Showing papers by "Natasha V. Raikhel published in 2011"

A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis

Abstract: The interactions between phytohormones are crucial for plants to adapt to complex environmental changes. One example is the ethylene-regulated local auxin biosynthesis in roots, which partly contributes to ethylene-directed root development and gravitropism. Using a chemical biology approach, we identified a small molecule, l-kynurenine (Kyn), which effectively inhibited ethylene responses in Arabidopsis thaliana root tissues. Kyn application repressed nuclear accumulation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Moreover, Kyn application decreased ethylene-induced auxin biosynthesis in roots, and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE RELATEDs (TAA1/TARs), the key enzymes in the indole-3-pyruvic acid pathway of auxin biosynthesis, were identified as the molecular targets of Kyn. Further biochemical and phenotypic analyses revealed that Kyn, being an alternate substrate, competitively inhibits TAA1/TAR activity, and Kyn treatment mimicked the loss of TAA1/TAR functions. Molecular modeling and sequence alignments suggested that Kyn effectively and selectively binds to the substrate pocket of TAA1/TAR proteins but not those of other families of aminotransferases. To elucidate the destabilizing effect of Kyn on EIN3, we further found that auxin enhanced EIN3 nuclear accumulation in an EIN3 BINDING F-BOX PROTEIN1 (EBF1)/EBF2-dependent manner, suggesting the existence of a positive feedback loop between auxin biosynthesis and ethylene signaling. Thus, our study not only reveals a new level of interactions between ethylene and auxin pathways but also offers an efficient method to explore and exploit TAA1/TAR-dependent auxin biosynthesis.

Clusters of bioactive compounds target dynamic endomembrane networks in vivo

Abstract: Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana. Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.

Article A Molecular Switch for Initiating Cell Differentiation in Arabidopsis

Abstract: Centro de Biologi´a Molecular Severo Ochoa, CSIC-UAM,Cantoblanco, 28049 Madrid, SpainSummaryBackground:Theonsetofdifferentiationentailsmodifyingthegene expression state of cells, to allow activation of develop-mentalprogramsthataremaintainedrepressedintheundiffer-entiated precursor cells [1, 2]. This requires a mechanism tochangegeneexpression onagenome-scale. Recentevidencesuggeststhatinmammalianstemcells,derepressionofdevel-opmentalregulatorsduringdifferentiationinvolvesashiftfromstalled to productive elongation of their transcripts [3–5], butfactors mediating this shift have not been identified and theevidence remains correlative.Results:WereporttheidentificationoftheMINIYO(IYO)gene,a positive regulator of transcriptional elongation that is essen-tial for cells to initiate differentiation in Arabidopsis. IYO inter-actswithRNApolymeraseIIandtheElongator complexandisrequired to sustain global levels of transcriptional elongationactivity, specifically in differentiating tissues. Accordingly,IYO is expressed in embryos, meristems, and organ primordiaand not in mature tissues. Moreover, differential subcellularprotein distribution further refines the domain of IYO functionby directing nuclear accumulation, and thus its transcriptionalactivity,tocellsinitiatingdifferentiation.Importantly,IYOover-expression induces premature cell differentiation and leads tomeristem termination phenotypes.Conclusions: These findings identify IYO as a necessary andsufficient factor for initiating differentiation in Arabidopsisand suggest that the targeted nuclear accumulation of IYOfunctions as a transcriptional switch for this fate transition.IntroductionCell differentiation entails two sequential decisions byprecursor cells. The first one, shared by every event of differ-entiation,iswhethertoself-reneworinitiatethisfatetransition.Theseconddecision,specificforeachdifferentiation process,is which cell fate to attain. Genetic networks determiningprogression into specific fates have been described in bothanimals and plants [6]. In contrast, very little is known aboutthe mechanisms that trigger differentiation in the first place,possibly because this step is essential for organism viabilityand therefore is less amenable to genetic analysis. In plants,differentiation occurs in defined niches called meristems andis easily traceable due to the immobility of cells. In addition,plants have high developmental plasticity and tolerate drasticchanges in their body plan. This renders them a powerfulmodel system to search for genetic factors that determinethe timing of cell differentiation.It has been proposed that plant hormone gradients serve asinstructive signals toset theboundaries where celldifferentia-tion starts in meristems. For instance, crosstalk betweenauxins and cytokinins delimits the transition zone in root meri-stems[7]andauxinaccumulationdefinesthepositionoffutureorgan primordia in the shoot meristem [8]. Several genesinvolved in generating, perceiving, and transducing thesehormonal signals have been linked to these developmentalresponses [9]. However, it is unknown how the decision toinitiate differentiation is taken at the cellular level. Differentia-tion implies a global transcriptional reprogramming, so initia-tion factors probably target the transcriptional condition ofthe cell. In metazoans it is thought that the undifferentiatedstatus is the default transcriptional state maintained by self-reinforcement of networks that direct renewal and repressionof genes directing differentiation [2, 10]. Remarkably, inmammalian stem cells (SCs), chromatin is in an open state[10, 11], implying that the transcriptional machinery hasaccess to the promoters of genes directing differentiationeven though they remain transcriptionally inactive. In supportof this hypothesis, there is genome-wide transcriptional initia-tion,butproductiveelongationofdevelopmentalregulatorsdi-recting differentiation is prevented [3–5]. Differentiation inmammalian SCs may thus involve the active removal ofhindrances preventing transcriptional elongation of develop-mentalgenesor,alternatively,theaccumulationofanecessaryfactor for elongation in the progeny destined for differentia-tion. Interestingly, the results presented here suggest that inArabidopsis, the specific nuclear accumulation of a positiveelongation factor initiates differentiation.ResultsMINIYO Is Required for Initiating Cell Differentiationin the Shoot Apical MeristemThe shoot apical meristem (SAM) of Arabidopsis containsa pool of slow proliferating long-term SCs in the central zonesurrounded by faster proliferating transit-amplifying cells.The progeny from these cells is displaced to the periphery oftheSAMwhereitstartstodifferentiateandgeneratestheaerialorgans of the plant in a stereotypic phyllotactic pattern. In anArabidopsis EMS-mutagenized population, we identified theminiyo-1(iyo-1)mutantthathaddelayedemergenceofthefirstleaves and altered phyllotaxis (Figure 1A), suggesting thatorganogenesis in the SAM was perturbed. Moreover, theiyo-1 mutants had enlarged SAMs, ectopic meristems, andfasciated shoots (Figures 1B and 1C), which could be due toa defective transition of meristematic cells into differentiation.To clarify the function of IYO, we analyzed its genetic