Identification of potential Auxin Response Candidate genes for soybean rapid canopy coverage through comparative evolution and expression analysis.

Glycine max, soybean, is an abundantly cultivated crop worldwide. Efforts have been made over the past decades to improve soybean production in traditional and organic agriculture, driven by growing demand for soybean-based products. Rapid canopy cover development (RCC) increases soybean yields and suppresses early-season weeds. Genome-wide association studies have found natural variants associated with RCC, however causal mechanisms are unclear. Auxin modulates plant growth and development and has been implicated in RCC traits. Therefore, modulation of auxin regulatory genes may enhance RCC. Here, we focus on the use of genomic tools and existing datasets to identify auxin signaling pathway RCC candidate genes, using a comparative phylogenetics and expression analysis approach. We identified genes encoding 14 TIR1/AFB auxin receptors, 61 Aux/IAA auxin co-receptors and transcriptional co-repressors, and 55 ARF auxin response factors in the soybean genome. We used Bayesian phylogenetic inference to identify soybean orthologs of Arabidopsis thaliana genes, and defined an ortholog naming system for these genes. To further define potential auxin signaling candidate genes for RCC, we examined tissue-level expression of these genes in existing datasets and identified highly expressed auxin signaling genes in apical tissues early in development. We identified at least 4 TIR1/AFB, 8 Aux/IAA, and 8 ARF genes with highly specific expression in one or more RCC-associated tissues. We hypothesize that modulating the function of these genes through gene editing or traditional breeding will have the highest likelihood of affecting RCC while minimizing pleiotropic effects.

The TARGET System: Rapid Identification of Direct Targets of Transcription Factors by Gene Regulation in Plant Cells.

The TARGET system allows for the rapid identification of direct regulated gene targets of transcription factors (TFs). It employs the transient transformation of plant protoplasts with inducible nuclear entry of the TF and subsequent transcriptomic and/or ChIP-seq analysis. The ability to separate direct TF-target gene regulatory interactions from indirect downstream responses and the significantly shorter amount of time required to perform the assay, compared to the generation of transgenics, make this plant cell-based approach a valuable tool for a higher throughput approach to identify the genome-wide targets of multiple TFs, to build validated transcriptional networks in plants. Here, we describe the use of the TARGET system in Arabidopsis seedling root protoplasts to map the gene regulatory network downstream of transcription factors-of-interest.

Leaf cell-specific and single-cell transcriptional profiling reveals a role for the palisade layer in UV light protection.

Like other complex multicellular organisms, plants are composed of different cell types with specialized shapes and functions. For example, most laminar leaves consist of multiple photosynthetic cell types. These cell types include the palisade mesophyll, which typically forms one or more cell layers on the adaxial side of the leaf. Despite their importance for photosynthesis, we know little about how palisade cells differ at the molecular level from other photosynthetic cell types. To this end, we have used a combination of cell-specific profiling using fluorescence-activated cell sorting and single-cell RNA-sequencing methods to generate a transcriptional blueprint of the palisade mesophyll in Arabidopsis thaliana leaves. We find that despite their unique morphology, palisade cells are otherwise transcriptionally similar to other photosynthetic cell types. Nevertheless, we show that some genes in the phenylpropanoid biosynthesis pathway have both palisade-enriched expression and are light-regulated. Phenylpropanoid gene activity in the palisade was required for production of the ultraviolet (UV)-B protectant sinapoylmalate, which may protect the palisade and/or other leaf cells against damaging UV light. These findings improve our understanding of how different photosynthetic cell types in the leaf can function uniquely to optimize leaf performance, despite their transcriptional similarities.

Temporal Control of Morphogenic Factor Expression Determines Efficacy in Enhancing Regeneration.

BACKGROUND: Regeneration of fertile plants from tissue culture is a critical bottleneck in the application of new plant breeding technologies. Ectopic overexpression of morphogenic factors is a promising workaround for this hurdle. METHODS: Conditional overexpression of WUS and ARF5Δ was used to study the effect of timing the overexpression of these morphogenic factors during shoot regeneration from root explants in Arabidopsis. In addition, their effect on auxin-signaling activation was examined by visualization and cytometric quantification of the DR5:GFP auxin-signaling reporter in roots and protoplasts, respectively. RESULTS: The induced expression of both WUS and ARF5Δ led to an activation of auxin signaling in roots. Activation of auxin signaling by WUS and ARF5Δ was further quantified by transient transformation of protoplasts. Ectopic overexpression of both WUS and ARF5Δ enhanced regeneration efficiency, but only during the shoot-induction stage of regeneration and not during the callus-induction stage. CONCLUSIONS: The overexpression of WUS and ARF5Δ both lead to activation of auxin signaling. Expression during the shoot-induction stage is critical for the enhancement of shoot regeneration by WUS and ARF5Δ.

Protoplast Regeneration and Its Use in New Plant Breeding Technologies.

The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.

Characterization of the F Locus Responsible for Floral Anthocyanin Production in Potato.

Anthocyanins are pigmented secondary metabolites produced via the flavonoid biosynthetic pathway and play important roles in plant stress responses, pollinator attraction, and consumer preference. Using RNA-sequencing analysis of a cross between diploid potato (Solanum tuberosum L.) lines segregating for flower color, we identified a homolog of the ANTHOCYANIN 2 (AN2) gene family that encodes a MYB transcription factor, herein termed StFlAN2, as the regulator of anthocyanin production in potato corollas. Transgenic introduction of StFlAN2 in white-flowered homozygous doubled-monoploid plants resulted in a recovery of purple flowers. RNA-sequencing revealed the specific anthocyanin biosynthetic genes activated by StFlAN2 as well as expression differences in genes within pathways involved in fruit ripening, senescence, and primary metabolism. Closer examination of the locus using genomic sequence analysis revealed a duplication in the StFlAN2 locus closely associated with gene expression that is likely attributable to nearby genetic elements. Taken together, this research provides insight into the regulation of anthocyanin biosynthesis in potato while also highlighting how the dynamic nature of the StFlAN2 locus may affect expression.

Functional genomics identifies regulators of the phototransduction machinery in the Drosophila larval eye and adult ocelli.

Sensory perception of light is mediated by specialized Photoreceptor neurons (PRs) in the eye. During development all PRs are genetically determined to express a specific Rhodopsin (Rh) gene and genes mediating a functional phototransduction pathway. While the genetic and molecular mechanisms of PR development is well described in the adult compound eye, it remains unclear how the expression of Rhodopsins and the phototransduction cascade is regulated in other visual organs in Drosophila, such as the larval eye and adult ocelli. Using transcriptome analysis of larval PR-subtypes and ocellar PRs we identify and study new regulators required during PR differentiation or necessary for the expression of specific signaling molecules of the functional phototransduction pathway. We found that the transcription factor Krüppel (Kr) is enriched in the larval eye and controls PR differentiation by promoting Rh5 and Rh6 expression. We also identified Camta, Lola, Dve and Hazy as key genes acting during ocellar PR differentiation. Further we show that these transcriptional regulators control gene expression of the phototransduction cascade in both larval eye and adult ocelli. Our results show that PR cell type-specific transcriptome profiling is a powerful tool to identify key transcriptional regulators involved during several aspects of PR development and differentiation. Our findings greatly contribute to the understanding of how combinatorial action of key transcriptional regulators control PR development and the regulation of a functional phototransduction pathway in both larval eye and adult ocelli.

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 Arabidopsis, most of which are uncharacterized. TIR1 is among the best-studied plant FBPs and functions as a receptor for the plant hormone auxin. Here we use a yeast two-hybrid system to identify novel TIR1 mutants with altered properties. The analysis of these mutants reveals that TIR1 associates with the CULLIN1 (CUL1) subunit of the SCF through the N-terminal H1 helix of the F-box domain. Mutations that untether TIR1 from CUL1 stabilize the FBP and cause auxin resistance and associated growth defects, probably by protecting TIR1 substrates from degradation. Based on these results we propose that TIR1 is subject to autocatalytic degradation when assembled into an SCF. Further, our results suggest a general method for determining the physiological function of uncharacterized FBPs. Finally, we show that a key amino acid variation in the F-box domain of auxin signalling F-box (AFB1), a closely related FBP, reduces its ability to form an SCF, resulting in an increase in AFB1 levels.

Guard cell photosynthesis is critical for stomatal turgor production, yet does not directly mediate CO2 - and ABA-induced stomatal closing.

Stomata mediate gas exchange between the inter-cellular spaces of leaves and the atmosphere. CO2 levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO2 ]. [CO2 ] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll-deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll-deficient. Interestingly, approximately 45% of stomata had an unusual, previously not-described, morphology of thin-shaped chlorophyll-less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole-leaf photosynthetic parameters (PSII, qP, qN, FV '/FM' ) were comparable with wild-type plants. Time-resolved intact leaf gas-exchange analyses showed a reduction in stomatal conductance and CO2 -assimilation rates of the transgenic plants. Normalization of CO2 responses showed that stomata of transgenic plants respond to [CO2 ] shifts. Detailed stomatal aperture measurements of normal kidney-shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO2 ] elevation and abscisic acid (ABA), while thin-shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO2 ] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO2 and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll-less stomata cause a 'deflated' thin-shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.

Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes.

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1-5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, "hit-and-run" transcription model, which enables a "catalyst TF" to activate a large set of targets within minutes of signal perturbation.

Auxin perception: in the IAA of the beholder.

Auxin signaling through the SCF(TIR1)-Aux/IAA-ARF pathway is one of the best-studied plant hormone response pathways. Components of this pathway, from receptors through to transcription factors, have been identified and analyzed in detail. Although we understand elementary aspects of how the auxin signal is perceived and leads to a transcriptional response, many questions remain about the in vivo function of the pathway. Two crucial issues are the tissue specificity of the response, i.e. how distinct cell types can interpret the same auxin signal differently, and the response to a signaling gradient, i.e. how a graded distribution of auxin can elicit distinct expression patterns along its range. Here, we speculate on how signaling through the canonical SCF(TIR1)-Aux/IAA-ARF pathway may achieve divergent responses.

An undergraduate study of two transcription factors that promote lateral root formation.

We present a lab that enables students to test the role of genes involved in the regulation of lateral roots growth in the model plant Arabidopsis thaliana. Here, students design an experiment that follows the effects of the hormone auxin on the stimulation of genes involved in the formation of lateral root initials. These genes, known as lateral organ boundary domain containing protein (LBD) genes, are upregulated in the presence of auxin as part of a multistep molecular and biochemically controlled pathway. Depending on which LBD gene is tested, and the stage of root development, expression patterns are localized in a discrete and punctate fashion at the site of lateral root initials (LBD33), or reveal a broader localization pattern (LBD16). Students view expression using the reporter gene GUS (beta-glucuronidase). Before GUS staining, students view root growth in a "pseudo-aseptic" agar-based environment that allows complete visualization of whole root development to determine the proper stage to test molecular expression.

A map of cell type-specific auxin responses.

In plants, changes in local auxin concentrations can trigger a range of developmental processes as distinct tissues respond differently to the same auxin stimulus. However, little is known about how auxin is interpreted by individual cell types. We performed a transcriptomic analysis of responses to auxin within four distinct tissues of the Arabidopsis thaliana root and demonstrate that different cell types show competence for discrete responses. The majority of auxin-responsive genes displayed a spatial bias in their induction or repression. The novel data set was used to examine how auxin influences tissue-specific transcriptional regulation of cell-identity markers. Additionally, the data were used in combination with spatial expression maps of the root to plot a transcriptomic auxin-response gradient across the apical and basal meristem. The readout revealed a strong correlation for thousands of genes between the relative response to auxin and expression along the longitudinal axis of the root. This data set and comparative analysis provide a transcriptome-level spatial breakdown of the response to auxin within an organ where this hormone mediates many aspects of development.

Fluorescence-activated cell sorting for analysis of cell type-specific responses to salinity stress in Arabidopsis and rice.

Fluorescence-activated cell sorting (FACS) provides a rapid means of isolating large numbers of fluorescently tagged cells from a heterogeneous mixture of cells. Collections of transgenic plants with cell type-specific expression of fluorescent marker genes such as green fluorescent protein (GFP) are ideally suited for FACS-assisted studies of individual cell types. Here we describe the use of Arabidopsis and rice enhancer trap lines with tissue-specific GFP expression patterns in the root to isolate specific cell types of root tissues using FACS. Additionally, protocols are provided to impose a ramped salinity stress for 48 h prior to cell sorting.

High-throughput fluorescence-based isolation of live C. elegans larvae.

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.

Fluorescence activated cell sorting of plant protoplasts.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

Reassessing the role of phospholipase D in the Arabidopsis wounding response.

Plants respond to wounding by means of a multitude of reactions, with the purpose of stifling herbivore assault. Phospholipase D (PLD) has previously been implicated in the wounding response. Arabidopsis (Arabidopsis thaliana) AtPLDalpha1 has been proposed to be activated in intact cells, and the phosphatidic acid (PA) it produces to serve as a precursor for jasmonic acid (JA) synthesis and to be required for wounding-induced gene expression. Independently, PLD activity has been reported to have a bearing on wounding-induced MAPK activation. However, which PLD isoforms are activated, where this activity takes place (in the wounded or non-wounded cells) and what exactly the consequences are is a question that has not been comprehensively addressed. Here, we show that PLD activity during the wounding response is restricted to the ruptured cells using (32)P(i)-labelled phospholipid analyses of Arabidopsis pld knock-out mutants and PLD-silenced tomato cell-suspension cultures. pldalpha1 knock-out lines have reduced wounding-induced PA production, and the remainder is completely eliminated in a pldalpha1/delta double knock-out line. Surprisingly, wounding-induced protein kinase activation, AtLOX2 gene expression and JA biosynthesis were not affected in these knock-out lines. Moreover, larvae of the Cabbage White butterfly (Pieris rapae) grew equally well on wild-type and the pld knock-out mutants.