The embryonic transcriptome of Arabidopsis thaliana
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ABSTRACT: Cellular differentiation is associated with changes in transcript populations. Accurate quantification of transcriptomes during development can thus provide global insights into differentiation processes including the fundamental specification and differentiation events operating during plant embryogenesis. However, multiple technical challenges have limited the ability to obtain high quality early embryonic transcriptomes, namely the low amount of RNA obtainable and contamination from surrounding endosperm and seed-coat tissues. We compared the performance of three low-input mRNA sequencing (mRNA-seq) library preparation kits on 0.1 to 5 nanograms (ng) of total RNA isolated from Arabidopsis thaliana (Arabidopsis) embryos and identified a low-cost method with superior performance. This mRNA-seq method was then used to profile the transcriptomes of Arabidopsis embryos across eight developmental stages. By comprehensively comparing embryonic and post-embryonic transcriptomes, we found that embryonic transcriptomes do not resemble any other plant tissue we analyzed. Moreover, transcriptome clustering analyses revealed the presence of four distinct phases of embryogenesis which are enriched in specific biological processes. We also compared zygotic embryo transcriptomes with publicly available somatic embryo transcriptomes. Strikingly, we found little resemblance between zygotic embryos and somatic embryos derived from late-staged zygotic embryos suggesting that somatic and zygotic embryo transcriptomes are distinct from each other. In addition to the biological insights gained from our systematic characterization of the Arabidopsis embryonic transcriptome, we provide a data-rich resource for the community to explore.
Project description:Somatic embryogenesis closely resembles zygotic embryogenesis and hence, it is considered as a model system to explore dynamic events of embryogenesis, at a molecular level. We sequenced three district developmental time points of somatic embryo development in Arabidopsis thaliana with the aim of exploring transcriptomes at a global scale.
Project description:The microarray test aims to find the miRNAs involved in somatic embryogenesis in Arabidopsis. The plant microRNA array V2.0 (CapitalBio Corp., Beijing, China) contained 426 non-reduplicated plant miRNA probes, including 188 in Arabidopsis. Finally, 75 plant miRNAs expressed differentially, 36 increased and 39 decreased included. Two critical period samples of somatic embryogenesis were chosen for the microarray test: the edge of embryonic calli in embryonic callus-inducing medium (ECIM) for 14 days and the secondary somatic embryos protuberances in somatic embryo-inducing medium (SEIM) for 2 days (marked as “14D” and “J2D” accordingly) . Three chip were test in each sample.
Project description:We collected tissues from bent cotyledon stage zygotic embryos, proliferating tissue at day 7 and day 14 induction of somatic embryogenesis and mature somatic emrbyos in a wild type (Col-0) and vtc2 (SALK_146824) insertion. We used microarrays to identify global patterns of gene activity during somatic embryogenesis in a wild type (Col-0) and vitamin C deficient mutant (vtc2) RNA was extracted and amplified from four stages of somatic embryogenesis (bent cotyledon, day 7 induction, day 14 induction, and mature somatic embryos) in a wild type (Col-0) and vitamin C deficient mutant (vtc2) before being hybridized to the Arabidopsis ATH1 GeneChip in duplicate (two biological replicates).
Project description:The piRNA pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. piRNAs are maternally deposited and required for proper transposon silencing in adult offspring. However, a long-standing question in the field is the precise function of maternally deposited piRNAs and its associated factors during embryogenesis. Here, we probe the spatio-temporal expression patterns of several piRNA pathway components during early stages of development. Amongst those, factors required for transcriptional gene silencing (TGS) showed ubiquitous abundance in somatic and pole cells throughout the first half of embryogenesis. We further analysed the transcriptomes of various embryo stages and correlated these with the presence of selected chromatin marks. We found that a number of transposon families show bursts of transcription during early embryonic stages. Transposons heavily targeted by maternally deposited piRNAs accumulated repressive chromatin marks following their spike in expression. Furthermore, depletion of maternally deposited Piwi protein in early embryos resulted in increased expression of transposons targeted by inherited piRNAs and was accompanied by a strong loss of repressive chromatin marks at coding sequences. Overall, our data suggests a pivotal role for the piRNA pathway in transposon defence during Drosophila embryogenesis in somatic cells.
Project description:Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Correlations of stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary stages were performed. Comparative proteomic analysis revealed a total of 801 proteins, 392 of which were classified as differentially accumulated proteins in at least one of the developmental stages. The globular stage presented a higher number of unique proteins (16), 7 of which were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages, after which they decreased with increasing development, contributing to cell homeostasis in early somatic embryos. On the other hand, a progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins leading to somatic embryo maturation was observed. Additionally, the differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins correlated with increased contents of putrescine and spermidine, suggesting that polyamine metabolism is important to somatic embryo development. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins being more abundant during the early somatic embryo stages, while proteins involved in seed maturation are more abundant during the late stages.
Project description:Early embryogenesis is characterized by the maternal to zygotic transition (MTZ), in which maternally deposited messenger RNAs are translated and subsequently degraded while zygotic transcription begins. Posttranscriptional gene regulation by RNA-binding proteins (RBPs) is a dominant force controlling pre-zygotic gene expression. Here we describe the first in vivo mRNA-bound proteome in early Drosophila melanogaster embryos. mRNA interactome capture using conventional (254nm) and photoactivatable ribonucleoside-enhanced UV-crosslinking (365nm) was applied to detect RBPs bound to maternal and early zygotic polydenylated transcripts within the first two hours of embryogenesis. We identified a high confidence set of 476 putative RBPs and confirmed RNA-binding activity for most of the tested candidates. The majority of the identified proteins in the early fly mRNA interactome were known RBPs, harboring canonical RBPs features. Nearly hundred of the identified proteins were previously not known to bind RNA. Interestingly, mRNAs encoding RBPs and TFs exhibit time specific expression modules, in which RBPs dominate the first hours of embryonic development. Using fly-FISH data, we could show enriched RBP localization in the posterior embryo during these first hours of fly embryogenesis, suggesting general importance germ cell maturation.
Project description:With the goal to better understand somatic embryogenesis (SE) development and improve the efficiency of SE conversion in Theobroma cacao L. we examined the differences between zygotic and somatic embryo at gene expression level using a whole genome microarray. A gene expression microarray representing 28,752 genes was used to compare the expression profiles at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were identified which were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison of expression profiles of ZE and SE identified 10,175 differentially expressed genes. 463 and 714 transcription factor (TF) genes are differentially expressed during ZE and SE maturation respectively, of which, an overlapping set of 128 genes were regulated during both processes. Large numbers of TFs, putatively involved in ethylene metabolism and ethylene response, were expressed at higher levels during SE as compared to ZE. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. A 12-plex Nimblegen expression microarray representing 28,752 genes was manufactured on which only 46 of the predicted cacao genes were not represented with at least one probe. Total RNA was isolated from four biological replicates of six different types and stages of embryo development: zygotic embryo tissues: torpedo (T-ZE), early-full (EF-ZE), late-full (LF-ZE) and mature (M-ZE) embryos; and somatic embryos: late torpedo (LT-SE) and mature (M-SE) stages
Project description:Somatic embryos are very much similar to zygotic counterparts in many morphological aspects and the somatic embryos are derived from somatic cells by undergoing various metabolic regulations. The somatic embryos have been used in artificial seed technology, genetic engineering and germplasm conservation. Though somatic embryo development is an important topic in growth and developmental studies, the molecular mechanism underlying the developmental process remains unclear. Therefore, understanding the molecular basis behind somatic embryo development can provide insight on the signaling pathways integrating this process. Proteomic analysis of somatic embryo development in cv. Grand Naine (AAA) was carried out to identify the differentially accumulated protein using two dimensional gel electrophoresis coupled with mass spectrometry. In total, 25 protein spots were differentially accumulated in different developmental stages of somatic embryos. Among them, three proteins were uniquely present in 30 days globular stage somatic embryos and six proteins were uniquely present in 60 days matured somatic embryo. Functional annotation of identified spots showed that major proteins are involved in growth and developmental process (17 %) followed by defense response (12%) and signal transportation events (12 %). In early stage, cell division and growth related proteins were involved in the induction of somatic embryos whereas in late developmental stage, cell wall modification proteins along with stress related proteins like played a defense role against dehydration and osmotic stress and resulted in maturation of somatic embryo. Alongside some identified stage specific proteins are valuable indicators and have been used as genetic markers.
Project description:Embryogenesis entails dramatic shifts in mRNA translation and turnover to account for gene expression differences during proliferation and cellular differentiation. Codon identity modulates mRNA stability during early vertebrate embryogenesis, but how the composition of tRNA pools adapts to the embryo s translational demand is unknown. By quantitatively profiling the tRNA repertoires of zebrafish embryos during the maternal-to-zygotic transition, here we find that maternal and zygotic tRNA pools are distinct. We show that translational activation during embryogenesis and tRNA gene derepression are temporally coordinated by TORC1 activity, which increases at gastrulation and inactivates the RNA polymerase III repressor Maf1a/b in vivo. Reshaping of tRNA pools results in differential tRNA anticodon supply, but these changes do not alter decoding rates in zebrafish embryos. Instead, our data indicate that tRNA repertoires reflect the inherent codon bias of the zebrafish mRNA transcriptome, and tRNA levels are boosted at gastrulation to ensure efficient translation as embryos enter differentiation.
Project description:Land plants can reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. A key question is thus how embryo identity in plants is controlled, and how this process is modified during non-zygotic embryogenesis. The Arabidopsis zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we have used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We find that reprogramming is complex and accompanied by large transcriptomic changes prior to anatomic changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon re-establishment of cellular auxin levels or response. This suggests a remarkable degree of feedback regulation to create resilience in auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identify an auxin-dependent bHLH transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.