Functional Analysis of Plant Mitogen-activated Protein Kinase Cascades in Stress and Hormonal Signaling

本项目的主要目的是阐明植物对环境胁迫和激素信号作出反应的分子机制。大量研究表明，所谓的丝裂原活化蛋白激酶（MAPK）级联可被非生物胁迫（如寒冷、干旱、盐、伤害和触摸）、病原体和病原体衍生的激发子以及植物激素激活。MAPK级联是进化上保守的蛋白质模块，在多种真核生物（包括酵母、蠕虫、苍蝇、青蛙、哺乳动物和植物）的分子信号通路中起作用。mapk是一种蛋白激酶，通过磷酸化转录因子等调节蛋白来激活它们。MAPKs依次被MAPKKs磷酸化和激活，MAPKKs被MAPKKs磷酸化和激活。拟南芥基因组计划揭示了编码MAPKs、MAPKKs和MAPKKKs的大基因家族（称为MAPK级联基因）。然而，对于植物MAPK级联的构成以及特定的MAPK级联基因在特定的植物信号转导途径中所起的具体作用，人们知之甚少。基于基因组信息的综合方法将用于生成一套免费可用的MAPK相关资源，包括工程MAPK级联基因和过表达这些基因的转基因植物。工程MAPK级联基因将在拟南芥原生质体（单个细胞剥离其细胞壁以促进DNA摄取）中短暂表达，以确定拟南芥MAPK级联基因参与重要植物信号通路的功能。由于MAPK级联在植物信号转导途径中的功能可能是保守的，因此利用拟南芥基因组资源的研究将在包括重要作物在内的其他植物物种中具有广泛的意义和应用。该项目将重点研究四个MAPK级联，包括涉及渗透应激保护、细菌肽识别、激素信号传导和氧化应激反应的级联。总体策略将包括鉴定和克隆编码MAPKs、MAPKKs、MAPKKKs和相关蛋白磷酸酶的整套拟南芥基因。在拟南芥和玉米原生质体中，免疫标记蛋白激酶的瞬时表达将用于匹配特定的MAPK级联反应与特定的非生物或生物应激或激素信号。工程MAPKKKs和MAPKKs，被设计为组成活性或作为抑制剂，将与DNA微阵列技术结合使用，以鉴定在响应特定信号通路时激活的拟南芥基因。将开始鉴定与mapk相关的关键调控基因相对应的拟南芥敲除突变体。转基因拟南芥、玉米和大豆将产生过度表达特定工程MAPKKK基因的植株。这些转基因植物将进行试验，以展示其农艺上有用的性状。实验设计以拟南芥和玉米原生质体技术为基础。原生质体系统的瞬时特性使植物基因的直接功能分析能够以前所未有的高通量和相对较低的成本进行。该方法充分利用拟南芥基因组基础设施，拟南芥基因组基础设施是NSF植物基因组研究计划的一部分，包括DNA测序、基因表达技术和敲除突变文库。实验方法在揭示基因的功能方面尤其强大，传统的遗传和生化方法由于冗余、致命性或低表达水平而难以解决这些问题。阐明和操纵植物中的MAPK级联将揭示重要的调控过程，并为作物改良提供与抗逆性、抗病性和产量提高相关的新工具。该项目整合了三个实验室，它们在应激和激素信号、病原体反应以及转基因作物的产生和分析方面提供互补的专业知识。该项目还将在多学科的学术环境中提供良好的培训机会。从这个项目中产生的信息和材料，包括完整的克隆和工程MAPK基因，将通过一个可访问网络的数据库免费提供给植物界。

The major goal of this project is to elucidate the molecular mechanisms by which plants respond to environmental stress and hormonal signals. Numerous studies have shown that so-called mitogen-activated protein kinase (MAPK) cascades are activated by abiotic stresses (e.g., cold, drought, salt, wounding and touch), pathogens and pathogen-derived elicitors and plant hormones. MAPK cascades are evolutionarily conserved protein modules that function in molecular signaling pathways in a variety of eukaryotes, including yeasts, worms, flies, frogs, mammals, and plants. MAPKs are protein kinases that activate regulatory proteins such as transcription factors by phosphorylating them. MAPKs are in turn phosphorylated and activated by MAPKKs, and MAPKKs are phosphorylated and activated by MAPKKKs. The Arabidopsis genome project has revealed large gene families encoding MAPKs, MAPKKs and MAPKKKs (referred to as MAPK cascade genes). However, little is known about the constitution of plant MAPK cascades and the specific roles that particular MAPK cascade genes play in particular plant signal transduction pathways. A comprehensive approach based on genomic information will be employed to generate a freely available set of MAPK-related resources including engineered MAPK cascade genes and transgenic plants overexpressing these genes. Engineered MAPK cascade genes will be transiently expressed in Arabidopsis protoplasts (individual cells stripped of their cell walls to facilitate DNA uptake) to determine the function of Arabidopsis MAPK cascade genes involved in essential plant signaling pathways. Since the functions of MAPK cascades in plant signal transduction pathways are likely conserved, studies using the Arabidopsis genome resources will have broad implications and applications in other plant species including important crop plants.The four MAPK cascades that will be the focus of this project include those involved in osmotic stress protection, bacterial peptide recognition, hormone signaling, and oxidative stress responses. The overall strategy will involve the identification and cloning of the complete set of Arabidopsis genes encoding the MAPKs, MAPKKs, MAPKKKs and relevant protein phosphatases. Transient expression of immunologically-tagged protein kinases will be used to match particular MAPK cascades with particular abiotic or biotic stress or hormone signals in Arabidopsis and maize protoplasts. Engineered MAPKKKs and MAPKKs, designed to be constitutively active or to act as inhibitors, will be used in conjunction with DNA microarray technology to identify Arabidopsis genes that are activated in response to particular signaling pathways. Identification of Arabidopsis knockout mutants corresponding to key MAPK-related regulatory genes will be initiated. Transgenic Arabidopsis, maize and soybean plants that over-express particular engineered MAPKKK genes will be generated. The transgenic plants will be tested for the exhibition of agronomically useful traits. The experimental design is based on Arabidopsis and maize protoplast technology. The transient nature of the protoplast systems allows direct functional analysis of plant genes at an unprecedented high throughput rate and at a relatively low cost. This approach takes full advantage of the Arabidopsis genome infrastructure that is being created as a part of the NSF Plant Genome Research Program, including DNA sequencing, gene expression technology, and knockout mutant libraries. The experimental approaches are especially powerful in unraveling the functions of genes that are difficult to tackle by traditional genetic and biochemical approaches due to redundancy, lethality or low levels of expression. The elucidation and manipulation of MAPK cascades in plants will reveal fundamentally important regulatory processes and provide new tools for crop improvement related to stress tolerance, disease resistance, and yield enhancement. This project integrates three laboratories that provide complementary expertise in stress and hormonal signaling, pathogen responses, and transgenic crop plant generation and analysis. The project will also provide excellent training opportunities in a multidisciplinary academic environment. Information and materials generated from this project, including the full set of cloned and engineered MAPK genes, will be made freely available to the plant community via a web-accessible database.