Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri)

Journal Pre-proof Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri)

Chao Tang, Xiaoxuan Zhu, Xin Qiao, Hongrui Gao, Qionghou Li, Peng Wang, Juyou Wu, Shaoling Zhang PII:

S0888-7543(19)30461-6

DOI:

https://doi.org/10.1016/j.ygeno.2020.01.021

Reference:

YGENO 9459

To appear in:

Genomics

Received date:

18 July 2019

Revised date:

20 November 2019

Accepted date:

31 January 2020

Please cite this article as: C. Tang, X. Zhu, X. Qiao, et al., Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri), Genomics (2019), https://doi.org/10.1016/j.ygeno.2020.01.021

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© 2019 Published by Elsevier.

Journal Pre-proof Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri) Chao Tang# , Xiaoxuan Zhu# , Xin Qiao, Hongrui Gao, Qionghou Li, Peng Wang, Juyou Wu* and Shaoling Zhang* State Key Laboratory of Crop Genetics and Germplasm Enhancement, Centre of Pear Engineering Technology Research, Nanjing Agricultural University, Nanjing 210095, China Corresponding author

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E-mail: [email protected]; [email protected]

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*

Fax: +862584396485

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Tel: +862584396485

Journal Pre-proof Abstract Pectin methyl-esterases (PMEs) play crucial roles in plant growth. In this study, we identified 81 PbrPMEs in pear. Whole-genome duplication and purifying selection drove the evolution of PbrPME gene family. The expression of 47 PbrPMEs was detected in pear pollen tube, which were assigned to 13 clusters by an expression tendency analysis. One of the 13 clusters presented opposite expression trends towards the changes of methyl-esterified pectins at the apical cell wall. PbrPMEs were localized in the cytoplasm and plasma membrane. Repression of PbrPME11,

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PbrPME44, and PbrPME59 resulted in the inhibition of pear pollen tube growth and abnormal deposition of methyl-esterified pectins at pollen tube tip. Pharmacological

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analysis confirmed that reduced PbrPME activities repressed the pollen tube growth.

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Overall, we have explored the evolutionary characteristics of PbrPME gene family and found the key PbrPME genes that control the growth of pollen tube, which

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deepened the understanding of pear fertility regulation.

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Keywords: Pectin methyl-esterase; Pectin; Methyl-esterification; Pollen tube; Pear

Journal Pre-proof 1. Introduction In flowering plants, compatible pollen can germinate on a receptive stigma, developing a pollen tube to deliver the male gametes to ovules for successful double fertilization [1]. This process contains coordinated pollen-pistil interactions and pollen tube development. The pollen tube is one of the fastest growing organ in nature. During its rapid growth, the pollen tube needs to synthesize a large amount of cell wall materials to maintain its structure and balance of osmotic pressure. As previous studies demonstrate, two layers can be distinguished in the pollen tube cell wall [2].

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The primary layer mainly consists of polysaccharides (cellulose, hemicelluloses, and pectins) and the secondary layer is made up of callose. At the apex of the pollen tube,

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the wall is mostly composed of pectins [3, 4], and they determine mechanical

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properties of the pollen tube [5, 6]. Pectins are synthesized in cis-Golgi and methyl-esterified in medial-Golgi, and then delivered to the tip of the pollen tube in a

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highly methyl-esterified state. The methyl-esterified pectins were characterized to make the cell wall continuously extendable until they are demethyl-esterified by

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pectin methyl-esterases (PMEs; EC 3.1.1.11). PMEs catalyze the specific

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demethyl-esterification of the homogalacturonan (HGA) fraction [42, 43], then the

[7, 8].

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demethyl-esterified pectins are able to cross-link with Ca2+ and rigidifies the cell wall

Identified to express widely in higher plants, PMEs control multiple development stages from seed germination to fruit ripening [7, 9, 10]. Reduced PME activities are found to result in the reduction of germination time in Arabidopsis [11]. PME activities also contribute to the modification of seed coat mucilage by altering the interactions between the methyl-esterified pectins and the rhamnogalacturonic fractions [12]. PMEs play important roles in maintaining the mechanical properties of the pollen tube cell wall as mentioned, the loss of which would eventually trigger the instability and bursting of the pollen tube. The function loss of VGD1, a PME-homologous gene, leads to the formation of a barrier of pollen tube growth in the style and reduces male fertility [13]. AtPPME1, a pollen-specific PME gene, inhibits the growth rate by affecting the morpho logy of the pollen tube [14]. Moreover,

Journal Pre-proof the addition of exogenous PME has been proved to inhibit pollen tube growth by thickening the cell wall at the apex [6, 15]. According to an analysis of protein structures, PMEs can be classified into two main groups: Type I PME, which has an additional N-terminal pro-region (similar to PMEI domain), and Type II PME, which contains a single PME domain [7, 20]. Both Type I and Type II PMEs exhibit the activity of pectin methyl-esterification; however, the pro-region of Type I PMEs has been shown to affect the targeting of Type I PMEs via the exocytotic pathway and the pro-region exerts an auto- inhibitory activity of PME domain in tobacoo Type I PME

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[6, 61].

To date, genome-wide identification and comparative analysis of PME gene

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families have been reported in many plant species, including Linum usitatissimum

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[16], Brassica rapa [17], Gossypium arboretum [18], and Arabidopsis thaliana [19]. According to the systematic view of PME gene family in Arabidopsis, PMEs were

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subdivided into four distinct subfamilies. On the other hand, Whole-genome duplication (WGD) and single- gene duplication, such as tandem duplication and

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transposed duplication, can drive the expansion of PME gene families [17, 21].

remain unclear.

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However, the identification and evolutionary history of PME gene family in pear

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In this study, we performed genome-wide identification of PMEs in pear. The gene structure, evolutionary history, and expression pattern of PbrPME family genes were analyzed. PbrPME11, PbrPME44, or PbrPME59 were found to be involved in pectin methyl-esterification at the apex of pollen tube in pear. Suppressing the expression of PbrPME11,

PbrPME44,

or

PbrPME59

resulted

in

the

accumulation

of

methyl-esterified pectins at the pollen tube apex and inhibited its growth. 2. Materials and Methods 2.1. Plant growth conditions and RNA extraction All materials used in this study were collected from pear trees (Pyrus bretschneideri Rehd. cv. ‘Dangshansuli’) grown in the Nanjing Agricultural Fruit Experimental Field, China. Roots, stems, leaves, and fruits were collected and frozen immediately in liquid nitrogen and stored at -80ºC. Anthers were collected and dried for 24 h, then

Journal Pre-proof stored in silica gel at -20ºC. The pistils were isolated and stored in liquid nitrogen. Pollen was cultured using methods as described in our previous study [22]. Total RNA was extracted from frozen tissue using a Plant Total RNA Isolation Kit (FOREGENE, China) according to the manufacturer’s instructions (http://www.foregene.com/). 2.2. Identification of PME gene family sequences The sequences of 66 AtPME proteins were retrieved from the TAIR10 database (http://www.arabidopsis.org/). The sequences of PbrPME proteins were retained from the pear genome project (http://peargenome.njau.edu.cn/) [23]. The sequences of

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AtPME proteins AT1G02810 (group 1), AT2G47030 (group 2), AT5G09760 (group 3), and AT1G69940 (group 4) were used as queries to perform BLASTP searches against

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the pear genome with an E-value < 1 × 10-10 [19]. Hidden Markov Model (HMM)

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profiles for the PME domain (PF01095) and pectin methyl-esterase inhibitor (PMEI) domain (PF04043) were downloaded from the Pfam database (http://pfam.xfam.org/).

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Then, the two profiles were used as queries to search for the genes homologous to PME using HMMER3 software (E value < e-10 ). To validate the search results, all sequences

were

further

confirmed

using

SMART

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candidate

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(http://smart.embl-heidelberg.de/) and the Pfam database (http://pfam.xfam.org/) by

removed.

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checking the PME and PMEI domains. Redundant sequences were manually

2.3. Phylogenetic analysis

The gene ID of collected plant species is shown in Table S2. The taxonomic tree of investigated plant species was obtained

from the NCBI taxonomy tools

(https://www.ncbi.nlm.nih.gov/taxonomy). The tree was drawn and graphically edited using FigTree version 1.4.3. Phylogenetic trees of PMEs were constructed using the maximum likelihood (ML) method with MEGA 7.0 software [24]. The complete amino acid sequences of PbrPMEs were aligned using the ClustalW program. A bootstrap test with 1,000 replications was carried out. The tree was further graphically edited using FigTree version 1.4.3. 2.4. Protein motif and gene structure analysis

Journal Pre-proof The conserved motifs of PMEs in Arabidopsis and pear were investigated using Multiple Expectation-Maximization for Motif Elicitation (MEME) tool version 5.0.5 (http://meme-suite.org/tools/meme) [25]. The results were plotted using TB tools. The details of the motifs are shown in Fig. S4. The CDS and genomic sequences of PME family genes were parsed from the TAIR10 database and pear genome project. The gene structure of PMEs was analyzed using the Gene Structure Display Server (GSDS version 2.0) (http://gsds.cbi.pku.edu.cn/) [26]. 2.5. Chromosomal localization and synteny analysis

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The chromosomal locations of PMEs was obtained based on genome annotation data. The syntenic relationship between Arabidopsis and pear genomes was constructed

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using a method similar to that developed for the Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication/) [27, 28]. Initially, potential homologous

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gene pairs were detected using the BLASTP algorithm (E < 1 e−5 , top five matches)

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between two genomes. Secondly, the BLASTP results and the gene location information were input into MCScanX software to identify the syntenic chains. Whole

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genome, tandem, proximal, and dispersed duplications were identified by MCScanX.

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Syntenic relationship and gene location data were presented using Circos software [29]. Genes located on unanchored scaffolds were not included.

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2.6. Calculating Ka and Ks

MCScanX downstream analysis tools were used to estimate the nonsynonymous (Ka) and synonymous (Ks) substitution rates between syntenic gene pairs with the Nei-Gojobori (NG) method. KaKs_Calculator version 2.0 was used to calculate the Ka and Ks of duplicated gene pairs in different PME subfamilies with the NG method [30]. 2.7. RT-PCR The expression patterns of 81 PbrPMEs in various tissues were determined using RT-PCR. Total RNA was extracted from collected samples as mentioned above. The purified cDNA was reverse transcribed using the RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific, USA). The PCR reaction was conducted in a total volume of 20 μ L containing 0.1 μL cDNA sample, 4 μL of 0.1 μM gene-specific

Journal Pre-proof primer mixture, 10 μ L of 2×Taq Plus Master Mix (Vazyme, China), and 5.9 μL double distilled water. The PCR reaction was performed with the following program: 3 min at 94°C, 25 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C, and a final extension of 10 min at 72°C. The PbrTUB-2 was used as the reference gene. PCR products were fractionated on 2% agarose gel. Image J software was used to measure the gray-scale of the image and the expression patterns of 81 PbrPMEs in various tissues were presented using Morpheus software (https://software.broadinstitute.org/morpheus). Primers are listed in Table S1.

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2.8. Quantitative real-time PCR

The expression analysis of 47 PbrPMEs in the growth period of pear pollen tube (1–5

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h) was quantified by quantitative real-time PCR (qPCR). The qPCR reaction was conducted in a total volume of 20 μL containing 0.2 μL cDNA, 5 μL of 0.5 μM

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gene-specific primer mixture, 10 μL of 2 × SYBR Green Master Mix, and 4.8 μL

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water. The qPCR was performed using the LightCycler® SYBR Green I Master (Roche, Germany). Data was calculated using the 2 -ΔΔCT method [31]. PbrTUB-2 was

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used as the internal standard. The melting curve was implemented to verify the

result.

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efficiency of all qPCR primers. Three biological replicates were employed for each

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2.9. Subcellular localization analysis For the subcellular localization analysis of PbrPMEs, the CDS of PbrPME11, PbrPME20, PbrPME29, PbrPME30, PbrPME44, PbrPME46, PbrPME59, and PbrPME78 without the termination codon were amplified and then cloned into a pCAMBIA1300-35S: CDS-GFP vector [32]. The recombinant plasmids were transformed into tobacco (Nicotiana benthamiana) leaf using a previously published protocol [33]. The red- fluorescent dye FM4-64 (Invitrogen, USA) was used as a membrane marker. The fluorescence was imaged using a confocal microscope LSM780 (Zeiss, Germany). 2.10. LM20 immunohistochemical analysis of the pollen tube In vitro pollen germination in liquid medium (1-5 h) was performed following a published protocol [34]. Samples were centrifuged (3,000 rpm, 3 min) to remove the

Journal Pre-proof supernatant and then fixed with 4% paraformaldehyde. The immunohistochemistry of methyl-esterified pectin was performed [35]. The fluorescence was imaged using an LSM780 confocal microscope (Zeiss, Germany). Three independent biological replicates were set and assayed for each experiment. ZEISS ZEN imaging software (https://www.zeiss.com) was used for quantification. LM20

primary

antibodies,

which

can

recognize

methyl-esterified

homogalacturonan (diluted 1:20), were acquired from the Knox Cell Wall Lab

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Sigma, USA) was employed as the secondary antibody

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(http://www.plants.leeds.ac.uk/pk/antibodies.htm). Anti-rat IgG-FITC (diluted 1:500;

2.11. Gallic acid treatment

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Gallic acid (Macklin, China) was dissolved in alcohol to make a 10% mother solution

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for further assay. The pollen sample was pre-cultured in liquid germination media for 1 h, followed by adding gallic acid with a final concentration of 0.1%. Data was

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acquired using a Nikon TE100 microscope. The length of the pollen tubes was measured by Image-Pro Plus 6.0 (http://www.mediacy.com/).

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2.12. Measurement of PME activity

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Protein extraction from pear pollen tubes was performed following the methods described by a previous study [36]. PME activity was measured according to a

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published study [37]. Three biological replicates were employed for each treatment. 2.13. Antisense oligodeoxynucleotide experiment Both phosphorothioate antisense oligo deoxynucleotide (as-ODN) and sense control (s-ODN)

were

designed

using

RNA

fold

Web

Server

(http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Soligo software was used to calculate efficient

candidate

as-ODN

sequences

for

suitable

target

regions

(http://sfold.wadsworth.org/soligo.pl). The antisense oligonucleotide experiment was performed as previously described [38, 39]. The length of the pollen tubes was measured using a Nikon TE100 microscope with Image-Pro Plus 6.0. The sequences of oligonucleotide primers are listed in Table S1. 2.14. Analysis of expressional tendency

Journal Pre-proof Gene expression tendency analysis was performed by Short Time-series Expression Miner software (STEM) on the OmicShare tools platform [40], a free online platform for data analysis (www.omicshare.com/tools). The parameters were set as follows: Maximum Unit Change in model profiles between time points was 1; Maximum output profiles number was set to 20 (similar profiles will be merged); Minimum ratio of fold changes of differentially expressed genes was no less than 2.0. 2.15. Statistical analysis All experimental data was analyzed by SPSS version 22. Two groups of samples were

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compared using Student’s t-test. Multiple samples were analyzed using ANOVA followed by LSD, Duncan’s, or Turkey’s difference test.

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3. Results

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3.1. Genome-wide identification and classification of PMEs In this study, 81 PbrPME candidates were identified in pear, including 56 type I

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PbrPMEs with a single PME domain and 25 type II PbrPMEs with both a PME and PMEI domain (Fig. 1B). The evolutionary relationships of investigated species and

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the number of PME genes are shown in Fig. 1A and Table S2. Based on the common

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taxonomic tree, we found that PMEs were not detected in green algae, but diverged in the genomes after the initial existence of pectin in the plant cell wall. In addition, we

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found that PMEs were widely dispersed among the basal angiosperm Amborella trichopoda, monocot and eudicot. The size of the PME gene family in different species was influenced by WGD events. A maximum- likelihood phylogenetic tree was built using the protein sequences of PMEs from pear and Arabidopsis (Fig. 2). According to the previous definition of AtPMEs [19], PbrPMEs were divided into four distinct subfamilies (A, B, C, and D). PbrPMEs containing a PMEI domain were clustered into subfamilies A, B, and D, whereas PbrPMEs without the PMEI domain were clustered into subfamily C. 3.2. Gene features and conserved motifs of PMEs The exon- intron structures of 81 PbrPMEs were compared according to their phylogenetic relationships (Fig. 3). Gene features, such as gene ID, gene length, and exon length, are shown in Table S3. The gene structures of PMEs varied in the

Journal Pre-proof different subfamilies. The length of the exons and average length of exons in subfamily C were less than those in the other three subfamilies, and the number of exons in subfamily C was greater than that in the other three subfamilies (Fig. S1 A, B, D). Furthermore, the members of subfamily C possessed shorter gene lengths than the members of subfamily A, B, and D due to the loss of the PMEI domain (Fig. 4C) In total, 30 conserved motifs were identified, the logos of which were acquired from the MEME Suite web server (Fig. 3 and Fig. S4). Conserved motifs 1, 2, 3, 4, 5, 7, and 17, which are characterized as the PME domain, were present in all PME genes,

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yet motifs 9, 12 ,13, 15, and 24, forming the PMEI domain, were identified to exist only in the PMEs of subfamilies A, B, and D. Members of subfamily C (8-15) had

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fewer motifs than subfamilies A (8-24), B (14-18), and D (18-22). In addition, the

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length of peptides in subfamily C was shorter than in subfamilies A, B, and D (Fig. S1D). Motif 5 was widely present in subfamilies A, B, and D, but was rarely present

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in subfamily C. Members of subfamily C generally had similar motifs, except for motif 25, which was only found in a few genes including AT5G07420, AT5G07430, AT1G69940,

AT5G07410,

PbrPME16,

PbrPME80,

PbrPME33,

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AT5G61680,

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PbrPME32, PbrPME40, and PbrPME41.

3.3. Chromosomal localization and evolutionary analysis of the PME gene family

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Most PbrPMEs were mapped onto 16 out of 17 pear chromosomes; 19 genes were located on the scaffolds (Fig. 4 and Table S3). AtPMEs were mapped onto five out of the six Arabidopsis chromosomes and mainly located on three chromosomes (Chromosome 2, 3, and 5).

Gene duplication can drive the expansion of gene families and provide raw material for generating new functions. Therefore, we investigated the different types of gene duplications in the PbrPME gene family. Each gene was allotted to a single duplication mode: singleton, WGD/segmental, tandem, proximal, or dispersed (Table S5). WGD followed by dispersed duplication largely accounted for the expansion of the PbrPME gene family. PbrPMEs in subfamily B only underwent a WGD and a tandem duplication event (Fig. 4A).

Journal Pre-proof To explore the evolutionary trajectory of PbrPMEs, the syntenic relationship among PME family genes was analyzed using the MCScanX package. Twenty-seven gene pairs were found in the PbrPME gene family (Table S4). We calculated the Ka/Ks value for each gene pair to detect the selection pressure on PbrPMEs. Overall, most

Ka/Ks

values

were

less

than

one,

except

for

two

gene

pairs,

PbrPME33-PbrPME32 (Ka/Ks = 1.8523) and PbrPME76-PbrPME5 (Ka/Ks = 1.2025), suggesting that purifying selection was the primary evolutionary force acting on PbrPMEs. We also observed that subfamily D possessed a higher Ka/Ks value than

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other subfamilies, even though subfamily C had one gene pair possessing a relatively high Ka/Ks value (Fig. 4B). These results indicated that the evolutionary rate of

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PMEs in subfamilies A, B, and C was slower than the evolutionary rate of subfamily

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D.

3.4. Expression pattern of PME family genes

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We made a heat map using the results of the RT-PCR analysis to investigate the expression patterns of PbrPMEs in five different tissues of pear. As presented in Fig.

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5 and S3, genes in subfamily A were preferentially expressed in the pistil compared to

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genes in subfamily B. Genes in subfamily B were expressed in all tissues, except for PbrPME12 which was only expressed in the pollen tube and pistil. Additionally,

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genes in subfamilies C and D expressed in both vegetative and reproductive tissues but occupied a larger proportion in reproductive tissues than vegetative tissues, except PbrPME34 and PbrPME50.

3.5. Expression of PbrPMEs in pear pollen tube growth To explore the roles of PbrPMEs in plant reproduction, we investigated the expression levels and corresponding methyl-esterified pectins in pear pollen tubes. Here, we focused on the change in methyl-esterified pectin during the first 5 hours of pollen tube growth. As shown in Fig. 6 A and B, methyl-esterified pectin primarily spread at the tip of the pollen tube, along the first 5 μm of meridional length. Additionally, methyl-esterified pectin exhibited varying distributions at the tip of the pollen tube. The measuring fluorescence became weak at 2 h, but was intense at 1 h, 4 h, and 5 h (Fig. 6C, S5). To further investigate the relationship between

Journal Pre-proof methyl-esterified pectin and pear pollen tube growth, a PME inhibitor, gallic acid, was used to assess the influence [44]. The activity of PME decreased to approximately 28% of the control value, and the pollen tube growth was suppressed in the gallic acid challenge (Fig. 6D, S7). These results suggested that PbrPMEs played important roles in promoting the growth of pear pollen tubes. To explore the major expression trends of PbrPMEs from 1 to 5 hour time points, qPCR was performed using gene-specific primers (Table S1). Genes that shared a similar expression pattern were assigned to 13 clusters (Table S6). Among them,

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cluster 1 was found to be statistically up-regulated from 1 h to 2 h and down-regulated from 3 h to 5 h; cluster 2 was found to be statistically down-regulated from 3 h to 4 h

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and up-regulated from 4 h to 5 h (p- value < 0.05). We also observed that clusters 7, 8,

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10, 11, 12, and 13 were up-regulated from 1 h to 2 h. In addition, clusters 1, 2, 3, 4, 5, 8, 9, and 12 were down-regulated from 3 h to 4 h (Fig. 6E).

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3.6. Subcellular localization of PbrPMEs expressed in pear pollen tubes The protein structure analysis of eight PbrPMEs expressed in pear pollen tubes

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showed that both PbrPME11 and PbrPME78 had a signal peptide at the N terminal.

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However, the other six PbrPMEs possessed a transmembrane domain at the N terminal. The corresponding subcellular localization of eight PbrPMEs were further

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detected. As seen in Fig. 7, PbrPME11, PbrPME29, and PbrPME78 were localized to the cytoplasm and plasma membrane. PbrPME20, PbrPME30, PbrPME44, PbrPME46, and PbrPME59 were localized to the plasma membrane, which were in accordance with the protein structures. 3.7. Functional analysis of PbrPME11, PbrPME44 and PbrPME59 in pear pollen tube growth To confirm the contribution of PbrPMEs to pear pollen tube growth and pectin dimethyl-esterification, the oligo deoxynucleotide (ODN) technique was used to specifically knock down PbrPME11, PbrPME44, and PbrPME59 in pear pollen, respectively. ODN is a method that has been previously employed to specifically suppress target gene expression in pollen tubes [38, 39, 45, 46]. As shown in Fig. S2, the expression levels of PbrPME11, PbrPME44, and PbrPME59 were decreased by

Journal Pre-proof ODN. Moreover, either the specific knockdown of PbrPME11, PbrPME44, or PbrPME59 led to a significant decrease in pollen tube length (Fig. 8A). We then observed the degree of pectin methyl-esterification at the tip of the pollen tube, and found that the fluorescence became strong after as-ODN treatments (Fig. 8B and C). Unexpectedly, the specific knockdown of either PbrPME44 or PbrPME59 resulted in the swelling of the pollen tube tip (Fig. 8B), and the specific knockdown of either PbrPME44 or PbrPME59 showed no effects of mRNA degradation on other PbrPMEs (Fig. S8 and Fig. S9). These results indicated that methyl-esterified pectin

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could be mediated by PbrPME11, PbrPME44, and PbrPME59 in the pollen tubes of pear.

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4. Discussion

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As a significant modifier of methyl-esterified pectins in the plant cell wall, PMEs are involved in many physiological processes, such as fruit maturation [47], cellular

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adhesion [48], stem elongation [49], and pollen tube growth [6, 14]. In this study, we identified 81 PME genes in pear. The phylogenetic analysis showed that PbrPME

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family genes could be classified into four subfamilies (A, B, C, and D), which is

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consistent with a previous report [19]. WGD was the major driving force for the expansion of the PbrPME gene family. Evolutionary analysis indicated that purifying

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selection played a central role in driving the evolution of PbrPMEs. In addition, the expression analysis showed that PbrPMEs were expressed in different tissues and developmental stages of pollen tube. Importantly, the changes of methyl-esterified pectins at the tip during the first 5 h of pear pollen tube growth were observed. The growth of the pear pollen tube was inhibited by reduced methyl-esterified pectins. Finally, the co-expression analysis showed that the expression of eight PbrPMEs was associated with the changes of methyl-esterified pectins. Knocking down method with antisense oligonucleotides suggested that PbrPME11, PbrPME44, and PbrPME59 could accelerate pear pollen tube growth. Almost

all

plants

have

undergone

paleo-polyploidization

[50,

51].

Polyploidization contributes to the proliferation of specific gene families and provides chances for generating diverged functions between duplicated genes over their long

Journal Pre-proof evolutionary history [52, 53]. In Brassica rapa, Linum usitatissimum, and Gossypium arboretum, PME gene families have also experienced WGD [16-18]. Here, the taxonomic tree suggested that PMEs were prevalent across the plant kingdom, except in green algae. Forty-seven PMEs were identified in Physcomitrella patens but no PMEs were found in green algae, suggesting that the PME gene families appeared after the divergence of the bryophytes and accompanied the appearance of pectins. The results of the phylogenetic analysis, gene/motif structural analysis, and intron/exon analysis showed that three subfamilies (A, B, and D) were different from

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subfamily C; this may result from the loss and modification of the PMEI domain at the genome level [17]. WGD leads to the expansion of different gene families in pear

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[27, 54, 55]. Brassica rapa experienced an additional whole-genome triplication event

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and had more PMEs than Arabidopsis thaliana [41]. Each of the duplicated genes generated by WGD may then show subfunctionalization or neofunctionalization [56,

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57]. In this study, WGD occupied the majority of duplication events throughout four subfamilies, indicating that the expansion of the PbrPME gene family was driven by

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WGD. Most Ka/Ks ratios of PbrPME gene pairs were less than one, implying that

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they might evolve under purifying selection, however, further analysis, such as the branch-specific model of Phylogenetic Analysis by Maximum Likelihood (PAML)

selection.

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[62], could help to verify whether PbrPME gene pairs have underwent positive

PME participates in the pollen tube growth by modifying the plasticity of the cell wall at the tip of the pollen tube [36, 37, 58-60]. In our study, we first investigated the change of methyl-esterified pectins at the apical cell wall during the first 5 h of pollen tube growth, and we found that methyl-esterified pectins reflected a dynamic and recurring deposition, which may be related to the oscillatory growth of the pollen tube [42]. We repressed the PME activity by gallic acid and found that the length of the pollen tube was reduced. These results suggested that the methyl-esterified pectins at the apical cell wall were important for the growth of the pear pollen tube. Thus, which PbrPMEs might be involved in this process? To determine this, we then selected eight PbrPMEs with remarkable change and opposite expression trends from the change of

Journal Pre-proof methyl-esterified pectins at the apical cell wall. Subcellular localization showed that eight PbrPMEs had the plasma membrane localization, which may be important for their functions. Meanwhile, the ODN assay showed that the repression of PbrPME11, PbrPME44, or PbrPME59 could accelerate the deposition of methyl-esterified pectins at the apical cell wall, and even the swelling of the tips of the pollen tube. Thus, we proposed that PbrPME11, PbrPME44, and PbrPME59 were the candidate genes functioning in the dynamic change of methyl-esterified pectins and pollen tube growth in pear. Further studies should focus on verifying this hypothesis.

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In conclusion, this work presented the phylogenetic relationships, evolutionary history, and expression patterns of PbrPME family genes. PbrPMEs directly affected

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the growth of pear pollen tubes. During the early developmental stages of the pollen

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tube, eight PbrPMEs were selected as candidate genes due to their expression trends associated with the changes of methyl-esterified pectins at the apical cell wall. Plasma

PbrPME44,

and

PbrPME59

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membrane localization was common among the eight PbrPMEs. PbrPME11, could

directly

regulate

the

distribution

of

pollen tube. Author contributions

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methyl-esterified pectins, suggesting their important roles in the growth of the pear

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Conceived and designed the experiments: Juyou Wu and Shaoling Zhang. Experiments performance: Chao Tang, Xiaoxuan Zhu and Qionghou Li. Manuscript writing: Chao Tang and Xiaoxuan Zhu. Manuscript revision and confirmation: Xin Qiao, Chao Tang, Xiaoxuan Zhu, Peng Wang, Shaoling Zhang and Juyou Wu. All the participators have read and confirmed the paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (31772276); A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions; and China Postdoctoral Science Foundation (2017M620213). Bioinformatic analysis was supported by the Bioinformatics Center of Nanjing Agricultural University. Conflict of interest

Journal Pre-proof The authors declare that they have no conflict of interest. Figure legends Fig. 1 The evolutionary relations hips of the collected species and the nume rical details of the PME superfamily of each species. (A) The red/yellow/green dots represents the whole- genome duplication events in the species, the blue box indicates the appearance of pectins in the cell wall of different organisms. ‘ - ’ indicates no detection, and the number of PME subfamily members is given in red. (B) The illustrative diagram of type I and type II PMEs. SP: signal

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peptide, TM: transmembrane domain.

Fig. 2 Maximum-likelihood phylogeny of PME family proteins from Pyrus and

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Arabidopsis.

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The phylogenetic tree was constructed by MEGA 7.0 software using full- length protein sequences. A bootstrap analysis was conducted with 1,000 replicates. The

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yellow, blue, red, and green backgrounds indicate the four subfamilies (A, B, C, D) of PMEs in Pyrus (orange lines) and Arabidopsis (green lines), respectively. The scale

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bar is 2.0.

and Arabidopsis.

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Fig. 3 The exon-intron structure and conserved motif analysis of PMEs in Pyrus

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The maximum- likelihood tree was built using MEGA 7.0 software. Exons and introns are shown by yellow rectangles and thin lines, respectively. The untranslated regions (UTRs) are indicated by blue rectangles. Each motif is marked with a number in a colored box (motifs 1 to 30). Settings: number of motifs: 30, minimum motif width: 10, and maximum motif width: 50. The yellow, blue, red, and green backgrounds indicate the four subfamilies (A, B, C, D) of PME members in Pyrus (orange circles) and Arabidopsis (green triangles). The orange circle represents PbrPMEs and the green circle represents AtPMEs. Fig. 4 Synteny and expansion analysis of the PME gene family in the pear genome. (A) Quantification statistics for different duplication events in four PbrPME subfamilies. (B) Evolutionary rate variance analysis of four PbrPME subfamilies. Ks

Journal Pre-proof synonymous

substitution

rate,

Ka

non-synonymous

substitution

rate.

(C)

Chromosomes of Pyrus (green) and Arabidopsis (blue) are shown and chromosome numbers are shown on the inner side. The approximate positions of PMEs are marked with short red lines on the circles. Gene pairs with syntenic relationships are joined by the colored lines. Fig. 5 Expression analysis of PbrPMEs in six tissues and four developmental stages of pear fruit. Sources of the tissue samples were as follows: root, stem, leaf, pollen (pollen tube),

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fruit, and pistil. Dark purple indicates high expression, light purple indicates intermediate expression, and white indicates no expression.

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Fig. 6 PbrPMEs contributed to pear pollen tube growth.

(A) Relative spatial distribution of methyl-esterified pectin in a pear pollen tube.

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Bars=20 μm. (B) Illustrative diagram of nomenclature of distance measurements. (C)

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Corresponding quantification of LM20 staining for the pollen tubes shown in A. Different line colors represent fluorescence intensity for 1–5 h samples. The values on

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the x-axis represent the distance along the meridional cell surface from the pole. Data

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are the mean ± SEM, n = 3 biological replicates. (D) The pollen tube length under 0.1% gallic acid. Different letters indicate significant differences, as determined by

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ANOVA followed by LSD and Tukey’s multiple comparison test (p < 0.05, n = 3). Data are shown as mean ± s.e.m. (E) Short Time-series Expression Miner (STEM) and cluster analyses of PbrPMEs expressed in pollen tubes. The top number is the gene number allocated in each cluster and the bottom number represents the p-value. Fig. 7 Subcellular localization of eight PbrPME-GFP proteins in tobacco leaf. The

PbrPPA11-GFP,

PbrPPA20-GFP,

PbrPPA29-GFP,

PbrPPA30-GFP,

PbrPPA44-GFP, PbrPPA46-GFP, PbrPPA59-GFP, and PbrPPA78-GFP fusion proteins and the GFP control were transiently expressed in transformed tobacco leaf epidermal cells, and were observed under a confocal microscope. Green represents the fluorescence intensity of GFP; red represents the fluorescence intensity of FM4-64. Bars = 20 μm.

Journal Pre-proof Fig. 8 Knockdown of the expression level of PbrPME11, PbrPME44 or PbrPME59 caused the

inhibition of both

pear pollen tube

growth and pectin

demethyl-esterification. (A) Treatment with PbrPME11/44/59 antisense oligonucleotides (30 μm as-ODN) resulted in the inhibition of pollen tube growth. Cytofectin (Lipofectamine 2000) was the reagent used for the ODN treatment. s-ODN was transfected with sense oligonucleotides in the presence of cytofectin for the negative control. The pollen tube length (three replications with 50 pollen tubes. * p < 0.05 by Student’s t-test) was

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measured by Image-Pro Plus 6.0. Data are the mean ± s.e.m., n = 3 biological replicates. (B) LM20 staining of growing pollen tubes under s-ODN and as-ODN

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treatments. FITC, fluorescein isothiocyanate. Bars = 20 μm. (C) Graphs show

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fluorescence intensity from the position (denoted by white dashed lines); the x-axis values represent the distance from the first dash above (in pixels) along the dashed

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lines.

Suppleme ntary Fig. S1 Boxplot for the length of exons (A), number of exons (B),

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length of genes (C), average exon length (D), and length of peptide s (E) of four

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distinct subfamilies in AtPMEs and PbrPMEs genes. Subfamilies A, B, C, and D are highlighted in yellow, blue, red, and green,

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respectively. Different letters indicate significant differences, as determined by ANOVA followed by Duncan’s Test (p < 0.05). Suppleme ntary Fig. S2 qPCR expression analysis of as-ODN-mediated knockdown of PbrPME11/44/59 expression. (A) PbrPME11-knockdown pollen exhibited a marked decrease in expression level. PbrTUB-2 was used as the reference gene. Data are the mean ± SEM.

**

p < 0.01 by

Student’s t-test. (B) PbrPME44-knockdown pollen showed marked decrease in expression level. (C) PbrPME59-knockdown pollen showed marked decrease in expression level. Supplementary Fig. S3 RT-PCR expression analysis of PbrPMEs. Six tissues (root, stem, leaf, pollen, pistil, and fruit) were used for this analysis.

Journal Pre-proof PbrTUB-2 was used as the reference gene. Experiments were performed with at least three biological replicates. Supplementary Fig. S4 Logo of motifs in PbrPMEs using MEME. The settings were: maximum numbers of different motifs, 30; minimum motif width, 10; and maximum motif width, 50. Suppleme ntary Fig. S5 Quantification of the peak fluorescence intensity for LM20 staining in figure 6A. Different letters indicate significant differences, as determined by an ANOVA

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followed by LSD and Tukey’s multiple comparison test (p < 0.05). Data are the mean ± s.e.m.

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Suppleme ntary Fig. S6 Quantification of the peak fluorescence intensity for

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LM20 staining in Figure 8B.

Different letters indicate significant differences, as determined by ANOVA followed

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by LSD and Tukey’s multiple comparison test (p < 0.05). Data are the mean ± s.e.m. Suppleme ntary Fig. S7 Measure ment of PME activity in the presence of 0.1%

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gallic acid.

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The PME activity in the control was used as a calibrator (set as 100%). Data are the mean ± SEM. * p < 0.05 by Student’s t-test. Experiments were performed with at least

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three biological replicates.

Suppleme ntary Fig. S8 qPCR detection of antisence-ODN-mediated knockdown of PbrPME44 expression.

qPCR detection of antisence-ODN-mediated knockdown of PbrPME44 affects target PME expression. PbrPME44-knockdown pollen exhibited a marked decrease in mRNA levels. There were no effects of mRNA degradation on other PbrPMEs during antisence-mediated

knockdown of targeted

PbrPME44.

"s-ODN-PbrPME44"

represents "sence-oligo deoxynucleotide-PbrPME44", and "as-ODN-PbrPME44" represents "antisence-oligo deoxynucleotide-PbrPME44". Suppleme ntary Fig. S9 qPCR detection of as-ODN-mediated knockdown of PbrPME59 expression.

Journal Pre-proof qPCR detection of antisence-ODN-mediated knockdown of PbrPME59 affects target PME expression. PbrPME59-knockdown pollen exhibited a marked decrease in mRNA levels. There were no effects of mRNA degradation on other PbrPMEs during antisence-mediated

knockdown of targeted

PbrPME59.

"s-ODN-PbrPME59"

represents "sence-oligo deoxynucleotide-PbrPME59", and "as-ODN-PbrPME59" represents "antisence-oligo deoxynucleotide-PbrPME59". Supplementary Table S1 List of primers used in the paper. Supplementary Table S2 Gene ID of the collected species.

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Supplementary Table S3 Information of the PMEs in Pyrus and Arabidopsis. Supplementary Table S4 Inference of Ka and Ks in paralogous pairs.

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Suppleme ntary Table S5 Investigation of different duplication events in PbrPMEs.

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Supplementary Table S6 Data used in the STEM analysis.

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Journal Pre-proof Author statement: Conceived and designed the experiments: Juyou Wu and Shaoling Zhang. Experiments performance: Chao Tang, Xiaoxuan Zhu and Qionghou Li. Manuscript writing: Chao Tang and Xiaoxuan Zhu. Manuscript revision and confirmation: Xin Qiao, Chao Tang, Xiaoxuan Zhu, Peng Wang, Shaoling Zhang and Juyou Wu. All the

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participators have read and confirmed the paper.

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The authors declared no conflict of competing interest.

Journal Pre-proof Highlights: 

Identification and evolutionary analysis of the pectin methyl-esterase gene family in pear



Dynamic changes of the methyl-esterified pectins at the pollen tube tip are presented Three pectin methyl-esterase genes are closely related to the growth of pear

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pollen tube

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

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