CAREER: Initiation of Cell Size Patterning in Arabidopsis

随着人口的快速增长和耕地的减少，世界将需要大幅提高粮食产量。玉米、豆类、大米、小麦和豌豆的可食用部分的细胞经历了一种称为内复制的特殊过程，细胞多次复制它们的DNA。虽然单个细胞经历不同数量的内复制，但细胞的比例在植物间保持不变。当遗传学家试图增加内复制时，他们发现看似相同的细胞的反应有些随机：有些细胞反应强烈，有些反应适度，有些则完全没有。这一观察提出了发育生物学中最大的问题之一：相同的细胞如何做出不同的决定？拟议的研究将调查一个反直觉的假设，即单个植物细胞利用小的、随机的分子差异来决定多少内复制。这个假设令人惊讶：生物学家通常会期望随机性被抑制，因为发育是高度可复制的，总是产生相同比例的内复制细胞。相反，实际上可能需要随机性来产生这部分正常的内复制细胞。要验证这一假设，需要先进的计算机技术来检测单个细胞随时间的变化。大学生和中学生将同时参与这种分析，并接受计算技能的培训。农作物的核内复制细胞深埋在种皮中，在实验上是无法接近的。因此，拟南芥萼片的内复制将被研究，其表皮细胞在进行内复制时可以进行实时成像。拟南芥MERISTEM LAYER1 （ATML1）转录因子驱动萼片内重复，并可能导致其随机发生。由于ATML1在所有表皮细胞中表达，目前尚不清楚ATML1如何仅引起少数细胞内复制。初步数据显示，ATML1以剂量依赖的方式触发内复制：高ATML1使所有表皮细胞内复制，低ATML1阻止任何内复制，并且单个萼片细胞具有不同水平的ATML1。这里测试的假设是随机波动的ATML1在不同细胞中引起不同水平的内复制；少数细胞超过ATML1的阈值，触发内复制，而其他细胞保持在阈值以下，不进行内复制。在Aim 1中，我们将确定ATML1波动是否启动内复制。相关的教育目标将是与本科生一起分析在这个目标中产生的图像。对于目标2，我们将确定ATML1反馈回路是否通过放大ATML1表达的差异来驱动内重复，并且在相关的教育目标中，学生将编写并发布计算建模术语表，以更好地理解新生成的模型。最后，在Aim 3中，我们将确定ATML1以剂量依赖性方式诱导内重复的分子机制。我们还将在一个基于网络的实践图像分析研讨会中，为中学生提供对Aim 3图像的分析。

With a rapidly growing population and decreasing farmland, the world will need to dramatically increase crop yields. The cells in the edible parts of corn, beans, rice, wheat, and peas undergo a specialized process called endoreduplication where the cells replicate their DNA many times. Although individual cells undergo different amounts of endoreduplication, the proportion of cells remains constant from plant to plant. When geneticists try to increase endoreduplication, they find that the response of seemingly identical cells is somewhat random: some cells respond strongly, some moderately, and some not at all. This observation raises one of the biggest questions in developmental biology: how do identical cells make different decisions? The proposed research will investigate the counterintuitive hypothesis that individual plant cells make these decisions about how much to endoreduplicate utilizing small, random molecular differences. This hypothesis is surprising: biologists would normally expect randomness to be suppressed, since development is highly reproducible, always creating the same proportion of endoreduplicated cells. Instead, randomness may in fact be required to produce this regular portion of endoreduplicated cells. Testing this hypothesis will require advanced use of computers to examine individual cells over time. University students and middle school girls will simultaneously be involved in this analysis as well as being trained in computational skills.Endoreduplicating cells of crop plants are buried deep within the seed coat where they are experimentally inaccessible. Endoreduplication will thus be studied in Arabidopsis thaliana sepals, whose epidermal cells are accessible for live imaging as they endoreduplicate. The transcription factor Arabidopsis thaliana MERISTEM LAYER1 (ATML1) drives sepal endoreduplication, and may cause it to occur randomly. Since ATML1 is expressed in all epidermal cells, it is unclear how ATML1 causes only a few cells to endoreduplicate. Preliminary data show that ATML1 triggers endoreduplication in a dosage-dependent manner: high ATML1 makes all epidermal cells endoreduplicate, low ATML1 prevents any endoreduplication, and individual sepal cells have varying levels of ATML1. The hypothesis tested here is that randomly fluctuating ATML1 causes different levels of endoreduplication in different cells; a few cells exceed ATML1's threshold, triggering endoreduplication, while other cells remain below the threshold and do not endoreduplicate. In Aim 1, we will determine whether ATML1 fluctuations initiate endoreduplication. The associated education goal will be to work with undergraduate students to analyze images generated in this aim. For Aim 2, we will determine whether ATML1 feedback loops drive endoreduplication by amplifying differences in ATML1 expression and, in an associated educational aim, students will write and publish a computational modeling glossary to better understand the newly generated models. Finally, in Aim 3 we will determine the molecular mechanism through which ATML1 induces endoreduplication in a dosage-dependent manner. We will also develop an outreach to middle school students for the analysis of images from Aim 3 in a hands-on, web-based image analysis workshop.