Genes Genet. Syst. (2010) 85, p. 97–106
Functional characterization of pectin methylesterase inhibitor (PMEI) in wheat Min Jeong Hong, Dae Yeon Kim, Tong Geon Lee, Woong Bae Jeon and Yong Weon Seo* College of Life Sciences and Biotechnology, Korea University, Seoul, 136-713, Republic of Korea (Received 21 December 2009, accepted 5 May 2010)
Pectin, one of the main components of plant cell wall, is deesterified by the pectin methylesterase (PME). PME activity is regulated by inhibitor proteins known as the pectin methylesterase inhibitor (PMEI), which plays a key role in wounding, osmotic stress, senescence and seed development. However, the role of PMEI in many plant species still remains to be elucidated, especially in wheat. To facilitate the expression analysis of the TaPMEI gene, RT-PCR was performed using leaf, stem and root tissues that were treated with exogeneous application of phytohormones and abiotic stresses. High transcription was detected in salicylic acid (SA) and hydrogen peroxide treatments. To elucidate the subcellular localization of the TaPMEI protein, the TaPMEI:GFP fusion construct was transformed into onion epidermal cells by particle bombardment. The fluorescence signal was exclusively detected in the cell wall. Using an enzyme assay, we confirmed that PME was completely inhibited by TaPMEI. These results indicated that TaPMEI was involved in inhibition of pectin methylesterification and may play a role in the plant defense mechanism via cell wall fortification. Key words: 2BS/2RL translocation line, cell wall, pectin methylesterase (PME), pectin methylesterase inhibitor (PMEI) INTRODUCTION The plant cell wall, which serves as a protective barrier, is composed of highly complex structures such as celluloses, hemicelluloses and pectins (Juge, 2006; Willats et al., 2001a). Pectin, the major component of primary cell wall, is related to plant growth, cell expansion, wall porosity, morphogenesis, fruit development and plant defense (Ridley et al., 2001). Made up of highly heterogenous groups of polymers including homogalacturonans and rhamnogalacturonans I and II (Willats et al., 2001b), pectins are synthesized in the cis-Golgi, methylesterified in the medial Golgi, swapped with side chains in the trans-Golgi cisternae, and then released into the cell wall (Grsic-Rausch and Rausch, 2004). Homogalacturonans are major components of pectin which are demethylesterified by pectin methylesterase (PME) (Pelloux et al., 2007). This enzyme belongs to class 8 of the carbohydrate esterases (CAZy website, http://www.cazy.org/fam/ CE8.html) and removes methyl ester groups from homogalacturonans within plant cell walls (O’Neill et al., 1990). Edited by Koji Murai * Corresponding author. E-mail:
[email protected]
PMEs are involved in diverse physiological process such as cell wall extension, cellular separation, seed germination, internode stem growth, root tip elongation, dry fruit dehiscence and soft fruit ripening (Al-Qsous et al., 2004; Sobry et al., 2005; Wen et al., 1999; Ren and Kermode, 2000). Also, pectin methylesterase plays a key role in pectin remodeling. PMEs affect pectin properties and cell wall rigidity by demethylesterification of homogalacturonans (Micheli, 2001; Willats et al., 2001b). PME activity affects the properties of the pectin matrix by influencing the calcium-pectate interactions and, thereby, increasing wall porosity and reducing the apoplastic pH, which is thought to activate local hydrolases. (Willats et al., 2001b). PMEs have been found in higher plants such as arabidopsis, tomato, potato and kiwi (LyNguyen et al., 2004). In arabidopsis, 67 PME-related genes are known to be existed (Markovic and Janecek, 2004). Pectin methylesterase is particularly regulated by inhibitor proteins such as the pectin methylesterase inhibitor (PMEI) (Camardella et al., 2000). The PMEIs inhibit demethylesterification of homogalacturonans and are similar to invertase inhibitor related proteins, which play key roles in metabolic enzymes (Koch, 1996). Functions of invertase inhibitor related proteins have a
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connection with plant defense mechanism and plant stress reactions such as wounding, senescence, drought stress and osmotic stress (An et al., 2008; Greiner et al., 1998, 1999). Fourteen inhibitor related protein genes were discovered by a homology search in arabidopsis (Rausch and Greiner, 2004) and two PMEI genes had been identified to reduce PME activity in vivo (Lionetti et al., 2007; Raiola et al., 2004; Wolf et al., 2003). These enzymes interact specifically with plant PMEs (including kiwi, orange, apple, tomato, apricot, carrot, potato and banana) (Ly-Nguyen et al., 2004). The PMEI is active against plant PMEs conforming to 1:1 non-covalent complex. The PME-PMEI complex has been identified at the structural level (Di matteo et al., 2005; Hao et al., 2008; Hothorn et al., 2004), with PMEI protein sequences containing four Cys conserved positions to construct two disulfide bridges (Camardella et al., 2000). Genes encoding for PMEI proteins have been revealed and functionally characterized from various plant species. (An et al., 2008; Lionetti et al., 2007; Mei et al., 2007; Raiola et al., 2004). However, there is little known about function of PMEIs in vivo especially in monocotyledon species. In this study, we isolated and functionally characterized a pectin methylesterase inhibitor gene (TaPMEI) in 2BS/2RL wheat-rye translocation line (Lee et al., 2009). GFP fusion protein was constructed to demonstrate the localization of TaPMEI, and abiotic stress and exogenous hormone treatment were applied to young seedlings of wheat to predict the transcript pattern of TaPMEI. Also, an enzyme assay was performed to verify the inhibitory role of TaPMEI on PME activity. MATERIALS AND METHODS Plant materials Near-isogenic lines (NILs) that are differed by the presence or absence of 2RL (Seo et al., 1997), each of three wheat progenitor (AA, SS ≒ BB, and DD genome), rye were incorporated in this study. Seeds were germinated for 36 h at room temperature and transferred to a Magenta box (6.5 × 6.5 × 20 cm, Greenpia Technology Inc.) containing polypro mesh (polypropylene mesh opening 0.1 cm, Small Parts Inc.) as described by previous paper (Lee et al., 2007). The seedlings were grown in a Magenta box filled with 180 ml of a half strength of Hoagland’s nutrient solution no. 2 (H2395, Sigma). Seedlings were grown in the growth chambers (Vison Scientific VS-91G09M) set 23/20°C (day/night) and 13/11 h (day/night). The Hoagland’s nutrient solution was exchanged each day. Application of phytohormones and abiotic stress treatments In order to examine the responses of the TaPMEI gene to phytohormones, 14 day-old seedlings were transferred to 180 ml of a half strength of
Hoagland’s nutrient solution, which contains each 100 mM of salicylic acid (SA), 100 μM of methyl jasmonic acid (MeJA) and 100 μM of abscisic acid (ABA) (An et al., 2008). The wheat seedlings (fully expanded 2nd leaf stage) were treated via dissolving root parts in 180 ml of a half strength of Hoagland’s solution dissolved with each of 25% (w/v) polyethylene glycol (PEG, MW 10,000) and 250 mM NaCl (Jung et al., 2008). Hydrogen peroxide (H2O2) treatment was applied by dissolving 100 mM of hydrogen peroxide (H2O2) in a half strength of Hoagland’s solution (An et al., 2008). The treated plant samples were immediately frozen in liquid nitrogen at –80°C until used for RNA extraction. Extraction of total RNA and RT-PCR Total RNA was extracted from the fully expanded 1st and 2nd leaf, stem and root with Trizol reagent (Invitrogen). Total RNA was treated with DNase I to avoid genomic DNA contamination. For RT-PCR, total RNA (3 μg) of each sample was used for the first-strand cDNA synthesis as described by manufacturer (iNtRON biotechnology) with some modification (Jung et al., 2008). Gene specific primers for RT-PCR were designed on the basis of cDNA sequence of TaPMEI (5’-ATCGACCACCGGGTCGTTATC3’ and 5’-CCGCCTCGTACCGCTCCCCAC-3’). The RTPCR primer was designed using 2RL specific sequences comparing to nucleotide sequences from each of wheat progenitor (AA, SS ≒ BB, DD), rye, NIL (2BS/2RL translocation), and NIL (non-translocation). The amplification for the RT-PCR was programmed as follows: 1 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 56°C, 1 min at 72°C and a final extension cycle of 72°C for 4 min. The wheat actin gene (accession no. AB181991, 5’-GCCACACTGTTCCAATCTATGA-3’ and 5’-TGAAATTGTATGTCGCTTC-3’) was used as a control. The amplified products were separated in 1% agarose in 1 × TAE buffer and stained with ethidium bromide. Cloning and sequencing of TaPMEI In a previous study, we discovered 2RL-specific markers using crossspecies clusters of unigene sequences from wheat, rice, barley and rye (Lee et al., 2009). The PMEI that was identified as a 2RL-specific marker (NSFT03P2_Contig16317) and is composed of about 300 bp nucleotides with absence of a start codon. In order to obtain the full sequence of the PMEI, primer sets were designed from the contig sequences downloaded from the GrainGenes 2.0 (wheat sequence, http://wheat.pw.usda.gov/GG2/index.shtml), TIGR database (rice sequence, http://rice.plantbiology.msu.edu/) and NCBI database (rye sequence, http://www.ncbi.nlm.nih.gov/). The forward (5’-ATGGCATCCTTCTACGCAGCCA-3’) and reverse (5’-TCATCAACGGGGGATATGCTTGGCG-3’) primers were used for the PCR amplification. The PCR cycling conditions were consisted of 32 cycles of 94°C for 60 sec, 56°C for 60 sec and 72°C for 90 sec. Amplification
Functional analysis of PMEI in wheat products were cloned into the T&A cloning vector (Real Biotech Cooperation) and sequenced. Sequencing results were analyzed using NCBI and GrainGenes database. Subcellular localization of TaPMEI:smGFP fusion protein The full-length PMEI ORF without the stop codon was isolated via PCR using primer sets, 5’-TCTAGAATGGCATCCTTCTACGCAGCC-3’ and 5’-GGATCCACGGGGGATAAGCTTGGCGATA-3’. The amplified DNA was inserted into T&A cloning kit (Real Biotech Cooperation) and digested with Xba I and BamH I. To generate 35S-smGFP:TaPMEI, the fragment was ligated with the 326GFP vector. Using the biolistic particle delivery system (Bio-Rad), onion epidermal cells were transformed using gold particles (1.0 μm) coated with 35SsmGFP:TaPMEI. The transformed onion epidermal cells were incubated for 24 h at 25°C in dark condition. The plain 326GFP vector was used as a control. Subcellular localization of the GFP fusion protein was observed using Carl-Zeiss, LSM 5 Exciter confocal laser scanning microscope (Carl Zeiss Canada, Don Mills, ON, CA). Expression and purification of recombinant TaPMEI protein The TaPMEI coding region was amplified by PCR with forward (5’-GGATCCATGGCATCCTTCTACGCAGCCA-3’) and reverse (5’-TCTAGATCAACGGGGGATATGCTTGGCG-3’) primers. Amplification products were ligated by the T&A cloning kit (Real Biotech Cooperation) and its sequence was confirmed with the ABI PRISM 310 Genetic Analyzer (Perkin Elmer). After digestion with BamH I and Xba I, amplified products were ligated into the pMAL-c4x vector (New England Biolabs). The constructed vector (MBP:TaPMEI) was transformed into the E. coli strain BL21 pLysS (DE3) (Invitrogen), grown in rich broth to 2 × 108 cells/ml (A600 of ~0.5) at 37°C, and induced with 0.3 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) for 2 h. After incubation, cells were centrifuged at 4000 × g for 20 min, and extracted with 25 ml of column buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 10 mM 2-mercaptoethanol) and lysed by sonication. The fusion protein was passed by an amylose resin column. The amylose resin column was washed with 12 column volumes of column buffer. Proteins were released with elution buffer (column buffer containing 10 mM maltose). MBP (maltose binding protein) was used as a negative control. The eluted protein was quantified by the Bradford Assay and size confirmed by SDS-PAGE. Enzyme assay of pectin methylesterase inhibitor To determine the inhibitory effect of TaPMEI, PME activity in the presence of TaPMEI was measured as described by Grsic-Rausch and Rausch (2004). The reaction mixture contains 894 μl 0.4 mM NAD (nicotinamide adenine
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dinucleotide) in 50 mM phosphate buffer (pH 7.5), 80 μl 5% (w/v) pectin (~90% esterified; Sigma) in H2O, 8 μl (0.35 U) formaldehyde dehydrogenase (Sigma) and 8 μl (1.0 U) alcohol oxidase (Sigma). After mixing, 10 μl (7.8 mU) of PME from orange peel (Sigma) was added to the mixture. Inhibitor solution of 10 μl (0.5 μg TaPMEI fusion protein, 20 mM Tris buffer [pH 7.5], 0.2 mM NaCl) was mixed with 10 μl PME and 0.5 μl 3 M K-acetate buffer (pH 5.3, Sigma). The reaction rate was measured constantly at 340 nm using a UV-Vis Spectrophotometer (Optizen 2120UV) at 25°C after 20 min pre-incubation. RESULTS Isolation and identification of TaPMEI In order to identify the sequence of TaPMEI (Triticum aestivum pectin methylesterase inhibitor) (accession no. FJ006730), gene-specific primers from interspecies conserved regions across four species (wheat, rye, barley and rice), three wheat progenitors (AA, SS ≒ BB, and DD genome), rye (RR genome), NIL (2BS/2RL translocation), and NIL (non-translocation) were prepared. Using this primer, we isolated TaPMEI which was derived specifically from 2RL. The genomic DNA sequence of TaPMEI was 677 bp from start to stop coding regions, which contain 98 bp of an intron sequence. The cDNA sequence of TaPMEI comprised of 579 bp open reading frame, which encodes 192 amino acid residues with a predicted molecular weight of 20.62 kDa and isoelectric point of 4.14 (Fig. 1). Most of the TaPMEI amino acid residues were hydrophobic. The N-terminal of deduced TaPMEI was analysized using the SignalIP program (http://www. cbs.dtu.dk/services/SignalP/). Deduced TaPMEI amino acids contain 28 signal peptide sequences and a transmembrane region. Homology searches for protein sequences were performed using the blast program at the National Center for Biotechnology Information (NCBI). The predicted amino acid sequence of TaPMEI was found to be 31% identical to that of Arabidopsis thaliana pectinesterase inhibitor (NP_201267) and to possess 31% identity to A. thaliana invertase inhibitor homologues (CAA73335, AAO50452), 26% with Actinidia deliciosa pectinmethylesterase inhibitor (BAC54966) and 29% with Oryza sativa pectinesterase inhibitor domain containing protein (ABA97456). Amino acid sequence homologies among PMEI ranged from 25% to 31%. We found that TaPMEI contains a conserved pectin methylesterase inhibitor domain of 150 amino acids (PF04043; http://www.ebi.ac.uk/Tools/InterProScan/). The protein sequence of TaPMEI represents four Cys which are at positions conserved in all sequences, and these are expected to be involved in the construction of two disulfide bridges (Fig. 2).
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Fig. 1. TaPMEI nucleotide sequence. peptide sequences.
The red arrow indicates the intron site (98 bp) and the blue line shows signal
Expression of TaPMEI gene in response to exogenous hormone and abiotic stress treatments At the molecular level, the PMEI gene may be involved in the early stages of the active defense responses to exogenous treatment with plant hormones (An et al., 2008). In order to investigate the expression of TaPMEI at the transcriptional level in wheat, RT-PCR was performed using leaf (fully expanded 1st and 2nd leaf), stem and root tissues that were treated with exogeneous application of phytohormone and abiotic stress treatments. TaPMEI was expressed in all tissues (i.e. fully expanded 1st and 2nd leaf, stem and root) (Fig. 3). The TaPMEI gene was continuously induced up to 24 h and peaked 48 h after treatment with hydrogen preroxide (H2O2). TaPMEI transcripts began to increase 12 h after the NaCl treatment and then diminished to initial expression levels in the leaf. With NaCl treatment in the stem and root,
TaPMEI transcripts were induced at 6 h, peaked at 12 h, and then decreased at 24 h. In the PEG treated wheat, TaPMEI transcripts were not changed in the leaf, but induced at 24 h in stems (Fig. 4). Salicylic acid (SA) strongly increased expression of TaPMEI at 6 h in leaves after treatment but, decreased gradually thereafter. In stem and root, TaPMEI transcripts accumulated at 24 h and then declined thereafter. Transcription levels of TaPMEI were declined slightly after ABA treatment in leaf and increased at 12 h in stem and root. Following treatment with methyl jasmonic acid (MeJA), a strong induction of TaPMEI was observed at 24 h in leaf and 6 h in root. In stems, TaPMEI transcripts were detected at a lower level in comparison to the expression levels in the control (Fig. 5). TaPMEI did not show tissue specific expression in response to exogenous application of phytohormones and abiotic stress treatments. However, the
Functional analysis of PMEI in wheat
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Fig. 2. Pectin methylesterase inhibitor domain was compared with plant species from different families. Gray color indicates pectin methylesterase inhibitor domain. Blue bars represented conserved Cys sequences among various plant species.
Fig. 3. lings.
TaPMEI expression in different tissues of wheat seed-
TaPMEI gene may play a key role in a plant’s defensive response to SA and hydrogen peroxide treatments. Subcellular localization TaPMEI:smGFP fusion protein To elucidate the subcellular localization of the TaPMEI protein, the TaPMEI:smGFP fusion construct
was prepared and transformed into onion epidermal cells by particle bombardment. The 326-GFP plain vector was used as a control (Fig. 6A). While the fluorescence signal of the cells transformed with the 326-GFP plain vector was dispersed throughout the cell, those of the cells transformed with the TaPMEI:smGFP fusion protein construct were exclusively detected in cell walls and plasma membrane (Fig. 6B). Purification of recombinant TaPMEI protein in E. coli and inhibition of PME assay In order to obtain recombinant TaPMEI protein, the TaPMEI coding region was cloned into pMAL-c4x vector and transformed into the E. coli strain BL21 pLysS. The recombinant single cell was grown at 37°C and induced with different concen-
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Fig. 4.
Transcription levels of the TaPMEI gene in different tissues at various time intervals after abiotic treatments.
Fig. 5. Expression levels of TaPMEI in wheat seedlings at various time intervals after exogenous application of phytohormones.
tration of IPTG (Fig. 7). A significant band at the expected size of 64 kDa was identified only in the induced cells. The bands consisted of maltose binding protein (MBP, ~42.5 kDa) fused to the TaPMEI protein (~20.63
kDa). The soluble fraction was purified by an amylose resin column. After purification by affinity chromatography, the recombinant protein showed a single band of ~64 kDa (Fig. 8, lane 5). This recombinant protein was
Functional analysis of PMEI in wheat
Fig. 6. Subcellular localization of the TaPMEI protein in onion epidermal cells. A: smGFP (control), B: TaPMEI:smGFP fusion protein. Onion epidermal cells were shown at a magnification of either 400 X (A) or 200 X (B).
Fig. 7. Recombinant TaPMEI protein expression in E. coli BL21 (DE3) pLysS. Cells were grown in rich-media, and recombinant protein expression was induced with IPTG. lane 1: uninduced cells, lane 2: 0.05 mM IPTG, lane 3: 0.1 mM IPTG, lane 4: 0.3 mM IPTG, lane 5: 0.7 mM IPTG, lane 6: 1 mM IPTG. Red arrow indicates TaPMEI fusion protein after IPTG induction.
composed TaPMEI protein with maltose binding protein of N-terminal region. The concentration of MBP:PMEI fusion protein was estimated to be about 0.5 μg/ml by the Bradford Assay. MBP was used as a negative control (Fig. 8, lane 6). TaPMEI includes pectin methylesterase inhibitor domain and PMEI is known to play a role in inhibiting pectin methylesterase (Camardella et al., 2000). In silico comparison of pectin methylesterase inhibitor domains from different plant species has denoted that the PMEI protein family is related to the regulation of pectin degradation (Raiola et al., 2004), even thought their protein sequences
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Fig. 8. Purification of recombinant TaPMEI protein. Lane 1: uninduced E.coli BL21 cell extracts, lane 2: induced E. coli BL21 cell extracts, lane 3: insoluble protein extracts, lane 4: crude protein extracts of E. coli cells producing the MBP:TaPMEI fusion protein, lane 5: purified MBP-TaPMEI fusion protein, lane 6: purified MBP (negative control). Red arrow indicates TaPMEI fusion protein.
are only about 31% identical. In order to study inhibition of PME (from orange peel) by TaPMEI protein, we used the coupled PME enzyme assay to estimate the formation of NADH by PME inhibitor (Rausch and Greiner, 2004). The TaPMEI fusion protein inhibited PME (from orange peels) enzyme activity, and a higher concentration of TaPMEI fusion protein exhibited stronger inhibition. Maltose binding protein (MBP) did not show any effect on NADH emission and PME and MBP activities were nearly the same. NADH were not detected in denature form of PME by heating. Maltose binding protein (negative control) did not affected PME activity (Fig. 9). However, when the TaPMEI fusion protein was added, NADH concentrations were decreased in comparison to that in PME enzyme activity. Also, NADH emissions were reduced right after PMEI added in the middle of assay (Fig. 9). These results indicated that TaPMEI may function as an inhibitor against PME activity. DISCUSSION Diverse range of cell wall enzymes involves in the plant cell wall synthesis or modification (Vorwerk et al., 2004). Most cell wall degradation enzymes (CWDEs) exist as multigene families which demonstrate various changes of expression and functional division (Coutinho et al., 2003). A number of proteins have been found to inhibit the activity of other CWDEs on pectin. In silico analysis have shown several PMEI related proteins that share structural homology with invertase inhibitors although the sequence homology is at a very
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Fig. 9. Inhibition of pectin methylesterase activity by the wheat pectin methylesterase inhibitor.PME : pectin methylesterase, PME-Heat : denature form of PME (Incubation of 95°C), PMEI : pectin methylesterase inhibitor, PME + PMEImedium : PMEI was added to the complete reaction mixture 12 min after the reaction was started (see arrow), MBP : maltose binding protein (negative control).
low level (range of 31–25%). The TaPMEI protein contains the four Cys residues, which are at positions conserved among other species of PMEI proteins. The four Cys residues involved in the two disulfide bridges are linked up first with second cysteines and third and fourth cysteines. These bridges play key roles in the maintenance of the protein structure (Camardella et al., 2000; Rausch and Greiner, 2004). The TaPMEI coding region contains one signal peptide at the N-terminal which is removed in the secretory pathway. At the transcriptional level, we compared the expression of TaPMEI in different tissues of 14 day-old wheat plants (i.e. fully expanded 1st and 2nd leaf, stem, root). In different tissues, There was no great difference in the expression levels of the different tissues (Fig. 3). In a previous report, the PMEI gene responded to the stresses including SA, MeJA, ethylene and ABA in pepper (An et al., 2008). Transcription of the TaPMEI gene was significantly induced with NaCl, H2O2 and SA treatments, and reduced when plants were treated with ABA. In particular, a strong expression of TaPMEI was observed 6 h after SA treatment. This result showed that the TaPMEI gene is connected with SA signaling, where SA application induced the accumulation of pathogenesis-related (PR) proteins (Loake and Grant, 2007). Following treatment with H2O2, TaPMEI1 transcription was induced at 48 h (Fig.4). Transcription level of the TaPMEI gene indicated its involvement in oxidative stress and regulation of various antioxidant enzymes (Guo et al., 1997;
Pastori and Trippi, 1992; Sairam and Srivastava, 2000). TaPMEI protein was detected in the cell wall and plasma membrane. When plasmolysis occurred in the onion cells, the fluorescence signal was detected only in the plasma membrane. In a previous study, the PMEPMEI complex was localized in the cell wall according to their signal peptide sequences toward final cellular localization (Camardella et al., 2000). However, PMEI does not appear to tightly adhere to the cell wall, which is in accordance with the localization of PMEI in a layer close to the cell membrane (Camardella et al., 2000). Plant cell walls have been related to diverse defensive responses including inhibitors of pectin degrading enzymes. PMEI might be involved in the control of pectin metabolism that affected plant cell wall porosity (Juge, 2006; Juge and Svensson, 2006). Especially, wheat-rye translocation line possessing 2RL have conferred useful genetic sources of disease and pest resistance, which may indicate that the wheat-rye translocation line might have a special response mechanism to pathogen attacks and different expressions of resistant genes for cell wall fortification (Friebe et al., 1990; Jang et al., 2003; Seo et al., 2001). PMEI was derived from a wheat-rye translocation (2BS/2RL) that possesses resistance and defenses against various stresses such wounding and infections by plant pathogens. The increase of PMEI activity may obstruct the demethylesterification of the highly heterogeneous pectin polymers in Arabidopsis (Lionetti et al., 2007).
Functional analysis of PMEI in wheat Inhibition of endogenous PME by PMEI keeps up highly methylated pectins. Methylesterification of pectin has a potential constitutive resistance to fungal and bacterial pathogens (Le Cam et al., 1994). Also, the cell wall mechanical properties are influenced by degree of methylesterification of pectins which related to interaction of PME and PMEI (Peaucelle et al., 2008). We infer that TaPMEI gene affects pectin methylesterification and plant disease resistance against several pathogen through the forming a high affinity complex with PME. In this report, we demonstrated that TaPMEI from wheat played an inhibitory effect on PME. In addition, we characterized the unknown function of PMEI in monocots, especially in wheat. Localization of TaPMEI may be important not only for the understanding of cell wall porosity by methylesterification, but also for the defensive mechanism against plant pathogen attack in wheat. This work was supported by the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry. This work was also supported by grant from the BioGreen 21 Program (20070301034016), Rural Development Administration, Rupublic of Korea.
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