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: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:Arabidopsis thaliana is a well-established model system for the analysis of the basic physiological and metabolic pathways of plants. The presented model is a new semi-quantitative mathematical model of the metabolism of Arabidopsis thaliana. The Petri net formalism was used to express the complex reaction system in a mathematically unique manner. To verify the model for correctness and consistency concepts of network decomposition and network reduction such as transition invariants, common transition pairs, and invariant transition pairs were applied. Based on recent knowledge from literature, including the Calvin cycle, glycolysis and citric acid cycle, glyoxylate cycle, urea cycle, sucrose synthesis, and the starch metabolism, the core metabolism of Arabidopsis thaliana was formulated. Each reaction (transition) is experimentally proven. The complete Petri net model consists of 134 metabolites, represented by places, and 243 reactions, represented by transitions. Places and transitions are connected via 572 edges.
Project description:Small RNA sequences from Arabidopsis thaliana Col-0 inflorescence tissues of three biological replicates. The data were analyzed to identify non-templated nucleotides in Arabidopsis small RNAs.
Project description:High-throughput sequencing of Arabidopsis thaliana endogenous small RNAs by 454 pyrosequencing. Keywords: high-throughput sequencing