Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotianatabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno–independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno–independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site. Protein biosynthesis (translation) is a highly regulated process in gene expression. In all organisms, initiation of translation depends on molecular recognition of the messenger RNA by ribosomes. In prokaryotes (bacteria, mitochondria, and chloroplasts), this recognition is mediated by a specific sequence motif in the 5′ untranslated region of the mRNA, called “ribosome-binding site” or “Shine-Dalgarno sequence.” However, many messenger RNAs lack Shine-Dalgarno sequences, and it is currently unknown how the correct translation initiation site is recognized in these mRNAs. Here, we provide insights into the mechanism of translation initiation in the absence of a ribosome-binding site. We have performed genome-wide searches for Shine-Dalgarno–independent translation in bacterial and organellar genomes and report that a large fraction of transcripts is translated in a Shine-Dalgarno–independent manner in all prokaryotic systems. We find that Shine-Dalgarno–independent translation initiation is strongly correlated with the presence of a local minimum in RNA secondary structure around the translational start codon. The significance of RNA unfoldedness as the key determinant of start codon recognition in Shine-Dalgarno–independent translation initiation was confirmed experimentally by employing reporter gene fusions in the bacterium Escherichia coli. In conclusion, our work suggests an intriguing mechanism for translation initiation on mRNAs that lack a ribosome-binding site.

Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but—contrary to previous suggestions—it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus. Reduced genomes of parasitic bacteria, chloroplasts, and mitochondria do not encode the full set of 32 tRNAs required to read all triplets of the genetic code according to Francis Crick's wobble rules. tRNAs with U in the wobble position of their anticodon might be able to make up for the deficit by pairing with any of the four bases at the third position of the codon via a mechanism called superwobbling. We have investigated the feasibility of superwobbling in the chloroplast genome of tobacco plants. We find that superwobbling occurs in all codon families where it is theoretically possible, demonstrating that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA utilizing standard base pairing or wobbling and by a second tRNA employing superwobbling. We also show that the efficiency of superwobbling is variable in different codon families. Finally, our data reveal that the minimum number of tRNAs required to sustain protein biosynthesis is 25.

Plastid transformation has become an attractive tool in biotechnology. Because of the prokaryotic nature of the plastid's gene expression machinery, expression elements (promoters and untranslated regions) that trigger high-level foreign protein accumulation in plastids usually also confer high expression in bacterial cloning hosts. This can cause problems, for example, when production of antimicrobial compounds is attempted. Their bactericidal activity can make the cloning of the corresponding genes in plastid transformation vectors impossible. Here, we report a general solution to this problem. We have designed a strategy (referred to as toxin shuttle) that allows the expression in plastids of proteins that are toxic to Escherichia coli . The strategy is based on blocking transcription in E. coli by bacterial transcription terminators upstream of the gene of interest, which subsequently are excised in planta by site-specific recombination. We demonstrate the applicability of the strategy by the high-level expression in plastids (to up to 30% of the plant's total soluble protein) of 2 phage-derived protein antibiotics that are toxic to E. coli . We also show that the plastid-produced antibiotics efficiently kill pathogenic strains of Streptococcus pneumoniae , the causative agent of pneumonia, thus providing a promising strategy for the production of next-generation antibiotics in plants.

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant‐derived products. In the coming years, plant‐based research will be among the major drivers ensuring food security and the expansion of the bio‐based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.