Motif models proposing independent and interdependent impacts of nucleotides are related to high and low affinity transcription factor binding sites in Arabidopsis.

Position weight matrix (PWM) is the traditional motif model representing the transcription factor (TF) binding sites. It proposes that the positions contribute independently to TFs binding affinity, although this hypothesis does not fit the data perfectly. This explains why PWM hits are missing in a substantial fraction of ChIP-seq peaks. To study various modes of the direct binding of plant TFs, we compiled the benchmark collection of 111 ChIP-seq datasets for Arabidopsis thaliana, and applied the traditional PWM, and two alternative motif models BaMM and SiteGA, proposing the dependencies of the positions. The variation in the stringency of the recognition thresholds for the models proposed that the hits of PWM, BaMM, and SiteGA models are associated with the sites of high/medium, any, and low affinity, respectively. At the medium recognition threshold, about 60% of ChIP-seq peaks contain PWM hits consisting of conserved core consensuses, while BaMM and SiteGA provide hits for an additional 15% of peaks in which a weaker core consensus is compensated through intra-motif dependencies. The presence/absence of these dependencies in the motifs of alternative/traditional models was confirmed by the dependency logo DepLogo visualizing the position-wise partitioning of the alignments of predicted sites. We exemplify the detailed analysis of ChIP-seq profiles for plant TFs CCA1, MYC2, and SEP3. Gene ontology (GO) enrichment analysis revealed that among the three motif models, the SiteGA had the highest portions of genes with the significantly enriched GO terms among all predicted genes. We showed that both alternative motif models provide for traditional PWM greater extensions in predicted sites for TFs MYC2/SEP3 with condition/tissue specific functions, compared to those for TF CCA1 with housekeeping functions. Overall, the combined application of standard and alternative motif models is beneficial to detect various modes of the direct TF-DNA interactions in the maximal portion of ChIP-seq loci.

Tissue-specific transcriptome profiling of the Arabidopsis inflorescence stem reveals local cellular signatures.

Genome-wide gene expression maps with a high spatial resolution have substantially accelerated plant molecular science. However, the number of characterized tissues and growth stages is still small due to the limited accessibility of most tissues for protoplast isolation. Here, we provide gene expression profiles of the mature inflorescence stem of Arabidopsis thaliana covering a comprehensive set of distinct tissues. By combining fluorescence-activated nucleus sorting and laser-capture microdissection with next-generation RNA sequencing, we characterized the transcriptomes of xylem vessels, fibers, the proximal and distal cambium, phloem, phloem cap, pith, starch sheath, and epidermis cells. Our analyses classified more than 15,000 genes as being differentially expressed among different stem tissues and revealed known and novel tissue-specific cellular signatures. By determining overrepresented transcription factor binding regions in the promoters of differentially expressed genes, we identified candidate tissue-specific transcriptional regulators. Our datasets predict the expression profiles of an exceptional number of genes and allow hypotheses to be generated about the spatial organization of physiological processes. Moreover, we demonstrate that information about gene expression in a broad range of mature plant tissues can be established at high spatial resolution by nuclear mRNA profiling. Tissue-specific gene expression values can be accessed online at https://arabidopsis-stem.cos.uni-heidelberg.de/.

Cell Dynamics in WOX5-Overexpressing Root Tips: The Impact of Local Auxin Biosynthesis.

Root stem cell niche functioning requires the formation and maintenance of the specific "auxin-rich domain" governed by directional auxin transport and local auxin production. Auxin maximum co-localizes with the WOX5 expression domain in the quiescent center that separates mitotically active proximal and distal root meristems. Here we unravel the interconnected processes happening under WOX5 overexpression by combining in vivo experiments and mathematical modeling. We showed that WOX5-induced TAA1-mediated auxin biosynthesis is the cause, whereas auxin accumulation, PIN transporters relocation, and auxin redistribution between proximal and distal root meristems are its subsequent effects that influence the formation of the well-described phenotype with an enlarged root cap. These findings helped us to clarify the role of WOX5, which serves as a local QC-specific regulator that activates biosynthesis of non-cell-autonomous signal auxin to regulate the distal meristem functioning. The mathematical model with WOX5-mediated auxin biosynthesis and auxin-regulated cell growth, division, and detachment reproduces the columella cells dynamics in both wild type and under WOX5 dysregulation.

metaRE R Package for Meta-Analysis of Transcriptome Data to Identify the cis-Regulatory Code behind the Transcriptional Reprogramming.

At the molecular level, response to an external factor or an internal condition causes reprogramming of temporal and spatial transcription. When an organism undergoes physiological and/or morphological changes, several signaling pathways are activated simultaneously. Examples of such complex reactions are the response to temperature changes, dehydration, various biologically active substances, and others. A significant part of the regulatory ensemble in such complex reactions remains unidentified. We developed metaRE, an R package for the systematic search for cis-regulatory elements enriched in the promoters of the genes significantly changed their transcription in a complex reaction. metaRE mines multiple expression profiling datasets generated to test the same organism's response and identifies simple and composite cis-regulatory elements systematically associated with differential expression of genes. Here, we showed metaRE performance for the identification of low-temperature-responsive cis-regulatory code in Arabidopsis thaliana and Danio rerio. MetaRE identified potential binding sites for known as well as unknown cold response regulators. A notable part of cis-elements was found in both searches discovering great conservation in low-temperature responses between plants and animals.

Fold-Change-Specific Enrichment Analysis (FSEA): Quantification of Transcriptional Response Magnitude for Functional Gene Groups.

Gene expression profiling data contains more information than is routinely extracted with standard approaches. Here we present Fold-Change-Specific Enrichment Analysis (FSEA), a new method for functional annotation of differentially expressed genes from transcriptome data with respect to their fold changes. FSEA identifies Gene Ontology (GO) terms, which are shared by the group of genes with a similar magnitude of response, and assesses these changes. GO terms found by FSEA are fold-change-specifically (e.g., weakly, moderately, or strongly) affected by a stimulus under investigation. We demonstrate that many responses to abiotic factors, mutations, treatments, and diseases occur in a fold-change-specific manner. FSEA analyses suggest that there are two prevailing responses of functionally-related gene groups, either weak or strong. Notably, some of the fold-change-specific GO terms are invisible by classical algorithms for functional gene enrichment, Singular Enrichment Analysis (SEA), and Gene Set Enrichment Analysis (GSEA). These are GO terms not enriched compared to the genome background but strictly regulated by a factor within specific fold-change intervals. FSEA analysis of a cancer-related transcriptome suggested that the gene groups with a tightly coordinated response can be the valuable source to search for possible regulators, markers, and therapeutic targets in oncogenic processes. Availability and Implementation: FSEA is implemented as the FoldGO Bioconductor R package and a web-server.

3D Analysis of Mitosis Distribution Pattern in the Plant Root Tip with iRoCS Toolbox.

The protocol allows to define and characterize mitosis distribution patterns in the plant root meristem. The method does not require genetic markers, which makes it applicable to plants of different non-transgenic genotypes, including ecotypes, mutants, and non-model plant species. Computer analysis of the mitosis distribution in three dimensions with iRoCS Toolbox identifies statistically significant changes in proliferation activity within specific root tissues and cell lineages.

Salicylic Acid Affects Root Meristem Patterning via Auxin Distribution in a Concentration-Dependent Manner.

The phytohormone salicylic acid (SA) is well known for its induction of pathogenesis-related proteins and systemic acquired resistance; SA also has specific effects on plant growth and development. Here we analyzed the effect of SA on Arabidopsis (Arabidopsis thaliana) root development. We show that exogenous SA treatment at low (below 50 µM) and high (greater than 50 µM) concentrations affect root meristem development in two different PR1-independent ways. Low-concentration SA promoted adventitious roots and altered architecture of the root apical meristem, whereas high-concentration SA inhibited all growth processes in the root. All exposures to exogenous SA led to changes in auxin synthesis and transport. A wide range of SA treatment concentrations activated auxin synthesis, but the effect of SA on auxin transport was dose dependent. Mathematical modeling of auxin synthesis and transport predicted auxin accumulation or depletion in the root tip following low- or high-concentration SA treatments, respectively. SA-induced auxin accumulation led to the formation of more layers of columella initials, an additional cortical cell layer (middle cortex), and extra files of epidermis, cortex, and endodermis cells. Suppression of SHORT ROOT and activation of CYCLIN D6;1 mediated the changes in radial architecture of the root. We propose that low-concentration SA plays an important role in shaping root meristem structure and root system architecture.

Deciphering Auxin-Ethylene Crosstalk at a Systems Level.

The auxin and ethylene pathways cooperatively regulate a variety of developmental processes in plants. Growth responses to ethylene are largely dependent on auxin, the key regulator of plant morphogenesis. Auxin, in turn, is capable of inducing ethylene biosynthesis and signaling, making the interaction of these hormones reciprocal. Recent studies discovered a number of molecular events underlying auxin-ethylene crosstalk. In this review, we summarize the results of fine-scale and large-scale experiments on the interactions between the auxin and ethylene pathways in Arabidopsis. We integrate knowledge on molecular crosstalk events, their tissue specificity, and associated phenotypic responses to decipher the crosstalk mechanisms at a systems level. We also discuss the prospects of applying systems biology approaches to study the mechanisms of crosstalk between plant hormones.

3D analysis of mitosis distribution highlights the longitudinal zonation and diarch symmetry in proliferation activity of the Arabidopsis thaliana root meristem.

To date CYCB1;1 marker and cortex cell lengths have been conventionally used to determine the proliferation activity of the Arabidopsis root meristem. By creating a 3D map of mitosis distribution we showed that these markers overlooked that stele and endodermis save their potency to divide longer than the cortex and epidermis. Cessation of cell divisions is not a random process, so that mitotic activity within the endodermis and stele shows a diarch pattern. Mitotic activity of all root tissues peaked at the same distance from the quiescent center (QC); however, different tissues stopped dividing at different distances, with cells of the protophloem exiting the cell cycle first and the procambial cells being the last. The robust profile of mitotic activity in the root tip defines the longitudinal zonation in the meristem with the proliferation domain, where all cells are able to divide; and the transition domain, where the cell files cease to divide. 3D analysis of cytokinin deficient and cytokinin signaling mutants showed that their proliferation domain is similar to that of the wild type, but the transition domain is much longer. Our data suggest a strong inhibitory effect of cytokinin on anticlinal cell divisions in the stele.

A Sacrifice-for-Survival Mechanism Protects Root Stem Cell Niche from Chilling Stress.

Temperature has a profound influence on plant and animal development, but its effects on stem cell behavior and activity remain poorly understood. Here, we characterize the responses of the Arabidopsis root to chilling (low but above-freezing) temperature. Chilling stress at 4°C leads to DNA damage predominantly in root stem cells and their early descendants. However, only newly generated/differentiating columella stem cell daughters (CSCDs) preferentially die in a programmed manner. Inhibition of the DNA damage response in these CSCDs prevents their death but makes the stem cell niche more vulnerable to chilling stress. Mathematical modeling and experimental validation indicate that CSCD death results in the re-establishment of the auxin maximum in the quiescent center (QC) and the maintenance of functional stem cell niche activity under chilling stress. This mechanism improves the root's ability to withstand the accompanying environmental stresses and to resume growth when optimal temperatures are restored.

Meta-analysis of transcriptome data identified TGTCNN motif variants associated with the response to plant hormone auxin in Arabidopsis thaliana L.

Auxin is the major regulator of plant growth and development. It regulates gene expression via a family of transcription factors (ARFs) that bind to auxin responsive elements (AuxREs) in the gene promoters. The canonical AuxREs found in regulatory regions of many auxin responsive genes contain the TGTCTC core motif, whereas ARF binding site is a degenerate TGTCNN with TGTCGG strongly preferred. Thereby two questions arise: which TGTCNN variants are functional AuxRE cores and whether different TGTCNN variants have distinct functional roles? In this study, we performed meta-analysis of microarray data to reveal TGTCNN variants essential for auxin response and to characterize their functional features. Our results indicate that four TGTCNN motifs (TGTCTC, TGTCCC, TGTCGG, and TGTCTG) are associated with auxin up-regulation and two (TGTCGG, TGTCAT) with auxin down-regulation, but to a lesser extent. The genes having some of these motifs in their regulatory regions showed time-specific auxin response. Functional annotation of auxin up- and down-regulated genes also revealed GO terms specific for the auxin-regulated genes with certain TGTCNN variants in their promoters. Our results provide an idea that various TGTCNN motifs may play distinct roles in the auxin regulation of gene expression.

The Interplay of Chromatin Landscape and DNA-Binding Context Suggests Distinct Modes of EIN3 Regulation in Arabidopsis thaliana.

The plant hormone ethylene regulates numerous developmental processes and stress responses. Ethylene signaling proceeds via a linear pathway, which activates transcription factor (TF) EIN3, a primary transcriptional regulator of ethylene response. EIN3 influences gene expression upon binding to a specific sequence in gene promoters. This interaction, however, might be considerably affected by additional co-factors. In this work, we perform whole genome bioinformatics study to identify the impact of epigenetic factors in EIN3 functioning. The analysis of publicly available ChIP-Seq data on EIN3 binding in Arabidopsis thaliana showed bimodality of distribution of EIN3 binding regions (EBRs) in gene promoters. Besides a sharp peak in close proximity to transcription start site, which is a common binding region for a wide variety of TFs, we found an additional extended peak in the distal promoter region. We characterized all EBRs with respect to the epigenetic status appealing to previously published genome-wide map of nine chromatin states in A. thaliana. We found that the implicit distal peak was associated with a specific chromatin state (referred to as chromatin state 4 in the primary source), which was just poorly represented in the pronounced proximal peak. Intriguingly, EBRs corresponding to this chromatin state 4 were significantly associated with ethylene response, unlike the others representing the overwhelming majority of EBRs related to the explicit proximal peak. Moreover, we found that specific EIN3 binding sequences predicted with previously described model were enriched in the EBRs mapped to the chromatin state 4, but not to the rest ones. These results allow us to conclude that the interplay of genetic and epigenetic factors might cause the distinct modes of EIN3 regulation.

Computational analysis of auxin responsive elements in the Arabidopsis thaliana L. genome.

Auxin responsive elements (AuxRE) were found in upstream regions of target genes for ARFs (Auxin response factors). While Chip-seq data for most of ARFs are still unavailable, prediction of potential AuxRE is restricted by consensus models that detect too many false positive sites. Using sequence analysis of experimentally proven AuxREs, we revealed both an extended nucleotide context pattern for AuxRE itself and three distinct types of its coupling motifs (Y-patch, AuxRE-like, and ABRE-like), which together with AuxRE may form the composite elements. Computational analysis of the genome-wide distribution of the predicted AuxREs and their impact on auxin responsive gene expression allowed us to conclude that: (1) AuxREs are enriched around the transcription start site with the maximum density in 5'UTR; (2) AuxREs mediate auxin responsive up-regulation, not down-regulation. (3) Directly oriented single AuxREs and reverse multiple AuxREs are mostly associated with auxin responsiveness. In the composite AuxRE elements associated with auxin response, ABRE-like and Y-patch are 5'-flanking or overlapping AuxRE, whereas AuxRE-like motif is 3'-flanking. The specificity in location and orientation of the coupling elements suggests them as potential binding sites for ARFs partners.

Mathematical modeling of auxin transport in protoxylem and protophloem of Arabidopsis thaliana root tips.

Phytohormone auxin is the main regulator of plant growth and development. Nonuniform auxin distribution in plant tissue sets positional information, which determines morphogenesis. Auxin is transported in tissue by means of diffusion and active transport through the cell membrane. There is a number of auxin carriers performing its influx into a cell (AUX\LAX family) or efflux from a cell (PIN, PGP families). The paper presents mathematical models for auxin transport in vascular tissues of Arabidopsis thaliana L.root tip, namely protophloem and protoxylem. Tissue specificity of auxin active transport was considered in these models. There is PIN-mediated auxin efflux in both protoxylem and protophloem, but AUX1-mediated influx exists only in protophloem. Optimal values of parameters were adjusted for model solutions to fit the experimentally observed auxin distributions in the root tip. Based on simulation results we predicted that shoot-derived auxin flow to protophloem is lower than one to protoxylem, and the efficiency of PIN-mediated auxin transport in protophloem is higher than in protoxylem. In summary, our simulation showed that despite the same auxin distribution pattern, provascular tissues in the root tip differ in dynamics of auxin transport.

How multiple auxin responsive elements may interact in plant promoters: a reverse problem solution.

Plant hormone auxin is a key regulator of growth and development. Auxin affects gene expression through ARF transcription factors, which bind specifically auxin responsive elements (AuxREs). Auxin responsive genes usually have more than one AuxRE, for example, a widely used auxin sensor DR5 contains seven AuxREs. Auxin responsive regions of several plant genes have been studied using sets of transgenic constructions in which the activity of one or several AuxREs were abolished. Here we present the method for analysis of the datasets on promoter activity assays having promoter sequences, namely, number and sequences of AuxREs, altogether with their measured auxin induction level. The method for a reverse problem solution considers two extreme models of AuxRE cooperation. Additive model describes auxin induction level of a gene as a sum of the individual AuxREs impacts. Multiplicative model considers pure cooperation between the AuxREs, where the combined effect is the multiplication of the individual AuxRE impacts. The reverse problem solution allows estimating the impact of an individual AuxRE into the induction level and the model for their cooperation. For promoters of three genes belonging to different plant species we showed that the multiplicative model fits better than additive. The reverse problem solution also suggests repressive state of auxin responsive promoters before auxin induction. The developed method provides possibility to investigate AuxRE structure-activity relationship and may be used as the basis for a novel approach for AuxRE recognition.

A plausible mechanism for auxin patterning along the developing root.

BACKGROUND: In plant roots, auxin is critical for patterning and morphogenesis. It regulates cell elongation and division, the development and maintenance of root apical meristems, and other processes. In Arabidopsis, auxin distribution along the central root axis has several maxima: in the root tip, in the basal meristem and at the shoot/root junction. The distal maximum in the root tip maintains the stem cell niche. Proximal maxima may trigger lateral or adventitious root initiation. RESULTS: We propose a reflected flow mechanism for the formation of the auxin maximum in the root apical meristem. The mechanism is based on auxin's known activation and inhibition of expressed PIN family auxin carriers at low and high auxin levels, respectively. Simulations showed that these regulatory interactions are sufficient for self-organization of the auxin distribution pattern along the central root axis under varying conditions. The mathematical model was extended with rules for discontinuous cell dynamics so that cell divisions were also governed by auxin, and by another morphogen Division Factor which combines the actions of cytokinin and ethylene on cell division in the root. The positional information specified by the gradients of these two morphogens is able to explain root patterning along the central root axis. CONCLUSION: We present here a plausible mechanism for auxin patterning along the developing root, that may provide for self-organization of the distal auxin maximum when the reverse fountain has not yet been formed or has been disrupted. In addition, the proximal maxima are formed under the reflected flow mechanism in response to periods of increasing auxin flow from the growing shoot. These events may predetermine lateral root initiation in a rhyzotactic pattern. Another outcome of the reflected flow mechanism - the predominance of lateral or adventitious roots in different plant species - may be based on the different efficiencies with which auxin inhibits its own transport in different species, thereby distinguishing two main types of plant root architecture: taproot vs. fibrous.

A cellular automaton to model the development of primary shoot meristems of Arabidopsis thaliana.

Development of organisms is a very complex process in which a lot of gene networks of different cell types are integrated. Development of a cellular automaton (Ermentrout and Edelshtein-Keshet, J Theor Biol 160:97-133, 1993) that models the morphodynamics of different cell types is the first step in understanding and analysis of the regulatory mechanisms underlying the functioning of developmental gene networks. A model of a cellular automaton has been developed, which simulates the embryonic development of shoot meristem in Arabidopsis thaliana. The model adequately describes the basic stages in development of this organ in wild and mutant types.