Nonessential Plastid-Encoded Ribosomal Proteins in Tobacco: A Developmental Role for Plastid Translation and Implications for Reductive Genome Evolution  

Abstract

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 Drps15 plants showed only a mild phenotype, Drpl36 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.

Influence of plastids on light signalling and development

“…Dysfunctional plastids caused other developmental abnormalities that are consistent with plastid signals contributing to development, such as pale and elongated leaves, reduced apical dominance [68] and lack of aerial organ development [69]. Blocking chloroplast biogenesis with a norflurazon treatment appeared to affect leaf size in part by inhibiting the transition from cell-proliferation-based morphogenesis to cell-expansion-based morphogenesis [38].…”

Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops inChlamydomonas

“…In this respect, reverse genetic analysis in tobacco revealed that inactivation of the chloroplast ribosomal protein genes rps15 and rpl36 gave rise to homoplasmic transplastomic lines, indicating that these proteins are nonessential (Fleischmann et al, 2011). However, photosynthetic activity and growth were strongly impaired in the absence of Rpl36 and affected to a lesser extent in the absence of Rps15, indicating that translational activity is decreased in the absence of either of these two proteins.…”

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

“…The hybridization probe used for DNA gel blot analysis (derived from the psaJ coding region) is also indicated. (B) Map of the genomic region containing rps15 and map of the transformed plastid genome in Drps15 mutants (Fleischmann et al, 2011). In the Drps15 single mutant and the Drps15/Drpl36 double knockout, the spectinomycin resistance marker aadA disrupts the rps15 gene, whereas in the Drps15/Drpl33 plants, rps15 is inactivated with the kanamycin resistance gene aphA-6 (Huang et al, 2002).…”

“…Not all components of the 70S ribosome are essential for translation; in both bacteria and plastids, several ribosomal proteins have been identified as nonessential (Baba et al, 2006;Rogalski et al, 2008;Fleischmann et al, 2011). The genes for the Rps15 protein (S15) of the small ribosomal subunit and the Rpl33 (L33) and Rpl36 (L36) proteins of the large ribosomal subunit can be inactivated in E. coli (Maguire and Wild, 1997;Ikegami et al, 2005;Ikegami et al, 2005) and in plastids (Rogalski et al, 2008;Fleischmann et al, 2011) without losing viability.…”

“…The genes for the Rps15 protein (S15) of the small ribosomal subunit and the Rpl33 (L33) and Rpl36 (L36) proteins of the large ribosomal subunit can be inactivated in E. coli (Maguire and Wild, 1997;Ikegami et al, 2005;Ikegami et al, 2005) and in plastids (Rogalski et al, 2008;Fleischmann et al, 2011) without losing viability. While the rpl36 gene knockout in tobacco (Nicotiana tabacum) plastids displays a severe mutant phenotype, rpl33 and rps15 knockout plants are nearly indistinguishable from the wild type (Rogalski et al, 2008;Fleischmann et al, 2011).…”