The functional connectome in obsessive-compulsive disorder: resting-state mega-analysis and machine learning classification for the ENIGMA-OCD consortium.

Current knowledge about functional connectivity in obsessive-compulsive disorder (OCD) is based on small-scale studies, limiting the generalizability of results. Moreover, the majority of studies have focused only on predefined regions or functional networks rather than connectivity throughout the entire brain. Here, we investigated differences in resting-state functional connectivity between OCD patients and healthy controls (HC) using mega-analysis of data from 1024 OCD patients and 1028 HC from 28 independent samples of the ENIGMA-OCD consortium. We assessed group differences in whole-brain functional connectivity at both the regional and network level, and investigated whether functional connectivity could serve as biomarker to identify patient status at the individual level using machine learning analysis. The mega-analyses revealed widespread abnormalities in functional connectivity in OCD, with global hypo-connectivity (Cohen's d: -0.27 to -0.13) and few hyper-connections, mainly with the thalamus (Cohen's d: 0.19 to 0.22). Most hypo-connections were located within the sensorimotor network and no fronto-striatal abnormalities were found. Overall, classification performances were poor, with area-under-the-receiver-operating-characteristic curve (AUC) scores ranging between 0.567 and 0.673, with better classification for medicated (AUC = 0.702) than unmedicated (AUC = 0.608) patients versus healthy controls. These findings provide partial support for existing pathophysiological models of OCD and highlight the important role of the sensorimotor network in OCD. However, resting-state connectivity does not so far provide an accurate biomarker for identifying patients at the individual level.

Case report: Tumor collision in the colon, adenocarcinoma - lymphoma.

INTRODUCTION: Tumor collision is the cohesion in a time of 2 tumors in the same space or organ but of different cell lineage. PRESENTATION OF CASE: 72-year-old patient with a history of black stools, involuntary weight loss and moderate protein-calorie malnutrition, asthenia, and adynamia; with endoscopic studies with the finding of esophageal candidiasis that leads to taking a rapid test for HIV (human immunodeficiency virus) which is positive; CT (computerized axial tomography) of the abdomen is performed, showing thickening of the cecum and distal ileum, as well as striation of fat in the same area, findings related to a primary neoformative process, for which it is decided to carry out a right hemicolectomy laparoscopically with pathology that reports ulcerated moderately differentiated adenocarcinoma that invades up to the muscle layer, associated with lymphoid proliferation with immunohistochemical findings consistent with lymphoplasmablastic lymphoma, this is probably linked to his diagnosis of HIV, configuring the diagnosis of tumor collision; extension studies with no findings of metastatic neoplastic disease. DISCUSSION: Tumor collision is a rare entity, which implies failure in the genomic control and replication sites of different cell lines, which, due to their lineage, have different regulatory processes, all this occurring at the same time in the same tissue. CONCLUSION: The casuistry for collision tumors is scarce; even in the reference centers for oncology, the treatment is challenging given the multiple variables to consider, the particularity of each case, and the scarce evidence on the subject.

Ligand diversity contributes to the full activation of the jasmonate pathway in Marchantia polymorpha.

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 (5Z,8Z)-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 (Δ4-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 Δ4-dn-OPDA. Moreover, using a double mutant compromised in the production of both Δ4-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.

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.

An evolutionarily ancient fatty acid desaturase is required for the synthesis of hexadecatrienoic acid, which is the main source of the bioactive jasmonate in Marchantia polymorpha.

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.

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.

Characteristics and Prognosis of Patients Who Receive Noninvasive Ventilation and Present Limitation of Life Support Treatment: The LLST-NIVCat Multicenter Cohort Study.

BACKGROUND: Patients who receive noninvasive ventilation (NIV) in the emergency department (ED) sometimes have a limitation of life support treatment (LLST). The characteristics and prognosis in these patients may be worse, however, few studies have been carried out in this respect. OBJECTIVE: Analyze the differences between patients receiving NIV in the ED with LLST (NIV-LLST) or without LLST (NIV-noLLST) and their impact on in-hospital mortality, as well as investigate in-hospital mortality in the NIV-LLST group. METHOD: We performed a secondary analysis of data from the NIVCat registry. This was a prospective, multicenter, analytical cohort study with consecutive inclusion of patients receiving NIV from February to March 2015 in 11 hospital EDs in Spain. Data on the baseline characteristics, the acute episode, and final patient destination were collected. The dependent variable was all-cause in-hospital mortality. RESULTS: We analyzed 152 cases receiving NIV, 66 (43.4%) of whom had NIV-LLTS. Age ≥ 75 years was associated with NIV-LLST. In-hospital mortality was higher in the NIV-LLST group, with an adjusted hazard ratio of 2.50 (95% confidence interval [CI] 1.03-6.06). Patients with NIV-LLST presenting an exacerbation of chronic obstructive pulmonary disease (COPD) presented the lowest mortality, with an odds ratio of 0.27 (95% confidence interval 0.08-0.93), compared with the remaining patients. CONCLUSION: In our cohort of patients receiving NIV in the ED, the presence of LLST is frequent and is associated with high hospital mortality. The NIV-LLST patients with a COPD exacerbation have a better prognosis than NIV-LLST patients with other diseases.

Estudio SEPHCAT: análisis de los servicios de emergencias prehospitalarios en Cataluña / Prehospital emergency services in Catalonia: the SEPHCAT análisis

Objetivo: Describir las principales características de todos los servicios de emergencias prehospitalarios (SEPH) existentes en Cataluña. Método: Una encuestadora profesional entrevistó a los responsables clínicos de todos los SEPH de Cataluña. La encuesta fue preparada por los autores, y abordaba diversos aspectos organizativos, profesionales, laborales, formativos, docentes y de investigación. Las preguntas contenidas en la encuesta solo permitían respuestas cerradas, y hacían referencia a la situación en 2015. Resultados: Se identificaron 13 SEPH (11 públicos, 2 privados), que recibieron 2.482.627 consultas (16,4% a SEPH privados) y realizaron 943.849 atenciones (11,8% por SEPH privados). Había 336 bases de soporte vital básico y 73 de avanzado, con instalaciones mayoritariamente consideradas de tamaño suficiente y calidad buena. Se contrataron 1.374.845 horas anuales (753.995 de médico y 620.850 de enfermero), el 23,4% de ellas por SEPH privados, que globalmente corresponderían a 815 puestos de trabajo a jornada completa (447 de médico, 368 de enfermero). La dotación de médicos/enfermeros era relativamente estable durante el día, pero decaía un 31%/9% de madrugada. La especialidad médica mayoritaria era medicina familiar y comunitaria (56,8%), el 21,3% no tenía formación especializada, y el 6,5% tenía título de doctor. Había médicos/enfermeros profesores universitarios de grado en el 61,5%/46,2% de los SEPH; y de postgrado en el 46,2%/84,6%. Recibían estudiantes de medicina/enfermería en prácticas de grado el 15,4%/15,4% de los SEPH, y de postgrado el 69,2%/76,9%. Conclusiones: La actividad de los SEPH en Cataluña es elevada; un 12% la desarrollan SEPH privados, y globalmente implica a un número alto de médicos y enfermeros, los cuales además desarrollan un rol docente y, en menor medida, investigador

Objective: To describe the main characteristics of all prehospital emergency services (SEPHs, the Catalan acronym) in Catalonia (the SEPHCAT study). Methods: A professional survey researcher interviewed the medical directors of all services in Catalonia, using a questionnaire prepared by the authors. Questions covered aspects related to organization, professional staffing and employment conditions, as well as the staff’s training, instructional activity and research. Only closed answers were collected. The survey reflected the situation in 2015. Results: We identified 13 SEPHs (11 in the public health service and 2 private companies). Together they received 2 482 627 calls (16.4% to private services) and attended 943 849 emergencies (11.8% attended by private companies). Three hundred thirty-six basic life support units and 73 advanced life support units were reported. They were mostly considered to be of sufficient size and quality. The SEPHs contracted 1 374 845 person-hours/y (753 995 physician-hours and 620 850 nurse-hours; 23.4% in private companies). These figures correspond to 815 full-time staff positions (447 for physicians and 368 for nurses). The numbers of physicians and nurses working were relatively stable during the morning, afternoon and evening shifts but decreased during the midnight-to-early-morning shift (physicians, by 31%; nurses, by 9%). A majority of the physicians employed were trained in family and community medicine (56.8%), but 21.3% had no specialized training; 6.5% had PhD degrees. SEPH physicians (61.5%) and nurses (46.2%) also taught undergraduate medical students; 46.2% of physicians and 84.6% of nurses taught postgraduate medical courses. Both undergraduate medical and nursing students were received in the same measure for practical training by 15.4% of the SEPHs; 69.2% also offered practical training for physicians at the postgraduate level and 76.9% trained postgraduate nurses. Conclusions: SEPHs in Catalonia are very active, and private companies account for nearly 12% of the activity. Together the public and private sectors employ a large number of physicians and nurses. Staff members are involved in training others but are less involved in research

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.

CUL3BPM E3 ubiquitin ligases regulate MYC2, MYC3, and MYC4 stability and JA responses.

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 CUL3BPM 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 CUL3BPM-mediated degradation of MYC transcription factors.

Prehospital emergency services in Catalonia: the SEPHCAT analysis. / Estudio SEPHCAT: análisis de los servicios de emergencias prehospitalarios en Cataluña.

OBJECTIVES: To describe the main characteristics of all prehospital emergency services (SEPHs, the Catalan acronym) in Catalonia (the SEPHCAT study). MATERIAL AND METHODS: A professional survey researcher interviewed the medical directors of all services in Catalonia, using a questionnaire prepared by the authors. Questions covered aspects related to organization, professional staffing and employment conditions, as well as the staff's training, instructional activity and research. Only closed answers were collected. The survey reflected the situation in 2015. RESULTS: We identified 13 SEPHs (11 in the public health service and 2 private companies). Together they received 2 482 627 calls (16.4% to private services) and attended 943 849 emergencies (11.8% attended by private companies). Three hundred thirty-six basic life support units and 73 advanced life support units were reported. They were mostly considered to be of sufficient size and quality. The SEPHs contracted 1374845 person-hours/y (753995 physician-hours and 620 850 nurse-hours; 23.4% in private companies). These figures correspond to 815 full-time staff positions (447 for physicians and 368 for nurses). The numbers of physicians and nurses working were relatively stable during the morning, afternoon and evening shifts but decreased during the midnight-to-early-morning shift (physicians, by 31%; nurses, by 9%). A majority of the physicians employed were trained in family and community medicine (56.8%), but 21.3% had no specialized training; 6.5% had PhD degrees. SEPH physicians (61.5%) and nurses (46.2%) also taught undergraduate medical students; 46.2% of physicians and 84.6% of nurses taught postgraduate medical courses. Both undergraduate medical and nursing students were received in the same measure for practical training by 15.4% of the SEPHs; 69.2% also offered practical training for physicians at the postgraduate level and 76.9% trained postgraduate nurses. CONCLUSION: SEPHs in Catalonia are very active, and private companies account for nearly 12% of the activity. Together the public and private sectors employ a large number of physicians and nurses. Staff members are involved in training others but are less involved in research.

OBJETIVO: Describir las principales características de todos los servicios de emergencias prehospitalarios (SEPH) existentes en Cataluña. METODO: Una encuestadora profesional entrevistó a los responsables clínicos de todos los SEPH de Cataluña. La encuesta fue preparada por los autores, y abordaba diversos aspectos organizativos, profesionales, laborales, formativos, docentes y de investigación. Las preguntas contenidas en la encuesta solo permitían respuestas cerradas, y hacían referencia a la situación en 2015. RESULTADOS: Se identificaron 13 SEPH (11 públicos, 2 privados), que recibieron 2.482.627 consultas (16,4% a SEPH privados) y realizaron 943.849 atenciones (11,8% por SEPH privados). Había 336 bases de soporte vital básico y 73 de avanzado, con instalaciones mayoritariamente consideradas de tamaño suficiente y calidad buena. Se contrataron 1.374.845 horas anuales (753.995 de médico y 620.850 de enfermero), el 23,4% de ellas por SEPH privados, que globalmente corresponderían a 815 puestos de trabajo a jornada completa (447 de médico, 368 de enfermero). La dotación de médicos/enfermeros era relativamente estable durante el día, pero decaía un 31%/9% de madrugada. La especialidad médica mayoritaria era medicina familiar y comunitaria (56,8%), el 21,3% no tenía formación especializada, y el 6,5% tenía título de doctor. Había médicos/enfermeros profesores universitarios de grado en el 61,5%/46,2% de los SEPH; y de postgrado en el 46,2%/84,6%. Recibían estudiantes de medicina/enfermería en prácticas de grado el 15,4%/15,4% de los SEPH, y de postgrado el 69,2%/76,9%. CONCLUSIONES: La actividad de los SEPH en Cataluña es elevada; un 12% la desarrollan SEPH privados, y globalmente implica a un número alto de médicos y enfermeros, los cuales además desarrollan un rol docente y, en menor medida, investigador.

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.

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.

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.

Autoregulation of RCO by Low-Affinity Binding Modulates Cytokinin Action and Shapes Leaf Diversity.

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.

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.

PthA4AT , a 7.5-repeats transcription activator-like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance.

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 AT (X. citri AT ) 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. PthA4AT was identified within the pthA repertoire of X. citri AT followed by assay of the effects on different hosts. The mode of action of PthA4AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4AT 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.