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 SCF(COI1) 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 SCF(COI1) 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.

SUMMARYPlant 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 shadeavoidance 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.

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.T ranscription factors (TFs) mediate cellular responses through recognizing specific cis-regulatory DNA sequences at the promoters of their targets genes. In plants, organ development is a continuous process that expands beyond the embryonic phase and, as sessile organisms, plants have to face with a wide range of environmental stresses. Signaling cascades governing developmental and stress switches converge at the gene expression level. Pioneering work (1) suggested that transcriptional regulation may play more important roles in plants than in animals, given the large number of TF-coding genes in plant genomes, ranging from 6% to 10%, depending on the database.During the last few years, the advance in the determination of TF-binding sites, including both in vivo and in vitro techniques, is helping to decipher the transcriptional regulatory code (2-4). In vivo approaches involving immunoprecipitation of TF-bound chromatin followed by microarray or sequencing analysis (ChIPchip and ChIP-seq, respectively) are contributing to the knowledge of the transcriptional networks associated with a TF. ChIP-based techniques revealed that TFs may bind to thousands of genomic fragments, suggesting that the TF is interacting indirectly with DNA or that the binding requires additional cooperative factors (5, 6). The situation may not be different in the case of plant genomes. To date, only a limited number of studies have deepened in the discovery of the targets of some TFs and found that, similar to TFs in animals, TFs in plants bind to hundreds or thousands of DNA fragments; in some cases, only a small proportion of targets respond transcriptionally to the TF, obscuring the identification of actual binding sites (7-9). In this context, the precise identification of the DNA-binding sequence of each TF may be ins...

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.

Genomic integrity requires faithful chromosome duplication. Origins of replication, the genomic sites where DNA replication initiate, are scattered throughout the genome Their mapping at a genomic scale in multicellular organisms has been challenging. Here we have profiled origins in Arabidopsis by high-throughput sequencing of newly-synthesized DNA and identified ~1500 putative origins genome-wide. This was supported by ChIP-chip experiments to identify ORC1 and CDC6 binding sites. Origin activity was validated independently by measuring the abundance of nascent DNA strands. The midpoints of most Arabidopsis origin regions are preferentially located within the 5’ half of genes, slightly enriched in G+C, histone H2A.Z, H3K4me2/3 and H4K5ac, and depleted of H3K4me1 and H3K9me2. Our data establish the basis for understanding the epigenetic specification of DNA replication origins in Arabidopsis and have implications for other eukaryotes.

Eugenol is a volatile phenylpropanoid that contributes to flower and ripe fruit scent. In ripe strawberry (Fragaria 3 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.The octoploid cultivated strawberry (Fragaria 3 ananassa) is one of the most economically important, nonclimacteric, soft fruits, in which volatile compounds influence fruit flavor and aroma. Both characteristics contribute to the fruit organoleptic traits and are crucial factors to determine fruit quality.At present, extensive surveys on the components that contribute to strawberry flavor have been performed. In these studies, more than 360 volatiles have been identified (Latrasse, 1991;Nijssen, 1996;Zabetakis and Holden, 1997), but only 15 to 20 of them in wild varieties of strawberry are believed to be essential for sensory quality, together with nonvolatile sugars and organic acids (Schieberle and Hofmann, 1997). In contrast, in cultivated varieties of strawberry, only about six odor-active compounds have been identified as contributors to fruit flavor (Raab et al., 2006;Ulrich et al., 2007). Strawberry aroma is the result of the combined perception of fruity (ethyl butanoate, ethyl hexanoate, and methyl 2-methylbutanoat), green (Z-3-hexenal), sweaty (butanoic acid and 2-methylbutanoic acid), peach-like (g-decalactone), and caramel-like [4-hydroxy-2,5-dimethyl-3(2H)-furanone and 2,5-dimethyl-4-methoxy-3(2H)-furanone] flavor notes (Pyysalo et al., 1979;Larsen and Poll, 1992). Several wild strawb...

SUMMARYThe 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.

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.