Functional Genomics of Chloroplast Biogenesis

叶绿体是一种重要的细胞器，其生物发生是一个复杂的、协调的过程，对植物的生长和发育至关重要。数百个核基因参与核编码蛋白质的输入、核和叶绿体编码蛋白质的细胞器内分选、叶绿体基因的表达、叶绿体酶的组装以及这些过程的调节。然而，只有一小部分这样的基因已经被详细描述。该项目的目标是开发，使用和传播一套互补的和强大的资源，用于这一复杂过程的遗传和生化解剖。 玉米为这个项目提供了一组理想的属性。除了其出色的遗传工具和Mu转座子高频率产生突变的能力外，该项目还利用了可以很容易地获得非光合突变组织用于生化分析的事实。 将开发的核心资源是转座子标记的叶绿体缺陷玉米突变体的饱和集合。 突变体将基于其叶绿素缺乏的叶片和/或增加的叶绿素荧光（光合电子传递受阻的指示）从Mu活性玉米品系中选择。 以前的研究支持的概念，这些容易识别的表型之一，将导致从叶绿体生物发生的大多数方面（进口的蛋白质进入细胞器，脂质，色素和辅基合成，叶绿体基因表达，叶绿体内蛋白质分选，组装的光合机构）的破坏。突变体收集将用于两种方式：（i）确定已知序列但功能未知的基因的作用;（ii）发现在叶绿体生物发生和功能中发挥关键作用的新基因。 (i)基因组测序项目已经揭示了数千种预测的叶绿体定位蛋白，其中大多数没有已知的功能。 为了确定这些蛋白质的作用，突变体收集将用于开发反向遗传资源，称为光合作用突变体搜索（PMS）。 PMS已经在小范围内发挥作用，由来自植物的DNA池组成，这些植物的叶绿体缺陷是由Mu插入引起的。 目前有1200个独立产生的突变体在收集中;这一数字将增加到约2000个，预计在这一点上，收集将饱和。少量的DNA池可以以具有成本效益的方式进行筛选，以发现在叶绿体生物发生中具有可疑作用的已知序列的基因的突变等位基因。突变体将被识别并作为服务提供。 用户将分析突变表型，以阐明被破坏基因的功能。(ii)为了发现在叶绿体生物发生和功能中起关键作用的新基因，相同的突变株系将经历视觉表型和叶绿体蛋白质和RNA缺陷的“快照”表征。每一个突变体的描述将被纳入一个网站，突变体将提供给其他研究人员。 从这些快照中，用户将订购特定的品系，用于进一步研究和克隆被破坏的基因。此外，合作者团队将形成协同伙伴关系，利用这些工具专注于叶绿体生物发生的一个方面：叶绿体基因表达及其控制。将产生的独特遗传资源将促进整个社区对叶绿体生物学许多其他方面的研究。除了在叶绿体过程中发挥作用的基因的农学相关性外，预计该项目将影响从进化生物学到基础细胞和分子生物学的各个领域。

The chloroplast is an essential organelle whose biogenesis is a complex, orchestrated process central to plant growth and development. Many hundreds of nuclear genes are involved in the import of nucleus-encoded proteins, the intra-organellar sorting of nucleus- and chloroplast-encoded proteins, the expression of chloroplast genes, the assembly of chloroplast enzymes, and the regulation of these processes. However, only a small proportion of such genes have been characterized in any detail. The goal of this project is to develop, use, and disseminate a set of complementary and powerful resources for the genetic and biochemical dissection of this complex process. Maize offers an ideal set of attributes for this project. In addition to its superb genetic tools and the capacity to generate mutations at high frequency with Mu transposons, this project exploits the fact that non-photosynthetic mutant tissue can readily be obtained for biochemical analysis. The core resource that will be developed is a saturated collection of transposon-tagged chloroplast-defective maize mutants. Mutants will be selected from Mu-active maize lines based upon their chlorophyll-deficient leaves and/or increased chlorophyll fluorescence, an indication of a block in photosynthetic electron transport. Previous studies support the notion that one of these easily identified phenotypes will result from a disruption of most aspects of chloroplast biogenesis (import of proteins into the organelle, lipid, pigment, and prosthetic group synthesis, chloroplast gene expression, intra-chloroplast protein sorting, assembly of the photosynthetic apparatus). The mutant collection will be used in two ways: (i) To determine the role of genes of known sequence but unknown function; and (ii) To discover new genes that play critical roles in chloroplast biogenesis and function. (i) Genome sequencing projects have unmasked thousands of predicted chloroplast-localized proteins, the majority of which have no known function. To determine the roles of such proteins, the mutant collection will be used to develop a reverse genetics resource, called Photosynthesis Mutant Search (PMS). PMS, already functioning on a small scale, consists of DNA pools from plants with chloroplast defects caused by Mu insertions. 1200 independently-arising mutants are currently in the collection; this number will be increased to ~2000, at which point it is predicted that the collection will be saturated. The small number of DNA pools can be screened in a cost-effective manner to find mutant alleles of genes of known sequence with suspected roles in chloroplast biogenesis. Mutants will be identified and provided as a service. Users will analyze the mutant phenotypes to elucidate the function of the disrupted gene.(ii) To discover new genes that play critical roles in chloroplast biogenesis and function, the same mutant lines will undergo "snapshot" characterization of visual phenotype and chloroplast protein and RNA defects. The description of each mutant will be incorporated into a web site and the mutants will be made available to other researchers. From these snapshots, users will order specific lines for further study and cloning of the disrupted gene. In addition, the team of collaborators will form a synergistic partnership to use these tools to focus on one aspect of chloroplast biogenesis: chloroplast gene expression and its control. The study of many other aspects of chloroplast biology by the community-at-large will be facilitated by the unique genetic resources that will be produced. In addition to the agronomic relevance of genes that function in chloroplast processes, it is anticipated that this project will impact fields ranging from evolutionary biology to basic cell and molecular biology.