Transcriptional control of growth regulatory modules by AIL transcription factors

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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.