Final Report Summary - DARK GROWTH (Identification and characterization of novel plant growth regulators acting downstream of the phytochrome-interacting bHLH transcription factors in the dark)
Plants modulate their growth and development to adjust to the continuous variations in their light environment. During early development, seedlings emerging from buried seeds grow vigorously upward in the subterranean darkness toward the soil surface (etiolated growth), a developmental strategy promoted by several members of the phytochrome (phy)-interacting factor (PIF) subfamily of bHLH transcription factors (PIF1,3,4 and 5) in Arabidopsis thaliana. Upon reaching the soil surface, light signals perceived by members of the red (R)/far-red (FR) family of phy photoreceptors (phyA-E in Arabidopsis) reverse etiolation (deetiolation) by inducing rapid degradation of the PIF proteins. Thus, an antagonistic functional interaction between active phy and PIFs determines seedling growth during deetiolation. A similar mechanism regulates rhythmic growth in seedlings growing under diurnal conditions (in dark and light cycles), where growth is restricted to the end of the night through phy-regulation of PIFs abundance. Altogether, PIFs are central regulators of plant growth in the dark both during seedling deetiolation and under diurnal conditions. Unravelling how PIFs function in the dark during these two dark-light transitions is of great interest to understand regulation of plant growth. While recent data have started to provide some insight into the transcriptional network regulated by the PIFs in the dark, little is known on which of the identified genes are direct targets or implement downstream cellular and morphogenic facets of growth.
The research proposal aims to identify and characterize novel regulators of plant growth in the dark acting downstream of the PIFs. First, a novel genetic screen has been performed to identify suppressors of the pif1pif3pif4pif5 (pifq) quadruple mutant phenotype in etiolated seedlings. For this, we have set up the conditions for a novel suppressor screen of the constitutively photomorphogenic (cop)-like phenotype of the pifq mutant, based on the FOX (Full-length cDNA Over-eXpressing) gene hunting system (Ichikawa et al (2006).The Plant Journal 48, 974-985). We have generated 13260 pifq transformants by using a library of 10000 independent full-length Arabidopsis cDNAs under the control of the CaMV 35S promoter. Among the transformants, we originally identified 173 suppressor of pifq (sop) mutants in the T1 generation, and 28 of them were validated in the T2 generation. Sequencing of the transgenes of the 28 sop mutants resulted in the identification of a single cDNA in 23 lines, two cDNAs in one line, and an empty vector in the remaining 4 lines. A majority of the identified cDNAs, potentially causing the pifq-suppressor phenotype, corresponded to genes whose expression was regulated by the PIFs, and are thus strong candidates to implement cellular and morphogenic facets of growth downstream of the PIFs. We are currently performing a functional characterization of one of these genes, a DOF transcription factor that is direct target of the PIFs, which will result in a publication that is currently in preparation. The rest of the candidates are a valuable resource that will be the ground for future research.
Second, we have conducted a biochemical and genomic approach to identify PIF3-regulated genes at the end of the night in seedlings growing under diurnal conditions. We established that PIF3, together with PIF1, PIF4 and PIF5, plays a prominent role in implementing growth at the end of the night (Soy et al (2012). The Plant Journal 71, 390-401; Soy et al (2014).Journal of Experimental Botany 65, 2925-36). Importantly, we have defined that a dynamic antagonism is established between the phyB photoreceptor and PIF3 and related PIFs to regulate growth responses to light under diurnal conditions, as well as in two other experimental conditions: during seedling deetiolation and in response to low R/FR ratio (Leivar et al (2012). Molecular Plant 5, 208-223). The data suggest that phy repress growth in response to different light conditions through a common mechanism involving the proteolytic degradation of the PIF transcription factors. In addition, we performed comparative transcriptomic analysis of seedlings grown under these experimental conditions (deetiolation, diurnal and low R/FR), and have identified a core set of PIF-regulated genes, enriched in transcription factors and hormone-related genes, and containing G-boxes in their promoters, that are similarly regulated by the PIFs in at least two of the growth responses tested (Leivar et al (2012). The Plant Cell 24, 1398-1419). These genes are thus strong candidate primary targets of the phy/PIFs regulatory network. Consistent with this view, we have performed chromatin immunoprecipitation (ChIP-qPCR) analysis and have shown that PIF3 binds to the promoter regions of at least three of the identified genes, timing their expression to peak at the end of the night under diurnal conditions (Soy et al (2012). The Plant Journal 71, 390-401). In order to refine and narrow down the list of PIF-regulated genes potentially implementing downstream facets of morphogenesis under diurnal conditions, we have extended the comparative transcriptomic analysis to include the lists of defined PIF-target genes. For this, we took advantage of recent publications that define the genomic binding sites for PIF1, PIF3, PIF4 and PIF5 in chip-chip and chip-seq experiments. Our integrated new analysis defines a core set of 22 PIF-induced PIF-target genes during deetiolation, diurnal and low R/FR, which might comprise components of a common cellular machinery that implement cell growth responses downstream of the PIFs under different phy-regulated growth conditions (Leivar and Monte (2014). The Plant Cell 26, 56-78).We have confirmed for several of these genes that PIF3 binds to their promoter regions and times their expression to peak at the end of the night under diurnal conditions, which will be included in a manuscript that is in preparation.
The ongoing research focuses on the elucidation of novel PIF-regulated components that regulate plant growth during deetiolation and under diurnal conditions. PIF transcription factors are emerging as central regulators of plant growth and development throughout the life cycle of the plant, acting as cellular hubs that integrate diverse external (light, temperature) and internal (circadian clock, hormones) signals, acting as systems integrators in plant development as proposed in 2 influential reviews that resulted from this project (Leivar and Quail (2011). Trends in Plant Science 16, 19-28; Leivar and Monte (2014).The Plant Cell 26, 56-78). In addition to their role during seedling deetiolation and under diurnal growth conditions, PIFs are being unveiled as instrumental regulators of multiple additional responses such as seed germination, flowering, neighbour-sensing shade avoidance, adaptations of plant architecture in response to high temperature, or stomatal development. Because of the wide range of PIF-regulated responses and because the mechanism of PIF action in different processes appears to share common components, it is expected that our results will be relevant to understand critical aspects of plant growth and development throughout the life cycle of the plant.
These studies will also contribute to understand molecularly how changes in the light environment perceived by the phy photoreceptors are transduced to the downstream regulatory network to trigger the proper adjustments in plant growth and development for optimal fitness. Because plant growth is of great agronomical importance, and because phy and PIFs are conserved in major crops, it is expected that this work will provide potential targets for comparative genomics and genetic modification in suitable crops. Because crops are often grown in open fields under diurnal conditions (day and night cycles), understanding the molecular mechanisms underlying plant growth under these conditions may allow manipulation of the system with the long-term goal of increasing crop productivity to satisfy the increasing demand of a growing population.
The researcher has been involved in several activities that have completed his training and have increased his competitiveness towards the goal of gaining an independent research position. First, the researcher has initiated a strong project in Europe which has resulted in 8 publications related to the project, some of them in top plant journals. Second, the researcher has supervised the work of two PhD students and two Master students, thus gaining experience in leadership and project management abilities. Third, the researcher has attended and presented the results of the project in 9 conferences, which allowed positive feedback from the international community and new networking opportunities in Europe. Fourth, the researcher has also been able to maintain a close collaboration with the former laboratory at University of California at Berkeley, thus keeping a solid network of international collaborations that is having a strong feedback on the project. Finally, the researcher has participated in reviewing papers in international journals, as well as in evaluating thesis by participating in thesis committees. Together, these additional training has been instrumental for the researcher to find an independent position in Europe.
The research proposal aims to identify and characterize novel regulators of plant growth in the dark acting downstream of the PIFs. First, a novel genetic screen has been performed to identify suppressors of the pif1pif3pif4pif5 (pifq) quadruple mutant phenotype in etiolated seedlings. For this, we have set up the conditions for a novel suppressor screen of the constitutively photomorphogenic (cop)-like phenotype of the pifq mutant, based on the FOX (Full-length cDNA Over-eXpressing) gene hunting system (Ichikawa et al (2006).The Plant Journal 48, 974-985). We have generated 13260 pifq transformants by using a library of 10000 independent full-length Arabidopsis cDNAs under the control of the CaMV 35S promoter. Among the transformants, we originally identified 173 suppressor of pifq (sop) mutants in the T1 generation, and 28 of them were validated in the T2 generation. Sequencing of the transgenes of the 28 sop mutants resulted in the identification of a single cDNA in 23 lines, two cDNAs in one line, and an empty vector in the remaining 4 lines. A majority of the identified cDNAs, potentially causing the pifq-suppressor phenotype, corresponded to genes whose expression was regulated by the PIFs, and are thus strong candidates to implement cellular and morphogenic facets of growth downstream of the PIFs. We are currently performing a functional characterization of one of these genes, a DOF transcription factor that is direct target of the PIFs, which will result in a publication that is currently in preparation. The rest of the candidates are a valuable resource that will be the ground for future research.
Second, we have conducted a biochemical and genomic approach to identify PIF3-regulated genes at the end of the night in seedlings growing under diurnal conditions. We established that PIF3, together with PIF1, PIF4 and PIF5, plays a prominent role in implementing growth at the end of the night (Soy et al (2012). The Plant Journal 71, 390-401; Soy et al (2014).Journal of Experimental Botany 65, 2925-36). Importantly, we have defined that a dynamic antagonism is established between the phyB photoreceptor and PIF3 and related PIFs to regulate growth responses to light under diurnal conditions, as well as in two other experimental conditions: during seedling deetiolation and in response to low R/FR ratio (Leivar et al (2012). Molecular Plant 5, 208-223). The data suggest that phy repress growth in response to different light conditions through a common mechanism involving the proteolytic degradation of the PIF transcription factors. In addition, we performed comparative transcriptomic analysis of seedlings grown under these experimental conditions (deetiolation, diurnal and low R/FR), and have identified a core set of PIF-regulated genes, enriched in transcription factors and hormone-related genes, and containing G-boxes in their promoters, that are similarly regulated by the PIFs in at least two of the growth responses tested (Leivar et al (2012). The Plant Cell 24, 1398-1419). These genes are thus strong candidate primary targets of the phy/PIFs regulatory network. Consistent with this view, we have performed chromatin immunoprecipitation (ChIP-qPCR) analysis and have shown that PIF3 binds to the promoter regions of at least three of the identified genes, timing their expression to peak at the end of the night under diurnal conditions (Soy et al (2012). The Plant Journal 71, 390-401). In order to refine and narrow down the list of PIF-regulated genes potentially implementing downstream facets of morphogenesis under diurnal conditions, we have extended the comparative transcriptomic analysis to include the lists of defined PIF-target genes. For this, we took advantage of recent publications that define the genomic binding sites for PIF1, PIF3, PIF4 and PIF5 in chip-chip and chip-seq experiments. Our integrated new analysis defines a core set of 22 PIF-induced PIF-target genes during deetiolation, diurnal and low R/FR, which might comprise components of a common cellular machinery that implement cell growth responses downstream of the PIFs under different phy-regulated growth conditions (Leivar and Monte (2014). The Plant Cell 26, 56-78).We have confirmed for several of these genes that PIF3 binds to their promoter regions and times their expression to peak at the end of the night under diurnal conditions, which will be included in a manuscript that is in preparation.
The ongoing research focuses on the elucidation of novel PIF-regulated components that regulate plant growth during deetiolation and under diurnal conditions. PIF transcription factors are emerging as central regulators of plant growth and development throughout the life cycle of the plant, acting as cellular hubs that integrate diverse external (light, temperature) and internal (circadian clock, hormones) signals, acting as systems integrators in plant development as proposed in 2 influential reviews that resulted from this project (Leivar and Quail (2011). Trends in Plant Science 16, 19-28; Leivar and Monte (2014).The Plant Cell 26, 56-78). In addition to their role during seedling deetiolation and under diurnal growth conditions, PIFs are being unveiled as instrumental regulators of multiple additional responses such as seed germination, flowering, neighbour-sensing shade avoidance, adaptations of plant architecture in response to high temperature, or stomatal development. Because of the wide range of PIF-regulated responses and because the mechanism of PIF action in different processes appears to share common components, it is expected that our results will be relevant to understand critical aspects of plant growth and development throughout the life cycle of the plant.
These studies will also contribute to understand molecularly how changes in the light environment perceived by the phy photoreceptors are transduced to the downstream regulatory network to trigger the proper adjustments in plant growth and development for optimal fitness. Because plant growth is of great agronomical importance, and because phy and PIFs are conserved in major crops, it is expected that this work will provide potential targets for comparative genomics and genetic modification in suitable crops. Because crops are often grown in open fields under diurnal conditions (day and night cycles), understanding the molecular mechanisms underlying plant growth under these conditions may allow manipulation of the system with the long-term goal of increasing crop productivity to satisfy the increasing demand of a growing population.
The researcher has been involved in several activities that have completed his training and have increased his competitiveness towards the goal of gaining an independent research position. First, the researcher has initiated a strong project in Europe which has resulted in 8 publications related to the project, some of them in top plant journals. Second, the researcher has supervised the work of two PhD students and two Master students, thus gaining experience in leadership and project management abilities. Third, the researcher has attended and presented the results of the project in 9 conferences, which allowed positive feedback from the international community and new networking opportunities in Europe. Fourth, the researcher has also been able to maintain a close collaboration with the former laboratory at University of California at Berkeley, thus keeping a solid network of international collaborations that is having a strong feedback on the project. Finally, the researcher has participated in reviewing papers in international journals, as well as in evaluating thesis by participating in thesis committees. Together, these additional training has been instrumental for the researcher to find an independent position in Europe.