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.

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/.

Specification and regulation of vascular tissue identity in the Arabidopsis embryo.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.

A PLETHORA/PIN-FORMED/auxin network mediates prehaustorium formation in the parasitic plant Striga hermonthica.

The parasitic plant Striga (Striga hermonthica) invades the host root through the formation of a haustorium and has detrimental impacts on cereal crops. The haustorium results from the prehaustorium, which is derived directly from the differentiation of the Striga radicle. The molecular mechanisms leading to radicle differentiation shortly after germination remain unclear. In this study, we determined the developmental programs that regulate terminal prehaustorium formation in S. hermonthica at cellular resolution. We showed that shortly after germination, cells in the root meristem undergo multiplanar divisions. During growth, the meristematic activity declines and associates with reduced expression of the stem cell regulator PLETHORA1 and the cell cycle genes CYCLINB1 and HISTONE H4. We also observed a basal localization of the PIN-FORMED (PIN) proteins and a decrease in auxin levels in the meristem. Using the structural layout of the root meristem and the polarity of outer-membrane PIN proteins, we constructed a mathematical model of auxin transport that explains the auxin distribution patterns observed during S. hermonthica root growth. Our results reveal a fundamental molecular and cellular framework governing the switch of S. hermonthica roots to form the invasive prehaustoria.

Rocks in the auxin stream: Wound-induced auxin accumulation and ERF115 expression synergistically drive stem cell regeneration.

Plants are known for their outstanding capacity to recover from various wounds and injuries. However, it remains largely unknown how plants sense diverse forms of injury and canalize existing developmental processes into the execution of a correct regenerative response. Auxin, a cardinal plant hormone with morphogen-like properties, has been previously implicated in the recovery from diverse types of wounding and organ loss. Here, through a combination of cellular imaging and in silico modeling, we demonstrate that vascular stem cell death obstructs the polar auxin flux, much alike rocks in a stream, and causes it to accumulate in the endodermis. This in turn grants the endodermal cells the capacity to undergo periclinal cell division to repopulate the vascular stem cell pool. Replenishment of the vasculature by the endodermis depends on the transcription factor ERF115, a wound-inducible regulator of stem cell division. Although not the primary inducer, auxin is required to maintain ERF115 expression. Conversely, ERF115 sensitizes cells to auxin by activating ARF5/MONOPTEROS, an auxin-responsive transcription factor involved in the global auxin response, tissue patterning, and organ formation. Together, the wound-induced auxin accumulation and ERF115 expression grant the endodermal cells stem cell activity. Our work provides a mechanistic model for wound-induced stem cell regeneration in which ERF115 acts as a wound-inducible stem cell organizer that interprets wound-induced auxin maxima.

Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis.

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF-DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Mechanisms of stress response in the root stem cell niche.

As plants are sessile organisms unable to escape from environmental hazards, they need to adapt for survival. The stem cell niche in the root apical meristem is particularly sensitive to DNA damage induced by environmental stresses such as chilling, flooding, wounding, UV, and irradiation. DNA damage has been proven to cause stem cell death, with stele stem cells being the most vulnerable. Stress also induces the division of quiescent center cells. Both reactions disturb the structure and activity of the root stem cell niche temporarily; however, this preserves root meristem integrity and function in the long term. Plants have evolved many mechanisms that ensure stem cell niche maintenance, recovery, and acclimation, allowing them to survive in a changing environment. Here, we provide an overview of the cellular and molecular aspects of stress responses in the root stem cell niche.

A single ChIP-seq dataset is sufficient for comprehensive analysis of motifs co-occurrence with MCOT package.

Recognition of composite elements consisting of two transcription factor binding sites gets behind the studies of tissue-, stage- and condition-specific transcription. Genome-wide data on transcription factor binding generated with ChIP-seq method facilitate an identification of composite elements, but the existing bioinformatics tools either require ChIP-seq datasets for both partner transcription factors, or omit composite elements with motifs overlapping. Here we present an universal Motifs Co-Occurrence Tool (MCOT) that retrieves maximum information about overrepresented composite elements from a single ChIP-seq dataset. This includes homo- and heterotypic composite elements of four mutual orientations of motifs, separated with a spacer or overlapping, even if recognition of motifs within composite element requires various stringencies. Analysis of 52 ChIP-seq datasets for 18 human transcription factors confirmed that for over 60% of analyzed datasets and transcription factors predicted co-occurrence of motifs implied experimentally proven protein-protein interaction of respecting transcription factors. Analysis of 164 ChIP-seq datasets for 57 mammalian transcription factors showed that abundance of predicted composite elements with an overlap of motifs compared to those with a spacer more than doubled; and they had 1.5-fold increase of asymmetrical pairs of motifs with one more conservative 'leading' motif and another one 'guided'.

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.

Meta-Analysis of Transcriptome Data Detected New Potential Players in Response to Dioxin Exposure in Humans.

Dioxins are one of the most potent anthropogenic poisons, causing systemic disorders in embryonic development and pathologies in adults. The mechanism of dioxin action requires an aryl hydrocarbon receptor (AhR), but the downstream mechanisms are not yet precisely clear. Here, we performed a meta-analysis of all available transcriptome datasets taken from human cell cultures exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Differentially expressed genes from different experiments overlapped partially, but there were a number of those genes that were systematically affected by TCDD. Some of them have been linked to toxic dioxin effects, but we also identified other attractive targets. Among the genes that were affected by TCDD, there are functionally related gene groups that suggest an interplay between retinoic acid, AhR, and Wnt signaling pathways. Next, we analyzed the upstream regions of differentially expressed genes and identified potential transcription factor (TF) binding sites overrepresented in the genes responding to TCDD. Intriguingly, the dioxin-responsive element (DRE), the binding site of AhR, was not overrepresented as much as other cis-elements were. Bioinformatics analysis of the AhR binding profile unveils potential cooperation of AhR with E2F2, CTCFL, and ZBT14 TFs in the dioxin response. We discuss the potential implication of these predictions for further dioxin studies.

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.

Diversity of cis-regulatory elements associated with auxin response in Arabidopsis thaliana.

The phytohormone auxin regulates virtually every developmental process in land plants. This regulation is mediated via de-repression of DNA-binding auxin response factors (ARFs). ARFs bind TGTC-containing auxin response cis-elements (AuxREs), but there is growing evidence that additional cis-elements occur in auxin-responsive regulatory regions. The repertoire of auxin-related cis-elements and their involvement in different modes of auxin response are not yet known. Here we analyze the enrichment of nucleotide hexamers in upstream regions of auxin-responsive genes associated with auxin up- or down-regulation, with early or late response, ARF-binding domains, and with different chromatin states. Intriguingly, hexamers potentially bound by basic helix-loop-helix (bHLH) and basic leucine zipper (bZIP) factors as well as a family of A/T-rich hexamers are more highly enriched in auxin-responsive regions than canonical TGTC-containing AuxREs. We classify and annotate the whole spectrum of enriched hexamers and discuss their patterns of enrichment related to different modes of auxin response.

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.

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.

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.

Meet your MAKR: the membrane-associated kinase regulator protein family in the regulation of plant development.

A small family composed of BRI1 KINASE INHIBITOR1 (BKI1) and MEMBRANE-ASSOCIATED KINASE REGULATORS (MAKRs) has recently captured the attention of plant biologists, due to their involvement in developmental processes downstream of hormones and Receptor-Like Kinases (RLK) signalling. BKI1/MAKRs are intrinsically disordered proteins (so-called unstructured proteins) and as such lack specific domains. Instead, they are defined by the presence of two conserved linear motifs involved in the interaction with lipids and proteins, respectively. Here, we first relate the discovery of the MAKR gene family. Then, we review the individual function of characterized family members and discuss their shared and specific modes of action. Finally, we explore and summarize the structural, comparative and functional genomics data available on this gene family. Together, this review aims at building a comprehensive reference about BKI1/MAKR protein function in plants.

CisCross: A gene list enrichment analysis to predict upstream regulators in Arabidopsis thaliana.

Having DNA-binding profiles for a sufficient number of genome-encoded transcription factors (TFs) opens up the perspectives for systematic evaluation of the upstream regulators for the gene lists. Plant Cistrome database, a large collection of TF binding profiles detected using the DAP-seq method, made it possible for Arabidopsis. Here we re-processed raw DAP-seq data with MACS2, the most popular peak caller that leads among other ones according to quality metrics. In the benchmarking study, we confirmed that the improved collection of TF binding profiles supported a more precise gene list enrichment procedure, and resulted in a more relevant ranking of potential upstream regulators. Moreover, we consistently recovered the TF binding profiles that were missing in the previous collection of DAP-seq peak sets. We developed the CisCross web service (https://plamorph.sysbio.ru/ciscross/) that gives more flexibility in the analysis of potential upstream TF regulators for Arabidopsis thaliana genes.

Transcriptional regulation in plants: Using omics data to crack the cis-regulatory code.

Innovative omics technologies, advanced bioinformatics, and machine learning methods are rapidly becoming integral tools for plant functional genomics, with tremendous recent advances made in this field. In transcriptional regulation, an initial lag in the accumulation of plant omics data relative to that of animals stimulated the development of computational methods capable of extracting maximum information from the available data sets. Recent comprehensive studies of transcription factor-binding profiles in Arabidopsis and maize and the accumulation of uniformly processed omics data in public databases have brought plant biologists into the big leagues, with many cutting-edge methods available. Here, we summarize the state-of-the-art bioinformatics approaches used to predict or infer the cis-regulatory code behind transcriptional gene regulation, focusing on their plant research applications.

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.

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.