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Integrative analysis of pectin methylesterase (PME) and PME inhibitors in tomato (Solanum lycopersicum): Identification, tissue-specific expression, and biochemical characterization
Plant Physiology and Biochemistry 132 (2018) 557–565
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Integrative analysis of pectin methylesterase (PME) and PME inhibitors in tomato (Solanum lycopersicum): Identification, tissue-specific expression, and biochemical characterization
T
Ho Young Jeonga, Hong Phuong Nguyena, Seok Hyun Eomc,∗∗, Chanhui Leea,b,∗ a
Graduate School of Biotechnology, Kyung Hee University, Yongin, 446-701, South Korea Department of Plant and Environmental New Resources, Kyung Hee University, Yongin, 446-701, South Korea c Department of Horticultural Biotechnology, Kyung Hee University, Yongin, 446-701, South Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pectin Pectin methylesterases Pectin methylesterase inhibitors Tomato Plant cell walls Cell wall modifications
Although previous studies have demonstrated that the degree of demethylesterification of pectin polysaccharides is modulated during tomato fruit ripening, its involvement in vegetative organ development has been seldom investigated. As a first step in understanding the importance of pectin modification during vegetative stages, we used chemical, biochemical, and molecular approaches to analyze PMEs and PMEIs in tomato plants. We found that tomato cell walls isolated from vegetative tissues as well as the fruit contain substantial quantities of pectin, and different degrees of methylesterification were evident in different tissues. Our chemical study was further substantiated by immunolocalization analysis, which showed that selective removal of pectin-bound methyl groups is required for proper organ development and growth. In the tomato genome, there exists 79 PMEs and 48 PMEIs with temporally and spatially regulated expression. As a case study, we showed that two tomato PMEIs (SolycPMEI13 and SolycPMEI14) exhibited PMEI activities. This is the first report regarding the genome-wide identification and expression profiling of PME/PMEIs in tomato and the first chemical evidence of the differential degrees of pectin methylesterification in vegetative and reproductive tissues. Taken together, our findings provide an important tool to unravel the molecular and physiological functions of tomato PME and PMEI in further study.
1. Introduction Pectin is a main composite of the primary cell wall and the middle lamella and is found in nearly all vascular plants. A structural investigation revealed that plant cell wall pectins are the most complex polysaccharides in nature, requiring over 60 glycosyltransferases and many cell wall modifying enzymes (methyltransferases and acetyltransferases) for biosynthesis in the Golgi apparatus (Mohnen, 2008; Wang et al., 2013). Thus, a newly synthesized pectin is heavily acetylated and methylated. Pectin is a family of polysaccharides classified into four different types based on backbone structure and glycosidic linkages of substitution, homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII), and xylogalacturonan (XGA) (Harholt et al., 2010; Mohnen, 2008; Oh et al., 2013). HGA is the most dominant pectic domain, accounting for approximately 65% of pectin. HGA is a linear polymer of (1,4)-linked-α-galacturonic acids (GalUA) that is acetylated at O-2/O-3 and methylated at C-6. Fully ∗
synthesized pectins are transported to the cell walls and assembled with other cell wall polysaccharides by chemical bonds. Selective removal of a methyl group by PMEs (EC 3.1.1.11) belonging to class 8 carbohydrate esterases (CAZy, http://www.cazy.org/ fam/CE8.html) results in partially demethylesterified HGA. PMEs are classified into either Type-1 (with a PMEI domain at the N-terminus) or Type-2 (no PMEI domain) (Jolie et al., 2010). Higher PME activity affects cell wall biomechanical properties (rigidity, elasticity, and permeability) and has been known to play multiple functions in plant growth, development, disease resistance, and abiotic tolerance (Pelloux et al., 2007). Partially demethylesterified HGA facilitates the degradation of polygalacturonic acid chains by polygalacturonases, promotes the formation of calcium cross-linkages, and increases wall porosity and extension in plant cells. Because such modification by PME activities leads to cell wall loosening and increased wall permeability, higher PME activities are found in cell types involved in cell expansion and elongation such as germinating tissues, root hair cells, and pollen tube
Corresponding author. Graduate School of Biotechnology, Kyung Hee University, Yongin, 446-701, South Korea. Corresponding author. E-mail addresses: [email protected] (S.H. Eom), [email protected] (C. Lee).
∗∗
https://doi.org/10.1016/j.plaphy.2018.10.006 Received 28 March 2018; Received in revised form 8 October 2018; Accepted 8 October 2018 Available online 09 October 2018 0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved.
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and lyophilized. The uronic acid content was measured as described by Filisetti-Cozzi and Carpita (1991). Lyophilized PEFs were dissolved in 0.4 ml of 0.5 M H2SO4 at room temperature for 1 h, following the addition of 40 μl of 4 M sulfamic acid-potassium sulfamate (pH 1.6). Samples were hydrolyzed in 2.4 ml of 75 mM Na2B4O7 dissolved in concentrated H2SO4 at 100 °C for 20 min. After cooling, 80 μl of 0.15% (w/v) m-hydroxybiphenyl in 0.5% (w/v) NaOH was added. The solution was mixed and incubated at room temperature for 30 min. Absorbance at 525 nm was measured, and the uronic acid content was determined using galacturonic acid as a standard.
cells (Jeong et al., 2014; Müller et al., 2013a). In addition, demethylesterification of the fruit cell walls by PMEs is a critical cellular process during fruit ripening (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). Enzymatic action of PMEs is posttranslationally inhibited by PMEIs that exert an effect by directly or indirectly binding to the catalytic domain of PMEs. Thus, the degree of HGA methylesterification is determined by the combined effect of PME and PMEI activities. Specific pairs of PME/PMEI are known to be present and modulate pectin modification (Sénéchal et al., 2015; Pelloux et al., 2007). PMEs and PMEIs are multigene members in higher plants. There are 66 PMEs and 76 PMEIs in Arabidopsis, 43 PMEs and 49 PMEIs in Oryza sativa, 105 PMEs and 95 PMEIs in Linum usitatissimum, and 79 PMEs and 48 PMEIs in Solanum lycopersicum (Jeong et al., 2014, Nguyen et al., 2016, Pelloux et al., 2007, Pinzon-Latorre and Deyholos, 2013, Vandevenne et al., 2008). Molecular and genetic approaches contribute to our understanding of the major physiological roles of PMEs and PMEIs in plants. In Arabidopsis, genetic mutations of PME genes cause a defect in pollen tube growth and pollen tetrad formation, abnormal secondary cell wall formation, and seed coat mucilage deficiency (Bosch et al., 2005; Francis et al., 2006; Hongo et al., 2012). Representative phenotypes caused by down- or up-regulation of PMEI genes include the delayed release of seed mucilage, increased germination rate, twisted stems, and organ fusion (Saez-Aguayo et al., 2013. Müller et al., 2013a, 2013b). In tomato, several reports have focused only on cellular events during fruit formation (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). Thus, investigations have focused on the importance of the combined actions of pectin-modifying enzymes including PMEs, PMEIs, and polygalacturonases, which result in a decrease of fruit firmness. Transgenic plants that down-regulated a fruit-specific PME (SolycPME2) displayed a loss of tissue integrity (Tieman et al., 1992). However, as seen in other plant species, pectin demethylesterification by PMEs and PMEIs could also play a critical role in vegetative organ development. In this report, as a first step to understanding the importance of pectin modification during vegetative stages, we analyzed tomato PMEs and PMEIs by chemical, biochemical, and molecular approaches.
2.4. Measurement of cell wall-bound methyl ester contents AIRs were treated with 100 μL of 0.5 M NaOH for 1 h at room temperature and then neutralized with 50 μL of 1 M HCl. Soluble residues were collected by centrifugation, and 50 μL of supernatant was mixed with 0.03 units of alcohol oxidase (Sigma-Aldrich; Cat No. A2404) in 50 μL of 20 mm phosphate buffer, pH 7.5 for 15 min at room temperature. The resultant extract was then developed for 15 min at 60 °C in 20 mM acetylacetone, 50 mM acetic acid, and 2 M ammonium acetate. Absorbance was measured at 412 nm using a spectrophotometer and compared with a standard curve generated with a methanol dilution series (Klavons and Bennett, 1986). 2.5. Soluble protein isolation and PME and PMEI activity assay Soluble extracts were prepared from 1 month old root, one month old leaf, one month stem, young flower, green fruit, and red fruit. Tissues were homogenized with equal volumes (w/v) of extraction buffer (100 mM Tris-HCl, pH 7.5, 500 mM NaCl containing protease inhibitor cocktail) and incubated at 4 °C for 120 min. The samples were centrifuged at 11,500 g at 4 °C for 20 min, and then the supernatant was used immediately for all enzyme assays. The protein concentration of the soluble protein was measured using the BioRad protein assay kit with bovine serum albumin (BSA) as the standard. PME and PMEI activity assays were performed following our previous reports (GrsicRausch and Rausch, 2004; Jeong et al., 2014; Nguyen et al., 2016). Briefly, for PMEI activity, 100 μg of soluble protein were first incubated with a commercial PME (Orange peel PME, P5400; Sigma-Aldrich) protein for 10 min and then the reaction mixture (a total volume of 1 ml) containing 0.4 mM NAD in 50 mM phosphate buffer, pH 7.5, 5% (w/v) pectin (P9135; Sigma), 0.35 unit formaldehyde dehydrogenase (F1879; Sigma), and 1.0 unit alcohol oxidase was added. Heat-denatured soluble proteins of each sample were used as a negative control. After incubation at 21 °C for 1 h, the reaction was stopped by heating in a boiling water bath. The inhibitory activities of soluble proteins and two recombinant proteins to commercial PME were measured at 340 nm in a spectrophotometer. To check the effect of temperature on SolycPMEI13 and SolycPMEI14 activities, all components in the reaction mixture were kept and incubated in indicated temperature conditions, keeping the pH value constant at 8.5. The effect of pH on SolycPMEI13 and SolycPMEI14 activities was determined for the values of 5.5, 6.5, 7.5, 8, 8.5, 9, or 9.5 at RT.
2. Materials and methods 2.1. Plant materials Tomato seeds (S. lycopersicum cv. Moneymaker) were germinated at 26 °C and grown in a cultivation chamber under 16 h light/8 h dark. Samples for RNA and cell wall protein extraction were collected at the indicated developmental stages, frozen in liquid nitrogen, and stored at −80 °C until analysis. 2.2. Preparation of alcohol insoluble residues (AIRs) AIRs were isolated as previously described (Lee et al., 2007; Zhong et al., 2005). AIRs were prepared with 1 month old root, 1 month old leaf, 2 month old leaf, 2 month old stems, young flowers, whole mature green fruit and whole mature red fruit. Tomato tissues were ground under liquid nitrogen and suspended in 70% (v/v) ethanol using a Polytron homogenizer three times. The insoluble residues were collected by centrifugation, followed by sequential washing with absolute ethanol and 100% acetone. The final residues were dried in a vacuum oven at 60 °C.
2.6. Quantitative real time PCR (qRT-PCR) RNA was isolated from 6 different tissues from three biological replicates. Each RNA replicate was analyzed independently. Root, leaf, and stem were collected from vegetative stage plants 1 month after germination. Young flowers were collected from plants 2 months after germination. Whole mature green and red fruits were collected and used for RNA isolation. Total RNA was isolated from different tomato tissues using a Qiagen RNA isolation kit following the manufacturer's protocol. Real-time PCR analysis was performed using the first-strand cDNA as template with the QuantiTect SyBR Green PCR kit (Clonetech). For each analysis, three technical replicates were performed. The PCR
2.3. Measurement of uronic acid contents In order to obtain pectin enriched fractions (PEFs), the AIRs (100 mg) were treated sequentially with ammonium oxalate solution (50 mM) and 4 N potassium hydroxide, for 24 h at room temperature. Soluble fractions after each treatment were collected by centrifugation 558
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Fig. 1. Chemical and biochemical analyses of pectin modification in tomato cell walls. (A) Quantitative analysis of uronic acid content in pectin-enriched fractions of cell walls isolated from different tissues. (B) Quantification of cell wall-bound methanol in different tissues. Error bars represent the SE of five biological replicates. (C) Soluble proteins were isolated and used for a pectin methylesterase (PME) activity assay. The enzyme activity of green fruit was taken as 100, and the activity in other tissues was expressed as a percentage of that of the green fruit. Error bars represent the SE of three biological replicates. (D) Pectin methylesterase inhibitor (PMEI) activity. The enzyme activity of 2-month-old leaf was taken as 100, and the activity in other tissues was expressed as a percentage of that of the 2-month-old leaf. Error bars represent the SE of three biological replicates.
2.8. Genome-wide identification of SolycPMEs and SolycPMEIs and in silico analysis
threshold cycle number of each gene was normalized with expression level of the tomato Ubiquitin13 (SolycUbi13, SGN-U593552) as a reference gene. The data were calculated and remade heatmap with R program (http://cran.r-project.org/).
We searched putative PMEs and PMEIs in the Sol Genomics Network (SGN) (http://www.solgenomics.net) using Pfam ID (PF01095) and PMEI (PF4043), respectively. Each class of PME protein sequence of rice and Arabidopsis was queried in BLASTp search to identify all classes of PME members in S. lycopersicum. All of the SolycPMEs/SolycPMEIs identified by BLASTp were also confirmed by HMM-alignment to the PFAM domains. PME proteins with both a PMEI and a PME domain were designated Type 1, and proteins with a PME domain were designated Type 2 PMEs. The presence of signal peptide and transmembrane domains was predicted with SignalP 4.0 and TMHMM v.2.0, respectively. WoLF PSORT and Plant-mPLoc were used to predict the protein subcellular localization. Cleavage sites were predicted as described by Pelloux et al. (2007) and Wolf et al. (2009). The predicted isoelectric points of the complete and the mature proteins were calculated using Vector NTI 10.
2.7. Histology and immunodetection Root, leaf, and stems of 1-month-old plants were fixed in 2% glutaraldehyde in 1× PBS at 4 °C overnight. Tissues were dehydrated through a gradient of ethanol, embedded in LR white resin (Ted Pella Inc.) and polymerized. 0.5 μm thick sections were cut with a ultramicrotome. Sections were blocked with 3% BSA dissolved in 1X PBS for 30 min and then incubated with LM19 and LM20 monoclonal antibodies (Plantprobes, Leeds University, UK) for 2 h at RT. After washing, sections were incubated with fluorescein isothiocyanate-conjugated secondary antibodies. The fluorescent-labeled sections were observed using a confocal microscope.
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Fig. 2. Immunodetection of methyl esterified homogalacturonans of tomato root, leaf, and stem. Thin sections (0.5 μm) of tissues were probed with the LM19 and LM20 monoclonal antibodies. (A) A tomato root section immunolabeled with LM19. (B) A tomato root section immunolabeled with LM20. (C) A tomato leaf section immunolabeled with LM19. (D) A tomato leaf section immunolabeled with LM20. (E) A tomato top-stem section immunolabeled with LM19. (F) A tomato top-stem section immunolabeled with LM20. (G) A tomato bottom-stem section immunolabeled with LM19. (H) A tomato bottom-stem section immunolabeled with LM20. Scale bar indicates 50 μm.
plays an essential role in the cell walls during tomato fruit ripening processes (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). However, a comprehensive analysis of pectin and its modification in vegetative tissues remains to be determined. AIRs were isolated from representative vegetative and reproductive tissues and subjected to uronic acid quantification. Uronic acid quantification is known to be an indirect measurement of pectin content because at least 80% of the total mass of pectin is composed of uronic acid. In order to obtain PEFs from AIRs, we subsequently treated cell walls with ammonium oxalate and then potassium hydroxide (see Materials and methods). Soluble fractions after ammonium oxalate- and KOH-treatment were collected and pooled. The quantitative measurement of total uronic acid indicated that higher levels were observed in fruit tissues (green and red fruit) compared with other tissues (Fig. 1A). A substantial amount of uronic acid was also found in all vegetative tissues tested, albeit to a lesser extent. We also found that pectin fractions isolated in vegetative tissues and flowers contained higher levels of cell wall-bound methanol compared with those of fruit tissues (Fig. 1B). Given that the majority of cell wall-bound methanol is found in HGAs, our data suggested that methyl groups bound to HGAs are intensively removed by PMEs during the fruit maturation process.
2.9. Cloning, expression, and purification of SolycPMEI13 and SolycPMEI14 Because an intron structure was not predicted, full-length cDNA of SolycPMEI13 and SolycPMEI14 was amplified by PCR from genomic DNA using primers containing restriction sites (see Table S1). The amplification products were cloned into pTOP TA V2 (Enzynomics), and sequences were confirmed by sequencing. The insertions were excised with the restriction enzymes and ligated into pMAL-c2X vector (NEB) to generate recombinant proteins fused to the C-terminus of maltose binding protein (MBP). The MBP-tagged plasmids were transformed into Escherichia coli strain BL21 CodonPlus (DE3)-RIPL (Agilent Technologies, USA). Overnight cultures of recombinant strains from single colonies were diluted, induced, and purified according to the manufacturer's protocols. The purified proteins were quantified using a Bradford assay (Bio-Rad, USA).
3. Results 3.1. Chemical analysis of pectin modification in tomato vegetative and reproductive tissues Previously, several studies demonstrated that pectin modification 560
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47, 48, 78, and 79) were predominantly expressed in flowers, and rootspecific expression was also found in six SolycPMEs (SolycPMEs22, 25, 28, 33, 45, and 58). High levels of expression of seven SolycPMEs (SolycPMEs2, 31, 35, 49, 51, 52, and 59) were detected in both green and red fruits. Among 48 SolycPMEIs, 23 (48%) were significantly upregulated in at least one organ (Fig. 4). Particularly, SolycPMEI1, 7, 22, 37, and 41 showed specific expression in flowers, whereas the SolycPMEI5 transcript was only highly expressed in red fruit.
3.2. PME and PMEI enzyme activities of different tomato tissues Pectin modification by PME/PMEI action during plant development and biotic/abiotic stresses is one of the most well-characterized mechanisms in various higher plants (Pelloux et al., 2007). Thus, we next determined the PME activity in tomato tissues. Soluble proteins were isolated from nine different tissues and used for PME and PMEI activity assays in vitro. In accordance with previous studies, the highest PME activity was detected in samples isolated from green fruits, whereas soluble proteins isolated from green and red fruit possessed the lowest PMEI activity (Fig. 1C and D). Notably, root and stems possessed high PME activities, while low activities were detected in leaf tissues, suggesting that vegetative tissues also undergo dynamic pectin modification. Our biochemical data strongly indicated that considerable PME/ PMEI activities also contribute to the normal growth and development of vegetative tissues.
3.6. PMEI activity assay of two tomato PMEIs (SolycPMEI13 and 14) In order to determine if tomato PME and PMEI indeed possess enzymatic activities, we selected 10 fruit-specific SolycPME and six ubiquitously expressed SolycPMEI genes. Full-length cDNA was successfully cloned and expressed in E.Coli strain BL21 CodonPlus (DE3)-RIPL, a heterologous system with extra copies of rare codons. Although five SolycPME were successfully purified from E. coli crude proteins, we failed to observe any PME activity in the assay reaction. This could be due to the lack of post-translational modification in our recombinant proteins expressed in E. coli. Out of six SolycPMEIs, considerable amounts of the soluble recombinant proteins of two genes (SolycPMEI13 and 14) were recovered and purified (Fig. 5A and B). In our assay system, the recombinant proteins exhibited high inhibitory activity against a commercial PME protein compared to a recombinant protein with empty vector (Fig. 5C). Higher PMEI activity was detected in a SolycPMEI13 recombinant protein even at an early time point. Next, we investigated which pH and temperature are optimal for PMEI activity and found that both recombinant proteins showed the highest activity at pH 8.5 and 20 °C (Fig. 6).
3.3. Immunolocalization of pectin status by LM19 and LM20 monoclonal antibodies Previous study demonstrated that dynamic alteration of the degree of pectin demethylesterification occurs during fruit ripening process. However, the status of pectin modification during vegetative organ development remains to be determined. We performed immunolocalization experiment using two epitopes raised against demethylesterified pectin (LM19) and methylesterified pectin (LM20). LM 19 recognizes demethylesterified pectin whereas LM20 binds methylesterified pectin. Four representative vegetative tissues (root, leaf, top stem, and bottom stem) were cross-reacted with epitopes (Fig. 2). Immunolabeling of root, leaf, and bottom stem by LM20 revealed much stronger fluorescence signals compared with that by LM19 (Fig. 2). Only weak signals were detected in top stem for both LM19 and LM20 (Fig. 2E and F). In accordance with our chemical data, vegetative tissues also contain highly methylated pectins in their cell walls.
4. Discussion Genome-wide analysis has identified PMEs and PMEIs in nearly all vascular plants and in multiple gene members (Pelloux et al., 2007). However, the majority of them have not been functionally characterized. Our integrative analysis indicated that the dynamic structural modification of pectin HGAs was observed in both vegetative and reproductive tissues. Previous studies have demonstrated that selective removal of a methyl group from HGAs as well as partial digestion of the pectin backbone by polygalacturonases play an important role during tomato fruit ripening (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). Accordingly, our chemical and biochemical data clearly showed that extensive enzymatic removal of HGA-bound methyl groups by PMEs is required in the fruit ripening stage (Fig. 1). The degree of methylesterification on HGAs mediated by PMEs and PMEIs affects cell and tissue properties. Pectin demethylation by PMEs results in a calcium-linked gel structure by the localized aggregation of pectin backbone structures (uronic acid). Consequently, this gel structure becomes susceptible to polygalacturonase-mediated digestion. Thus, selective removal of HGA-bound methyl groups by tomato PMEs and concomitant partial digestion by fruit-specific polygalacturonases play a critical role in fruit softening. Bioinformatics analysis indicated that tomato contains 79 putative PMEs and 48 putative PMEIs. Our expression profiling indicated that tissue-specific or ubiquitous expression patterns of PMEs and PMEIs. To our knowledge, this is first comprehensive expression analysis of tomato PMEs/PMEIs in vegetative tissues. The importance of PME and PMEI in vegetative growth and development has been reported in several plant species. In Arabidopsis, in which pectin is mainly found in the primary cell walls, demethylesterification by PME35 is required for providing mechanical support to the stem (Hongo et al., 2012). Significant differential expression and enzyme activities of PMEs/PMEIs were observed during the first 24 h of Arabidopsis seed germination, and overexpression of PMEI5 resulted in faster seed germination and a reduced sensitivity of seeds to ABA, an inhibitory phytohormone on germination, indicating that the reduced PME activities make cell walls
3.4. Identification and phylogenetic analysis of tomato PMEs and PMEIs The availability of S. lycopersicum genome sequence facilitated genome-wide identification and analysis of PME and PMEI families from cultivated tomato. To identify proteins encoding putative PMEs and PMEIs in S. lycopersicum, we searched proteins containing PME domains (PF1095) and PMEI domains (PF0404), respectively, and a total of 79 putative PMEs (SolycPMEs) and 48 putative PMEIs (SolycPMEIs) were identified (Table S2). The identified PME proteins were confirmed using InterProScan, which showed the presence of PMEs (IPR000070). The presence of a PMEI-conserved domain (cd15797) in the identified PMEI proteins was validated using the NCBI conserved domain database. A comparative phylogenetic tree using amino sequences of 79 SolycPMEs and 66 previously annotated Arabidopsis PMEs (AtPMEs) indicated clustering into six different clades/ groups (Groups 1 to 6) (Fig. S1). Additionally, we constructed a phylogenetic tree containing 48 SolycPMEIs and 78 AtPMEIs (Fig. S2). In silico analysis of the exon/intron organization of SolycPMEs and SolycPMEIs revealed that, different from SolycPME genes, only seven SolycPMEIs (SolycPMEI2, 22, 31, 36, 41, 47, and 48) have an intron (Figs. S3 and S4). 3.5. Tissue-specific expression of SolycPMEs and SolycPMEIs We next analyzed organ-specific expression of the 79 SolycPMEs and 48 SolycPMEIs in six representative organs (root, leaf, stem, flower, green fruit, and red fruit) by qRT-PCR. We integrated the phylogenetic tree into our heatmap to compare the transcriptional levels between paralogs. Expression analysis of SolycPMEs showed that, among the 78 genes, 39 (50%) were highly expressed in at least one organ (Fig. 3). Interestingly, 13 SolycPMEs (SolycPMEs4, 5, 9, 12, 18, 30, 39, 40, 42, 561
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Fig. 3. Heat map of the abundance of SolycPME transcripts in different tissues. The color of the cell represents transcript abundance. Gray cells indicate no transcripts were detected. Green colored boxes denote a low level of expression, and red colored boxes denote a high level of expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Although a large number of genes encoding PME/PMEI and associated regulators are expressed in both vegetative and reproductive organs in tomato, none of them have been directly linked to a precise physiological role. Our biochemical data including enzyme activities and cell wall analysis as well as immunolocalization by the LM19 and LM20 monoclonal antibodies clearly indicated that the degree of pectin methylesterification is actively regulated by two pectin-modifying enzymes. We found that PME activities in vegetative tissues were
of seeds more permeable to water (Müller et al., 2013a). In addition, tight regulation of PME activity by AtPMEI6 contributes to the accumulation of mucilage polysaccharides in the apoplast of seed coat epidermal cells (Saez-Aguayo et al., 2013). We previously reported that the overexpression of rice PMEI28 (OsPMEI28) caused a higher degree of methylesterification and a dwarf phenotype (Nguyen et al., 2017). Significant inhibition of intrusive apical growth was observed in transgenic plants overexpressing poplarPME1 (Siedlecka et al., 2008). 562
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Fig. 4. Heat map of the abundance of SolycPMEI transcripts in different tissues. The color of the cell represents transcript abundance. Gray cells indicate no transcripts were detected. Green colored boxes denote a low level of expression, and red colored boxes denote a high level of expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
modulated by cell wall properties (Yang et al., 2013). Therefore, we speculated that those genes with low or no detectable expression in representative tissues might function as regulators to cope with external environmental factors. Taken together, our findings provide an important tool to unravel the molecular and physiological functions of tomato PME and PMEI in further studies.
relatively lower than that of proteins isolated in green and red fruit, whereas PMEI activities were higher. Accordingly, several PME genes were expressed specifically or ubiquitously in fruits. It should be noted that low or no detectable expression was observed in more than half of PMEs and PMEIs. As mentioned in the Introduction, it is well known that biotic or abiotic stresses lead to structural modification of cell walls (An et al., 2008). Moreover, differential sensitivity to heavy metals is
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Fig. 5. Assay of the pectin methylesterase inhibitor (PMEI) activity of recombinant SolycPMEI13 and SolycPMEI14 proteins. Maltose binding protein (MBP)-tagged full-length proteins were expressed in Escherichia coli, purified, and then subjected to an assay to measure their inhibitory activity on commercial pectin methylesterase (PME) protein (orange peel PME). (A) SDS–PAGE detection of recombinant SolycPMEI13 expressed in E. coli. (B) SDS–PAGE detection of recombinant SolycPMEI14 expressed in E. coli. Lane 1, Uninduced E. coli BL21 Codon Plus (RILP) harboring MPB:SoPMEI fusion protein cell extracts; Lane 2, Induced cell extracts; Lane 3, Insoluble protein extracts; Lane 4, Crude protein extracts; Lane 5, Purified MBP:SolycPMEI fusion proteins; Lane 6, Purified MBP expressed from the pMALc2x vector with no insert (∼51 KDa). Molecular weight markers are shown on the left. (C) Time course PMEI activity assay of the recombinant SolycPMEIs. No inhibitory activity was detected for MBP. Error bars denote the SE of five independent assays.
Fig. 6. Biochemical properties of recombinant SoPMEI13 and SoPMEI14 proteins. (A) The effect of temperature on SoPMEI13 activity. (B) The effect of temperature on SoPMEI14 activity. The reaction with the highest activity was taken as 100%. (C) The effect of pH on SoPMEI13 activity. (D) The effect of pH on SoPMEI14 activity. The effect of pH was presented as the number of reaction product NADH formation after reactions. Error bars represent the SE of three biological replicates. 564
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Contribution
The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis. Plant Cell Physiol. 48, 1659–1672. Mohnen, D., 2008. Pectin structure and biosynthesis. Curr. Opin. Plant Biol. 11, 266–277. Müller, K., Levesque-Tremblay, G., Bartels, S., Weitbrecht, K., Wormit, A., Usadel, B., Haughn, G., Kermode, A.R., 2013a. Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination. Plant Physiol. 161, 305–316. Müller, K., Levesque-Tremblay, G., Fernandes, A., Wormit, A., Bartels, S., Usadel, B., Kermode, A., 2013b. Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation. Plant Signal. Behav. 8 (12), e26464. Nguyen, H.P., Jeong, H.Y., Kim, H., Kim, Y.,C., Lee, C., 2016. Molecular and biochemical characterization of rice pectin methylesterase inhibitors (OsPMEIs). Plant Physiol. Biochem. 101, 105–112. Nguyen, H.P., Jeong, H.Y., Jeon, S.H., Kim, D., Lee, C., 2017. Rice pectin methylesterase inhibitor28 (OsPMEI28) encodes a functional PMEI and its overexpression results in a dwarf phenotype through increased pectin methylesterification levels. J. Plant Physiol. 208, 17–25. Oh, C.S., Kim, H., Lee, C., 2013. Rice cell wall polysaccharides: structure and biosynthesis. J. Plant Biol. 56, 274–282. Pelloux, J., Rusterucci, C., Mellerowicz, E.J., 2007. New insights into pectin methylesterase structure and function. Trends Plant Sci. 12, 267–277. Pinzon-Latorre, D., Deyholos, M.K., 2013. Characterization and transcript profiling of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) gene families in flax (Linum usitatissimum). BMC Genomics 14, 742. Reca, I.B., Lionetti, V., Camardella, L., D'Avino, R., Giardina, T., Cervone, F., Bellincampi, D., 2012. A functional pectin methylesterase inhibitor protein (SolyPMEI) is expressed during tomato fruit ripening and interacts with PME1. Plant Mol. Biol. 79, 429–442. Rose, J.K., 2003. Plant cell wall disassembly. Plant Cell Wall 8, 264–324. Saez-Aguayo, S., Ralet, M.C., Berger, A., Botran, L., Ropartz, D., Marion-Poll, A., North, H.M., 2013. PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells. Plant Cell 25, 308–323. Sénéchal, F., L'Enfant, M., Domon, J.M., Rosiau, E., Crépeau, M.J., Surcouf, O., EsquivelRodriguez, J., Marcelo, P., Mareck, A., Guérineau, F., Kim, H.R., Mravec, J., Bonnin, E., Jamet, E., Kihara, D., Lerouge, P., Ralet, M.C., Pelloux, J., Rayon, C., 2015. Tuning of pectin methylesterification: PECTIN METHYLESTERASE INHIBITOR7 modulate the processive activity of co-expressed PECTIN METHYLESTERASE3 in a pH-dependent manner. J. Biol. Chem. 290, 23320–23335. Siedlecka, A., Wiklund, S., Péronne, M.A., Micheli, F., Lesniewska, J., Sethson, I., Edlund, U., Richard, L., Sundberg, B., Mellerowicz, E.J., 2008. Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus. Plant Physiol. 146, 554–565. Tieman, D.M., Harriman, R.W., Ramamohan, G., Handa, A.K., 1992. An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. Plant Cell 4, 667–679. Vandevenne, E., Van Buggenhout, S., Duvetter, T., Brouwers, E., Declerck, P.J., Hendrickx, M.E., Van Loey, A., Gils, A., 2009. Development and evaluation of monoclonal antibodies as probes to assess the differences between two tomato pectin methylesterase isoenzymes. J. Immunol. Methods 349, 18–27. Wang, M., Yuan, D., Gao, W., Li, Y., Tan, J., Zhang, X., 2013. A comparative genome analysis of PME and PMEI families reveals the evolution of pectin metabolism in plant cell walls. PloS One 8, e72082. Wolf, S., Rausch, T., Greiner, S., 2009. The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus. Plant J. 58 (3), 361–375. Yang, X.Y., Zeng, Z.H., Yan, J.Y., Fan, W., Bian, H.W., Zhu, M.Y., Yang, J.L., Zheng, S.J., 2013. Association of specific pectin methylesterases with Al-induced root elongation inhibition in rice. Physiol. Plant 148 (4), 502–511. Zhong, R., Peña, M.J., Zhou, G.K., Nairn, C.J., Wood-Jones, A., Richardson, E.A., Morrison, W.H., Darvill, A.G., York, W.S., Ye, Z.H., 2005. Arabidopsis fragile fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis. Plant Cell 17 (12), 3390–3408.
Study conception and design: Chanhui Lee, Seok Hyun Eom, Ho Young Jeong. Acquisition of data: Ho Young Jeong, Hong Phuong Nguyen, Chanhui Lee. Analysis and interpretation of data: Ho Young Jeong, Hong Phuong Nguyen, Seok Hyun Eom, Chanhui Lee. Drafting of manuscript: Ho Young Jeong, Hong Phuong Nguyen, Seok Hyun Eom, Chanhui Lee. Acknowledgments This work was supported by a grant from the Kyung Hee University (KHU-20150735). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.plaphy.2018.10.006. References An, S.H., Sohn, K.H., Choi, H.W., Hwang, I.S., Lee, S.C., Hwang, B.K., 2008. Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance. Planta 228, 61–78. Bosch, M., Cheung, A.Y., Hepler, P.K., 2005. Pectin methylesterase, a regulator of pollen tube growth. Plant Physiol. 38, 1334–1346. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311–340. Filisetti-Cozzi, T.M., Carpita, N.C., 1991. Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197 (1), 157–162. Francis, K.E., Lam, S.Y., Copenhaver, G.P., 2006. Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene. Plant Physiol. 142, 1004–1013. Grsic-Rausch, S., Rausch, T.A., 2004. Coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors. Anal. Biochem. 333, 14–18. Harholt, J., Suttangkakul, A., Scheller, H.V., 2010. Biosynthesis of pectin. Plant Physiol. 153, 384–395. Hongo, S., Sato, K., Yokoyama, R., Nishitani, K., 2012. Demethylesterification of the primary wall by PECTIN METHYLESTERASE35 provides mechanical support to the Arabidopsis stem. Plant Cell 24, 2624–2634. Hyodo, H., Terao, A., Furukawa, J., Sakamoto, N., Yurimoto, H., Satoh, S., Iwai, H., 2013. Tissue specific localization of pectin–Ca2+ cross linkages and pectin methyl-esterification during fruit ripening in tomato (Solanum lycopersicum). PLoS One 8 (11), e78949. https://doi.org/10.1371/journal.pone.0078949. Jeong, H.Y., Nguyen, H.P., Lee, C., 2014. Genome-wide identification and expression analysis of rice pectin methylesterases: implication of functional roles of pectin modification in rice physiology. J. Plant Physiol. 183, 23–29. Jolie, R.P., Duvetter, T., Van Loey, A.M., Hendrickx, M.E., 2010. Pectin methylesterase and its proteinaceous inhibitor: a review. Carbohydr. Res. 345, 2583–2595. Klavons, J.A., Bennett, R.D., 1986. Determination of methanol using alcohol oxidase and its application to methyl ester content of pectins. J. Agric. Food Chem. 34, 597–599. Lee, C., Zhong, R., Richardson, E.A., Himmelsbach, D.S., McPhail, B.T., Ye, Z.H., 2007.
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Research article
Integrative analysis of pectin methylesterase (PME) and PME inhibitors in tomato (Solanum lycopersicum): Identification, tissue-specific expression, and biochemical characterization
T
Ho Young Jeonga, Hong Phuong Nguyena, Seok Hyun Eomc,∗∗, Chanhui Leea,b,∗ a
Graduate School of Biotechnology, Kyung Hee University, Yongin, 446-701, South Korea Department of Plant and Environmental New Resources, Kyung Hee University, Yongin, 446-701, South Korea c Department of Horticultural Biotechnology, Kyung Hee University, Yongin, 446-701, South Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Pectin Pectin methylesterases Pectin methylesterase inhibitors Tomato Plant cell walls Cell wall modifications
Although previous studies have demonstrated that the degree of demethylesterification of pectin polysaccharides is modulated during tomato fruit ripening, its involvement in vegetative organ development has been seldom investigated. As a first step in understanding the importance of pectin modification during vegetative stages, we used chemical, biochemical, and molecular approaches to analyze PMEs and PMEIs in tomato plants. We found that tomato cell walls isolated from vegetative tissues as well as the fruit contain substantial quantities of pectin, and different degrees of methylesterification were evident in different tissues. Our chemical study was further substantiated by immunolocalization analysis, which showed that selective removal of pectin-bound methyl groups is required for proper organ development and growth. In the tomato genome, there exists 79 PMEs and 48 PMEIs with temporally and spatially regulated expression. As a case study, we showed that two tomato PMEIs (SolycPMEI13 and SolycPMEI14) exhibited PMEI activities. This is the first report regarding the genome-wide identification and expression profiling of PME/PMEIs in tomato and the first chemical evidence of the differential degrees of pectin methylesterification in vegetative and reproductive tissues. Taken together, our findings provide an important tool to unravel the molecular and physiological functions of tomato PME and PMEI in further study.
1. Introduction Pectin is a main composite of the primary cell wall and the middle lamella and is found in nearly all vascular plants. A structural investigation revealed that plant cell wall pectins are the most complex polysaccharides in nature, requiring over 60 glycosyltransferases and many cell wall modifying enzymes (methyltransferases and acetyltransferases) for biosynthesis in the Golgi apparatus (Mohnen, 2008; Wang et al., 2013). Thus, a newly synthesized pectin is heavily acetylated and methylated. Pectin is a family of polysaccharides classified into four different types based on backbone structure and glycosidic linkages of substitution, homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII), and xylogalacturonan (XGA) (Harholt et al., 2010; Mohnen, 2008; Oh et al., 2013). HGA is the most dominant pectic domain, accounting for approximately 65% of pectin. HGA is a linear polymer of (1,4)-linked-α-galacturonic acids (GalUA) that is acetylated at O-2/O-3 and methylated at C-6. Fully ∗
synthesized pectins are transported to the cell walls and assembled with other cell wall polysaccharides by chemical bonds. Selective removal of a methyl group by PMEs (EC 3.1.1.11) belonging to class 8 carbohydrate esterases (CAZy, http://www.cazy.org/ fam/CE8.html) results in partially demethylesterified HGA. PMEs are classified into either Type-1 (with a PMEI domain at the N-terminus) or Type-2 (no PMEI domain) (Jolie et al., 2010). Higher PME activity affects cell wall biomechanical properties (rigidity, elasticity, and permeability) and has been known to play multiple functions in plant growth, development, disease resistance, and abiotic tolerance (Pelloux et al., 2007). Partially demethylesterified HGA facilitates the degradation of polygalacturonic acid chains by polygalacturonases, promotes the formation of calcium cross-linkages, and increases wall porosity and extension in plant cells. Because such modification by PME activities leads to cell wall loosening and increased wall permeability, higher PME activities are found in cell types involved in cell expansion and elongation such as germinating tissues, root hair cells, and pollen tube
Corresponding author. Graduate School of Biotechnology, Kyung Hee University, Yongin, 446-701, South Korea. Corresponding author. E-mail addresses: [email protected] (S.H. Eom), [email protected] (C. Lee).
∗∗
https://doi.org/10.1016/j.plaphy.2018.10.006 Received 28 March 2018; Received in revised form 8 October 2018; Accepted 8 October 2018 Available online 09 October 2018 0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved.
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and lyophilized. The uronic acid content was measured as described by Filisetti-Cozzi and Carpita (1991). Lyophilized PEFs were dissolved in 0.4 ml of 0.5 M H2SO4 at room temperature for 1 h, following the addition of 40 μl of 4 M sulfamic acid-potassium sulfamate (pH 1.6). Samples were hydrolyzed in 2.4 ml of 75 mM Na2B4O7 dissolved in concentrated H2SO4 at 100 °C for 20 min. After cooling, 80 μl of 0.15% (w/v) m-hydroxybiphenyl in 0.5% (w/v) NaOH was added. The solution was mixed and incubated at room temperature for 30 min. Absorbance at 525 nm was measured, and the uronic acid content was determined using galacturonic acid as a standard.
cells (Jeong et al., 2014; Müller et al., 2013a). In addition, demethylesterification of the fruit cell walls by PMEs is a critical cellular process during fruit ripening (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). Enzymatic action of PMEs is posttranslationally inhibited by PMEIs that exert an effect by directly or indirectly binding to the catalytic domain of PMEs. Thus, the degree of HGA methylesterification is determined by the combined effect of PME and PMEI activities. Specific pairs of PME/PMEI are known to be present and modulate pectin modification (Sénéchal et al., 2015; Pelloux et al., 2007). PMEs and PMEIs are multigene members in higher plants. There are 66 PMEs and 76 PMEIs in Arabidopsis, 43 PMEs and 49 PMEIs in Oryza sativa, 105 PMEs and 95 PMEIs in Linum usitatissimum, and 79 PMEs and 48 PMEIs in Solanum lycopersicum (Jeong et al., 2014, Nguyen et al., 2016, Pelloux et al., 2007, Pinzon-Latorre and Deyholos, 2013, Vandevenne et al., 2008). Molecular and genetic approaches contribute to our understanding of the major physiological roles of PMEs and PMEIs in plants. In Arabidopsis, genetic mutations of PME genes cause a defect in pollen tube growth and pollen tetrad formation, abnormal secondary cell wall formation, and seed coat mucilage deficiency (Bosch et al., 2005; Francis et al., 2006; Hongo et al., 2012). Representative phenotypes caused by down- or up-regulation of PMEI genes include the delayed release of seed mucilage, increased germination rate, twisted stems, and organ fusion (Saez-Aguayo et al., 2013. Müller et al., 2013a, 2013b). In tomato, several reports have focused only on cellular events during fruit formation (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). Thus, investigations have focused on the importance of the combined actions of pectin-modifying enzymes including PMEs, PMEIs, and polygalacturonases, which result in a decrease of fruit firmness. Transgenic plants that down-regulated a fruit-specific PME (SolycPME2) displayed a loss of tissue integrity (Tieman et al., 1992). However, as seen in other plant species, pectin demethylesterification by PMEs and PMEIs could also play a critical role in vegetative organ development. In this report, as a first step to understanding the importance of pectin modification during vegetative stages, we analyzed tomato PMEs and PMEIs by chemical, biochemical, and molecular approaches.
2.4. Measurement of cell wall-bound methyl ester contents AIRs were treated with 100 μL of 0.5 M NaOH for 1 h at room temperature and then neutralized with 50 μL of 1 M HCl. Soluble residues were collected by centrifugation, and 50 μL of supernatant was mixed with 0.03 units of alcohol oxidase (Sigma-Aldrich; Cat No. A2404) in 50 μL of 20 mm phosphate buffer, pH 7.5 for 15 min at room temperature. The resultant extract was then developed for 15 min at 60 °C in 20 mM acetylacetone, 50 mM acetic acid, and 2 M ammonium acetate. Absorbance was measured at 412 nm using a spectrophotometer and compared with a standard curve generated with a methanol dilution series (Klavons and Bennett, 1986). 2.5. Soluble protein isolation and PME and PMEI activity assay Soluble extracts were prepared from 1 month old root, one month old leaf, one month stem, young flower, green fruit, and red fruit. Tissues were homogenized with equal volumes (w/v) of extraction buffer (100 mM Tris-HCl, pH 7.5, 500 mM NaCl containing protease inhibitor cocktail) and incubated at 4 °C for 120 min. The samples were centrifuged at 11,500 g at 4 °C for 20 min, and then the supernatant was used immediately for all enzyme assays. The protein concentration of the soluble protein was measured using the BioRad protein assay kit with bovine serum albumin (BSA) as the standard. PME and PMEI activity assays were performed following our previous reports (GrsicRausch and Rausch, 2004; Jeong et al., 2014; Nguyen et al., 2016). Briefly, for PMEI activity, 100 μg of soluble protein were first incubated with a commercial PME (Orange peel PME, P5400; Sigma-Aldrich) protein for 10 min and then the reaction mixture (a total volume of 1 ml) containing 0.4 mM NAD in 50 mM phosphate buffer, pH 7.5, 5% (w/v) pectin (P9135; Sigma), 0.35 unit formaldehyde dehydrogenase (F1879; Sigma), and 1.0 unit alcohol oxidase was added. Heat-denatured soluble proteins of each sample were used as a negative control. After incubation at 21 °C for 1 h, the reaction was stopped by heating in a boiling water bath. The inhibitory activities of soluble proteins and two recombinant proteins to commercial PME were measured at 340 nm in a spectrophotometer. To check the effect of temperature on SolycPMEI13 and SolycPMEI14 activities, all components in the reaction mixture were kept and incubated in indicated temperature conditions, keeping the pH value constant at 8.5. The effect of pH on SolycPMEI13 and SolycPMEI14 activities was determined for the values of 5.5, 6.5, 7.5, 8, 8.5, 9, or 9.5 at RT.
2. Materials and methods 2.1. Plant materials Tomato seeds (S. lycopersicum cv. Moneymaker) were germinated at 26 °C and grown in a cultivation chamber under 16 h light/8 h dark. Samples for RNA and cell wall protein extraction were collected at the indicated developmental stages, frozen in liquid nitrogen, and stored at −80 °C until analysis. 2.2. Preparation of alcohol insoluble residues (AIRs) AIRs were isolated as previously described (Lee et al., 2007; Zhong et al., 2005). AIRs were prepared with 1 month old root, 1 month old leaf, 2 month old leaf, 2 month old stems, young flowers, whole mature green fruit and whole mature red fruit. Tomato tissues were ground under liquid nitrogen and suspended in 70% (v/v) ethanol using a Polytron homogenizer three times. The insoluble residues were collected by centrifugation, followed by sequential washing with absolute ethanol and 100% acetone. The final residues were dried in a vacuum oven at 60 °C.
2.6. Quantitative real time PCR (qRT-PCR) RNA was isolated from 6 different tissues from three biological replicates. Each RNA replicate was analyzed independently. Root, leaf, and stem were collected from vegetative stage plants 1 month after germination. Young flowers were collected from plants 2 months after germination. Whole mature green and red fruits were collected and used for RNA isolation. Total RNA was isolated from different tomato tissues using a Qiagen RNA isolation kit following the manufacturer's protocol. Real-time PCR analysis was performed using the first-strand cDNA as template with the QuantiTect SyBR Green PCR kit (Clonetech). For each analysis, three technical replicates were performed. The PCR
2.3. Measurement of uronic acid contents In order to obtain pectin enriched fractions (PEFs), the AIRs (100 mg) were treated sequentially with ammonium oxalate solution (50 mM) and 4 N potassium hydroxide, for 24 h at room temperature. Soluble fractions after each treatment were collected by centrifugation 558
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Fig. 1. Chemical and biochemical analyses of pectin modification in tomato cell walls. (A) Quantitative analysis of uronic acid content in pectin-enriched fractions of cell walls isolated from different tissues. (B) Quantification of cell wall-bound methanol in different tissues. Error bars represent the SE of five biological replicates. (C) Soluble proteins were isolated and used for a pectin methylesterase (PME) activity assay. The enzyme activity of green fruit was taken as 100, and the activity in other tissues was expressed as a percentage of that of the green fruit. Error bars represent the SE of three biological replicates. (D) Pectin methylesterase inhibitor (PMEI) activity. The enzyme activity of 2-month-old leaf was taken as 100, and the activity in other tissues was expressed as a percentage of that of the 2-month-old leaf. Error bars represent the SE of three biological replicates.
2.8. Genome-wide identification of SolycPMEs and SolycPMEIs and in silico analysis
threshold cycle number of each gene was normalized with expression level of the tomato Ubiquitin13 (SolycUbi13, SGN-U593552) as a reference gene. The data were calculated and remade heatmap with R program (http://cran.r-project.org/).
We searched putative PMEs and PMEIs in the Sol Genomics Network (SGN) (http://www.solgenomics.net) using Pfam ID (PF01095) and PMEI (PF4043), respectively. Each class of PME protein sequence of rice and Arabidopsis was queried in BLASTp search to identify all classes of PME members in S. lycopersicum. All of the SolycPMEs/SolycPMEIs identified by BLASTp were also confirmed by HMM-alignment to the PFAM domains. PME proteins with both a PMEI and a PME domain were designated Type 1, and proteins with a PME domain were designated Type 2 PMEs. The presence of signal peptide and transmembrane domains was predicted with SignalP 4.0 and TMHMM v.2.0, respectively. WoLF PSORT and Plant-mPLoc were used to predict the protein subcellular localization. Cleavage sites were predicted as described by Pelloux et al. (2007) and Wolf et al. (2009). The predicted isoelectric points of the complete and the mature proteins were calculated using Vector NTI 10.
2.7. Histology and immunodetection Root, leaf, and stems of 1-month-old plants were fixed in 2% glutaraldehyde in 1× PBS at 4 °C overnight. Tissues were dehydrated through a gradient of ethanol, embedded in LR white resin (Ted Pella Inc.) and polymerized. 0.5 μm thick sections were cut with a ultramicrotome. Sections were blocked with 3% BSA dissolved in 1X PBS for 30 min and then incubated with LM19 and LM20 monoclonal antibodies (Plantprobes, Leeds University, UK) for 2 h at RT. After washing, sections were incubated with fluorescein isothiocyanate-conjugated secondary antibodies. The fluorescent-labeled sections were observed using a confocal microscope.
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Fig. 2. Immunodetection of methyl esterified homogalacturonans of tomato root, leaf, and stem. Thin sections (0.5 μm) of tissues were probed with the LM19 and LM20 monoclonal antibodies. (A) A tomato root section immunolabeled with LM19. (B) A tomato root section immunolabeled with LM20. (C) A tomato leaf section immunolabeled with LM19. (D) A tomato leaf section immunolabeled with LM20. (E) A tomato top-stem section immunolabeled with LM19. (F) A tomato top-stem section immunolabeled with LM20. (G) A tomato bottom-stem section immunolabeled with LM19. (H) A tomato bottom-stem section immunolabeled with LM20. Scale bar indicates 50 μm.
plays an essential role in the cell walls during tomato fruit ripening processes (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). However, a comprehensive analysis of pectin and its modification in vegetative tissues remains to be determined. AIRs were isolated from representative vegetative and reproductive tissues and subjected to uronic acid quantification. Uronic acid quantification is known to be an indirect measurement of pectin content because at least 80% of the total mass of pectin is composed of uronic acid. In order to obtain PEFs from AIRs, we subsequently treated cell walls with ammonium oxalate and then potassium hydroxide (see Materials and methods). Soluble fractions after ammonium oxalate- and KOH-treatment were collected and pooled. The quantitative measurement of total uronic acid indicated that higher levels were observed in fruit tissues (green and red fruit) compared with other tissues (Fig. 1A). A substantial amount of uronic acid was also found in all vegetative tissues tested, albeit to a lesser extent. We also found that pectin fractions isolated in vegetative tissues and flowers contained higher levels of cell wall-bound methanol compared with those of fruit tissues (Fig. 1B). Given that the majority of cell wall-bound methanol is found in HGAs, our data suggested that methyl groups bound to HGAs are intensively removed by PMEs during the fruit maturation process.
2.9. Cloning, expression, and purification of SolycPMEI13 and SolycPMEI14 Because an intron structure was not predicted, full-length cDNA of SolycPMEI13 and SolycPMEI14 was amplified by PCR from genomic DNA using primers containing restriction sites (see Table S1). The amplification products were cloned into pTOP TA V2 (Enzynomics), and sequences were confirmed by sequencing. The insertions were excised with the restriction enzymes and ligated into pMAL-c2X vector (NEB) to generate recombinant proteins fused to the C-terminus of maltose binding protein (MBP). The MBP-tagged plasmids were transformed into Escherichia coli strain BL21 CodonPlus (DE3)-RIPL (Agilent Technologies, USA). Overnight cultures of recombinant strains from single colonies were diluted, induced, and purified according to the manufacturer's protocols. The purified proteins were quantified using a Bradford assay (Bio-Rad, USA).
3. Results 3.1. Chemical analysis of pectin modification in tomato vegetative and reproductive tissues Previously, several studies demonstrated that pectin modification 560
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47, 48, 78, and 79) were predominantly expressed in flowers, and rootspecific expression was also found in six SolycPMEs (SolycPMEs22, 25, 28, 33, 45, and 58). High levels of expression of seven SolycPMEs (SolycPMEs2, 31, 35, 49, 51, 52, and 59) were detected in both green and red fruits. Among 48 SolycPMEIs, 23 (48%) were significantly upregulated in at least one organ (Fig. 4). Particularly, SolycPMEI1, 7, 22, 37, and 41 showed specific expression in flowers, whereas the SolycPMEI5 transcript was only highly expressed in red fruit.
3.2. PME and PMEI enzyme activities of different tomato tissues Pectin modification by PME/PMEI action during plant development and biotic/abiotic stresses is one of the most well-characterized mechanisms in various higher plants (Pelloux et al., 2007). Thus, we next determined the PME activity in tomato tissues. Soluble proteins were isolated from nine different tissues and used for PME and PMEI activity assays in vitro. In accordance with previous studies, the highest PME activity was detected in samples isolated from green fruits, whereas soluble proteins isolated from green and red fruit possessed the lowest PMEI activity (Fig. 1C and D). Notably, root and stems possessed high PME activities, while low activities were detected in leaf tissues, suggesting that vegetative tissues also undergo dynamic pectin modification. Our biochemical data strongly indicated that considerable PME/ PMEI activities also contribute to the normal growth and development of vegetative tissues.
3.6. PMEI activity assay of two tomato PMEIs (SolycPMEI13 and 14) In order to determine if tomato PME and PMEI indeed possess enzymatic activities, we selected 10 fruit-specific SolycPME and six ubiquitously expressed SolycPMEI genes. Full-length cDNA was successfully cloned and expressed in E.Coli strain BL21 CodonPlus (DE3)-RIPL, a heterologous system with extra copies of rare codons. Although five SolycPME were successfully purified from E. coli crude proteins, we failed to observe any PME activity in the assay reaction. This could be due to the lack of post-translational modification in our recombinant proteins expressed in E. coli. Out of six SolycPMEIs, considerable amounts of the soluble recombinant proteins of two genes (SolycPMEI13 and 14) were recovered and purified (Fig. 5A and B). In our assay system, the recombinant proteins exhibited high inhibitory activity against a commercial PME protein compared to a recombinant protein with empty vector (Fig. 5C). Higher PMEI activity was detected in a SolycPMEI13 recombinant protein even at an early time point. Next, we investigated which pH and temperature are optimal for PMEI activity and found that both recombinant proteins showed the highest activity at pH 8.5 and 20 °C (Fig. 6).
3.3. Immunolocalization of pectin status by LM19 and LM20 monoclonal antibodies Previous study demonstrated that dynamic alteration of the degree of pectin demethylesterification occurs during fruit ripening process. However, the status of pectin modification during vegetative organ development remains to be determined. We performed immunolocalization experiment using two epitopes raised against demethylesterified pectin (LM19) and methylesterified pectin (LM20). LM 19 recognizes demethylesterified pectin whereas LM20 binds methylesterified pectin. Four representative vegetative tissues (root, leaf, top stem, and bottom stem) were cross-reacted with epitopes (Fig. 2). Immunolabeling of root, leaf, and bottom stem by LM20 revealed much stronger fluorescence signals compared with that by LM19 (Fig. 2). Only weak signals were detected in top stem for both LM19 and LM20 (Fig. 2E and F). In accordance with our chemical data, vegetative tissues also contain highly methylated pectins in their cell walls.
4. Discussion Genome-wide analysis has identified PMEs and PMEIs in nearly all vascular plants and in multiple gene members (Pelloux et al., 2007). However, the majority of them have not been functionally characterized. Our integrative analysis indicated that the dynamic structural modification of pectin HGAs was observed in both vegetative and reproductive tissues. Previous studies have demonstrated that selective removal of a methyl group from HGAs as well as partial digestion of the pectin backbone by polygalacturonases play an important role during tomato fruit ripening (Brummell and Harpster, 2001; Hyodo et al., 2013, Reca et al., 2012, Rose, 2003). Accordingly, our chemical and biochemical data clearly showed that extensive enzymatic removal of HGA-bound methyl groups by PMEs is required in the fruit ripening stage (Fig. 1). The degree of methylesterification on HGAs mediated by PMEs and PMEIs affects cell and tissue properties. Pectin demethylation by PMEs results in a calcium-linked gel structure by the localized aggregation of pectin backbone structures (uronic acid). Consequently, this gel structure becomes susceptible to polygalacturonase-mediated digestion. Thus, selective removal of HGA-bound methyl groups by tomato PMEs and concomitant partial digestion by fruit-specific polygalacturonases play a critical role in fruit softening. Bioinformatics analysis indicated that tomato contains 79 putative PMEs and 48 putative PMEIs. Our expression profiling indicated that tissue-specific or ubiquitous expression patterns of PMEs and PMEIs. To our knowledge, this is first comprehensive expression analysis of tomato PMEs/PMEIs in vegetative tissues. The importance of PME and PMEI in vegetative growth and development has been reported in several plant species. In Arabidopsis, in which pectin is mainly found in the primary cell walls, demethylesterification by PME35 is required for providing mechanical support to the stem (Hongo et al., 2012). Significant differential expression and enzyme activities of PMEs/PMEIs were observed during the first 24 h of Arabidopsis seed germination, and overexpression of PMEI5 resulted in faster seed germination and a reduced sensitivity of seeds to ABA, an inhibitory phytohormone on germination, indicating that the reduced PME activities make cell walls
3.4. Identification and phylogenetic analysis of tomato PMEs and PMEIs The availability of S. lycopersicum genome sequence facilitated genome-wide identification and analysis of PME and PMEI families from cultivated tomato. To identify proteins encoding putative PMEs and PMEIs in S. lycopersicum, we searched proteins containing PME domains (PF1095) and PMEI domains (PF0404), respectively, and a total of 79 putative PMEs (SolycPMEs) and 48 putative PMEIs (SolycPMEIs) were identified (Table S2). The identified PME proteins were confirmed using InterProScan, which showed the presence of PMEs (IPR000070). The presence of a PMEI-conserved domain (cd15797) in the identified PMEI proteins was validated using the NCBI conserved domain database. A comparative phylogenetic tree using amino sequences of 79 SolycPMEs and 66 previously annotated Arabidopsis PMEs (AtPMEs) indicated clustering into six different clades/ groups (Groups 1 to 6) (Fig. S1). Additionally, we constructed a phylogenetic tree containing 48 SolycPMEIs and 78 AtPMEIs (Fig. S2). In silico analysis of the exon/intron organization of SolycPMEs and SolycPMEIs revealed that, different from SolycPME genes, only seven SolycPMEIs (SolycPMEI2, 22, 31, 36, 41, 47, and 48) have an intron (Figs. S3 and S4). 3.5. Tissue-specific expression of SolycPMEs and SolycPMEIs We next analyzed organ-specific expression of the 79 SolycPMEs and 48 SolycPMEIs in six representative organs (root, leaf, stem, flower, green fruit, and red fruit) by qRT-PCR. We integrated the phylogenetic tree into our heatmap to compare the transcriptional levels between paralogs. Expression analysis of SolycPMEs showed that, among the 78 genes, 39 (50%) were highly expressed in at least one organ (Fig. 3). Interestingly, 13 SolycPMEs (SolycPMEs4, 5, 9, 12, 18, 30, 39, 40, 42, 561
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Fig. 3. Heat map of the abundance of SolycPME transcripts in different tissues. The color of the cell represents transcript abundance. Gray cells indicate no transcripts were detected. Green colored boxes denote a low level of expression, and red colored boxes denote a high level of expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Although a large number of genes encoding PME/PMEI and associated regulators are expressed in both vegetative and reproductive organs in tomato, none of them have been directly linked to a precise physiological role. Our biochemical data including enzyme activities and cell wall analysis as well as immunolocalization by the LM19 and LM20 monoclonal antibodies clearly indicated that the degree of pectin methylesterification is actively regulated by two pectin-modifying enzymes. We found that PME activities in vegetative tissues were
of seeds more permeable to water (Müller et al., 2013a). In addition, tight regulation of PME activity by AtPMEI6 contributes to the accumulation of mucilage polysaccharides in the apoplast of seed coat epidermal cells (Saez-Aguayo et al., 2013). We previously reported that the overexpression of rice PMEI28 (OsPMEI28) caused a higher degree of methylesterification and a dwarf phenotype (Nguyen et al., 2017). Significant inhibition of intrusive apical growth was observed in transgenic plants overexpressing poplarPME1 (Siedlecka et al., 2008). 562
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Fig. 4. Heat map of the abundance of SolycPMEI transcripts in different tissues. The color of the cell represents transcript abundance. Gray cells indicate no transcripts were detected. Green colored boxes denote a low level of expression, and red colored boxes denote a high level of expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
modulated by cell wall properties (Yang et al., 2013). Therefore, we speculated that those genes with low or no detectable expression in representative tissues might function as regulators to cope with external environmental factors. Taken together, our findings provide an important tool to unravel the molecular and physiological functions of tomato PME and PMEI in further studies.
relatively lower than that of proteins isolated in green and red fruit, whereas PMEI activities were higher. Accordingly, several PME genes were expressed specifically or ubiquitously in fruits. It should be noted that low or no detectable expression was observed in more than half of PMEs and PMEIs. As mentioned in the Introduction, it is well known that biotic or abiotic stresses lead to structural modification of cell walls (An et al., 2008). Moreover, differential sensitivity to heavy metals is
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Fig. 5. Assay of the pectin methylesterase inhibitor (PMEI) activity of recombinant SolycPMEI13 and SolycPMEI14 proteins. Maltose binding protein (MBP)-tagged full-length proteins were expressed in Escherichia coli, purified, and then subjected to an assay to measure their inhibitory activity on commercial pectin methylesterase (PME) protein (orange peel PME). (A) SDS–PAGE detection of recombinant SolycPMEI13 expressed in E. coli. (B) SDS–PAGE detection of recombinant SolycPMEI14 expressed in E. coli. Lane 1, Uninduced E. coli BL21 Codon Plus (RILP) harboring MPB:SoPMEI fusion protein cell extracts; Lane 2, Induced cell extracts; Lane 3, Insoluble protein extracts; Lane 4, Crude protein extracts; Lane 5, Purified MBP:SolycPMEI fusion proteins; Lane 6, Purified MBP expressed from the pMALc2x vector with no insert (∼51 KDa). Molecular weight markers are shown on the left. (C) Time course PMEI activity assay of the recombinant SolycPMEIs. No inhibitory activity was detected for MBP. Error bars denote the SE of five independent assays.
Fig. 6. Biochemical properties of recombinant SoPMEI13 and SoPMEI14 proteins. (A) The effect of temperature on SoPMEI13 activity. (B) The effect of temperature on SoPMEI14 activity. The reaction with the highest activity was taken as 100%. (C) The effect of pH on SoPMEI13 activity. (D) The effect of pH on SoPMEI14 activity. The effect of pH was presented as the number of reaction product NADH formation after reactions. Error bars represent the SE of three biological replicates. 564
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Contribution
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