Elucidating novel mechanisms of plastid gene function and regulation by an in-depth characterization of the Oenothera (evening-primrose) plastome mutant collection
Plant Biochemistry and Biophysics
Final Report Abstract
A primary aim of the present project was to understand mechanisms of occurrence and spectrum of inheritable mutations in the chloroplast genome. This was achieved by an in-depth characterization of the evening primrose (Oenothera) plastome mutants collection, based on full chloroplast genome sequences. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms, but are insertions/deletions originating from DNA replication slippage events that are biased to particular regions in the genome. Our analyses identified the spectrum of spontaneous mutations in plastids and revealed that this spectrum is very different from that in the nucleus. The present collection represents a unique assortment of spontaneous mutants of the chloroplast genome. Its characterization then allowed: (i) The identification of ribosomal frameshifting as novel mechanism of chloroplast gene expression. Here we could show that the process enables correction of frameshift mutations in the chloroplast genome. It can restore photoautotrophic growth in the presence of mutation that otherwise would be lethal. (ii) Further characterization of mutants of the collection yielded mutant alleles that allow elucidation of novel features of plastid gene regulation and function. Of particular interest are the five mutants we identified that display defects in chloroplast RNA metabolism (splicing/processing deficiencies). With this material we could show, that defects in mRNA metabolism can be induced by mutations in coding regions, conferred by so far unknow sequence motives and, probably, unknow binding proteins. In addition, the mutant I-tau uncovered developmental regulation of chloroplast translation. Last, (iii) characterization of the plastome mutants collection generated a genetic resource to address questions of chloroplast inheritance. The resulting paper was highlighted in Nature Plants (“Lipids control to win”) and the Laborjournal (“Kampf der Organellen”). Derived from the technical demands of the project, systematic high-throughput sequencing of 65 chloroplast genomes, there was need to develop state-of-the-art chloroplast genomes annotation and submission software. The powerful automated annotation and sequence processing software (GeSeq and GB2sequin, respectively) are now freely available and part of the CHLOROBOX toolbox, hosted at the Max Planck Institute of Molecular Plant Physiology. The software are the most advanced and heavily used in the field, currently setting the standards. The GeSeq software was highlighted by Laborjournal Online (“Feinschliff für Organellengenome“).
Publications
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(2015) No evidence that sex and transposable elements drive genome size variation in evening primroses. Evolution 69: 1053-1062
Ågren JA, Greiner S, Johnson MTJ, Wright SI
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(2016) Spontaneous chloroplast mutants mostly occur by replication slippage and show a biased pattern in the plastome of Oenothera. The Plant Cell 28: 911-929
Massouh A, Schubert J, Yaneva-Roder L, Johnson MTJ, Wright SI, Pellizzer T, Ulbricht-Jones ES, Zupok A, Sobanski J, Bock R, Greiner S
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(2017) GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Research 45: W6-W11
Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, Greiner S
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(2019) GB2sequin - a file converter preparing custom GenBank files for database submission. Genomics 111: 759-761
Lehwark P, Greiner S
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(2021) Correction of frameshift mutations in the atpB gene by ribosomal frameshifting in chloroplasts of Oenothera and tobacco. The Plant Cell
Malinova I, Zupok A, Massouh A, Schöttler MA, Meyer EH, Yaneva-Roder L, Szymanski W, Rößner M, Ruf S, Bock R, Greiner S
