouplana L, Ribas De, Martínez-Castilla L, ., Yanofsky MF, %J Proceedings of the National Academy of Sciences of

e-

the United States of America: An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. 2000, 97(10):5328-5333. Zhu Y, Wu N, Song W, Yin G, Qin Y, Yan Y, Hu YJBPB: Soybean (Glycine max) expansin gene

Pr

58.

superfamily origins: segmental and tandem duplication events followed by divergent selection among subfamilies. 2014, 14(1):93.

Guo C, Guo R, Xu X, Gao M, Li X, Song J, Zheng Y, Wang XJJoEB: Evolution and expression

al

59.

analysis of the grape (Vitis vinifera L.) WRKY gene family. 2014, 65(6):1513-1528. Li J, Qin M, Qiao X, Cheng Y, Li X, Zhang H, Wu JJP, Physiology C: A New Insight into the

rn

60.

Evolution and Functional Divergence of SWEET Transporters in Chinese White Pear (Pyrus 61.

Jo u

bretschneideri). 2017, 58(4):839. Wang GM, Yin H, Qiao X, Tan X, Gu C, Wang BH, Cheng R, Wang YZ, Zhang SLJPSAIJoEPB: F-box genes: Genome-wide expansion, evolution and their contribution to pollen growth in pear ( Pyrus bretschneideri ). 2016, 253:164-175. 62.

Yingli Y, Feng Z, Meigui Z, Lizhe A, Lixin Z, Nianlai CJPCR: Properties of plasma membrane H+ -ATPase in salt-treated Populus euphratica callus. 2007, 26(2):229-235.

63.

Sibole JV, Cabot C, Michalke W, Poschenrieder C, Barceló JJP: Relationship between expression of the PM H + -ATPase, growth and ion partitioning in the leaves of salt-treated Medicago species. 2005, 221(4):557-566.

64.

Moriau L, ., Michelet B, ., Bogaerts P, ., Lambert L, ., Michel A, ., Oufattole M, ., Boutry M, . %J Plant Journal: Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme. 2010, 19(1):31-41.

65.

Burkle L, Hibberd JM, Quick WP, Kuhn C, Hirner B, Frommer WB: The H+-sucrose cotransporter NtSUT1 is essential for sugar export from tobacco leaves. Plant Physiol 1998, 118(1):59-68. 28

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Figure 1 Phylogenetic tree of P-type ATPase genes in seven Rosaceae species and Arabidopsis. The IQ-TREE was used to construct maximum likelihood phylogenetic tree with 1000 bootstrap replicates. Different colors indicate different subfamilies of P-type ATPase. The bootstrap support values showing the confidence level are given

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at the clade nodes as percentages.

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Figure 2 The conserved motifs in P-type ATPase genes of Chinese white pear

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identified by MEME. Thirty motifs were indicated by different colors.

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Figure 3 Comparison of different gene duplication modes in seven Rosaceae species. The x-axis shows the five Rosaceae species. The y-axis shows the percentage

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of different gene duplication modes. Whole-genome duplication (WGD), tandem

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duplication (DSD)

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duplication (TD), proximal duplication (PD), transposed duplication (TRD), dispersed

Figure 4 Synteny analysis of Chinese white pear and six other Rosaceae species. The chromosomes of different species are portrayed as blocks of different colors. The lines show the details of collinear gene pairs between seven Rosaceae species; the gray background represents all the collinearity pairs

Figure 5 Localization and synteny of the P-type ATPase genes in seven Rosaceae genomes. P-type ATPase genes in seven Rosaceae species were mapped on the 29

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different chromosomes. Gene pairs with a syntenic relationship were connected by a gray line

Figure 6 Comparison of Ka/Ks values for different modes of gene duplications. WGD: whole-genome duplicates; TD: tandem duplicates; PD: proximal duplicates;

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TRD: transposed duplicates; DSD: dispersed duplicates.

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Figure 7 A. Heatmap of expression levels of P-type ATPase genes in Chinese white

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pear. The color scale represents expression values, red means high expression, white

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means low expression, and blue means no expression detected. DAFB (Days after full bloom)

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B. Malic acid content at different development stages of Chinese white pear. The

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x-axis indicates different days after full bloom of pear, and the y-axis indicates malic

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content, the error bars indicate SE; 15, 45, 90, and 120 represent four different developmental stages: on 15 days after full bloom (DAFB), 45 DAFB, 90 DAFB and 120 DAFB.

C. qRT-PCR analysis of the P3A-ATPase genes at four different development stages of pear. The x-axis represents the four stages of pear fruit development. The left y-axis represents the FPKM values of RNA-seq, the right y-axis represents the relative expression levels calculated using the 2−△△Ct method. The error bars indicate standard deviation.

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Figure 8 Impact of transient transformation of Pbr029767.1 expression on malic acid of ‘Dangshansuli’ pear fruit. A. Expression profiles of Pbr029767.1 after transient silence. B. Impact of transient silence of Pbr029767.1 expression on the content of malic acid.

C. Expression profiles of Pbr029767.1 after transient

overexpression. D. Impact of transient overexpression of Pbr029767.1 expression

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on the content of malic acid. Data represent the mean ± SD of three biological

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replicates. The expression level of Pbr029767.1 in control fruit was set as 1.0.

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*represents P-value＜ 0.05 and ** represents P-value＜ 0.01.

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Figure 9 A. Co-expression relationships between 16 P-type ATPase genes and 78 transcription factor genes. The small circle on the left shows the P-type ATPase

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genes, and the large circle on the right shows the transcription factor genes.

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B. Gene Co-expression sub-network. The orange dots represent P-type ATPase

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genes and the blue dots represent transcription factors

Figure.S1 Phylogenetic tree of P-type ATPase genes in seven Rosaceae species and Arabidopsis. The phylogenetic tree was obtained by neighbor-joining (NJ) using MEGA 7 software with 1000 bootstrap replicates. Different colors indicate different subfamilies of P-type ATPase. The bootstrap support values showing the confidence level are given at the clade nodes as percentages.

Figure.S2 Analysis of cis-acting elements of P-type ATPase genes in Chinese white 31

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pear. Different cis-elements are shown in different colors.

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Figure.S3 Comparison of Ka，Ks values for different modes of gene duplications.

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author statement Yuxin Zhang, Qionghou Li and Linlin Xu conceived and designed the experiments. Yuxin Zhang and Qionghou Li carried out the experimental design. Yuxin Zhang analyzed data and drafted the manuscript. Yuxin Zhang and Linlin Xu performed the qRT-PCR experiment.

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Chunxin Liu provided experimental methods. Linlin Xu and Xin Qiao contributed to proofreading and critical review of this

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manuscript.

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Shaoling Zhang managed the research and experiments.

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All authors have read and approved the final manuscript. Bioinformatic analysis

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supported by the Bioinformatics Center of Nanjing Agricultural University

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We are the first to identify P-type ATPase gene family in seven Rosaceae species. The P-type ATPase gene family was found to have dispersed duplication as a common evolutionary mechanism.

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Integrated with bioinformatic analysis and experimental verification, Pbr029767.1 plays an important role in fruit acid transport and

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accumulation.

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Using a co-expression network, we identified several transcription factors

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family.

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that may have regulatory relationships with the P-type ATPase gene

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Journal Pre-proof Table1 Features of P-type ATPase genes identified in Chinese white pear CDS length Gene id

PI

M W (kDa)

Chr

start

end

（bp）

Amino acid length

genomic length

5.81

108.24

Chr2

11839751

11844936

2982

994

5186

Pbr001237.1

6.94

143.52

Chr12

3998690

4004073

3822

1274

5384

Pbr002050.1

6.47

114.26

Chr14

7904316

7911231

3141

1047

6916

Pbr002140.1

7.33

103.75

scaffold1079

32935

38532

2850

950

5598

Pbr002476.1

5.04

108.99

Chr5

21439117

21445459

3057

1019

6343

Pbr002671.1

8.63

94.15

Chr15

1208414

1213241

2559

853

4828

Pbr002730.1

8.37

113.23

Chr15

1613339

1616425

3087

1029

3087

Pbr004298.1

8.75

126.49

Chr8

6064640

6078098

3450

1150

13459

Pbr004369.1

8.63

113.74

Chr12

2082081

2085185

3105

1035

3105

Pbr004738.1

5.83

85.30

scaffold1224

64417

66753

2337

779

2337

Pbr004742.1

6.6

83.96

scaffold1224

86043

88958

2292

764

2916

Pbr004851.1

7.16

119.33

Chr17

14972044

14981018

3294

1098

8975

Pbr006318.1

6.66

106

scaffold132

397677

401905

2832

944

4229

Pbr007143.1

5.39

133.75

Chr14

14817495

14822844

Pbr007323.1

6.61

103.69

Chr2

Pbr008201.1

8.44

110.02

scaffold1493

Pbr008252.1

5.39

114.74

Chr12

Pbr009309.3

6.67

192.36

Pbr009695.1

6.59

Pbr011498.1

1181

5350

20719466

2844

948

4532

3051

8752

3006

1002

5702

9736331

9740826

3150

1050

4496

Chr15

3644584

3676412

5247

1749

31829

111.16

Chr7

1754543

1761826

3081

1027

7284

6.53

105.58

Chr6

1405044

1410632

2883

961

5589

Pbr012902.1

6.6

130

Chr3

2451637

2457750

3549

1183

6114

Pbr013224.1

7.48

135.52

Chr3

21638966

21644050

3594

1198

5085

Pbr014696.1

6.67

106.47

Chr13

4925599

4929031

2898

966

3433

Pbr015276.1

7.2

138.77

Chr9

7666192

7672510

3684

1228

6319

Pbr015310.1

6.69

54.59

Chr15

23993930

23998941

1530

510

5012

Pbr017956.1

7.81

134.46

Chr17

19628520

19634235

3543

1181

5716

Pbr017990.2

7.34

113.86

Chr17

19858425

19870124

3195

1065

11700

Pbr019744.1

7.07

110.61

Chr15

7233193

7237938

3051

1017

4746

Pbr019767.1

6.19

111.64

Chr15

7059830

7071896

3057

1019

12067

Pbr019915.1

6.08

94.53

Chr15

6029314

6035170

2667

889

5857

Pbr020230.1

5.5

133.71

Chr6

4141439

4146791

3549

1183

5353

Pbr021391.1

7.54

142.25

Chr10

2462279

2467290

3753

1251

5012

Pbr021467.1

4.99

107.13

Chr10

1856142

1862220

3009

1003

6079

Pbr021950.2

8.36

138.6

Chr8

13299710

13313837

3744

1248

14128

Pbr021970.1

6.92

110.94

Chr8

13038033

13044460

3051

1017

6428

Pbr022936.1

6.58

113.09

Chr2

6916727

6922104

3093

1031

5378

Pbr023454.1

5.21

40.75

Chr11

28603232

28604344

1113

371

1113

Pbr023540.3

5.61

110.56

Chr15

22674621

22680002

3048

1016

5382

Pbr023904.1

7.07

101.17

Chr4

11077973

11083770

2772

924

5798

Jo u

rn

Pr

e-

3543

20714935

al

pr

oo

f

Pbr001038.1

35

6.66

121.8

Chr9

14496287

14505609

3348

1116

9323

Pbr024238.1

5.87

113.53

Chr12

13533464

13540358

3120

1040

6895

Pbr024412.1

7.8

135.07

Chr9

21535214

21540992

3570

1190

5779

Pbr024501.1

6.11

116.02

scaffold384

308867

314450

3156

1052

5584

Pbr024716.1

8.17

144.69

Chr2

8218211

8223690

3948

1316

5480

Pbr024958.1

6.67

103.26

Chr9

4791339

4795904

2817

939

4566

Pbr025200.1

7.47

89.85

Chr2

12896634

12903127

2493

831

6494

Pbr025730.1

5.5

104.92

Chr11

8469380

8476060

2916

972

6681

Pbr026279.1

6.71

105.46

Chr9

20870860

20876711

2865

955

5852

Pbr027045.1

7.71

135.78

Chr17

24681484

24687634

3579

1193

6151

Pbr027296.1

7.06

138.33

scaffold449

24233

29107

3684

1228

4875

Pbr027297.1

7.06

138.33

scaffold449

71507

76381

3684

1228

4875

Pbr027300.1

7.05

138.28

scaffold449

226647

232654

3684

1228

6008

Pbr029113.1

5.89

114.04

Chr16

1418528

1421763

3105

1035

3236

Pbr029767.1

7.63

104.34

Chr8

11186888

11193754

2838

946

6867

Pbr030065.1

7.41

136.35

Chr13

3906532

3911347

3627

1209

4816

Pbr030078.1

7.41

136.35

Chr13

3784780

3789595

1209

4816

Pbr031014.1

6.69

106.83

Chr6

14286935

14295990

2958

986

9056

Pbr031016.1

5.46

111.34

Chr6

pr

3627

14215818

14261485

3081

1027

45668

Pbr031025.1

6.2

106.98

Chr6

14026441

14032656

2964

988

6216

Pbr031602.1

4.99

107.16

Chr10

12316315

12322074

3009

1003

5760

Pbr031825.2

6.88

118.39

Chr15

18875610

18884781

3246

1082

9172

Pbr032010.1

7.2

138.77

scaffold570

67089

73236

3684

1228

6148

Pbr032213.1

5.27

113.81

Chr8

3103806

3108882

3138

1046

5077

Pbr033845.1

6.61

103.65

Chr2

20916723

20921255

2844

948

4533

Pbr033851.1

6.61

103.65

Chr2

20966036

20970568

2844

948

4533

Pbr036112.1

6.8

105.53

Chr14

17310200

17315865

2883

961

5666

Pbr036737.1

8.3

124.37

scaffold731

58124

70066

3432

1144

11943

oo

e-

Pr

rn

Jo u

f

Pbr024083.1

al

Journal Pre-proof

Pbr036902.1

8.2

113.31

Chr14

13799675

13802857

3087

1029

3183

Pbr037297.1

7.81

101.2

scaffold757

158102

160867

2766

922

2766

Pbr037298.2

6.52

91.01

scaffold757

172589

175891

2493

831

3303

Pbr037631.1

7.92

117.37

Chr16

15898975

15918738

3231

1077

19764

Pbr038135.1

7.55

112.71

Chr16

15160526

15168624

3132

1044

8099

Pbr039171.1

8.43

137.37

Chr13

3380162

3391009

3624

1208

10848

Pbr040918.1

5.5

104.93

Chr11

8132081

8138161

2916

972

6081

Pbr041824.1

6.56

105.32

scaffold955

32971

41061

2868

956

8091

Pbr041968.1

7.1

113.14

Chr15

35494613

35497696

3084

1028

3084

M W (molecular weight), pI (isoelectric point)

36

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9