Abstract
Mutations that eliminate chloroplast translation in Arabidopsis (Arabidopsis thaliana) result in embryo lethality. The stage of embryo arrest, however, can be influenced by genetic background. To identify genes responsible for improved growth in the absence of chloroplast translation, we examined seedling responses of different Arabidopsis accessions on spectinomycin, an inhibitor of chloroplast translation, and crossed the most tolerant accessions with embryo-defective mutants disrupted in chloroplast ribosomal proteins generated in a sensitive background. The results indicate that tolerance is mediated by ACC2, a duplicated nuclear gene that targets homomeric acetyl-coenzyme A carboxylase to plastids, where the multidomain protein can participate in fatty acid biosynthesis. In the presence of functional ACC2, tolerance is enhanced by a second locus that maps to chromosome 5 and heightened by additional genetic modifiers present in the most tolerant accessions. Notably, some of the most sensitive accessions contain nonsense mutations in ACC2, including the "Nossen" line used to generate several of the mutants studied here. Functional ACC2 protein is therefore not required for survival in natural environments, where heteromeric acetyl-coenzyme A carboxylase encoded in part by the chloroplast genome can function instead. This work highlights an interesting example of a tandem gene duplication in Arabidopsis, helps to explain the range of embryo phenotypes found in Arabidopsis mutants disrupted in essential chloroplast functions, addresses the nature of essential proteins encoded by the chloroplast genome, and underscores the value of using natural variation to study the relationship between chloroplast translation, plant metabolism, protein import, and plant development.
Highlights
Accession Embryo PhenotypeEmbryo Sizeb emb3126-1 L1 emb3126-3 emb3137-1 S13 emb3137-2 RIKEN JICc RIKENSalk “Nossen” Ler “Nossen” Columbia mm Preglobular
Different results were obtained with tobacco and Arabidopsis seedlings, which were much more sensitive to spectinomycin. In light of this reported variation in seedling responses to spectinomycin and the known duplication of ACC1 in the Brassicaceae, we decided to explore whether natural accessions of Arabidopsis differed in their ability to tolerate a loss of chloroplast translation and whether genetic analysis in Arabidopsis could uncover some of the genes involved
To rule out the possibility that accession-specific differences were unique to spectinomycin, we evaluated the effect of another inhibitor of chloroplast translation with a different mode of action on tolerant and sensitive accessions
Summary
Several factors were considered before deciding which accessions to evaluate on spectinomycin: geographical location, genetic diversity (McKhann et al, 2004; Nordborg et al, 2005; Clark et al, 2007), inclusion among mutants defective in chloroplast translation (Bryant et al, 2011), availability of genomic sequence data (Weigel and Mott, 2009), and flowering time. We pursued a second, complementary approach that involved crosses between tolerant accessions and embryo-defective mutants disrupted in chloroplast ribosomal proteins in a sensitive background This approach led to the identification of distinct genetic loci that impacted the extent of embryo development in the absence of chloroplast translation and later to the demonstration that variation in these same genes contributed to the tolerance of seedlings from different accessions on spectinomycin. Plants with more advanced embryos should be included among the late class if additional modifiers are present Using this approach, we identified a single dominant suppressor in the Tsu-0 accession that significantly increases the size of mutant seeds and supports embryo development to a globular stage. To determine whether the suppressor impacts both embryo development in the absence of chloroplast translation and seedling response to spectinomycin, we PCR genotyped sensitive and tolerant F2 seedlings from Tsu-0 3 “Nossen” populations using the ACC2linked markers noted above. To determine whether the suppressor found in the Tsu-0 accession is present in other tolerant accessions, we genotyped F2 seedlings on spectinomycin derived from a “Nossen” cross with the tolerant Be-1 accession
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