Functional Characterization of Pectin Methylesterase Inhibitors in Rice (Oryza sativa)

Nguyen Thi Hong Phuong 경희대학교 대학원 2016 국내박사

Pectin, an enriched component in primary cell walls and middle lamellae, is an essential polysaccharide in all higher plants. Homogalacturonan (HG), a major form of pectin, is synthesized and methyl-esterified by enzymes localized in the Golgi apparatus and transported into the cell wall. Depending on cell type, the degree and pattern of pectin methyl-esterification are strictly regulated by cell wall-localized pectin methylesterases (PMEs) which are governed by multiple pectin methylesterase inhibitors (PMEIs) in vivo. Pectin methylesterases (PMEs, EC 3.1.1.11), belonging to carbohydrate esterase family 8, cleave the ester bond between a galacturonic acid and a methyl group, resulting change in pectin methyl-esterification status which clearly impacts diverse plant developmental processes and stress responses. PMEs play major roles in modification of pectin properties, such as the stiffening by forming Ca2+-pectate cross-link complexes or loosening by triggering CWDEs to break out cell wall components including pectins and its interactions with other cell wall components. By inhibition of PME activity, the action of PMEIs results in opposite consequences on pectin properties, leading to contrary phenotypes between the actions of each individual in PME-PMEI pairs. Optimal pectin methyl-esterification status in each cell type is determined by the balance between PME activity and post-translational PME inhibition by PME inhibitors (PMEIs). The detail information is explained in Chapter1 in which the PMEs and PMEIs were introduced from the structures to biological functions or biotechnological applications with an update knowledge, suggesting ideal for the further studies on models of PMEs and PMEIs and trying to answer opening questions from the previous researches, finally, to put an overview of PMEs and PMEIs functioning as a whole pathway in biology. Despite the importance of the PMEs and PMEIs in plant developments, environmental responses and biotechnology applications, there are only a few genes of these large families characterized in dicot plants and little is known about the physiological functions of pectins and its properties controlled by pectin methyl-esterification status in rice (Oryza sativa), an essential staple crop utilized by human being. Cultivation of this crop is now facing a big challenge when coping with diverse abiotic and/or biotic stresses and the increasing demands due to population growth. Thus, study of pectin methyl-esterification status in rice (Oryza sativa) can contribute precious genetic information to improve agronomic traits, sustaining food safety. Gene redundancy and the complicated regulation mechanisms in the OsPME family are the big challenges for the study of pectin methyl-esterification status in rice (Oryza sativa) by using the knockout approach due to the recovery of PME activity by other isoforms. Therefore, employing the overexpression of the inhibitor OsPMEIs, the endogenous OsPME activity is reduced by which phenotypes related to modification of pectin methyl-esterification status in rice can be observed and elucidated. Following this ideal, my research started with the elucidation of functional pectin in rice and pectin modification by de-methyl-esterification via PME activity. These works were done and reported in the Chapter2. The presence of pectin in rice cell walls was substantiated by uronic acid quantification and immunodetection of JIM7 monoclonal antibodies. PME activity assays were performed with cell wall proteins isolated from different rice tissues. In accordance with data from Arabidopsis, the highest activity was observed in germinating tissues, young culm, and spikelets. Transcriptional profiling of OsPMEs, containing 43 different isoforms, by real-time PCR and meta-analysis indicated that PMEs exhibit spatial- and stress-specific expression patterns during rice developments. Based on in silico analysis, subcellular compartments, isoelectric point, and cleavage sites of OsPMEs were identified. Taken together, these findings provided an important tool for further studies seeking to unravel the functional importance of pectin modification during plant development and abiotic and biotic responses in grass plants. In the Chapter3, OsPMEIs were functional characterized by biochemical approaches and further analyzed the potential inhibitory on local PME activity using subcellular localization and transcriptional analysis employing the quantitative real-time PCR (qRT-PCR) method. There were 49 PMEI members found in PMEI family in the rice genome. Analysis of their transcript levels by qRT-PCR and meta-expression analysis showed that they were regulated spatially and temporally, as well as in response to diverse stresses. Quantification of cell wall bound methyl-esters indicated that the degree of pectin methyl-esterification was developmentally regulated; in particular, higher PMEI activities were detected in cell wall proteins prepared from young leaves. Furthermore, an activity assay demonstrated that two recombinant OsPMEI proteins (OsPMEI8 and 12) were able to inhibit the enzymatic activity of a commercial PME protein. Subcellular localization showed that OsPMEI8 was targeted to the middle lamella and OsPMEI12 was localized in the plasma membrane, suggesting the involvement of OsPMEI8 in cell adhesion and OsPMEI12 in inhibitory activity of PMEs working on the walls closely located at the plasma membrane such as secondary cell walls. In sum, these results were the first molecular and biochemical evidences of functional characterization of PMEIs in rice growth and development. Rice contains 49 PMEIs and none of them are functionally characterized. Genomic sequence analysis led to the identification of rice PMEI28 (OsPMEI28). In the chapter4, the phenotype of overexpression of OsPMEI28 was introduced as the first evidence of OsPMEIs function in plant morphology, particularly, the phenotype related to modification of pectin methyl-esterification status in rice (Oryza sativa). Overexpression of OsPMEI28 in rice resulted in an increased level of cell-wall-bound methyl-ester 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. Recombinant OsPMEI28 exhibited inhibitory activity against commercial PME protein with the highest activities detected at pH 8.5, suggesting OsPMEI28 was a typical PMEI working on inhibition of endogenous OsPME activity in vivo. The dwarf phenotype was a result of inhibited activity of an OsPME functioning in cell wall loosening in rice culm tissues, resulting cell extension. In summary, OsPMEI28 functioned as a critical structural modulator by regulating the degree of pectin methyl-esterification by which physiochemical properties of the cell wall components were affected, leading to abnormal cell extensibility in rice culm tissues. In conclusion, in rice the modification of pectin methyl-esterification also affects the plant morphology as observed on the overexpression of OsPMEI28 resulted in dwarf phenotype. However, the cognate PME or PMEs which specifically inhibited by OsPMEI28 has/have not been elucidated in my study. It seems like the PME(s) maybe interact with BR signaling to control plant height as in AtPMEI5 overexpression (Wolf et al. 2012) or it (they) can act as the cell loosening factors by their own ways, de-methyl-esterification of pectin. The de-esterified pectin will be the substrate of cell-wall-degrading enzymes (CWDEs), resulting in loosening the cell wall, thus, facilitating cell extension. The number of candidate PMEs will be narrowed down by in silico analysis of tissue specific expression of both OsPMEI28 and candidate PMEs, and, finally, experimentally performed co-expression analysis using reporter genes in transplant tissues. These are the further studies not only for OsPMEI28 and its PME partner but also for other OsPMEs and OsPMEIs in the big families which showed dynamic expressions during plant development and in response to diverse stresses, promising multi-functions of these families in plant biology, specially, in rice researches. The contents of this thesis are being prepared / have been published in the following papers: Chapter 1: Hong Phuong Nguyen, and Chanhui Lee*, “A review of Pectin Methylesterase (PME) and Pectin Methylesterase Inhibitor (PMEI): Roles of PMEs and PMEIs in plant physiology and biotechnological applications” (in preparation) Chapter 2: Ho Young Jeong§, Hong Phuong Nguyen§, and Chanhui Lee*, “Genome-wide identification and expression analysis of rice pectin methylesterases: Implication of functional roles of pectin modification in rice physiology”, Journal of plant physiology. 2015, 183:23-29 (§: equal contribuition) Chapter 3: Hong Phuong Nguyen, Ho Young Jeong, Hun Kim, Young Chang Kim, and Chanhui Lee*, “Molecular and biochemical characterization of rice pectin methylesterase inhibitors (OsPMEIs)”, Plant Physiology and Biochemistry. 2016, 101: 105-112 Chapter 4: Hong Phuong Nguyen, Ho Young Jeong and Chanhui Lee*, “Rice Pectin Methylesterase Inhibitor28 (OsPMEI28) encodes a functional PMEI and its overexpression results in a dwarf phenotype through reduced pectin methylesterfication levels” (submitted)

Light-dependent responses in rice and Xanthomonas oryzae pathovar oryzae

Hong-Phuong Thi Nguyen 경희대학교 2013 국내석사

Light response of living organism is the interesting major for scientists, especially for botanists and pathologists. Despite of the useful roles in photosynthesis, light could give the harmful effect on life because of reactive properties. To check the harmful respects of light and the responses of life to the stress, the UV-treated rice leaves were used to check rice responses as well as their metabolic and regulation network. By using transcriptomic analysis, the phenylalanie biosynthetic genes were found immediately up-regulated by UV treatment. Phenylpropanoid pathway genes and flavonoid biosynthetic genes were also found to be up-regulated. These findings suggest that early aromatic amino acid biosynthetic pathway and flavonoid biosynthetic pathway are coordinately activated for flavonoid phytoalexin biosynthesis. The putative signaling components predicted based on functional gene network analysis using the RiceNet are suggested to participate in the regulation of phytoalexin biosynthesis induced by UV-C stress. Besides the harmful effects, the light could give some information for living organism. The photo-morphogenesis was well known for plants, while the light-responses of phyto-pathogens were not well understood. For those reasons, the Xanthomonas oryzae pv. oryzae were used for checking the light-response base on the study of bacteriophytochrome (XoBphP). Transcriptomic analysis on wild-type under monochromic light exposures and phenotypic studies of XoBphP mutant showed XoBphP is a photo-sensor, which may involve in photo-taxis and be important at the first infection stage.