Role of LEAFY in Determining Floral Identity

花发育的一个核心步骤是一群未分化的细胞决定接受花的命运。LEAFY基因在这一过程中起着关键作用，它的活性是促进花过梢发育所必需的。这位研究者从拟南芥中克隆了LEAFY基因，并表明它编码一种新型的核蛋白，这是已知的最早在新生花原基中被激活的基因产物。他建议通过阐明LEAFY蛋白如何在分子水平上发挥其功能，并通过解剖LEAFY启动子的顺式调控元件来详细研究LEAFY如何控制花的命运。由于LEAFY编码一种核蛋白，因此将确定它是否编码一种新型的转录调节因子。该蛋白含有两个结构域，与已知转录调节因子的转录激活结构域相似。这些结构域的体内活性将通过将LEAFY的体外工程缺失变体引入转基因植物并测试这些构建物来补充LEAFY突变来确定。LEAFY作为一种新型的DNA结合蛋白的可能性将通过测试体外合成的LEAFY蛋白与DNA结合的能力来探索。此外，还将研究LEAFY与其他已知在花发育中起作用的蛋白质的分子相互作用。未来，基于它们在体外结合叶子蛋白的能力，将分离出新的蛋白质。将通过三种策略来确定介导LEAFY表达的时空调控的顺式作用元件：首先，将测试含有5‘和3’序列组合以及内含子序列的构建体与LEAFY基因的融合，以在转基因植物中补充lfy突变表型。其次，将Snapgon FLORICAULA的LEAFY同源同源物的启动子序列与LEAFY启动子的启动子序列进行比较，以确定保守的序列元件。第三，5.4kb片段的不同部分，包括5‘和3’以及内含子序列，将在转基因植物中测试它们对报告基因表达的影响。对带有报告基因结构的LEAFY调控序列的分析将通过研究两个花控制基因APETALA1和AGAMOUS的花特异启动子控制下的LEAFY表达是否能够完全或部分补充LEAFY突变表型来补充。这些实验将允许评估在APETALA1和AGAMOUS之前激活的LEAFY的早期表达是否确实是其体内功能所必需的。这项拟议的研究将进一步加深我们对花是如何启动的理解，并将是朝着实现对这一过程的机械理解的最终目标迈出的重要一步。在未来，这将不仅允许在拟南芥等模式系统中，而且也在其他重要的经济开花植物中，对开花进行直接控制。这应该会改善光同化物在生殖器官和营养器官之间的分配，从而提高作物产量。

A central step in flower development is the decision of a group of undifferentiated cells to adopt a floral fate. A key role in this process is played by the LEAFY gene, whose activity is required to promote floral over shoot development. This investigator has cloned the LEAFY gene from Arabidopsis and shown that it encodes a novel type of nuclear protein, which is the earliest known gene product to be activated in nascent flower primordia. He proposes to study in detail how LEAFY controls floral fate by elucidating how LEAFY protein exerts its function at the molecular level, and by dissecting the cis-regulatory elements of the LEAFY promoter. Since LEAFY encodes a nuclear protein, it will be determined whether it encodes a novel type of transcriptional regulator. The protein contains two domains with similarity to transcriptional activation domains of known transcriptional regulators. The in vivo activity of these domains will be determined by introducing in vitro engineered deletion variants of LEAFY into transgenic plants and testing these constructs for complementation of leafy mutations. The possibility that LEAFY acts as a novel type of DNA-binding protein will be explored by testing in vitro synthesized LEAFY protein for its ability to bind DNA. In addition, the molecular interaction of LEAFY with other proteins known to have a role in floral development will be studied. In the future, new proteins will be isolated based on their ability to bind LEAFY protein in vitro. Cis-acting elements mediating the temporal and spatial control of LEAFY expression will be identified by three strategies: First, constructs containing combinations of 5' and 3' sequences as well as intron sequences fused to a LEAFY cDNA will be tested for complementation of the lfy mutant phenotype in transgenic plants. Second, the promoter sequence of the LEAFY cognate homolog from snapdragon, FLORICAULA, will be compared to that of the LEAFY promoter, in order to identify co nserved sequence elements. Third, various parts of the 5.4 kb fragment, including 5' and 3' as well as intron sequences, will be tested in transgenic plants for their effects on the expression of a reporter gene. The analysis of LEAFY regulatory sequences with reporter gene constructs will be complemented by studying whether expression of LEAFY under the control of two floral specific promoters, those of the two floral control genes APETALA1 and AGAMOUS, can completely or partially complement the leafy mutant phenotype. These experiments will allow for assessing whether the early expression of LEAFY, which is activated before APETALA1 and AGAMOUS, is indeed required for its in vivo function. The proposed research will further our understanding of how flowers are initiated, and will be an important step toward the ultimate goal of achieving a mechanistic understanding of this process. In the future, this will permit for directed manipulation of flowering not only in model systems such as Arabidopsis, but also in other, economically important flowering plants. This should lead to improved partitioning of photoassimilates between reproductive and vegetative organs, and thus to better crop yields.