TDTHub, a web server tool for the analysis of transcription factor binding sites in plants.

Transcriptional regulation underlies most developmental programs and physiological responses to environmental changes in plants. Transcription factors (TFs) play a key role in the regulation of gene expression by binding specifically to short DNA sequences in the regulatory regions of genes: the TF binding sites (TFBSs). In recent years, several bioinformatic tools have been developed to detect TFBSs in candidate genes, either by de novo prediction or by directly mapping experimentally known TFBSs. However, most of these tools contain information for only a few species or require multi-step procedures, and are not always intuitive for non-experienced researchers. Here we present TFBS-Discovery Tool Hub (TDTHub), a web server for quick and intuitive studies of transcriptional regulation in plants. TDTHub uses pre-computed TFBSs in 40 plant species and allows the choice of two mapping algorithms, providing a higher versatility. Besides the main TFBS enrichment tool, TDTHub includes additional tools to assist in the analysis and visualization of data. In order to demonstrate the effectiveness of TDTHub, we analyzed the transcriptional regulation of the anthocyanin biosynthesis pathway. We also analyzed the transcriptional cascades in response to jasmonate and wounding in Arabidopsis and tomato (Solanum lycopersicum), respectively. In these studies, TDTHub helped to verify the most relevant TF nodes and to propose new ones with a prominent role in these pathways. TDTHub is available at http://acrab.cnb.csic.es/TDTHub/, and it will be periodically upgraded and expanded for new species and gene annotations.

DNA features beyond the transcription factor binding site specify target recognition by plant MYC2-related bHLH proteins.

Transcription factors (TFs) regulate gene expression by binding to cis-regulatory sequences in the promoters of target genes. Recent research is helping to decipher in part the cis-regulatory code in eukaryotes, including plants, but it is not yet fully understood how paralogous TFs select their targets. Here we addressed this question by studying several proteins of the basic helix-loop-helix (bHLH) family of plant TFs, all of which recognize the same DNA motif. We focused on the MYC-related group of bHLHs, that redundantly regulate the jasmonate (JA) signaling pathway, and we observed a high correspondence between DNA-binding profiles in vitro and MYC function in vivo. We demonstrated that A/T-rich modules flanking the MYC-binding motif, conserved from bryophytes to higher plants, are essential for TF recognition. We observed particular DNA-shape features associated with A/T modules, indicating that the DNA shape may contribute to MYC DNA binding. We extended this analysis to 20 additional bHLHs and observed correspondence between in vitro binding and protein function, but it could not be attributed to A/T modules as in MYCs. We conclude that different bHLHs may have their own codes for DNA binding and specific selection of targets that, at least in the case of MYCs, depend on the TF-DNA interplay.

DAP-Seq Identification of Transcription Factor-Binding Sites in Potato.

Plant growth and adaptation to environmental fluctuations involve a tight control of cellular processes which, to a great extent, are mediated by changes at the transcriptional level. This regulation is exerted by transcription factors (TFs), a group of regulatory proteins that control gene expression by directly binding to the gene promoter regions via their cognate TF-binding sites (TFBS). The nature of TFBS defines the pattern of expression of the various plant loci, the precise combinatorial assembly of these elements being key in conferring plant's adaptation ability and in domestication. As such, TFs are main potential targets for biotechnological interventions, prompting in the last decade notable protein-DNA interaction efforts toward definition of their TFBS. Distinct methods based on in vivo or in vitro approaches defined the TFBS for many TFs, mainly in Arabidopsis, but comprehensive information on the transcriptional networks for many regulators is still lacking, especially in crops. In this chapter, detailed protocols for DAP-seq studies to unbiased identification of TFBS in potato are provided. This methodology relies on the affinity purification of genomic DNA-protein complexes in vitro, and high-throughput sequencing of the eluted DNA fragments. DAP-seq outperforms other in vitro DNA-motif definition strategies, such as protein-binding microarrays and SELEX-seq, since the protein of interest is directly bound to the genomic DNA extracted from plants yielding all the potential sites bound by the TF in the genome. Actually, data generated from DAP-seq experiments are highly similar to those out of ChIP-seq methods, but are generated much faster. We also provide a standard procedure to the analysis of the DAP-seq data, addressed to non-experienced users, that involves two consecutive steps: (1) processing of raw data (trimming, filtering, and read alignment) and (2) peak calling and identification of enriched motifs. This method allows identification of the binding profiles of dozens of TFs in crops, in a timely manner.

Potato CYCLING DOF FACTOR 1 and its lncRNA counterpart StFLORE link tuber development and drought response.

Plants regulate their reproductive cycles under the influence of environmental cues, such as day length, temperature and water availability. In Solanum tuberosum (potato), vegetative reproduction via tuberization is known to be regulated by photoperiod, in a very similar way to flowering. The central clock output transcription factor CYCLING DOF FACTOR 1 (StCDF1) was shown to regulate tuberization. We now show that StCDF1, together with a long non-coding RNA (lncRNA) counterpart, named StFLORE, also regulates water loss through affecting stomatal growth and diurnal opening. Both natural and CRISPR-Cas9 mutations in the StFLORE transcript produce plants with increased sensitivity to water-limiting conditions. Conversely, elevated expression of StFLORE, both by the overexpression of StFLORE or by the downregulation of StCDF1, results in an increased tolerance to drought through reducing water loss. Although StFLORE appears to act as a natural antisense transcript, it is in turn regulated by the StCDF1 transcription factor. We further show that StCDF1 is a non-redundant regulator of tuberization that affects the expression of two other members of the potato StCDF gene family, as well as StCO genes, through binding to a canonical sequence motif. Taken together, we demonstrate that the StCDF1-StFLORE locus is important for vegetative reproduction and water homeostasis, both of which are important traits for potato plant breeding.

Molecular mechanisms of Evening Complex activity in Arabidopsis.

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

An Ancient COI1-Independent Function for Reactive Electrophilic Oxylipins in Thermotolerance.

The jasmonate signaling pathway regulates development, growth, and defense responses in plants. Studies in the model eudicot, Arabidopsis thaliana, have identified the bioactive hormone (jasmonoyl-isoleucine [JA-Ile]) and its Coronatine Insensitive 1 (COI1)/Jasmonate-ZIM Domain (JAZ) co-receptor. In bryophytes, a conserved signaling pathway regulates similar responses but uses a different ligand, the JA-Ile precursor dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), to activate a conserved co-receptor. Jasmonate responses independent of JA-Ile and COI1, thought to be mediated by the cyclopentenone OPDA, have also been suggested, but experimental limitations in Arabidopsis have hindered attempts to uncouple OPDA and JA-Ile biosynthesis. Thus, a clear understanding of this pathway remains elusive. Here, we address the role of cyclopentenones in COI1-independent responses using the bryophyte Marchantia polymorpha, which is unable to synthesize JA-Ile but does accumulate OPDA and dn-OPDA. We demonstrate that OPDA and dn-OPDA activate a COI1-independent pathway that regulates plant thermotolerance genes, and consequently, treatment with these oxylipins protects plants against heat stress. Furthermore, we identify that these molecules signal through their electrophilic properties. By performing comparative analyses between M. polymorpha and two evolutionary distant species, A. thaliana and the charophyte alga Klebsormidium nitens, we demonstrate that this pathway is conserved in streptophyte plants and pre-dates the evolutionary appearance of the COI1-dependent jasmonate pathway, which later co-opted the pre-existing dn-OPDA as its ligand. Taken together, our data indicate that cyclopentenone-regulated COI1-independent signaling is an ancient conserved pathway, whose ancestral role was to protect plants against heat stress. This pathway was likely crucial for plants' successful land colonization and will be critical for adaption to current climate warming.

The JA-pathway MYC transcription factors regulate photomorphogenic responses by targeting HY5 gene expression.

Jasmonates are key regulators of the balance between defence and growth in plants. However, the molecular mechanisms by which activation of defence reduces growth are not yet fully understood. Here, we analyze the role of MYC transcription factors (TFs) and jasmonic acid (JA) in photomorphogenic growth. We found that multiple myc mutants share light-associated phenotypes with mutants of the phytochrome B photoreceptor, such as delayed seed germination in the dark and long hypocotyl growth. Overexpression of MYC2 in a phyB background partially suppressed its long hypocotyl phenotype. Transcriptomic analysis of multiple myc mutants confirmed that MYCs are required for full expression of red (R) light-regulated genes, including the master regulator HY5. ChIP-seq analyses revealed that MYC2 and MYC3 bind directly to the promoter of HY5 and that HY5 gene expression and protein levels are compromised in multiple myc mutants. Altogether, our results pinpoint MYCs as photomorphogenic TFs that control phytochrome responses by activating HY5 expression. This has important implications in understanding the trade-off between growth and defence as the same TFs that activate defence responses are photomorphogenic growth regulators.

Basal differences in the transcriptional profiles of tomato leaves associated with the presence/absence of the resistance gene Mi-1 and changes in these differences after infestation by the whitefly Bemisia tabaci.

The tomato Mi-1 gene mediates plant resistance to whitefly Bemisia tabaci, nematodes, and aphids. Other genes are also required for this resistance, and a model of interaction between the proteins encoded by these genes was proposed. Microarray analyses were used previously to identify genes involved in plant resistance to pests or pathogens, but scarcely in resistance to insects. In the present work, the GeneChip™ Tomato Genome Array (Affymetrix®) was used to compare the transcriptional profiles of Motelle (bearing Mi-1) and Moneymaker (lacking Mi-1) cultivars, both before and after B. tabaci infestation. Ten transcripts were expressed at least twofold in uninfested Motelle than in Moneymaker, while other eight were expressed half or less. After whitefly infestation, differences between cultivars increased to 14 transcripts expressed more in Motelle than in Moneymaker and 14 transcripts less expressed. Half of these transcripts showed no differential expression before infestation. These results show the baseline differences in the tomato transcriptomic profile associated with the presence or absence of the Mi-1 gene and provide us with valuable information on candidate genes to intervene in either compatible or incompatible tomato-whitefly interactions.

Jasmonate-Related MYC Transcription Factors Are Functionally Conserved in Marchantia polymorpha.

The lipid-derived phytohormone jasmonoyl-isoleucine regulates plant immunity, growth and development in vascular plants by activating genome-wide transcriptional reprogramming. In Arabidopsis (Arabidopsis thaliana), this process is largely orchestrated by the master regulator MYC2 and related transcription factors (TFs). However, the TFs activating this pathway in basal plant lineages are currently unknown. We report the functional conservation of MYC-related TFs between the eudicot Arabidopsis and the liverwort Marchantia polymorpha, a plant belonging to an early diverging lineage of land plants. Phylogenetic analysis suggests that MYC function first appeared in charophycean algae and therefore predates the evolutionary appearance of any other jasmonate pathway component. M. polymorpha possesses two functionally interchangeable MYC genes, one in females and one in males. Similar to AtMYC2, MpMYCs showed nuclear localization, interaction with JASMONATE-ZIM-DOMAIN PROTEIN repressors, and regulation by light. Phenotypic and molecular characterization of loss- and gain-of-function mutants demonstrated that MpMYCs are necessary and sufficient for activating the jasmonate pathway in M. polymorpha, but unlike their Arabidopsis orthologs, do not regulate fertility. Therefore, despite 450 million years of independent evolution, MYCs are functionally conserved between bryophytes and eudicots. Genetic conservation in an early diverging lineage suggests that MYC function existed in the common ancestor of land plants and evolved from a preexisting MYC function in charophycean algae.

The MYB transcription factor Emission of Methyl Anthranilate 1 stimulates emission of methyl anthranilate from Medicago truncatula hairy roots.

Plants respond to herbivore or pathogen attacks by activating specific defense programs that include the production of bioactive specialized metabolites to eliminate or deter the attackers. Volatiles play an important role in the interaction of a plant with its environment. Through transcript profiling of jasmonate-elicited Medicago truncatula cells, we identified Emission of Methyl Anthranilate (EMA) 1, a MYB transcription factor that is involved in the emission of the volatile compound methyl anthranilate when expressed in M. truncatula hairy roots, giving them a fruity scent. RNA sequencing (RNA-Seq) analysis of the fragrant roots revealed the upregulation of a methyltransferase that was subsequently characterized to catalyze the O-methylation of anthranilic acid and was hence named M. truncatula anthranilic acid methyl transferase (MtAAMT) 1. Given that direct activation of the MtAAMT1 promoter by EMA1 could not be unambiguously demonstrated, we further probed the RNA-Seq data and identified the repressor protein M. truncatula plant AT-rich sequence and zinc-binding (MtPLATZ) 1. Emission of Methyl Anthranilate 1 binds a tandem repeat of the ACCTAAC motif in the MtPLATZ1 promoter to transactivate gene expression. Overexpression of MtPLATZ1 in transgenic M. truncatula hairy roots led to transcriptional silencing of EMA1, indicating that MtPLATZ1 may be part of a negative feedback loop to control the expression of EMA1. Finally, application of exogenous methyl anthranilate boosted EMA1 and MtAAMT1 expression dramatically, thus also revealing a positive amplification loop. Such positive and negative feedback loops seem to be the norm rather than the exception in the regulation of plant specialized metabolism.

A Single JAZ Repressor Controls the Jasmonate Pathway in Marchantia polymorpha.

JAZ proteins are negative regulators of jasmonate responses, acting both as repressors of transcription factors and as co-receptors of JA-Ile. The high redundancy of JAZ genes in angiosperms has hindered the characterization of a complete depletion of JAZ function. Moreover, the recent discovery that dn-OPDA is the jasmonate ligand in Marchantia polymorpha demonstrates that JA-Ile is not the sole COI1/JAZ ligand in land plants and highlights the importance of studying JAZ co-receptors in bryophytes. Here, we have exploited the low gene redundancy of the liverwort M. polymorpha to characterize the single MpJAZ in this early diverging plant lineage. We clarify the phylogenetic history of the TIFY family, demonstrate that MpJAZ is the ortholog of AtJAZ with a conserved function, and characterize its repressor activity of dn-OPDA responses. Our results show that, consistent with previous findings in Arabidopsis, MpJAZ represses jasmonates biosynthesis, senescence, and plant defenses, and promotes cell growth and reproductive fitness, highlighting the power of studies in Marchantia.

Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants.

Evolutionary mechanisms underlying innovation of cell types have remained largely unclear. In multicellular eukaryotes, the evolutionary molecular origin of sperm differentiation is unknown in most lineages. Here, we report that in algal ancestors of land plants, changes in the DNA-binding domain of the ancestor of the MYB transcription factor DUO1 enabled the recognition of a new cis-regulatory element. This event led to the differentiation of motile sperm. After neo-functionalization, DUO1 acquired sperm lineage-specific expression in the common ancestor of land plants. Subsequently the downstream network of DUO1 was rewired leading to sperm with distinct morphologies. Conjugating green algae, a sister group of land plants, accumulated mutations in the DNA-binding domain of DUO1 and lost sperm differentiation. Our findings suggest that the emergence of DUO1 was the defining event in the evolution of sperm differentiation and the varied modes of sexual reproduction in the land plant lineage.

PIF4-induced BR synthesis is critical to diurnal and thermomorphogenic growth.

The Arabidopsis PIF4 and BES1/BZR1 transcription factors antagonize light signaling by facilitating co-activated expression of a large number of cell wall-loosening and auxin-related genes. While PIF4 directly activates expression of these targets, BES1 and BZR1 activity switch from a repressive to an activator function, depending on interaction with TOPLESS and other families of regulators including PIFs. However, the complexity of this regulation and its role in diurnal control of plant growth and brassinosteroid (BR) levels is little understood. We show by using a protein array that BES1, PIF4, and the BES1-PIF4 complex recognize different DNA elements, thus revealing a distinctive cis-regulatory code beneath BES1-repressive and PIF4 co-activation function. BES1 homodimers bind to conserved BRRE- and G-box elements in the BR biosynthetic promoters and inhibit their expression during the day, while elevated PIF4 competes for BES1 homodimer formation, resulting in de-repressed BR biosynthesis at dawn and in response to warmth. Our findings demonstrate a central role of PIF4 in BR synthesis activation, increased BR levels being essential to thermomorphogenic hypocotyl growth.

Coordination of Chloroplast Development through the Action of the GNC and GLK Transcription Factor Families.

Fundamental questions regarding how chloroplasts develop from proplastids remain poorly understood despite their central importance to plant life. Two families of nuclear transcription factors, the GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and GOLDEN TWO-LIKE (GLK) families, have been implicated in directly and positively regulating chloroplast development. Here, we determined the degree of functional overlap between the two transcription factor families in Arabidopsis (Arabidopsis thaliana), characterizing their ability to regulate chloroplast biogenesis both alone and in concert. We determined the DNA-binding motifs for GNC and GLK2 using protein-binding microarrays; the enrichment of these motifs in transcriptome datasets indicates that GNC and GLK2 are repressors and activators of gene expression, respectively. ChIP-seq analysis of GNC identified PHYTOCHROME INTERACTING FACTOR and brassinosteroid activity genes as targets whose repression by GNC facilitates chloroplast biogenesis. In addition, GNC targets and represses genes involved in ERECTA signaling and thereby facilitates stomatal development. Our results define key regulatory features of the GNC and GLK transcription factor families that contribute to the control of chloroplast biogenesis and photosynthetic activity, including areas of independence and cross talk.

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

Arabidopsis SWC4 Binds DNA and Recruits the SWR1 Complex to Modulate Histone H2A.Z Deposition at Key Regulatory Genes.

Deposition of the H2A.Z histone variant by the SWR1 complex (SWR1-C) in regulatory regions of specific loci modulates transcription. Characterization of mutations in Arabidopsis thaliana homologs of yeast SWR1-C has revealed a role for H2A.Z exchange in a variety of developmental processes. Nevertheless, the exact composition of plant SWR1-C and how it is recruited to target genes remains to be established. Here we show that SWC4, the Arabidopsis homolog of yeast SANT domain protein Swc4/Eaf2, is a DNA-binding protein that interacts with SWR1-C subunits. We demonstrate that the swc4-1 knockout mutant is embryo-lethal, while SWC4 RNAi knockdown lines display pleiotropic phenotypic alterations in vegetative and reproductive traits, including acceleration of flowering time, indicating that SWC4 controls post-embryonic processes. Transcriptomic analyses and genome-wide profiling of H2A.Z indicate that SWC4 represses transcription of a number of genes, including the floral integrator FT and key transcription factors, mainly by modulating H2A.Z deposition. Interestingly, SWC4 silencing does not affect H2A.Z deposition at the FLC locus nor expression of this gene, a master regulator of flowering previously shown to be controlled by SWR1-C. Importantly, we find that SWC4 recognizes specific AT-rich DNA elements in the chromatin regions of target genes and that SWC4 silencing impairs SWR1-C binding at FT. Collectively, our data suggest that SWC4 regulates plant growth and development by aiding SWR1-C recruitment and modulating H2A.Z deposition.

The Solanum lycopersicum WRKY3 Transcription Factor SlWRKY3 Is Involved in Salt Stress Tolerance in Tomato.

Salinity threatens productivity of economically important crops such as tomato (Solanum lycopersicum L.). WRKY transcription factors appear, from a growing body of knowledge, as important regulators of abiotic stresses tolerance. Tomato SlWRKY3 is a nuclear protein binding to the consensus CGTTGACC/T W box. SlWRKY3 is preferentially expressed in aged organs, and is rapidly induced by NaCl, KCl, and drought. In addition, SlWRKY3 responds to salicylic acid, and 35S::SlWRKY3 tomatoes showed under salt treatment reduced contents of salicylic acid. In tomato, overexpression of SlWRKY3 impacted multiple aspects of salinity tolerance. Indeed, salinized (125 mM NaCl, 20 days) 35S::SlWRKY3 tomato plants displayed reduced oxidative stress and proline contents compared to WT. Physiological parameters related to plant growth (shoot and root biomass) and photosynthesis (stomatal conductance and chlorophyll a content) were retained in transgenic plants, together with lower Na+ contents in leaves, and higher accumulation of K+ and Ca2+. Microarray analysis confirmed that many stress-related genes were already up-regulated in transgenic tomatoes under optimal conditions of growth, including genes coding for antioxidant enzymes, ion and water transporters, or plant defense proteins. Together, these results indicate that SlWRKY3 is an important regulator of salinity tolerance in tomato.

Cytokinin induces genome-wide binding of the type-B response regulator ARR10 to regulate growth and development in Arabidopsis.

The plant hormone cytokinin affects a diverse array of growth and development processes and responses to the environment. How a signaling molecule mediates such a diverse array of outputs and how these response pathways are integrated with other inputs remain fundamental questions in plant biology. To this end, we characterized the transcriptional network initiated by the type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs) that mediate the cytokinin primary response, making use of chromatin immunoprecipitation sequencing (ChIP-seq), protein-binding microarrays, and transcriptomic approaches. By ectopic overexpression of ARR10, Arabidopsis lines hypersensitive to cytokinin were generated and used to clarify the role of cytokinin in regulation of various physiological responses. ChIP-seq was used to identify the cytokinin-dependent targets for ARR10, thereby defining a crucial link between the cytokinin primary-response pathway and the transcriptional changes that mediate physiological responses to this phytohormone. Binding of ARR10 was induced by cytokinin with binding sites enriched toward the transcriptional start sites for both induced and repressed genes. Three type-B ARR DNA-binding motifs, determined by use of protein-binding microarrays, were enriched at ARR10 binding sites, confirming their physiological relevance. WUSCHEL was identified as a direct target of ARR10, with its cytokinin-enhanced expression resulting in enhanced shooting in tissue culture. Results from our analyses shed light on the physiological role of the type-B ARRs in regulating the cytokinin response, mechanism of type-B ARR activation, and basis by which cytokinin regulates diverse aspects of growth and development as well as responses to biotic and abiotic factors.

Identification of plant transcription factor target sequences.

Regulation of gene expression depends on specific cis-regulatory sequences located in the gene promoter regions. These DNA sequences are recognized by transcription factors (TFs) in a sequence-specific manner, and their identification could help to elucidate the regulatory networks that underlie plant physiological responses to developmental programs or to environmental adaptation. Here we review recent advances in high throughput methodologies for the identification of plant TF binding sites. Several approaches offer a map of the TF binding locations in vivo and of the dynamics of the gene regulatory networks. As an alternative, high throughput in vitro methods provide comprehensive determination of the DNA sequences recognized by TFs. These advances are helping to decipher the regulatory lexicon and to elucidate transcriptional network hierarchies in plants in response to internal or external cues. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Abscisic acid signaling is controlled by a BRANCHED1/HD-ZIP I cascade in Arabidopsis axillary buds.

Shoot-branching patterns determine key aspects of plant life and are important targets for crop breeding. However, we are still largely ignorant of the genetic networks controlling locally the most important decision during branch development: whether the axillary bud, or branch primordium, grows out to give a lateral shoot or remains dormant. Here we show that, inside the buds, the TEOSINTE BRANCHED1, CYCLOIDEA, PCF (TCP) transcription factor BRANCHED1 (BRC1) binds to and positively regulates the transcription of three related Homeodomain leucine zipper protein (HD-ZIP)-encoding genes: HOMEOBOX PROTEIN 21 (HB21), HOMEOBOX PROTEIN 40 (HB40), and HOMEOBOX PROTEIN 53 (HB53). These three genes, together with BRC1, enhance 9-CIS-EPOXICAROTENOID DIOXIGENASE 3 (NCED3) expression, lead to abscisic acid accumulation, and trigger hormone response, thus causing suppression of bud development. This TCP/HD-ZIP genetic module seems to be conserved in dicot and monocotyledonous species to prevent branching under light-limiting conditions.