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Mannan-related enzymes of wheat endosperm

Periodic Reporting for period 1 - MANAN (Mannan-related enzymes of wheat endosperm)

Reporting period: 2016-11-01 to 2018-10-31

The MANAN project focuses on plant cell walls. These matrices, mainly made of polysaccharides (complex sugars), encompass plant cells. Cell wall polysaccharides are fundamental to plant biology and have a strong impact on the use of plant products in industrial processes. Studying cell wall polysaccharides helps to adapt crops to climate change, to improve the production of biofuels, and to increase the productivity of food and feed what is necessary to support the incoming growth of the global population. A better understanding of the biosynthesis and the functions of cell wall polysaccharides present in wheat grain can contribute to increase in a sustainable manner the yield of this staple food and have positive outcomes for its use and its processing. The MANAN project was carried out in INRA Nantes, France, and aimed to determine how one class of polysaccharides, named mannan, is biosynthesized in wheat grain and how this class impacts wheat grain development and the quality of flours.
To understand how mannan are synthesized in wheat endosperm, the grain tissue that corresponds to white flour, we first had to determine the structure of the polysaccharide in this tissue. We used biochemical approaches and analyzed wheat grain harvested at distinct developmental stages. We purified mannan from wheat flour and through chemical reactions and measurements by colorimetry, sophisticated chromatography and spectroscopy techniques, we have determined that mannan in wheat endosperm are exclusively made of mannose. This finding was unexpected as previous studies had hypothesized more complex structures for mannan in wheat grain.

To discover how mannan are synthesized and to gain knowledge on their biological function, and based on endosperm-targeted gene expression survey (transcriptomics and proteomics data), we selected four genes that are believed to be involved in the mannan synthesis. We produced RNAi transformants of wheat or obtained mutagenized wheat lines for these genes. Unfortunately, these procedures that started before the beginning of MANAN had not been a success. We did not obtain enough transformed lines in our third generation of transformants and our genes of interest were not or barely downregulated. As an alternative, we expressed our genes in other species. Their expression in tobacco showed that our genes of interest produced proteins located in the Golgi apparatus. Using a fungus, we have been able to show a mannan synthase activity for one of our gene candidate. We have also expressed our genes of interest in Arabidopsis, a plant model, and are currently working on the third generation to confirm the activity observed in the fungus. We have carried out histochemical analysis of wheat grain that shows mannan start to be synthesized at an early stage of endosperm development. Studying the Arabidopsis transformants should tell us more about how mannan contribute to plant life.

In terms of dissemination, our results have been presented at one national and three international conferences. We have published one methodology article, the writing of a second manuscript is in progress and two additional manuscripts should follow it. Moreover, a summary of the project has been presented to a broad audience using several social media.

This work has been made possible through collaborations with our partners from the Rothamsted Research (UK), The University of Leeds (UK), the University of Dusseldorf (Ger) and the Joint BioEnergy Institute (US).
We have successfully characterized the biochemical structure of mannan in the tissue used for the production of wheat flour. This structural characterization provides the base to study the mannan biosynthesis as it allows to determine which type of genes/enzymes can or cannot be expected to be involved in the mannan synthesis. In this regard, we have studied four genes and have successfully demonstrated the activity of one of these genes in the mannan synthesis. We studied the physiological function of mannan in wheat grain and our results suggest mannan may impact wheat grain development.

Overall, our project contributes to fundamental progress needed to adapt crops to climate change, to improve the production of biofuels, and to increase the productivity of food and feed.
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