Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1.

Plants possessing dysfunctional plastids due to defects in pigment biosynthesis or translation are known to repress photosynthesis-associated nuclear genes via retrograde signals from the disturbed organelles toward the nucleus. These signals are thought to be essential for proper biogenesis and function of the plastid. Mutants lacking plastid-encoded RNA polymerase-associated proteins (PAPs) display a genetic arrest in eoplast-chloroplast transition leading to an albino phenotype in the light. Retrograde signaling in these mutants, therefore, could be expected to be similar as under conditions inducing plastid dysfunction. To answer this question, we performed plastome- and genomewide array analyses in the pap7-1 mutant of Arabidopsis (Arabidopsis thaliana). In parallel, we determined the potential overlap with light-regulated expression networks. To this end, we performed a comparative expression profiling approach using light- and dark-grown wild-type plants as relative control for the expression profiles obtained from light-grown pap7-1 mutants. Our data indicate a specific impact of retrograde signals on metabolism-related genes in pap7-1 mutants reflecting the starvation situation of the albino seedlings. In contrast, light regulation of PhANGs and other nuclear gene groups appears to be fully functional in this mutant, indicating that a block in chloroplast biogenesis per se does not repress expression of them as suggested by earlier studies. Only genes for light harvesting complex proteins displayed a significant repression indicating an exclusive retrograde impact on this gene family. Our results indicate that chloroplasts and arrested plastids each emit specific signals that control different target gene modules both in positive and negative manner.

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.

Dual Targeting of the Protein Methyltransferase PrmA Contributes to Both Chloroplastic and Mitochondrial Ribosomal Protein L11 Methylation in Arabidopsis.

Methylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function.

Uncovering the protein lysine and arginine methylation network in Arabidopsis chloroplasts.

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the ß-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.

Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants.

In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.