A transcriptomic time-series reveals differing trajectories during pre-floral development in the apex and leaf in winter and spring varieties of Brassica napus.

Oilseed rape (Brassica napus) is an important global oil crop, with spring and winter varieties grown commercially. To understand the transcriptomic differences between these varieties, we collected transcriptomes from apex and leaf tissue from a spring variety, Westar, and a winter variety, Tapidor, before, during, and after vernalisation treatment, until the plants flowered. Large transcriptomic differences were noted in both varieties during the vernalisation treatment because of temperature and day length changes. Transcriptomic alignment revealed that the apex transcriptome reflects developmental state, whereas the leaf transcriptome is more closely aligned to the age of the plant. Similar numbers of copies of genes were expressed in both varieties during the time series, although key flowering time genes exhibited expression pattern differences. BnaFLC copies on A2 and A10 are the best candidates for the increased vernalisation requirement of Tapidor. Other BnaFLC copies show tissue-dependent reactivation of expression post-cold, with these dynamics suggesting some copies have retained or acquired a perennial nature. BnaSOC1 genes, also related to the vernalisation pathway, have expression profiles which suggest tissue subfunctionalisation. This understanding may help to breed varieties with more consistent or robust vernalisation responses, of special importance due to the milder winters resulting from climate change.

Oxonium ion scanning mass spectrometry for large-scale plasma glycoproteomics.

Protein glycosylation, a complex and heterogeneous post-translational modification that is frequently dysregulated in disease, has been difficult to analyse at scale. Here we report a data-independent acquisition technique for the large-scale mass-spectrometric quantification of glycopeptides in plasma samples. The technique, which we named 'OxoScan-MS', identifies oxonium ions as glycopeptide fragments and exploits a sliding-quadrupole dimension to generate comprehensive and untargeted oxonium ion maps of precursor masses assigned to fragment ions from non-enriched plasma samples. By applying OxoScan-MS to quantify 1,002 glycopeptide features in the plasma glycoproteomes from patients with COVID-19 and healthy controls, we found that severe COVID-19 induces differential glycosylation in IgA, haptoglobin, transferrin and other disease-relevant plasma glycoproteins. OxoScan-MS may allow for the quantitative mapping of glycoproteomes at the scale of hundreds to thousands of samples.

Positional motif analysis reveals the extent of specificity of protein-RNA interactions observed by CLIP.

BACKGROUND: Crosslinking and immunoprecipitation (CLIP) is a method used to identify in vivo RNA-protein binding sites on a transcriptome-wide scale. With the increasing amounts of available data for RNA-binding proteins (RBPs), it is important to understand to what degree the enriched motifs specify the RNA-binding profiles of RBPs in cells. RESULTS: We develop positionally enriched k-mer analysis (PEKA), a computational tool for efficient analysis of enriched motifs from individual CLIP datasets, which minimizes the impact of technical and regional genomic biases by internal data normalization. We cross-validate PEKA with mCross and show that the use of input control for background correction is not required to yield high specificity of enriched motifs. We identify motif classes with common enrichment patterns across eCLIP datasets and across RNA regions, while also observing variations in the specificity and the extent of motif enrichment across eCLIP datasets, between variant CLIP protocols, and between CLIP and in vitro binding data. Thereby, we gain insights into the contributions of technical and regional genomic biases to the enriched motifs, and find how motif enrichment features relate to the domain composition and low-complexity regions of the studied proteins. CONCLUSIONS: Our study provides insights into the overall contributions of regional binding preferences, protein domains, and low-complexity regions to the specificity of protein-RNA interactions, and shows the value of cross-motif and cross-RBP comparison for data interpretation. Our results are presented for exploratory analysis via an online platform in an RBP-centric and motif-centric manner ( https://imaps.goodwright.com/apps/peka/ ).

Comparative transcriptomics reveals desynchronisation of gene expression during the floral transition between Arabidopsis and Brassica rapa cultivars.

Comparative transcriptomics can be used to translate an understanding of gene regulatory networks from model systems to less studied species. Here, we use RNA-Seq to determine and compare gene expression dynamics through the floral transition in the model species Arabidopsis thaliana and the closely related crop Brassica rapa. We find that different curve registration functions are required for different genes, indicating that there is no single common 'developmental time' between Arabidopsis and B. rapa. A detailed comparison between Arabidopsis and B. rapa and between two B. rapa accessions reveals different modes of regulation of the key floral integrator SOC1, and that the floral transition in the B. rapa accessions is triggered by different pathways. Our study adds to the mechanistic understanding of the regulatory network of flowering time in rapid cycling B. rapa and highlights the importance of registration methods for the comparison of developmental gene expression data.

Total FLC transcript dynamics from divergent paralogue expression explains flowering diversity in Brassica napus.

Flowering time is a key adaptive and agronomic trait. In Arabidopsis, natural variation in expression levels of the floral repressor FLOWERING LOCUS C (FLC) leads to differences in vernalization. In Brassica napus there are nine copies of FLC. Here, we study how these multiple FLC paralogues determine vernalization requirement as a system. We collected transcriptome time series for Brassica napus spring, winter, semi-winter, and Siberian kale crop types. Modelling was used to link FLC expression dynamics to floral response following vernalization. We show that relaxed selection pressure has allowed expression of FLC paralogues to diverge, resulting in variation of FLC expression during cold treatment between paralogues and accessions. We find that total FLC expression dynamics best explains differences in cold requirement between cultivars, rather than expression of specific FLC paralogues. The combination of multiple FLC paralogues with different expression dynamics leads to rich behaviour in response to cold and a wide range of vernalization requirements in B. napus. We find evidence for different strategies to determine the response to cold in existing winter rapeseed accessions.

The oilseed rape developmental expression resource: a resource for the investigation of gene expression dynamics during the floral transition in oilseed rape.

BACKGROUND: Transcriptome time series can be used to track the expression of genes during development, allowing the timing, intensity, and dynamics of genetic programmes to be determined. Furthermore, time series analysis can reveal causal relationships between genes, leading to an understanding of how the regulatory networks are rewired during development. Due to its impact on yield, a developmental transition of agricultural interest in crops is the switch from vegetative to floral growth. We previously reported the collection of genome-wide gene expression data during the floral transition in the allopolyploid crop Brassica napus (oilseed rape, OSR). To provide the OSR research community with easy access to this dataset, we have developed the Oilseed Rape Developmental Expression Resource (ORDER; http://order.jic.ac.uk ). RESULTS: ORDER enables users to search for genes of interest and plot expression patterns during the floral transition in both a winter and a spring variety of OSR. We illustrate the utility of ORDER using two case studies: the first investigating the interaction between transcription factors, the second comparing genes that mediate the vernalisation response between OSR and radish (Raphanus sativus L.). All the data is downloadable and the generic website platform underlying ORDER, called AionPlot, is made freely and openly available to facilitate the dissemination of other time series datasets. CONCLUSIONS: ORDER provides the OSR research community with access to a dataset focused on a period of OSR development important for yield. AionPlot, the platform on which ORDER is built, will allow researchers from all fields to share similar time series datasets.

QTL-seq identifies BnaFT.A02 and BnaFLC.A02 as candidates for variation in vernalization requirement and response in winter oilseed rape (Brassica napus).

Winter, spring and biennial varieties of Brassica napus that vary in vernalization requirement are grown for vegetable and oil production. Here, we show that the obligate or facultative nature of the vernalization requirement in European winter oilseed rape is determined by allelic variation at a 10 Mbp region on chromosome A02. This region includes orthologues of the key floral regulators FLOWERING LOCUS C (BnaFLC.A02) and FLOWERING LOCUS T (BnaFT.A02). Polymorphism at BnaFLC.A02 and BnaFT.A02, mostly in cis-regulatory regions, results in distinct gene expression dynamics in response to vernalization treatment. Our data suggest allelic variation at BnaFT.A02 is associated with flowering time in the absence of vernalization, while variation at BnaFLC.A02 is associated with flowering time under vernalizing conditions. We hypothesize selection for BnaFLC.A02 and BnaFT.A02 gene expression variation has facilitated the generation of European winter oilseed rape varieties that are adapted to different winter climates. This knowledge will allow for the selection of alleles of flowering time regulators that alter the vernalization requirement of oilseed rape, informing the generation of new varieties with adapted flowering times and improved yields.

Identification and evolution of gene regulatory networks: insights from comparative studies in plants.

The availability of genome sequences, genome-wide assays of transcription factor binding, and accessible chromatin maps have unveiled gene regulatory landscapes in plants. This understanding has ushered in comparative gene regulatory network studies that assess network rewiring between species, across time, and between biological tissues. Comparisons of cis-regulatory elements across the plant kingdom have uncovered examples of conserved sequences, but also of divergence, indicating that selective pressures can vary in different plant families. Transcription factor duplication, followed by spatiotemporal expression divergence of the duplicates, also appears to be a key mechanism of network evolution. Here, we review recent literature describing the regulation of gene expression in plants, and how comparative studies provide insights into how these regulatory interactions change and lead to gene regulatory network rewiring.

Enhanced Maps of Transcription Factor Binding Sites Improve Regulatory Networks Learned from Accessible Chromatin Data.

Determining where transcription factors (TFs) bind in genomes provides insight into which transcriptional programs are active across organs, tissue types, and environmental conditions. Recent advances in high-throughput profiling of regulatory DNA have yielded large amounts of information about chromatin accessibility. Interpreting the functional significance of these data sets requires knowledge of which regulators are likely to bind these regions. This can be achieved by using information about TF-binding preferences, or motifs, to identify TF-binding events that are likely to be functional. Although different approaches exist to map motifs to DNA sequences, a systematic evaluation of these tools in plants is missing. Here, we compare four motif-mapping tools widely used in the Arabidopsis (Arabidopsis thaliana) research community and evaluate their performance using chromatin immunoprecipitation data sets for 40 TFs. Downstream gene regulatory network (GRN) reconstruction was found to be sensitive to the motif mapper used. We further show that the low recall of Find Individual Motif Occurrences, one of the most frequently used motif-mapping tools, can be overcome by using an Ensemble approach, which combines results from different mapping tools. Several examples are provided demonstrating how the Ensemble approach extends our view on transcriptional control for TFs active in different biological processes. Finally, a protocol is presented to effectively derive more complete cell type-specific GRNs through the integrative analysis of open chromatin regions, known binding site information, and expression data sets. This approach will pave the way to increase our understanding of GRNs in different cellular conditions.

Spatio-temporal expression dynamics differ between homologues of flowering time genes in the allopolyploid Brassica napus.

Polyploidy is a recurrent feature of eukaryotic evolution and has been linked to increases in complexity, adaptive radiation and speciation. Within angiosperms such events have occurred repeatedly in many plant lineages. Here we investigate the retention and spatio-temporal expression dynamics of duplicated genes predicted to regulate the floral transition in Brassica napus (oilseed rape, OSR). We show that flowering time genes are preferentially retained relative to other genes in the OSR genome. Using a transcriptome time series in two tissues (leaf and shoot apex) across development we show that 67% of these retained flowering time genes are expressed. Furthermore, between 64% (leaf) and 74% (shoot apex) of the retained gene homologues show diverged expression patterns relative to each other across development, suggesting neo- or subfunctionalization. A case study of homologues of the shoot meristem identity gene TFL1 reveals differences in cis-regulatory elements that could explain this divergence. Such differences in the expression dynamics of duplicated genes highlight the challenges involved in translating gene regulatory networks from diploid model systems to more complex polyploid crop species.

Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle.

Vinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.

An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

ELF3 controls thermoresponsive growth in Arabidopsis.

Plant development is highly responsive to ambient temperature, and this trait has been linked to the ability of plants to adapt to climate change. The mechanisms by which natural populations modulate their thermoresponsiveness are not known. To address this, we surveyed Arabidopsis accessions for variation in thermal responsiveness of elongation growth and mapped the corresponding loci. We find that the transcriptional regulator EARLY FLOWERING3 (ELF3) controls elongation growth in response to temperature. Through a combination of modeling and experiments, we show that high temperature relieves the gating of growth at night, highlighting the importance of temperature-dependent repressors of growth. ELF3 gating of transcriptional targets responds rapidly and reversibly to changes in temperature. We show that the binding of ELF3 to target promoters is temperature dependent, suggesting a mechanism where temperature directly controls ELF3 activity.