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).
