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Plant Cell Wall Dynamics During Cell Cycle

Final Report Summary - CEWALDYN (Plant Cell Wall Dynamics During Cell Cycle)

The plant cell wall is a special rigid layer covering plant cells. This highly complex structure which consists of various biopolymers like polysaccharides sometimes determines but also limits the cellular activities. However, little is known how cell walls are arranged in order to perform desired functionalities. Cell division in plants supposes a high level of the regulation of the cell wall build-up and turn over. In the project CeWalDyn we took the challenge to address the questions: How plant cell walls are dynamically adjusted according the stage of the cell cycle? What are the molecular players of this process?

The project has been divided into three work packages. In the first work package we used the state-of-the art biochemical method to analyse the cell wall composition at different stages of the cell cycle. Our key model was synchronous Arabidopsis cell culture which provided us with relatively homogenous samples of cells at different stages of the cell cycle. We employed a unique technology called comprehensive microarray polymer profiling (CoMPP). This method enables a high-throughput analysis of cell wall composition in large number of samples. We identified several potential novel modes of cell wall regulation during cell division. For instance we observed that the highly decorated pectin type rhamnogalacturonan I is upregulated at the stage when the cell plate is formed (cytokinesis). On the other hand cell wall arabinogalactan proteins (AGPs) are upregulated at the stage when cell cease their division (stationary phase). This observation has to be further confirmed by additional biochemical methods like FTIR, monosaccharide compositional analyses and by in situ visualization.
In the work package 2 we studied the distribution of different cell wall components in plant meristems (e.g. Arabidopsis root tips or Brachypodium embryos). Conventional methods to analyse the cell wall components in situ are precarious. For instance the anti-glycan antibodies show limited penetration capacity and are often prone to masking (the blockage of the epitopes by other cell wall components). As a part of the work package 2 we developed novel types of glycan probes. The binding of the probes to their target is governed by a multiple polar interaction of the amine groups on chitosan and carboxy groups on the de-esterified homogalacturonan. These probing tools have some superior characteristics over the antibodies for instance better penetration and specificity. We demonstrated the proficiency of the probes by studying the process of maturation and sloughing of Arabidopsis root tips. We could show the elevated level of pectin de-esterification in root cap cells destined to be sloughed off. This cell wall modification is important for the cell separation process, because when inhibited by chemical inhibitor of pectin methyl-esterases (PMEs) epigallocatechin gallate (EGCG), cells do not detach and accumulate as multiple layers around the root tip. The probes are an efficient tool for animal and fungal glycobiology as well. For instance we for the first time demonstrated the presence of chitosan in the joint area of Drosophila legs. The chitosan present in the joints may serve for instance as a lubricant. These results have been published in Development in 2014.
In the work package 3 we aimed to characterise several cell wall related genes. By promoter-GUS constructs we confirmed the cell cycle expression of some pectin related genes e.g. PMEs and polygalacturonase inhibitory protein (PGIP1). We also initiated the phenotyping of the respective mutants.
However, the genetic redundancy is a common problem in cell wall-related genes and the generation of the multiple mutants would be necessary to overcome this obstacle. One of the possibilities would be to use specific chemical inhibitors. The compound EGCG proved again to be useful in elucidating the function of PMEs in cell division; external application of EGCG caused phenotypes in the cell plate formation suggesting the cell plate-localized activity of PMEs.
The main output and conclusions of the project
• The CeWalDyn project provided the cell wall research community by novel types of glycan probes enabling in vivo and real time imaging of the two structural polysaccharides.
• We confirmed the hypothesis that some of the cell wall components are highly dynamic during the cell cycle. Pectins (homogalacturonan as well as rhamnogalacturoan I) are the most dynamic cell wall components in the plant meristems and may be involved in the construction of the cell plate. However, more analyses, especially the genetic and molecular would be necessary to elucidate the functional relevance of these observations.
Impact of the results
The results of the CeWalDyn project provided an original insight in the biology of plant cell wall. Cell walls are the major part of the utilizable biomass. Large effort is currently made to overcome the bottlenecks of the production of biofuels and other emerging biomaterials like bioplastics. The basic knowledge about the biosynthesis of cell walls will provide the new possible means to manipulate the cell walls to acquire the desired characteristics, for instance more efficient saccharification performance for the bioethanol production. Part of the projects has been already published in a highly recognized peer-reviewed journal with impact factor over 6. Novel oligosaccharide probes have been introduced on the recognized international conference CELL WALL in Nantes, France (over 300 attendees).We already received numeral requests for the probes and we applied for the pre-seed grant from private funds to fully commercialize the probes. Several other manuscripts are in the preparation.