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

丝裂原活化蛋白激酶（MAPKs）是参与植物发育调控的进化保守信号转导。在根毛中，SIMK（应力诱导的MAPK）、肌动蛋白细胞骨架和囊泡运输之间的串扰对根毛生长至关重要（Samaj et al. 2002,2004a,b）。已知顶端生长还受其他信号分子的控制，如Rop GTPases （Rop2/4）、活性氧（ROS）和磷脂酶D (PLD)/磷脂酸（PA） （Jones et al. 2002, Foreman et al. 2003, Ohashi et al. 2003）以及肌动蛋白和肌动蛋白结合蛋白谱（Baluska et al. 2000, Ramachandran et al. 2000, Ringli et al. 2002）。在这一建议的背景下，重要的是，ROS和PA都激活植物中的MAPKs，并在细胞水平上引起肌动蛋白细胞骨架和囊泡运输的重组。此外，PA和ROS可以通过ox1 /AGC2-1激酶整合到涉及MPK3和MPK6的MARK级联中，ox1 /AGC2-1激酶是最近被证明参与拟南芥根毛尖生长的一种新型蛋白激酶（Anthony et al. 2004, Rentel et al. 2004）。我们发现少量的MPK4和MPK6，而不是MPK3，与核内体构成相关。然而，MPK3被活性氧、盐和热激活，并在激活状态下重新定位到内体腔室。应激激活后，MPK6迁移到更广泛的内膜，包括内质网（ER）和核内体。此外，SIMK与肌动蛋白结合，MPK6与肌动蛋白丝结合。这可能与肌动蛋白在内质网和核内体的运动和其他功能中的作用有关，内质网和核内体代表两个不同的内膜室。另一方面，MPK4被热激活，而不是被ROS激活，这种活性形式也被重新定位到核内体中。本研究旨在研究拟南芥根毛中ROS-、PA-和oxil介导的MAPKs调控、囊泡运输和细胞骨架之间的交叉对话。我们将分离两个mapk （MPK3和MPK6）的新功能缺失（敲除）突变体，并（使用遗传学，细胞生物学，分子生物学和生化方法）表征其中显示根毛表型的突变体。我们还将进一步表征已知的mpk4突变体，这些突变体显示出异位和分枝的根毛，以及其他在潜在的上游信号成分中有缺陷的突变体，如asyoda、apn2xapn3、oxil和rhd2，以及rop2构成活性和显性阴性的功能突变体，这些突变体都显示出根毛表型（长/短或分枝根毛）。最后，我们将研究作为MPK3/4/6以及MPK6与肌动蛋白相互作用和MPK3/4与内体Rab gtpase相互作用的潜在下游靶点的两种谱蛋白PRN1和PRN2。我们期望在这个项目过程中收集的数据将对我们对植物和其他真核细胞极性控制的理解产生强烈的影响。

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.