263 research outputs found

    Functional analysis of plant Mei2-like proteins : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

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    Molecular techniques were used to analyse the function of a novel class of RNA-bindmg proteins in plants, termed Mei2-like. The biochemical function of this class of proteins is unclear Although the conserved presence of three RNA recognition motifs (RRMs) in all members of the family suggests the importance of an RNA binding activity, the precise biochemical mechanism by which these proteins act is unknown. Genetic and molecular analyses of the founding member of the family. Schizosaccharomyces pombe Mei2p, provide of a conceptual framework for the studies of the plant Mei2-like proteins presented here. Therefore, the aims of this thesis were to 1) study the cellular localisation of Mei2p in plant cells, and 2) deduce the functions of plant Mei2-like genes by identifying the protein(s) that physically interact(s) with Mei2-like proteins. Transient expression of GFP-fused Mei2p in onion epidermal cells was performed to show that Mei2p localised into the nucleus in the presence of meiRNA, a non-coding mRNA. Thus plants seem to share the capacity with S. pombe for meiRNA-dependent nuclear localisation of Mei2p. Moreover, intracellular localisation of one of the plant Mei2-like proteins, TERMINAL EAR-Like 2 (TEL2), was studied in onion epidermal cells. The GFP-fused TEL2 localised into the nucleus without co-expression of any special RNA, suggesting that either some RNA species that assist nuclear localisation of TEL2 are already present in onion epidermal cells, or the mechanism of intracellular localisation of TEL2 is different from Mei2p. The yeast two-hybrid system was utilised to identify protein interactors with TEL2. Six proteins were identified, including the well-studied KORRIGAN (KOR) protein. Based on the proteins identified, speculation is offered on how these proteins interact with TEL2. Since TEL genes are expressed in the central zone (CZ) of meristems, and mitotic activity of cells in the CZ is low, TEL2 may be involved in controlling cell division in the CZ via interactions with these proteins

    A Formal Architectural Description Language based on Symbolic Transition Systems and Modal Logic

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    International audienceComponent Based Software Engineering has now emerged as a discipline for system development. After years of battle between component platforms, the need for means to abstract away from specific implementation details is now recognized. This paves the way for model driven approaches (such as MDE) but also for the more older Architectural Description Language (ADL) paradigm. In this paper we present KADL, an ADL based on the Korrigan formal language which supports the following features: integration of fully formal behaviours and data types, expressive component composition mechanisms through the use of modal logic, specification readability through graphical notations, and dedicated architectural analysis techniques. Key Words: Architectural Description Language, Component Based Software Engineering, Mixed Formal Specifications, Symbolic Transition Systems, Abstract Data Types, Modal Logic Glue, Graphical Notations, Verification

    Cellulose and callose synthesis and organization in focus, what's new?

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    Plant growth and development are supported by plastic but strong cell walls. These walls consist largely of polysaccharides that vary in content and structure. Most of the polysaccharides are produced in the Golgi apparatus and are then secreted to the apoplast and built into the growing walls. However, the two glucan polymers cellulose and callose are synthesized at the plasma membrane by cellulose or callose synthase complexes, respectively. Cellulose is the most common cell wall polymer in land plants and provides strength to the walls to support directed cell expansion. In contrast, callose is integral to specialized cell walls, such as the cell plate that separates dividing cells and growing pollen tube walls, and maintains important functions during abiotic and biotic stress responses. The last years have seen a dramatic increase in our understanding of how cellulose and callose are manufactured, and new factors that regulate the synthases have been identified. Much of this knowledge has been amassed via various microscopy-based techniques, including various confocal techniques and super-resolution imaging. Here, we summarize and synthesize recent findings in the fields of cellulose and callose synthesis in plant biology

    Analyzing the complex machinery of cell wall biosynthesis

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    The plant cell wall polymers make up most of the plant biomass and provide the raw material for many economically important products including food, feed, bio-materials, chemicals, textiles, and biofuel. This broad range of functions and applications make the biosynthesis of these polysaccharides a highly interesting target of scientific research. In this thesis a protein-protein interaction strategy was used to gain insight in the cell wall biosynthesis of Arabidopsis thaliana and to identify additional genes involved in this process. Using the membrane based yeast two hybrid system several distinct goals have been reached in this thesis, i) the characterization of the rosette structure by resolving the organization of the different cellulose synthase proteins in the complex, ii) the identification of unknown components of the cellulose synthezing machinery, iii) the confirmation of a xyloglucan synthesizing complex and the identification of several of its components. On the whole, this work has generated an effective tool in cell wall research and identified new players in the biosynthesis of both cellulose and xyloglucan. <br/

    Microwave Measurements Part I: Linear Measurements

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    An Overview of the most relevant issues concerning RF and microwave linear measurements is presented. Vector Network Analyzer foremost used instrumentation for this kind of measures is describe

    Deciphering the Molecular Functions of Sterols in Cellulose Biosynthesis

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    Sterols play vital roles in plant growth and development, as components of membranes and as precursors to steroid hormones. Analysis of Arabidopsis mutants indicates that sterol composition is crucial for cellulose biosynthesis. Sterols are widespread in the plasma membrane (PM), suggesting a possible link between sterols and the multimeric cellulose synthase complex. In one possible scenario, molecular interactions in sterol-rich PM microdomains or another form of sterol-dependent membrane scaffolding may be critical for maintaining the correct subcellular localization, structural integrity and/or activity of the cellulose synthase machinery. Another possible link may be through steryl glucosides, which could act as primers for the attachment of glucose monomers during the synthesis of Ī²āˆ’(1ā€‰ā†’ā€‰4) glucan chains that form the cellulose microfibrils. This mini-review examines genetic and biochemical data supporting the link between sterols and cellulose biosynthesis in cell wall formation and explores potential approaches to elucidate the mechanism of this association

    The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis

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    Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalogue of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites

    Metabolism of polysaccharides in dynamic middle lamellae during cotton fibre development

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    Main conclusion: Evidence is presented that cotton fibre adhesion and middle lamella formation are preceded by cutin dilution and accompanied by rhamnogalacturonan-I metabolism. Cotton fibres are single cell structures that early in development adhere to one another via the cotton fibre middle lamella (CFML) to form a tissue-like structure. The CFML is disassembled around the time of initial secondary wall deposition, leading to fibre detachment. Observations of CFML in the light microscope have suggested that the development of the middle lamella is accompanied by substantial cell-wall metabolism, but it has remained an open question as to which processes mediate adherence and which lead to detachment. The mechanism of adherence and detachment were investigated here using glyco-microarrays probed with monoclonal antibodies, transcript profiling, and observations of fibre auto-digestion. The results suggest that adherence is brought about by cutin dilution, while the presence of relevant enzyme activities and the dynamics of rhamnogalacturonan-I side-chain accumulation and disappearance suggest that both attachment and detachment are accompanied by rhamnogalacturonan-I metabolism

    GLUCAN SYNTHASE-LIKE 8: A Key Player in Early Seedling Development in Arabidopsis

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    Plantsā€™ cell walls have unique chemical composition and features which enable them to play essential roles during plant development as shaping the cells and providing intercellular communication between adjacent cells. Polysaccharides, including callose, and glycoproteins are known as the main constituents of the cell wall. Callose, a linear Ī²-1,3-glucan polymer, is accumulated at the cell plate during cytokinesis, in plasmodesmata, where it regulates cell-to-cell communication, in dormant phloem, where it seals sieve plates after mechanical injury and pathogen attack, and in male and female gametophytes. GLUCAN SYNTHASE-LIKE (GSL) genes in Arabidopsis comprise a family of 12 members. A new allele of GSL8, essp8, was identified as having seedling-lethal phenotype through a genetic screen for Arabidopsis mutants showing ectopic expression of seed storage proteins (essp). The gene responsible for the observed mutant phenotype was detected using a combination of bulked-segregant analysis, rough-mapping, and next-generation mapping. An EMS-induced point mutation was identified at an intron splice site of GSL8, predicted to introduce a premature STOP-codon. essp8 seedlings exhibit pleiotropic phenotypic defects, including disruption of root tissue patterning, dwarfism and seedling lethality. Histochemical detection of callose and cell-to-cell diffusion assays showed reduction of callose deposition at the cell plates and plasmodesmata, cytokinesis defects and significant increase in size exclusion limit of plasmodesmata in essp8 seedlings. Further investigation showed that the increase in size exclusion limit leads to an alteration in symplastic trafficking in primary roots of essp8 seedlings. Plasmodesmata defects in essp8 induce ectopic movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development. Attempts to identify the components of a hypothetical callose synthase complex revealed the interaction of GSL8 with two plasmodesmata-associated proteins, PLASMODESMATA-LOCALIZED PROTEIN 5 and Ī²-1,3-GLUCANASE, as well as SUCROSE SYNTHASE 1, suggesting that they all might be parts of a single complex. The proposed putative complex might regulate callose deposition at the plasmodesmata and thereby determines the size exclusion limit. In summary, my findings suggest that GSL8 is required for cell wall integrity, maintaining the basic ploidy level and regulation of symplastic movement during early seedling development in Arabidopsis
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