Ultra-high-density QTL markers mapping for seedling photomorphogenesis mediating Arabidopsis establishment in southern Patagonia
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ABSTRACT: The intent of the experiment was to construct molecular SNP-binning markers of a Col-0 x Pat RIL population, for sensitive QTL mapping. We performed Illumina low-coverage DNA sequencing of plant tissues.
Project description:The environmental “light” plays a vital role in regulating the plant growth and development. Transcriptomic profilings were widely used to examine how light regulates the changes of mRNA populations at a genome-wide scale. However, it remains unclear if translational regulation represents a new dimension of gene expression regulation in response to the light signal. Through a transcriptomic comparison of steady-state and polysome-bound mRNAs, we revealed an increased translational efficiency in de-etiolating Arabidopsis seedlings. Over 3,500 genes are subjected to translational regulation whereas only about 770 genes have increased mRNA abundances in response to the light signal. This result suggests a stronger impact of translational control over transcriptomic changes during photomorphogenesis. Genes encoding ribosomal protein are preferentially regulated at the translational level, possibly contributing to the enhancement of translation efficiency as observed. We also uncovered mRNAs regulated at the translational level share characteristics of longer half-lives and shorter cDNA length. The presence of a cis-element, TAGGGTTT, in the 5’untranslated region of a transcript renders its translational regulation by light signals. Taken together, our study revealed a previously neglected aspect of gene expression regulation during Arabidopsis photomorphogenesis. The identities and molecular signatures associated with mRNAs regulated at the translational level also offer new directions to perform mechanistic studies of light-trigged translational enhancement in Arabidopsis. Three biological replicates for 4-d-old etiolated seedlings with or without 0. 5 h or 4 h of white-light treatment.
Project description:Arabidopsis thaliana shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, Arabidopsis has a high potential for the identification of genes underlying ecologically important complex traits, thus providing new insights on genome evolution. Previous research suggested that distinct light responses were crucial for Arabidopsis establishment in a peculiar ecological niche of southern Patagonia. The aim of this study was to explore the genetic basis of contrasting light-associated physiological traits that may have mediated the rapid adaptation to this new environment. From a biparental cross between the photomorphogenic contrasting accessions Patagonia (Pat) and Columbia (Col-0), we generated a novel recombinant inbred line (RIL) population, which was entirely next-generation sequenced to achieve ultra-high-density saturating molecular markers resulting in supreme mapping sensitivity. We validated the quality of the RIL population by quantitative trait loci (QTL) mapping for seedling de-etiolation, finding seven QTLs for hypocotyl length in the dark and continuous blue light (Bc), continuous red light (Rc), and continuous far-red light (FRc). The most relevant QTLs, Rc1 and Bc1, were mapped close together to chromosome V; the former for Rc and Rc/dark, and the latter for Bc, FRc, and dark treatments. The additive effects of both QTLs were confirmed by independent heterogeneous inbred families (HIFs), and we explored TZP and ABA1 as potential candidate genes for Rc1 and Bc1QTLs, respectively. We conclude that the Pat × Col-0 RIL population is a valuable novel genetic resource to explore other adaptive traits in Arabidopsis.
Project description:Flowers have a species-specific fertile period during which pollination and fertilization have to occur to initiate seed and fruit development. Within the flower, the functional life span of the ovule containing the female gametophyte is decisive for fertilization and the initiation of seed development. Here we performed an RNA-sequencing based transcriptome analysis of senescing unfertilized ovules during in a time series. We isolated ovules from Arabidopsis thaliana flowers emasculated at stage 12c at three different time points: 2 days after emasculation (DAE), 3 DAE, and 4 DAE. These time points correspond to intact mature ovules (2DAE), early ovule senescence (3 DAE), and late ovule senescence (4 DAE). We extracted total RNA from the ovules in 3 independent biological replicates, thus generating 9 RNA samples in total, for RNA-sequencing by Illumina HiSeq.
Project description:Environmental conditions contributing to abiotic stresses such as drought and salinity result in large annual economic losses around the world. As sessile organisms, plants cannot escape the environmental stresses they encounter, but instead must adapt to survive. Previous studies investigating osmotic and/or salt responses have largely focused on understanding short-term responses (0-1h) at the transcriptomic, proteomic and phosphoproteomic level; however, our understanding of intermediate to longer-term adaptation (24h - days) is relatively limited. In addition to protein abundance and phosphorylation changes, recent evidence suggests reversible protein acetylation may be important for abiotic stress responses. Therefore, to characterize the effects of osmotic and salt stress, we undertook a label-free proteomic and PTMomic analysis of Arabidopsis roots exposed to 300mM Mannitol and 150mM NaCl for 24 h. We quantitatively assessed protein abundance, phosphorylation and acetylation.
Project description:Arabidopsis seedlings undergo photomorphogenic development even in darkness when the function of De-etiolated 1 (DET1), a repressor of photomorphogenesis, is disrupted. Our results indicate that DET1 directly interacts with a group of transcription factors known as the phytochrome-interacting factors (PIFs). Furthermore, our results suggest that DET1 positively regulates PIF protein levels primarily by stabilizing PIF proteins in the dark. Genomic analysis also revealed that DET1 may control the expression of light-regulated genes to mediate photomorphogenesis partially through PIFs. Total of twelve samples, two treatments and three genotypes, and each have three replicates.
Project description:Stem cell differentiation strategies and optimization for generating lineage-specific cells and tissues most frequently rely on a three-dimensional embryoid body (EB) intermediate. We previously applied nanotechnology tools of photolithography to generate custom microarrays that allow high throughput uniform formation of EBs of custom size for precise downstream analysis. Formation of EBs of 200 or 500 micron size revealed distinct morphological differences that are single or multicystic cores, respectively, independent of method of formation from single cells or two-dimensional (2D) clusters. Here we utilize photolithographic array generated EBs to obtain 3D cultures under a standardized platform for transcriptome analysis to compare EB size and the method of EB formation from single cells or mechanically passaged 2D clusters. Our analysis evaluates RNA expression in EBs formed from the human embryonic stem cell (hESC) line WA09 and from ethnically diverse human induced pluripotent stem cell lines (ED-iPSC) of African American and Hispanic Latino ethnicity recently derived in our laboratory. This is the first comprehensive study on EB transcriptomes including multiple size parameters, EB formation methodologies, and ethnicities. Our analysis indicates upregulation of genes involved in wound healing for mechanically passaged cells and of genes for embryonic tube formation in 500 micron multicystic EBs. We propose that EB maturation may be a longer process then previously realized. In addition, the type or extent of maturation possible may be influenced by EB size, with larger EBs capable of more extensive remodeling as revealed by multicystic morphology and initiation of early tube formation pathways while retaining pluripotency status. We anticipate that this information will be broadly useful to the stem cell and bioengineering communities in optimization of tissue engineering with pluripotent stem cells and understanding sources of variation. mRNA profiles by RNA-seq from embryoid bodies generated by different methods
Project description:Hordeum vulgare is one of the first domesticated grains in the world and it has been reported that variations in the light environment have a substantial effect on barley plant development and biological processes. High-throughput RNA-Seq study was performed to investigate the complex transcriptome network required for photomorphogenesis in barley. Seedlings were grown in dark and light conditions and three biological replicates were sampled from each condition. Six libraries from poly-A rich mRNA fraction were subjected to 51bp single-end RNA-seq sequencing.
Project description:The levels of acetylated and non-acetylated histone peptide forms were quantified in A. thaliana plants with loss of function of HDA6, and plants exposed to HDAs inhibitors (trichostatin and sodium butyrate)during germination .