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Transcription factors of the CYCLING DOF FACTOR (CDF) family activate in potato the SP6A FT tuberization signal in leaves. In modern cultivars, truncated StCDF1.2 alleles override strict SD control by stabilizing the StCDF1 protein, which leads to StCOL1 suppression and impaired activation of the antagonic SP5G paralog. By using DAP-seq and RNA-seq studies, we here show that StCDF1 not only acts as an upstream regulator of the day length pathway but also directly regulates several N assimilation and transport genes. StCDF1 directly represses expression of NITRATE REDUCTASE (NR/NIA), which catalyses the first reduction step in nitrate assimilation, and is encoded by a single potato locus. StCDF1 knock-down lines performed better in N-limiting conditions, and this phenotype correlated with derepressed StNR expression. Also, deletion of the StNR DAP-seq region abolished repression by StCDF1, while it did not affect NLP7-dependent activation of the StNR promoter. We identified multiple nucleotide polymorphisms in the DAP-seq region in potato cultivars with early StCDF1 alleles, suggesting that this genetic variation was selected as compensatory mechanism to the negative impact of StCDF1 stabilization. Thereby, directed modification of the StCDF1-recognition elements emerges as a promising strategy to enhance limiting StNR activity in potato., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Arsenic poses a global threat to living organisms, compromising crop security and yield. Limited understanding of the transcriptional network integrating arsenic-tolerance mechanisms with plant developmental responses hinders the development of strategies against this toxic metalloid. Here, we conducted a high-throughput yeast one-hybrid assay using as baits the promoter region from the arsenic-inducible genes ARQ1 and ASK18 from Arabidopsis thaliana, coupled with a transcriptomic analysis, to uncover novel transcriptional regulators of the arsenic response. We identified the GLABRA2 (GL2) transcription factor as a novel regulator of arsenic tolerance, revealing a wider regulatory role beyond its established function as a repressor of root hair formation. Furthermore, we found that ANTHOCYANINLESS2 (ANL2), a GL2 subfamily member, acts redundantly with this transcription factor in the regulation of arsenic signaling. Both transcription factors act as repressors of arsenic response. gl2 and anl2 mutants exhibit enhanced tolerance and reduced arsenic accumulation. Transcriptional analysis in the gl2 mutant unveils potential regulators of arsenic tolerance. These findings highlight GL2 and ANL2 as novel integrators of the arsenic response with developmental outcomes, offering insights for developing safer crops with reduced arsenic content and increased tolerance to this hazardous metalloid., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Background and Aims: SPATULA (SPT) encodes a basic Helix-Loop-Helix transcription factor in Arabidopsis thaliana that functions in the development of the style, stigma and replum tissues, all of which arise from the carpel margin meristem (CMM) of the gynoecium. Here, we use a comparative approach to investigate the evolutionary history of SPT and identify changes that potentially contributed to its role in gynoecium development., Methods: We investigate SPT's molecular and functional evolution using phylogenetic reconstruction, yeast-2-hybrid analyses of protein-protein interactions, microarray-based analyses of protein-DNA interactions, plant transformation assays, RNA in-situ hybridization, and in-silico analyses of promoter sequences., Key Results: We demonstrate the SPT lineage to have arisen at the base of euphyllophytes from a clade of potentially light-regulated transcription factors through gene duplication followed by the loss of an Active Phytochrome Binding (APB) domain. We also clarify the more recent evolutionary history of SPT and its paralog ALCATRAZ (ALC), which appear to have arisen through a large-scale duplication within Brassicales. We find that SPT orthologs from diverse groups of seed plants share strikingly similar capacities for protein-protein and protein-DNA interactions, and that SPT coding regions from a wide taxonomic range of plants are able to complement loss-of-function spt mutations in transgenic Arabidopsis. However, the expression pattern of SPT appears to have evolved significantly within angiosperms, and we identify structural changes in SPT's promoter region that correlate with the acquisition of high expression levels in tissues arising from the CMM in Brassicaeae., Conclusions: We conclude that changes to SPT's expression pattern made a major contribution to the evolution of its developmental role in the gynoecium of Brassicaeae. By contrast, the main biochemical capacities of SPT, as well as many of its immediate transcriptional targets, appear to have been conserved at least since the base of living angiosperms., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

In plants, jasmonate signaling regulates a wide range of processes from growth and development to defense responses and thermotolerance. Jasmonates, such as jasmonic acid (JA), (+)-7- iso -jasmonoyl-l-isoleucine (JA-Ile), 12-oxo-10,15( Z )-phytodienoic acid (OPDA), and dinor-12-oxo-10,15( Z )-phytodienoic acid (dn-OPDA), are derived from C18 (18 Carbon atoms) and C16 polyunsaturated fatty acids (PUFAs), which are found ubiquitously in the plant kingdom. Bryophytes are also rich in C20 and C22 long-chain polyunsaturated fatty acids (LCPUFAs), which are found only at low levels in some vascular plants but are abundant in organisms of other kingdoms, including animals. The existence of bioactive jasmonates derived from LCPUFAs is currently unknown. Here, we describe the identification of an OPDA-like molecule derived from a C20 fatty acid (FA) in the liverwort Marchantia polymorpha (Mp), which we term (5 Z ,8 Z )-10-(4-oxo-5-(( Z )-pent-2-en-1-yl)cyclopent-2-en-1-yl)deca-5,8-dienoic acid (C20-OPDA). This molecule accumulates upon wounding and, when applied exogenously, can activate known Coronatine Insensitive 1 (COI1) -dependent and -independent jasmonate responses. Furthermore, we identify a dn-OPDA-like molecule (Δ 4 -dn-OPDA) deriving from C20-OPDA and demonstrate it to be a ligand of the jasmonate coreceptor (MpCOI1-Mp Jasmonate-Zinc finger inflorescence meristem domain [MpJAZ]) in Marchantia . By analyzing mutants impaired in the production of LCPUFAs, we elucidate the major biosynthetic pathway of C20-OPDA and Δ 4 -dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ 4 -dn-OPDA and dn-OPDA, we demonstrate the additive nature of these molecules in the activation of jasmonate responses. Taken together, our data identify a ligand of MpCOI1 and demonstrate LCPUFAs as a source of bioactive jasmonates that are essential to the immune response of M. polymorpha .

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., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Jasmonates are fatty acid-derived hormones that regulate multiple aspects of plant development, growth and stress responses. Bioactive jasmonates, defined as the ligands of the conserved COI1 receptor, differ between vascular plants and bryophytes (jasmonoyl-l-isoleucine (JA-Ile) and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), respectively). The biosynthetic pathways of JA-Ile in the model vascular plant Arabidopsis thaliana have been elucidated. However, the details of dn-OPDA biosynthesis in bryophytes are still unclear. Here, we identify an orthologue of Arabidopsis fatty-acid-desaturase 5 (AtFAD5) in the model liverwort Marchantia polymorpha and show that FAD5 function is ancient and conserved between species separated by more than 450 million years (Myr) of independent evolution. Similar to AtFAD5, MpFAD5 is required for the synthesis of 7Z-hexadecenoic acid. Consequently, in Mpfad5 mutants, the hexadecanoid pathway is blocked, dn-OPDA concentrations are almost completely depleted and normal chloroplast development is impaired. Our results demonstrate that the main source of wounding-induced dn-OPDA in Marchantia is the hexadecanoid pathway and the contribution of the octadecanoid pathway (i.e. from OPDA) is minimal. Remarkably, despite extremely low concentrations of dn-OPDA, MpCOI1-mediated responses to wounding and insect feeding can still be activated in Mpfad5, suggesting that dn-OPDA may not be the only bioactive jasmonate and COI1 ligand in Marchantia., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

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., (© 2021 The Author(s).)

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., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4 are central nodes in plant signaling networks integrating environmental and developmental signals to fine-tune JA defenses and plant growth. Continuous activation of MYC activity is potentially lethal. Hence, MYCs need to be tightly regulated in order to optimize plant fitness. Among the increasing number of mechanisms regulating MYC activity, protein stability is arising as a major player. However, how the levels of MYC proteins are modulated is still poorly understood. Here, we report that MYC2, MYC3, and MYC4 are targets of BPM (BTB/POZ-MATH) proteins, which act as substrate adaptors of CUL3-based E3 ubiquitin ligases. Reduction of function of CUL3 BPM in amiR-bpm lines, bpm235 triple mutants, and cul3ab double mutants enhances MYC2 and MYC3 stability and accumulation and potentiates plant responses to JA such as root-growth inhibition and MYC-regulated gene expression. Moreover, MYC3 polyubiquitination levels are reduced in amiR-bpm lines. BPM3 protein is stabilized by JA, suggesting a negative feedback regulatory mechanism to control MYC activity, avoiding harmful runaway responses. Our results uncover a layer for JA-pathway regulation by CUL3 BPM -mediated degradation of MYC transcription factors., Competing Interests: The authors declare no competing interest.

Mechanisms through which the evolution of gene regulation causes morphological diversity are largely unclear. The tremendous shape variation among plant leaves offers attractive opportunities to address this question. In cruciferous plants, the REDUCED COMPLEXITY (RCO) homeodomain protein evolved via gene duplication and acquired a novel expression domain that contributed to leaf shape diversity. However, the molecular pathways through which RCO regulates leaf growth are unknown. A key question is to identify genome-wide transcriptional targets of RCO and the DNA sequences to which RCO binds. We investigate this question using Cardamine hirsuta, which has complex leaves, and its relative Arabidopsis thaliana, which evolved simple leaves through loss of RCO. We demonstrate that RCO directly regulates genes controlling homeostasis of the hormone cytokinin to repress growth at the leaf base. Elevating cytokinin signaling in the RCO expression domain is sufficient to both transform A. thaliana simple leaves into complex ones and partially bypass the requirement for RCO in C. hirsuta complex leaf development. We also identify RCO as its own target gene. RCO directly represses its own transcription via an array of low-affinity binding sites, which evolved after RCO duplicated from its progenitor sequence. This autorepression is required to limit RCO expression. Thus, evolution of low-affinity binding sites created a negative autoregulatory loop that facilitated leaf shape evolution by defining RCO expression and fine-tuning cytokinin activity. In summary, we identify a transcriptional mechanism through which conflicts between novelty and pleiotropy are resolved during evolution and lead to morphological differences between species., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2019 American Society of Plant Biologists. All rights reserved.)

Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant A T (X. citri A T ) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4 AT was identified within the pthA repertoire of X. citri A T followed by assay of the effects on different hosts. The mode of action of PthA4 AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4 AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4 AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to 'classical' TALEs., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

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., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

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., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

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.

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., (© 2018 The Authors.)

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., (© 2018 American Society of Plant Biologists. All rights reserved.)

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., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

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.

In plants, perception of vegetation proximity by phytochrome photoreceptors activates a transcriptional network that implements a set of responses to adapt to plant competition, including elongation of stems or hypocotyls. In Arabidopsis thaliana, the homeodomain-leucine zipper (HD-Zip) transcription factor ARABIDOPSIS THALIANA HOMEOBOX 4 (ATHB4) regulates this and other responses, such as leaf polarity. To better understand the shade regulatory transcriptional network, we have carried out structure-function analyses of ATHB4 by overexpressing a series of truncated and mutated forms and analyzing three different responses: hypocotyl response to shade, transcriptional activity and leaf polarity. Our results indicated that ATHB4 has two physically separated molecular activities: that performed by HD-Zip, which is involved in binding to DNA-regulatory elements, and that performed by the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR-associated amphiphilic repression (EAR)-containing N-terminal region, which is involved in protein-protein interaction. Whereas both activities are required to regulate leaf polarity, DNA-binding activity is not required for the regulation of the seedling responses to plant proximity, which indicates that ATHB4 works as a transcriptional cofactor in the regulation of this response. These findings suggest that transcription factors might employ alternative mechanisms of action to regulate different developmental processes., (© 2017 The Authors New Phytologist © 2017 New Phytologist Trust.)

Grape ( Vitis vinifera ) color somatic variants that can be used to develop new grapevine cultivars occasionally appear associated with deletion events of uncertain origin. To understand the mutational mechanisms generating somatic structural variation in grapevine, we compared the Tempranillo Blanco (TB) white berry somatic variant with its black berry ancestor, Tempranillo Tinto. Whole-genome sequencing uncovered a catastrophic genome rearrangement in TB that caused the hemizygous deletion of 313 genes, including the loss of the functional copy for the MYB transcription factors required for anthocyanin pigmentation in the berry skin. Loss of heterozygosity and decreased copy number delimited interspersed monosomic and disomic regions in the right arm of linkage groups 2 and 5. At least 11 validated clustered breakpoints involving intrachromosomal and interchromosomal translocations between three linkage groups flanked the deleted fragments, which, according to segregation analyses, are phased in a single copy of each of the affected chromosomes. These hallmarks, along with the lack of homology between breakpoint joins and the randomness of the order and orientation of the rearranged fragments, are all consistent with a chromothripsis-like pattern generated after chromosome breakage and illegitimate rejoining. This unbalanced genome reshuffling has additional consequences in reproductive development. In TB, lack of sexual transmission of rearranged chromosomes associates with low gamete viability, which compromises fruit set and decreases fruit production. Our findings show that catastrophic genome rearrangements arise spontaneously and stabilize during plant somatic growth. These dramatic rearrangements generate new interesting phenotypes that can be selected for the improvement of vegetatively propagated plant species., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

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 Ca 2+ . 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.

Only a few transcription factors have been described in the regulation of the strawberry (Fragaria x ananassa) fruit ripening process. Using a transcriptomic approach, we identified and functionally characterized FaDOF2, a DOF-type ripening-related transcription factor, which is hormonally regulated and specific to the receptacle, though high expression levels were also found in petals. The expression pattern of FaDOF2 correlated with eugenol content, a phenylpropanoid volatile, in both fruit receptacles and petals. When FaDOF2 expression was silenced in ripe strawberry receptacles, the expression of FaEOBII and FaEGS2, two key genes involved in eugenol production, were down-regulated. These fruits showed a concomitant decrease in eugenol content, which confirmed that FaDOF2 is a transcription factor that is involved in eugenol production in ripe fruit receptacles. By using the yeast two-hybrid system and bimolecular fluorescence complementation, we demonstrated that FaDOF2 interacts with FaEOBII, a previously reported regulator of eugenol production, which determines fine-tuning of the expression of key genes that are involved in eugenol production. These results provide evidence that FaDOF2 plays a subsidiary regulatory role with FaEOBII in the expression of genes encoding enzymes that control eugenol production. Taken together, our results provide new insights into the regulation of the volatile phenylpropanoid pathway in ripe strawberry receptacles., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: The authors declare no conflict of interest.

Diatoms are amongst the most important marine microalgae in terms of biomass, but little is known concerning the molecular mechanisms that regulate their versatile metabolism. Here, the pennate diatom Phaeodactylum tricornutum was studied at the metabolite and transcriptome level during nitrogen starvation and following imposition of three other stresses that impede growth. The coordinated upregulation of the tricarboxylic acid (TCA) cycle during the nitrogen stress response was the most striking observation. Through co-expression analysis and DNA binding assays, the transcription factor bZIP14 was identified as a regulator of the TCA cycle, also beyond the nitrogen starvation response, namely in diurnal regulation. Accordingly, metabolic and transcriptional shifts were observed upon overexpression of bZIP14 in transformed P. tricornutum cells. Our data indicate that the TCA cycle is a tightly regulated and important hub for carbon reallocation in the diatom cell during nutrient starvation and that bZIP14 is a conserved regulator of this cycle., (© 2017 The Authors.)

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., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Cytokinin activates transcriptional cascades important for development and the responses to biotic and abiotic stresses. Most of what is known regarding cytokinin-regulated gene expression comes from studies of the dicotyledonous plant Arabidopsis thaliana. To expand the understanding of the cytokinin-regulated transcriptome, we employed RNA-Seq to analyze gene expression in response to cytokinin in roots and shoots of the monocotyledonous plant rice., Results: We identified over 4,600 and approximately 2,400 genes differentially expressed in response to cytokinin in roots and shoots respectively. There were some similarities in the sets of cytokinin-regulated genes identified in rice and Arabidopsis, including an up-regulation of genes that act to reduce cytokinin function. Consistent with this, we found that the preferred DNA-binding motif of a rice type-B response regulator is similar to those from Arabidopsis. Analysis of the genes regulated by cytokinin in rice revealed a large number of transcription factors, receptor-like kinases, and genes involved in protein degradation, as well as genes involved in development and the response to biotic stress. Consistent with the over-representation of genes involved in biotic stress, there is a substantial overlap in the genes regulated by cytokinin and those differentially expressed in response to pathogen infection, suggesting that cytokinin plays an integral role in the transcriptional response to pathogens in rice, including the induction of a large number of WRKY transcription factors., Conclusions: These results begin to unravel the complex gene regulation after cytokinin perception in a crop of agricultural importance and provide insight into the processes and responses modulated by cytokinin in monocots.

In Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3 domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2 [AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlying molecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements (with a 5'-CATG-3' core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrella patens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1) promoter by the B3 AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation of the promoter, suggesting that several proteins are involved. Consistent with this idea, LEC2 and ABI3 showed synergistic effects on the activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex) further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2 proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanation for their functional interactions. Taken together, these results allow us to propose a molecular model for the transcriptional regulation of seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry a specific aspartate-55 residue, and B3 transcription factors., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The CONSTANS-FT pathway defines a core module for reproductive transition in both long-day (LD) and short-day (SD) plants. Changes in the transcriptional function of the CONSTANS (CO) protein have been proposed to mediate differential SD activation of FLOWERING LOCUS T (FT) orthologs in SD plants. Potato Andigena genotypes have an obligate SD requirement for tuber formation, and this photoperiodic response correlates with activation of the FT StSP6A gene in leaves. The potato StCOL1 factor represses expression of this mobile tuberization signal, but the control mechanism is poorly understood. Here, we analyzed StCOL1 diurnal oscillation and protein accumulation at different photoperiods and light wavelengths. We observed that the potato StCOL1 gene peaked at dawn and that, in contrast to the Arabidopsis AtCO homolog, the light receptor phyB is necessary for protein stabilization in the light. Reduced StCOL1 levels in RNAi lines strongly correlated with downregulated expression of an additional potato FT family member, StSP5G. Co-regulated StCOL1 and StSP5G expression suggested that StCOL1 activates this target directly rather than controlling StSP6A expression. By hybridization of a universal protein-binding microarray, we established that StCOL1 binds a TGTGGT element, and we found that immunoprecipitated StCOL1 protein fractions were enriched in StSP5G promoter fragments bearing this element. We show that StSP5G represses tuberization in LD conditions and that this FT-like homolog suppresses StSP6A gene expression. Rewiring StCOL1 transcriptional function from direct activation of the StSP6A inducer signal to the control of an FT-like repressor thus mediates the strict SD requirement of Andigena plants for tuberization., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression., (© 2016. Published by The Company of Biologists Ltd.)

The cytokinin response factors (CRFs) are a group of related AP2/ERF transcription factors that are transcriptionally induced by cytokinin. Here we explore the role of the CRFs in Arabidopsis thaliana growth and development by analyzing lines with decreased and increased CRF function. While single crf mutations have no appreciable phenotypes, disruption of multiple CRFs results in larger rosettes, delayed leaf senescence, a smaller root apical meristem (RAM), reduced primary and lateral root growth, and, in etiolated seedlings, shorter hypocotyls. In contrast, overexpression of CRFs generally results in the opposite phenotypes. The crf1,2,5,6 quadruple mutant is embryo lethal, indicating that CRF function is essential for embryo development. Disruption of the CRFs results in partially insensitivity to cytokinin in a root elongation assay and affects the basal expression of a significant number of cytokinin-regulated genes, including the type-A ARRs, although it does not impair the cytokinin induction of the type-A ARRs. Genes encoding homeobox transcription factors are mis-expressed in the crf1,3,5,6 mutant, including STIMPY/WOX9 that is required for root and shoot apical meristem maintenance roots and which has previously been linked to cytokinin. These results indicate that the CRF transcription factors play important roles in multiple aspects of plant growth and development, in part through a complex interaction with cytokinin signaling., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes., (© 2015 American Society of Plant Biologists. All rights reserved.)

The plant hormone jasmonate (JA) plays an important role in regulating growth, development, and immunity. Activation of the JA-signaling pathway is based on the hormone-triggered ubiquitination and removal of transcriptional repressors (JASMONATE-ZIM DOMAIN [JAZ] proteins) by an SCF receptor complex (SCF(COI1)/JAZ). This removal allows the rapid activation of transcription factors (TFs) triggering a multitude of downstream responses. Identification of TFs bound by the JAZ proteins is essential to better understand how the JA-signaling pathway modulates and integrates different responses. In this study, we found that the JAZ3 repressor physically interacts with the YABBY (YAB) family transcription factor FILAMENTOUS FLOWER (FIL)/YAB1. In Arabidopsis thaliana, FIL regulates developmental processes such as axial patterning and growth of lateral organs, shoot apical meristem activity, and inflorescence phyllotaxy. Phenotypic analysis of JA-regulated responses in loss- and gain-of-function FIL lines suggested that YABs function as transcriptional activators of JA-triggered responses. Moreover, we show that MYB75, a component of the WD-repeat/bHLH/MYB complex regulating anthocyanin production, is a direct transcriptional target of FIL. We propose that JAZ3 interacts with YABs to attenuate their transcriptional function. Upon perception of JA signal, degradation of JAZ3 by the SCF(COI1) complex releases YABs to activate a subset of JA-regulated genes in leaves leading to anthocyanin accumulation, chlorophyll loss, and reduced bacterial defense., (© 2015 American Society of Plant Biologists. All rights reserved.)

Amplification and diversification of transcriptional regulators that control development is a driving force of morphological evolution. A major source of protein diversity is alternative splicing, which leads to the generation of different isoforms from a single gene. The mechanisms and timing of intron evolution nonetheless remain unclear, and the functions of alternative splicing-generated protein isoforms are rarely studied. In Solanum tuberosum, the BRANCHED1a (BRC1a) gene encodes a TCP transcription factor that controls lateral shoot outgrowth. Here, we report the recent evolution in Solanum of an alternative splice site in BRC1a that leads to the generation of two BRC1a protein isoforms with distinct C-terminal regions, BRC1a(Long) and BRC1a(Short), encoded by unspliced and spliced mRNA, respectively. The BRC1a(Long) C-terminal region has a strong activation domain, whereas that of BRC1a(S) lacks an activation domain and is predicted to form an amphipathic helix, the H domain, which prevents protein nuclear targeting. BRC1a(Short) is thus mainly cytoplasmic, while BRC1a(Long) is mainly nuclear. BRC1a(Long) functions as a transcriptional activator, whereas BRC1a(Short) appears to have no transcriptional activity. Moreover, BRC1a(Short) can heterodimerize with BRC1a(Long) and act as a dominant-negative factor; it increases BRC1a(Long) concentration in cytoplasm and reduces its transcriptional activity. This alternative splicing mechanism is regulated by hormones and external stimuli that control branching. The evolution of a new alternative splicing site and a novel protein domain in Solanum BRC1a led to a multi-level mechanism of post-transcriptional and post-translational BRC1a regulation that effectively modulates its branch suppressing activity in response to environmental and endogenous cues., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Eugenol is a volatile phenylpropanoid that contributes to flower and ripe fruit scent. In ripe strawberry (Fragaria × ananassa) fruit receptacles, eugenol is biosynthesized by eugenol synthase (FaEGS2). However, the transcriptional regulation of this process is still unknown. We have identified and functionally characterized an R2R3 MYB transcription factor (emission of benzenoid II [FaEOBII]) that seems to be the orthologous gene of PhEOBII from Petunia hybrida, which contributes to the regulation of eugenol biosynthesis in petals. The expression of FaEOBII was ripening related and fruit receptacle specific, although high expression values were also found in petals. This expression pattern of FaEOBII correlated with eugenol content in both fruit receptacle and petals. The expression of FaEOBII was repressed by auxins and activated by abscisic acid, in parallel to the ripening process. In ripe strawberry receptacles, where the expression of FaEOBII was silenced, the expression of cinnamyl alcohol dehydrogenase1 and FaEGS2, two structural genes involved in eugenol production, was down-regulated. A subsequent decrease in eugenol content in ripe receptacles was also observed, confirming the involvement of FaEOBII in eugenol metabolism. Additionally, the expression of FaEOBII was under the control of FaMYB10, another R2R3 MYB transcription factor that regulates the early and late biosynthetic genes from the flavonoid/phenylpropanoid pathway. In parallel, the amount of eugenol in FaMYB10-silenced receptacles was also diminished. Taken together, these data indicate that FaEOBII plays a regulating role in the volatile phenylpropanoid pathway gene expression that gives rise to eugenol production in ripe strawberry receptacles., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

To cope with growth in low-phosphate (Pi) soils, plants have evolved adaptive responses that involve both developmental and metabolic changes. Phosphate Starvation Response 1 (PHR1) and related transcription factors play a central role in the control of Pi starvation responses (PSRs). How Pi levels control PHR1 activity, and thus PSRs, remains to be elucidated. Here, we identify a direct Pi-dependent inhibitor of PHR1 in Arabidopsis, SPX1, a nuclear protein that shares the SPX domain with yeast Pi sensors and with several Pi starvation signaling proteins from plants. Double mutation of SPX1 and of a related gene, SPX2, resulted in molecular and physiological changes indicative of increased PHR1 activity in plants grown in Pi-sufficient conditions or after Pi refeeding of Pi-starved plants but had only a limited effect on PHR1 activity in Pi-starved plants. These data indicate that SPX1 and SPX2 have a cellular Pi-dependent inhibitory effect on PHR1. Coimmunoprecipitation assays showed that the SPX1/PHR1 interaction in planta is highly Pi-dependent. DNA-binding and pull-down assays with bacterially expressed, affinity-purified tagged SPX1 and ΔPHR1 proteins showed that SPX1 is a competitive inhibitor of PHR1 binding to its recognition sequence, and that its efficiency is highly dependent on the presence of Pi or phosphite, a nonmetabolizable Pi analog that can repress PSRs. The relative strength of the SPX1/PHR1 interaction is thus directly influenced by Pi, providing a link between Pi perception and signaling.

The zinc finger superfamily includes transcription factors that regulate multiple aspects of plant development and were recently shown to regulate abiotic stress tolerance. Cultivated tomato (Solanum lycopersicum Zinc Finger2 [SIZF2]) is a cysteine-2/histidine-2-type zinc finger transcription factor bearing an ERF-associated amphiphilic repression domain and binding to the ACGTCAGTG sequence containing two AGT core motifs. SlZF2 is ubiquitously expressed during plant development, and is rapidly induced by sodium chloride, drought, and potassium chloride treatments. Its ectopic expression in Arabidopsis (Arabidopsis thaliana) and tomato impaired development and influenced leaf and flower shape, while causing a general stress visible by anthocyanin and malonyldialdehyde accumulation. SlZF2 enhanced salt sensitivity in Arabidopsis, whereas SlZF2 delayed senescence and improved tomato salt tolerance, particularly by maintaining photosynthesis and increasing polyamine biosynthesis, in salt-treated hydroponic cultures (125 mm sodium chloride, 20 d). SlZF2 may be involved in abscisic acid (ABA) biosynthesis/signaling, because SlZF2 is rapidly induced by ABA treatment and 35S::SlZF2 tomatoes accumulate more ABA than wild-type plants. Transcriptome analysis of 35S::SlZF2 revealed that SlZF2 both increased and reduced expression of a comparable number of genes involved in various physiological processes such as photosynthesis, polyamine biosynthesis, and hormone (notably ABA) biosynthesis/signaling. Involvement of these different metabolic pathways in salt stress tolerance is discussed.

Transcription factors (TFs) regulate gene expression through binding to cis-regulatory specific sequences in the promoters of their target genes. In contrast to the genetic code, the transcriptional regulatory code is far from being deciphered and is determined by sequence specificity of TFs, combinatorial cooperation between TFs and chromatin competence. Here we addressed one of these determinants by characterizing the target sequence specificity of 63 plant TFs representing 25 families, using protein-binding microarrays. Remarkably, almost half of these TFs recognized secondary motifs, which in some cases were completely unrelated to the primary element. Analyses of coregulated genes and transcriptomic data from TFs mutants showed the functional significance of over 80% of all identified sequences and of at least one target sequence per TF. Moreover, combining the target sequence information with coexpression analysis we could predict the function of a TF as activator or repressor through a particular DNA sequence. Our data support the correlation between cis-regulatory elements and the sequence determined in vitro using the protein-binding microarray and provides a framework to explore regulatory networks in plants.

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cell reprogramming in response to jasmonates requires a tight control of transcription that is achieved by the activity of JA-related transcription factors (TFs). Among them, MYC2, MYC3 and MYC4 have been described as activators of JA responses. Here we characterized the function of bHLH003, bHLH013 and bHLH017 that conform a phylogenetic clade closely related to MYC2, MYC3 and MYC4. We found that these bHLHs form homo- and heterodimers and also interact with JAZ repressors in vitro and in vivo. Phenotypic analysis of JA-regulated processes, including root and rosette growth, anthocyanin accumulation, chlorophyll loss and resistance to Pseudomonas syringae, on mutants and overexpression lines, suggested that these bHLHs are repressors of JA responses. bHLH003, bHLH013 and bHLH017 are mainly nuclear proteins and bind DNA with similar specificity to that of MYC2, MYC3 and MYC4, but lack a conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Moreover, expression of bHLH017 is induced by JA and depends on MYC2, suggesting a negative feed-back regulation of the activity of positive JA-related TFs. Our results suggest that the competition between positive and negative TFs determines the output of JA-dependent transcriptional activation.

In this report, we focus on two different array-based technologies that enable large-scale screening of protein interactions. First, protein arrays focus on the identification of protein-protein interactions (PPIs). Second, DNA arrays have also evolved to explore the identification of protein-DNA interactions (PDIs), offering novel tools to control key biological processes. Such a tool is termed protein-binding DNA arrays (also protein-DNA arrays or protein-binding microarrays). These two array-based technologies share unrivaled screening capabilities and constitute valid approaches to address biological questions at the molecular level and, eventually, may be used in biomedical applications. Outstanding achievements of these technologies and their eventual application in biomedicine are discussed here, including the identification and characterization of biomarkers, screening of PPIs, detection of protein posttranslational modifications and biofluid profiling. Advantages and limitations of protein arrays, protein-binding arrays, and other proteomic technologies are also discussed here. Finally, we built a list of dedicated databases and on-line resources comprising updated information on human PPIs and PDIs that can serve as a toolbox for researchers in the field., (© 2014 Elsevier Inc. All rights reserved.)

Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro.