The Role of Copper and Transcriptional Regulatory Networks Governing Copper Homeostasis in Pollen Fertility in A. thaliana.

由于目前的人口增长，全球对高产作物的需求正在增加，这一趋势迫使将边际土地用于农业目的。例如，土壤中铜的可用性有限导致作物缺铜，这种情况导致植物不育，从而导致低产量；然而，在严重缺铜的情况下，可以预期作物全部歉收。尽管我们对植物如何从土壤中获取铜，以及铜在植物体内是如何被调动的了解很多，但对铜如何影响植物的肥力却知之甚少。该项目将采用跨学科的方法，提供铜在模式植物拟南芥花粉发育和生育力中的作用的基本见解。更具体地说，该项目还将有助于解释SPL7和新发现的CITF1两种蛋白质如何协调铜的摄取和传递到拟南芥的生殖器官，以及它们如何协调花粉的发育和繁殖。这一新知识和认识将有助于在目前正在耕种的土壤或边缘土壤中培育出产量更高的作物。将向本科生和研究生提供研究机会，并特别努力吸引妇女和代表性不足的少数民族。将开发一个多媒体网络模块“矿物质营养：从植物到人类”，目的是提高高中学生和K-12教师对微量营养素在植物生长发育中的作用的认识，因为它关系到食品安全和人类福祉。虽然铜被认为是植物生育所必需的微量营养素，但人们对铜向生殖器官的输送，以及协调铜稳态和植物繁殖的转录调控网络知之甚少。在拟南芥中，SPL7是已知唯一在铜稳态中起作用的转录因子。这个项目是基于PI实验室最近发现的CITF1。CITF1是TFs bHLH家族的成员，它使拟南芥能够在缺铜中存活。初步证据表明，CITF1与spl7通路相互作用。破坏这种相互作用会导致幼苗死亡和花粉不育。通过对不同铜水平下生长的突变系的分析，揭示铜在花药和花粉中的作用位点，以及SPL7- CITF1在花粉发育中的作用。同步加速器x射线荧光显微镜将用于成像（2D & 3D）铜在花粉发育过程中的分布。空气扫描电子显微镜和组织学分析将用于表征铜水平对对照和突变系的解剖和形态的影响。靶向染色质免疫沉淀和功能遗传学分析将确定CITF1的直接上游调节因子。结合计算共表达网络分析的全球表达谱将揭示SPL7和CITF1可能的下游铜响应靶点、调控成分的层次结构及其与铜摄取、器官分配和传递的关系。长期目标是了解铜稳态的调控和信号网络，并将这些知识应用于生产适应低铜可用性土壤的品种。

The global demand for high-yielding crops is increasing due to current population growth, a trend that is forcing the utilization of marginal lands for agricultural purposes. For instance, limited copper availability in the soil causes copper deficiency in crop plants, a condition that leads to plant infertility and consequently low yields; however, total crop failure can be expected under acute copper deficiencies. Although a great deal is known about how plants acquire copper from the soil, and how it is mobilized within the plant, not much is understood about how copper affects plant fertility. This project will use interdisciplinary approaches to provide fundamental insights on the role copper plays in pollen development and fertility in the model plant Arabidopsis thaliana. More specifically, this project will also help to explain how two proteins, SPL7 and the newly discovered CITF1, coordinate copper uptake and delivery to the reproductive organs of A. thaliana, and how they orchestrate pollen development and fertility. This new knowledge and understanding will facilitate the development of crops with better grain yields in soils currently under cultivation or marginal soils. Research opportunities will be offered to undergraduate and graduate students with a special effort to attract women and underrepresented minorities. A multi-media web module "Mineral nutrition: from plants to humans" will be developed with the intent to increase the awareness of high school students and K-12 teachers about the role of micronutrients in plant growth and development as it pertains to food safety and human well-being. Although copper is recognized as an essential micronutrient for plant fertility, very little is known about copper delivery to reproductive organs, or about transcriptional regulatory networks that coordinate copper homeostasis and plant reproduction. In Arabidopsis thaliana, SPL7 is the only transcription factor known to function in copper homeostasis. This project is based on the recent discovery of CITF1 in the PI's lab. CITF1 is a member of the bHLH family of TFs that enables A. thaliana to survive copper deficiency. Preliminary evidence indicates that CITF1 interacts with the SPL7-pathway. Disrupting this interaction causes seedling lethality and pollen sterility. The sites of copper action in anthers and pollen, and the role played by SPL7- CITF1 in pollen development will be characterized through the analysis of mutant lines grown under different copper levels. Synchrotron X-ray fluorescent microscopy will be used to image (2D & 3D) copper distribution during pollen development. In-air scanning electron microscopy and histological analyses will be used to characterize copper-level effects on the anatomy and morphology of control and mutant lines. Targeted chromatin-immunoprecipitation and functional genetic assays will identify direct upstream regulators of CITF1. Global expression profiling coupled with computational co-expression network analyses will reveal likely downstream copper-responsive targets of SPL7 and CITF1, the hierarchy of regulatory components and their relationship to copper uptake, and organ partitioning and delivery. The long-term goals are to understand the regulatory and signaling networks underlying copper homeostasis, and to apply this knowledge to produce cultivars adapted to soils with low copper availability.