@article{3018, abstract = {The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of auxin transport.}, author = {Kleine-Vehn, Jürgen and Dhonukshe, Pankaj and Swarup, Ranjan and Bennett, Malcolm and Jirí Friml}, journal = {Plant Cell}, number = {11}, pages = {3171 -- 3181}, publisher = {American Society of Plant Biologists}, title = {{Subcellular trafficking of the Arabidopsis auxin influx carrier AUX1 uses a novel pathway distinct from PIN1}}, doi = {10.1105/tpc.106.042770}, volume = {18}, year = {2006}, } @article{3020, abstract = {High throughput microarray transcription analyses provide us with the expression profiles for large amounts of plant genes. However, their tissue and cellular resolution is limited. Thus, for detailed functional analysis, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here, we present an in situ mRNA hybridization method that is routinely used for the analysis of plant gene expression patterns. The protocol is optimized for whole mount mRNA localizations in Arabidopsis seedling tissues including embryos, roots, hypocotyls and young primary leaves. It can also be used for comparable tissues in other species. Part of the protocol can also be automated and performed by a liquid handling robot. Here we present a detailed protocol, recommended controls and troubleshooting, along with examples of several applications. The total time to carry out the entire procedure is ∼7 d, depending on the tissue used.}, author = {Hejátko, Jan and Blilou, Ikram and Brewer, Philip B and Jirí Friml and Scheres, Ben and Eva Benková}, journal = {Nature Protocols}, number = {4}, pages = {1939 -- 1946}, publisher = {Nature Publishing Group}, title = {{In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples}}, doi = {10.1038/nprot.2006.333}, volume = {1}, year = {2006}, } @article{3015, abstract = {As the field of plant molecular biology is swiftly advancing, a need has been created for methods that allow rapid and reliable in situ localization of proteins in plant cells. Here we describe a whole-mount 'immunolocalization' technique for various plant tissues, including roots, hypocotyls, cotyledons, young primary leaves and embryos of Arabidopsis thaliana and other species. The detailed protocol, recommended controls and troubleshooting are presented, along with examples of applications. The protocol consists of five main procedures: tissue fixation, tissue permeation, blocking, primary and secondary antibody incubation. Notably, the first procedure (tissue fixation) includes several steps (4-12) that are absolutely necessary for protein localization in hypocotyls, cotyledons and young primary leaves but should be omitted for other tissues. The protocol is usually done in 3 days, but could also be completed in 2 days.}, author = {Sauer, Michael and Paciorek, Tomasz and Eva Benková and Jirí Friml}, journal = {Nature Protocols}, number = {1}, pages = {98 -- 103}, publisher = {Nature Publishing Group}, title = {{Immunocytochemical techniques for whole mount in situ protein localization in plants}}, doi = {10.1038/nprot.2006.15}, volume = {1}, year = {2006}, } @article{3013, abstract = {There is a growing demand for methods that allow rapid and reliable in situ localization of proteins in plant cells. The immunocytochemistry protocol presented here can be used routinely to observe protein localization patterns in tissue sections of various plant species. This protocol is especially suitable for plant species with more-complex tissue architecture (such as maize, Zea mays), which makes it difficult to use an easier whole-mount procedure for protein localization. To facilitate the antibody-antigen reaction, it is necessary to include a wax-embedding and tissue-sectioning step. The protocol consists of the following procedures: chemical fixation of tissue, dehydration, wax embedding, sectioning, dewaxing, rehydration, blocking and antibody incubation. The detailed protocol, recommended controls and troubleshooting are presented here, along with examples of applications.}, author = {Paciorek, Tomasz and Sauer, Michael and Balla, Jozef and Wiśniewska, Justyna and Jirí Friml}, journal = {Nature Protocols}, number = {1}, pages = {104 -- 107}, publisher = {Nature Publishing Group}, title = {{Immunocytochemical technique for protein localization in sections of plant tissues}}, doi = {10.1038/nprot.2006.16}, volume = {1}, year = {2006}, } @article{3014, abstract = {Plant biology is currently confronted with an overflow of expression profile data provided by high-throughput microarray transcription analyses. However, the tissue and cellular resolution of these techniques is limited. Thus, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here we present an in situ mRNA hybridization method that is routinely used in the analysis of gene expression patterns. The protocol is optimized for mRNA localizations in sectioned tissue of Arabidopsis seedlings including embryos, roots, hypocotyls, young primary leaves and flowers. The detailed protocol, recommended controls and troubleshooting are presented along with examples of application. The total time for the process is 10 days.}, author = {Brewer, Philip B and Heisler, Marcus G and Hejátko, Jan and Jirí Friml and Eva Benková}, journal = {Nature Protocols}, number = {3}, pages = {1462 -- 1467}, publisher = {Nature Publishing Group}, title = {{In situ hybridization for mRNA detection in Arabidopsis tissue sections}}, doi = {10.1038/nprot.2006.226}, volume = {1}, year = {2006}, }