Project description:RNAseq profiling of 10 time points during germination in Arabidopsis, from freshly harvested seed, through mature seed, stratification, germination and to post-germination.
Project description:sRNA-seq profiling of 10 time points during germination in Arabidopsis, from freshly harvested seed, through mature seed, stratification, germination and to post-germination.
Project description:methylC-seq profiling of 4 time points during germination in Arabidopsis, from mature seed, through stratification, germination and to post-germination.
Project description:During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes of the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes of primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.
Project description:The developmental program of seed formation and seedling development requires not only tight regulation of cell division and metabolism but also the adaption of organelles in structure and function. Therefore, changes in organellar protein composition is one crucial factor in development. Of particular interest in plants is the switch to photoautotrophic growth, for which biosynthesis and degradation of lipid droplets (LDs) play a critical role. We present here a bottom-up proteomics study analyzing eight different developmental phases during silique development, seed germination and seedling establishment. We investigated both total protein fractions and LD-enriched fractions for each time point. The overall changes in the seed and seedling proteome during germination and seedling establishment monitored in this study present a rich resource for researchers interested in different questions of early seedling biology. The analysis of the proteome of LDs using LD-enrichment factors allowed the identification of four LD-associated protein families, which were subsequently confirmed by a cell biological approach. In addition to protein discovery, our dataset allows for the study of the dynamics of LD proteins throughout the developmental phases analyzed. We found that the relative levels of oleosin stay stable, while many other proteins accumulate on LDs at later stages of seedling establishment. The methodology described here is shown to be well suited for describing a comprehensive and quantitative view of the Arabidopsis proteome across time, with a particular focus on proteins associated with LDs.
Project description:In response to environmental light signals, transcriptomic adjustment plays an important role in Arabidopsis seed germination and seedling development. G-box cis-element is commonly present in promoters of genes positively or negatively responding to the light signal. For the pursuit of additional transcriptional regulator modulating light-mediated transcriptome changes, we have identified AtbZIP16, a basic region/leucine zipper motif transcription factor, via G-box DNA affinity chromatography. We have confirmed that AtbZIP16 possesses G-box-specific binding activity. Analyses of atbzip16 mutants indicate that AtbZIP16 is a negative regulator in phyB-mediated inhibition of cell elongation, but a positive regulator in phytochrome-mediated seed germination process. Transcriptomic analysis supports that AtbZIP16 is primarily a transcriptional repressor regulating light-, GA- and ABA-responsive genes. Chromatin immunoprecipitation study revealed that AtbZIP16 could directly target RGL2, a DELLA gene, and indirectly repress the expression of PIL5 gene, which encodes a bHLH protein inhibiting seed germination in Arabidopsis. Our study indicated that, through repressing the expression of RGL2 and the antagonizing the expression of PIL5, AtbZIP16 functions to promote seed germination and hypocotyl elongation during early stages of Arabidopsis seedling development. In response to environmental light signals, transcriptomic adjustment plays an important role in Arabidopsis seed germination and seedling development. G-box cis-element is commonly present in promoters of genes positively or negatively responding to the light signal. For the pursuit of additional transcriptional regulator modulating light-mediated transcriptome changes, we have identified AtbZIP16, a basic region/leucine zipper motif transcription factor, via G-box DNA affinity chromatography. We have confirmed that AtbZIP16 possesses G-box-specific binding activity. Analyses of atbzip16 mutants indicate that AtbZIP16 is a negative regulator in phyB-mediated inhibition of cell elongation, but a positive regulator in phytochrome-mediated seed germination process. Transcriptomic analysis supports that AtbZIP16 is primarily a transcriptional repressor regulating light-, GA- and ABA-responsive genes. Chromatin immunoprecipitation study revealed that AtbZIP16 could directly target RGL2, a DELLA gene, and indirectly repress the expression of PIL5 gene, which encodes a bHLH protein inhibiting seed germination in Arabidopsis. Our study indicated that, through repressing the expression of RGL2 and the antagonizing the expression of PIL5, AtbZIP16 functions to promote seed germination and hypocotyl elongation during early stages of Arabidopsis seedling development.
Project description:The developmental program of seed formation and seedling development requires not only tight regulation of cell division and metabolism but also the adaption of organelles in structure and function. Therefore, changes in organellar protein composition is one crucial factor in development. Of particular interest in plants is the switch to photoautotrophic growth, for which biosynthesis and degradation of lipid droplets (LDs) play a critical role. We present here a bottom-up proteomics study analyzing eight different developmental phases during silique development, seed germination and seedling establishment. We investigated both total protein fractions and LD-enriched fractions for each time point. The overall changes in the seed and seedling proteome during germination and seedling establishment monitored in this study present a rich resource for researchers interested in different questions of early seedling biology. The analysis of the proteome of LDs using LD-enrichment factors allowed the identification of four LD-associated protein families, which were subsequently confirmed by a cell biological approach. In addition to protein discovery, our dataset allows for the study of the dynamics of LD proteins throughout the developmental phases analyzed. We found that the relative levels of oleosin stay stable, while many other proteins accumulate on LDs at later stages of seedling establishment. The methodology described here is shown to be well suited for describing a comprehensive and quantitative view of the Arabidopsis proteome across time, with a particular focus on proteins associated with LDs.
Project description:Background: Heat Stress Factor A9 (A9), a seed-specific transcription factor contributing to seed longevity, also enhances phytochrome-dependent seedling greening Results: The RNA-seq analyses of imbibed-seed transcripts here reported indicated, potential, additional effects of A9 on cryptochrome-mediated blue-light responses. These analyses also suggested that in contrast to the A9 effects on longevity, which require coactivation by additional factors as A4a, A9 alone might suffice for the enhancement of photomorphogenesis at the seedling stage. We find that upon its seed-specific over-expression, A9 indeed enhanced the expected blue-light responses. Comparative loss-of-function analyses of longevity and greening, performed by similar expression of dominant-negative and inactive forms of A9, not only confirmed the additional greening effects of A9, but also were consistent with A9 not requiring A4a (or additional factors) for the greening effects Conclusion: Our results strongly indicate that A9 would differentially regulate seed longevity and photomorphogenesis at the seedling stage, A9 alone sufficing for both the phytochrome- and cryptochrome-dependent greening enhancement effects
Project description:Seed germination involves transition from desiccation tolerance to sensitivity during early stages of seedling establishment accompanied by comprehensive changes of metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes of primary metabolites. Six metabolites in dry seeds important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. In the heat stable proteome an almost equal fractioning of ordered and intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were assigned to functional bins development, protein, RNA and stress. Thermal stable proteins were related to a multitude of active biological processes. The majority of LEA proteins decreased during germination and seedling establishment in parallel to reduced desiccation tolerance. Five germination specific dehydrins were identified most likely providing stress tolerance. Network analysis of common proteins and metabolites over all time points revealed a tight network of LEA proteins with five hub LEAs with high connectivity. Connections to proteins such as Rubisco large subunit and seed storage proteins were discovered. Insights into the heat-stable proteome – metabolome network during seed germination are provided.