Genome modification in plant mitochondria.

Mitochondria are an indispensable component of every plant cell and are inextricably linked to many vital functions. One of their key characteristics is that they have their own genome. This genome, although greatly reduced, encodes several essential genes.

Reverse genetics in the Arabidopsis chloroplast genome identifies rps16 as a transcribed pseudogene.

The plastid (chloroplast) genomes of seed plants contain a conserved set of ribosomal protein genes. The rps16 gene represents an exception: It has been lost from the plastid genomes of gymnosperms and several lineages of angiosperms, and may have undergone pseudogenization in a few other lineages, including members of the Brassicaceae family. Here we report a reverse genetic approach to test the annotated rps16 gene in the Arabidopsis plastid genome for functionality.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome.

Targeted knockout of a conserved plant mitochondrial gene by genome editing.

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I.

Mitochondrial ferredoxin-like is essential for forming complex I-containing supercomplexes in Arabidopsis.

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I-V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like).

Expression strategies for the efficient synthesis of antimicrobial peptides in plastids.

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants.

DNA base editing in nuclear and organellar genomes.

Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities.

Targeted introduction of heritable point mutations into the plant mitochondrial genome.

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut.

WUSCHEL triggers innate antiviral immunity in plant stem cells.

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones.

Germline-Transmitted Genome Editing Methodology in Arabidopsis thaliana Using TAL Effector Nucleases.

TAL effector nucleases (TALENs) are powerful tools to create specific knockout mutants in plants. The use of an optimized TALEN backbone and the choice of promoters that are strongly active in the stem cells of the shoot apical meristem are key to a high rate of heritable targeted mutations. Recommendations for construct design and screening for mutants are given in this chapter.

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance.

High-efficiency generation of fertile transplastomic Arabidopsis plants.

The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation.

Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in .

Shoot regeneration can be achieved through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for shoot regeneration.

A Comprehensive Toolkit for Inducible, Cell Type-Specific Gene Expression in Arabidopsis.

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis () based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems.

Control of plant cell fate transitions by transcriptional and hormonal signals.

Plant meristems carry pools of continuously active stem cells, whose activity is controlled by developmental and environmental signals. After stem cell division, daughter cells that exit the stem cell domain acquire transit amplifying cell identity before they are incorporated into organs and differentiate. In this study, we used an integrated approach to elucidate the role of () genes in regulating developmental trajectories of shoot stem cells in .

RETINOBLASTOMA RELATED1 mediates germline entry in .

To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1-dependent inactivation of the Retinoblastoma homolog RBR1.

Integration of light and metabolic signals for stem cell activation at the shoot apical meristem.

A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling.

Germline-transmitted genome editing in Arabidopsis thaliana Using TAL-effector-nucleases.

Transcription activator-like effector nucleases (TALENs) are custom-made bi-partite endonucleases that have recently been developed and applied for genome engineering in a wide variety of organisms. However, they have been only scarcely used in plants, especially for germline-modification. Here we report the efficient creation of small, germline-transmitted deletions in Arabidopsis thaliana via TALENs that were delivered by stably integrated transgenes.

GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.

Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project.

RNA PROCESSING FACTOR3 is crucial for the accumulation of mature ccmC transcripts in mitochondria of Arabidopsis accession Columbia.

RNA PROCESSING FACTOR1 (RPF1) and RPF2 are pentatricopeptide repeat (PPR) proteins involved in 5' processing of different mitochondrial mRNAs in Arabidopsis (Arabidopsis thaliana). Both factors are highly similar to RESTORERS OF FERTILITY (RF), which are part of cytoplasmic male sterility/restoration systems in various plant species. These findings suggest a predominant role of RF-like PPR proteins in posttranscriptional 5' processing.

A RESTORER OF FERTILITY-like PPR gene is required for 5'-end processing of the nad4 mRNA in mitochondria of Arabidopsis thaliana.

Processing of 5'-ends is a frequently observed step during maturation of plant mitochondrial mRNAs. Up to now, very little is known about the biochemistry of this process and the proteins involved in the removal of 5' leader sequences. Based on natural genetic variation we have used linkage mapping and complementation studies to identify a nuclear gene required for the efficient generation of a 5'-end 228 nucleotides upstream of the mitochondrial nad4 gene encoding subunit 4 of the NADH dehydrogenase complex.

Transcriptional control of a plant stem cell niche.

Despite the independent evolution of multicellularity in plants and animals, the basic organization of their stem cell niches is remarkably similar. Here, we report the genome-wide regulatory potential of WUSCHEL, the key transcription factor for stem cell maintenance in the shoot apical meristem of the reference plant Arabidopsis thaliana. WUSCHEL acts by directly binding to at least two distinct DNA motifs in more than 100 target promoters and preferentially affects the expression of genes with roles in hormone signaling, metabolism, and development.

RNA PROCESSING FACTOR2 is required for 5' end processing of nad9 and cox3 mRNAs in mitochondria of Arabidopsis thaliana.

In mitochondria of higher plants, the majority of 5' termini of mature mRNAs are generated posttranscriptionally. To gain insight into this process, we analyzed a natural 5' end polymorphism in the species Arabidopsis thaliana. This genetic approach identified the nuclear gene At1g62670, encoding a pentatricopeptide repeat protein.

Arabidopsis encodes four tRNase Z enzymes.

Functional transfer RNA (tRNA) molecules are a prerequisite for protein biosynthesis. Several processing steps are required to generate the mature functional tRNA from precursor molecules. Two of the early processing steps involve cleavage at the tRNA 5' end and the tRNA 3' end.