Rice pectin methylesterase inhibitor28 (OsPMEI28) encodes a functional PMEI and its overexpression results in a dwarf phenotype through increased pectin methylesterification levels

Abstract

Pectin methylesterases (PMEs, EC 3.1.1.11) belonging to carbohydrate esterase family 8 cleave the ester bond between a galacturonic acid and an methyl group and the resulting change in methylesterification level plays an important role during the growth and development of plants. Optimal pectin methylesterification status in each cell type is determined by the balance between PME activity and post-translational PME inhibition by PME inhibitors (PMEIs). Rice contains 49 PMEIs and none of them are functionally characterized. Genomic sequence analysis led to the identification of rice PMEI28 (OsPMEI28). Recombinant OsPMEI28 exhibited inhibitory activity against commercial PME protein with the highest activities detected at pH 8.5. Overexpression of OsPMEI28 in rice resulted in an increased level of cell wall bound methylester groups and differential changes in the composition of cell wall neutral monosaccharides and lignin content in culm tissues. Consequently, transgenic plants overexpressing OsPMEI28 exhibited dwarf phenotypes and reduced culm diameter. Our data indicate that OsPMEI28 functions as a critical structural modulator by regulating the degree of pectin methylesterification and that an impaired status of pectin methylesterification affects physiochemical properties of the cell wall components and causes abnormal cell extensibility in rice culm tissues.

Introduction

Determination of the final plant organ size depends on both cell division and expansion. Combinatory effects of multiple endogenous phytohormones and exogenous environmental signals dictate the expression levels of genes involved in cell proliferation and expansion (Vanstraelen and Benková, 2012). Once cell division takes place, a number of enzymes that participate in cell wall loosening exert their effects on cell expansion. Cell expansion mediated by glycosyl hydrolases and cell wall modifying enzymes is critical as it allows the enlargement in volume that determines the final organ size (Bashline et al., 2014, Gilbert, 2010). Cell wall loosening is caused by the specific disruption of chemical bonds between cellulose and other cell wall polymers, microtubule reorganization, and the incorporation of de novo synthesized polysaccharides required for cell wall extensibility. Previous studies have led to the identification of a wide range of cell wall modifying enzymes such as glycosyl hydrolases including expansin and xyloglucan endotransglucosylase/hydrolases (XTHs), carbohydrate esterases, and carbohydrate lyases (Minic and Jouanin, 2006, Minic et al., 2009).

Pectin is a major polysaccharide of primary cell walls and appears to be involved in wall plasticity and cellular adhesion (Mohnen, 2008). Compositional analysis indicates that the primary cell walls of typical grasses contain less than 5% pectin (Carpita, 1996, Oh et al., 2013, Vogel, 2008). Recent studies using chemical and immunochemical labeling methods demonstrated that rice has significant amounts of pectins in primary cell walls and middle lamella (Nguyen et al., 2016). Heavily methylated forms of homogalactruonan (HGA), which is a dominant form of pectin polymers, are synthesized in the Golgi apparatus and deposited in cell walls. An important aspect of enzyme-mediated cell wall weakening is the demethylesterification of HGA by the action of pectin methylesterases (PMEs). As a result of this activity, the local demethylesterified HGA residues can form Ca²⁺ cross linkages, which decrease wall extensibility and wall elasticity in plant cells (Jolie et al., 2010, Pelloux et al., 2007, Sénéchal et al., 2014). Thus, fine tuning of PME activities is crucial to maintain cell wall mechanics.

PME activities are known to be post-translationally regulated by the action of PME inhibitors (PMEIs). Sequence and transcriptome analyses in plants indicate that PMEI is a multigene family that shows differential expression patterns during development and in response to diverse stresses (An et al., 2008, Hongo et al., 2012, Jeong et al., 2014, Jithesh et al., 2012, Pinzon-Latorre and Deyholos, 2013, Wang et al., 2013). Studies have identified 76 PMEIs in Arabidopsis thaliana, 49 in rice, and 48 in Solanum lycopersicum. Although experimental evidence for the action of PMEIs on PMEs remains scarce, it is generally believed that specific PME/PMEI pairs modulate the degree of methylesterification in a microdomain of cell walls. Sénéchal et al. reported the possible interaction of AtPME17 with AtPMEI4 based on co-expression analysis and characterization of a pmei4 mutant (Sénéchal et al., 2015a). Recently, detailed in vitro biochemical characterization of a co-expressed pair (PME/PMEI) demonstrated that the enzymatic activity of AtPME3 is directly regulated by AtPMEI7 in a pH-dependent manner (Sénéchal et al., 2015b). The physiological role of PMEIs and PMEs has been studied in Arabidopsis and other plant species by gene knockout and overexpression approaches (An et al., 2008, Bosch et al., 2005, Francis et al., 2006, Hongo et al., 2012, Siedlecka et al., 2008). Overexpression of a PMEI gene (AtPMEI5) in Arabidopsis caused abnormal developmental phenotypes including increased germination rate, twisted stems, and organ fusion (Müller et al., 2013a, Müller et al., 2013b). In addition, transgenic lines overexpressing AtPMEI3 exhibited hypermethylesterification of HGA in the meristem tissues and affected phyllotaxis (Peaucelle et al., 2008). A T-DNA knockout mutation in seed coat-specific AtPMEI6 resulted in delayed release of seed mucilage polysaccharide, demonstrating that PMEI6 promotes mucilage release during germination (Saez-Aguayo et al., 2013). Derbyshire et al. showed a consistent relationship between the degree of methylesterification and the degree of hypocotyl elongation (Derbyshire et al., 2007). They concluded that the reduced hypocotyl length in two gibberellic acid (GA) mutants was closely associated with the reduction in pectin methylesterification status. It has been demonstrated that a tomato PMEI (SolyPMEI) is specifically expressed during fruit ripening and its protein interacted with fruit-specific SolyPME1, indicating that different regulation of methylesterification degree by PMEI plays an crucial role in tomato fruit development (Reca et al., 2012). Molecular and biochemical characterization of a grapevine PMEI (VvPMEI1) also demonstrated that VvPMEI1 is tightly associated with grape berry development (Lionetti et al., 2015).

Few data are available regarding the role of PMEI proteins in growth and development in rice (Nguyen et al., 2016, Yang et al., 2013). The present study characterized the biochemical function and physiological role of OsPMEI28. OsPMEI28 transcripts were ubiquitously expressed and the protein was shown to possess PME inhibitory activity. Transformation experiments showed that OsPMEI28 overexpression resulted in an increased level of pectin methylesterification and consequently a shortened culm length. These results demonstrate the importance of fine-tuned methylesterification status in rice development.