Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein-flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1-positive vesicle formation. Vesicle size, as outlined by the gold particles, was ;100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chainmOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFPFlot1-positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

). † These two authors contributed equally to this work. SUMMARYThe proper spatial and temporal expression and localization of mitogen-activated protein kinases (MAPKs) is essential for developmental and cellular signalling in all eukaryotes. Here, we analysed expression, subcellular localization and function of MPK6 in roots of Arabidopsis thaliana using wild-type plants and three mpk6 knock-out mutant lines. The MPK6 promoter showed two expression maxima in the most apical part of the root meristem and in the root transition zone. This expression pattern was highly consistent with 'no root' and 'short root' phenotypes, as well as with ectopic cell divisions and aberrant cell division planes, resulting in disordered cell files in the roots of these mpk6 knock-out mutants. In dividing root cells, MPK6 was localized on the subcellular level to distinct fine spots in the pre-prophase band and phragmoplast, representing the two most important cytoskeletal structures controlling the cell division plane. By combining subcellular fractionation and microscopic in situ and in vivo co-localization methods, MPK6 was localized to the plasma membrane (PM) and the trans-Golgi network (TGN). In summary, these data suggest that MPK6 localizing to mitotic microtubules, secretory TGN vesicles and the PM is involved in cell division plane control and root development in Arabidopsis.

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Immunogold electron microscopy (EM) study of Arabidopsis root apices analyzed using specific IAA antibody and high-pressure freeze fixation technique allowed, for the first time, vizualization of subcellular localization of IAA in cells assembled intactly within plant tissues. Our quantitative analysis reveals that there is considerable portion of IAA gold particles that clusters within vesicles and membraneous compartments in all root apex cells. There are clear tissue-specific and developmental differences of clustered IAA in root apices. These findings have significant consequences for our understanding of this small molecule which is controlling plant growth, development and behavior.