Cytokinins regulate spatially-specific ethylene production to control root growth in Arabidopsis.

The two principal growth regulators cytokinins and ethylene are known to interact in the regulation of plant growth. However, information about underlying molecular mechanism and positional specificity of the cytokinin/ethylene crosstalk in root growth control is scarce. We have identified spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be dependent on ethylene biosynthesis. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. In contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis, the production of ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) by ACC SYNTHASEs (ACSs), and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3 and ACO4 as being responsible for ethylene biosynthesis and the ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling is involved in the cytokinin-induced, ethylene-mediated control of ACO4. We propose tight cooperation between cytokinin and ethylene signaling in the spatial-specific regulation of ethylene biosynthesis as a key aspect of hormonal control over root growth.

AtSNP_TATAdb: Candidate Molecular Markers of Plant Advantages Related to Single Nucleotide Polymorphisms within Proximal Promoters of Arabidopsis thaliana L.

The mainstream of the post-genome target-assisted breeding in crop plant species includes biofortification such as high-throughput phenotyping along with genome-based selection. Therefore, in this work, we used the Web-service Plant_SNP_TATA_Z-tester, which we have previously developed, to run a uniform in silico analysis of the transcriptional alterations of 54,013 protein-coding transcripts from 32,833 Arabidopsis thaliana L. genes caused by 871,707 SNPs located in the proximal promoter region. The analysis identified 54,993 SNPs as significantly decreasing or increasing gene expression through changes in TATA-binding protein affinity to the promoters. The existence of these SNPs in highly conserved proximal promoters may be explained as intraspecific diversity kept by the stabilizing natural selection. To support this, we hand-annotated papers on some of the Arabidopsis genes possessing these SNPs or on their orthologs in other plant species and demonstrated the effects of changes in these gene expressions on plant vital traits. We integrated in silico estimates of the TBP-promoter affinity in the AtSNP_TATAdb knowledge base and showed their significant correlations with independent in vivo experimental data. These correlations appeared to be robust to variations in statistical criteria, genomic environment of TATA box regions, plants species and growing conditions.

N6-adenosine methylation of mRNA integrates multilevel auxin response and ground tissue development in Arabidopsis.

N6-methyl adenosine (m6A) is a widespread internal mRNA modification impacting the expression of numerous genes. Here, we characterize auxin-related defects among the pleiotropic phenotypes of hypomorphic Arabidopsis thaliana mutants with impaired m6A status and reveal that they show strong resistance to exogenously applied auxin. By combining major published m6A datasets, we propose that among high-confidence target transcripts emerge those encoding the main components required for auxin signaling, including the TIR1/AFB auxin receptors and ARF transcriptional regulators. We also observe subtle changes in endogenous levels of indole-3-acetic acid metabolites in these hypomorphic lines, which correlate with the methylation status of indole-3-acetic acid amidohydrolase transcripts. In addition, we reveal that reduced m6A levels lead to defects in endodermal patterning in the primary root arising from impaired timing of periclinal cell divisions. These defects can be reverted by inhibition of auxin signaling. Together, our data underline that m6A likely affects auxin-dependent processes at multiple levels.

Hormone-regulated expansins: Expression, localization, and cell wall biomechanics in Arabidopsis root growth.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

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.

Web-MCOT Server for Motif Co-Occurrence Search in ChIP-Seq Data.

(1) Background: The widespread application of ChIP-seq technology requires annotation of cis-regulatory modules through the search of co-occurred motifs. (2) Methods: We present the web server Motifs Co-Occurrence Tool (Web-MCOT) that for a single ChIP-seq dataset detects the composite elements (CEs) or overrepresented homo- and heterotypic pairs of motifs with spacers and overlaps, with any mutual orientations, uncovering various similarities to recognition models within pairs of motifs. The first (Anchor) motif in CEs respects the target transcription factor of the ChIP-seq experiment, while the second one (Partner) can be defined either by a user or a public library of Partner motifs being processed. (3) Results: Web-MCOT computes the significances of CEs without reference to motif conservation and those with more conserved Partner and Anchor motifs. Graphic results show histograms of CE abundance depending on orientations of motifs, overlap and spacer lengths; logos of the most common CE structural types with an overlap of motifs, and heatmaps depicting the abundance of CEs with one motif possessing higher conservation than another. (4) Conclusions: Novel capacities of Web-MCOT allow retrieving from a single ChIP-seq dataset with maximal information on the co-occurrence of motifs and potentiates planning of next ChIP-seq experiments.

Plant_SNP_TATA_Z-Tester: A Web Service That Unequivocally Estimates the Impact of Proximal Promoter Mutations on Plant Gene Expression.

Synthetic targeted optimization of plant promoters is becoming a part of progress in mainstream postgenomic agriculture along with hybridization of cultivated plants with wild congeners, as well as marker-assisted breeding. Therefore, here, for the first time, we compiled all the experimental data-on mutational effects in plant proximal promoters on gene expression-that we could find in PubMed. Some of these datasets cast doubt on both the existence and the uniqueness of the sought solution, which could unequivocally estimate effects of proximal promoter mutation on gene expression when plants are grown under various environmental conditions during their development. This means that the inverse problem under study is ill-posed. Furthermore, we found experimental data on in vitro interchangeability of plant and human TATA-binding proteins allowing the application of Tikhonov's regularization, making this problem well-posed. Within these frameworks, we created our Web service Plant_SNP_TATA_Z-tester and then determined the limits of its applicability using those data that cast doubt on both the existence and the uniqueness of the sought solution. We confirmed that the effects (of proximal promoter mutations on gene expression) predicted by Plant_SNP_TATA_Z-tester correlate statistically significantly with all the experimental data under study. Lastly, we exemplified an application of Plant_SNP_TATA_Z-tester to agriculturally valuable mutations in plant promoters.

Salicylic Acid in Root Growth and Development.

In plants, salicylic acid (SA) is a hormone that mediates a plant's defense against pathogens. SA also takes an active role in a plant's response to various abiotic stresses, including chilling, drought, salinity, and heavy metals. In addition, in recent years, numerous studies have confirmed the important role of SA in plant morphogenesis. In this review, we summarize data on changes in root morphology following SA treatments under both normal and stress conditions. Finally, we provide evidence for the role of SA in maintaining the balance between stress responses and morphogenesis in plant development, and also for the presence of SA crosstalk with other plant hormones during this process.

Characterization of BisI Homologs.

BisI is a sequence-specific and 5-methylcytosine (m5C)-dependent restriction endonuclease (REase), that cleaves the modified DNA sequence Gm5CNGC (G indicates that the cytosine opposite to G is modified). We expressed and purified a number of BisI homologs from sequenced bacterial genomes and used Illumina sequencing to determine the Pam7902I (Esp638I-like) cleavage sites in phage Xp12 DNA. One BisI homolog KpnW2I is EcoBLMcrX-like, cleaving GCNGC/RCNGY/RCNRC sites with m5C. We also cloned and expressed three BisI homologs from metagenome sequences derived from thermophilic sources. One enzyme EsaTMI is active at 37 to 65°C. EsaHLI cleaves GCNGC sites with three to four m5C and is active up to 50°C. In addition, we determined the number and position of m5C in BisI sites for efficient cleavage. BisI cleavage efficiency of GCNGC site is as following: Gm5CNGC (two internal m5C) > Gm5CNGC (one internal m5C) > GCNGm5C (one external m5C) > > GCNGC (unmodified). Three or four m5C in GCNGC site also supports BisI cleavage although partial inhibition was observed on duplex oligos with four m5C. BisI can be used to partially cleave a desired GCNGC site targeted with a complementary oligonucleotide (hemi-methylated). The m5C-dependent BisI variants will be useful for epigenetic research.

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.

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.

Asymmetric Conservation within Pairs of Co-Occurred Motifs Mediates Weak Direct Binding of Transcription Factors in ChIP-Seq Data.

(1) Background: Transcription factors (TFs) are main regulators of eukaryotic gene expression. The cooperative binding to genomic DNA of at least two TFs is the widespread mechanism of transcription regulation. Cooperating TFs can be revealed through the analysis of co-occurrence of their motifs. (2) Methods: We applied the motifs co-occurrence tool (MCOT) that predicted pairs of spaced or overlapped motifs (composite elements, CEs) for a single ChIP-seq dataset. We improved MCOT capability for the prediction of asymmetric CEs with one of the participating motifs possessing higher conservation than another does. (3) Results: Analysis of 119 ChIP-seq datasets for 45 human TFs revealed that almost for all families of TFs the co-occurrence with an overlap between motifs of target TFs and more conserved partner motifs was significantly higher than that for less conserved partner motifs. The asymmetry toward partner TFs was the most clear for partner motifs of TFs from the ETS (E26 Transformation Specific) family. (4) Conclusion: Co-occurrence with an overlap of less conserved motif of a target TF and more conserved motifs of partner TFs explained a substantial portion of ChIP-seq data lacking conserved motifs of target TFs. Among other TF families, conservative motifs of TFs from ETS family were the most prone to mediate interaction of target TFs with its weak motifs in ChIP-seq.

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.

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'.

Shaping Ethylene Response: The Role of EIN3/EIL1 Transcription Factors.

EIN3/EIL1 transcription factors are the key regulators of ethylene signaling that sustain a variety of plant responses to ethylene. Since ethylene regulates multiple aspects of plant development and stress responses, its signaling outcome needs proper modulation depending on the spatiotemporal and environmental conditions. In this review, we summarize recent advances on the molecular mechanisms that underlie EIN3/EIL1-directed ethylene signaling in Arabidopsis. We focus on the role of EIN3/EIL1 in tuning transcriptional regulation of ethylene response in time and space. Besides, we consider the role of EIN3/EIL1-independent regulation of ethylene signaling.

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.

Click chemistry-based tracking reveals putative cell wall-located auxin binding sites in expanding cells.

Auxin is a key plant regulatory molecule, which acts upon a plethora of cellular processes, including those related to cell differentiation and elongation. Despite the stunning progress in all disciplines of auxin research, the mechanisms of auxin-mediated rapid promotion of cell expansion and underlying rearrangement of cell wall components are poorly understood. This is partly due to the limitations of current methodologies for probing auxin. Here we describe a click chemistry-based approach, using an azido derivative of indole-3-propionic acid. This compound is as an active auxin analogue, which can be tagged in situ. Using this new tool, we demonstrate the existence of putative auxin binding sites in the cell walls of expanding/elongating cells. These binding sites are of protein nature but are distinct from those provided by the extensively studied AUXIN BINDING PROTEIN 1 (ABP1). Using immunohistochemistry, we have shown the apoplastic presence of endogenous auxin epitopes recognised by an anti-IAA antibody. Our results are intriguingly in line with previous observations suggesting some transcription-independent (non-genomic) activity of auxin in cell elongation.

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.