Melting the wall: plant parasitism entails pectin modification.

Phtheirospermum japonicum shows induced expression of PjPME and PjPMEI genes during haustoria development in rice and Arabidopsis with increased PME activity, which leads to the modulated cell wall during parasitism. Moreover, how PME and PMEI proteins interact and balance during haustoria development remains elusive.

MYB-bHLH-TTG1 in a Multi-tiered Pathway Regulates Arabidopsis Seed Coat Mucilage Biosynthesis Genes Including PECTIN METHYLESTERASE INHIBITOR14 Required for Homogalacturonan Demethylesterification.

MYB-bHLH-TTG1 (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat biosynthesis pathways via a multi-tiered regulatory mechanism. The MYB genes include MYB5, MYB23 and TRANSPARENT TESTA2 (TT2), which regulate GLABRA2 (GL2), HOMEODOMAIN GLABROUS2 (HDG2) and TRANSPARENT TESTA GLABRA2 (TTG2). Here, we examine the role of PECTIN METHYLESTERASE INHIBITOR14 (PMEI14) in seed coat mucilage pectin methylesterification and provide evidence in support of multi-tiered regulation of seed coat mucilage biosynthesis genes including PMEI14. The PMEI14 promoter was active in the seed coat and developing embryo. A pmei14 mutant exhibited stronger attachment of the outer layer of seed coat mucilage, increased mucilage homogalacturonan demethylesterification and reduced seed coat radial cell wall thickness, results consistent with decreased PMEI activity giving rise to increased PME activity. Reduced mucilage release from the seeds of myb5, myb23, tt2 and gl2, hdg2, ttg2 triple mutants indicated that HDG2 and MYB23 play minor roles in seed coat mucilage deposition. Chromatin immunoprecipitation analysis found that MYB5, TT8 and seven mucilage pathway structural genes are directly regulated by MYB5. Expression levels of GL2, HDG2, TTG2 and nine mucilage biosynthesis genes including PMEI14 in the combinatorial mutant seeds indicated that these genes are positively regulated by at least two of those six TFs and that TTG1 and TTG2 are major regulators of PMEI14 expression. Our results show that MYB-bHLH-TTG1 complexes regulate mucilage biosynthesis genes, including PMEI14, both directly and indirectly via a three-tiered mechanism involving GL2, HDG2 and TTG2.

Overexpression of MePMEI1 in Arabidopsis enhances Pb tolerance.

Pb is one of the most ubiquitously distributed heavy metal pollutants in soils and has serious negative effects on plant growth, food safety, and public health. Pectin methylesterase inhibitors (PMEIs) play a pivotal role in regulating the integrity of plant cell walls; however, the molecular basis by which PMEIs promote plant resistance to abiotic stress remains poorly understood. In this study, we identified a novel PMEI gene, MePMEI1, from Manihot esculenta, and determined its role in plant resistance to Pb stress. The expression of MePMEI1 was remarkably upregulated in the roots, stems, and leaves of cassava plants following exposure to Pb stress. An analysis of subcellular localization revealed that the MePMEI1 protein was localized in the cell wall. MePMEI1 inhibited commercial orange peel pectin methyltransferase (PME), and the expression of MePMEI1 in Arabidopsis decreased the PME activity, indicating that MePMEI1 can inhibit PME activity in the cell wall. Additionally, the overexpression of MePMEI1 in Arabidopsis reduced oxidative damage and induced the thickening of cell walls, thus contributing to Pb tolerance. Altogether, the study reports a novel mechanism by which the MePMEI1 gene, which encodes the PMEI protein in cassava, plays an essential role in promoting tolerance to Pb toxicity by regulating the thickness of cell walls. These results provide a theoretical basis for the MePMEI1-mediated plant breeding for increasing heavy metal tolerance and provide insights into controlling Pb pollution in soils through phytoremediation in future studies.

Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana.

The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG de-methylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion.

Transcriptome-Guided Identification of Pectin Methyl-Esterase-Related Enzymes and Novel Molecular Processes Effectuating the Hard-to-Cook Defect in Common Bean (Phaseolus vulgaris L.).

The hard-to-cook defect in common beans is dictated by the ability to achieve cell separation during cooking. Hydrolysis of pectin methyl-esters by the pectin methyl-esterase (PME) enzyme influences cell separation. However, the contributions of the PME enzyme and the cell wall to the hard-to-cook defect have not been studied using molecular tools. We compared relevant molecular processes in fast- and slow-cooking bean varieties to understand the mechanisms underpinning the hard-to-cook defect. A PME spectrophotometric assay showed minor differences in enzyme activity between varieties. Meanwhile, a PME HMMER search in the P. vulgaris genome unveiled 113 genes encoding PMEs and PME inhibitors (PMEIs). Through RNA sequencing, we compared the gene expression of the PME-related genes in both varieties during seed development. A PME (Phvul010g080300) and PMEI gene (Phvul005g007600) showed the highest expression in the fast- and slow-cooking beans, respectively. We further identified 2132 differentially expressed genes (DEGs). Genes encoding cell-wall-related enzymes, mainly glycosylphosphatidylinositol mannosyltransferase, xyloglucan O-acetyltransferase, pectinesterase, and callose synthase, ranked among the top DEGs, indicating novel relations to the hard-to-cook defect. Gene ontology mapping revealed hydrolase activity and protein phosphorylation as functional categories with the most abundant upregulated DEGs in the slow-cooking bean. Additionally, the cell periphery contained 8% of the DEGs upregulated in the slow-cooking bean. This study provides new insights into the role of pectin methyl-esterase-related genes and novel cell wall processes in the occurrence of the hard-to-cook defect.

Amplification, sequencing and characterization of pectin methyl esterase inhibitor 51 gene in Tectona grandis L.f.

Tectona grandis L.f. (Teak), a very important source of incomparable timber, withstands a wide range of tropical deciduous conditions. We achieved partial amplification of pectin methylesterase inhibitor 51 (PMEI) gene in teak by E. pilularis cinnamoyl Co-A reductase (CCR) gene specific primer. The amplified teak gene was of 750 bp, 79% identity and 97% query cover with PMEI of Sesamum indicum. The phylogenetic tree clustered the amplified gene with PMEI of database plant species, Erythranthe guttata and Sesamum indicum (87% bootstrap value). On conversion to amino acid sequence, the obtained protein comprised 237 amino acids. However, PMEI region spanned from 24 to 171 amino acids, 15.94 kDa molecular weight, 8.97 pI value and C697H1117N199O211S9 molecular formula with four conserved cysteine residues as disulfide bridges. 25.9 % protein residues were hydrophilic, 42.7% hydrophobic and 31.2% neutral. Teak 3D PMEI protein structure corresponded well with Arabidopsis thaliana and Actinidia deliciosa PMEIs. The gene maintains integrity of pectin component of middle lamella of primary cell wall and confers tolerance against various kinds of stresses. Teak conferred with overexpression of PMEI may secure a wide adaptability as well as luxuriant timber productivity and quality in adverse/ fluctuating/ scarce climatic and environmental conditions of tropical forests.

AtENO2 functions in the development of male gametophytes in Arabidopsis thaliana.

Pollen fertility is an important factor affecting the seed setting rate and seed yield of plants. The Arabidopsis thaliana enolase gene ENO2 (AtENO2) can affect the pollen morphology, germination, and pollen tube growth. AtENO2 encodes two proteins AtENO2 and AtMBP-1. To examine the effect of AtENO2 protein on pollen development, the 2nd ATG of the AtENO2 coding sequence for AtMBP-1 was mutated by site-directed mutagenesis, and transgenic plants expressing only AtENO2 but not AtMBP-1 were obtained. Phenotypic analysis indicated that AtENO2 was essential in the pollen development. The mechanisms of AtENO2 on pollen development were analyzed. AtENO2 can affect development of the pollen intine, and the mechanism may be that AtENO2 regulated the methyl esterification of pectin in pollen intine through ARF3 and AtPMEI-pi. The -734 ∼ -573 sequence of AtENO2 promoter is the main transcriptional regulatory region of AtENO2 affecting pollen development. The functional cis-acting element may be GTGANTG10(GTGA), and the trans-acting factors may be KAN, AS2 and ARF3/ETT. Moreover, the deletion of AtENO2 can cause significant difference in the expression of multiple genes related to pollen exine development. These results are useful for further studying the function of AtENO2 and exploring the mechanism of plant pollen development.

Mechanochemical feedback mediates tissue bending required for seedling emergence.

Tissue bending is vital to plant development, as exemplified by apical hook formation during seedling emergence by bending of the hypocotyl. How tissue bending is coordinated during development remains poorly understood, especially in plants where cells are attached via rigid cell walls. Asymmetric distribution of the plant hormone auxin underlies differential cell elongation during apical hook formation. Yet the underlying mechanism remains unclear. Here, we demonstrate spatial correlation between asymmetric auxin distribution, methylesterified homogalacturonan (HG) pectin, and mechanical properties of the epidermal layer of the hypocotyl in Arabidopsis. Genetic and cell biological approaches show that this mechanochemical asymmetry is essential for differential cell elongation. We show that asymmetric auxin distribution underlies differential HG methylesterification, and conversely changes in HG methylesterification impact the auxin response domain. Our results suggest that a positive feedback loop between auxin distribution and HG methylesterification underpins asymmetric cell wall mechanochemical properties to promote tissue bending and seedling emergence.

Gene-wide identification and expression analysis of the PMEI family genes in soybean (Glycine max).

Pectin Methylesterase Inhibitors (PMEI) gene family is widely spread in plants and plays crucial roles in plant development as well as biotic and abiotic stress response. However, little information was known about the function of PMEI genes in soybean. Herein, we identified 170 PMEI genes in soybean. These PMEI genes were divided into four groups (I-IV) based on phylogenetic analysis, and they were unevenly distributed in 18 soybean chromosomes. Gene structures and motif pattern analyses revealed that the PMEI genes in the same group showed the same characteristics. For the GmPMEI genes, gene duplication events occurred broadly, 52 pairs tandem duplication events and 55 pairs segmental duplication events suggested that the GmPMEI genes had high homology. Besides, the PMEI genes presented different expression patterns in different tissues, while several of these genes were expressed only in flowers. Under the biotic and abiotic stresses, PMEI genes had significant positive impact on the tolerance ability of soybean, and the ABA-responsive elements and SA-responsive elements played vital roles in responding to a variety of stresses. This study provides insights into the evolution and potential functions of GmPMEIs.

Genome-Wide Identification, Characterization and Expression Patterns of the Pectin Methylesterase Inhibitor Genes in Sorghum bicolor.

Cell walls are basically complex with dynamic structures that are being involved in several growth and developmental processes, as well as responses to environmental stresses and the defense mechanism. Pectin is secreted into the cell wall in a highly methylesterified form. It is able to perform function after the de-methylesterification by pectin methylesterase (PME). Whereas, the pectin methylesterase inhibitor (PMEI) plays a key role in plant cell wall modification through inhibiting the PME activity. It provides pectin with different levels of degree of methylesterification to affect the cell wall structures and properties. The PME activity was analyzed in six tissues of Sorghum bicolor, and found a high level in the leaf and leaf sheath. PMEI families have been identified in many plant species. Here, a total of 55 pectin methylesterase inhibitor genes (PMEIs) were identified from S. bicolor whole genome, a more detailed annotation of this crop plant as compared to the previous study. Chromosomal localization, gene structures and sequence characterization of the PMEI family were analyzed. Moreover, cis-acting elements analysis revealed that each PMEI gene was regulated by both internal and environmental factors. The expression patterns of each PMEI gene were also clustered according to expression pattern analyzed in 47 tissues under different developmental stages. Furthermore, some SbPMEIs were induced when treated with hormonal and abiotic stress. Taken together, these results laid a strong foundation for further study of the functions of SbPMEIs and pectin modification during plant growth and stress responses of cereal.

Pectin Demethylesterification Generates Platforms that Anchor Peroxidases to Remodel Plant Cell Wall Domains.

Plant cell walls are made of polysaccharidic-proteinaceous complex matrices. Molecular interactions governing their organization remain understudied. We take advantage of the highly dynamic cell walls of Arabidopsis seed mucilage secretory cells to propose a hierarchical multi-molecular interaction model within a cell wall domain. We show that the PECTINMETHYLESTERASE INHIBITOR6 activity creates a partially demethylesterified pectin pattern acting as a platform allowing positioning of PEROXIDASE36 in a remote primary cell wall domain during early development. This allows triggering the loosening of this domain during later development, in turn leading to proper physiological function upon mature seed imbibition and germination. We anticipate that this pioneer example of molecular scaffold within a cell wall domain is more widespread through other combinations of the individual molecular players all belonging to large multigenic families. These results highlight the role of cell wall polysaccharide-protein interactions in the organization of cell wall domains.

Phenylephrine, a small molecule, inhibits pectin methylesterases.

Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1 µM. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.

The Multifaceted Role of Pectin Methylesterase Inhibitors (PMEIs).

Plant cell walls are complex and dynamic structures that play important roles in growth and development, as well as in response to stresses. Pectin is a major polysaccharide of cell walls rich in galacturonic acid (GalA). Homogalacturonan (HG) is considered the most abundant pectic polymer in plant cell walls and is partially methylesterified at the C6 atom of galacturonic acid. Its degree (and pattern) of methylation (DM) has been shown to affect biomechanical properties of the cell wall by making pectin susceptible for enzymatic de-polymerization and enabling gel formation. Pectin methylesterases (PMEs) catalyze the removal of methyl-groups from the HG backbone and their activity is modulated by a family of proteinaceous inhibitors known as pectin methylesterase inhibitors (PMEIs). As such, the interplay between PME and PMEI can be considered as a determinant of cell adhesion, cell wall porosity and elasticity, as well as a source of signaling molecules released upon cell wall stress. This review aims to highlight recent updates in our understanding of the PMEI gene family, their regulation and structure, interaction with PMEs, as well as their function in response to stress and during development.

Influence of pH on the Structure and Function of Kiwi Pectin Methylesterase Inhibitor.

Pectin methylesterase is a pectin modifying enzyme that plays a key role in plant physiology. It is also an important quality-related enzyme in plant-based food products. The pectin methylesterase inhibitor (PMEI) from kiwifruit inhibits this enzyme activity and is widely used as an efficient tool for research purposes and also recommended in the context of fruit and vegetable processing. Using several methodologies of protein biochemistry, including circular dichroism and fluorescence spectroscopy, chemical modifications, direct protein-sequencing, enzyme activity, and bioinformatics analysis of the crystal structure, this study demonstrates that conformational changes occur in kiwi PMEI by the pH rising over 6.0 bringing about structure loosening, exposure, and cleavage of a natively buried disulfide bond, unfolding and aggregation, ultimately determining the loss of ability of kiwi PMEI to bind and inhibit PME. pH-induced structural changes are prevented when PMEI is already engaged in complex or is in a solution of high ionic strength.

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.

Arabidopsis PME17 Activity can be Controlled by Pectin Methylesterase Inhibitor4.

The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.

How do pectin methylesterases and their inhibitors affect the spreading of tobamovirus?

After replication in the cytoplasm, viruses spread from the infected cell into the neighboring cells through plasmodesmata, membranous channels embedded by the cell wall. As obligate parasites, viruses have acquired the ability to utilize host factors that unwillingly cooperate for the viral infection process. For example, the viral movement proteins (MP) interacts with the host pectin methylesterase (PME) and both proteins cooperate to sustain the viral spread. However, how and where PMEs interact with MPs and how the PME/MP complexes favor the viral translocation is not well understood. Recently, we demonstrated that the overexpression of PME inhibitors (PMEIs) in tobacco and Arabidopsis plants limits the movement of Tobacco mosaic virus and Turnip vein clearing virus and reduces plant susceptibility to these viruses. Here we discuss how overexpression of PMEI may reduce tobamovirus spreading.

Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification.

Claviceps purpurea is a biotrophic fungal pathogen of grasses causing the ergot disease. The infection process of C. purpurea on rye flowers is accompanied by pectin degradation and polygalacturonase (PG) activity represents a pathogenicity factor. Wheat is also infected by C. purpurea and we tested whether the presence of polygalacturonase inhibiting protein (PGIP) can affect pathogen infection and ergot disease development. Wheat transgenic plants expressing the bean PvPGIP2 did not show a clear reduction of disease symptoms when infected with C. purpurea. To ascertain the possible cause underlying this lack of improved resistance of PvPGIP2 plants, we expressed both polygalacturonases present in the C. purpurea genome, cppg1 and cppg2 in Pichia pastoris. In vitro assays using the heterologous expressed PGs and PvPGIP2 showed that neither PG is inhibited by this inhibitor. To further investigate the role of PG in the C. purpurea/wheat system, we demonstrated that the activity of both PGs of C. purpurea is reduced on highly methyl esterified pectin. Finally, we showed that this reduction in PG activity is relevant in planta, by inoculating with C. purpurea transgenic wheat plants overexpressing a pectin methyl esterase inhibitor (PMEI) and showing a high degree of pectin methyl esterification. We observed reduced disease symptoms in the transgenic line compared with null controls. Together, these results highlight the importance of pectin degradation for ergot disease development in wheat and sustain the notion that inhibition of pectin degradation may represent a possible route to control of ergot in cereals.