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

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

PIF3 (phytochrome·interaeting factor 3) and Circadian Clock interaction in the regulation of plant growth

[cat] El desenvolupament i creixement de plàntules d’Arabidopsis thaliana està influenciat tant per factors ambientals, com la llum o la temperatura, com per factors interns, com per exemple les hormones o el rellotge circadià. Es coneix que, en condicions de dia curt (cicles de 8h de llum i 16h de foscor), tant la llum com el rellotge circadià realitzen un paper clau en la regulació de l’elongació de l’hipocòtil ja que mutacions en components de les dues vies tenen com a conseqüència alteracions en la llargada d’aquest òrgan. L’objectiu principal d’estudi en aquesta tesi ha estat determinar la funció dels factors de transcripció PIF, intermediaris de la via de senyalització lumínica, en la regulació de l’elongació de l’hipocòtil en plàntules crescudes en dia curt i la connexió que existeix entre aquestes proteïnes i la proteïna TOC1, component central del rellotge circadià. Les dades fenotípiques i moleculars obtingudes al llarg de la tesi ens han permès concloure que les proteïnes PIF1 i PIF3, conjuntament amb PIF4 i PIF5 les quals ja havien estat prèviament involucrades en el procés, actuen com a reguladors positius de l’elongació de l’hipocòtil al final del període de foscor en condicions de dia curt. A més, diferents estudis fenotípics i anàlisis genòmics i bioquímics, conjuntament amb experiments d’immunoprecipitació de la cromatina, realitzats en diferents combinacions de mutants PIFs i TOC1 han permès determinar que existeix un nou punt de relació directa entre la llum i el rellotge circadià en la regulació de l’elongació de l’hipocòtil. Aquest treball ha permès demostrar l’existència d’un nou mecanisme de regulació pel qual el rellotge circadià restringeix el creixement de l’hipocòtil just al final del període de foscor en plàntules crescudes en condicions de dia curt. Tal mecanisme regulatori es basa en la repressió feta per la proteïna TOC1 sobre l’activitat transcripcional de la proteïna PIF3 durant la primera meitat del període de foscor.[eng] Arabidopsis thaliana seedling growth and development is influenced by both external (such as light and temperature) and internal (like hormones or circadian clock) factors. In short-day (SD) growing conditions, it is known that both light and clock have a pivotal role in the regulation of hypocotyl elongation, as mutations in members of the two pathways result in altered lengthening of the hypocotyl. The main objective of the thesis was to determine the function of PIF transcription factors, as light-signaling pathway intermediates, in the regulation of hypocotyl elongation of SD-grown seedlings, together with the possible interplay between them and TOC1 protein, a central component of the circadian clock. Phenotypic and molecular data obtained along the thesis allowed us to conclude that PIF1 and PIF3 proteins, together with the other members PIF4 and PIF5 which had previously been related with the process, act as positive regulators of hypocotyl growth at the end of the dark period in SD conditions. Moreover, several phenotypic studies, gene expression and protein accumulation analysis, together with chromatin immunoprecipitation assays performed in multiple combinations of PIFs and TOC1 mutants allows us to determine that there is a previously unknown direct crosstalk point between light and circadian clock in the regulation of hypocotyl elongation. Together, the work indicates the existence of a new regulatory mechanism by which clock gates hypocotyl elongation at the end of the dark period in SD-grown seedlings by repressing the transcriptional activity of PIF3 protein during the rest of the night

Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes

Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to “growth” and “no growth” conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes.This work was supported by grants from the Spanish “Ministerio de Ciencia e Innovación” BIO2009-07675, “Ministerio de Economía” BIO2012-31672, and from the Generalitat de Catalunya 2014-SGR-1406 to EM.Peer reviewedPeer Reviewe

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