Investigation of the Action Patterns of Pectinmethylesterase Isoforms through Kinetic Analyses and NMR Spectroscopy

Abstract

Well characterized pectin samples were incubated with cell wall-bound and -solubilized pure isoforms of pectinmethylesterase from mung bean hypocotyls (Vigna radiata). Both enzyme activity and average product structure were determined at intervals along the deesterification pathway at pH 5.6 and 7.6. The latter analyses were performed by 13 C NMR spectroscopy, and the degree of esterification was probed by both 13 C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the ␣ and ␥ isoforms when compared with that of the ␤ isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the ␣ and ␥ isoforms, a single chain mechanism associated with a free carboxyl group at the second nearest neighbor position could be postulated at pH 5.6, whereas some multiple attack character was required to reproduce the data at pH 7.6. Several mechanisms that differed from the preceding ones were compatible with the data for the ␤ isoform at the two pH values. Both the nature of the polysaccharides produced in these reactions and the role of pectinmethylesterase in the cell wall-stiffening process along the growth gradient are discussed.Pectins that represent around 30% of the primary plant cell walls play a key role in plant physiology as well as in plant pathology. The general structure of pectic polymers consists of homogalacturonan linear chains (smooth regions) interspersed with highly branched galacturonic chains (hairy regions). Some of the galacturonic residues linked by ␣-1,4 glycosidic bonds are methyl-esterified at the carboxyl group. The degree of methylesterification (DE) 1 varies greatly depending on the plant organ and the degree of differentiation of the cells. Young, plastic cell walls are generally characterized by a high content of highly methylated pectin that decreases in parallel with the loss of extensibility of the walls, whereas the amount of acidic residues increases (1-3). Moreover, the balance between high and low methylated pectins varies inside the wall of a single cell (4) generating microdomains. Not only the number but also the distribution of free, unesterified galacturonate carboxyl groups within the galacturonan regions will control the gellforming capacity of the pectin and thereby the porosity and the extensibility of the apoplasm (5, 6). It is commonly accepted that the polygalacturonic backbone is polymerized in the cisGolgi cisternae, methylesterified in the medial-Golgi, substituted with side chains in the trans-Golgi, and exported to the cell walls as a highly methylated polygalacturonan (7). At a later stage, it is deesterified in muro by cell wall pectinmethylesterases (PMEs). Many proteins exhibiting PME activity have been purified, and their biochemical features such as molecular weight, optimal pH, pI, and substrate specificity have been established (8). In some cases, the corresponding genes have also been cloned and sequenced (9 -11...

PECTIN METHYLESTERASE48 Is Involved in Arabidopsis Pollen Grain Germination

“…As discussed by Jolie et al (2010), the pH, the DM, and the pattern of methylesterification are known to modify the mode of action of PMEs (Catoire et al, 1998;Denès et al, 2000;Sénéchal et al, 2014). Thus, we can also hypothesize that, upon random action of the PME (PME48 or others from the 13 other pollen-specific PMEs), the partial removal of methylester groups may allow other pectin-degrading enzymes such as PGases and/or PLs to cleave the HG, affecting the rigidity of the cell wall (Micheli, 2001;Sénéchal et al, 2014).…”

Modulation of the Degree and Pattern of Methyl-esterification of Pectic Homogalacturonan in Plant Cell Walls

“…PME genes occur in multigene families and encode isoforms with differing action patterns with respect to the removal of methyl esters. However, the specific functions of PME populations in the context of cell expansion and other processes are not well understood (15)(16)(17). Methyl esters can be distributed in diverse patterns along HG chains and it is clear that the action patterns of plant PMEs (pPMEs) can be influenced by local cell wall pH, the existing balance of methyl and free carboxyl groups on HG substrates, and metal ion concentration (16, 18 -22).…”

Global Identification of DELLA Target Genes during Arabidopsis Flower Development

“…Thus, it is possible that DELLA proteins affect the expression of genes involved in cell expansion, a process that is largely dependent on cell wall modification in plants. We have identified a group of RGAregulated genes that encode proteins involved in the remodeling and modification of cell wall structure, such as Pro-rich cell wall proteins, HRGPs, GRPs, and expansins (Showalter, 1993;Catoire et al, 1998;Micheli, 2001). The expression of these genes is distributed widely in floral organs, including stamens, pollen, carpels, petals, and sepals (Supplemental Tables S5 and S6), indicating that RGA regulation of cell expansion is a general mechanism for various floral organs.…”

“…encoding cell wall proteins (Pro-rich cell wall proteins, extensins, Hyp-rich glycoproteins [HRGPs], Gly-rich proteins [GRPs], expansins), glycoproteins (arabinogalactan, xyloglucan transferase, polygalacturonase), and pectin-related enzymes (Showalter, 1993;Catoire et al, 1998;Micheli, 2001). Compared with the percentage of metabolic genes (around 27%) in the Arabidopsis genome (The Arabidopsis Information Resource; www.…”