A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5–Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response

A modular analysis of the Auxin signalling network

Auxin is essential for plant development from embryogenesis onwards. Auxin acts in large part through regulation of transcription. The proteins acting in the signalling pathway regulating transcription downstream of auxin have been identified as well as the interactions between these proteins, thus identifying the topology of this network implicating 54 Auxin Response Factor (ARF) and Aux/IAA (IAA) transcriptional regulators. Here, we study the auxin signalling pathway by means of mathematical modeling at the single cell level. We proceed analytically, by considering the role played by five functional modules into which the auxin pathway can be decomposed: the sequestration of ARF by IAA, the transcriptional repression by IAA, the dimer formation amongst ARFs and IAAs, the feedback loop on IAA and the auxin induced degradation of IAA proteins. Focusing on these modules allows assessing their function within the dynamics of auxin signalling. One key outcome of this analysis is that there are both specific and overlapping functions between all the major modules of the signaling pathway. This suggests a combinatorial function of the modules in optimizing the speed and amplitude of auxin-induced transcription. Our work allows identifying potential functions for homo- and hetero-dimerization of transcriptional regulators, with ARF:IAA, IAA:IAA and ARF:ARF dimerization respectively controlling the amplitude, speed and sensitivity of the response and a synergistic effect of the interaction of IAA with transcriptional repressors on these characteristics of the signaling pathway. Finally, we also suggest experiments which might allow disentangling the structure of the auxin signaling pathway and analysing further its function in plants

Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

Background: The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. Results: Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 59-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. Conclusion: All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner

Binding profile of the endogenous novel heptapeptide Met-enkephalin-Gly-tyr in zebrafish and rat brain

6 pages, 3 figures, 2 tables.-- PMID: 15901806 [PubMed].-- Printed version published Aug 2005.Zebrafish is considered a model organism, not only for the study of the biological functions of vertebrates but also as a tool to analyze the effects of some drugs or toxic agents. Five opioid precursor genes homologous to the mammalian opioid propeptide genes have recently been identified; one of these, the zebrafish proenkephalin, codes a novel heptapeptide, the Met-enkephalin-Gly-Tyr (MEGY). To analyze the pharmacological properties of this novel ligand, we have labeled it with tritium ([3H]MEGY). In addition, we have also synthesized two analogs: (D-Ala2)-MEGY (Y-D-Ala-GFMGY) and (D-Ala2, Val5)-MEGY (Y-D-Ala-GFVGY). The binding profile of these three agents has been studied in zebrafish and rat brain membranes. [3H]MEGY presents one binding site in zebrafish, as well as in rat brain membranes, although it shows a slight higher affinity in zebrafish brain. The observed saturable binding is displaced by naloxone, thus confirming the opioid nature of the binding sites. Competition binding assays indicate that the methionine residue is essential for high-affinity binding of MEGY and probably of other peptidic agonists in zebrafish, whereas the change of a Gly for a D-Ala does not dramatically affect the ligand affinity. Our results show that the percentage of [3H]MEGY displaced by all the ligands studied is higher than 100%, thus inferring that naloxone (used to determine nonspecific binding) does not bind to all the sites labeled by [3H]MEGY. Therefore, we can deduct that some of the MEGY binding sites should not be considered classical opioid sites.This study was supported by Ministerio de Educación y Ciencia (SAF2004-05144), Junta de Castilla y León (SA031/03/00B), and Hungarian National Scientific Research Foundation (Hungary) Grant T046514Peer reviewe

Beyond an associative conception of automatic self-evaluations: applying the relational responding task to measure self-esteem

According to dual-process models, implicit self-esteem (SE) is based on automatic self-associations that can be measured with indirect techniques based on an associative conception of implicit cognition (e.g., Implicit Association Test; IAT). However, alternative theoretical proposals (e.g., relational frame theory; RFT) propose that implicit SE might not be based on automatic self-associations, but on implicit propositional self-evaluations that can be captured only with nonassociative implicit measures (e.g., Implicit Relational Assessment Procedure; IRAP). In the present study, both reliability and validity of a new propositional measure of implicit self-esteem (relational responding task; RRT) were assessed, and compared with the SE-IAT and with two self-report scales of self-esteem. In the first study, two alternative self-esteem RRTs (SE-RRT and RSE-RRT) were administered along with a SE-IAT and other scales, to assess reliability and validity issues. The results showed: 1) acceptable, though not optimal, reliability for both RRTs; 2) an adequate support for convergent validity, with significant correlations between implicit and explicit measures of SE; 3) the criterion validity was supported for the RSE-RRT (with significant correlations with all theoretically linked scales), although only partially supported for the SE-RRT (with a significant correlation only with depression; 4) RRTs were not significantly correlated with impression management and self-deception; and 5) incremental validity of implicit propositional SE on depression, controlling for automatic SE associations and explicit self-esteem. In a second study, it was experimentally demonstrated that SE-RRT showed levels of “fakeability” similar to a classical implicit self-esteem measure like the SE-IAT, and considerably lower than SE scales

Binding of AT4 receptor ligands to insulin regulated aminopeptidase (IRAP) in intact Chinese hamster ovary cells

International audienceInsulin regulated aminopeptidase (IRAP) recognises "AT-receptor" ligands like angiotensin IV (Ang IV) and peptidomimetics like AL-11. The metabolic stability and high affinity of [H]AL-11 for catalytically active IRAP allowed its detection in Chinese hamster ovary (CHO-K1) cell membranes in the absence of chelators (Demaegdt et al., 2009). Here, we show that, contrary to [H]Ang IV, [H]AL-11 displays high affinity and specificity for IRAP in intact CHO-K1 cells as well. After binding to IRAP at the surface, [H]AL-11 is effectively internalized by an endocytotic process. Unexpectedly, surface binding and internalization of [H]AL-11 was not affected by pretreating the cells with Ang IV but declined with AL-11. In the latter case surface expression of IRAP even increased. After elimination of simpler explanations, it is proposed that metabolically stable "AT-receptor" ligands undergo semi-continuous cycling between the cell surface and endosomal compartments. The efficacy of stable and unstable "AT-receptor" ligands could therefore differ

Identification of MEDIATOR16 as the Arabidopsis COBRA suppressor MONGOOSE1.

We performed a screen for genetic suppressors of cobra, an Arabidopsis mutant with defects in cellulose formation and an increased ratio of unesterified/esterified pectin. We identified a suppressor named mongoose1 (mon1) that suppressed the growth defects of cobra, partially restored cellulose levels, and restored the esterification ratio of pectin to wild-type levels. mon1 was mapped to the MEDIATOR16 (MED16) locus, a tail mediator subunit, also known as SENSITIVE TO FREEZING6 (SFR6). When separated from the cobra mutation, mutations in MED16 caused resistance to cellulose biosynthesis inhibitors, consistent with their ability to suppress the cobra cellulose deficiency. Transcriptome analysis revealed that a number of cell wall genes are misregulated in med16 mutants. Two of these genes encode pectin methylesterase inhibitors, which, when ectopically expressed, partially suppressed the cobra phenotype. This suggests that cellulose biosynthesis can be affected by the esterification levels of pectin, possibly through modifying cell wall integrity or the interaction of pectin and cellulose