AuxSynBio: synthetic biology tools to understand and engineer auxin.

The plant hormone auxin is a crucial coordinator of nearly all plant growth and development processes. Because of its centrality to plant physiology and the modular nature of the signaling pathway, auxin has played a critical role at the forefront of plant synthetic biology. This review will highlight how auxin is both a subject and an object of synthetic biology.

The Systems and Synthetic Biology of Auxin.

Auxin biology as a field has been at the forefront of advances in delineating the structures, dynamics, and control of plant growth networks. Advances have been enabled by combining the complementary fields of top-down, holistic systems biology and bottom-up, build-to-understand synthetic biology. Continued collaboration between these approaches will facilitate our understanding of and ability to engineer auxin's control of plant growth, development, and physiology.

A Synthetic Approach Allows Rapid Characterization of the Maize Nuclear Auxin Response Circuit.

Auxin plays a key role across all land plants in growth and developmental processes. Although auxin signaling function has diverged and expanded, differences in the molecular functions of signaling components have largely been characterized in Arabidopsis (). Here, we used the nuclear Auxin Response Circuit recapitulated in yeast () system to functionally annotate maize () auxin signaling components, focusing on genes expressed during the development of ear and tassel inflorescences.

Accelerating structure-function mapping using the ViVa webtool to mine natural variation.

Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of these data remain inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the 1001 Genomes Project (http://1001genomes.org).

Functional analysis of molecular interactions in synthetic auxin response circuits.

Auxin-regulated transcription pivots on the interaction between the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressor proteins and the AUXIN RESPONSE FACTOR (ARF) transcription factors. Recent structural analyses of ARFs and Aux/IAAs have raised questions about the functional complexes driving auxin transcriptional responses. To parse the nature and significance of ARF-DNA and ARF-Aux/IAA interactions, we analyzed structure-guided variants of synthetic auxin response circuits in the budding yeast Saccharomyces cerevisiae Our analysis revealed that promoter architecture could specify ARF activity and that ARF19 required dimerization at two distinct domains for full transcriptional activation.

Oligomerization of SCFTIR1 Is Essential for Aux/IAA Degradation and Auxin Signaling in Arabidopsis.

The phytohormone auxin is a key regulator of plant growth and development. Molecular studies in Arabidopsis have shown that auxin perception and signaling is mediated via TIR1/AFB-Aux/IAA co-receptors that assemble as part of the SCFTIR1/AFB E3 ubiquitin-ligase complex and direct the auxin-regulated degradation of Aux/IAA transcriptional repressors. Despite the importance of auxin signaling, little is known about the functional regulation of the TIR1/AFB receptor family.

A Live-imaging, Heat Shock-inducible System to Measure Aux/IAA Degradation Rates in Planta.

An emerging theme in biology is the importance of cellular signaling dynamics. In addition to monitoring changes in absolute abundance of signaling molecules, many signal transduction pathways are sensitive to changes in temporal properties of signaling components (Purvis and Lahav, 2013). The phytohormone auxin regulates myriad processes in plant development.

Evaluation to Improve a High School Summer Science Outreach Program.

The goal of the Young Scientist Program (YSP) at Washington University School of Medicine in St. Louis (WUSM) is to broaden science literacy and recruit talent for the scientific future. In particular, YSP seeks to expose underrepresented minority high school students from St.

Auxin signaling modules regulate maize inflorescence architecture.

In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation.

Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response.

Plant genomes encode large numbers of F-box proteins (FBPs), the substrate recognition subunit of SKP1-CULLIN-F-box (SCF) ubiquitin ligases. There are ~700 FBPs in , most of which are uncharacterized. TIR1 is among the best-studied plant FBPs and functions as a receptor for the plant hormone auxin.

Rate Motifs Tune Auxin/Indole-3-Acetic Acid Degradation Dynamics.

Ubiquitin-mediated protein degradation is a common feature in diverse plant cell signaling pathways; however, the factors that control the dynamics of regulated protein turnover are largely unknown. One of the best-characterized families of E3 ubiquitin ligases facilitates ubiquitination of auxin (aux)/indole-3-acetic acid (IAA) repressor proteins in the presence of auxin. Rates of auxin-induced degradation vary widely within the Aux/IAA family, and sequences outside of the characterized degron (the minimum region required for auxin-induced degradation) can accelerate or decelerate degradation.

Auxin-induced degradation dynamics set the pace for lateral root development.

Auxin elicits diverse cell behaviors through a simple nuclear signaling pathway initiated by degradation of Aux/IAA co-repressors. Our previous work revealed that members of the large Arabidopsis Aux/IAA family exhibit a range of degradation rates in synthetic contexts. However, it remained an unresolved issue whether differences in Aux/IAA turnover rates played a significant role in plant responses to auxin.

Tuning the auxin transcriptional response.

How does auxin provoke such a diverse array of responses? This long-standing question is further complicated by a remarkably short nuclear auxin signalling pathway. To crack the auxin code, several potential sources of specificity need to be evaluated. These include: specificity of interactions among the core auxin response components, specificity resulting from higher order complex dynamics, and specificity in interactions with global factors controlling protein turnover and transcriptional repression.

Mutations in the TIR1 auxin receptor that increase affinity for auxin/indole-3-acetic acid proteins result in auxin hypersensitivity.

The phytohormone auxin regulates virtually every aspect of plant development. The hormone directly mediates the interaction between the two members of the auxin coreceptor complex, a TRANSPORT INHIBITOR RESPONSE (TIR1)/AUXIN SIGNALING F-BOX protein and an AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressor. To learn more about the interaction between these proteins, a mutant screen was performed using the yeast (Saccharomyces cerevisiae) two-hybrid system in Arabidopsis (Arabidopsis thaliana).

Interrogation of inhibitor of nuclear factor κB α/nuclear factor κB (IκBα/NF-κB) negative feedback loop dynamics: from single cells to live animals in vivo.

Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled IκBα/NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a κB(5)→IκBα-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ΙκBα degradation and subsequent NF-κB transcriptional activity in response to diverse modes of TNFα stimulation.

Bioluminescence imaging of myeloperoxidase activity in vivo.

The myeloperoxidase (MPO) system of activated phagocytes is central to normal host defense mechanisms, and dysregulated MPO contributes to the pathogenesis of inflammatory disease states ranging from atherosclerosis to cancer. Here we show that upon systemic administration, the small molecule luminol enables noninvasive bioluminescence imaging (BLI) of MPO activity in vivo. Luminol-BLI allowed quantitative longitudinal monitoring of MPO activity in animal models of acute dermatitis, mixed allergic contact hypersensitivity, focal arthritis and spontaneous large granular lymphocytic tumors.

Advances in bioluminescence imaging of live animal models.

Many of the obligate steps of physiology and disease are dynamic in time and space, and thus, end-point assays do not always provide a full understanding of these processes. Comprehensive understanding of the functional complexity of protein interactions and cell trafficking requires mapping of cellular and molecular function within complex systems over biologically relevant time scales. New approaches to bioluminescence imaging of cell migration, signaling pathways, drug action, and interacting protein partners in vivo allow the study of biology and disease within the context of living animals.

Identification of a ligand-induced transient refractory period in nuclear factor-kappaB signaling.

In response to a variety of extracellular ligands, nuclear factor-kappaB (NF-kappaB) signaling regulates inflammation, cell proliferation, and apoptosis. It is likely that cells are not continuously exposed to stimulating ligands in vivo but rather experience transient pulses. To study the temporal regulation of NF-kappaB and its major regulator, inhibitor of NF-kappaBalpha (IkappaBalpha), in real time, we utilized a novel transcriptionally coupled IkappaBalpha-firefly luciferase fusion reporter and characterized the dynamics and responsiveness of IkappaBalpha processing upon a short 30-s pulse of tumor necrosis factor alpha (TNFalpha) or a continuous challenge of TNFalpha following a 30-s preconditioning pulse.

Veni, vidi, vici: in vivo molecular imaging of immune response.

"I came, I saw, I conquered," Julius Caesar proclaimed, highlighting the importance of direct visualization as a winning strategy. Continuing the "From the Field" series (see Editorial [2007] 26, 131), Gross et al. summarize how modern molecular imaging techniques can successfully dissect the complexities of immune response in vivo.

Tumor shedding of laminin binding protein modulates angiostatin production in vitro and interferes with plasmin-derived inhibition of angiogenesis in aortic ring cultures.

The growth of solid tumors is largely controlled by the process of angiogenesis. A 67 kDa protein, the laminin binding protein (LBP), is shed from malignant cells in significant amounts and binds to laminin-1 (Starkey et al., Cytometry 1999;35:37-47; Karpatová et al.