Subcellular localization and characterisation of MAPKs, and cytoskeleton adaptation to stress in <em>Arabidopsis</em> roots

Abstract

In plants, development and morphogenesis but also adaptation to dynamic environmental changes strongly depend on regulated perception and transmission of stimuli causing a highly specific cellular response. Amongst others, mitogen-activated protein kinases (MAPKs) are main players, known to be responsible for proper signalling and regulation of subcellular changes in all eukaryotes. In this context, cytoskeletal and endomembrane rearrangements are commonly described as a result of MAPK activation. Since a limited number of MAPKs seem to be able to mediate the responses to a large variety of stimuli, specificity may not only reside in the molecular diversity of the MAPKs but also in the spatial and temporal organization of MAPK-modules.<br /> This work aims to characterize the subcellular organization of three MAPKs in Arabidopsis thaliana, namely MPK3, MPK4 and MPK6 with the main focus on MPK6. With the help of biochemical fractionation methods, this work shows for the first time that all three MAPKs not only reside in the cytosol but also associate with the microsomal fraction. Using specific antibodies against MAPKs and a set of antibodies against organellar marker proteins, it is shown that MPK3, MPK4 and MPK6 co-fractionate with the plasma membrane, trans-Golgi network (TGN) and with clathrin. Additionally, using brefeldin A as a well established inhibitor of membrane trafficking, this work provides further evidence that all three MAPKs indeed are associated with the early endocytotic/secretory pathway. The specific localization is further confirmed by the use of GFP-, and immunofluorescence co-localization techniques especially for MPK6 and it is shown that not only cytosolic but also microsomal MPK6 gets activated by flagellin, which is inducing pathogen related responses.<br /> Double-immunolabelling experiments also show that MPK6 is associated with cortical and subcortical microtubules (MTs) and that MTs and partially also actin co-localize with MPK6 on cytokinetic structures such as the preprophase band (PPB) and the phragmoplast. Moreover, the behavior of the cytoskeleton as a consequence of specific inhibition of MAPK signalling and endosomal trafficking is investigated.<br /> In order to support the previous results, a mpk6 knockout mutant line is phenotypically characterized and it´s cytoskeletal organization is compared to the wild type plants. It is shown, that the mpk6-2 knock-out mutant is disturbed in root elongation and early post-embryogenic root development. On the cellular level, mpk6-2 mutant plants show several defects in the alignment of cell-files and cell-division planes in the root tip.<br /> Finally, this work characterizes the cell type specific changes of the actin- and microtubule- cytoskeleton in course of heat shock, a type of physical stress, which is also known to activate MPK6. Live cell imaging using Arabidopsis lines transformed with GFP-fusion reporter constructs, revealed that epidermal root cells can cope with short-term heat stress showing disruption and subsequent recovery of microtubules and actin filaments in a time-dependent manner. Further analysis revealed that the strength of disruption of microtubules, but not of actin filaments, is dependent on cell type and developmental stage. On the other hand, recovery of actin filaments, but not microtubules, from heat shock stress is dependent on the same parameters.