Transcriptional control of growth regulatory modules by AIL transcription factors

AIL转录因子对生长调节模块的转录控制

• 批准号: 2211715
• 负责人: Beth Krizek
• 金额: $ 54.92万
• 依托单位: University of South Carolina at Columbia
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2211715&HistoricalAwards=false
• 关键词: Transcriptional; control; growth; regulatory; modules

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.

植物器官的大小取决于组成细胞的数量和大小。发育中器官内的生长由细胞数量增加的早期阶段和细胞尺寸扩大的后期阶段组成。调节细胞分裂或扩增速率或任一生长阶段持续时间的途径已被确定。然而，这些途径的调节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。这里提出的工作将研究模式植物拟南芥生长途径的调节。了解调节叶子和花朵生长的机制可以在提高植物产量和生物量方面产生重要的经济效益。叶类蔬菜作物中较大的叶子和较大的花朵及其产生的产品（水果、种子和谷物）可以有助于增加农业产量，以满足不断增长的世界人口的需求。此外，该项目旨在通过开发分子生物学实验室技能研讨会来扩大对科学的参与，该研讨会将在大学第一学期向第一代和代表性不足的学生提供。作为南卡罗来纳州教师研究经验 (RET) 计划的一部分，该项目还将邀请当地 K-12 教师进行暑期研究经验。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子和花朵，而 ANT 的过度表达会产生较大的叶子和花朵。 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。该项目将测试以下假设：ANT 和 AIL6 直接调节来自几个不同生长调节模块的基因表达，并且这些不同模块之间的反馈整合生长信息以控制最终的器官大小。将研究 ANT 和 AIL6 在调节 miR396/GRF-GIF、DA1/BB 和 KLU 生长模块（控制细胞增殖阶段的持续时间）的六个基因表达中的作用。 RT-qPCR、原位杂交、启动子：GUS 报告基因和激光捕获显微切割将用于表征这些生长基因响应 ANT 和 AIL6 活性变化的表达水平和模式。 ANT 和 AIL6 结合的顺式调控元件将使用电泳迁移率变动测定来确定。突变启动子：GUS 报告基因、CRISPR-Cas 基因组编辑和 ChIP-qPCR 检测将探讨这些已识别的顺式元件在体内的重要性。此外，该项目还将调查 ANT/AIL6 与这些其他增长模块之间是否发生相互交叉监管。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。