Transcriptional control of growth regulatory modules by AIL transcription factors

植物器官的大小取决于组成器官的细胞的数量和大小。发育器官的生长包括细胞数量增加的早期阶段和细胞大小扩大的后期阶段。已经确定了调节细胞分裂或扩增速度或生长阶段持续时间的途径。然而，这些途径的调控以及这些不同途径的输出如何整合以控制植物器官的最终大小尚不清楚。本文提出的工作将研究模式植物拟南芥生长途径的调控。了解叶片和花朵生长的调控机制对提高植物产量和生物量具有重要的经济效益。叶菜作物的大叶子和大花朵以及它们所产生的产品（水果、种子和谷物）有助于提高农业产量，以养活不断增长的世界人口。此外，该项目旨在通过开发分子生物学实验室技能讲习班来扩大科学参与，该讲习班将在大学第一学期提供给第一代和代表性不足的学生。该项目还将涉及一名当地K-12教师的夏季研究经验，作为南卡罗来纳州教师研究经验（RET）计划的一部分。植物器官生长的关键启动子是转录因子AINTEGUMENTA （ANT）。ANT突变体产生较小的叶和花，而过表达的ANT导致较大的叶和花。ChIP-Seq研究表明，ANT和相关转录因子AINTEGUMENTA-LIKE 6/ PLETHORA3 （AIL6/PLT3）在发育中的花原基中与许多不同的生长基因结合。本项目将验证ANT和AIL6直接调控来自几种不同生长调节模块的基因表达，这些不同模块之间的反馈整合了生长信息以控制最终器官大小的假设。我们将研究ANT和AIL6在调节miR396/GRF-GIF、DA1/BB和KLU生长模块中控制细胞增殖期持续时间的6个基因表达中的作用。RT-qPCR、原位杂交、启动子：GUS报告子和激光捕获显微解剖将用于表征这些生长基因的表达水平和模式，以响应ANT和AIL6活性的变化。由ANT和AIL6结合的顺式调控元件将通过电泳迁移转移测定来确定。突变启动子：GUS报告，CRISPR-Cas基因组编辑和ChIP-qPCR检测将探讨这些鉴定的顺式元件在体内的重要性。此外，该项目将研究ANT/AIL6与这些其他生长模块之间是否存在相互交叉调节。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The size of plant organs depends on both the number and size of the constituent cells. Growth within developing organs consists of an early phase in which there is an increase in cell number followed by a later phase in which cells expand in size. Pathways that regulate the rate of cell division or expansion, or the duration of either phase of growth have been identified. However, the regulation of these pathways and how output from these different pathways is integrated to control the final size of a plant organ is not well understood. The work proposed here will investigate regulation of growth pathways in the model plant Arabidopsis thaliana. Understanding the mechanisms regulating the growth of leaves and flowers can have important economic benefits in increasing plant yield and biomass. Larger leaves in leaf vegetable crops and larger flowers and the products they give rise to (fruits, seeds, and grains) can contribute to increased agricultural production needed to feed a growing world population. In addition, the project aims to broaden participation in science by developing a molecular biology lab skills workshop that will be offered to first generation and underrepresented students in their first semester of college. The project will also involve a local K-12 teacher for a summer research experience as part of the South Carolina Research Experience for Teachers (RET) program. A key promoter of organ growth in plants is the transcription factor AINTEGUMENTA (ANT). ANT mutants produce smaller leaves and flowers while overexpression of ANT results in larger leaves and flowers. ChIP-Seq studies show that ANT and the related transcription factor AINTEGUMENTA-LIKE 6/ PLETHORA3 (AIL6/PLT3) are bound to a number of different growth genes in developing floral primordia. This project will test the hypothesis that ANT and AIL6 directly regulate expression of genes from several different growth regulatory modules and that feedback among these different modules integrates growth information to control final organ size. The roles of ANT and AIL6 in regulating the expression of six genes from the miR396/GRF-GIF, DA1/BB, and KLU growth modules, which control the duration of the cell proliferation phase of growth, will be investigated. RT-qPCR, in situ hybridization, promoter:GUS reporters, and laser capture microdissection will be used to characterize the expression levels and patterns of these growth genes in response to changes in ANT and AIL6 activity. Cis-regulatory elements bound by ANT and AIL6 will be determined using electrophoretic mobility shift assays. Mutated promoter:GUS reporters, CRISPR-Cas genome editing, and ChIP-qPCR assays will probe the importance of these identified cis-elements in vivo. In addition, the project will investigate whether reciprocal cross-regulation occurs between ANT/AIL6 and these other growth modules.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.