A novel pectin methylesterase inhibitor, PMEI3, in common bean suggests a key role of pectin methylesterification in Pseudomonas resistance.

The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3, was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack.

Genome-wide survey and evolutionary history of the pectin methylesterase (PME) gene family in the Dothideomycetes class of fungi.

Once deposited in the plant cell wall, pectin undergoes demethylesterification by endogenous pectin methylesterases (PMEs), which play various roles in growth and development, including defense against pathogen attacks. Pathogen PMEs can alter pectin's methylesterification pattern, increasing its susceptibility to degradation by other fungal pectinases and thus playing a critical role as virulence factors during early infection stages. To investigate the evolutionary history of PMEs in the Dothideomycetes class of fungi, we obtained genomic data from 15 orders (79 species) and added genomic data from 61 isolates of Corynespora cassiicola. Our analyses involved maximum likelihood phylogenies, gene genealogies, and selection analyses. Additionally, we measured PME gene expression levels of C. cassiicola using soybean as a host through RT-qPCR assays. We recovered 145 putative effector PMEs and 57 putative non-effector PMEs from across the Dothideomycetes. The PME gene family exhibits a small size (up to 5 members per genome) and comprises three major clades. The evolutionary patterns of the PME1 and PME2 clades were largely shaped by duplications and recurring gene retention events, while biased gene loss characterized the small-sized PME3 clade. The presence of five members in the PME gene family of C. cassiicola suggests that the family may play a key role in the evolutionary success of C. cassiicola as a polyphagous plant pathogen. The haplogroups Cc_PME1.1 and Cc_PME1.2 exhibited an accelerated rate of evolution, whereas Cc_PME2.1, Cc_PME2.2, and Cc_PME2.3 seem to be under strong purifying selective constraints. All five PME genes were expressed during infection of soybean leaves, with the highest levels during from six to eight days post-inoculation. The highest relative expression level was measured for CC_29_g7533, a member of the Cc_PME2.3 clade, while the remaining four genes had relatively lower levels of expression.

Selection and Molecular Response of AHL-lactonase (aiiA) Producing Bacillus sp. Under Penicillin G-induced Conditions.

Quorum sensing (QS) is the process by which microorganisms employ chemicals called autoinducers (AIs) to communicate with their population. The QS mechanism generally controls the expression of the virulence related genes in bacteria. N-acyl homoserine lactones (AHLs) are the most widespread QS molecules. Due to their diverse AHL-lactonase activities, Bacillus species make particularly suitable candidates for procedures such as demolition of pathogenic bacterial QS signals and bioremediation of ß-lactam antibiotics from contaminated environments. In this study, seven Bacillus strains with Quorum quenching (QQ) activity were isolated using an enrichment medium supplemented with Penicillin G (PenG). The AHL-lactonase encoding gene (aiiA) was amplified by PCR and sequenced. Amino acid sequences underwent multiple sequence alignment. Docking studies were carried out with both C6HSL and PenG ligand using AutoDock tools. The aiiA amino acid sequences of the isolates were found to be well conserved. Furthermore, amino acid sequence alignment revealed that 74.9% of amino acid sequences were conserved in the genus Bacillus. Docking of the C6HSL to wild type (3DHA) and H97D variant reduced the docking score by only 0.1 kcal/mol for the mutated protein. When PenG docked with a higher (1.5 kcal/mol) score as a ligand to wild-type and mutant receptors, the docking score for the mutated protein likewise decreased by 0.1 kcal/mol. This research contributed to the diversification of organisms with QQ activity and beta-lactam antibiotic resistance. It also clarified the binding score of the PenG ligand to the Bacillus AHL lactonase molecule for the first time.

The cotton pectin methyl esterase gene GhPME21 functions in microspore development and fertility in Gossypium hirsutum L.

Pectin widely exists in higher plants' cell walls and intercellular space of higher plants and plays an indispensable role in plant growth and development. We identified 55 differentially expressed genes related to pectin degradation by transcriptomic analysis in the male sterile mutant, ms1. A gene encoding pectin methylesterase (GhPME21) was found to be predominantly expressed in the developing stamens of cotton but was significantly down-regulated in ms1 stamens. The tapetal layer of GhPME21 interfered lines (GhPME21i) was significantly thickened compared to that of WT at the early stage; anther compartment morphology of GhPME21i lines was abnormal, and the microspore wall was broken at the middle stage; Alexander staining showed that the pollen grains of GhPME21i lines differed greatly in volume at the late stage. The mature pollen surfaces of GhPME21i lines were deposited with discontinuous and broken sheets and prickles viewed under SEM. Fewer pollen tubes were observed to germinate in vitro in GhPME21i lines, while tiny of those in vivo were found to elongate to the ovary. The seeds harvested from GhPME21i lines as pollination donors were dry and hollow. The changes of phenotypes in GhPME21i lines at various stages illustrated that the GhPME21 gene played a vital role in the development of cotton stamens and controlled plant fertility by affecting stamen development, pollen germination, and pollen tube elongation. The findings of this study laid the groundwork for further research into the molecular mechanisms of PMEs involved in microspore formation and the creation of cotton male sterility materials.

New insights into the function of plant tannase with promiscuous acyltransferase activity.

Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterases in tannin-rich plants, are involved in the degalloylation of two important groups of secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins. In this paper, we have made new progress in studying the function of tea (Camellia sinensis) (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity in vitro and in vivo and promotes the synthesis of simple galloyl glucoses and flavan-3-ol gallates in plants. We studied the functions of CsTA through enzyme analysis, protein mass spectrometry, and metabolic analysis of genetically modified plants. Firstly, CsTA was found to be not only a hydrolase but also an acyltransferase. In the two-step catalytic reaction where CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate or 1,2,3,4,6-penta-O-galloyl-ß-d-glucose into their degalloylated forms, a long-lived covalently bound Ser159-linked galloyl-enzyme intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptor (such as water, methanol, flavan-3-ols, and simple galloyl glucoses). Then, metabolic analysis suggested that transient overexpression of TAs in young strawberry (Fragaria × ananassa) fruits, young leaves of tea plants, and young leaves of Chinese bayberry (Myrica rubra) actually increased the total contents of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant TA.

Research Progress of Pectin Methylesterase and its Inhibitors.

As an important pectin enzyme, pectin methylesterase (PME) can hydrolyze methyl esters, release methanol and reduce esterification. It is essential in regulating pollen tube development, root extension, and fruit ripening. Pectin methylesterase inhibitors (PMEI) can specifically bind PME and inhibit its activity, which jointly determines the esterification degree of pectin. PMEI has important application prospects in plant pest control, fruits and vegetable processing fields. In this paper, the gene families, crystal structures, molecular recognition, and applications in plants and industry are reviewed for the PME and PMEI systems. Finally, the semi-rational design of PMEI is discussed and discussed prospected.

Methylesterification of cell-wall pectin controls the diurnal flower-opening times in rice.

Flowers are the core reproductive organ of plants, and flowering is essential for cross-pollination. Diurnal flower-opening time is thus a key trait influencing reproductive isolation, hybrid breeding, and thermostability in plants. However, the molecular mechanisms controlling this trait remain unknown. Here, we report that rice Diurnal Flower Opening Time 1 (DFOT1) modulates pectin methylesterase (PME) activity to regulate pectin methylesterification levels of the lodicule cell walls, which affect lodicule swelling to control diurnal flower-opening time. DFOT1 is specifically expressed in the lodicules, and its expression gradually increases with the approach to flowering but decreases with flowering. Importantly, a knockout of DFOT1 showed earlier diurnal flower opening. We demonstrate that DFOT1 interacts directly with multiple PMEs to promote their activity. Knockout of PME40 also resulted in early diurnal flower opening, whereas overexpression of PME42 delayed diurnal flower opening. Lower PME activity was observed to be associated with higher levels of pectin methylesterification and the softening of cell walls in lodicules, which contribute to the absorption of water by lodicules and cause them to swell, thus promoting early diurnal flower opening. Higher PME activity had the opposite effect. Collectively, our work uncovers a molecular mechanism underlying the regulation of diurnal flower-opening time in rice, which would help reduce the costs of hybrid breeding and improve the heat tolerance of flowering plants by avoiding higher temperatures at anthesis.

Homogalacturonan and xylogalacturonan region specificity of self-cloning vector-expressed pectin methylesterases (AoPME1-3) in Aspergillus oryzae.

Aspergillus oryzae is a safe microorganism that is commonly used in food production. We constructed a self-cloning vector capable of high expression in A. oryzae. Using the vector, three putative pectin methylesterase (PME) genes belonging to Carbohydrate Esterase family 8 derived from A. oryzae were expressed, and several characteristics of the gene products were examined. The effects of temperature and pH on the three enzymes (AoPME1, 2, and 3) were similar, with optimal reaction temperatures of 50 - 60 °C and optimal reaction pH range of 5 - 6. The specific activities of AoPME1, 2, and 3 for apple pectin were significantly different (34, 7,601, and 2 U/mg, respectively). When the substrate specificity was examined, AoPME1 showed high activity towards pectin derived from soybean and pea. Although AoPME2 showed little activity towards these pectins, it showed very high activity towards apple- and citrus-derived pectins. AoPME3 showed low specific activity towards all substrates tested. Sugar composition analysis revealed that apple- and citrus-derived pectins were rich in homogalacturonan, while soybean- and pea-derived pectins were rich in xylogalacturonan. When pea pectin was treated with endo-polygalacturonase or endo-xylogalacturonase in the presence of each PME, specific synergistic actions were observed (endo-polygalacturonase with AoPME1 or AoPME2 and endo-xylogalacturonase with AoPME1 or AoPME3). Thus, AoPME1 and AoPME3 hydrolyzed the methoxy group in xylogalacturonan. This is the first report of this activity in microbial enzymes. Our findings on the substrate specificity of PMEs should lead to the determination of the distribution of methoxy groups in pectin and the development of new applications in the field of food manufacturing.

Characterization of the pectin methylesterase inhibitor gene family in Rosaceae and role of PbrPMEI23/39/41 in methylesterified pectin distribution in pear pollen tube.

MAIN CONCLUSION: Pectin methylesterase inhibitor gene family in the seven Rosaceae species (including three pear cultivars) is characterized and three pectin methylesterase inhibitor genes are identified to regulate pollen tube growth in pear. Pectin methylesterase inhibitor (PMEI) participates in a variety of biological processes in plants. However, the information and function of PMEI genes in Rosaceae are largely unknown. In this study, a total of 423 PMEI genes are identified in the genomes of seven Rosaceae species. The PMEI genes in pear are categorized into five subfamilies based on structural analysis and evolutionary analysis. WGD and TD are the main duplication events in the PMEI gene family of pear. Quantitative real-time PCR analysis indicates that PbrPMEI23, PbrPMEI39, and PbrPMEI41 are increasingly expressed during pear pollen tube growth. Under the treatment of recombinant proteins PbrPMEI23, PbrPMEI39 or PbrPMEI41, the content of methylesterified pectin at the region 5-20 µm from the pollen tube tip significantly increases, and the growth of pear pollen tubes is promoted. These results indicate that PMEI regulates the growth of pollen tubes by changing the distribution of methylesterified pectin in the apex.

New insights into the specificity and processivity of two novel pectinases from Verticillium dahliae.

Pectin, the major non-cellulosic component of primary cell wall can be degraded by polygalacturonases (PGs) and pectin methylesterases (PMEs) during pathogen attack on plants. We characterized two novel enzymes, VdPG2 and VdPME1, from the fungal plant pathogen Verticillium dahliae. VdPME1 was most active on citrus methylesterified pectin (55-70%) at pH 6 and a temperature of 40 °C, while VdPG2 was most active on polygalacturonic acid at pH 5 and a temperature of 50 °C. Using LC-MS/MS oligoprofiling, and various pectins, the mode of action of VdPME1 and VdPG2 were determined. VdPME1 was shown to be processive, in accordance with the electrostatic potential of the enzyme. VdPG2 was identified as endo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, when flax roots were used as substrate, acetylated OGs were detected. The comparisons of OGs released from Verticillium-susceptible and partially resistant flax cultivars identified new possible elicitor of plant defence responses.

Tea Polyphenol Attenuates Oxidative Stress-Induced Degeneration of Intervertebral Discs by Regulating the Keap1/Nrf2/ARE Pathway.

OBJECTIVE: Intervertebral disc degeneration (IDD) and low back pain caused by IDD have attracted public attention owing to their extremely high incidence and disability rate. Oxidative stress is a major cause of IDD. Tea polyphenols (TP) are natural-derived antioxidants extracted from tea leaves. This study explored the protective role of TP on the nucleus pulposus cells (NPCs) of intervertebral discs and their underlying mechanism. METHODS: An in vitro model of H2O2-induced degeneration of NPCs was established. RT-qPCR and western blotting were used to detect the mRNA and protein expression of the targets. An in vivo model of IDD was established via acupuncture of the intervertebral disc. Radiological imaging and histological staining were performed to evaluate the protective role of TP. RESULTS: H2O2 contributed to NPC degeneration by inducing high levels of oxidative stress. TP treatment effectively increased the expression of nucleus pulposus matrix-associated genes and reduced the expression of degeneration factors. Further mechanistic studies showed that TP delayed H2O2-mediated NPC degeneration by activating the Keap1/Nrf2/ARE pathway. In vivo experiments showed that TP delayed the degeneration of NPCs in rats through the Keap1/Nrf2/ARE pathway. CONCLUSION: Our study confirmed that TP activates the Keap1/Nrf2/ARE pathway to exert an antioxidative stress role, ultimately delaying the degeneration of intervertebral discs.

Complete genome sequencing and comparative CAZyme analysis of Rhodococcus sp. PAMC28705 and PAMC28707 provide insight into their biotechnological and phytopathogenic potential.

Study of carbohydrate-active enzymes (CAZymes) can reveal information about the lifestyle and behavior of an organism. Rhodococcus species is well known for xenobiotic metabolism; however, their carbohydrate utilization ability has been less discussed till date. This study aimed to present the CAZyme analysis of two Rhodococcus strains, PAMC28705 and PAMC28707, isolated from lichens in Antarctica, and compare them with other Rhodococcus, Mycobacterium, and Corynebacterium strains. Genome-wide computational analysis was performed using various tools. Results showed similarities in CAZymes across all the studied genera. All three genera showed potential for significant polysaccharide utilization, including starch, cellulose, and pectin referring their biotechnological potential. Keeping in mind the pathogenic strains listed across all three genera, CAZymes associated to pathogenicity were analyzed too. Cutinase enzyme, which has been associated with phytopathogenicity, was abundant in all the studied organisms. CAZyme gene cluster of Rhodococcus sp. PAMC28705 and Rhodococcus sp. PAMC28707 showed the insertion of cutinase in the cluster, further supporting their possible phytopathogenic properties.

Chemoproteomics-Enabled De Novo Discovery of Photoswitchable Carboxylesterase Inhibitors for Optically Controlled Drug Metabolism.

Herein, we report arylazopyrazole ureas and sulfones as a novel class of photoswitchable serine hydrolase inhibitors and present a chemoproteomic platform for rapid discovery of optically controlled serine hydrolase targets in complex proteomes. Specifically, we identify highly potent and selective photoswitchable inhibitors of the drug-metabolizing enzymes carboxylesterases 1 and 2 and demonstrate their pharmacological application by optically controlling the metabolism of the immunosuppressant drug mycophenolate mofetil. Collectively, this proof-of-concept study provides a first example of photopharmacological tools to optically control drug metabolism by modulating the activity of a metabolizing enzyme. Our arylazopyrazole ureas and sulfones offer synthetically accessible scaffolds that can be expanded to identify specific photoswitchable inhibitors for other serine hydrolases, including lipases, peptidases, and proteases. Our chemoproteomic platform can be applied to other photoswitches and scaffolds to achieve optical control over diverse protein classes.

Allergen Homologues, Pathogenesis-Related 1, Polygalacturonase, and Pectin Methyl Esterase from a Japanese Hop.

BACKGROUND: Japanese hop is an important cause of weed pollinosis in East Asia. Its pollen is abundant in autumn. This pollen is known to be the cause of many allergic diseases. However, molecular characteristics of its allergens have not been elucidated. OBJECTIVE: In this study, we produced recombinant proteins of allergen homologues from Japanese hop by the analysis of expressed sequence tags (EST), and evaluated its allergenicity. METHODS: cDNA library was constructed using as little as 50 ng of total RNA from Japanese hop pollen. Allergen homologues were identified by the initial screening of 963 EST clones. Recombinant proteins were overexpressed in the E. coli expression system and purified using Ni-nitrilotriacetic acid-agarose. Purified proteins were analyzed by ELISA. RESULTS AND DISCUSSION: Japanese hop pathogenesis-related 1 protein (PR-1) shares 37.0 to 44.4% of amino acid sequence identity with Art v 2, Cuc m 3, and Cyn d 24. Pectin methyl esterase (PME) shows 23.2 to 50.2% of identities to Act d 7, Ole e 11, and Sal k 1. Polygalacturonase (PGs) shows 16.7 to 19.3% of identities to Phl p 13, Cry j 2, Cha o 2, Jun a 2, Pla a 2, and Pla or 2. IgE antibodies from Japanese hop allergy patients' sera recognized PR-1 (3.4%), PME (13.8%), PGs (3.7%), and profilin (13.8%), respectively. CONCLUSION: Novel allergenic components were identified, even though low IgE reactivity was displayed reflecting the low degree of cross-reactivity with other pollen allergens. We believe that these molecules have worth further studies.

Characterization of an acidic pectin methylesterase from Paenibacillus xylanexedens and its application in fruit processing.

A pectinase-producing bacterial isolate, identified as Paenibacillus xylanexedens SZ 29, was screened by using the soil dilution plate with citrus pectin and congo red. A pectin methylesterase gene (Pxpme) was cloned and expressed in Escherichia coli. The gene coded for a protein with 334 amino acids and a calculated molecular mass of 36.76 kDa. PxPME showed the highest identity of 32.4% with the characterized carbohydrate esterase family 8 pectin methylesterase from Daucus carota. The recombined PxPME showed a specific activity with 39.38 U/mg against citrus pectin with >65% methylesterification. The optimal pH and temperature for PxPME activity were 5.0 and 45 °C. Its Km and Vmax value were determined to be 1.43 mg/mL and 71.5 µmol/mg·min, respectively. Moreover, PxPME could increase the firmness of pineapple cubes by 114% when combined with CaCl2. The acidic and mesophilic properties make PxPME a potential candidate for application in the fruit processing.

Ascorbic acid during the suckling period is required for proper DNA demethylation in the liver.

Ascorbic acid (AA, vitamin C) serves as a cofactor for ten-eleven translocation (TET) enzymes and induces DNA demethylation in vitro. However, its role in DNA demethylation in vivo remains unclear. We previously reported that DNA demethylation in the mouse liver was enhanced during the suckling period. Therefore, we hypothesized that DNA demethylation is enhanced in an AA-dependent manner during the suckling period. To examine our hypothesis, we employed wild-type (WT) mice, which synthesize AA, and senescence marker protein-30/gluconolactonase (SMP30/GNL) knockout (KO) mice, which cannot synthesize AA, and analyzed the DNA methylation status in the livers of offspring in both the suckling period and adulthood. SMP30/GNL KO offspring showed DNA hypermethylation in the liver possibly due to low plasma and hepatic AA levels during the suckling period despite the administration of rescue-dose AA to dams. Furthermore, DNA hypermethylation of the fibroblast growth factor 21 gene (Fgf21), a PPARα target gene, persisted into adulthood. In contrast, a high-dose AA administration to SMP30/GNL KO dams during the lactation period restored DNA demethylation in the livers of offspring. Even though a slight increase was observed in plasma AA levels with the administration of rescue-dose AA to WT dams during the gestation and lactation periods, DNA demethylation in the livers of offspring was minimally enhanced. The present results demonstrate that AA intake during the suckling period is required for proper DNA demethylation in the liver.

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.

Physiological Importance of Pectin Modifying Genes During Rice Pollen Development.

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

A GDSL-type esterase/lipase gene, GELP77, is necessary for pollen dissociation and fertility in Arabidopsis.

Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.

The Homogalacturonan Deconstruction System of Paenibacillus amylolyticus 27C64 Requires No Extracellular Pectin Methylesterase and Has Significant Industrial Potential.

Paenibacillus amylolyticus 27C64, a Gram-positive bacterium with diverse plant cell wall polysaccharide deconstruction capabilities, was isolated previously from an insect hindgut. Previous work suggested that this organism's pectin deconstruction system differs from known systems in that its sole pectin methylesterase is cytoplasmic, not extracellular. In this work, we have characterized the specific roles of key extracellular pectinases involved in homogalacturonan deconstruction, including four pectate lyases and one pectin lyase. We show that one newly characterized pectate lyase, PelC, has a novel substrate specificity, with a lower Km for highly methylated pectins than for polygalacturonic acid. PelC works synergistically with PelB, a high-turnover exo-pectate lyase that releases Δ4,5-unsaturated trigalacturonate as its major product. It is likely that PelC frees internal stretches of demethylated homogalacturonan which PelB can degrade. We also show that the sole pectin lyase has a high kcat value and rapidly depolymerizes methylated substrates. Three cytoplasmic GH105 hydrolases were screened for the ability to remove terminal unsaturated galacturonic acid residues from oligogalacturonide products produced by the action of extracellular lyases, and we found that two are active on demethylated oligogalacturonides. This work confirms that efficient homogalacturonan deconstruction in P. amylolyticus 27C65 does not require extracellular pectin methylesterase activity. Three of the extracellular lyases studied in this work are also thermostable, function well over a broad pH range, and have significant industrial potential.IMPORTANCE Pectin is an important structural polysaccharide found in most plant cell walls. In the environment, pectin degradation is part of the decomposition process that turns over dead plant material and is important to organisms that feed on plants. Industrially, pectinases are used to improve the quality of fruit juices and can also be used to process coffee cherries or tea leaves. These enzymes may also prove useful in reducing the environmental impact of paper and cotton manufacturing. This work is significant because it focuses on a Gram-positive bacterium that is evolutionarily distinct from other well-studied pectin-degrading organisms and differs from known systems in key ways. Most importantly, a simplified extracellular deconstruction process in this organism is able to break down pectins without first removing the methyl groups that inhibit other systems. Moreover, some of the enzymes described here have the potential to improve industrial processes that rely on pectin deconstruction.