J Plant Biotechnol 2022; 49(4): 271-291
Published online December 31, 2022
https://doi.org/10.5010/JPB.2022.49.4.271
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: hobang@ibiomedic.co.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pectin methylesterase (PME) plays an important role in vegetative and reproductive development and biotic/abiotic stress responses by regulating the degree of methyl-esterification of pectic polysaccharides in the plant cell wall. PMEs are encoded by a large multigene family in higher land plant genomes. In general, the expression of plant PME genes shows tissue- or cell-specific patterns and is induced by endogenous and exogenous stimuli. In this study, we identified PME multigene family members (CsPMEs) from the sweet orange genome and report detailed molecular characterization and expression profiling in different citrus tissues and two fruit developmental stages. We also discussed the possible functional roles of some CsPME genes by comparing them with the known functions of PMEs from other plant species. We identified 48 CsPME genes from the citrus genome. A phylogenetic tree analysis revealed that the identified CsPMEs were divided into two groups/types. Some CsPMEs showed very close phylogenetic relationships with the PMEs whose functions were formerly addressed in Arabidopsis, tomato, and maize. Expression profiling showed that some CsPME genes are highly or specifically expressed in the leaf, root, flower, or fruit. Based on the phylogenetic relationships and gene expression profiling results, we suggest that some CsPMEs could play functional roles in pollen development, pollen tube growth, cross incompatibility, root development, embryo/seed development, stomata movement, and biotic/abiotic stress responses. Our results shed light on the biological roles of individual CsPME isoforms and contribute to the search for genetic variations in citrus genetic resources.
Keywords Citrus, Genome, Multigene Family, Pectin Methylesterase, Plant Cell Wall, Transcriptome
Plant cell wall is a highly sophisticated structure composed of diverse polysaccharides, enzymes, and structural proteins, and it helps to determine cell size and shape, growth and development, intercellular communication and interaction with the external environment (Micheli 2001; Pelloux et al. 2007). Two classes of polysaccharides, pectins and hemicelluloses, are major components of the middle lamellae and primary cell walls. Pectins compose 30-35% of the cell wall dry weight in dicots and non-grass monocots, 2-10% of grass primary walls, and up to 5% of wood tissues. Pectins are important for both cellular adhesion and cell wall plasticity and are a highly complex and heterogenous group of polymers that can be divided into five classes of pectic polysaccharides: homogalacturonan (HGA), rhamnogalacturonans I and II, xylogalacturonan and apiogalacturonan (Pelloux et al. 2007; Wolf et al. 2009; Wu et al. 2018).
HGA is a linear homopolymer of 1,4-linked α-D-galacturonic acid (GalA) and the most abundant of the pectic polysaccharides, constituting 65% of all pectins (Wolf et al. 2009). Plant genes encoding HGA biosynthetic enzymes have been identified from
A primary structure analysis of
In higher plant species, PME isoforms are encoded by a large multigene family that is differentially expressed in different tissues and in response to environmental stresses (Louvet et al. 2006; Sénéchal et al. 2014b; Wang et al. 2021; Wen et al. 2020; Zhang et al. 2019). In recent years, whole-genome sequencing technologies and bioinformatic analyses have led to the genome-wide identification and characterization of the
Citrus is a global economic fruit crop with the largest fruit production in the world. Citrus has a relatively small genome with compact gene content (~30,00 genes). The average haploid genome size in citrus species ranges from 360 Mb (mandarin) to 398 Mb (citron) (Gmitter et al. 2012), and several reference genomes are currently available (https://www.citrusgenomedb.org/). Citrus has several fascinating botanical features, such as long juvenility, self-/cross-incompatibility, sterility in pollen/ovule, polyembryony, and high citrus-specific flavonoid content in the fruits (Kim et al. 2016; Woo et al. 2020), which attract scientists to study its growth and development. In this study, we identify
PME protein sequences were retrieved from the sweet orange (
Full-length PME protein sequences derived from citrus (this study),
To analyze the expression profiles of
To identify putative
The amino acid sequence alignments of PMEs from plants, fungi and bacteria showed the five conserved sequence regions in the PME domain: 44_GxYxE, 113_QAVAL, 135_QDTL, 157_DFIFG, and 223_LGRPW (according to the carrot PME numbering) (Markovič and Janeček 2004). Six strictly conserved residues have been identified in those segments: Gly44, Gly154, Asp157, Gly161, Arg225, and Trp227. To investigate whether the five segments are highly conserved among the PME domains of 48 citrus PMEs, we performed a multiple sequence alignment using the ClustalW tool embedded in MEGA7.0 software (Supplementary Fig. 1). The sequence alignment revealed that amino acid residues consisting of an active site, two Asp residues (D136 in region III and D157 in region IV), two Gln residues (Q113 in region II and Q135 in region III) and an Arg residue (R225 in region V) were highly conserved among the citrus PMEs, although amino acid substitutions were found in the conserved Q113, Q135, and D136 sites. Amino acid substitutions were also found in two of the six conserved residues, Gly44 and Trp227.
A molecular phylogenetic analysis was performed for the 48 full-length CsPMEs by using MEGA 7.0 with the Neighbor Joining method. The resulting phylogenetic tree revealed that the 48 CsPMEs are divided into 2 groups/ types, group 1/type II and group 2/type I, containing 18 and 30 members, respectively (Fig. 1A). The tree indicated that CsPME46 belongs to group 2/type I, although no PMEI domain was found in its protein sequence (Fig. 1A and Supplementary Table 3). Functional roles of several plant PMEs have been extensively studied in
According to evolutionary research using eukaryotic genome sequences, the exon-intron structure of protein-coding genes appears to have evolved concomitantly with eukaryotic cells, and introns are a major driving force in genome evolution and the functional divergence of gene family members (Liu et al. 2021; Rogozin et al. 2012). To better understand the structural diversity of citrus
To further explore the spatial and temporal expression profiles of the 48 identified
As cell wall-localized enzymes, plant
Up to now, genome-wide analyses of higher land plant genomes revealed that PME isoforms are encoded by a large multigene family, ranging from 41 (rice and
The 3D crystallographic structure of plant and bacterial PMEs and their co-crystallization with HGA have revealed various biophysical properties, such as their folding topology, carbohydrate-binding sites, catalytic sites, and action mechanism of hydrolysis (Pelloux et al. 2007; Sénéchal et al. 2014b). Previous amino acid sequence alignments of PMEs from plants and microorganisms revealed that six amino acid residues in five characteristic sequence segments are strictly conserved, supporting the crystallization data (Markovič and Janeček 2004). The alignments of the PME domains of the 48 CsPMEs reported here indicate that the segments and amino acid residues are also highly conserved. Three amino acid residues, Gly154, Gly161, and Arg225 were completely conserved among the 48 CsPMEs (Supplementary Fig. 1). However, changes in the Gly44, Asp157, and Trp227 residues were found in some CsPMEs, and Gly44 was missing from CsPME7. Future studies are needed to confirm whether the CsPMEs with variations in the conserved residues have biochemical PME activity. The importance of the Gln135Asp136 residues in the active site of a tobacco PME was confirmed in transgenic plants expressing a mutated form (Dorokhov et al. 2006).
Studies during the past two decades have used sense/ antisense transgenic plants and mutants and found that
Table 1 List of
CsPMEs | Tissues highly expressed in citrus | Possible functional orthologs in plant species | Functional roles in plant species |
---|---|---|---|
Flower | Cross incompatibility | ||
N.D.* | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Root | Root growth and development. Defense responses against biotic stresses | ||
Leaf | Embryo and seed development. Stomatal movement | ||
Mature green fruit | Fruit ripening | ||
Flower | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Flower | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Root | Root growth and development | ||
Flower | Cross incompatibility | ||
Root, Flower | Fruit ripening |
*N.D. indicates that transcripts were not detected in the tissues investigated in this study.
Plenty of
Several studies using
The roles of pectin modification during fruit development and maturation have been extensively studied in tomato (Wang et al. 2018). Fruit softening is also an important issue in post-harvest storage in the citrus industry. The cDNAs of several
Increased biotic and abiotic stresses caused by global climate change threatens the sustainable production of most crops, including citrus. Plant cells have been shown to develop elaborate systems for responding to a variety of challenges, including environmental stresses. The expression of plant
Guard cells perceive a multitude of endogenous and environmental stimuli, including hormonal cues, light, CO2 concentration, pathogen, drought, and heat. The opening/ closure of stomata pores is strictly regulated by an integration of environmental stimuli and endogenous hormonal signals. The highly specialized walls of guard cells enable them to undergo large and reversible deformation during the construction of stomata (Wu et al. 2018). Guard cell walls are rich in unesterified HGA (Amsbury et al. 2016).
Plant
This research was supported by the Cooperative Research Program for Agriculture Science & Technology Development, RDA (Project No. PJ01514103), and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) (Grant No. 322072031HD040), Republic of Korea. The authors appreciate Grace Kim (Yieun Kim) for her illustration work.
J Plant Biotechnol 2022; 49(4): 271-291
Published online December 31, 2022 https://doi.org/10.5010/JPB.2022.49.4.271
Copyright © The Korean Society of Plant Biotechnology.
Ho Bang Kim · Chang Jae Oh · Nam-Hoon Kim · Cheol Woo Choi · Minju Kim · Sukman Park · Seong Beom Jin · Su-Hyun Yun · Kwan Jeong Song
Life Sciences Research Institute, Biomedic Co., Ltd., Bucheon 14548, Republic of Korea
PHYZEN Genomics Institute, Seongnam, 13558, Republic of Korea
Citrus Research Institute, National Institute of Horticultural & Herbal Science, Seogwipo, 63607, Republic of Korea
Major of Horticultural Science, Faculty of Bioscience and Industry, Jeju National University, Jeju, 63243, Republic of Korea
Research Institute for Subtropical Agriculture & Biotechnology, Jeju National University, Jeju, 63243, Republic of Korea
Correspondence to:e-mail: hobang@ibiomedic.co.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pectin methylesterase (PME) plays an important role in vegetative and reproductive development and biotic/abiotic stress responses by regulating the degree of methyl-esterification of pectic polysaccharides in the plant cell wall. PMEs are encoded by a large multigene family in higher land plant genomes. In general, the expression of plant PME genes shows tissue- or cell-specific patterns and is induced by endogenous and exogenous stimuli. In this study, we identified PME multigene family members (CsPMEs) from the sweet orange genome and report detailed molecular characterization and expression profiling in different citrus tissues and two fruit developmental stages. We also discussed the possible functional roles of some CsPME genes by comparing them with the known functions of PMEs from other plant species. We identified 48 CsPME genes from the citrus genome. A phylogenetic tree analysis revealed that the identified CsPMEs were divided into two groups/types. Some CsPMEs showed very close phylogenetic relationships with the PMEs whose functions were formerly addressed in Arabidopsis, tomato, and maize. Expression profiling showed that some CsPME genes are highly or specifically expressed in the leaf, root, flower, or fruit. Based on the phylogenetic relationships and gene expression profiling results, we suggest that some CsPMEs could play functional roles in pollen development, pollen tube growth, cross incompatibility, root development, embryo/seed development, stomata movement, and biotic/abiotic stress responses. Our results shed light on the biological roles of individual CsPME isoforms and contribute to the search for genetic variations in citrus genetic resources.
Keywords: Citrus, Genome, Multigene Family, Pectin Methylesterase, Plant Cell Wall, Transcriptome
Plant cell wall is a highly sophisticated structure composed of diverse polysaccharides, enzymes, and structural proteins, and it helps to determine cell size and shape, growth and development, intercellular communication and interaction with the external environment (Micheli 2001; Pelloux et al. 2007). Two classes of polysaccharides, pectins and hemicelluloses, are major components of the middle lamellae and primary cell walls. Pectins compose 30-35% of the cell wall dry weight in dicots and non-grass monocots, 2-10% of grass primary walls, and up to 5% of wood tissues. Pectins are important for both cellular adhesion and cell wall plasticity and are a highly complex and heterogenous group of polymers that can be divided into five classes of pectic polysaccharides: homogalacturonan (HGA), rhamnogalacturonans I and II, xylogalacturonan and apiogalacturonan (Pelloux et al. 2007; Wolf et al. 2009; Wu et al. 2018).
HGA is a linear homopolymer of 1,4-linked α-D-galacturonic acid (GalA) and the most abundant of the pectic polysaccharides, constituting 65% of all pectins (Wolf et al. 2009). Plant genes encoding HGA biosynthetic enzymes have been identified from
A primary structure analysis of
In higher plant species, PME isoforms are encoded by a large multigene family that is differentially expressed in different tissues and in response to environmental stresses (Louvet et al. 2006; Sénéchal et al. 2014b; Wang et al. 2021; Wen et al. 2020; Zhang et al. 2019). In recent years, whole-genome sequencing technologies and bioinformatic analyses have led to the genome-wide identification and characterization of the
Citrus is a global economic fruit crop with the largest fruit production in the world. Citrus has a relatively small genome with compact gene content (~30,00 genes). The average haploid genome size in citrus species ranges from 360 Mb (mandarin) to 398 Mb (citron) (Gmitter et al. 2012), and several reference genomes are currently available (https://www.citrusgenomedb.org/). Citrus has several fascinating botanical features, such as long juvenility, self-/cross-incompatibility, sterility in pollen/ovule, polyembryony, and high citrus-specific flavonoid content in the fruits (Kim et al. 2016; Woo et al. 2020), which attract scientists to study its growth and development. In this study, we identify
PME protein sequences were retrieved from the sweet orange (
Full-length PME protein sequences derived from citrus (this study),
To analyze the expression profiles of
To identify putative
The amino acid sequence alignments of PMEs from plants, fungi and bacteria showed the five conserved sequence regions in the PME domain: 44_GxYxE, 113_QAVAL, 135_QDTL, 157_DFIFG, and 223_LGRPW (according to the carrot PME numbering) (Markovič and Janeček 2004). Six strictly conserved residues have been identified in those segments: Gly44, Gly154, Asp157, Gly161, Arg225, and Trp227. To investigate whether the five segments are highly conserved among the PME domains of 48 citrus PMEs, we performed a multiple sequence alignment using the ClustalW tool embedded in MEGA7.0 software (Supplementary Fig. 1). The sequence alignment revealed that amino acid residues consisting of an active site, two Asp residues (D136 in region III and D157 in region IV), two Gln residues (Q113 in region II and Q135 in region III) and an Arg residue (R225 in region V) were highly conserved among the citrus PMEs, although amino acid substitutions were found in the conserved Q113, Q135, and D136 sites. Amino acid substitutions were also found in two of the six conserved residues, Gly44 and Trp227.
A molecular phylogenetic analysis was performed for the 48 full-length CsPMEs by using MEGA 7.0 with the Neighbor Joining method. The resulting phylogenetic tree revealed that the 48 CsPMEs are divided into 2 groups/ types, group 1/type II and group 2/type I, containing 18 and 30 members, respectively (Fig. 1A). The tree indicated that CsPME46 belongs to group 2/type I, although no PMEI domain was found in its protein sequence (Fig. 1A and Supplementary Table 3). Functional roles of several plant PMEs have been extensively studied in
According to evolutionary research using eukaryotic genome sequences, the exon-intron structure of protein-coding genes appears to have evolved concomitantly with eukaryotic cells, and introns are a major driving force in genome evolution and the functional divergence of gene family members (Liu et al. 2021; Rogozin et al. 2012). To better understand the structural diversity of citrus
To further explore the spatial and temporal expression profiles of the 48 identified
As cell wall-localized enzymes, plant
Up to now, genome-wide analyses of higher land plant genomes revealed that PME isoforms are encoded by a large multigene family, ranging from 41 (rice and
The 3D crystallographic structure of plant and bacterial PMEs and their co-crystallization with HGA have revealed various biophysical properties, such as their folding topology, carbohydrate-binding sites, catalytic sites, and action mechanism of hydrolysis (Pelloux et al. 2007; Sénéchal et al. 2014b). Previous amino acid sequence alignments of PMEs from plants and microorganisms revealed that six amino acid residues in five characteristic sequence segments are strictly conserved, supporting the crystallization data (Markovič and Janeček 2004). The alignments of the PME domains of the 48 CsPMEs reported here indicate that the segments and amino acid residues are also highly conserved. Three amino acid residues, Gly154, Gly161, and Arg225 were completely conserved among the 48 CsPMEs (Supplementary Fig. 1). However, changes in the Gly44, Asp157, and Trp227 residues were found in some CsPMEs, and Gly44 was missing from CsPME7. Future studies are needed to confirm whether the CsPMEs with variations in the conserved residues have biochemical PME activity. The importance of the Gln135Asp136 residues in the active site of a tobacco PME was confirmed in transgenic plants expressing a mutated form (Dorokhov et al. 2006).
Studies during the past two decades have used sense/ antisense transgenic plants and mutants and found that
Table 1 . List of
CsPMEs | Tissues highly expressed in citrus | Possible functional orthologs in plant species | Functional roles in plant species |
---|---|---|---|
Flower | Cross incompatibility | ||
N.D.* | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Root | Root growth and development. Defense responses against biotic stresses | ||
Leaf | Embryo and seed development. Stomatal movement | ||
Mature green fruit | Fruit ripening | ||
Flower | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Flower | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Root | Root growth and development | ||
Flower | Cross incompatibility | ||
Root, Flower | Fruit ripening |
*N.D. indicates that transcripts were not detected in the tissues investigated in this study..
Plenty of
Several studies using
The roles of pectin modification during fruit development and maturation have been extensively studied in tomato (Wang et al. 2018). Fruit softening is also an important issue in post-harvest storage in the citrus industry. The cDNAs of several
Increased biotic and abiotic stresses caused by global climate change threatens the sustainable production of most crops, including citrus. Plant cells have been shown to develop elaborate systems for responding to a variety of challenges, including environmental stresses. The expression of plant
Guard cells perceive a multitude of endogenous and environmental stimuli, including hormonal cues, light, CO2 concentration, pathogen, drought, and heat. The opening/ closure of stomata pores is strictly regulated by an integration of environmental stimuli and endogenous hormonal signals. The highly specialized walls of guard cells enable them to undergo large and reversible deformation during the construction of stomata (Wu et al. 2018). Guard cell walls are rich in unesterified HGA (Amsbury et al. 2016).
Plant
This research was supported by the Cooperative Research Program for Agriculture Science & Technology Development, RDA (Project No. PJ01514103), and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) (Grant No. 322072031HD040), Republic of Korea. The authors appreciate Grace Kim (Yieun Kim) for her illustration work.
Table 1 . List of
CsPMEs | Tissues highly expressed in citrus | Possible functional orthologs in plant species | Functional roles in plant species |
---|---|---|---|
Flower | Cross incompatibility | ||
N.D.* | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Root | Root growth and development. Defense responses against biotic stresses | ||
Leaf | Embryo and seed development. Stomatal movement | ||
Mature green fruit | Fruit ripening | ||
Flower | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Flower | Reproductive development (pollen, pollen grains, and pollen tube growth) | ||
Root | Root growth and development | ||
Flower | Cross incompatibility | ||
Root, Flower | Fruit ripening |
*N.D. indicates that transcripts were not detected in the tissues investigated in this study..
Ho Bang Kim, Sanghyun Lim, Jae Joon Kim, Young Cheol Park, Su-Hyun Yun, and Kwan Jeong Song
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