FLC and SVP Are Key Regulators of Flowering Time in the Biennial/Perennial Species Noccaea caerulescens.

The appropriate timing of flowering is crucial for plant reproductive success. Studies of the molecular mechanism of flower induction in the model plant Arabidopsis thaliana showed long days and vernalization as major environmental promotive factors. Noccaea caerulescens has an obligate vernalization requirement that has not been studied at the molecular genetics level. Here, we characterize the vernalization requirement and response of four geographically diverse biennial/perennial N. caerulescens accessions: Ganges (GA), Lellingen (LE), La Calamine (LC), and St. Felix de Pallières (SF). Differences in vernalization responsiveness among accessions suggest that natural variation for this trait exists within N. caerulescens. Mutants which fully abolish the vernalization requirement were identified and were shown to contain mutations in the FLOWERING LOCUS C (NcFLC) and SHORT VEGETATIVE PHASE (NcSVP) genes, two key floral repressors in this species. At high temperatures, the non-vernalization requiring flc-1 mutant reverts from flowering to vegetative growth, which is accompanied with a reduced expression of LFY and AP1. This suggested there is "crosstalk" between vernalization and ambient temperature, which might be a strategy to cope with fluctuations in temperature or adopt a more perennial flowering attitude and thus facilitate a flexible evolutionary response to the changing environment across the species range.

Genetic complexity of miscanthus cell wall composition and biomass quality for biofuels.

BACKGROUND: Miscanthus sinensis is a high yielding perennial grass species with great potential as a bioenergy feedstock. One of the challenges that currently impedes commercial cellulosic biofuel production is the technical difficulty to efficiently convert lignocellulosic biomass into biofuel. The development of feedstocks with better biomass quality will improve conversion efficiency and the sustainability of the value-chain. Progress in the genetic improvement of biomass quality may be substantially expedited by the development of genetic markers associated to quality traits, which can be used in a marker-assisted selection program. RESULTS: To this end, a mapping population was developed by crossing two parents of contrasting cell wall composition. The performance of 182 F1 offspring individuals along with the parents was evaluated in a field trial with a randomized block design with three replicates. Plants were phenotyped for cell wall composition and conversion efficiency characters in the second and third growth season after establishment. A new SNP-based genetic map for M. sinensis was built using a genotyping-by-sequencing (GBS) approach, which resulted in 464 short-sequence uniparental markers that formed 16 linkage groups in the male map and 17 linkage groups in the female map. A total of 86 QTLs for a variety of biomass quality characteristics were identified, 20 of which were detected in both growth seasons. Twenty QTLs were directly associated to different conversion efficiency characters. Marker sequences were aligned to the sorghum reference genome to facilitate cross-species comparisons. Analyses revealed that for some traits previously identified QTLs in sorghum occurred in homologous regions on the same chromosome. CONCLUSION: In this work we report for the first time the genetic mapping of cell wall composition and bioconversion traits in the bioenergy crop miscanthus. These results are a first step towards the development of marker-assisted selection programs in miscanthus to improve biomass quality and facilitate its use as feedstock for biofuel production.

Splicing-related genes are alternatively spliced upon changes in ambient temperatures in plants.

Plants adjust their development and architecture to small variations in ambient temperature. In a time in which temperatures are rising world-wide, the mechanism by which plants are able to sense temperature fluctuations and adapt to it, is becoming of special interest. By performing RNA-sequencing on two Arabidopsis accession and one Brassica species exposed to temperature alterations, we showed that alternative splicing is an important mechanism in ambient temperature sensing and adaptation. We found that amongst the differentially alternatively spliced genes, splicing related genes are enriched, suggesting that the splicing machinery itself is targeted for alternative splicing when temperature changes. Moreover, we showed that many different components of the splicing machinery are targeted for ambient temperature regulated alternative splicing. Mutant analysis of a splicing related gene that was differentially spliced in two of the genotypes showed an altered flowering time response to different temperatures. We propose a two-step mechanism where temperature directly influences alternative splicing of the splicing machinery genes, followed by a second step where the altered splicing machinery affects splicing of downstream genes involved in the adaptation to altered temperatures.

Low Temperature Affects Stem Cell Maintenance in Brassica oleracea Seedlings.

Most of the above ground tissues in higher plants originate from stem cells located in the shoot apical meristem (SAM). Several plant species can suffer from spontaneous stem cell arrest resulting in lack of further shoot development. In Brassica oleracea this SAM arrest is known as blindness and occurs in an unpredictable manner leading to considerable economic losses for plant raisers and farmers. Detailed analyses of seedlings showed that stem cell arrest is triggered by low temperatures during germination. To induce this arrest reproducibly and to study the effect of the environment, an assay was developed. The role of genetic variation on the susceptibility to develop blind seedlings was analyzed by a quantitative genetic mapping approach, using seeds from a double haploid population from a cross between broccoli and Chinese kale, produced at three locations. The analysis revealed, besides an effect of the seed production location, a region on linkage group C3 associated with blindness sensitivity. A subsequent dynamic genome-wide transcriptome analysis resulted in the identification of around 3000 differentially expressed genes early after blindness induction. A large number of cell cycle genes were en masse induced early during the development of blindness, whereas shortly after, all were down-regulated. This miss-regulation of core cell cycle genes is accompanied with a strong reduction of cells reaching the DNA replication phase. From the differentially expressed genes, 90 were located in the QTL region C3. Among them are two genes belonging to the MINICHROMOSOMAL MAINTENANCE gene family, known to be involved in DNA replication, a RETINOBLASTOMA-RELATED gene, a key regulator for cell cycle initiation, and several MutS homologs genes, involved in DNA repair. These genes are potential candidates for being involved in the development of blindness in Brassica oleracea sensitive genotypes.

Orphan Crops Browser: a bridge between model and orphan crops.

Many important crops have received little attention by the scientific community, either because they are not considered economically important or due to their large and complex genomes. De novo transcriptome assembly, using next-generation sequencing data, is an attractive option for the study of these orphan crops. In spite of the large amount of sequencing data that can be generated, there is currently a lack of tools which can effectively help molecular breeders and biologists to mine this type of information. Our goal was to develop a tool that enables molecular breeders, without extensive bioinformatics knowledge, to efficiently study de novo transcriptome data from any orphan crop (http://www.bioinformatics.nl/denovobrowser/db/species/index). The Orphan Crops Browser has been designed to facilitate the following tasks (1) search and identification of candidate transcripts based on phylogenetic relationships between orthologous sequence data from a set of related species and (2) design specific and degenerate primers for expression studies in the orphan crop of interest. To demonstrate the usability and reliability of the browser, it was used to identify the putative orthologues of 17 known lignin biosynthetic genes from maize and sugarcane in the orphan crop Miscanthus sinensis. Expression studies in miscanthus stem internode tissue differing in maturation were subsequently carried out, to follow the expression of these genes during lignification. Our results showed a negative correlation between lignin content and gene expression. The present data are in agreement with recent findings in maize and other crops, and it is further discussed in this paper.

A comprehensive set of transcript sequences of the heavy metal hyperaccumulator Noccaea caerulescens.

Noccaea caerulescens is an extremophile plant species belonging to the Brassicaceae family. It has adapted to grow on soils containing high, normally toxic, concentrations of metals such as nickel, zinc, and cadmium. Next to being extremely tolerant to these metals, it is one of the few species known to hyperaccumulate these metals to extremely high concentrations in their aboveground biomass. In order to provide additional molecular resources for this model metal hyperaccumulator species to study and understand the mechanism of adaptation to heavy metal exposure, we aimed to provide a comprehensive database of transcript sequences for N. caerulescens. In this study, 23,830 transcript sequences (isotigs) with an average length of 1025 bp were determined for roots, shoots and inflorescences of N. caerulescens accession "Ganges" by Roche GS-FLEX 454 pyrosequencing. These isotigs were grouped into 20,378 isogroups, representing potential genes. This is a large expansion of the existing N. caerulescens transcriptome set consisting of 3705 unigenes. When translated and compared to a Brassicaceae proteome set, 22,232 (93.2%) of the N. caerulescens isotigs (corresponding to 19,191 isogroups) had a significant match and could be annotated accordingly. Of the remaining sequences, 98 isotigs resembled non-plant sequences and 1386 had no significant similarity to any sequence in the GenBank database. Among the annotated set there were many isotigs with similarity to metal homeostasis genes or genes for glucosinolate biosynthesis. Only for transcripts similar to Metallothionein3 (MT3), clear evidence for an additional copy was found. This comprehensive set of transcripts is expected to further contribute to the discovery of mechanisms used by N. caerulescens to adapt to heavy metal exposure.

Beyond genomic variation--comparison and functional annotation of three Brassica rapa genomes: a turnip, a rapid cycling and a Chinese cabbage.

BACKGROUND: Brassica rapa is an economically important crop species. During its long breeding history, a large number of morphotypes have been generated, including leafy vegetables such as Chinese cabbage and pakchoi, turnip tuber crops and oil crops. RESULTS: To investigate the genetic variation underlying this morphological variation, we re-sequenced, assembled and annotated the genomes of two B. rapa subspecies, turnip crops (turnip) and a rapid cycling. We then analysed the two resulting genomes together with the Chinese cabbage Chiifu reference genome to obtain an impression of the B. rapa pan-genome. The number of genes with protein-coding changes between the three genotypes was lower than that among different accessions of Arabidopsis thaliana, which can be explained by the smaller effective population size of B. rapa due to its domestication. Based on orthology to a number of non-brassica species, we estimated the date of divergence among the three B. rapa morphotypes at approximately 250,000 YA, far predating Brassica domestication (5,000-10,000 YA). CONCLUSIONS: By analysing genes unique to turnip we found evidence for copy number differences in peroxidases, pointing to a role for the phenylpropanoid biosynthesis pathway in the generation of morphological variation. The estimated date of divergence among three B. rapa morphotypes implies that prior to domestication there was already considerably divergence among B. rapa genotypes. Our study thus provides two new B. rapa reference genomes, delivers a set of computer tools to analyse the resulting pan-genome and uses these to shed light on genetic drivers behind the rich morphological variation found in B. rapa.

Over-expression of Arabidopsis AtCHR23 chromatin remodeling ATPase results in increased variability of growth and gene expression.

BACKGROUND: Plants are sessile organisms that deal with their -sometimes adverse- environment in well-regulated ways. Chromatin remodeling involving SWI/SNF2-type ATPases is thought to be an important epigenetic mechanism for the regulation of gene expression in different developmental programs and for integrating these programs with the response to environmental signals. In this study, we report on the role of chromatin remodeling in Arabidopsis with respect to the variability of growth and gene expression in relationship to environmental conditions. RESULTS: Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type. Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation. The reduced-growth phenotype requires sufficient light and is magnified by applying deliberate abiotic (salt, osmotic) stress. In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects. In addition, we show that over-expression of AtCHR23 increases the variability of growth in populations of genetically identical plants. These data indicate that accurate and controlled expression of AtCHR23 contributes to the stability or robustness of growth. Detailed RNAseq analyses demonstrate that upon AtCHR23 over-expression also the variation of gene expression is increased in a subset of genes that associate with environmental stress. The larger variation of gene expression is confirmed in individual plants with the help of independent qRT-PCR analysis. CONCLUSIONS: Over-expression of AtCHR23 gives Arabidopsis a phenotype that is markedly different from the growth arrest phenotype observed upon over-expression of AtCHR12, the paralog of AtCHR23, in response to abiotic stress. This demonstrates functional sub-specialization of highly similar ATPases in Arabidopsis. Over-expression of AtCHR23 increases the variability of growth among genetically identical individuals in a way that is consistent with increased variability of expression of a distinct subset of genes that associate with environmental stress. We propose that ATCHR23-mediated chromatin remodeling is a potential component of a buffer system in plants that protects against environmentally-induced phenotypic and transcriptional variation.

Predicting the impact of alternative splicing on plant MADS domain protein function.

Several genome-wide studies demonstrated that alternative splicing (AS) significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function. One example is the plant MADS-domain transcription factor family, members of which interact to form protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the protein-protein interaction capabilities of MIKC-type MADS-domain proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1-3) subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP), resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the potential impact on the interaction capabilities of the encoded MIKC proteins.

Assessing the contribution of alternative splicing to proteome diversity in Arabidopsis thaliana using proteomics data.

BACKGROUND: Large-scale analyses of genomics and transcriptomics data have revealed that alternative splicing (AS) substantially increases the complexity of the transcriptome in higher eukaryotes. However, the extent to which this complexity is reflected at the level of the proteome remains unclear. On the basis of a lack of conservation of AS between species, we previously concluded that AS does not frequently serve as a mechanism that enables the production of multiple functional proteins from a single gene. Following this conclusion, we hypothesized that the extent to which AS events contribute to the proteome diversity in Arabidopsis thaliana would be lower than expected on the basis of transcriptomics data. Here, we test this hypothesis by analyzing two large-scale proteomics datasets from Arabidopsis thaliana. RESULTS: A total of only 60 AS events could be confirmed using the proteomics data. However, for about 60% of the loci that, based on transcriptomics data, were predicted to produce multiple protein isoforms through AS, no isoform-specific peptides were found. We therefore performed in silico AS detection experiments to assess how well AS events were represented in the experimental datasets. The results of these in silico experiments indicated that the low number of confirmed AS events was the consequence of a limited sampling depth rather than in vivo under-representation of AS events in these datasets. CONCLUSION: Although the impact of AS on the functional properties of the proteome remains to be uncovered, the results of this study indicate that AS-induced diversity at the transcriptome level is also expressed at the proteome level.

Comparative analysis indicates that alternative splicing in plants has a limited role in functional expansion of the proteome.

BACKGROUND: Alternative splicing (AS) is a widespread phenomenon in higher eukaryotes but the extent to which it leads to functional protein isoforms and to proteome expansion at large is still a matter of debate. In contrast to animal species, for which AS has been studied extensively at the protein and functional level, protein-centered studies of AS in plant species are scarce. Here we investigate the functional impact of AS in dicot and monocot plant species using a comparative approach. RESULTS: Detailed comparison of AS events in alternative spliced orthologs from the dicot Arabidopsis thaliana and the monocot Oryza sativa (rice) revealed that the vast majority of AS events in both species do not result from functional conservation. Transcript isoforms that are putative targets for the nonsense-mediated decay (NMD) pathway are as likely to contain conserved AS events as isoforms that are translated into proteins. Similar results were obtained when the same comparison was performed between the two more closely related monocot species rice and Zea mays (maize).Genome-wide computational analysis of functional protein domains encoded in alternatively and constitutively spliced genes revealed that only the RNA recognition motif (RRM) is overrepresented in alternatively spliced genes in all species analyzed. In contrast, three domain types were overrepresented in constitutively spliced genes. AS events were found to be less frequent within than outside predicted protein domains and no domain type was found to be enriched with AS introns. Analysis of AS events that result in the removal of complete protein domains revealed that only a small number of domain types is spliced-out in all species analyzed. Finally, in a substantial fraction of cases where a domain is completely removed, this domain appeared to be a unit of a tandem repeat. CONCLUSION: The results from the ortholog comparisons suggest that the ability of a gene to produce more than one functional protein through AS does not persist during evolution. Cross-species comparison of the results of the protein-domain oriented analyses indicates little correspondence between the analyzed species. Based on the premise that functional genetic features are most likely to be conserved during evolution, we conclude that AS has only a limited role in functional expansion of the proteome in plants.