Leaf Development in the Reproductive Phase of Arabidopsis: Investigating the Mechanics of Cauline Leaf Identity and Bract Suppression

Darren Paul Manuela, University of South Carolina

Abstract

Leaves in the reproductive phase subtend inflorescences or flowers and exhibit diverse morphological modifications specialized for reproductive success, such as attracting pollinators, protecting flowers/fruit, and photosynthesis. Using the model organism Arabidopsis, we investigated the development of these specialized leaves. In Arabidopsis, uniquely shaped cauline leaves subtend inflorescences, protect young floral buds and exhibit increased photosynthetic activity crucial for seed production. As form affects function, we analyzed gene expression patterns by RT-qPCR and GUS reporter genes in cauline leaves and examined mutant phenotypes with defective cauline leaf morphology. Our results reveal that redundant miR156-targeted SPLs function to promote cauline leaf identity by controlling cauline leaf morphology and associated number of leaves produced on the secondary inflorescence. As cauline leaf development represents an intermediate phase between vegetative and reproductive development, we found that flowering activators SOC1 and FUL promote cauline leaf identity upstream by directly activating SPL9 and SPL15 in cauline leaves during floral induction, whereas transcription factor SPL9 directly promotes BRI1 expression in cauline leaves. Our results reveal a SOC1/FUL-SPL-BRI1 module: upregulation of SOC1/FUL during floral induction partially activates SPLs in cauline leaves, and these SPLs, which control leaf heteroblasty, promote cauline leaf identity partially through regulation of the BR pathway, such as the BR receptor BRI1. Though Arabidopsis produces cauline leaves subtending the inflorescences during the intermediate reproductive phase, leaves subtending flowers (bracts) in the reproductive phase are suppressed and do not grow out. The floral meristem identity regulator LFY, the organ boundary genes BOP1 and BOP2, and floral activators SOC1, FUL, and AGL24 (despite their roles in cauline leaves) suppress floral bract development in Arabidopsis, as mutations in these genes result in bract outgrowth. To investigate the mechanisms that promote bract outgrowth, we characterized these bracteate mutant phenotypes and analyzed gene expression patterns in stage 2-3 flower primordium by GUS reporter genes and in situ hybridization. Our results show that organ growth regulators ANT and AIL6 are misexpressed to the developing bracts of bracteate mutants. We then ectopically expressed ANT and AIL6 in wild-type and found that AIL6 is sufficient to promote bract outgrowth. To determine whether ANT/AIL6 are necessary for bract outgrowth, we analyzed loss of function ant/ail6 in these bracteate mutants, resulting in the disappearance of bracts. Through ChIP-qPCR analysis, we found that bract suppressors bind directly to the ANT promoter and AIL6 regulatory intron sequence. Our data reveals a mechanism of phytomer modification where bract suppressors regulate ANT/AIL6 to arrest bract growth and promote flower development without leaves. This work provides insight on phytomer modification and changes in inflorescence architecture, and a potential tool for modifying leaf development in the phytomers of agriculturally important crops.