CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana

Pectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell‐wall stiffness, cell‐to‐cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)‐like proteins and an unrelated plant‐specific protein, cotton Golgi‐related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss‐of‐function mutants and over‐expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell‐wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over‐expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell‐wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus.     

Expanding roles for pectins in plant development

Pectins are complex cell wall polysaccharides important for many aspects of plant development. Recent studies have discovered extensive physical interactions between pectins and other cell wall components, implicating pectins in new molecular functions. Pectins are often localized in spatially‐restricted patterns, and some of these non‐uniform pectin distributions contribute to multiple aspects of plant development, including the morphogenesis of cells and organs. Furthermore, a growing number of mutants affecting cell wall composition have begun to reveal the distinct contributions of different pectins to plant development. This review discusses the interactions of pectins with other cell wall components, the functions of pectins in controlling cellular morphology, and how non‐uniform pectin composition can be an important determinant of developmental processes.     

Simultaneous in vivo truncation of pectic side chains

Despite the wide occurrence of pectin in nature only a few source materials have been used to produce commercial pectins. One of the reasons for this is that many plant species contain pectins with high levels of neutral sugar side chains or that are highly substituted with acetyl or other groups. These modifications often prevent gelation, which has been a major functional requirement of commercial pectins until recently. We have previously shown that modification of pectin is possible through heterologous expression of pectin degrading enzymes in planta. To test the effect of simultaneous modification of the two main neutral pectic side chains in pectic rhamnogalacturonan I (RGI), we constitutively expressed two different enzymes in Arabidopsis thaliana that would either modify the galactan or the arabinan side chains, or both side chains simultaneously. Our analysis showed that the simultaneous truncation of arabinan and galactan side chains is achievable and does not severely affect the growth of Arabidopsis thaliana.     

A conserved functional role of pectic polymers in stomatal guard cells from a range of plant species

Guard cell walls combine exceptional strength and flexibility in order to accommodate the turgor pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores. To investigate the molecular basis of these exceptional qualities, we have used a combination of compositional and functional analyses in three different plant species. We show that comparisons of FTIR spectra from stomatal guard cells and those of other epidermal cells indicate a number of clear differences in cell-wall composition. The most obvious characteristics are that stomatal guard cells are enriched in phenolic esters of pectins. This enrichment is apparent in guard cells from Vicia faba (possessing a type I cell wall) and Commelina communis and Zea mays (having a type II wall). We further show that these common defining elements of guard cell walls have conserved functional roles. As previously reported in C. communis, we show that enzymatic modification of the pectin network in guard cell walls in both V. faba and Z. mays has profound effects on stomatal function. In all three species, incubation of epidermal strips with a combination of pectin methyl esterase and endopolygalacturonase (EPG) caused an increase in stomatal aperture on opening. This effect was not seen when strips were incubated with EPG alone indicating that the methyl-esterified fraction of homogalacturonan is key to this effect. In contrast, arabinanase treatment, and incubation with feruloyl esterase both impeded stomatal opening. It therefore appears that pectins and phenolic esters have a conserved functional role in guard cell walls even in grass species with type II walls, which characteristically are composed of low levels of pectins.     

The state of cell wall pectin monitored by wall associated kinases: A model

The Wall Associated Kinases (WAKs) bind to both cross-linked polymers of pectin in the plant cell wall, but have a higher affinity for smaller fragmented pectins that are generated upon pathogen attack or wounding. WAKs are required for cell expansion during normal seedling development and this involves pectin binding and a signal transduction pathway involving MPK3 and invertase induction. Alternatively WAKs bind pathogen generated pectin fragments to activate a distinct MPK6 dependent stress response. Evidence is provided for a model for how newly generated pectin fragments compete for longer pectins to alter the WAK dependent responses.     

Functional characterization of the gels prepared with pectin methylesterase (PME)-treated pectins

High- and low-methoxyl pectins were treated with pectin methylesterase (PME) and the functional properties of the resulting pectin gels were characterized. The degree of esterification of high- and low-methoxyl pectins decreased from 74.5% to 6.3% and 40.0% to 6.5%, respectively while not changing their molecular weight. Also, the addition of glucono-delta-lactone (GDL) dramatically affected the gel strength and the pH reduction by the GDL led to the increased syneresis of the pectin gels, which was also observed in the PME-treated samples. When flavor compounds were incorporated into the pectin gels, the flavor release from the gels increased with decreasing the degree of esterification due to increased hydrophilic properties.     

Polypotency of the immunomodulatory effect of pectins

Pectins are the major component of plant cell walls, and they display diverse biological activities including immunomodulation. The pectin macromolecule contains fragments of linear and branched regions of polysaccharides such as homogalacturonan, rhamnogalacturonan-I, xylogalacturonan, and apiogalacturonan. These structural features determine the effect of pectins on the immune system. The backbones of pectic macromolecules have immunosuppressive activity. Pectins containing greater than 80% galacturonic acid residues were found to decrease macrophage activity and inhibit the delayed-type hypersensitivity reaction. Branched galacturonan fragments result in a biphasic immunomodulatory action. The branched region of pectins mediates both increased phagocytosis and antibody production. The fine structure of the galactan, arabinan, and apiogalacturonan side chains determines the stimulating interaction between pectin and immune cells. This review summarizes data regarding the relationship between the structure and immunomodulatory activity of pectins isolated from the plants of the European north of Russia and elucidates the concept of polypotency of pectins in native plant cell walls to both stimulate and suppress the immune response. The possible mechanisms of the immunostimulatory and anti-inflammatory effects of pectins are also discussed.     

Enzymatically and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties.

A series of pectins with different levels and patterns of methyl esterification was produced by treatment of a very highly methylated pectin with acid, alkali, plant pectin methyl esterase and fungus pectin methyl esterase. The intrinsic pK values, as well as the free fractions of monovalent and calcium counterions, were determined on pectin salt-free solutions. The variations of pK(a) versus the ionisation degree were found to depend on the de-esterification process but a unique value of 2.90+/-0.15 was estimated for the intrinsic pK value. Calcium binding properties of chemically and enzymatically de-esterified pectins were investigated and experimental results were compared to Manning's theoretical values. A progressive dimerisation process for pectins with a blockwise distribution of carboxyl groups in the presence of calcium ions is hypothesised.     

Synthesis of sulfated pectins and their anticoagulant activity

The following pectins were sulfated: bergenan BC (the pectin of Bergenia crassifolia L), lemnan LM (the pectin of Lemna minor L), and galacturonan as a backbone of pectins. Pyridine monomethyl sulfate, pyridine sulfotrioxide, and chlorosulfonic acid were used as reagents for sulfation. Chlorosulfonic acid proved to be the optimal reagent for sulfation of galacturonan and other pectins. Galacturonan and pectin derivatives with different degrees of sulfation were synthesized and their anticoagulant activities were shown to depend on the quantity of sulfate groups in the pectin macromolecules.     

Structural characterization and surface activity of hydrophobically functionalized extracted pectins

The technological properties of pectins are generally influenced by their chemical modification. Thus, amidated pectins are important derivatives with good emulsifying properties at low concentrations. The present article focuses on the comparative study of physicochemical properties of three modified pectin derivatives. Various amphiphilic derivatives in which pectin is associated with hydrophobic amines chains were prepared. The reaction was carried out in heterogeneous medium in methanol at 20 °C for 7 days and with 0.5 pectin/alkylamine mass ratio. The degrees of amidation (DA) of the derivatives were calculated based on the results of FTIR spectroscopy. The surface-active properties of the modified pectins were determined by surface tension (air/water) and interfacial tension (oil/water) measurements. The aminolysis of pectins appears to be an interesting way to produce pectin derivatives with new properties able to stabilize oil-in-water emulsions.     

Immunocytochemical localization of pectins in the maturing anther of Allium cepa L.

Distribution of pectins in cell walls of maturing anther of Allium cepa L. was investigated. The monoclonal antibodies against defined epitopes of pectin were used: JIM5 recognizing unesterified pectin and JIM7 recognizing esterified pectin. It has been found that the cell walls of all anther tissues mainly contain esterified pectins. In the somatic tissues only small amounts of unesterified pectins are present in the cell wall junctions and adjacent middle lamellae and in the cell walls of the connective tissue. Thickening of the epiderm cell walls and growth of trabeculae in endothecium are completed through deposition of esterified pectins. In the cell walls of the middle layer and tapetum, unesterified pectins have been found only prior to their disintegration. The primary wall of microsporocytes is made up mainly of esterified pectins. Unesterified pectins occur outside microsporocytes only prior to the callose isolation stage. The presence of esterified pectins has also been detected on the surface of the callose wall surrounding dividing microsporocytes. Lysis of those pectins takes place after microsporogenesis, simultaneously with the lysis of the callosic walls. Before these processes pectins are unesterified. In the sporoderm of pollen grains mainly esterified pectins occur. They have been localized in the intine and aperture. The level of unesterified pectins in the intine is markedly lower.     

Study of the methyl ester distribution in pectin with endo-polygalacturonase and high-performance size-exclusion chromatography

A method was developed that enables the study of the methyl ester distribution in the polymers of pectin on a molecular level. Endo-polygalacturonase was used to extensively degrade three 70% methyl esterified pectins. The molecular weight distribution of the non- and enzymatically degraded pectins was determined with high-performance size-exclusion chromatography. Next, the molecular weight distribution was converted into a degree of polymerization distribution of galacturonan fragments. Monte Carlo methods were employed for the reconstruction of the parental polymers from their enzymatic degradation products. The results for the random methyl esterified pectin revealed that the enzyme-degradable sites were indeed randomly distributed, which confirmed the correctness of the procedure developed. The two other pectins studied differed greatly in the amount of non-, low-, and high-esterified regions present in the reconstructed pectic molecules of a given molecular mass. That the approach developed is able to reveal such detailed information makes it unique. The information on the fragmental composition of pectic polymers obtained is an important addition to the study of the methyl ester distribution and the functional properties of pectin.     

New insights into pectin methylesterase structure and function

In bacteria, fungi and plants, pectin methylesterases are ubiquitous enzymes that modify the degree of methylesterification of pectins, which are major components of plant cell walls. Such changes in pectin structure are associated with changes in cellular adhesion, plasticity, pH and ionic contents of the cell wall and influence plant development and stress responses. In plants, pectin methylesterases belong to large multigene families, are regulated in a highly specific manner, and are involved in vegetative and reproductive processes, including wood and pollen formation, in addition to plant-pathogen interactions. Although, overall, protein structures are highly conserved between isoforms, recent data indicate that structural variations might be associated with the targeting and functions of specific pectin methylesterases.     

Chemically methylated and reduced pectins: preparation,characterisation by 1H NMR spectroscopy,enzymatic degradation,and gelling properties

The gelling properties of pectins are known to be closely related to the degree of methylation (DM) and the distribution of the ester groups. In order to investigate this dependency, a natural citrus pectin (DM 64%) has been methylated to pectins with higher DM or saponified to achieve pectins with lower DM. A simple method for determination of DM by 1H NMR spectroscopy is presented. New modified pectins have been prepared by treatment of pectins having different DM with NaBH(4) to reduce selectively the methyl esters to primary alcohols in the presence of free acids. The degree of reduction (DR) and the DM of the remaining carboxylic acids could likewise be determined by 1H NMR spectroscopy. The new reduced pectins are recognized by the pectin degrading enzymes polygalacturonase PGI and PGII as well as by pectin lyase, all from Aspergillus niger, but the enzymes exhibit lower specific activities as compared with unmodified pectin. The new reduced pectins exhibit high gelling properties.     

Cellular localization and levels of pectins and arabinogalactan proteins in olive (Olea europaea L.) pistil tissues during development: implications for pollen–pistil interaction

Cell wall components in the pistil are involved in cell–cell recognition, nutrition and regulation of pollen tube growth. The aim of this work was to study the level, whole-organ distribution, and subcellular localization of pectins and arabinogalactan proteins (AGPs) in the olive developing pistil. Western blot analyses and immunolocalization with fluorescence and electron microscopy were carried out using a battery of antibodies recognizing different types of pectin epitopes (JIM7, JIM5, LM5, and LM6) and one anti-AGPs antibody (JIM13). In the olive pistil, highest levels of acid esterified and de-esterified pectins were observed at pollination. Moreover, pollination was accompanied by a slight decrease of the galactose-rich pectins pool, whereas arabinose-rich pectins were more abundant at that time. An increased expression of AGPs was also observed during pollination, in comparison to the pistil at the pre-anthesis stage. After pollination, the levels of pectins and AGPs declined significantly. Inmunofluorescence localization of pectins showed their different localization in the olive pistil. Pectins with galactose residues were located mainly in the cortical zones of the pistil, similar to the neutral pectins, which were found in the parenchyma and epidermis. In turn, the neutral pectins, which contain arabinose residues and AGPs, were localized predominantly in the stigmatic exudate, in the cell wall of secretory cells of the stigma, as well as in the transmitting tissue of the pistil during the pollination period. The differences in localization of pectins and AGPs are discussed in relation to their roles during olive pistil developmental course.     

Plant protein inhibitors of cell wall degrading enzymes

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.     

Anti-inflammatory activity of pectins and their galacturonan backbone

It has been shown that pectin polysaccharides from different plants, depending on their structure, can either protect the intestinal walls of mammals against damage and inhibit the development of inflammation or, on the contrary, have proinflammatory effects. At the same time, galacturonan isolated from any pectin, being the main carbohydrate chain (backbone) of its macromolecule, shows a marked anti-inflammatory effect. A decrease in the quantity of neutrophiles in the intestinal wall after induced inflammation indicates that the anti-inflammatory effects of pectins can be based on their influence on the functional activity of leukocytes.     

Characterization of citrus pectin samples extracted under different conditions: influence of acid type and pH of extraction

Background and Aims

Pectin is a complex macromolecule, the fine structure of which is influenced by many factors. It is used as a gelling, thickening and emulsifying agent in a wide range of applications, from food to pharmaceutical products. Current industrial pectin extraction processes are based on fruit peel, a waste product from the juicing industry, in which thousands of tons of citrus are processed worldwide every year. This study examines how pectin components vary in relation to the plant source (orange, lemon, lime, grapefruit) and considers the influence of extraction conditions on the chemical and macromolecular characteristics of pectin samples.

Methods

Citrus peel (orange, lemon, lime and grapefruit) from a commercial supplier was used as raw material. Pectin samples were obtained on a bulk plant scale (kilograms; harsh nitric acid, mild nitric acid and harsh oxalic acid extraction) and on a laboratory scale (grams; mild oxalic acid extraction). Pectin composition (acidic and neutral sugars) and physicochemical properties (molar mass and intrinsic viscosity) were determined.

Key Results

Oxalic acid extraction allowed the recovery of pectin samples of high molecular weight. Mild oxalic acid-extracted pectins were rich in long homogalacturonan stretches and contained rhamnogalacturonan I stretches with conserved side chains. Nitric acid-extracted pectins exhibited lower molecular weights and contained rhamnogalacturonan I stretches encompassing few and/or short side chains. Grapefruit pectin was found to have short side chains compared with orange, lime and lemon. Orange and grapefruit pectin samples were both particularly rich in rhamnogalacturonan I backbones.

Conclusions

Structural, and hence macromolecular, variations within the different citrus pectin samples were mainly related to their rhamnogalacturonan I contents and integrity, and, to a lesser extent, to the length of their homogalacturonan domains.     

Process for selective extraction of pectins from plant material by differential pH

Extraction of natural hydrocolloid carbohydrate polymers, such as pectin, from plant matter is accomplished at somewhat elevated temperature and controlled conditions of acidity/alkalinity. In many cases the plant material contains a variety of different extractables, including non-polymeric carbohydrates (sugars) in addition to the pectins. Very recently two different kinds of pectin, a calcium-sensitive pectin (CSP) and a non-calcium-sensitive pectin (NCSP), have become interesting commercially. What is described in this work is a process to selectively extract NCSP and CSP by varying the pH of the extracting solution. In a first extraction with acidic aqueous solution, a pH between 3.0 and 3.3 without addition of polyvalent salt is sufficient to extract NCSP pectin. A second extraction under stronger acid conditions (pH of about 2.0) is sufficient to extract the remaining pectin, which is primarily CSP.