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Genome-wide identification and expression analysis of rice pectin methylesterases: Implication of functional roles of pectin modification in rice physiology
Journal of Plant Physiology 183 (2015) 23–29
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Genome-wide identification and expression analysis of rice pectin methylesterases: Implication of functional roles of pectin modification in rice physiology Ho Young Jeong a,1 , Hong Phuong Nguyen a,1 , Chanhui Lee a,b,∗ a b
Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Republic of Korea Department of Plant and Environmental New Resources, Kyung Hee University, Yongin 446-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 April 2015 Received in revised form 9 May 2015 Accepted 10 May 2015 Available online 2 June 2015 Keywords: Pectin Pectin methylesterases Rice Plant cell walls Cell wall modifications
a b s t r a c t Pectin, which is enriched in primary cell walls and middle lamellae, is an essential polysaccharide in all higher plants. Homogalacturonans (HGA), a major form of pectin, are synthesized and methylesterified by enzymes localized in the Golgi apparatus and transported into the cell wall. Depending on cell type, the degree and pattern of pectin methylesterification are strictly regulated by cell wall-localized pectin methylesterases (PMEs). Despite its importance in plant development and growth, little is known about the physiological functions of pectin in rice, which contains 43 different types of PME. The presence of pectin in rice cell walls has been substantiated by uronic acid quantification and immunodetection of JIM7 monoclonal antibodies. We performed PME activity assays with cell wall proteins isolated from different rice tissues. In accordance with data from Arabidopsis, the highest activity was observed in germinating tissues, young culm, and spikelets, where cells are actively elongating. Transcriptional profiling of OsPMEs by real-time PCR and meta-analysis indicates that PMEs exhibit spatial- and stress-specific expression patterns during rice development. Based on in silico analysis, we identified subcellular compartments, isoelectric point, and cleavage sites of OsPMEs. Our findings provide an important tool for further studies seeking to unravel the functional importance of pectin modification during plant growth and abiotic and biotic responses of grass plants. © 2015 Elsevier GmbH. All rights reserved.
Introduction Pectin, one of the major cell wall components found in the primary cell wall and middle lamellae, is classified into four different domains based on backbone structure and monosaccharide compositions and glycosidic linkages of substitution: homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII), and xylogalacturonan (XGA) (Harholt et al., 2010; Mohnen, 2008). Structural analyses of pectin reveal that HGA, a linear polymer of (1,4)-linked-␣-galacturonic acids (GalUA), accounts for approximately 65% of pectin composition, and that the dominant monosaccharide of pectin is GalUA. GalUA in HGA is methylesterified at the C-6 carboxyl by pectin methyltransferases in the Golgi apparatus (Pelloux et al., 2007). Once synthesized, methylesterified
∗ Corresponding author at: Department of Plant and Environmental New Resources, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu, Yongin 446701, Republic of Korea. Tel.: +82 31 201 3480; fax: +82 31 204 8116. E-mail address: [email protected] (C. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jplph.2015.05.001 0176-1617/© 2015 Elsevier GmbH. All rights reserved.
HGA is targeted to the cell walls and undergoes further modifications depending on cell types and their positions in the pectin network. Pectin methyltesterases (PME, EC 3.1.1.11) belonging to class 8 carbohydrate esterases (CAZy, http://www.cazy.org/fam/CE8. html) catalyze the demethylesterification of GalUA in HGA (Cantarel et al., 2009). Partially demethylesterified HGA is then degraded by polygalacturonases, resulting in changes of wall properties such as rigidity, elasticity, and permeability (Moustacas et al., 1991). Since such structural remodeling of HGA by PMEs generally leads to cell wall loosening, cell types programed for cell expansion and elongation during cell development are required to increase the activity of PMEs. Moreover, recent studies showed that the methanol produced after PME action is an important signaling molecule in plant defense against biotic and abiotic stresses (Komarova et al., 2014). Sequence analyses of several model species showed that PMEs are a large multigene family; there are 66 PMEs in Arabidopsis thaliana, 89 in Poplus trichocarpa, 43 in Oryza sativa, 105 in Linum usitatissimum, and 79 in Lycoperscicon esculentum (Pelloux et al., 2007; Pinzon-Latorre and Deyholos, 2013; Vandevenne et al.,
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2009). Depending on the presence or absence of the PME inhibitor (PMEI) domain at the N-terminus (also known as the PRO region), PMEs are grouped into either Type-1 PME (with PMEI domain) or Type-2 PME (without PMEI domain) (Jolie et al., 2010). Genome analysis of bacteria, fungi, and moss Physcomitrella, revealed the absence of PMEI domain-containing PME proteins (Pelloux et al., 2007). Although there is no experimental evidence of the functional role of PMEI domains in Type-1 PME proteins, it is postulated to play a role in self-inhibitory activity to prevent premature demethoxylation (Bosch et al., 2005). Over the last two decades, mutational and transgenic approaches have led to significant progress in understanding the major biological roles of PMEs in plants (Francis et al., 2006; Bosch et al., 2005; Hongo et al., 2012). Representative phenotypes caused by mutations in PME isoforms in Arabidopsis include a defect in pollen tube growth and pollen tetrad formation and pendant stem growth. Transgenic plants overexpressing the Arabidopsis PMEI gene exhibit reduced PME activities and in increased pectin methylesterification (Müller et al., 2013). These transgenic plants also have faster germination rates than the wild type and reduced seed yields. In addition to its roles in plant development, several lines of evidence indicated that PME proteins function as a key regulator in plant-microbe interactions. Down-regulation of tobacco PME expression via antisense suppression resulted in delayed systemic movement of tobacco mosaic virus (Chen and Citovsky, 2003). Also, Hewezi et al. reported that a cyst nematode (Heterodera schachtii) secretory protein directly interacts with AtPME3 and this interaction is required for enhanced susceptibility of plants to the pathogen. Moreover, AtPME3 is rapidly induced upon infection of Pectobacterium carotovorum and Botrytis cinerea and acts as a susceptibility factor for the initial colonization by the pathogens (Raiola et al., 2011). The importance of pectin modification by PME was further demonstrated by comparing changes in the activities of PME during cold acclimation. The increase in PME activity was observed in cold-acclimated oilseed rape plants (Solecka et al., 2008). A previous study also showed that PMEs are involved in drought stress tolerance as overexpression of a pepper PMEI protein in Arabidopsis caused enhanced tolerance to drought stress (An et al., 2008). PMEs are also likely to be an important determinant of salt stress response. An Arabidopsis mutant with T-DNA insertion within the promoter of a PMEI gene (At1g62760) exhibited reduced sensitivity to NaCl stress (Jithesh et al., 2012). Taken together, previous studies demonstrate the importance of temporal and spatial regulation of PME activities in plant physiology as well as plant-microbe interactions. The functional role of rice PMEs is poorly understood. A previous study explored the association of aluminum toxicity with altered transcriptional expressions of eight rice PMEs (Yang et al., 2013). Transgenic rice plants overexpressing OsPME14 gene displayed increased sensitivity to aluminum stress. In this report, an initial step to characterize PME functions in rice, we found that rice cell walls contain substantial quantities of pectin through chemical and immunolocalization analysis, and that cell wall proteins exhibit PME enzyme activities. Transcriptional profiling of OsPMEs indicates that PMEs exhibit spatial- and stress-specific expression patterns during rice development. Materials and methods Plant materials Rice seeds (Oryza sativa cv. Dongjinbyeo) were soaked in water and pregerminated at 28 ◦ C under a light cycle of 16 h light/8 h dark for three days and transferred to soil. Rice plants were grown in a temperature-controlled glasshouse with natural lighting conditions. For RNA and total protein extraction, various tissues from germination to reproductive stages were collected, frozen in liquid
nitrogen, and stored at −80 ◦ C until analysis. Germinating tissues (shoot and root) were collected after 4 days of pregermination and immediately used for RNA and protein extraction. Young culm indicates samples collected from the entire third and fourth internode at 14 days before heading. Old culm indicates samples collected from the bottom parts of internode at 7 days after heading. Preparation of alcohol insoluble residues (AIRs) and measurement of uronic acid contents AIRs were prepared as described previously (Zhong et al., 2005; Lee et al., 2007). Briefly, frozen materials of each tissue were ground under liquid nitrogen. Fine powders were suspended in 70% (v/v) ethanol using a Polytron homogenizer three times. The AIRs collected were washed sequentially in absolute ethanol and 100% acetone. The resulting residues were dried in a vacuum oven at 60 ◦ C. In order to obtain pectin enriched fractions (PEFs), the AIRs (100 mg) were stirred to ammonium oxalate solution (50 mM) for 24 h at room temperature. Soluble fractions were collected by centrifugation and lyophilized. Uronic acid content was determined as described by Filisetti-Cozzi and Carpita (1991). Lyophilized PEFs were dissolved in 0.4 ml of 0.5 M H2 SO4 at room temperature for 1 h 40 l of 4 M sulfamic acid-potassium sulfamate (pH1.6) was added and mixed thoroughly. Samples were hydrolyzed in 2.4 ml of 75 mM Na2 B4 O7 dissolved in concentrated H2 SO4 at 100 ◦ C for 20 min. After cooling, 80 l of 0.15% (w/v) m-hydroxybipheneyl 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 by using galacturonic acid as a standard. PME activity assay Cell wall protein extracts were generated from germinating shoot, germinating root, 1 month old shoot, 1 month old root, 2 month old leaf, 2 month old root, young culm, spikelet, and flag leaf. Tissues were collected in liquid nitrogen and homogenized with equal volumes (w/v) of extraction buffer (100 mm Tris–HCl, pH 7.5, 500 mm NaCl containing protease inhibitor cocktail) and homogenized samples were then rotated at 4 ◦ C for 120 min. The samples were centrifuged at 11,500 × g at 4 ◦ C for 20 min and the supernatant were used immediately for all enzyme assays. A coupled enzymatic assay was performed under standard conditions as described by Grsic-Rausch and Rausch (2004) using a spectrophotometric plate reader. Heat-denatured cell wall proteins of each sample were used as a negative control. Histology and immunodetection of recognized methyl esterified homogalacturonans Young culm and leaves of 9-week-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.). 0.5 m thick sections were cut with a microtome and stained with 0.1% toluidine blue solution. For immunodetection, 0.5 m thick sections were incubated with JIM 7 monoclonal antibody (Plantprobes, Leeds University, UK) and then with fluorescein isothiocyanate-conjugated secondary antibodies. The fluorescence-labeled sections were observed using confocal microscope. Primer design and real time PCR Using Pfam ID (PF01095), we searched Putative Function Search Tool in Rice Genome Annotation Project (rice.plantbiology.msu.edu/). Then, we used Primer-BLAST in
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NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index. cgi?LINK LOC=BlastHome) for designing primers (Table. S1). In the primer-BLAST result, we had chosen a pair of primer that doesn’t match unspecific binding to other gene. Total RNA was isolated from different rice 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). The PCR threshold cycle number of each gene was normalized with expression level of the rice Ubiquitin 5 (OsUbi5, LOC Os01g22490) as a reference gene. We used Rice Oligonucleotide Array Database (http://www. ricearray.org/) to obtain microarray data of PMEs in response to abiotic and biotic stressed. In case of expression patterns of PMEs for abiotic stresses, data from GSE7256 were used. In case of expression patterns of PMEs for biotic stresses, data from GSE 19844 were used. The data were calculated and remake heatmap with R program (http://cran.r-project.org/). Meta-analysis of stress responsive expression of OsPMEs We used meta-analysis of stress specific expression profiles based on 231 Affymetrix array data for abiotic stresses and 85 Affymetrix array data for biotic stresses, which were downloaded from the NCBI gene expression omnibus (GEO, http://www.ncbi. nlm.nih.gov/geo/) (Nguyen et al., 2014). We then uploaded the log2 normalized intensity data in tab-delimited text format into Multi Experiment Viewer (MEV, http://www.tm4.org/mev/). We generated a heat map image based on log2 fold-change data in response to abiotic and biotic stresses and then integrated the heat map into the phylogenetic tree context. Phylogenetic analysis For phylogenetic analysis, we used PMEs from rice and Arabidopsis. PME amino acid sequences for Arabidopsis were extracted from Phytozome. We use ClustalW2 (http://www.ebi.ac.uk/Tools/ msa/clustalw2/) for drawing a comparative phylogenetic tree. In order to analyze tree groups of each PME protein, sequence files were loaded onto dendroscope (http://ab.inf.uni-tuebingen. de/software/dendroscope/). The tree groups were assigned as previously described (Louvet et al., 2006). In silico analysis of rice PMEs The presence of signal peptide was predicted with SignalP 4.0. Transmembrane domains were predicted using TMHMM v.2.0. Two different prediction programs, WoLF PSORT and Plant-mPLoc, were used for the protein subcellular localization. Cleavage sites were predicted as described by Pelloux et al. (2007) and Wolf et al. (2009). The predicted isoelectric point of the complete and the mature proteins was calculated using Vector NTI 10. Results Measurements of rice pectin quantity and PME activity A previous study reported that monocot plants possess less pectin in cell walls than dicot plants (Carpita, 1996; Vogel, 2008; Oh et al., 2013). In order to quantitatively determine pectin content in rice, cell walls from different tissues were prepared at different stages of development and treated with ammonium oxalate solution, which selectively extracts pectin (Ismail and Ramli, 2012). Since a major monosaccharide component in pectin is galacturonic acid, we performed sulfamate/m-hydroxydiphenyl assay to determine uronic acid content in rice tissues (Filisetti-Cozzi and Carpita, 1991). The highest quantities of uronic acid were observed in 40 d
Fig. 1. Quantitative analysis of uronic acid contents for pectin enriched fractions of cell walls isolated from different tissues (A) and PME activities in different tissues in rice (B). (A) AIRs were treated with 50 mM ammonium oxalate for 8 h and used for uronic acid quantification. Error bars represent the SE of three biological replicates. (B) Cell wall proteins were isolated and used for PME activity assay. The enzyme activity of germinating shoot was taken as 100, and the activity in other tissues was expressed as a percentage of the germinating shoot. Error bars represent the SE of three biological replicates.
young culm (Fig. 1A). Overall, cell walls of aerial tissues (leaves and culms) contain greater quantities of uronic acids than do roots. The presence of pectin in rice tissues was also examined by immunolocalization using monoclonal antibody JIM7, which binds to methyl esterified homogalacturonan (Fig. 2C and D). Immunolabeling of young rice culm and leaf sections showed intense signals in both parenchyma and sclerenchyma cell types, demonstrating significant deposition of pectin in rice primary and secondary cell walls. Next, we investigated whether pectin in rice cell walls also undergoes PME-mediated demethylesterification. Apoplastic proteins of different tissues were prepared and used to analyze PME activities (Fig. 1B). Interestingly, the highest levels of activity were detected in germinating rice shoots and spikelets. These data indicate that rice cell walls contain significant amounts of pectin and that rice PMEs play potentially diverse physiological roles. Identification of rice PMEs and tissue-specific transcriptional analysis In order to identify proteins encoding putative PMEs in rice, we searched for proteins containing PME domains (PF1095) and identified 43 putative PMEs (Table S1 and Fig. S2). We produced a phylogenetic tree consisting of 43 rice and 66 Arabidopsis PMEs (Fig. S1). Five major subfamilies of rice and Arabidopsis PMEs (labeled as groups A to E) were generated. Seventeen rice PMEs belong to group A, 8 to group B, 6 to group C, 5 to group D, and 7 to group E (Table S3). Twenty rice PMEs (47%) were Type-1 (proteins with both PMEI and PME domains) and 23 (53%) were Type-2 (proteins with only PME domains). In order to examine tissue- or organ-specific expressions of the 43 rice PMEs, we performed real-time PCR using 10 different tissues: germinating shoot, 1 month shoot, 1 month root, 2 month leaf, young culm (2 month), old culm (3 month), young spikelet,
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Fig. 2. Immunodetection of methyl esterified homogalacturonans of rice culm and leaf. Culm and leaf thin sections (1 m) were probed with the JIM7 monoclonal antibody. JIM7 antibody recognized methyl esterified homogalacturonans. (A) A rice culm section stained with toluidine blue staining solution. (B) A rice leaf section stained with toluidine blue staining solution. (C) A rice culm section immunostained with JIM7. (D) A rice leaf section immunostained with JIM7. cf—cortical fiber; bs—bundle sheath fiber; mx—metaxylem. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
old spikelet, flag leaf, and panicles with flowers. Integration of the phylogenetic tree into our transcriptional analysis would be a good reference to compare the expression levels between paralogs. Expression analyses showed that all rice PMEs were expressed in at least one tissue and that a number of rice PMEs (27/43) displayed high levels of gene expression during a vegetative stage and in young culm, in which cells are actively elongating (Fig. 3). Overall, ubiquitous expression was found in five PMEs (OsPME3, 6, 8, 10, and 32), while seven PMEs (OsPME18, 21, 25, 28, 29, 41, and 42) showed one or two forms of tissue-specific expression. In order to examine the expression profile change of OsPMEs during plant defense responses, we also performed a metatranscriptional analysis of OsPMEs under five abiotic (drought, salt, cold, heat, and anaerobic condition) and four biotic (Magnaporthe grisia, rice stripe virus, Xanthomonas oryzae pv. oryzae, and brown planthopper) stresses, utilizing publicly available microarray data (Fig. 4). Interestingly, a total of 13 OsPME genes (OsPME2, 3, 4, 6, 8, 10, 17, 20, 26, 27, 28, 34, and 41) were significantly upregulated (>4-fold) in at least one of the abiotic stresses, whereas a total of 9 OsPME genes (OsPME3, 6, 14, 15, 16, 19, 23, 28, and 35) were down-regulated (<4-fold) in at least one of the abiotic stresses. Particularly, a number of OsPME genes are transcriptionally responsive to drought, heat, and anaerobic stresses while a few OsPME genes are up- or down-regulated by salt and cold stresses. Based on the microarray data, we found similar expression patterns of OsPMEs in response to Magnaporthe grisea and X. oryzae pv. oryzae, which cause rice blast and rice bacterial blight diseases, respectively. OsPME2 and 28 showed elevated transcript levels in response to both pathgens. X. oryzae pv. oryzae infection. More dynamic transcriptional regulation of OsPMEs was found in response to rice stripe virus and brown planthopper. Nine OsPME genes (OsPME9, 15, 16, 20, 27, 28, 34, 41, and 43) are significantly up-regulated whereas four OsPME genes (OsPME2, 6, 7, 11, and 17) are down-regulated in response to rice stripe virus. Eight OsPME genes (OsPME2, 6, 8, 10, 17, 27, 28, and 32) are significantly upregulated whereas five OsPME genes (OsPME9, 15, 16, 19, and 36)
are down-regulated in response to brown planthopper. OsPME28, which is highly expressed in 1-month-old shoot and young culm (Fig. 1), showed increased transcript levels upon all biotic stresses examined and three abiotic stresses (drought, cold and heat). Taken together, the existence of numerous PMEs in the rice genome and their spatial- and stress-specific expression patterns indicate that the enzymatic actions of pectin modifications such as the removal of methyl groups by PMEs play important roles in rice growth and development.
In silico analysis of rice PMEs Proteins involved in cell wall modifications, including the removal of methyl groups in pectin by PMEs, are generally believed to be targeted to cell walls (Gilbert, 2010). Thus, such proteins should have either N-terminal signal peptides or transmembrane domains. All rice PMEs examined in this study have an N-terminal signal peptide, a transmembrane domain, or both, with the exceptions of OsPME38, 39, 40, and 41 (Table S3). Sequence analyses using Plant-mPLoc for the prediction of protein localization predicted all rice PMEs to be cell-wall localized proteins (Table S3). Previous studies of protein localization demonstrated that some Type-1 PMEs undergo proteolytic cleavages of the PMEI domain at specific sites (binding motif 1 and binding motif 2) through the action of subtilisin-like serine proteases (Von Groll et al., 2002). We searched for the motif [KKLR][RRMA][RRLL][RHLL][RRML][KLDL][RKLL] in the Type-1 PMEs using “Protein Pattern Find”. Out of 20 Type-1 PMEs, we found that 10 possessed a single motif site and that 3 had two motif sites. As expected, no cleavage sites were found in the Type-2 PMEs (Table S3). Another important feature of the enzymatic action of PMEs is the isoelectric point. Processed PME amino acid sequences were generated and the isoelectric point was calculated using Vector NTI 10. Most (26/43) of PMEs had a basic pI (above pG8.0), while 5 had an acidic pI (Table S3).
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Fig. 3. Heat map of transcript abundances of OsPMEs 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.)
Discussion Cell wall modifications by different classes of cell wall hydrolases and esterases are postulated to play important roles in fine tuning the composition and distribution of cell wall polysaccharides (Jamet et al., 2006). Compared to other model plants such as Arabidopsis, poplar, and tomato, the biological implications of such changes in rice, a monocot model, remain unexplored. Moreover, how these enzymes modulate the developmental and physiological responses of rice is poorly understood. In order to shed light on this issue, it is imperative to collect general molecular and biochemical evidence of cell wall modifying enzymes. Here, we provide data showing that rice cell walls from different tissues contain substantial quantities of pectin and identifying 43 different rice PMEs. Enzyme activity assays using cell wall proteins demonstrate the presence of PME activity in diverse tissues. Transcriptional analyses indicate diverse temporal and spatial expression patterns. These findings provide an experimental platform for future studies. Previous chemical studies reported that typical grass cell walls contain five-fold lower quantities of pectin than those of dicots, but are enriched with mixed glucans and phenolics (ferulic acid and pcoumaric acid), particularly the primary cell walls (Vogel, 2008; Carpita, 1996). However, we observed significant accumulations of pectin in rice primary and secondary cell walls. First, the quantification of uronic acid contents in pectin-enriched fractions indicates
comparable amounts of pectin in different tissues (Fig. 1A). Second, immunolocalization analysis demonstrated the presence of methyl esterified pectin in both primary and secondary cell walls (Fig. 2). These results are different from results for other types of dicot plants, in that pectin is a major polysaccharide predominantly found in primary cell types, but not in secondary cell walls. Since pectin serves as a gelling and stabilizing polymer of other cell wall polysaccharides, it is likely that pectin is a structurally important component in both types of cell walls in rice. Tissues in which cell elongation is actively occurring showed the highest PME activity (Fig. 1B). This observation is consistent with previous findings regarding the functional roles of PMEs in several species. PME activity increases during early stages of seed germination, root tip growth, leaf growth polarity, internode stem growth, and pollen tube growth. Demethylesterification of cell wall pectins by PMEs results in the formation of gel-like structures between HGAs and thereby decreases the mechanical restraint of neighbor cell types, steps that are required for cell elongation (Pelloux et al., 2007). Transcriptional analyses of rice PMEs using real-time PCR, combined with the analysis of publicly available micro array data, shows dynamic expression patterns during growth and in response to abiotic and biotic stresses (Figs. 3 and 4). In particular, similar to the enzyme activity assay, 23 and 13 OsPMEs are highly expressed in young vegetative tissues and young culm, respectively, where cell wall extension occurs. Taken together, our findings of higher PME activities and increased transcript levels in young vegetative
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Fig. 4. Expression pattern of OsPMEs in response to abiotic and biotic conditions. The heat map was drawn using Affymetrix meta-analysis of stress-specific expression database based on 983 array data. Transcriptional expression of OsPMEs in response to abiotic stresses (drought-root, drought-shoot, salt, cold, heat, and anaerobic condition) and to biotic stresses (Magnaporthe grisea, rice stripe virus, Xanthomonas oryzae pv. oryzae, and brown planthopper) was shown. Magnaporthe grisea causes rice blast disease and Xanthomonas oryzae pv. oryzae causes bacterial blight disease. Green colored boxes denote a low level of expression and red colored boxes denote a high level of expression. Gray color boxes indicate the data not analyzed due to the absence of respective probes on Affymetrix array. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
tissues indicate that PME expression and enzyme activity in rice are prerequisites for cell elongation. HGA polymers are synthesized in the Golgi apparatus and passively transported to cell walls, where PMEs exert enzymatic action to remove methyl groups depending on developmental and environmental signals (Mohnen, 2008). Previous localization experiments in flax and tomato demonstrated that processed (removal of PRO regions) or non-processed PMEs are predominantly found in cell walls (Al-Qsous et al., 2004; Micheli, 2001). In accordance with these previous findings, our in-silico results indicate that all 43 OsPMEs are localized in the cell walls. Definitive proof would depend on the actual localization of OsPMEs using fluorescent protein-tagged methods. We have successfully constructed GFP-fused constructs of five PMEs and will infiltrate these into tobacco leaves to confirm cell wall localization in future studies. The difficulty of performing enzyme activity assays using purified PME proteins has been the limiting step to a greater understanding of PMEs, largely due to the difficulty of expressing plant PMEs in heterologous systems (Pelloux et al., 2007). We initially attempted to detect PME activity by the use of recombinant OsPMEs in Escherichia coli (E. coli) and successfully purified these proteins (data not shown). However, we failed to detect any PME activity compared with the positive control (commercial purified PME). This could be due to the lack of posttranslation modification systems, including glycosylation, in bacteria. It would be necessary to express plant PMEs in other host systems such as Pichia pastoris to obtain further insights in PME enzymology. Transcriptional profiling using different tissues demonstrated overlaps of the expression patterns of many OsPMEs. Thus, it is unlikely that we will detect observable phenotypes when only a single OsPME gene is mutated, due to gene redundancy (Müller et al., 2013). Our attempts to demonstrate the biological importance of pectin demethylesterification will be pursued by generating transgenic plants overexpressing the PMEI genes. So far, we have
generated overexpressing transgenic lines from four different PMEI genes (PMEI8, 12, 24, and 26) and transferred these to the soil. The identification and molecular characterization of OsPMEs provides an important tool for further studies of pectin modification during plant growth and of the abiotic and biotic responses of grass plants. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) (Grant no. NRF-2014R1A1A1004615) funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A1A1004615) and a grant by High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (Grant no. 114027-3). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph.2015.05. 001 References Al-Qsous S, Carpentier E, Klein-Eude D, Burel C, Mareck A, Dauchel H, et al. Identification and isolation of a pectin methylesterase isoform that could be involved in flax cell wall stiffening. Planta 2004;219:369–78. An SH, Sohn KH, Choi HW, Hwang IS, Lee SC, Hwang BK. Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance. Planta 2008;228:61–78. Bosch M, Cheung AY, Hepler PK. Pectin methylesterase, a regulator of pollen tube growth. Plant Physiol 2005;38(3):1334–46. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 2009;37:D233–8. Carpita NC. Structure and biogenesis of the cell walls of grasses. Annu Rev Plant Physiol Plant Mol Biol 1996;47:445–76.
H.Y. Jeong et al. / Journal of Plant Physiology 183 (2015) 23–29 Chen MH, Citovsky V. Systemic movement of a tobamovirus requires host cell pectin methyl-esterase. Plant J 2003;35:386–92. Filisetti-Cozzi TM, Carpita NC. Measurement of uronic acids without interference from neutral sugars. Anal Biochem 1991;197(1):157–62. Francis KE, Lam SY, Copenhaver GP. Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene. Plant Physiol 2006;142(3):1004–13. Gilbert HJ. The biochemistry and structural biology of plant cell wall deconstruction. Plant Physiol 2010;153(2):444–55. Grsic-Rausch S, Rausch T. A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors. Anal Biochem 2004;333(1):14–8. Harholt J, Suttangkakul A, Scheller HV. Biosynthesis of pectin. Plant Physiol 2010;153:384–95. Hongo S, Sato K, Yokoyama R, Nishitani K. Demethylesterification of the primary wall by PECTIN METHYLESTERASE35 provides mechanical support to the Arabidopsis stem. Plant Cell 2012;24(6):2624–34. Ismail NSM, Ramli N. Extraction and characterization of pectin from Dragon fruit (Hylocereus polyrhizus) using various extraction conditions. Sains Malays 2012;41(1):41–5. Jamet E, Canut H, Boudart G, Pont-Lezica RF. Cell wall proteins: a new insight through proteomics. Trends Plant Sci 2006;11(1):33–9. Jithesh MN, Wally ODS, Manfield I, Critchley AT, Hiltz D, Prithiviraj B. Analysis of seaweed extract-induced transcriptome leads to identification of a negative regulator of salt tolerance in Arabidopsis. HortScience 2012;47:704–9. Jolie RP, Duvetter T, Van Loey AM, Hendrickx ME. Pectin methylesterase and its proteinaceous inhibitor: a review. Carbohydr Res 2010;345(18):2583–95. Komarova TV, Sheshukova EV, Dorokhov YL. Cell wall methanol as a signal in plant immunity. Front Plant Sci 2014;5:101, http://dx.doi.org/10.3389/ fpls.2014.00101. Lee C, Zhong R, Richardson EA, Himmelsbach DS, McPhail BT, Ye ZH. The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis. Plant Cell Physiol 2007;48(12):1659–72. Louvet R, Cavel E, Gutierrez L, Guénin S, Roger D, Gillet F, et al. Comprehensive expression profiling of the pectin methylesterase gene family during siliquedevelopment in Arabidopsis thaliana. Planta 2006;224:782–91. Micheli F. Pectin methylesterases: cell wall enzymes with important roles in plant physiology. Trends Plant Sci 2001;6:414–9.
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Mohnen D. Pectin structure and biosynthesis. Curr Opin Plant Biol 2008;11: 266–77. Moustacas AM, Nari J, Borel M, Noat G, Ricard J. Pectin methylesterase, metal ions and plant cell-wall extension. Biochem J 1991;279:351–4. Nguyen VN, Moon S, Jung KH. Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses. J Plant Physiol 2014;171(14):1276–88. Oh CS, Kim H, Lee C. Rice cell wall polysaccharides: structure and biosynthesis. J Plant Biol 2013;56(5):274–82. Pelloux J, Rusterucci C, Mellerowicz EJ. New insights into pectin methylesterase structure and function. Trends Plant Sci 2007;12:267–77. Pinzon-Latorre D, Deyholos MK. Characterization and transcript profiling of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) gene families in flax (Linum usitatissimum). BMC Genomics 2013;14: 742. Raiola A, Lionetti V, Elmaghraby I, Immerzeel P, Mellerowicz EJ, Salvi G, et al. Pectin methylesterase is induced in Arabidopsis upon infection and is necessary for a successful colonization by necrotrophic pathogens. Mol Plant Microbe Interact 2011;24(4):432–40. Solecka D, Zebrowski J, Kacperska A. Are involved in cold acclimation and de-acclimation of winter oil-seed rape plants? Ann Bot 2008;101: 521–30. Vandevenne E, Van Buggenhout S, Duvetter T, Brouwers E, Declerck PJ, Hendrickx ME, et al. Development and evaluation of monoclonal antibodies as probes to assess the differences between two tomato pectin methylesterase isoenzymes. J Immunol Methods 2009;349:18–27. Vogel J. Unique aspects of the grass cell wall. Curr Opin Plant Biol 2008;11(3):301–7. Von Groll U, Berger D, Altmann T. The subtilisin-like serine protease SDD1 mediates cell-to-cell signaling during Arabidopsis stomatal development. Plant Cell 2002;14(7):1527–39. Wolf S, Rausch T, Greiner S. The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus. Plant J 2009;58(3):361–75. Yang XY, Zeng ZH, Yan JY, Fan W, Bian HW, Zhu MY, et al. Association of specific pectin methylesterases with Al-induced root elongation inhibition in rice. Physiol Plant 2013;148(4):502–11. ˜ MJ, Zhou GK, Nairn CJ, Wood-Jones A, Richardson EA, et al. Arabidopsis Zhong R, Pena fragile fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis. Plant Cell 2005;17(12):3390–408.
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Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short communication
Genome-wide identification and expression analysis of rice pectin methylesterases: Implication of functional roles of pectin modification in rice physiology Ho Young Jeong a,1 , Hong Phuong Nguyen a,1 , Chanhui Lee a,b,∗ a b
Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701, Republic of Korea Department of Plant and Environmental New Resources, Kyung Hee University, Yongin 446-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 April 2015 Received in revised form 9 May 2015 Accepted 10 May 2015 Available online 2 June 2015 Keywords: Pectin Pectin methylesterases Rice Plant cell walls Cell wall modifications
a b s t r a c t Pectin, which is enriched in primary cell walls and middle lamellae, is an essential polysaccharide in all higher plants. Homogalacturonans (HGA), a major form of pectin, are synthesized and methylesterified by enzymes localized in the Golgi apparatus and transported into the cell wall. Depending on cell type, the degree and pattern of pectin methylesterification are strictly regulated by cell wall-localized pectin methylesterases (PMEs). Despite its importance in plant development and growth, little is known about the physiological functions of pectin in rice, which contains 43 different types of PME. The presence of pectin in rice cell walls has been substantiated by uronic acid quantification and immunodetection of JIM7 monoclonal antibodies. We performed PME activity assays with cell wall proteins isolated from different rice tissues. In accordance with data from Arabidopsis, the highest activity was observed in germinating tissues, young culm, and spikelets, where cells are actively elongating. Transcriptional profiling of OsPMEs by real-time PCR and meta-analysis indicates that PMEs exhibit spatial- and stress-specific expression patterns during rice development. Based on in silico analysis, we identified subcellular compartments, isoelectric point, and cleavage sites of OsPMEs. Our findings provide an important tool for further studies seeking to unravel the functional importance of pectin modification during plant growth and abiotic and biotic responses of grass plants. © 2015 Elsevier GmbH. All rights reserved.
Introduction Pectin, one of the major cell wall components found in the primary cell wall and middle lamellae, is classified into four different domains based on backbone structure and monosaccharide compositions and glycosidic linkages of substitution: homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII), and xylogalacturonan (XGA) (Harholt et al., 2010; Mohnen, 2008). Structural analyses of pectin reveal that HGA, a linear polymer of (1,4)-linked-␣-galacturonic acids (GalUA), accounts for approximately 65% of pectin composition, and that the dominant monosaccharide of pectin is GalUA. GalUA in HGA is methylesterified at the C-6 carboxyl by pectin methyltransferases in the Golgi apparatus (Pelloux et al., 2007). Once synthesized, methylesterified
∗ Corresponding author at: Department of Plant and Environmental New Resources, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu, Yongin 446701, Republic of Korea. Tel.: +82 31 201 3480; fax: +82 31 204 8116. E-mail address: [email protected] (C. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jplph.2015.05.001 0176-1617/© 2015 Elsevier GmbH. All rights reserved.
HGA is targeted to the cell walls and undergoes further modifications depending on cell types and their positions in the pectin network. Pectin methyltesterases (PME, EC 3.1.1.11) belonging to class 8 carbohydrate esterases (CAZy, http://www.cazy.org/fam/CE8. html) catalyze the demethylesterification of GalUA in HGA (Cantarel et al., 2009). Partially demethylesterified HGA is then degraded by polygalacturonases, resulting in changes of wall properties such as rigidity, elasticity, and permeability (Moustacas et al., 1991). Since such structural remodeling of HGA by PMEs generally leads to cell wall loosening, cell types programed for cell expansion and elongation during cell development are required to increase the activity of PMEs. Moreover, recent studies showed that the methanol produced after PME action is an important signaling molecule in plant defense against biotic and abiotic stresses (Komarova et al., 2014). Sequence analyses of several model species showed that PMEs are a large multigene family; there are 66 PMEs in Arabidopsis thaliana, 89 in Poplus trichocarpa, 43 in Oryza sativa, 105 in Linum usitatissimum, and 79 in Lycoperscicon esculentum (Pelloux et al., 2007; Pinzon-Latorre and Deyholos, 2013; Vandevenne et al.,
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2009). Depending on the presence or absence of the PME inhibitor (PMEI) domain at the N-terminus (also known as the PRO region), PMEs are grouped into either Type-1 PME (with PMEI domain) or Type-2 PME (without PMEI domain) (Jolie et al., 2010). Genome analysis of bacteria, fungi, and moss Physcomitrella, revealed the absence of PMEI domain-containing PME proteins (Pelloux et al., 2007). Although there is no experimental evidence of the functional role of PMEI domains in Type-1 PME proteins, it is postulated to play a role in self-inhibitory activity to prevent premature demethoxylation (Bosch et al., 2005). Over the last two decades, mutational and transgenic approaches have led to significant progress in understanding the major biological roles of PMEs in plants (Francis et al., 2006; Bosch et al., 2005; Hongo et al., 2012). Representative phenotypes caused by mutations in PME isoforms in Arabidopsis include a defect in pollen tube growth and pollen tetrad formation and pendant stem growth. Transgenic plants overexpressing the Arabidopsis PMEI gene exhibit reduced PME activities and in increased pectin methylesterification (Müller et al., 2013). These transgenic plants also have faster germination rates than the wild type and reduced seed yields. In addition to its roles in plant development, several lines of evidence indicated that PME proteins function as a key regulator in plant-microbe interactions. Down-regulation of tobacco PME expression via antisense suppression resulted in delayed systemic movement of tobacco mosaic virus (Chen and Citovsky, 2003). Also, Hewezi et al. reported that a cyst nematode (Heterodera schachtii) secretory protein directly interacts with AtPME3 and this interaction is required for enhanced susceptibility of plants to the pathogen. Moreover, AtPME3 is rapidly induced upon infection of Pectobacterium carotovorum and Botrytis cinerea and acts as a susceptibility factor for the initial colonization by the pathogens (Raiola et al., 2011). The importance of pectin modification by PME was further demonstrated by comparing changes in the activities of PME during cold acclimation. The increase in PME activity was observed in cold-acclimated oilseed rape plants (Solecka et al., 2008). A previous study also showed that PMEs are involved in drought stress tolerance as overexpression of a pepper PMEI protein in Arabidopsis caused enhanced tolerance to drought stress (An et al., 2008). PMEs are also likely to be an important determinant of salt stress response. An Arabidopsis mutant with T-DNA insertion within the promoter of a PMEI gene (At1g62760) exhibited reduced sensitivity to NaCl stress (Jithesh et al., 2012). Taken together, previous studies demonstrate the importance of temporal and spatial regulation of PME activities in plant physiology as well as plant-microbe interactions. The functional role of rice PMEs is poorly understood. A previous study explored the association of aluminum toxicity with altered transcriptional expressions of eight rice PMEs (Yang et al., 2013). Transgenic rice plants overexpressing OsPME14 gene displayed increased sensitivity to aluminum stress. In this report, an initial step to characterize PME functions in rice, we found that rice cell walls contain substantial quantities of pectin through chemical and immunolocalization analysis, and that cell wall proteins exhibit PME enzyme activities. Transcriptional profiling of OsPMEs indicates that PMEs exhibit spatial- and stress-specific expression patterns during rice development. Materials and methods Plant materials Rice seeds (Oryza sativa cv. Dongjinbyeo) were soaked in water and pregerminated at 28 ◦ C under a light cycle of 16 h light/8 h dark for three days and transferred to soil. Rice plants were grown in a temperature-controlled glasshouse with natural lighting conditions. For RNA and total protein extraction, various tissues from germination to reproductive stages were collected, frozen in liquid
nitrogen, and stored at −80 ◦ C until analysis. Germinating tissues (shoot and root) were collected after 4 days of pregermination and immediately used for RNA and protein extraction. Young culm indicates samples collected from the entire third and fourth internode at 14 days before heading. Old culm indicates samples collected from the bottom parts of internode at 7 days after heading. Preparation of alcohol insoluble residues (AIRs) and measurement of uronic acid contents AIRs were prepared as described previously (Zhong et al., 2005; Lee et al., 2007). Briefly, frozen materials of each tissue were ground under liquid nitrogen. Fine powders were suspended in 70% (v/v) ethanol using a Polytron homogenizer three times. The AIRs collected were washed sequentially in absolute ethanol and 100% acetone. The resulting residues were dried in a vacuum oven at 60 ◦ C. In order to obtain pectin enriched fractions (PEFs), the AIRs (100 mg) were stirred to ammonium oxalate solution (50 mM) for 24 h at room temperature. Soluble fractions were collected by centrifugation and lyophilized. Uronic acid content was determined as described by Filisetti-Cozzi and Carpita (1991). Lyophilized PEFs were dissolved in 0.4 ml of 0.5 M H2 SO4 at room temperature for 1 h 40 l of 4 M sulfamic acid-potassium sulfamate (pH1.6) was added and mixed thoroughly. Samples were hydrolyzed in 2.4 ml of 75 mM Na2 B4 O7 dissolved in concentrated H2 SO4 at 100 ◦ C for 20 min. After cooling, 80 l of 0.15% (w/v) m-hydroxybipheneyl 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 by using galacturonic acid as a standard. PME activity assay Cell wall protein extracts were generated from germinating shoot, germinating root, 1 month old shoot, 1 month old root, 2 month old leaf, 2 month old root, young culm, spikelet, and flag leaf. Tissues were collected in liquid nitrogen and homogenized with equal volumes (w/v) of extraction buffer (100 mm Tris–HCl, pH 7.5, 500 mm NaCl containing protease inhibitor cocktail) and homogenized samples were then rotated at 4 ◦ C for 120 min. The samples were centrifuged at 11,500 × g at 4 ◦ C for 20 min and the supernatant were used immediately for all enzyme assays. A coupled enzymatic assay was performed under standard conditions as described by Grsic-Rausch and Rausch (2004) using a spectrophotometric plate reader. Heat-denatured cell wall proteins of each sample were used as a negative control. Histology and immunodetection of recognized methyl esterified homogalacturonans Young culm and leaves of 9-week-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.). 0.5 m thick sections were cut with a microtome and stained with 0.1% toluidine blue solution. For immunodetection, 0.5 m thick sections were incubated with JIM 7 monoclonal antibody (Plantprobes, Leeds University, UK) and then with fluorescein isothiocyanate-conjugated secondary antibodies. The fluorescence-labeled sections were observed using confocal microscope. Primer design and real time PCR Using Pfam ID (PF01095), we searched Putative Function Search Tool in Rice Genome Annotation Project (rice.plantbiology.msu.edu/). Then, we used Primer-BLAST in
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NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index. cgi?LINK LOC=BlastHome) for designing primers (Table. S1). In the primer-BLAST result, we had chosen a pair of primer that doesn’t match unspecific binding to other gene. Total RNA was isolated from different rice 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). The PCR threshold cycle number of each gene was normalized with expression level of the rice Ubiquitin 5 (OsUbi5, LOC Os01g22490) as a reference gene. We used Rice Oligonucleotide Array Database (http://www. ricearray.org/) to obtain microarray data of PMEs in response to abiotic and biotic stressed. In case of expression patterns of PMEs for abiotic stresses, data from GSE7256 were used. In case of expression patterns of PMEs for biotic stresses, data from GSE 19844 were used. The data were calculated and remake heatmap with R program (http://cran.r-project.org/). Meta-analysis of stress responsive expression of OsPMEs We used meta-analysis of stress specific expression profiles based on 231 Affymetrix array data for abiotic stresses and 85 Affymetrix array data for biotic stresses, which were downloaded from the NCBI gene expression omnibus (GEO, http://www.ncbi. nlm.nih.gov/geo/) (Nguyen et al., 2014). We then uploaded the log2 normalized intensity data in tab-delimited text format into Multi Experiment Viewer (MEV, http://www.tm4.org/mev/). We generated a heat map image based on log2 fold-change data in response to abiotic and biotic stresses and then integrated the heat map into the phylogenetic tree context. Phylogenetic analysis For phylogenetic analysis, we used PMEs from rice and Arabidopsis. PME amino acid sequences for Arabidopsis were extracted from Phytozome. We use ClustalW2 (http://www.ebi.ac.uk/Tools/ msa/clustalw2/) for drawing a comparative phylogenetic tree. In order to analyze tree groups of each PME protein, sequence files were loaded onto dendroscope (http://ab.inf.uni-tuebingen. de/software/dendroscope/). The tree groups were assigned as previously described (Louvet et al., 2006). In silico analysis of rice PMEs The presence of signal peptide was predicted with SignalP 4.0. Transmembrane domains were predicted using TMHMM v.2.0. Two different prediction programs, WoLF PSORT and Plant-mPLoc, were used for the protein subcellular localization. Cleavage sites were predicted as described by Pelloux et al. (2007) and Wolf et al. (2009). The predicted isoelectric point of the complete and the mature proteins was calculated using Vector NTI 10. Results Measurements of rice pectin quantity and PME activity A previous study reported that monocot plants possess less pectin in cell walls than dicot plants (Carpita, 1996; Vogel, 2008; Oh et al., 2013). In order to quantitatively determine pectin content in rice, cell walls from different tissues were prepared at different stages of development and treated with ammonium oxalate solution, which selectively extracts pectin (Ismail and Ramli, 2012). Since a major monosaccharide component in pectin is galacturonic acid, we performed sulfamate/m-hydroxydiphenyl assay to determine uronic acid content in rice tissues (Filisetti-Cozzi and Carpita, 1991). The highest quantities of uronic acid were observed in 40 d
Fig. 1. Quantitative analysis of uronic acid contents for pectin enriched fractions of cell walls isolated from different tissues (A) and PME activities in different tissues in rice (B). (A) AIRs were treated with 50 mM ammonium oxalate for 8 h and used for uronic acid quantification. Error bars represent the SE of three biological replicates. (B) Cell wall proteins were isolated and used for PME activity assay. The enzyme activity of germinating shoot was taken as 100, and the activity in other tissues was expressed as a percentage of the germinating shoot. Error bars represent the SE of three biological replicates.
young culm (Fig. 1A). Overall, cell walls of aerial tissues (leaves and culms) contain greater quantities of uronic acids than do roots. The presence of pectin in rice tissues was also examined by immunolocalization using monoclonal antibody JIM7, which binds to methyl esterified homogalacturonan (Fig. 2C and D). Immunolabeling of young rice culm and leaf sections showed intense signals in both parenchyma and sclerenchyma cell types, demonstrating significant deposition of pectin in rice primary and secondary cell walls. Next, we investigated whether pectin in rice cell walls also undergoes PME-mediated demethylesterification. Apoplastic proteins of different tissues were prepared and used to analyze PME activities (Fig. 1B). Interestingly, the highest levels of activity were detected in germinating rice shoots and spikelets. These data indicate that rice cell walls contain significant amounts of pectin and that rice PMEs play potentially diverse physiological roles. Identification of rice PMEs and tissue-specific transcriptional analysis In order to identify proteins encoding putative PMEs in rice, we searched for proteins containing PME domains (PF1095) and identified 43 putative PMEs (Table S1 and Fig. S2). We produced a phylogenetic tree consisting of 43 rice and 66 Arabidopsis PMEs (Fig. S1). Five major subfamilies of rice and Arabidopsis PMEs (labeled as groups A to E) were generated. Seventeen rice PMEs belong to group A, 8 to group B, 6 to group C, 5 to group D, and 7 to group E (Table S3). Twenty rice PMEs (47%) were Type-1 (proteins with both PMEI and PME domains) and 23 (53%) were Type-2 (proteins with only PME domains). In order to examine tissue- or organ-specific expressions of the 43 rice PMEs, we performed real-time PCR using 10 different tissues: germinating shoot, 1 month shoot, 1 month root, 2 month leaf, young culm (2 month), old culm (3 month), young spikelet,
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Fig. 2. Immunodetection of methyl esterified homogalacturonans of rice culm and leaf. Culm and leaf thin sections (1 m) were probed with the JIM7 monoclonal antibody. JIM7 antibody recognized methyl esterified homogalacturonans. (A) A rice culm section stained with toluidine blue staining solution. (B) A rice leaf section stained with toluidine blue staining solution. (C) A rice culm section immunostained with JIM7. (D) A rice leaf section immunostained with JIM7. cf—cortical fiber; bs—bundle sheath fiber; mx—metaxylem. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
old spikelet, flag leaf, and panicles with flowers. Integration of the phylogenetic tree into our transcriptional analysis would be a good reference to compare the expression levels between paralogs. Expression analyses showed that all rice PMEs were expressed in at least one tissue and that a number of rice PMEs (27/43) displayed high levels of gene expression during a vegetative stage and in young culm, in which cells are actively elongating (Fig. 3). Overall, ubiquitous expression was found in five PMEs (OsPME3, 6, 8, 10, and 32), while seven PMEs (OsPME18, 21, 25, 28, 29, 41, and 42) showed one or two forms of tissue-specific expression. In order to examine the expression profile change of OsPMEs during plant defense responses, we also performed a metatranscriptional analysis of OsPMEs under five abiotic (drought, salt, cold, heat, and anaerobic condition) and four biotic (Magnaporthe grisia, rice stripe virus, Xanthomonas oryzae pv. oryzae, and brown planthopper) stresses, utilizing publicly available microarray data (Fig. 4). Interestingly, a total of 13 OsPME genes (OsPME2, 3, 4, 6, 8, 10, 17, 20, 26, 27, 28, 34, and 41) were significantly upregulated (>4-fold) in at least one of the abiotic stresses, whereas a total of 9 OsPME genes (OsPME3, 6, 14, 15, 16, 19, 23, 28, and 35) were down-regulated (<4-fold) in at least one of the abiotic stresses. Particularly, a number of OsPME genes are transcriptionally responsive to drought, heat, and anaerobic stresses while a few OsPME genes are up- or down-regulated by salt and cold stresses. Based on the microarray data, we found similar expression patterns of OsPMEs in response to Magnaporthe grisea and X. oryzae pv. oryzae, which cause rice blast and rice bacterial blight diseases, respectively. OsPME2 and 28 showed elevated transcript levels in response to both pathgens. X. oryzae pv. oryzae infection. More dynamic transcriptional regulation of OsPMEs was found in response to rice stripe virus and brown planthopper. Nine OsPME genes (OsPME9, 15, 16, 20, 27, 28, 34, 41, and 43) are significantly up-regulated whereas four OsPME genes (OsPME2, 6, 7, 11, and 17) are down-regulated in response to rice stripe virus. Eight OsPME genes (OsPME2, 6, 8, 10, 17, 27, 28, and 32) are significantly upregulated whereas five OsPME genes (OsPME9, 15, 16, 19, and 36)
are down-regulated in response to brown planthopper. OsPME28, which is highly expressed in 1-month-old shoot and young culm (Fig. 1), showed increased transcript levels upon all biotic stresses examined and three abiotic stresses (drought, cold and heat). Taken together, the existence of numerous PMEs in the rice genome and their spatial- and stress-specific expression patterns indicate that the enzymatic actions of pectin modifications such as the removal of methyl groups by PMEs play important roles in rice growth and development.
In silico analysis of rice PMEs Proteins involved in cell wall modifications, including the removal of methyl groups in pectin by PMEs, are generally believed to be targeted to cell walls (Gilbert, 2010). Thus, such proteins should have either N-terminal signal peptides or transmembrane domains. All rice PMEs examined in this study have an N-terminal signal peptide, a transmembrane domain, or both, with the exceptions of OsPME38, 39, 40, and 41 (Table S3). Sequence analyses using Plant-mPLoc for the prediction of protein localization predicted all rice PMEs to be cell-wall localized proteins (Table S3). Previous studies of protein localization demonstrated that some Type-1 PMEs undergo proteolytic cleavages of the PMEI domain at specific sites (binding motif 1 and binding motif 2) through the action of subtilisin-like serine proteases (Von Groll et al., 2002). We searched for the motif [KKLR][RRMA][RRLL][RHLL][RRML][KLDL][RKLL] in the Type-1 PMEs using “Protein Pattern Find”. Out of 20 Type-1 PMEs, we found that 10 possessed a single motif site and that 3 had two motif sites. As expected, no cleavage sites were found in the Type-2 PMEs (Table S3). Another important feature of the enzymatic action of PMEs is the isoelectric point. Processed PME amino acid sequences were generated and the isoelectric point was calculated using Vector NTI 10. Most (26/43) of PMEs had a basic pI (above pG8.0), while 5 had an acidic pI (Table S3).
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Fig. 3. Heat map of transcript abundances of OsPMEs 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.)
Discussion Cell wall modifications by different classes of cell wall hydrolases and esterases are postulated to play important roles in fine tuning the composition and distribution of cell wall polysaccharides (Jamet et al., 2006). Compared to other model plants such as Arabidopsis, poplar, and tomato, the biological implications of such changes in rice, a monocot model, remain unexplored. Moreover, how these enzymes modulate the developmental and physiological responses of rice is poorly understood. In order to shed light on this issue, it is imperative to collect general molecular and biochemical evidence of cell wall modifying enzymes. Here, we provide data showing that rice cell walls from different tissues contain substantial quantities of pectin and identifying 43 different rice PMEs. Enzyme activity assays using cell wall proteins demonstrate the presence of PME activity in diverse tissues. Transcriptional analyses indicate diverse temporal and spatial expression patterns. These findings provide an experimental platform for future studies. Previous chemical studies reported that typical grass cell walls contain five-fold lower quantities of pectin than those of dicots, but are enriched with mixed glucans and phenolics (ferulic acid and pcoumaric acid), particularly the primary cell walls (Vogel, 2008; Carpita, 1996). However, we observed significant accumulations of pectin in rice primary and secondary cell walls. First, the quantification of uronic acid contents in pectin-enriched fractions indicates
comparable amounts of pectin in different tissues (Fig. 1A). Second, immunolocalization analysis demonstrated the presence of methyl esterified pectin in both primary and secondary cell walls (Fig. 2). These results are different from results for other types of dicot plants, in that pectin is a major polysaccharide predominantly found in primary cell types, but not in secondary cell walls. Since pectin serves as a gelling and stabilizing polymer of other cell wall polysaccharides, it is likely that pectin is a structurally important component in both types of cell walls in rice. Tissues in which cell elongation is actively occurring showed the highest PME activity (Fig. 1B). This observation is consistent with previous findings regarding the functional roles of PMEs in several species. PME activity increases during early stages of seed germination, root tip growth, leaf growth polarity, internode stem growth, and pollen tube growth. Demethylesterification of cell wall pectins by PMEs results in the formation of gel-like structures between HGAs and thereby decreases the mechanical restraint of neighbor cell types, steps that are required for cell elongation (Pelloux et al., 2007). Transcriptional analyses of rice PMEs using real-time PCR, combined with the analysis of publicly available micro array data, shows dynamic expression patterns during growth and in response to abiotic and biotic stresses (Figs. 3 and 4). In particular, similar to the enzyme activity assay, 23 and 13 OsPMEs are highly expressed in young vegetative tissues and young culm, respectively, where cell wall extension occurs. Taken together, our findings of higher PME activities and increased transcript levels in young vegetative
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H.Y. Jeong et al. / Journal of Plant Physiology 183 (2015) 23–29
Fig. 4. Expression pattern of OsPMEs in response to abiotic and biotic conditions. The heat map was drawn using Affymetrix meta-analysis of stress-specific expression database based on 983 array data. Transcriptional expression of OsPMEs in response to abiotic stresses (drought-root, drought-shoot, salt, cold, heat, and anaerobic condition) and to biotic stresses (Magnaporthe grisea, rice stripe virus, Xanthomonas oryzae pv. oryzae, and brown planthopper) was shown. Magnaporthe grisea causes rice blast disease and Xanthomonas oryzae pv. oryzae causes bacterial blight disease. Green colored boxes denote a low level of expression and red colored boxes denote a high level of expression. Gray color boxes indicate the data not analyzed due to the absence of respective probes on Affymetrix array. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
tissues indicate that PME expression and enzyme activity in rice are prerequisites for cell elongation. HGA polymers are synthesized in the Golgi apparatus and passively transported to cell walls, where PMEs exert enzymatic action to remove methyl groups depending on developmental and environmental signals (Mohnen, 2008). Previous localization experiments in flax and tomato demonstrated that processed (removal of PRO regions) or non-processed PMEs are predominantly found in cell walls (Al-Qsous et al., 2004; Micheli, 2001). In accordance with these previous findings, our in-silico results indicate that all 43 OsPMEs are localized in the cell walls. Definitive proof would depend on the actual localization of OsPMEs using fluorescent protein-tagged methods. We have successfully constructed GFP-fused constructs of five PMEs and will infiltrate these into tobacco leaves to confirm cell wall localization in future studies. The difficulty of performing enzyme activity assays using purified PME proteins has been the limiting step to a greater understanding of PMEs, largely due to the difficulty of expressing plant PMEs in heterologous systems (Pelloux et al., 2007). We initially attempted to detect PME activity by the use of recombinant OsPMEs in Escherichia coli (E. coli) and successfully purified these proteins (data not shown). However, we failed to detect any PME activity compared with the positive control (commercial purified PME). This could be due to the lack of posttranslation modification systems, including glycosylation, in bacteria. It would be necessary to express plant PMEs in other host systems such as Pichia pastoris to obtain further insights in PME enzymology. Transcriptional profiling using different tissues demonstrated overlaps of the expression patterns of many OsPMEs. Thus, it is unlikely that we will detect observable phenotypes when only a single OsPME gene is mutated, due to gene redundancy (Müller et al., 2013). Our attempts to demonstrate the biological importance of pectin demethylesterification will be pursued by generating transgenic plants overexpressing the PMEI genes. So far, we have
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