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.

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

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.

Comparative proteome and metabolome analyses of latex-exuding and non-exuding Taraxacum koksaghyz roots provide insights into laticifer biology.

Taraxacum koksaghyz has been identified as one of the most promising alternative rubber crops. Its high-quality rubber is produced in the latex of laticifers, a specialized cell type that is organized in a network of elongated tubules throughout the entire plant body. In order to gain insights into the physiological role(s) of latex and hence laticifer biology, we examine the effects of barnase-induced latex RNA degradation on the metabolite and protein compositions in the roots. We established high-quality datasets that enabled precise discrimination between cellular and physiological processes in laticifers and non-laticifer cell types of roots at different vegetative stages. We identified numerous latex-specific proteins, including a perilipin-like protein that has not been studied in plants yet. The barnase-expressing plants revealed a phenotype that did not exude latex, which may provide a valuable genetic basis for future studies of plant-environment interactions concerning latex and also help to clarify the evolution and arbitrary distribution of latex throughout the plant kingdom. The overview of temporal changes in composition and protein abundance provided by our data opens the way for a deeper understanding of the molecular interactions, reactions, and network relationships that underlie the different metabolic pathways in the roots of this potential rubber crop.

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.

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz - role of cis-prenyltransferase-like 1 protein.

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high-molecular-weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis-1,4-isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis-prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT-like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis-1,4-isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1-RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low-expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS-based comparison of latex proteomes from TkCPTL1-RNAi plants and T. koksaghyz wild-types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.

Chloroplast Acetyltransferase NSI Is Required for State Transitions in Arabidopsis thaliana.

The amount of light energy received by the photosynthetic reaction centers photosystem II (PSII) and photosystem I (PSI) is balanced through state transitions. Reversible phosphorylation of a light-harvesting antenna trimer (L-LHCII) orchestrates the association between L-LHCII and the photosystems, thus adjusting the amount of excitation energy received by the reaction centers. In this study, we identified the enzyme NUCLEAR SHUTTLE INTERACTING (NSI; AT1G32070) as an active lysine acetyltransferase in the chloroplasts of Arabidopsis thaliana Intriguingly, nsi knockout mutant plants were defective in state transitions, even though they had a similar LHCII phosphorylation pattern as the wild type. Accordingly, nsi plants were not able to accumulate the PSI-LHCII state transition complex, even though the LHCII docking site of PSI and the overall amounts of photosynthetic protein complexes remained unchanged. Instead, the nsi mutants showed a decreased Lys acetylation status of specific photosynthetic proteins including PSI, PSII, and LHCII subunits. Our work demonstrates that the chloroplast acetyltransferase NSI is needed for the dynamic reorganization of thylakoid protein complexes during photosynthetic state transitions.

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.

YODA MAP3K kinase regulates plant immune responses conferring broad-spectrum disease resistance.

Mitogen-activated protein kinases (MAPKs) cascades play essential roles in plants by transducing developmental cues and environmental signals into cellular responses. Among the latter are microbe-associated molecular patterns perceived by pattern recognition receptors (PRRs), which trigger immunity. We found that YODA (YDA) - a MAPK kinase kinase regulating several Arabidopsis developmental processes, like stomatal patterning - also modulates immune responses. Resistance to pathogens is compromised in yda alleles, whereas plants expressing the constitutively active YDA (CA-YDA) protein show broad-spectrum resistance to fungi, bacteria, and oomycetes with different colonization modes. YDA functions in the same pathway as ERECTA (ER) Receptor-Like Kinase, regulating both immunity and stomatal patterning. ER-YDA-mediated immune responses act in parallel to canonical disease resistance pathways regulated by phytohormones and PRRs. CA-YDA plants exhibit altered cell-wall integrity and constitutively express defense-associated genes, including some encoding putative small secreted peptides and PRRs whose impairment resulted in enhanced susceptibility phenotypes. CA-YDA plants show strong reprogramming of their phosphoproteome, which contains protein targets distinct from described MAPKs substrates. Our results suggest that, in addition to stomata development, the ER-YDA pathway regulates an immune surveillance system conferring broad-spectrum disease resistance that is distinct from the canonical pathways mediated by described PRRs and defense hormones.

Dimethyl-Labeling-Based Quantification of the Lysine Acetylome and Proteome of Plants.

Photorespiratory enzymes in different cellular compartments have been reported to be posttranslational modified by phosphorylation, disulfide formation, S-nitrosylation, glutathionylation, and lysine acetylation. However, not much is known yet about the function of these modifications to regulate the activities, localizations, or interactions of the proteins in this metabolic pathway. Hence, it will be of great importance to study these modifications and their temporal and conditional occurrence in more detail. Here, we focus on the analysis of lysine acetylation as a relatively newly discovered modification on plant metabolic enzymes. The acetylation of lysine residues within proteins is a highly conserved and reversible posttranslational modification which occurs in all living organisms. First discovered on histones and implied in the regulation of gene expression, lysine acetylation also occurs on a diverse set of cellular proteins in different subcellular compartments and is particularly abundant on metabolic enzymes. Upon lysine acetylation, the function of proteins can be modulated due to the loss of the positive charge of the lysine residue. Lysine acetylation was also discovered on proteins involved in photosynthesis and novel tools are needed to study the regulation of this modification in dependence on the environmental conditions, tissues, or plant genotype. This chapter describes a method for the identification and relative quantification of lysine-acetylated proteins in plant tissues using a dimethyl labeling technique combined with an anti-acetyl lysine antibody enrichment strategy. Here, we describe the protein purification, labeling of trypsinated peptides, as well as immuno-enrichment of lysine-acetylated peptides followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) data acquisition and analysis.

Lysine acetylation in mitochondria: From inventory to function.

Cellular signaling pathways are regulated in a highly dynamic fashion in order to quickly adapt to distinct environmental conditions. Acetylation of lysine residues represents a central process that orchestrates cellular metabolism and signaling. In mitochondria, acetylation seems to be the most prevalent post-translational modification, presumably linked to the compartmentation and high turnover of acetyl-CoA in this organelle. Similarly, the elevated pH and the higher concentration of metabolites in mitochondria seem to favor non-enzymatic lysine modifications, as well as other acylations. Hence, elucidating the mechanisms for metabolic control of protein acetylation is crucial for our understanding of cellular processes. Recent advances in mass spectrometry-based proteomics have considerably increased our knowledge of the regulatory scope of acetylation. Here, we review the current knowledge and functional impact of mitochondrial protein acetylation across species. We first cover the experimental approaches to identify and analyze lysine acetylation on a global scale, we then explore both commonalities and specific differences of plant and animal acetylomes and the evolutionary conservation of protein acetylation, as well as its particular impact on metabolism and diseases. Important future directions and technical challenges are discussed, and it is pointed out that the transfer of knowledge between species and diseases, both in technology and biology, is of particular importance for further advancements in this field.

Phosphoprotein Enrichment Combined with Phosphopeptide Enrichment to Identify Putative Phosphoproteins During Defense Response in Arabidopsis thaliana.

Phosphoprotein/peptide enrichment is an important technique to elucidate signaling components of defense responses with mass spectrometry. Normally, proteins can be detected easily by shotgun experiments but the low abundance of phosphoproteins hinders their detection. Here, we describe a combination of prefractionation with desalting, phosphoprotein and phosphopeptide enrichment to effectively accumulate phosphorylated proteins from leaf tissue of stressed Arabidopsis plants.

Cellular reprogramming through mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the "PEN" pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).

Targeted proteomics analysis of protein degradation in plant signaling on an LTQ-Orbitrap mass spectrometer.

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 µg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor.

Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants.

Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events.

A protein phosphatase 2C, responsive to the bacterial effector AvrRpm1 but not to the AvrB effector, regulates defense responses in Arabidopsis.

Using a proteomics approach, a PP2C-type phosphatase (renamed PIA1, for PP2C induced by AvrRpm1) was identified that accumulates following infection by Pseudomonas syringae expressing the type III effector AvrRpm1, and subsequent activation of the corresponding plant NB-LRR disease resistance protein RPM1. No accumulation of PIA1 protein was seen following infection with P. syringae expressing AvrB, another type III effector that also activates RPM1, although PIA transcripts were observed. Accordingly, mutation of PIA1 resulted in enhanced RPM1 function in response to P. syringae pathover tomato (Pto) DC3000 (avrRpm1) but not to Pto DC3000 (avrB). Thus, PIA1 is a protein marker that distinguishes AvrRpm1- and AvrB-dependent activation of RPM1. AvrRpm1-induced expression of the pathogenesis-related genes PR1, PR2 and PR3, and salicylic acid accumulation were reduced in two pia1 mutants. By contrast, expression of other defense-related genes, including PR5 and PDF1.2 (plant defensin), was elevated in unchallenged pia1 mutants. Hence, PIA1 is required for AvrRpm1-induced responses, and confers dual (both positive and negative) regulation of defense gene expression.