Acetylation of conserved lysines fine-tunes mitochondrial malate dehydrogenase activity in land plants.

Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a post-translational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of mitochondrial malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 strongly activated it. Comparative homology modeling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.

PsbS contributes to photoprotection in Chlamydomonas reinhardtii independently of energy dissipation.

Photosynthetic organisms are frequently exposed to excess light conditions and hence to photo-oxidative stress. To counteract photo-oxidative damage, land plants and most algae make use of non- photochemical quenching (NPQ) of excess light energy, in particular the rapidly inducible and relaxing qE-mechanism. In vascular plants, the constitutively active PsbS protein is the key regulator of qE. In the green algae C. reinhardtii, however, qE activation is only possible after initial high-light (HL) acclimation for several hours and requires the synthesis of LHCSR proteins which act as qE regulators. The precise function of PsbS, which is transiently expressed during HL acclimation in C. reinhardtii, is still unclear. Here, we investigated the impact of different PsbS amounts on HL acclimation characteristics of C. reinhardtii cells. We demonstrate that lower PsbS amounts negatively affect HL acclimation at different levels, including NPQ capacity, electron transport characteristics, antenna organization and morphological changes, resulting in an overall increased HL sensitivity and lower vitality of cells. Contrarily, higher PsbS amounts do not result in a higher NPQ capacity, but nevertheless provide higher fitness and tolerance towards HL stress. Strikingly, constitutively expressed PsbS protein was found to be degraded during HL acclimation. We propose that PsbS is transiently required during HL acclimation for the reorganization of thylakoid membranes and/or antenna proteins along with the activation of NPQ and adjustment of electron transfer characteristics, and that degradation of PsbS is essential in the fully HL acclimated state.

Posttranslational Modification of the NADP-Malic Enzyme Involved in C4 Photosynthesis Modulates the Enzymatic Activity during the Day.

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize (Zea mays) C4-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that Ser419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, probably to avoid CO2 leakiness from bundle sheath cells.

Molecular adaptations of NADP-malic enzyme for its function in C4 photosynthesis in grasses.

In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism.