New insights into the proteome of the transcriptionally active chromosome from chloroplasts

Plastiden besitzen eigene DNA (ptDNA), die zusammen mit einer Vielzahl an Proteinen in Strukturen verpackt ist, die den bakteriellen Chromosomen ähneln. Diese Nukleoproteinkomplexe werden als plastidäre Nukleoide oder Plastidenkerne bezeichnet. Im Unterschied zum Chromatin im Zellkern gibt es nur wenige Informationen über die Organisation und Dynamik der Nukleoide in den Plastiden. Zur Identifizierung von Proteinen des Transkriptionsaktiven Chromosoms (TAC) aus Spinatchloroplasten wurden Proteomanalysen durchgeführt. In einer voran-gegangenen Proteomanalyse mit einer TAC-Fraktion aus Senfchloroplasten wurde das DNA-Bindeprotein Whirly1 identifiziert. Mit dem spezifischen Antikörper konnte hier gezeigt werden, dass das Protein in der konventionell präparierten TAC I Fraktion, aber nicht in der hochaufgereinigten TAC-II-Fraktion von Gersten-chloroplasten vorkommt. Durch eine Proteomanalyse der hochaufgereinigten TAC II Fraktion konnten darüber hinaus sechs neue DNA-Bindeproteine identifiziert werden. Sie wurden als plastidäre nukleoidassoziierte Proteine (ptNAP) benannt. Eines der neu identifizierten ptNAP ist das Protein AtSWIB-1 (Arabidopsis thaliana SWIB domain-containing protein-1). Das Protein ist in der TAC-II-Fraktion ange-reichert und bindet an plastidäre DNA. Dies deutet darauf hin, dass das SWIB-1-Protein im Unterschied zum Whirly1-Protein eine integrale Komponente des TAC ist. Durch Fusionen mit dem GFP-Protein konnte eine duale Lokalisation des AtSWIB-1-Proteins im Zellkern und in den Nukleoiden der Plastiden gezeigt werden. Daten-bankrecherchen erlaubten die Identifizierung von drei weiteren SWIB-Domänen-proteinen von Arabidopsis thaliana, deren vorhergesagte plastidäre Lokalisation durch GFP-Fusionen bestätigt werden konnte. Zusammen mit SWIB-1 gehören sie zu den ersten Chromatinmodellierungsfaktoren, die außerhalb des Kerns vorkom-men. Aufgrund der Ergebnisse dieser Arbeit wird die Hypothese aufgestellt, dass Plastiden Chromatinmodellierungsproteine besitzen, die über Veränderungen in der Struktur der Nukleoide die Genexpression beeinflussen können.Chloroplasts possess their own DNA (ptDNA), which is packaged with proteins into structures analogous to bacterial chromosomes, termed nucleoids or plastid nuclei. In contrast to nuclear chromatin, there is only limited information on the organization and dynamics of the plastid nucleoids. In order to investigate the protein composition of the transcriptionally active chromosome (TAC) fractions from chloroplasts and to identify new components of it, proteomic analyses were performed. One of the DNA-binding proteins identified in a previous study was the Whirly1 protein. Immunological experiments with an antibody specific for HvWhirly1 allowed the detection of the protein in a conventionally prepared TAC-I fraction from barley chloroplasts but not in a highly purified TAC-II fraction. By proteome analyses with the highly purified TAC-II fraction, six new DNA-binding proteins were identified. They were named plastid nucleoid-associated proteins (ptNAP). One of newly identified ptNAPs is the AtSWIB 1 (Arabidopsis thaliana SWIB domain-containing protein-1) protein. Immuno-logical analyses with an antibody specific for AtSWIB-1 allowed the detection of the SWIB-1 protein in both the conventionally prepared TAC-I and the highly enriched TAC-II fraction from spinach chloroplasts indicating that SWIB-1 in contrast to Whirly1 is a core component of TAC. An AtSWIB-1:GFP fusion protein was shown to be dually located in the nucleus and in plastid nucleoids. Binding of the SWIB-1 protein to plastid DNA was confirmed by Southwestern analysis. Database searches revealed that SWIB-1 together with further five SWIB-domain proteins belong to a subgroup of the SWIB family being predicted to be targeted to organelles. Fusions with GFP protein confirmed a plastid location for three of them. Together with SWIB-1, these proteins are the first identified chromatin remodeling factors being imported into plastids. Based on these findings, it is hypothesized that plastids possess chromatin remodeling proteins in order to regulate the expression of their genes by introducing structural changes of the nucleoid

Plant organellar RNA maturation

Plant organellar RNA metabolism is run by a multitude of nucleus-encoded RNA-binding proteins (RBPs) that control RNA stability, processing, and degradation. In chloroplasts and mitochondria, these post-transcriptional processes are vital for the production of a small number of essential components of the photosynthetic and respiratory machinery—and consequently for organellar biogenesis and plant survival. Many organellar RBPs have been functionally assigned to individual steps in RNA maturation, often specific to selected transcripts. While the catalog of factors identified is ever-growing, our knowledge of how they achieve their functions mechanistically is far from complete. This review summarizes the current knowledge of plant organellar RNA metabolism taking an RBP-centric approach and focusing on mechanistic aspects of RBP functions and the kinetics of the processes they are involved in.Peer Reviewe

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.Peer reviewe

Shifting the limits in wheat research and breeding using a fully annotated reference genome

Introduction: Wheat (Triticum aestivum L.) is the most widely cultivated crop on Earth, contributing about a fifth of the total calories consumed by humans. Consequently, wheat yields and production affect the global economy, and failed harvests can lead to social unrest. Breeders continuously strive to develop improved varieties by fine-tuning genetically complex yield and end-use quality parameters while maintaining stable yields and adapting the crop to regionally specific biotic and abiotic stresses. Rationale: Breeding efforts are limited by insufficient knowledge and understanding of wheat biology and the molecular basis of central agronomic traits. To meet the demands of human population growth, there is an urgent need for wheat research and breeding to accelerate genetic gain as well as to increase and protect wheat yield and quality traits. In other plant and animal species, access to a fully annotated and ordered genome sequence, including regulatory sequences and genome-diversity information, has promoted the development of systematic and more time-efficient approaches for the selection and understanding of important traits. Wheat has lagged behind, primarily owing to the challenges of assembling a genome that is more than five times as large as the human genome, polyploid, and complex, containing more than 85% repetitive DNA. To provide a foundation for improvement through molecular breeding, in 2005, the International Wheat Genome Sequencing Consortium set out to deliver a high-quality annotated reference genome sequence of bread wheat. Results: An annotated reference sequence representing the hexaploid bread wheat genome in the form of 21 chromosome-like sequence assemblies has now been delivered, giving access to 107,891 high-confidence genes, including their genomic context of regulatory sequences. This assembly enabled the discovery of tissue- and developmental stage–related gene coexpression networks using a transcriptome atlas representing all stages of wheat development. The dynamics of change in complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. Aspects of the future value of the annotated assembly for molecular breeding and research were exemplarily illustrated by resolving the genetic basis of a quantitative trait locus conferring resistance to abiotic stress and insect damage as well as by serving as the basis for genome editing of the flowering-time trait. Conclusion: This annotated reference sequence of wheat is a resource that can now drive disruptive innovation in wheat improvement, as this community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding. Importantly, the bioinformatics capacity developed for model-organism genomes will facilitate a better understanding of the wheat genome as a result of the high-quality chromosome-based genome assembly. By necessity, breeders work with the genome at the whole chromosome level, as each new cross involves the modification of genome-wide gene networks that control the expression of complex traits such as yield. With the annotated and ordered reference genome sequence in place, researchers and breeders can now easily access sequence-level information to precisely define the necessary changes in the genomes for breeding programs. This will be realized through the implementation of new DNA marker platforms and targeted breeding technologies, including genome editing

Multiple wheat genomes reveal global variation in modern breeding

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome 1, and the lack of genome-assembly data for multiple wheat lines 2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses 4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm1 6, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars

Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars

Shifting the limits in wheat research and breeding using a fully annotated reference genome

Wheat is one of the major sources of food for much of the world. However, because bread wheat's genome is a large hybrid mix of three separate subgenomes, it has been difficult to produce a high-quality reference sequence. Using recent advances in sequencing, the International Wheat Genome Sequencing Consortium presents an annotated reference genome with a detailed analysis of gene content among subgenomes and the structural organization for all the chromosomes. Examples of quantitative trait mapping and CRISPR-based genome modification show the potential for using this genome in agricultural research and breeding. Ramírez-González et al. exploited the fruits of this endeavor to identify tissue-specific biased gene expression and coexpression networks during development and exposure to stress. These resources will accelerate our understanding of the genetic basis of bread wheat