Organization of Xylan Production in the Golgi During Secondary Cell Wall Biosynthesis

Secondary cell wall (SCW) production during xylem development requires massive upregulation of hemicellulose (e.g. glucuronoxylan) biosynthesis in the Golgi. While mutant studies have revealed much of the xylan biosynthetic machinery, the precise arrangement of these proteins and their products in the Golgi apparatus is largely unknown. We used a fluorescently-tagged xylan backbone biosynthetic protein (IRREGULAR XYLEM9; IRX9) as a marker of xylan production in the Golgi of developing protoxylem tracheary elements in Arabidopsis (Arabidopsis thaliana). Both live-cell confocal and transmission electron microscopy (TEM) revealed SCW deposition is accompanied by a significant proliferation of Golgi stacks. Furthermore, although Golgi stacks were randomly distributed, the organization of the cytoplasm ensured their close proximity to developing SCWs. Quantitative immuno-TEM revealed IRX9 is present in a specific sub-domain of the Golgi stack and was most abundant in the ring of the inner margins of medial cisternae where fenestrations are abundant. Conversely, the xylan product accumulated in swollen trans-cisternal margins and the trans-Golgi network (TGN). The irx9 mutant lacked this expansion for both the cisternal margins and the TGN, while Golgi stack proliferation was unaffected. Golgi in irx9 also displayed dramatic changes in their structure, with increases in cisternal fenestration and tubulation. Our data support a new model where xylan biosynthesis and packaging into secretory vesicles are localized in distinct structural and functional domains of the Golgi. Rather than polysaccharide biosynthesis occurring in the centre of the cisternae, IRX9 and the xylan product are arranged in successive concentric rings in Golgi cisternae.