Marc Lohse

Trimmomatic: a flexible trimmer for Illumina sequence data

Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.

RobiNA: a user-friendly, integrated software solution for RNA-Seq-based transcriptomics

Recent rapid advances in next generation RNA sequencing (RNA-Seq)-based provide researchers with unprecedentedly large data sets and open new perspectives in transcriptomics. Furthermore, RNA-Seq-based transcript profiling can be applied to non-model and newly discovered organisms because it does not require a predefined measuring platform (like e.g. microarrays). However, these novel technologies pose new challenges: the raw data need to be rigorously quality checked and filtered prior to analysis, and proper statistical methods have to be applied to extract biologically relevant information. Given the sheer volume of data, this is no trivial task and requires a combination of considerable technical resources along with bioinformatics expertise. To aid the individual researcher, we have developed RobiNA as an integrated solution that consolidates all steps of RNA-Seq-based differential gene-expression analysis in one user-friendly cross-platform application featuring a rich graphical user interface. RobiNA accepts raw FastQ files, SAM/BAM alignment files and counts tables as input. It supports quality checking, flexible filtering and statistical analysis of differential gene expression based on state-of-the art biostatistical methods developed in the R/Bioconductor projects. In-line help and a step-by-step manual guide users through the analysis. Installer packages for Mac OS X, Windows and Linux are available under the LGPL licence from http://mapman.gabipd.org/web/guest/robin.

OrganellarGenomeDRAW—a suite of tools for generating physical maps of plastid and mitochondrial genomes and visualizing expression data sets

Mitochondria and plastids (chloroplasts) are cell organelles of endosymbiotic origin that possess their own genetic information. Most organellar DNAs map as circular double-stranded genomes. Across the eukaryotic kingdom, organellar genomes display great size variation, ranging from ∼15 to 20 kb (the size of the mitochondrial genome in most animals) to >10 Mb (the size of the mitochondrial genome in some lineages of flowering plants). We have developed OrganellarGenomeDraw (OGDRAW), a suite of software tools that enable users to create high-quality visual representations of both circular and linear annotated genome sequences provided as GenBank files or accession numbers. Although all types of DNA sequences are accepted as input, the software has been specifically optimized to properly depict features of organellar genomes. A recent extension facilitates the plotting of quantitative gene expression data, such as transcript or protein abundance data, directly onto the genome map. OGDRAW has already become widely used and is available as a free web tool (http://ogdraw.mpimp-golm.mpg.de/). The core processing components can be downloaded as a Perl module, thus also allowing for convenient integration into custom processing pipelines.

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.

Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic

Plastids (chloroplasts) possess an enormous capacity to synthesize and accumulate foreign proteins. Here we have maximized chloroplast protein production by over-expressing a proteinaceous antibiotic against pathogenic group A and group B streptococci from the plastid genome. The antibiotic, a phage lytic protein, accumulated to enormously high levels (>70% of the plants total soluble protein), and proved to be extremely stable in chloroplasts. This massive over-expression exhausted the protein synthesis capacity of the chloroplast such that the production of endogenous plastid-encoded proteins was severely compromised. Our data suggest that this is due to translational rather than transcriptional limitation of gene expression. We also show that the chloroplast-produced protein antibiotic efficiently kills the target bacteria. These unrivaled expression levels, together with the chloroplasts insensitivity to enzymes that degrade bacterial cell walls and the elimination of the need to remove bacterial endotoxins by costly purification procedures, indicate that this is an effective plant-based production platform for next-generation antibiotics, which are urgently required to keep pace with rapidly emerging bacterial resistance.

Systems Biology of Tomato Fruit Development: Combined Transcript, Protein, and Metabolite Analysis of Tomato Transcription Factor (nor, rin) and Ethylene Receptor (Nr) Mutants Reveals Novel Regulatory Interactions

Tomato (Solanum lycopersicum) is an established model to study fleshy fruit development and ripening. Tomato ripening is regulated independently and cooperatively by ethylene and transcription factors, including nonripening (NOR) and ripening-inhibitor (RIN). Mutations of NOR, RIN, and the ethylene receptor Never-ripe (Nr), which block ethylene perception and inhibit ripening, have proven to be great tools for advancing our understanding of the developmental programs regulating ripening. In this study, we present systems analysis of nor, rin, and Nr at the transcriptomic, proteomic, and metabolomic levels during development and ripening. Metabolic profiling marked shifts in the abundance of metabolites of primary metabolism, which lead to decreases in metabolic activity during ripening. When combined with transcriptomic and proteomic data, several aspects of the regulation of metabolism during ripening were revealed. First, correlations between the expression levels of a transcript and the abundance of its corresponding protein were infrequently observed during early ripening, suggesting that posttranscriptional regulatory mechanisms play an important role in these stages; however, this correlation was much greater in later stages. Second, we observed very strong correlation between ripening-associated transcripts and specific metabolite groups, such as organic acids, sugars, and cell wall-related metabolites, underlining the importance of these metabolic pathways during fruit ripening. These results further revealed multiple ethylene-associated events during tomato ripening, providing new insights into the molecular biology of ethylene-mediated ripening regulatory networks.

Large‐scale transcriptome analysis in chickpea (Cicer arietinum L.), an orphan legume crop of the semi‐arid tropics of Asia and Africa

Chickpea (Cicer arietinum L.) is an important legume crop in the semi-arid regions of Asia and Africa. Gains in crop productivity have been low however, particularly because of biotic and abiotic stresses. To help enhance crop productivity using molecular breeding techniques, next generation sequencing technologies such as Roche/454 and Illumina/Solexa were used to determine the sequence of most gene transcripts and to identify drought-responsive genes and gene-based molecular markers. A total of 103 215 tentative unique sequences (TUSs) have been produced from 435 018 Roche/454 reads and 21 491 Sanger expressed sequence tags (ESTs). Putative functions were determined for 49 437 (47.8%) of the TUSs, and gene ontology assignments were determined for 20 634 (41.7%) of the TUSs. Comparison of the chickpea TUSs with the Medicago truncatula genome assembly (Mt 3.5.1 build) resulted in 42 141 aligned TUSs with putative gene structures (including 39 281 predicted intron/splice junctions). Alignment of ∼37 million Illumina/Solexa tags generated from drought-challenged root tissues of two chickpea genotypes against the TUSs identified 44 639 differentially expressed TUSs. The TUSs were also used to identify a diverse set of markers, including 728 simple sequence repeats (SSRs), 495 single nucleotide polymorphisms (SNPs), 387 conserved orthologous sequence (COS) markers, and 2088 intron-spanning region (ISR) markers. This resource will be useful for basic and applied research for genome analysis and crop improvement in chickpea.

Arbuscule-containing and non-colonized cortical cells of mycorrhizal roots undergo extensive and specific reprogramming during arbuscular mycorrhizal development.

Most vascular plants form a mutualistic association with arbuscular mycorrhizal (AM) fungi, known as AM symbiosis. The development of AM symbiosis is an asynchronous process, and mycorrhizal roots therefore typically contain several symbiotic structures and various cell types. Hence, the use of whole-plant organs for downstream analyses can mask cell-specific variations in gene expression. To obtain insight into cell-specific reprogramming during AM symbiosis, comparative analyses of various cell types were performed using laser capture microdissection combined with microarray hybridization. Remarkably, the most prominent transcriptome changes were observed in non-arbuscule-containing cells of mycorrhizal roots, indicating a drastic reprogramming of these cells during root colonization that may be related to subsequent fungal colonization. A high proportion of transcripts regulated in arbuscule-containing cells and non-arbuscule-containing cells encode proteins involved in transport processes, transcriptional regulation and lipid metabolism, indicating that reprogramming of these processes is of particular importance for AM symbiosis.

RNA Interference of LIN5 in Tomato Confirms Its Role in Controlling Brix Content, Uncovers the Influence of Sugars on the Levels of Fruit Hormones, and Demonstrates the Importance of Sucrose Cleavage for Normal Fruit Development and Fertility

It has been previously demonstrated, utilizing intraspecific introgression lines, that Lycopersicum Invertase5 (LIN5), which encodes a cell wall invertase, controls total soluble solids content in tomato (Solanum lycopersicum). The physiological role of this protein, however, has not yet been directly studied, since evaluation of data obtained from the introgression lines is complicated by the fact that they additionally harbor many other wild species alleles. To allow a more precise comparison, we generated transgenic tomato in which we silenced the expression of LIN5 using the RNA interference approach. The transformants were characterized by an altered flower and fruit morphology, displaying increased numbers of petals and sepals per flower, an increased rate of fruit abortion, and a reduction in fruit size. Evaluation of the mature fruit revealed that the transformants were characterized by a reduction of seed number per plant. Furthermore, detailed physiological analysis revealed that the transformants displayed aberrant pollen morphology and a reduction in the rate of pollen tube elongation. Metabolite profiling of ovaries and green and red fruit revealed that metabolic changes in the transformants were largely confined to sugar metabolism, whereas transcript and hormone profiling revealed broad changes both in the hormones themselves and in transcripts encoding their biosynthetic enzymes and response elements. These results are discussed in the context of current understanding of the role of sugar during the development of tomato fruit, with particular focus given to its impact on hormone levels and organ morphology.

Mapping Metabolic and Transcript Temporal Switches during Germination in Rice Highlights Specific Transcription Factors and the Role of RNA Instability in the Germination Process

Transcriptome and metabolite profiling of rice (Oryza sativa) embryo tissue during a detailed time course formed a foundation for examining transcriptional and posttranscriptional processes during germination. One hour after imbibition (HAI), independent of changes in transcript levels, rapid changes in metabolism occurred, including increases in hexose phosphates, tricarboxylic acid cycle intermediates, and γ-aminobutyric acid. Later changes in the metabolome, including those involved in carbohydrate, amino acid, and cell wall metabolism, appeared to be driven by increases in transcript levels, given that the large group (over 6,000 transcripts) observed to increase from 12 HAI were enriched in metabolic functional categories. Analysis of transcripts encoding proteins located in the organelles of primary metabolism revealed that for the mitochondrial gene set, a greater proportion of transcripts peaked early, at 1 or 3 HAI, compared with the plastid set, and notably, many of these transcripts encoded proteins involved in transport functions. One group of over 2,000 transcripts displayed a unique expression pattern beginning with low levels in dry seeds, followed by a peak in expression levels at 1 or 3 HAI, before markedly declining at later time points. This group was enriched in transcription factors and signal transduction components. A subset of these transiently expressed transcription factors were further interrogated across publicly available rice array data, indicating that some were only expressed during the germination process. Analysis of the 1-kb upstream regions of transcripts displaying similar changes in abundance identified a variety of common sequence motifs, potential binding sites for transcription factors. Additionally, newly synthesized transcripts peaking at 3 HAI displayed a significant enrichment of sequence elements in the 3′ untranslated region that have been previously associated with RNA instability. Overall, these analyses reveal that during rice germination, an immediate change in some metabolite levels is followed by a two-step, large-scale rearrangement of the transcriptome that is mediated by RNA synthesis and degradation and is accompanied by later changes in metabolite levels.