(M.P.) 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.Pectin is a structurally complex polysaccharide that accounts for nearly 35% of the dicot and nongraminaceous monocot primary cell wall. A main component of pectin is homogalacturonan (HGA) consisting of a backbone of 1,4-linked a-D-GalUA units, with variable amounts of methylester in the C 6 position. Pectins are secreted into the cell wall in a highly methylesterified form and, soon thereafter, are deesterified in muro by pectin methylesterase (PME; Brummell and Harpster, 2001;Willats et al., 2001). Demethylesterification produces free carboxyl groups and modifies the pH and charge of the wall, allowing the aggregation of polyuronides into a calcium-linked gel structure and increasing the wall firmness (Willats et al., 2001). In addition, the action of PMEs makes HGA susceptible to degradation by hydrolases such as endopolygalacturonases (endoPGs), contributing to the softening of the cell wall (Brummell and Harpster, 2001;Wakabayashi et al., 2003).Plant PMEs are involved in important physiological processes such as microsporogenesis, pollen growth, pollen separation, seed germination, root development, polarity of leaf growth, stem elongation, fruit ripening, and loss of tissue integrity

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-Å resolution. The enzyme folds into a right-handed parallel b-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long a-helices aligned in an antiparallel manner in a classical up-and-down fourhelical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel b-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.

No abstract

We have identi¢ed, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI-1 and AtPMEI-2) encoding functional inhibitors of pectin methylesterases. AtP-MEI-1 and AtPMEI-2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant-derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esteri¢cation by inhibition of endogenous PMEs is present in di¡erent plant species. ß