Irene López-Vidriero

The JAZ family of repressors is the missing link in jasmonate signalling.

Jasmonates are essential phytohormones for plant development and survival. However, the molecular details of their signalling pathway remain largely unknown. The identification more than a decade ago of COI1 as an F-box protein suggested the existence of a repressor of jasmonate responses that is targeted by the SCFCOI1 complex for proteasome degradation in response to jasmonate. Here we report the identification of JASMONATE-INSENSITIVE 3 (JAI3) and a family of related proteins named JAZ (jasmonate ZIM-domain), in Arabidopsis thaliana. Our results demonstrate that JAI3 and other JAZs are direct targets of the SCFCOI1 E3 ubiquitin ligase and jasmonate treatment induces their proteasome degradation. Moreover, JAI3 negatively regulates the key transcriptional activator of jasmonate responses, MYC2. The JAZ family therefore represents the molecular link between the two previously known steps in the jasmonate pathway. Furthermore, we demonstrate the existence of a regulatory feed-back loop involving MYC2 and JAZ proteins, which provides a mechanistic explanation for the pulsed response to jasmonate and the subsequent desensitization of the cell.

Phytochrome interacting factors 4 and 5 control seedling growth in changing light conditions by directly controlling auxin signaling

Plant growth is strongly influenced by the presence of neighbors that compete for light resources. In response to vegetational shading shade-intolerant plants such as Arabidopsis display a suite of developmental responses known as the shade-avoidance syndrome (SAS). The phytochrome B (phyB) photoreceptor is the major light sensor to mediate this adaptive response. Control of the SAS occurs in part with phyB, which controls protein abundance of phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) directly. The shade-avoidance response also requires rapid biosynthesis of auxin and its transport to promote elongation growth. The identification of genome-wide PIF5-binding sites during shade avoidance revealed that this bHLH transcription factor regulates the expression of a subset of previously identified SAS genes. Moreover our study suggests that PIF4 and PIF5 regulate elongation growth by controlling directly the expression of genes that code for auxin biosynthesis and auxin signaling components.

DNA-binding specificities of plant transcription factors and their potential to define target genes

Significance We described the high-throughput identification of DNA-binding specificities of 63 plant transcription factors (TFs) and their relevance as cis-regulatory elements in vivo. Almost half of the TFs recognized secondary motifs partially or completely differing from their corresponding primary ones. Analysis of coregulated genes, transcriptomic data, and chromatin hypersensitive regions revealed the biological relevance of more than 80% of the binding sites identified. Our combined analysis allows the prediction of the function of a particular TF as activator or repressor through a particular DNA sequence. The data support the correlation between cis-regulatory elements in vivo and the sequence determined in vitro. Moreover, it provides a framework to explore regulatory networks in plants and contributes to decipher the transcriptional regulatory code. 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.

Genome-wide mapping of Arabidopsis thaliana origins of DNA replication and their associated epigenetic marks

Genome integrity requires faithful chromosome duplication. Origins of replication, the genomic sites at which DNA replication initiates, are scattered throughout the genome. Their mapping at a genomic scale in multicellular organisms has been challenging. In this study we profiled origins in Arabidopsis thaliana by high-throughput sequencing of newly synthesized DNA and identified ~1,500 putative origins genome-wide. This was supported by chromatin immunoprecipitation and microarray (ChIP-chip) experiments to identify ORC1- and CDC6-binding sites. We validated origin activity independently by measuring the abundance of nascent DNA strands. The midpoints of most A. thaliana origin regions are preferentially located within the 5′ half of genes, enriched in G+C, histone H2A.Z, H3K4me2, H3K4me3 and H4K5ac, and depleted in H3K4me1 and H3K9me2. Our data help clarify the epigenetic specification of DNA replication origins in A. thaliana and have implications for other eukaryotes.

Analysis of gene transcription alterations at the blastocyst stage related to the long-term consequences of in vitro culture in mice

We have reported that in vitro culture (IVC) of preimplantation mouse embryos in the presence of FCS produces long-term effects (LTE) on development, growth and behaviour of the offspring at adult age. To analyse the mechanisms underlying this phenomenon, we have examined development and global alterations in gene expression in the mouse blastocysts produced in the presence of FCS, conditions known to be suboptimal and that generate LTE. Embryos cultured in vitro in KSOM and in KSOM+FCS had a reduced number of cells in the inner cell mass at the blastocyst stage compared with in vivo derived embryos; however, only culture in KSOM+FCS leads to a reduction in the number of trophoblast cells. Gene expression levels were measured by comparison among three groups of blastocysts (in vivo, IVC in KSOM and IVC in KSOM+FCS). Different patterns of gene expression and development were found between embryos cultured in vitro or in vivo. Moreover, when we compared the embryos produced in KSOM versus KSOM+FCS, we observed that the presence of FCS affected the expression of 198 genes. Metabolism, proliferation, apoptosis and morphogenetic pathways were the most common processes affected by IVC. However, the presence of FCS during IVC preferentially affected genes associated with certain molecular and biological functions related to epigenetic mechanisms. These results suggest that culture-induced alterations in transcription at the blastocyst stage related to epigenetic mechanisms provide a foundation for understanding the molecular origin at the time of preimplantation development of the long-term consequences of IVC in mammals.

bHLH003, bHLH013 and bHLH017 are new targets of JAZ repressors negatively regulating JA responses

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.

The cytokinin response factors modulate root and shoot growth and promote leaf senescence in Arabidopsis.

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.

The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula

Basic helix-loop-helix family transcription factors from subclade IVa specifically regulate the biosynthesis of the triterpene saponins in a model legume. 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.

A one-year follow-up study in anorexia nervosa. Dietary pattern and anthropometrical evolution.

Objective: To find out the modifications and the interrelationships between the energy and macronutrient intakes and anthropometric measurements of a group of anorexia nervosa patients (ANP), with homogenous diagnosis, age and treatment, during a 1 y follow-up.Design: Longitudinal study design.Subjects: Fourteen restricting-type ANP in the adolescent age (11–21 y) were recruited for the study and evaluated at four time intervals during 1 y of multidisciplinary treatment: (a) on admission (AN0); (b) after 1 month (AN1); (c) after 6 months (AN6); (d) after 1 y (AN12).Measures: Energy and macronutrient composition of the diet (48 h recall) and anthropometric measurements including skinfold thickness.Results: Mean weight and BMI were under the 3rd percentile in AN0. All anthropometrical parameters increased significantly in AN1 compared to AN0 and did not change in AN6 and AN12. Energy and macronutrient intakes increased significantly in AN1. The dietary changes were not maintained in the subsequent time intervals and there was an overall tendency to turn back to AN0 results. Energy intake adequacy to Spanish Recommended Intakes (RI) showed only an acceptable ratio in AN1 (94% RI); however, a preserved protein intake was found throughout the study (mean intakes between 128.5% and 230.0% RI). Negative correlations were found at AN12 between size of arms subcutaneous fat stores and energy and macronutrient intakes.Conclusion: In this 1 y follow-up study the decrease in the intake of energy and macronutrients after discharge seems to be related to anthropometric evolution, so that a return to restrictive eating behaviour had occurred in those subjects who had reached the highest values in their subcutaneous fat stores, probably related to dissatisfaction with self-body image.European Journal of Clinical Nutrition (2001) 55, 547–554

Inactivation of the ELIP1 and ELIP2 genes affects Arabidopsis seed germination

Light regulates Arabidopsis seed germination through the phyB/PIL5 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 5) transduction pathway, and we have previously shown that the Dof transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a component of this pathway. By means of microarray analysis of dag1 and wild type developing siliques, we identified the EARLY LIGHT-INDUCED PROTEIN1 and 2 (ELIP1 and ELIP2) genes among those deregulated in the loss-of-function dag1 mutant. We analysed seed germination of elip single and double mutants, of elip dag1 double mutants as well as of elip1 elip2 dag1 triple mutant under different environmental conditions. We show that ELIP1 and ELIP2 are involved in opposite ways in the control of this developmental process, in particular under abiotic (light, temperature, salt) stress conditions.