Polarity control and cross-talk between MAPK signalling, cascades, vesicular trafficking and the actin cytoskeleton in plant root hairs

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Mitogen-activated protein kinases (MAPKs) are evolutionary conserved signal transducers involved in the regulation of plant development. In root hairs, crosstalks between SIMK (stress-induced MAPK), the actin cytoskeleton, and vesicular trafficking are essential for tipgrowth (Samaj et al. 2002, 2004a,b). Tip-growth is known to be controlled also by other signalling molecules such as Rop GTPases (Rop2/4), reactive oxygen species (ROS) and phospholipase D (PLD)/phosphatidic acid (PA) (Jones et al. 2002, Foreman et al. 2003, Ohashi et al. 2003) as well as by actin and actin-binding protein profilin (Baluska et al. 2000, Ramachandran et al. 2000, Ringli et al. 2002). In the context of this proposal it is important, that both ROS and PA activate MAPKs in plants and cause reorganization of actin cytoskeleton and vesicular trafficking on the cellular level. Moreover, PA and ROS can be integrated into MARK cascade involving MPK3 and MPK6 via OXI1/AGC2-1 kinase, which is a novel protein kinase that has recently been shown to be involved in Arabidopsis root hair tip growth (Anthony et al. 2004, Rentel et al. 2004). We have found that small amounts of MPK4 and MPK6, but not MPK3, are constitutivelly associated with endosomes. However, MPK3 is activated by ROS, salt and heat, and relocated to an endosomal compartment in its activated state. Upon stress activation, MPK6 relocates to broader spectrum of endomembranes including endoplasmic reticulum (ER) and endosomes. Additionally, SIMK binds to actin and MPK6 associates with actin filaments. This could be related to the actin role in the motility and other functions of ER and endosomes, representing two distinct endomembrane compartments. On the other hand, MPK4 is activated by heat, but not by ROS, and this active form is also relocated to endosomes. Here we aim to study cross-talks among ROS-, PA- and OXIl-mediated regulation of MAPKs, vesicular trafficking and the cytoskeleton in root hairs of Arabidopsis. We will isolate new Ioss-of-function (knockout) mutants for two MAPKs (MPK3 and MPK6) and characterise (using genetic, cell biological, molecular biological and biochemical approaches) those of them which are showing root hair phenotypes. We will also further characterise known mpk4 mutant showing ectopic and branched root hairs as well as other mutants defective in potential upstream signalling components such asyoda, apn2xapn3, oxil and rhd2 as well as rop2 constitutive-active and dominat-negative functional mutants, which are all showing root hair phenotypes (longer/shorter or branched root hairs). Finally, we will study two profilins PRN1 and PRN2 as potential downstream targets of MPK3/4/6 as well as MPK6 interaction with actin and MPK3/4 interactions with endosomal Rab GTPases. We expect that the data gathered in the course of this project will have a strong impact on our understanding of polarity control in plant and other eukaryotic cells.