Regulation of Division and Differentiation of Plant Stem Cells.

A major challenge in developmental biology is unraveling the precise regulation of plant stem cell maintenance and the transition to a fully differentiated cell. In this review, we highlight major themes coordinating the acquisition of cell identity and subsequent differentiation in plants. Plant cells are immobile and establish position-dependent cell lineages that rely heavily on external cues. Central players are the hormones auxin and cytokinin, which balance cell division and differentiation during organogenesis. Transcription factors and miRNAs, many of which are mobile in plants, establish gene regulatory networks that communicate cell position and fate. Small peptide signaling also provides positional cues as new cell types emerge from stem cell division and progress through differentiation. These pathways recruit similar players for patterning different organs, emphasizing the modular nature of gene regulatory networks. Finally, we speculate on the outstanding questions in the field and discuss how they may be addressed by emerging technologies.

Characterizing Auxin Response Circuits in Saccharomyces cerevisiae by Flow Cytometry.

Recapitulation of the nuclear auxin response pathway in Saccharomyces cerevisiae (yeast) provides a means to functionally assay the contribution of individual signaling components to response dynamics. Here, we describe a time course assay for characterizing auxin response circuits using flow cytometry. This method allows for quantitative measurements of the dynamic response of up to 12 circuits (strains) at once. We also describe a steady-state assay and how to utilize an R package we developed to facilitate data analysis.

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.

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.

Development of a robust method for isolation of shiga toxin-positive Escherichia coli (STEC) from fecal, plant, soil and water samples from a leafy greens production region in California.

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the "Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.

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. Here, we review recent progress towards characterizing and quantifying these interactions and highlight key gaps that remain.

A synthetic approach reveals extensive tunability of auxin signaling.

Explaining how the small molecule auxin triggers diverse yet specific responses is a long-standing challenge in plant biology. An essential step in auxin response is the degradation of Auxin/Indole-3-Acetic Acid (Aux/IAA, referred to hereafter as IAA) repressor proteins through interaction with auxin receptors. To systematically characterize diversity in degradation behaviors among IAA|receptor pairs, we engineered auxin-induced degradation of plant IAA proteins in yeast (Saccharomyces cerevisiae). We found that IAA degradation dynamics vary widely, depending on which receptor is present, and are not encoded solely by the degron-containing domain II. To facilitate this and future studies, we identified a mathematical model able to quantitatively describe IAA degradation behavior in a single parameter. Together, our results demonstrate the remarkable tunability conferred by specific configurations of the auxin response pathway.