Characterisation of the Arabidopsis xylan synthase complex

The hemicellulose xylan is present in both primary and secondary cell walls and is the major non-cellulosic polysaccharide in industrially important biomass such as wood and grasses. Despite the central role of xylans in development, growth, cell wall strength and biomass resilience, we still know very little about the organisation and distribution of xylan biosynthetic proteins in the Golgi apparatus and how these factors influence the biosynthesis of xylan and the cell wall. To fill this gap, we have cloned Arabidopsis thaliana IRREGULAR XYLEM 9 (AtIRX9), AtIRX10, and AtIRX14, which are involved in the synthesis of the xylan backbone. Transient expression of fluorescent protein fusions in Nicotiana benthamiana showed that only simultaneous expression of AtIRX9, AtIRX10, and AtIRX14 results in robust and efficient Golgi localisation, and co-immunoprecipitation experiments clearly showed interactions between the three proteins, indicating the formation of a heterotrimeric protein complex. We hypothesise that the function of xylan biosynthetic enzymes is regulated by protein-protein interactions and different intra-Golgi localisations. We also hypothesise that AtIRX9, AtIRX10 and AtIRX14 are part of a larger multiprotein xylan synthase complex in the Golgi consisting of proteins with distinct functions in xylan biosynthesis. The aim of this project is (1) to identify the molecular and mechanistic determinants responsible for the localisation of AtIRX9/10/14 in the Golgi and the interactions between these proteins, (2) to investigate the effects of modulation of Golgi localisation and protein-protein interactions on xylan biosynthesis and cell wall composition in transgenic Arabidopsis Irx mutants and (3) to identify the interactome of the Arabidopsis xylan synthase complex in planta. The aim is to find means to modulate xylan biosynthesis and cell wall composition in the model organism A. thaliana.