Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), 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.     

Synthetic Lethality in the Tobacco Plastid Ribosome and Its Rescue at Elevated Growth Temperatures

Consistent with their origin from cyanobacteria, plastids (chloroplasts) perform protein biosynthesis on bacterial-type 70S ribosomes. The plastid genomes of seed plants contain a conserved set of ribosomal protein genes. Three of these have proven to be nonessential for translation and, thus, for cellular viability: rps15, rpl33, and rpl36. To help define the minimum ribosome, here, we examined whether more than one of these nonessential plastid ribosomal proteins can be removed from the 70S ribosome. To that end, we constructed all possible double knockouts for the S15, L33, and L36 ribosomal proteins by stable transformation of the tobacco (Nicotiana tabacum) plastid genome. We find that, although S15 and L33 function in different ribosomal particles (30S and 50S, respectively), their combined deletion from the plastid genome results in synthetic lethality under autotrophic conditions. Interestingly, the lethality can be overcome by growth under elevated temperatures due to an improved efficiency of plastid ribosome biogenesis. Our results reveal functional interactions between protein and RNA components of the 70S ribosome and uncover the interdependence of the biogenesis of the two ribosomal subunits. In addition, our findings suggest that defining a minimal set of plastid genes may prove more complex than generally believed.     

Comparative genomics of figworts (Scrophularia,Scrophulariaceae), with implications for the evolution of Scrophularia and Lamiales

The figwort genus Scrophularia L. (Scrophulariaceae) comprises 200–300 species and is widespread throughout the temperate Northern Hemisphere. Due to reticulate evolution resulting from hybridization and polyploidization, the taxonomy and phylogeny of Scrophularia is notoriously challenging. Here we report the complete chloroplast (cp) genome sequences of S. henryi Hemsl. and S. dentata Royle ex Benth. and compare them with those of S. takesimensis Nakai and S. buergeriana Miq. The Scrophularia cp genomes ranged from 152 425 to 153 631 bp in length. Each cp genome contained 113 unigenes, consisting of 78 protein‐coding genes, 31 transfer RNA genes, and 4 ribosomal RNA genes. Gene order, gene content, AT content and IR/SC boundary structure were nearly identical among them. Nine cpDNA markers (trnH‐psbA, rps15, rps18‐rpl20, rpl32‐trnL, trnS‐trnG, ycf15‐trnL, rps4‐trnT, ndhF‐rpl32, and rps16‐trnQ) with more than 2% variable sites were identified. Our phylogenetic analyses including 55 genera from Lamiales strongly supported a sister relationship between ((Bignoniaceae + Verbenaceae) + Pedaliaceae) and (Acanthaceae + Lentibulariaceae). Within Scrophulariaceae, a topology of (S. dentata+ (S. takesimensis+ (S. buergeriana+S. henryi))) was strongly supported. The crown age of Lamiales was estimated to be 85.1 Ma (95% highest posterior density, 70.6–99.8 Ma). The higher core Lamiales originated at 65.6 Ma (95% highest posterior density, 51.4–79.4 Ma), with a subsequent radiation that occurred in the Paleocene (between 55.4 and 62.3 Ma) and gave birth to the diversified families. Our study provides a robust phylogeny and a temporal framework for further investigation of the evolution of Lamiales.