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The B-class MADS-box transcription factors STERILE TASSEL SILKY EAR1 (STS1) and SILKY1 (SI1) specify floral organ identity in the grass Zea mays (maize). STS1 and SI1 bind DNA as obligate heterodimers. Obligate heterodimerization between STS1 and SI1 homologs, although common in flowering plants, arose very recently in the grass family. This recent emergence of obligate heterodimerization from STS1 homodimerization provided an opportunity to test the consequences of evolutionary shifts in MADS-box protein-protein interactions. We tested the ability of evolutionary variation in STS1 dimerization to impact floral development, downstream gene regulation, and protein complex formation in maize. We found that STS1 hetero- vs. homodimerization had subtle effects on protein localization and stamen development. In contrast, differential STS1 dimerization resulted in large-scale changes to gene expression and protein complex composition. We identified kinases and proteins involved in ubiquitylation as candidate interactors with MADS-box proteins, and found that STS1 was phosphorylated, and in a complex with ubiquitylated proteins. In addition, we found that STS1 homodimers were more abundant than STS1-SI1 heterodimers, independent of RNA levels. Thus, differential dimerization can affect both protein degradation dynamics and combinatorial assembly of MADS-box protein complexes. Our results highlight the robustness of floral development to some molecular change, which may contribute to the evolvability of floral form. Significance Statement Interactions between transcription factors can alter downstream gene expression patterns and, in turn, organismal development. In plants, MADS-box transcription factors specify floral organ identity as part of large complexes. Shifting interactions between MADS-box proteins have been proposed as drivers in the evolution of the flower, and in the diversification of floral form. However, the functional consequences of changes to individual MADS-box protein-protein interactions are unknown. Here, we show that floral development is subtly affected by altered transcription factor protein-protein interactions. We also show that shifting transcription factor interactions can contribute to differential protein degradation. This reveals an important consequence for evolutionary variation in transcription factor interactions, and adds a new layer of complexity to the evolution of developmental gene networks.

The rapid rise and diversification of the angiosperms has puzzled biologists for centuries; processes leading to current angiosperm diversity remain a key question in evolutionary biology, with particular focus on the morphological diversity of flowers. The Zingiberales are an order of tropical monocots that represent an ideal group of plants to study the evolution of floral morphology. The order contains approximately 2,500 species, many of which form specialized pollination relationships with bees, birds, bats, dung beetles, moths, butterflies, and primates (lemurs) via alterations in floral form. After developing a technique for visualizing and then studying gene expression in floral apices, I investigated the role of two candidate gene families, the GLOBOSA (GLO)-like genes and the CYCLOIDEA/ TEOSINTE BRANCHED 1 (CYC/TB1)-like genes, in the evolution of floral morphology in the Zingiberales. Evolutionary developmental biology often combines methods for examining morphology (e.g. Scanning Electron Microscopy) with analyses of gene expression (e.g. RNA in situ hybridization). Due to differences in tissue preparation for SEM and gene expression analyses, the same specimen cannot be used for both sets of techniques. I developed a method that couples extended-depth-of-field (EDF) epi-illumination microscopy to in situ hybridization in a sequential format, enabling both surface microscopy and gene expression analyses to be carried out on the same specimen (Chapter 1). I first created a digital image of inflorescence apices using epi-illumination microscopy and commercially available EDF software. I then performed RNA in situ hybridizations on photographed apices to assess expression of two developmental genes: Knotted1 (Kn1) in Zea mays (Poaceae) and a GLO homolog in Musa basjoo (Musaceae). I demonstrate that expression signal is neither altered nor reduced in the imaged apices as compared with unphotographed controls. The demonstrated method reduces the amount of sample material necessary for developmental research and enables individual floral development to be placed in the context of the entire inflorescence. While the technique presented is particularly relevant to floral developmental biology, it is applicable to any research where observation and description of external features can be fruitfully linked with analyses of gene expression.The MADS box transcription factor family has long been identified as an important contributor to the control of floral development. It is often hypothesized that the evolution of floral development across angiosperms and within specific lineages may occur as a result of duplication, functional diversification, and changes in regulation of MADS box genes. In Chapter 2 I examine the role of GLO-like genes, members of the B-class MADS box gene lineage, in the evolution of floral development within the monocot order Zingiberales. I assessed changes in perianth and stamen whorl morphology in a phylogenetic framework. I identified GLO homologs from 50 Zingiberales species and investigated the evolution of this gene lineage. Expression of two GLO homologs was assessed in Costus spicatus Swartz (Costaceae) and Musa basjoo Siebold (Musaceae). Based on the phylogenetic data and expression results, I propose several family-specific losses and gains of GLO homologs that appear to be associated with key morphological changes. The GLO-like gene lineage has diversified concomitant with the evolution of the dimorphic perianth and the staminodial labellum. Duplications and expression divergence within the GLO-like gene lineage may have played a role in floral diversification in the Zingiberales.In the Zingiberales, evolutionary shifts in symmetry occur in all floral whorls, making this an ideal group of plants in which to study the evolution of this important ecological and developmental trait. The CYC/TB1-like genes have been implicated in the development and evolution of floral symmetry in divergent angiosperm lineages, and I thus chose them as a candidate gene family to investigate their role in the evolution of floral symmetry within the Zingiberales (Chapter 3). I identified both Zingiberales-specific gene duplications and a duplication in the TB1-like (TBL) lineage that predates the divergence of the commelinid monocots. I examined the expression of two TBL genes in Costus spicatus (Costaceae) and Heliconia stricta (Heliconiaceae), two Zingiberales taxa with divergent floral symmetries. I found that TBL gene expression shifts concomitant with shifts in floral symmetry. Through this body of work we have gained some insight into the mechanics of angiosperm evolution. Duplications in the GLO-like gene lineage in the Zingiberales may have allowed for gene sub- or neofunctionalization and the evolution of new morphologies; in particular, the evolution of differentiated sepals and petals and of the staminodial labellum. In addition, this study adds to the growing body of evidence that CYC/TB1-like genes have been repeatedly recruited through the course of evolution to generate bilateral floral symmetry (zygomorphy). Although this work certainly doesn't preclude the involvement of as yet uncharacterized genes and gene families, it adds to the growing body of evidence that angiosperms as a group do indeed have a genetic `toolkit': a core set of genes that have been variously deployed through evolutionary time to generate both convergent and divergent floral morphologies.

Shoot apical meristems are stem cell niches that balance proliferation with the incorporation of daughter cells into organ primordia. This balance is maintained by CLAVATA-WUSCHEL feedback signaling between the stem cells at the tip of the meristem and the underlying organizing center. Signals that provide feedback from organ primordia to control the stem cell niche in plants have also been hypothesized, but their identities are unknown. Here we report FASCIATED EAR3 (FEA3), a leucine-rich-repeat receptor that functions in stem cell control and responds to a CLAVATA3/ESR-related (CLE) peptide expressed in organ primordia. We modeled our results to propose a regulatory system that transmits signals from differentiating cells in organ primordia back to the stem cell niche and that appears to function broadly in the plant kingdom. Furthermore, we demonstrate an application of this new signaling feedback, by showing that weak alleles of fea3 enhance hybrid maize yield traits.