Brassinosteroids regulate pectin methylesterase activity and AtPME41 expression in Arabidopsis under chilling stress

Pectin methylesterases (PMEs) are important cell wall enzymes that may play important roles in plant chilling/freezing tolerance. We investigated the possible roles of brassinosteroids (BRs) in regulation of PMEs under chilling stress. Chilling stress or 24-epibrassinolide (eBL) treatments induced significant increases in PME activity in wild type (Col-0) seedlings of Arabidopsis. The chilling-stress-induced increases in PME activity were also found in bzr1-D mutant, a BZR1 stabilized mutant with a constitutively active BR signaling pathway, but not in bri1-116, a BR insensitive null allele of the BR receptor BRI1. The results suggest that the regulation of PME activity in Arabidopsis under chilling stress depends on the BR signaling pathway. Furthermore, we showed that the effect of chilling stress on PME activity was impaired in pme41, a knockout mutant of AtPME41. Semi-quantitative RT-PCR results showed that expression of AtPME41 was induced by chilling stress in wild type plants but not in the bri1-116 mutant. The expression of AtPME41 increased in bzr1-D and eBL treated wild type seedlings, but decreased in bri1-116 seedlings. Furthermore, ion leakage induced by low temperature were dramatically increased in both bri1-116 and pme41, while lipid peroxidation was increased in bri1-116 only. The results suggest that BRs may modulate total PME activity in Arabidopsis under chilling stress by regulating AtPME41 expression. Regulation of PME activity may serve as one of the mechanisms that BR participates in chilling tolerance of plants.     

AtPME17 is a functional Arabidopsis thaliana pectin methylesterase regulated by its PRO region that triggers PME activity in the resistance to Botrytis cinerea

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid–ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.     

Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis

Pectin methylesterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell wall. Previous work indicated that plant PMEs may be subject to post-translational regulation. Here, we report the analysis of two proteinaceous inhibitors of PME in Arabidopsis thaliana (AtPMEI1 and 2). The functional analysis of recombinant AtPMEI1 and 2 proteins revealed that both proteins are able to inhibit PME activity from flowers and siliques. Quantitative RT-PCR analysis indicated that expression of AtPMEI1 and 2 mRNAs is tightly regulated during plant development with highest mRNA levels in flowers. Promotor::GUS fusions demonstrated that expression is mostly restricted to pollen.     

Novel role for pectin methylesterase in Arabidopsis: a new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.     

Effect of pectin methylesterase gene expression on pea root development.

Expression of an inducible gene with sequences common to genes encoding pectin methylesterase (PME) was found to be tightly correlated, both spatially and temporally, with border cell separation in pea root caps. Partial inhibition of the gene's expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. This phenotype was correlated with an increase in extracellular pH, reduced root elongation, and altered cellular morphology. The translation product of the gene exhibited PME activity in vitro. These results are consistent with the long-standing hypothesis that the demethylation of pectin by PME plays a key role in cell wall metabolism.     

Overexpression of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea

Pectin, one of the main components of plant cell wall, is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The action of PME is important in plant development and defense and makes pectin susceptible to hydrolysis by enzymes such as endopolygalacturonases. Regulation of PME activity by specific protein inhibitors (PMEIs) can, therefore, play a role in plant development as well as in defense by influencing the susceptibility of the wall to microbial endopolygalacturonases. To test this hypothesis, we have constitutively expressed the genes AtPMEI-1 and AtPMEI-2 in Arabidopsis (Arabidopsis thaliana) and targeted the proteins into the apoplast. The overexpression of the inhibitors resulted in a decrease of PME activity in transgenic plants, and two PME isoforms were identified that interacted with both inhibitors. While the content of uronic acids in transformed plants was not significantly different from that of wild type, the degree of pectin methylesterification was increased by about 16%. Moreover, differences in the fine structure of pectins of transformed plants were observed by enzymatic fingerprinting. Transformed plants showed a slight but significant increase in root length and were more resistant to the necrotrophic fungus Botrytis cinerea. The reduced symptoms caused by the fungus on transgenic plants were related to its impaired ability to grow on methylesterified pectins.     

Expression of fungal pectin methylesterase in transgenic tobacco leads to alteration in cell wall metabolism and a dwarf phenotype

A transgenic tobacco plant (Nicotiana tabacum L.) expressing a fungal pectin methylesterase (PME; EC 3.1.1.11) gene derived from a black filamentous fungus, Aspergillus niger was created. Fungal PME should have a wider range of adaptability to substrate pectin compared with plant PME. As expected, the proportion of methyl esters in pectin was reduced in the transgenic tobacco. Consequently, the transgenic plant showed short internodes, small leaves and a dwarf phenotype. At a cellular level, the longitudinal lengths of stem epidermal cells were shorter than those of control plants. This is the first report that fungal PME promotes dwarfism in plants. It is worth noting that in the PME-expressing dwarf plant, the expression levels of cell wall metabolism related genes that included endo-1,4-beta-glucanase, cellulose synthase, endo-xyloglucan transferase and expansin gene were decreased. These results suggest that the expression of fungal PME in plants affects the cell wall metabolism.     

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.     

VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.     

BoPMEI1, a pollen-specific pectin methylesterase inhibitor, has an essential role in pollen tube growth

Pectin methylesterase (PME) is known to have important roles in pollen development and pollen tube growth. As pivotal regulatory factors in PME activity modulation, PME inhibitors (PMEIs) are thought to be key regulators of cell wall stability at the tip of the pollen tube. We report on the cloning and characterization of a novel B. oleracea PMEI gene, BoPMEI1. Heterologously expressed BoPMEI1 showed PMEI activity. RT-PCR studies of different tissues and promoter-GUS fusions confirmed that BoPMEI1 was specifically expressed in mature pollen grains and pollen tubes. Based on in vivo transient assays, we found that BoPMEI1 appears to be largely localized to the plasma membrane. Transgenic Arabidopsis plants expressing antisense BoPMEI1 under the control of the CaMV 35S promoter suppressed the expression of the orthologous gene At1g10770, which led to partial male sterility and decreased seed set by inhibition of pollen tube growth. Our study demonstrates the involvement of BoPMEI1 in pollen tube growth.     

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.     

Strawberry pectin methylesterase (PME): purification,characterization, thermal and high-pressure inactivation

Pectin methylesterase (PME) was extracted from strawberries (Fragaria ananassa, cv Elsanta) and purified by affinity chromatography on a CNBr-Sepharose 4B-PME-inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molecular mass, pI, and kinetic parameters of strawberry PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified strawberry PME could be described by a fractional-conversion model. Purified strawberry PME is much more stable toward high-pressure treatments in comparison to those from oranges and bananas.     

Analysis of LuPME3, a pectin methylesterase from Linum usitatissimum,revealed a variability in PME proteolytic maturation

Pectin methylesterase (PME) catalyzes the de-methylesterification of pectin in plant cell walls during cell elongation.¹ Pectins are mainly composed of α(1, 4)-D-galacturonosyl acid units that are synthesized in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca²⁺ ions during their intracellular transport.² The highly methylesterified pectins are then secreted into the apoplasm³ and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca²⁺ crosslinking of neighboring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening.⁴ PMEs belong to a large multigene family. Sixty­six PME-related genes are predicted in the Arabidopsis genome.¹ Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting.⁵ In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the ortholog of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Site-directed mutagenesis of the active site of pectin methylesterase from Aspergillus niger RH5344

Summary The role of two histidine residues of pectin methylesterase (PME) were analysed by site-directed mutagenesis. Mutant and wild-type pmeA-cDNA were expressed in A. niger strain NRRL3. Both mutant enzymes exhibited the same mobility on SDS-polyacrylamide gel electrophoresis and gave similar circular dichroism spectra to that of the wild-type enzyme. Substitution of His-137 to alanine caused a loss of PME activity. In contrast, replacement of His-188 had no effect on the PME activity. These results revealed that the histidine residue at position 137 is essential for enzyme activity and probably located in the active site of PME.     

High‐throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray‐based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.     

Purification, characterization, thermal, and high-pressure inactivation of pectin methylesterase from bananas (cv Cavendish)

Pectin methylesterase (PME) was extracted from bananas (cv Cavendish) and purified by affinity chromatography on a CNBr-Sepharose-PME inhibitor (PMEI) column. A single protein and PME activity peak was obtained. For banana PME, a biochemical characterization in terms of molar mass (MM), pI, and kinetic parameters was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal inactivation of purified banana PME could be described by a first-order kinetic model in a temperature range of 65 degrees to 72.5 degrees C, whereas its isobaric-isothermal inactivation followed a fractional-conversion model. Banana PME was found to be more thermally stable compared with PMEs extracted from orange, tomato, and apple.