Analysis of Apetala3 and Pistillata Floral Homeotic Gene Function

在拟南芥中，两个花同源异型基因APETALA 3（AP 3）和PISTILLATA（PI）是花瓣和雄蕊形成所必需的。 AP 3和PI都在花瓣和雄蕊原基中表达，与它们提出的器官身份角色一致。 AP 3和PI都编码MADS盒蛋白; MADS盒是DNA结合所必需的，并且已经在许多其他酵母和哺乳动物转录因子中发现。 AP 3和PI结合DNA作为一个专性异二聚体，和几条线的证据表明，AP 3/PI行动的特异性可能取决于与其他辅因子的相互作用，以及对蛋白质的修饰。 该提案旨在研究AP 3和PI蛋白的作用是如何调节的。 提出了一种改进的单杂交筛选来鉴定与AP 3和PI相互作用以调节其生物学特异性的辅因子。 由于AP 3和PI都含有保守的潜在磷酸化位点，因此蛋白质修饰也可能在调节AP 3/PI作用中起作用。 免疫沉淀实验和转基因研究将用于确定AP 3和/或PI的磷酸化状态是否在修改其功能中起作用。 最后，AP 3和PI可能以非细胞自主的方式起作用，表明这些基因产物可能在细胞间信号传导中起作用。 将使用改良的Ac/Ds系统生成AP 3（或PI）的植物镶嵌图。 花个体发育不同时期产生的镶嵌斑块将决定AP 3（或PI）作用的时间参数。 此外，抗AP 3和抗PI抗体将用于评估AP 3和PI蛋白是否实际上在细胞之间运输以实现其非自主功能。 利用互补的生化，遗传和发育的方法来阐明AP 3和PI的作用机制，将提供一个框架，了解这些花同源异型基因产物如何获得功能特异性。 此外，这些分析应该是有价值的，在开始定义的机制，植物细胞相互沟通，以协调其生长和分化。

In Arabidopsis, two floral homeotic genes, APETALA3 (AP3) and PISTILLATA (PI), are required for the formation of petals and stamens. AP3 and PI are both expressed in petal and stamen primordia, consistent with their proposed organ identity roles. AP3 and PI both encode MADS-box proteins; the MADS-box is required for DNA binding and has been found in a number of other yeast and mammalian transcription factors. AP3 and PI bind to DNA as an obligate heterodimer, and several lines of evidence suggest that the specificity of AP3/PI action may depend on interactions with other cofactors as well as on protein modification. This proposal aims to investigate how the action of the AP3 and PI proteins is regulated. A modified one-hybrid screen is proposed to identify cofactors that interact with AP3 and PI to modulate their biological specificity. Since both AP3 and PI contain a conserved potential phosphorylation site, it is possible that protein modification may also play a role in modulating AP3/PI action. Immunoprecipitation experiments and transgenic studies will be used to ascertain whether the phosphorylation status of AP3 and/or PI plays a role in modifying their function. Finally, AP3 and PI may act in a non-cell autonomous fashion, indicating that these gene products may play a role in intercellular signaling. Plants mosaic for AP3 (or PI) will be generated using a modified Ac/Ds system. Mosaic patches generated at different times in floral ontogeny will define the temporal parameters of AP3 (or PI) action. In addition, anti-AP3 and anti-PI antibodies will be used to assess whether AP3 and PI proteins actually traffic between cells to effect their non-autonomous functions. Using complementary biochemical, genetic, and developmental approaches to elucidate the mechanisms by which AP3 and PI act will provide a framework for uderstanding how these floral homeotic gene products attain functional specificity. Furthermore, these analyses should be valuable in beginning to define the mechanisms by which plant cells communicate with each other to coordinate their growth and differentiation.