A single helix repression domain is functional across diverse eukaryotes.

The corepressor TOPLESS (TPL) and its paralogs coordinately regulate a large number of genes critical to plant development and immunity. As in many members of the larger pan-eukaryotic Tup1/TLE/Groucho corepressor family, TPL contains a Lis1 Homology domain (LisH), whose function is not well understood. We have previously found that the LisH in TPL-and specifically the N-terminal 18 amino acid alpha-helical region (TPL-H1)-can act as an autonomous repression domain. We hypothesized that homologous domains across diverse LisH-containing proteins could share the same function. To test that hypothesis, we built a library of H1s that broadly sampled the sequence and evolutionary space of LisH domains, and tested their activity in a synthetic transcriptional repression assay in Saccharomyces cerevisiae. Using this approach, we found that repression activity was highly conserved and likely the ancestral function of this motif. We also identified key residues that contribute to repressive function. We leveraged this new knowledge for two applications. First, we tested the role of mutations found in somatic cancers on repression function in two human LisH-containing proteins. Second, we validated function of many of our repression domains in plants, confirming that these sequences should be of use to synthetic biology applications across many eukaryotes.

Expansion and innovation in auxin signaling: where do we grow from here?

The phytohormone auxin plays a role in almost all growth and developmental responses. The primary mechanism of auxin action involves the regulation of transcription via a core signaling pathway comprising proteins belonging to three classes: receptors, co-receptor/co-repressors and transcription factors. Recent studies have revealed that auxin signaling can be traced back at least as far as the transition to land. Moreover, studies in flowering plants have highlighted how expansion of the gene families encoding auxin components is tied to functional diversification. As we review here, these studies paint a picture of auxin signaling evolution as a driver of innovation.

A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana.

Root architecture is a major determinant of plant fitness and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia in Arabidopsis thaliana and discovered many upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate and demonstrated that the expression of several of these targets is required for normal root development. We also discovered subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition.

Decoding and recoding plant development.

The development of multicellular organisms has been studied for centuries, yet many critical events and mechanisms of regulation remain challenging to observe directly. Early research focused on detailed observational and comparative studies. Molecular biology has generated insights into regulatory mechanisms, but only for a limited number of species. Now, synthetic biology is bringing these two approaches together, and by adding the possibility of sculpting novel morphologies, opening another path to understanding biology. Here, we review a variety of recently invented techniques that use CRISPR/Cas9 and phage integrases to trace the differentiation of cells over various timescales, as well as to decode the molecular states of cells in high spatiotemporal resolution. Most of these tools have been implemented in animals. The time is ripe for plant biologists to adopt and expand these approaches. Here, we describe how these tools could be used to monitor development in diverse plant species, as well as how they could guide efforts to recode programs of interest.

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 (Arabidopsis thaliana). Here, we used the nuclear Auxin Response Circuit recapitulated in yeast (Saccharomyces cerevisiae) system to functionally annotate maize (Zea mays) auxin signaling components, focusing on genes expressed during the development of ear and tassel inflorescences. All 16 maize auxin/indole-3-acetic acid repressor proteins were degraded in response to auxin with rates that depended on both receptor and repressor identities. When fused to the maize TOPLESS homolog RAMOSA1 ENHANCER LOCUS2, maize auxin/indole-3-acetic acids were able to repress AUXIN RESPONSE FACTOR transcriptional activity. A complete auxin response circuit comprising all maize components, including the ZmAFB2/3 b1 maize AUXIN SIGNALING F-BOX (AFB) receptor, was fully functional. The ZmAFB2/3 b1 auxin receptor was more sensitive to hormone than AtAFB2 and allowed for rapid circuit activation upon auxin addition. These results validate the conserved role of predicted auxin response genes in maize as well as provide evidence that a synthetic approach can facilitate broader comparative studies across the wide range of species with sequenced genomes.

Complex Relationships between Chromatin Accessibility, Sequence Divergence, and Gene Expression in Arabidopsis thaliana.

Variation in regulatory DNA is thought to drive phenotypic variation, evolution, and disease. Prior studies of regulatory DNA and transcription factors across animal species highlighted a fundamental conundrum: Transcription factor binding domains and cognate binding sites are conserved, while regulatory DNA sequences are not. It remains unclear how conserved transcription factors and dynamic regulatory sites produce conserved expression patterns across species. Here, we explore regulatory DNA variation and its functional consequences within Arabidopsis thaliana, using chromatin accessibility to delineate regulatory DNA genome-wide. Unlike in previous cross-species comparisons, the positional homology of regulatory DNA is maintained among A. thaliana ecotypes and less nucleotide divergence has occurred. Of the ∼50,000 regulatory sites in A. thaliana, we found that 15% varied in accessibility among ecotypes. Some of these accessibility differences were associated with extensive, previously unannotated sequence variation, encompassing many deletions and ancient hypervariable alleles. Unexpectedly, for the majority of such regulatory sites, nearby gene expression was unaffected. Nevertheless, regulatory sites with high levels of sequence variation and differential chromatin accessibility were the most likely to be associated with differential gene expression. Finally, and most surprising, we found that the vast majority of differentially accessible sites show no underlying sequence variation. We argue that these surprising results highlight the necessity to consider higher-order regulatory context in evaluating regulatory variation and predicting its phenotypic consequences.

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. In addition, monomeric Aux/IAAs were able to repress ARF activity in both yeast and plants. This systematic, quantitative structure-function analysis identified a minimal complex-comprising a single Aux/IAA repressing a pair of dimerized ARFs-sufficient for auxin-induced transcription.

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. Here we show that TIR1 can oligomerize in planta via a set of spatially clustered amino acid residues. While none of the residues identified reside in the interaction interface of the TIR1-Aux/IAA degron, they nonetheless regulate the binding of TIR1 to Aux/IAA substrate proteins and their subsequent degradation in vivo as an essential aspect of auxin signaling. We propose oligomerization of TIR1 as a novel regulatory mechanism in the regulation of auxin-mediated plant patterning and development.

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. Here, we use the well-established model of lateral root development to directly test the hypothesis that the rate of auxin-induced Aux/IAA turnover sets the pace for auxin-regulated developmental events. We did this by generating transgenic plants expressing degradation rate variants of IAA14, a crucial determinant of lateral root initiation. Progression through the well-established stages of lateral root development was strongly correlated with the engineered rates of IAA14 turnover, leading to the conclusion that Aux/IAAs are auxin-initiated timers that synchronize developmental transitions.

Phytochrome B regulates resource allocation in Brassica rapa.

Crop biomass and yield are tightly linked to how the light signaling network translates information about the environment into allocation of resources, including photosynthates. Once activated, the phytochrome (phy) class of photoreceptors signal and re-deploy carbon resources to alter growth, plant architecture, and reproductive timing. Most of the previous characterization of the light-modulated growth program has been performed in the reference plant Arabidopsis thaliana. Here, we use Brassica rapa as a crop model to test for conservation of the phytochrome-carbon network. In response to elevated levels of CO2, B. rapa seedlings showed increases in hypocotyl length, shoot and root fresh weight, and the number of lateral roots. All of these responses were dependent on nitrogen and polar auxin transport. In addition, we identified putative B. rapa orthologs of PhyB and isolated two nonsense alleles. BrphyB mutants had significantly decreased or absent CO2-stimulated growth responses. Mutant seedlings also showed misregulation of auxin-dependent genes and genes involved in chloroplast development. Adult mutant plants had reduced chlorophyll levels, photosynthetic rate, stomatal index, and seed yield. These findings support a recently proposed holistic role for phytochromes in regulating resource allocation, biomass production, and metabolic state in the developing plant.

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. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.

As above, so below: Auxin's role in lateral organ development.

Organogenesis requires the coordination of many highly-regulated developmental processes, including cell fate determination, cell division and growth, and cell-cell communication. For tissue- and organ-scale coordination, a network of regulators enables molecular events in individual cells to translate into multicellular changes in structure and functional capacity. One recurrent theme in plant developmental networks is a central role for plant hormones, especially auxin. Here, we focus first on describing recent advances in understanding lateral root development, one of the best-studied examples of auxin-mediated organogenesis. We then use this framework to examine the parallel process of emergence of lateral organs in the shoot-a process called phyllotaxy. This comparison reveals a high degree of conservation, highlighting auxin's pivotal role determining overall plant architecture.

Recapitulation of the forward nuclear auxin response pathway in yeast.

Auxin influences nearly every aspect of plant biology through a simple signaling pathway; however, it remains unclear how much of the diversity in auxin effects is explained by variation in the core signaling components and which properties of these components may contribute to diversification in response dynamics. Here, we recapitulated the entire Arabidopsis thaliana forward nuclear auxin signal transduction pathway in Saccharomyces cerevisiae to test whether signaling module composition enables tuning of the dynamic response. Sensitivity analysis guided by a small mathematical model revealed the centrality of auxin/indole-3-acetic acid (Aux/IAA) transcriptional corepressors in controlling response dynamics and highlighted the strong influence of natural variation in Aux/IAA degradation rates on circuit performance. When the basic auxin response circuit was expanded to include multiple Aux/IAAs, we found that dominance relationships between coexpressed Aux/IAAs were sufficient to generate distinct response modules similar to those seen during plant development. Our work provides a new method for dissecting auxin signaling and demonstrates the key role of Aux/IAAs in tuning auxin response dynamics.

Auxin 2012: a rich mea ho'oulu.

In December 2012, scientists from around the world gathered in Waikoloa, Hawaii for 'Auxin 2012', a meeting organized by Paula McSteen (University of Missouri, USA), Ben Scheres (Utrecht University, The Netherlands) and Yunde Zhao (University of California, San Diego, USA). At the meeting, participants discussed the latest advances in auxin biosynthesis, transport and signaling research, in addition to providing context for how these pathways intersect with other aspects of plant physiology and development. Fittingly, the meeting began with a traditional Hawaiian ceremony that recognized the centrality of the harvest of plant life ('mea ho'oulu' in Hawaiian) for continued human survival.

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. We have used synthetic auxin degradation assays in yeast (Saccharomyces cerevisiae) and in plants to characterize motifs flanking the degron that contribute to tuning the dynamics of Aux/IAA degradation. The presence of these rate motifs is conserved in phylogenetically distant members of the Arabidopsis (Arabidopsis thaliana) Aux/IAA family, as well as in their putative Brassica rapa orthologs. We found that rate motifs can act by enhancing interaction between repressors and the E3, but that this is not the only mechanism of action. Phenotypes of transgenic plants expressing a deletion in a rate motif in IAA28 resembled plants expressing degron mutations, underscoring the functional relevance of Aux/IAA degradation dynamics in regulating auxin responses.

The effects of DELLAs on growth change with developmental stage and brassinosteroid levels.

There are two stages in photomorphogenesis. First, seedlings detect light and open their cotyledons. Second, seedlings optimize their light environment by controlled elongation of the seedling stem or hypocotyl. In this study, we used time-lapse imaging to investigate the relationship between the brassinosteroid (BR) and gibberellin (GA) hormones across both stages of photomorphogenesis. During the transition between one stage and the other, growth promotion by BRs and GAs switched from an additive to a synergistic relationship. Molecular genetic analysis revealed unexpected roles for known participants in the GA pathway during this period. Members of the DELLA family could either repress or enhance BR growth responses, depending on developmental stage. At the transition point for seedling growth dynamics, the BR and GA pathways had opposite effects on DELLA protein levels. In contrast to GA-induced DELLA degradation, BR treatments increased the levels of REPRESSOR of ga1-3 (RGA) and mimicked the molecular effects of stabilizing DELLAs. In addition, DELLAs showed complex regulation of genes involved in BR biosynthesis, implicating them in BR homeostasis. Growth promotion by GA alone depended on the PHYTOCHROME INTERACTING FACTOR (PIF) family of master growth regulators. The effects of BR, including the synergistic effects with GA, were largely independent of PIFs. These results point to a multi-level, dynamic relationship between the BR and GA pathways.

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). Two tir1 mutations were identified that increased interaction with Aux/IAAs. The D170E and M473L mutations increase affinity between TIR1 and the degron motif of Aux/IAAs and enhance the activity of the SCF(TIR1) complex. This resulted in faster degradation of Aux/IAAs and increased transcription of auxin-responsive genes in the plant. Plants carrying the pTIR1:tir1 D170E/M473L-Myc transgene exhibit diverse developmental defects during plant growth and display an auxin-hypersensitive phenotype. This work demonstrates that changes in the leucine-rich repeat domain of the TIR1 auxin coreceptor can alter the properties of SCF(TIR1).

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.