PIF1 promotes phytochrome-regulated growth under photoperiodic conditions in Arabidopsis together with PIF3, PIF4, and PIF5

Seedlings growing under diurnal conditions display maximal growth at the end of the night in short-day (SD) photoperiods. Current evidence indicates that this behaviour involves the action of PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) together with PIF4 and PIF5, through direct regulation of growth-related genes at dawn coinciding with a PIF3 accumulation peak generated by phytochrome-imposed oscillations in protein abundance. Here, to assess how alterations in PIF3 levels impact seedling growth, the night-specific accumulation of PIF3 was modulated by releasing SD-grown seedlings into continuous light, or by exposing them to a phytochrome-inactivating end-of-day far-red pulse (EOD-FRp). The data show a strong direct correlation between PIF3 accumulation, PIF3-regulated induction of growth-related genes, and hypocotyl elongation, and suggest that growth promotion in SD conditions involves factors other than PIF3, PIF4, and PIF5. Using a pif1 mutant, evidence is provided that PIF1 also contributes to inducing hypocotyl elongation during the dark period under diurnal conditions. PIF1 displayed constitutive transcript levels in SD conditions, suggesting that phytochrome-imposed oscillations in PIF1 protein abundance determine its accumulation and action during the night, similar to PIF3 and in contrast to PIF4 and PIF5, which oscillate diurnally due to a combination of circadian clock-regulated transcription and light control of protein accumulation. Furthermore, using single and higher order pif mutants, the relative contribution of each member of the PIF quartet to the regulation of morphogenesis and the expression of selected growth marker genes under SD conditions, or under SD conditions supplemented with an EOD-FRp, is defined. Collectively, the data indicate that PIF1, PIF3, PIF4, and PIF5 act together to promote and optimize growth under photoperiodic conditions

Phytochrome-imposed inhibition of PIF7 activity shapes photoperiodic growth in Arabidopsis together with PIF1 , 3, 4 and 5

Under photoperiodic conditions, Arabidopsis thaliana seedling growth is inhibited in long days (LDs), but promoted under the extended nights of short days (SDs). This behavior is partly implemented by phytochrome (phy)-imposed oscillations in the abundance of the growth-promoting, phy-interacting bHLH transcription factors PHY-INTERACTING FACTOR 1 (PIF1), PIF3, PIF4 and PIF5 (PIF quartet or PIFq). However, the observation that a pifq mutant is still stimulated to elongate when given a phy-inactivating end-of-day far-red pulse (EODFR), suggests that additional factors are involved in the phy-mediated suppression of growth during the subsequent dark period. Here, by combining growth-analysis of pif7 single- and higher-order mutants with gene expression analysis under SD, LD, SD-EODFR, and LD-EODFR, we show that PIF7 promotes growth during the dark hours of SD, by regulating growth-related gene expression. Interestingly, the relative contribution of PIF7 in promoting growth is stronger under EODFR, whereas PIF3 role is more important under SD, suggesting that PIF7 is a prominent target of phy-suppression. Indeed, we show that phy imposes phosphorylation and inactivation of PIF7 during the light hours in SD, and prevents full dephosphorylation during the night. This repression can be lifted with an EODFR, which correlates with increased PIF7-mediated gene expression and elongation. In addition, our results suggest that PIF7 function might involve heterodimerization with PIF3. Furthermore, our data indicate that a pifqpif7 quintuple mutant is largely insensitive to photoperiod for hypocotyl elongation. Collectively, the data suggest that PIF7, together with the PIFq, is required for the photoperiodic regulation of seasonal growth

Molecular convergence of clock and photosensory pathways through PIF3–TOC1 interaction and co-occupancy of target promoters

This study defines a molecular mechanism for how clock- and light-signaling pathways converge in Arabidopsis. The data reveal that TOC1, an essential core component of the central oscillator, binds to and represses PIF transcriptional activators, which are also the direct molecular signaling partners of the phytochrome photosensory receptors. This finding shows that TOC1 functions as a clock output-transducer, directly linking the core oscillator to a pleiotopically-acting transcriptional network, through repression of target genes. Collectively, in the plant, these components comprise a transcriptionallycentered signaling hub that provides clock-imposed gating of PIF-mediated, photosensory-regulated diurnal growth patterns. These results provide a framework for future research aimed at understanding how circadian dynamics are integrated with other plant physiological processes important for optimal plant fitness.Fil: Soy, Judit. Universitat Autònoma de Barcelona; EspañaFil: Leivar, Pablo. Universitat Autònoma de Barcelona; EspañaFil: Gonzalez Schain, Nahuel Damian. Universitat Autònoma de Barcelona; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Martín, Guiomar. Universitat Autònoma de Barcelona; EspañaFil: Diaz, Céline. Universitat Autònoma de Barcelona; EspañaFil: Sentandreu, Maria. Universitat Autònoma de Barcelona; EspañaFil: Al-Sady, Bassem. University of California at Berkeley; Estados Unidos. United States Department of Agriculture; Estados UnidosFil: Quail, Peter H.. University of California at Berkeley; Estados Unidos. United States Department of Agriculture; Estados UnidosFil: Monte, Elena. Universitat Autònoma de Barcelona; Españ

Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate <i>N</i>-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development.

--- - i: - N - N - O - N - Plasmodium falciparum - Cryptosporidium parvum - P. falciparum - N - N - P. falciparum - C. parvum b: - IMPORTANCE content: - UDP- - "-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and " - "- and " - "-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate " - "-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual " - " parasites. Furthermore, we present the 1.95\xE2\x80\x89\xC3\x85 resolution structure of the GNA1 ortholog from " - ", an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for " - " GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria." - " Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite's ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate " - -acetyltransferase (GNA1), required for the biosynthesis of UDP- - "-acetylglucosamine (UDP-GlcNAc), is essential for " - " asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite " - ", used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

Circadian waves of transcriptional repression shape PIF-regulated photoperiod-responsive growth in a<i>rabidopsis</i>

Plants coordinate their growth and development with the environment through integration of circadian clock and photosensory pathways. In Arabidopsis thaliana, rhythmic hypocotyl elongation in short days (SD) is enhanced at dawn by the basic-helix-loop-helix (bHLH) transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) directly inducing expression of growth-related genes [1-6]. PIFs accumulate progressively during the night and are targeted for degradation by active phytochromes in the light, when growth is reduced. Although PIF proteins are also detected during the day hours [7-10], their growth-promoting activity is inhibited through unknown mechanisms. Recently, the core clock components and transcriptional repressors PSEUDO-RESPONSE REGULATORS PRR9/7/5 [11, 12], negative regulators of hypocotyl elongation [13, 14], were described to associate to G boxes [15], the DNA motifs recognized by the PIFs [16, 17], suggesting that PRR and PIF function might converge antagonistically to regulate growth. Here we report that PRR9/7/5 and PIFs physically interact and bind to the same promoter region of pre-dawn-phased, growth-related genes, and we identify the transcription factor CDF5 [18, 19] as target of this interplay. In SD, CDF5 expression is sequentially repressed from morning to dusk by PRRs and induced pre-dawn by PIFs. Consequently, CDF5 accumulates specifically at dawn, when it induces cell elongation. Our findings provide a framework for recent TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) data [5, 20] and reveal that the long described circadian morning-to-midnight waves of the PRR transcriptional repressors (PRR9, PRR7, PRR5, and TOC1) [21] jointly gate PIF activity to dawn to prevent overgrowth through sequential regulation of common PIF-PRR target genes such as CDF5

Anàlisi funcional de la regió N-terminal de l'HMG-CoA reductasa d'Arabidopsis

[cat] Les plantes sintetitzen una gran varietat de compostos isoprenoides a través d'una ruta metabòlica complexa i ramificada. L'enzim HMG-CoA reductasa (HMGR) catalitza la conversió d'HMG-CoA en mevalonat, una etapa limitant de flux per la biosíntesis d'isoprenoides en el citosol-reticle endoplasmàtic (RE). En totes les espècies de plantes analitzades existeixen vàries isoformes d'HMGR codificades per petites famílies multigèniques. En el cas d'Arabidopsis, existeixen dos gens ( HMG1 i HMG2 ) que codifiquen per tres isoformes de l'enzim: HMGR1L, HMGR1S i HMGR2. Múltiples estudis suggereixen que les isoformes d'HMGR de plantes estarien implicades en la síntesi d'isoprenoides particulars. Aquest model d'especialització funcional de les isoformes d'HMGR està basat en estudis d'expressió gènica en diferents estadis del desenvolupament o en resposta a diferents estímuls. Tot i així, a l'inici d'aquesta tesi, no es tenien dades contrastades de la distribució de les isoformes d'HMGR en diferents teixits ni de la seva distribució subcel·lular. Aquest darrer aspecte era particularment interessant, doncs s'havia suggerit que l'especialització funcional de les isoformes d'HMGR podria venir determinada, en part, per diferències en la seva localització subcel·lular. Amb aquests antecedents, es va iniciar un estudi de la distribució tissular i subcel·lular de les diferents isoformes d'HMGR d'Arabidopsis. Mitjançant assaigs de western blot utilitzant anticossos generats contra el domini catalític de l'enzim, es va comprovar que les isoformes HMGR1S i HMGR1L es distribuïen de manera diferencial en teixits d'Arabidopsis, fet que es correlacionava amb els estudis previs d'expressió dels gens corresponents. Tanmateix, estudis de fraccionament subcel·lular a partir de cèl·lules de la línia T87 van demostrar que aquestes isoformes es trobaven en fraccions diferents: si bé l'HMGR1S es trobava bàsicament en una fracció que sedimenta a 16.000xg, l'HMGR1L s'enriquia en una fracció que sedimenta a 105.000xg. Estudis de localització subcel·lular emprant fusions amb la proteïna fluorescent verda (GFP) van determinar que, si bé la isoforma HMGR1S es localitza principalment en estructures vesiculars de 0.5-2 mi/m, la isoforma HMGR1L es troba exclusivament en el RE. Aquesta localització subcel·lular diferencial entre ambdues isoformes ve determinada per la presència d'una extensió N-terminal de 50 aminoàcids (1Lextra) en la isoforma HMGR1L. Per tant, la regió N-terminal citosòlica constitueix una regió que determina la localització subcel·lular de l'enzim, funció que ha de venir mediada per proteïnes que hi interaccionin específicament. Amb l'objectiu de cercar proteïnes que interaccionin amb la regió N-terminal citosòlica de l'HMGR1L, es va realitzar un crivellatge per doble híbrid d'una genoteca d'expressió d'Arabidopsis. D'aquesta manera, es van identificar les proteïnes PR2A1, PR2A2 i KLC1, i es va procedir a la seva caracterització. D'una banda, es va verificar que PR2A1 i PR2A2 eren subunitats reguladores B'' de la proteïna fosfatasa 2A (PP2A). Tanmateix, es va determinar que PR2A1 i PR2A2 interaccionaven selectivament amb la regió N-terminal citosòlica de les isoformes HMGR1L i HMGR1S, però no amb la de l'HMGR2. Finalment, es va constatar que la interacció de les isoformes HMGR1L i HMGR1S amb la PR2A1 és modulada per calci. Les dades suggereixen que la PP2A podria formar part de la cascada de senyalització subcel·lular que permetria ajustar l'activitat HMGR als estímuls ambientals i de desenvolupament. D'altra banda, es va constatar que KLC1 presentava similitud amb la cadena lleugera de quinesina de tipus I, i en conservava una estructura modular equivalent. A més, es va determinar que KLC1 interacciona amb la regió N-terminal citosòlica de la isoforma HMGR1L, però no amb la de l'HMGR1S ni l'HMGR2. També es va comprovar que la regió 1Lextra, que determina la localització de l'HMGR1L en el RE, és imprescindible per aquesta interacció. Aquest fet suggereix que KLC1 podria jugar un paper en la localització subcel·lular de la isoforma HMGR1L. En conjunt, les dades de localització subcel·lular i d'interacció amb les proteïnes PR2A1, PR2A2 i KLC1, permeten postular un model d'especialització funcional de les isoformes HMGR1S i HMGR1L en el qual la biosíntesis de mevalonat es produiria segregada en dos compartiments subcel·lulars: el RE i les estructures vesiculars, les quals constituirien un nou compartiment de biosíntesis d'isoprenoides en plantes

PIFs: Systems integrators in plant development

Phytochrome-interacting factors (PIFs) are members of the Arabidopsis thaliana basic helix-loop-helix family of transcriptional regulators that interact specifically with the active Pfr conformer of phytochrome (phy) photoreceptors. PIFs are central regulators of photomorphogenic development that act to promote stem growth, and this activity is reversed upon interaction with phy in response to light. Recently, significant progress has been made in defining the transcriptional networks directly regulated by PIFs, as well as the convergence of other signaling pathways on the PIFs to modulate growth. Here, we summarize and highlight these findings in the context of PIFs acting as integrators of light and other signals. We discuss progress in our understanding of the transcriptional and posttranslational regulation of PIFs that illustrates the integration of light with hormonal pathways and the circadian clock, and we review seedling hypocotyl growth as a paradigm of PIFs acting at the interface of these signals. Based on these advances, PIFs are emerging as required factors for growth, acting as central components of a regulatory node that integrates multiple internal and external signals to optimize plant development.This work was supported by the “Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa” fellowship from the Generalitat de Catalunya (Beatriu de Pinós program) and Marie Curie IRG PIRG06-GA-2009-256420 Grant to P.L., as well as by grants from the Spanish “Ministerio de Economía” BIO2012-31672, from the “Ministerio de Educación, Cultura y Deporte” PRX12/00631, and from the Generalitat de Catalunya 2009-SGR-206 to E.M.Peer reviewe

Out of the dark: how the PIFs are unmasking a dual temporal mechanism of phytochrome signalling

9 pages.-- PMID: 17855731 [PubMed].Following light-induced nuclear translocation, the phytochromes induce changes in gene expression to regulate plant development. PIF3 and other PIFs (phytochrome-interacting factors), members of the bHLH (basic helix-loop-helix) family of transcriptional regulators, interact specifically with the active Pfr conformer of the phytochrome molecule, suggesting that the PIFs are key components of phytochrome signal transduction. The mechanism by which the PIFs transduce phytochrome signals is not understood. After initial studies that suggested that PIF3 was a positive regulator of phytochrome signalling, mutant studies indicated that the PIFs primarily act as negative regulators in the pathway. Furthermore, in some cases they accumulate in the dark and are degraded upon illumination by the ubiquitin-26S proteasome system. At least for PIF3, the protein degradation depends on direct interaction with the phytochrome molecule and is preceded by protein phosphorylation. In this review, the current understanding of the role of the PIFs in phytochrome-mediated photomorphogenesis will be summarized, and recent findings suggesting an unanticipated dual mechanism of action of the PIFs will be discussed.Peer reviewe