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Rapeseed (Brassica napus L.), one of the most important sources of vegetable oil and protein-rich meals worldwide, is adapted to different geographical regions by modification of flowering time. Rapeseed cultivars have different day length and vernalization requirements, which categorize them into winter, spring, and semiwinter ecotypes. To gain a deeper insight into genetic factors controlling floral transition in B. napus, we performed RNA sequencing (RNA-seq) in the semiwinter doubled haploid line, Ningyou7, at different developmental stages and temperature regimes. The expression profiles of more than 54,000 gene models were compared between different treatments and developmental stages, and the differentially expressed genes were considered as targets for association analysis and genetic mapping to confirm their role in floral transition. Consequently, 36 genes with association to flowering time, seed yield, or both were identified. We found novel indications for neofunctionalization in homologs of known flowering time regulators like VIN3 and FUL. Our study proved the potential of RNA-seq along with association analysis and genetic mapping to identify candidate genes for floral transition in rapeseed. The candidate genes identified in this study could be subjected to genetic modification or targeted mutagenesis and genotype building to breed rapeseed adapted to certain environments.

Rapeseed (Brassica napus L.), one of the most important sources of vegetable oil and protein-rich meals worldwide, is adapted to different geographical regions by modification of flowering time. Rapeseed cultivars have different day length and vernalization requirements, which categorize them into winter, spring, and semiwinter ecotypes. To gain a deeper insight into genetic factors controlling floral transition in B. napus, we performed RNA sequencing (RNA-seq) in the semiwinter doubled haploid line, Ningyou7, at different developmental stages and temperature regimes. The expression profiles of more than 54,000 gene models were compared between different treatments and developmental stages, and the differentially expressed genes were considered as targets for association analysis and genetic mapping to confirm their role in floral transition. Consequently, 36 genes with association to flowering time, seed yield, or both were identified. We found novel indications for neofunctionalization in homologs of known flowering time regulators like VIN3 and FUL. Our study proved the potential of RNA-seq along with association analysis and genetic mapping to identify candidate genes for floral transition in rapeseed. The candidate genes identified in this study could be subjected to genetic modification or targeted mutagenesis and genotype building to breed rapeseed adapted to certain environments.

Background Functions for RNA-binding proteins in orchestrating plant development and environmental responses are well established. However, the lack of a genome-wide view of their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) genome-wide to determine the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7. Results iCLIP identifies 858 transcripts with significantly enriched crosslink sites in plants expressing AtGRP7-GFP that are absent in plants expressing an RNA-binding-dead AtGRP7 variant or GFP alone. To independently validate the targets, we performed RNA immunoprecipitation (RIP)-sequencing of AtGRP7-GFP plants subjected to formaldehyde fixation. Of the iCLIP targets, 452 were also identified by RIP-seq and represent a set of high-confidence binders. AtGRP7 can bind to all transcript regions, with a preference for 3′ untranslated regions. In the vicinity of crosslink sites, U/C-rich motifs are overrepresented. Cross-referencing the targets against transcriptome changes in AtGRP7 loss-of-function mutants or AtGRP7-overexpressing plants reveals a predominantly negative effect of AtGRP7 on its targets. In particular, elevated AtGRP7 levels lead to damping of circadian oscillations of transcripts, including DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN2 and CCR-LIKE. Furthermore, several targets show changes in alternative splicing or polyadenylation in response to altered AtGRP7 levels. Conclusions We have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1332-x) contains supplementary material, which is available to authorized users.

Auxin plays a pivotal role in virtually every aspect of plant morphogenesis. It simultaneously orchestrates a diverse variety of processes such as cell wall biogenesis, transition through the cell cycle, or metabolism of a wide range of chemical substances. The coordination principles for such a complex orchestration are poorly understood at the systems level. Here, we perform an RNA-seq experiment to study the transcriptional response to auxin treatment within gene groups of different biological processes, molecular functions, or cell components in a quantitative fold-change-specific manner. We find for Arabidopsis thaliana roots treated with auxin for 6 h that (i) there are functional groups within which genes respond to auxin with a surprisingly similar fold changes and that (ii) these fold changes vary from one group to another. These findings make it tempting to conjecture the existence of some transcriptional logic orchestrating the coordinated expression of genes within functional groups in a fold-change-specific manner. To obtain some initial insight about this coordinated expression, we performed a motif enrichment analysis and found cis-regulatory elements TBX1-3, SBX, REG, and TCP/site2 as the candidates conferring fold-change-specific responses to auxin in Arabidopsis thaliana.

The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ±0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

The stabilization of the transcription factor and prognostic tumor marker hypoxia-inducible factor 1α (HIF1α) is considered to be crucial for cellular metabolic adaptations to hypoxia. However, HIF1α has also been shown to accumulate under normoxic conditions, although this phenomenon is poorly understood. We investigated the conditions for normoxic HIF1α stabilization in different tumor cell lines (e.g., two mammary carcinoma cell lines and three oral squamous cell carcinoma cell lines) via Western blot analysis or immunohistochemical staining. The transcriptional activity of HIF1 was demonstrated by analyzing the messenger RNA (mRNA) expression of the HIF1 target carbonic anhydrase 9 (CA9) via PCR. Our data demonstrate that the combined incubation of tumor cells with glutamine and growth factors (e.g., EGF, insulin, and serum) mediates the normoxic accumulation of HIF1α in vitro. Consequently, the inhibition of glutaminolysis by a glutaminase inhibitor blocked the normoxic accumulation of HIF1α. Additionally, the normoxic HIF1α protein displayed nuclear translocation and transcriptional activity, which was confirmed by the induction of CA9 mRNA expression. Furthermore, the normoxic accumulation of HIF1α was associated with impaired proliferation of tumor cells. Finally, ammonia, the toxic waste product of glutaminolysis, induced a normoxic accumulation of HIF1α to the same extent as glutamine. Our study suggests that HIF1α is involved in the regulation of glutamine metabolism and the cellular levels of the toxic metabolic waste product ammonia under normoxia. Hence, our results, together with data presented in the literature, support the hypothesis that HIF1α and its target genes play a crucial role in metabolic pathways, such as glutaminolysis and glycolysis, under both hypoxic and normoxic conditions. Therefore, the inhibition of HIF1α (and/or HIF1α target genes) could emerge as a promising therapeutic approach that would result in the accumulation of toxic metabolic waste products in tumor cells as well as the reduction of their nutrition and energy supply.

Initiating the project on sequencing the Arabidopsis thaliana L. genome at the end of the twentieth century, researchers one day wished to expand the accumulated knowledge on Arabidopsis genetics to horticultural plants. The future arrived with the appearance of high-throughput sequencing technologies that allowed the investigation of transcriptomes of non-model plants at an unprecedented pace. RNA-seq experiments provide a unique opportunity of studying in depth the molecular-genetic basis for plant response to environmental cues.