Analysis of Apetala3 and Pistillata Floral Homeotic Gene Function

在拟南芥中，花瓣和雄蕊的形成需要两个花同源异型基因APETALA3(AP3)和雌蕊(PI)。AP3和PI都在花瓣和雄蕊原基中表达，这与它们在器官识别中的作用一致。AP3和PI都编码MADS-box蛋白；MADS-box是DNA结合所必需的，已经在许多其他酵母和哺乳动物转录因子中发现。AP3和PI以专性异源二聚体的形式与DNA结合，一些证据表明AP3/PI作用的特异性可能取决于与其他辅因子的相互作用以及蛋白质的修饰。这项建议旨在研究AP3和PI蛋白的作用是如何调节的。提出了一种改良的单杂交筛选法来鉴定与AP3和PI相互作用以调节其生物学特异性的辅因子。由于AP3和PI都含有一个保守的潜在磷酸化位点，因此蛋白质修饰也可能在调节AP3/PI的作用中发挥作用。免疫沉淀实验和转基因研究将被用来确定AP3和/或PI的磷酸化状态是否在改变它们的功能方面起作用。最后，AP3和PI可能以非细胞自主的方式发挥作用，表明这些基因产物可能在细胞间信号转导中发挥作用。AP3(或PI)的植物马赛克将使用改进的Ac/DS系统生成。在花个体发育的不同时期产生的镶嵌斑块将定义AP3(或PI)作用的时间参数。此外，抗AP3和抗PI抗体将被用来评估AP3和PI蛋白是否实际上在细胞之间运输，以影响它们的非自主功能。利用互补的生化、遗传和发育方法来阐明AP3和PI的作用机制，将为理解这些花同源异型基因产物如何获得功能特异性提供一个框架。此外，这些分析在开始确定植物细胞之间相互沟通以协调它们的生长和分化的机制方面应该是有价值的。

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.