Project description:We were interested in changes in small RNA abundance changes in response to developmental transitions in Arabidopsis thaliana shoots, with special focus on vegetative phase change. We specifically wanted to separate the temporal changes in gene expression that result from vegetative phase change and those from flowering. Because of the close timing between the juvenile-to-adult and adult-to-reproductive developmental transitions in Arabidopsis grown under long day conditions, we used the late-flowering genotype FRI;FLC developed by the lab of Richard Amasino by introgressing the FRI allele from Sf-2 into the Col-0 genetic background, which is fri;FLC. For the early flowering genotype, we used FRI;flc-3, also developed by the Amasino lab by EMS-mutagenizing FRI;FLC, identifying early flowering mutants, and backcrossing multiple times to eliminate other EMS-induced mutations. The onset of vegetative phase change in FRI;FLC and FRI;flc-3 under our growth conditions was identical, but the progression was slower in FRI;FLC. By sequencing small RNAs from shoot apices at different time points and fully-expanded leaves at different positions on the shoot and comparing the results between the two genotypes, we were able to obtain a clear picture of changes in small RNA abundance in response to vegetative phase change and flowering in Arabidopsis. For the small RNA samples, we performed two replicates using two different indices in the 5'-adapter and ran each replicate pair on the same sequencing lane. For the cotyledon and leaf samples we only performed one replicate using the same index for all samples because we obtained significantly different results with the two adapters used for the shoot apices, preventing us from using them as true replicates.
Project description:Arabiposis plants with conbinations of different FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) alleles grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D). Genotypes:; Columbia wild type (Col-0): fri FLC; Columbia with introgressed FRI from Sf-2: FRI FLC; Columbia with introgressed FRI and deleted FLC (flc-3): FRI flc; Columbia with deleted FLC (flc-3): fri flc; Time points:; 0, 2, and 4 days after shift to long days
Project description:We were interested in identifying targets of novel putative miRNAs we identified from small RNA sequencing libraries of Arabidopsis shoots. The small RNA (smRNA) sequencing libraries were made to identify changes in abundance of specific smRNAs in response to developmental transitions in Arabidopsis thaliana shoots, with special focus on vegetative phase change. We specifically wanted to separate the temporal changes in gene expression that result from vegetative phase change and those from flowering. Because of the close timing between the juvenile-to-adult and adult-to-reproductive developmental transitions in Arabidopsis grown under long day conditions, we used the late-flowering genotype FRI;FLC developed by the lab of Richard Amasino by introgressing the FRI allele from Sf-2 into the Col-0 genetic background, which is fri;FLC. For the early flowering genotype, we used FRI;flc-3, also developed by the Amasino lab by EMS-mutagenizing FRI;FLC, identifying early flowering mutants, and backcrossing multiple times to eliminate other EMS-induced mutations. The onset of vegetative phase change in FRI;FLC and FRI;flc-3 under our growth conditions was identical, but the progression was slower in FRI;FLC. By sequencing small RNAs from shoot apices at different time points and fully-expanded leaves at different positions on the shoot and comparing the results between the two genotypes, we were able to obtain a clear picture of changes in small RNA abundance in response to vegetative phase change and flowering in Arabidopsis. We then used the remaining RNA to make genome-wide mapping of uncapped and cleaved transcripts (GMUCT) 2.0 libraries of a subset of our samples. GMUCT 2.0 allows you to identify RNAs that are 1) uncapped and in the process of 5’->3’ exonuclease degradation and 2) miRNA and siRNA-mediated cleavage products. We wanted to use these GMUCT 2.0 libraries to identify targets of novel putative miRNAs discovered by our smRNA sequencing, thereby supporting the idea that these novel putative miRNAs are in fact functional.
Project description:Plants of three different genotypes (FRI FLC, FRI flc and fri flc) were induced to flowering by shifting from short day conditions to long day conditions. FRI=FRIGIDA, FLC=FLOWERING LOCUS C.
Project description:Arabiposis plants with conbinations of different FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) alleles grown in short days (9L:15D) for 30 days at 21°C, then shifted to long days (16L:8D). Genotypes: Columbia wild type (Col-0): fri FLC Columbia with introgressed FRI from Sf-2: FRI FLC Columbia with introgressed FRI and deleted FLC (flc-3): FRI flc Columbia with deleted FLC (flc-3): fri flc Time points: 0, 2, and 4 days after shift to long days Keywords = flowering Keywords: time-course
Project description:We report that H3.3 stimulates the expression of FLC and its homologs, and promotes the active histone modifications at their loci. FRI directly interacts with the H3.3 chaperone HIRA and elevates H3.3 deposition towards the FLC 3’ region. The enriched H3.3 facilitates FLC 3’ end interaction with the 5’ end, promoting active histone modifications around the transcription start site (TSS) of FLC. In contrast, though coordinately activate FLC expression; H3.3 and H2A.Z antagonize each other at the FLC chromatin. In addition to FLC and its homologs, H3.3 is required for H3K4me3 at a subset of especially short genes. Our findings reveal the important function of H3.3 in the regulation of the active chromatin state, which enhances FLC transcription and floral repression
Project description:Quantitative variation in expression of the Arabidopsis floral repressor FLC influences whether plants overwinter before flowering or have a rapid cycling habit, enabling multiple generations a year. Genetic analysis has identified activators and repressors of FLC expression, but how they interact to set expression level is poorly understood. Here, we show that antagonistic functions of the FLC activator FRIGIDA (FRI), and the repressor FCA, at a specific stage of embryo development, determines FLC expression and flowering. FRI antagonizes an FCA-induced proximal polyadenylation to increase FLC expression and delay flowering. Sector analysis shows that FRI activity during the early heart stage of embryo development maximally delays flowering. Opposing functions of co-transcriptional regulators during an early embryonic developmental window thus set FLC expression levels and determine flowering time.
Project description:FLOWERING LOCUS C (FLC) is a MADS box transcription factor that plays a well characterised role in repressing the vegetative to floral transition of Arabidopsis thaliana. FLC has also been shown to affect the Arabidopsis circadian clock, with mutant seedlings showing short circadian periods. In a previous study, we identified the temperature-dependent circadian period QTL PerCv5b near the FLC locus on the top arm of Chromosome 5. PerCv5b caused a significant period effect at 27oC but not at 12oC or 22oC. Temperature-dependent circadian period phenotypes and a known polymorphism in the Ler allele made FLC a strong candidate gene for PerCv5b. The period effect of FLC was enhanced by combination with alleles of FRIGIDA (FRI), a gene shown to up-regulate FLC's expression. We were interested in identifying how FLC affects the circadian clock, so we decided to identify its target genes. Greatest phenotypic difference was observed between fri; flc and FRI; FLC genotype seedlings at 27oC, so expression was compared between these lines (previously described in Michaels and Amasino1999 and 2001) on the Affymetrix ATH1 microarray. Seedlings were grown on media (MS 1.5% Agar containing 3% Sucrose) for 6 days under constant cool white fluorescent light (55-60 micro EINSTEINS) at 22oC then entrained for 4 days under (12h , 12h) light , dark cycles at 22oC. At dawn on the fourth day of entrainment they were transferred to constant light (25-30 micro EINSTEINS) at 27oC. Four samples were taken at 6 hour intervals starting 24h after the transfer to continuous conditions at the times 24h, 30h, 36h and 42h. Equal amounts of total RNA were pooled from the three time points to produce one sample per genotype. The pooling strategy was employed to reduce the effect of circadian regulation on genes expression. This was particularly important in our case as some interesting genes would likely be regulated by the circadian clock and may only show expression differences at particular phases that could easily be missed if using just one time point. Experimenter name = Kieron Edwards; Experimenter phone = 0131 651 3326; Experimenter fax = 0131 650 5392; Experimenter department = Institute of Molecular Plant Sciences; Experimenter institute = University of Edinbugh; Experimenter address = Kings Buildings; Experimenter address = Mayfield Road; Experimenter address = Edinburgh; Experimenter zip/postal_code = EH9 3JH; Experimenter country = UK Experiment Overall Design: 4 samples were used in this experiment
Project description:This experiment was annotated by TAIR (http://arabidopsis.org).FRI FLC time series; Experimenter name = Markus Schmid; Experimenter phone = ++49-7071-601-1413; Experimenter fax = ++49-7071-601-1412; Experimenter department = Detlef Weigel Laboratory Germany; Experimenter institute = Max-Planck-Institute for Developmental Biology; Experimenter address = Dept. of Molecular Biology; Experimenter address = Spemannstr. 37-39; Experimenter address = Tübingen; Experimenter zip/postal_code = 72076; Experimenter country = Germnay Experiment Overall Design: 36 samples were used in this experiment