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Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellar RNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and external cues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wide analyses of chloroplast translation (i.e., ribosome profiling), and the discovery of RNA binding proteins involved in the control of translational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In this review, we provide an overview of the current knowledge of the chloroplast translation machinery, its structure, organization, and function. In addition, we summarize the techniques that are currently available to study chloroplast translation and describe how translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss how translational control contributes to the regulation of chloroplast gene expression in response to developmental, environmental, and physiological cues. We also illustrate the commonalities and the differences between the chloroplast and bacterial translation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems.

RNA-guided nucleases of the CRISPR/Cas type can be repurposed as programmable nucleotide deaminases to mediate targeted nucleotide substitutions. Such base editors have enormous potential in genome editing, gene therapy and precision breeding. However, current editors suffer from limited specificity in that they edit different and/or multiple bases within a larger sequence window. Using cytidine deaminase base editors that elicit C-to-T mutations, we show here that high editing precision can be achieved by engineering the connection between the deaminase domain and the Cas domain of the editor. By systematically testing different linker sequences and removing non-essential sequences from the deaminase, we obtain high-precision base editors with narrow activity windows that can selectively edit a single cytidine at a specific position with high accuracy and efficiency. These base editors will enable the use of genome editing in applications where single-nucleotide changes are required and off-target editing of adjacent nucleotides is not tolerable. Base editors can target multiple bases within a window around the target site, reducing their specificity. Here the authors engineer the connection between the deaminase and Cas domain to narrow the window of activity.

Rhodophytes (red algae) are a diverse group of algae with great ecological and economic importance. However, tools for post-genomic research on red algae are still largely lacking. Here, we report the development of an efficient genetic transformation system for the model rhodophyte Porphyridium purpureum . We show that transgenes can be expressed to unprecedented levels of up to 5% of the total soluble protein. Surprisingly, the transgenic DNA is maintained episomally, as extrachromosomal high-copy number plasmid. The bacterial replication origin confers replication in the algal nucleus, thus providing an intriguing example of a prokaryotic replication origin functioning in a eukaryotic system. The extended presence of bacterial episomal elements may provide an evolutionary explanation for the frequent natural occurrence of extrachromosomal plasmids in red algae, and may also have contributed to the high rate of horizontal gene transfer from bacteria to the nuclear genome of Porphyridium purpureum and other rhodophytes. Genetic tools for research on red algae (rhodophytes) are lacking. Here, Li and Bock present an efficient genetic transformation system for a model rhodophyte, and show that the transgenic DNA can be maintained as an extrachromosomal multi-copy plasmid in the algal nucleus.

Base editors (BEs) are RNA-guided CRISPR-Cas-derived genome editing tools that induce single-nucleotide changes. The limitations of current BEs lie in their low precision (especially when multiple target nucleotides of the deaminase are present within the activity window) and their restriction to targets that are in proper distance from the PAM sequence. We have recently developed high-precision cytidine BEs by engineering CDA1 truncations and nCas9 fusions that predominantly edit nucleotide C −18 relative to the PAM sequence NGG. Here, by testing fusions with Cas9 variants that recognize alternative PAMs, we provide a series of high-precision BEs that greatly expand the versatility of base editing. In addition, we obtained BEs that selectively edit C −15 or C −16 . We also show that our high-precision BEs can substantially reduce off-target effect. These improved base editing tools will be widely applicable in basic research, biotechnology and gene therapy. Base editors can be limited by precision and the size of the target window. Here the authors test Cas9s that recognise alternative PAMs to obtain a series of high-precision editors.

Plant-mediated RNA interference (RNAi) shows great potential in crop protection. It relies on plants stably expressing double-stranded RNAs (dsRNAs) that target essential genes in pest insects. Practical application of this strategy is challenging because producing sufficient amounts of stable dsRNA in plants has proven to be difficult to achieve with conventional transgenesis. In addition, many insects do not respond to exogenously applied dsRNAs, either degrading them or failing to import them into the cytoplasm. We summarize recent progress in RNAi-mediated insect pest control and discuss factors determining its efficacy. Expressing dsRNA in chloroplasts overcomes many of the difficulties previously encountered. We also highlight remaining challenges and discuss the environmental and biosafety issues involved in the use of this technology in agriculture. Plant-mediated RNAi that targets essential genes in insects and other pests is becoming a promising approach in crop protection. Expression of dsRNA targeting insect genes can potentially provide crop protection without chemical pesticides and offers the additional advantages that no foreign protein is made and the number of target genes is nearly unlimited. The length and amount of the dsRNA as well as its stability in planta and in the gut of the target insect are crucial factors determining the success of plant-mediated RNAi strategies. High-level expression of long dsRNAs from the genome of the chloroplast represents a particularly promising strategy for efficient RNAi-mediated crop protection.

Tissue grafting includes applications ranging from plant breeding to animal organ transplantation. Donor and recipient are generally believed to maintain their genetic integrity, in that the grafted tissues are joined but their genetic materials do not mix. We grafted tobacco plants from two transgenic lines carrying different marker and reporter genes in different cellular compartments, the nucleus and the plastid. Analysis of the graft sites revealed the frequent occurrence of cells harboring both antibiotic resistances and both fluorescent reporters. Our data demonstrate that plant grafting can result in the exchange of genetic information via either large DNA pieces or entire plastid genomes. This observation of novel combinations of genetic material has implications for grafting techniques and also provides a possible path for horizontal gene transfer.

The genomes of DNA-containing cell organelles (mitochondria, chloroplasts) can be laterally transmitted between organisms, a process known as organelle capture. Organelle capture often occurs in the absence of detectable nuclear introgression, and the capture mechanism is unknown. Here, we have considered horizontal genome transfer across natural grafts as a mechanism underlying chloroplast capture in plants. By grafting sexually incompatible species, we show that complete chloroplast genomes can travel across the graft junction from one species into another. We demonstrate that, consistent with reported phylogenetic evidence, replacement of the resident plastid genome by the alien genome occurs in the absence of intergenomic recombination. Our results provide a plausible mechanism for organelle capture in plants and suggest natural grafting as a path for horizontal gene and genome transfer between sexually incompatible species.

Carotenoids are essential pigments of the photosynthetic apparatus and an indispensable component of the human diet. In addition to being potent antioxidants, they also provide the vitamin A precursor β-carotene. In tomato (Solanum lycopersicum) fruits, carotenoids accumulate in specialized plastids, the chromoplasts. How the carotenoid biosynthetic pathway is regulated and what limits total carotenoid accumulation in fruit chromoplasts is not well understood. Here, we have introduced the lycopene β-cyclase genes from the eubacterium Erwinia herbicola and the higher plant daffodil (Narcissus pseudonarcissus) into the tomato plastid genome. While expression of the bacterial enzyme did not strongly alter carotenoid composition, expression of the plant enzyme efficiently converted lycopene, the major storage carotenoid of the tomato fruit, into provitamin A (β-carotene). In green leaves of the transplastomic tomato plants, more lycopene was channeled into the β-branch of carotenoid biosynthesis, resulting in increased accumulation of xanthophyll cycle pigments and correspondingly reduced accumulation of the α-branch xanthophyll lutein. In fruits, most of the lycopene was converted into β-carotene with provitamin A levels reaching 1 mg per g dry weight. Unexpectedly, transplastomic tomatoes also showed a >50% increase in total carotenoid accumulation, indicating that lycopene β-cyclase expression enhanced the flux through the pathway in chromoplasts. Our results provide new insights into the regulation of carotenoid biosynthesis and demonstrate the potential of plastids genome engineering for the nutritional enhancement of food crops.

The formation of a new species can occur by an asexual mechanism by transfer of entire nuclear genomes between plant cells as shown by the creation of a new allopolyploid plant from parental herbaceous and woody plant species, this mechanism is a potential new tool for crop improvement. Speciation by growing together Grafting is a common occurrence in nature and is familiar as a means of manipulating plants and trees for use in agriculture and horticulture. Here Ralph Bock and colleagues demonstrate that entire nuclear genomes can be transferred across the graft junction from plant to plant. In grafting experiments between the tree tobacco Nicotiana glauca and the cigarette tobacco N. tabacum (woody and herbaceous species, respectively), horizontal transfer of nuclear genomes can result in the formation of a new polyploid species — Nicotiana tabauca . This is an example of allopolyploidization, the combination of the genomes from two different species that has contributed to evolutionary innovation, adaptation, speciation and domestication. It is thought to occur through hybridization events between species, accompanied or followed by genome duplication, but this work shows that it can also occur through an asexual mechanism that is readily available as a potential tool for crop improvement. Allopolyploidization, the combination of the genomes from two different species, has been a major source of evolutionary innovation and a driver of speciation and environmental adaptation 1 , 2 , 3 , 4 . In plants, it has also contributed greatly to crop domestication, as the superior properties of many modern crop plants were conferred by ancient allopolyploidization events 5 , 6 . It is generally thought that allopolyploidization occurred through hybridization events between species, accompanied or followed by genome duplication 6 , 7 . Although many allopolyploids arose from closely related species (congeners), there are also allopolyploid species that were formed from more distantly related progenitor species belonging to different genera or even different tribes 8 . Here we have examined the possibility that allopolyploidization can also occur by asexual mechanisms. We show that upon grafting—a mechanism of plant–plant interaction that is widespread in nature—entire nuclear genomes can be transferred between plant cells. We provide direct evidence for this process resulting in speciation by creating a new allopolyploid plant species from a herbaceous species and a woody species in the nightshade family. The new species is fertile and produces fertile progeny. Our data highlight natural grafting as a potential asexual mechanism of speciation and also provide a method for the generation of novel allopolyploid crop species.