Electron Tomography of RabA4b‐ and PI‐4Kβ1‐Labeled Trans Golgi Network Compartments in Arabidopsis

Abstract

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin‐coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi‐associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30–35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi‐associated and free TGN compartments, but not trans Golgi cisternae, bind anti‐RabA4b and anti‐phosphatidylinositol‐4 kinase (PI‐4K) antibodies. RabA4b and PI‐4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ∼30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA‐a1‐green fluorescent protein (GFP) and SYP61‐cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI‐4Kβ1/2 regulates SV size.