The interplay of post-translational protein modifications in Arabidopsis leaves during photosynthesis induction.

Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM) and have been studied at depth at the level of individual proteins. In contrast, a global picture of the light-dependent PTMome dynamics is lacking, leaving the response of a large proportion of cellular function undefined. Here, we investigated the light-dependent metabolome and proteome changes in Arabidopsis rosettes in a time resolved manner to dissect their kinetic interplay, focusing on phosphorylation, lysine acetylation, and cysteine-based redox switches. Of over 24 000 PTM sites that were detected, more than 1700 were changed during the transition from dark to light. While the first changes, as measured 5 min after onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveal connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insight into the extent by which photosynthesis reprograms global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.

Mass Spectrometry-Based Quantitative Cysteine Redox Proteome Profiling of Isolated Mitochondria Using Differential iodoTMT Labeling.

Mitochondria are central hubs of redox biochemistry in the cell. An important role of mitochondrial carbon metabolism is to oxidize respiratory substrates and to pass the electrons down the mitochondrial electron transport chain to reduce oxygen and to drive oxidative phosphorylation. During respiration, reactive oxygen species are produced as a side reaction, some of which in turn oxidize cysteine thiols in proteins. Hence, the redox status of cysteine-containing mitochondrial proteins has to be controlled by the mitochondrial glutathione and thioredoxin systems, which draw electrons from metabolically derived NADPH. The redox status of mitochondrial cysteines can undergo fast transitions depending on the metabolic status of the cell, as for instance at early seed germination. Here, we describe a state-of-the-art method to quantify redox state of protein cysteines in isolated Arabidopsis seedling mitochondria of controlled metabolic and respiratory state by MS2-based redox proteomics using the isobaric thiol labeling reagent Iodoacetyl Tandem Mass Tag™ (iodoTMT). The procedure is also applicable to isolated mitochondria of other plant and nonplant systems.

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.

The functionality of plant mechanoproteins (forisomes) is dependent on the dual role of conserved cysteine residues.

Forisomes are giant polyprotein complexes that undergo reversible conformational rearrangements from a spindle-like to a plug-like state in response to Ca2+ or changes in pH. They act as valves in the plant vasculature, and reproduce this function in vitro to regulate flow in microfluidic capillaries controlled by electro-titration. Heterologous expression in yeast or plants allows the large-scale production of tailor-made artificial forisomes for technical applications. Here we investigated the unexpected disintegration of artificial forisomes in response to Ca2+ following the deletion of the M1 motif in the MtSEO-F1 protein or the replacement of all four conserved cysteine residues therein. This phenomenon could be mimicked in wild-type forisomes under reducing conditions by adding a thiol alkylating agent. We propose a model in which reversible changes in forisome structure depend on cysteine residues with ambiguous redox states, allowing the formation of intermolecular disulfide bridges (confirmed by mass spectrometry) as well as noncovalent thiol interactions to connect forisome substructures in the dispersed state. This is facilitated by the projection of the M1 motif from the MtSEO-F1 protein as part of an extended loop. Our findings support the rational engineering of disintegrating forisomes to control the release of peptides or enzymes in microfluidic systems.

Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis.

Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways. It is known that several plant mitochondrial proteins are phosphorylated in a reversible manner. However, the identities of the protein kinases/phosphatases involved in this mechanism and their roles in the regulation of the tricarboxylic acid (TCA) cycle remain unclear. In this study, we isolated and characterized plants lacking two mitochondrially targeted phosphatases (Sal2 and PP2c63) along with pyruvate dehydrogenase kinase (PDK). Protein-protein interaction analysis, quantitative phosphoproteomics, and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase complex (PDC), while PP2c63 nonspecifically regulates PDC. When recombinant PP2c63 and Sal2 proteins were added to mitochondria isolated from mutant plants, protein-protein interaction and enzymatic analyses showed that PP2c63 directly phosphorylates and modulates the activity of PDC, while Sal2 only indirectly affects TCA cycle enzymes. Characterization of steady-state metabolite levels and fluxes in the mutant lines further revealed that these phosphatases regulate flux through the TCA cycle, and that altered metabolism in the sal2 pp2c63 double mutant compromises plant growth. These results are discussed in the context of current models of the control of respiration in plants.

High-Resolution Lysine Acetylome Profiling by Offline Fractionation and Immunoprecipitation.

Acetylation of lysine side chains at their ε-amino group is a reversible posttranslational modification (PTM), which can affect diverse protein functions. Lysine acetylation was first described on histones, and nowadays gains more and more attention due to its more general occurrence in proteomes, and its possible crosstalk with other protein modifications. Here we describe a workflow to investigate the acetylation of lysine-containing peptides on a large scale. For this high-resolution lysine acetylome analysis, dimethyl-labeled peptide samples are pooled and offline-fractionated using hydrophilic interaction liquid chromatography (HILIC). The offline fractionation is followed by an immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for data acquisition and subsequent data analysis.

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics.

Mitochondria host vital cellular functions, including oxidative phosphorylation and co-factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity-based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage-Dependent Anion Channel 1 to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.

In vivo evidence for a regulatory role of phosphorylation of Arabidopsis Rubisco activase at the Thr78 site.

Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine-78 (Thr78) in low light and in the dark, suggesting a potential regulatory role in photosynthesis, but this has not been directly tested. To do so, we transformed an rca-knockdown mutant largely lacking redox regulation with wild-type Rca-ß or Rca-ß with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site-directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-ß wild-type control, as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II (ϕPSII) and reduced growth relative to control plants expressing wild-type Rca-ß under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-ß with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ϕPSII relative to Rca-ß controls. While expression of the wild-type Rca-ß or the T78A mutant fully rescued the slow-growth phenotype of the rca-knockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-ß control plants at low light (30 µmol photons m-2 s-1) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei.

The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.

T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis.

MAIN CONCLUSION: T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis and therefore, exerts little control over the photorespiratory pathway. T-protein is the aminomethyltransferase of the glycine cleavage multienzyme system (GCS), also known as the glycine decarboxylase complex, and essential for photorespiration and one-carbon metabolism. Here, we studied what effects varying levels of the GCS T-protein would have on GCS activity, the operation of the photorespiratory pathway, photosynthesis, and plant growth. To this end, we examined Arabidopsis thaliana T-protein overexpression lines with up to threefold higher amounts of leaf T-protein as well as one knockdown mutant with about 5% residual leaf T-protein and one knockout mutant. Overexpression did not alter photosynthetic CO2 uptake and plant growth, and the knockout mutation was lethal even in the non-photorespiratory environment of air enriched to 1% CO2. Unexpectedly in light of this very low T-protein content, however, the knockdown mutant was able to grow and propagate in normal air and displayed only some minor changes, such as a moderate glycine accumulation in combination with somewhat delayed growth. Neither overexpression nor the knockdown of T-protein altered the amounts of the other three GCS proteins, suggesting that the biosynthesis of the GCS proteins is not synchronized at this level. We also observed that the knockdown causes less T-protein mostly in leaf mesophyll cells, but not so much in the vasculature, and discuss this phenomenon in light of the dual involvement of the GCS and hence T-protein in plant metabolism. Collectively, this work shows that T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis and therefore exerts little control over the photorespiratory pathway.