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Prediction of protein localization plays an important role in understanding protein function and mechanisms. In this paper, we propose a general deep learning-based localization prediction framework, MULocDeep, which can predict multiple localizations of a protein at both subcellular and suborganellar levels. We collected a dataset with 44 suborganellar localization annotations in 10 major subcellular compartments—the most comprehensive suborganelle localization dataset to date. We also experimentally generated an independent dataset of mitochondrial proteins in Arabidopsis thaliana cell cultures, Solanum tuberosum tubers, and Vicia faba roots and made this dataset publicly available. Evaluations using the above datasets show that overall, MULocDeep outperforms other major methods at both subcellular and suborganellar levels. Furthermore, MULocDeep assesses each amino acid’s contribution to localization, which provides insights into the mechanism of protein sorting and localization motifs. A web server can be accessed at http://mu-loc.org.

The Genomes Uncoupled 4 (GUN4) is one of the retrograde signaling genes in Arabidopsis and its orthologs have been identified in oxygenic phototrophic organisms from cyanobacterium to higher plants. GUN4 is involved in tetrapyrrole biosynthesis and its mutation often causes chlorophyll-deficient phenotypes with increased levels of reactive oxygen species (ROS), hence it has been speculated that GUN4 may also play a role in photoprotection. However, the biological mechanism leading to the increased ROS accumulation in gun4 mutants remains largely unknown. In our previous studies, we generated an epi-mutant allele of OsGUN4 (gun4epi), which downregulated its expression to ∼0.5% that of its wild-type (WT), and a complete knockout allele gun4-1 due to abolishment of its translation start site. In the present study, three types of F2 plant derived from a gun4-1/gun4epi cross, i.e., gun4-1/gun4-1, gun4-1/gun4epi and gun4epi/gun4epi were developed and used for further investigation by growing them under photoperiodic condition (16 h/8 h light/dark) with low light (LL, 100 μmol photons m–2 s–1) or high light (HL, 1000 μmol photons m–2 s–1). The expression of OsGUN4 was light responsive and had two peaks in the daytime. gun4-1/gun4-1-F2 seeds showed defective germination and died within 7 days. Significantly higher levels of ROS accumulated in all types of OsGUN4 mutants than in WT plants under both the LL and HL conditions. A comparative RNA-seq analysis of WT variety LTB and its gun4epi mutant HYB led to the identification of eight peroxidase (PRX)-encoding genes that were significantly downregulated in HYB. The transcription of these eight PRX genes was restored in transgenic HYB protoplasts overexpressing OsGUN4, while their expression was repressed in LTB protoplasts transformed with an OsGUN4 silencing vector. We conclude that OsGUN4 is indispensable for rice, its expression is light- and oxidative-stress responsive, and it plays a role in ROS accumulation via its involvement in the transcriptional regulation of PRX genes.

Abstract Background Sample preparation is a critical process for proteomic studies. Many efficient and reproducible sample preparation methods have been developed for mass spectrometry-based proteomic analysis of human and animal tissues or cells, but no attempt has been made to evaluate these protocols for plants. We here present an LC–MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC–MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation. Results The proteomics workflow identified and quantified up to 1800 barley proteins based on sequencing of up to 6900 peptides per sample. The two spin filter-based protocols provided a 12–38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labelling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent. Conclusions We provide detailed protocols for efficient plant protein sample preparation for LC–MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of leaf samples for MS-based proteomics. However, a simple protocol provides comparable results although with different peptide digestion profile.

Abstract Targeted chemical modification of peptides and proteins by laser pulses in a biologically relevant environment, i.e. aqueous solvent at room temperature, allows for accurate control of biological processes. However, the traditional laser methods of control of chemical reactions are applicable only to a small class of photosensitive biomolecules because of strong and ultrafast perturbations from biomolecule-solvent interactions. Here, we report excitation of harmonics of vibration modes of solvent molecules by femtosecond laser pulses to produce controlled chemical modifications of non-photosensitive peptides and proteins in polar liquids under room conditions. The principal modifications included lysine formylation and methionine sulfoxidation both of which occur with nearly 100% yield under atmospheric conditions. That modification occurred only if the laser irradiance exceeded certain threshold level. The threshold, type, and extent of the modifications were completely controlled by solvent composition, laser wavelength, and peak irradiance of ultrashort laser pulses. This approach is expected to assist in establishing rigorous control over a broad class of biological processes in cells and tissues at the molecular level.

Copper ion-catalyzed oxidation of yeast SOD1 (ySOD1) was examined to determine early oxidative modifications, including oxidation of a crucial disulfide bond, and the structural and functional repercussions of these events. The study used distinct oxidative conditions: Cu2+/H2O2, Cu2+/H2O2/AscH− and Cu2+/H2O2/glucose. Capillary electrophoresis experiments and quantification of protein carbonyls indicate that ySOD1 is highly susceptible to oxidative modification and that changes can be detected within 0.1 min of the initiation of the reaction. Oxidation-induced structural perturbations, characterized by circular dichroism, revealed the formation of partially-unfolded ySOD1 species in a dose-dependent manner. Consistent with these structural changes, pyrogallol assay indicates a partial loss of enzymatic activity. ESI-MS analyses showed seven distinct oxidized ySOD1 species under mild oxidation within 0.1 min. LC/MS analysis after proteolytic digestion demonstrated that the copper-coordinating active site histidine residues, His47 and His49, were converted into 2-oxo-histidine. Furthermore, the Cu and Zn bridging residue, His64 is converted into aspartate/asparagine. Importantly, the disulfide-bond Cys58-Cys147 which is critical for the structural and functional integrity of ySOD1 was detected as being oxidized at Cys147. We propose, based on LC/MS analyses, that disulfide-bond oxidation occurs without disulfide bond cleavage. Modifications were also detected at Met85 and five surface-exposed Lys residues. Based on these data we propose that the Cys58-Cys147 bond may act as a sacrificial target for oxidants and protect ySOD1 from oxidative inactivation arising from exposure to Cu2+/H2O2 and auto-inactivation during extended enzymatic turnover. Keywords: Amyotrophic lateral sclerosis, Disulfide oxidation, Metal-ion catalyzed oxidation, Oxidative stress, Protein carbonyls, Superoxide dismutase

The xantha trait of a yellow leaf rice mutant (HYB), controlled epigenetically by elevated CHG methylation of the genomes uncoupled 4 (OsGUN4) promoter, has reduced chlorophyll content, altered tetrapyrrole biosynthesis, and deregulated transcription of photosynthesis-associated nuclear genes (PhANGs) compared to its wild-type progenitor Longtefu B (LTB). In the present study, we identified a suppressor mutant (CYB) of HYB and characterized its genetic, molecular, and physiological basis of the mutant phenotype. We found that the light-green phenotype of CYB was caused by a suppressor mutation in an unknown gene other than OsGUN4. Compared to HYB, the CHG methylation in the OsGUN4 promoter was reduced, while OsGUN4 transcript and protein abundance levels were increased in CYB. The contents of total chlorophyll and its intermediate metabolites (except protoporphyrin IX) in CYB plants were intermediate between HYB and LTB. The expression levels of 30 genes involved in tetrapyrrole biosynthesis in CYB were all partially reverted to those of LTB, so were the PhANGs. In summary, a suppressor mutation caused the reversion of the xantha trait via reducing CHG methylation in OsGUN4 promoter.

To function as a metabolic hub, plant mitochondria have to exchange a wide variety of metabolic intermediates as well as inorganic ions with the cytosol. As identified by proteomic profiling or as predicted by MU-LOC, a newly developed bioinformatics tool, Arabidopsis thaliana mitochondria contain 128 or 143 different transporters, respectively. The largest group is the mitochondrial carrier family, which consists of symporters and antiporters catalyzing secondary active transport of organic acids, amino acids, and nucleotides across the inner mitochondrial membrane. An impressive 97% (58 out of 60) of all the known mitochondrial carrier family members in Arabidopsis have been experimentally identified in isolated mitochondria. In addition to many other secondary transporters, Arabidopsis mitochondria contain the ATP synthase transporters, the mitochondria protein translocase complexes (responsible for protein uptake across the outer and inner membrane), ATP-binding cassette (ABC) transporters, and a number of transporters and channels responsible for allowing water and inorganic ions to move across the inner membrane driven by their transmembrane electrochemical gradient. A few mitochondrial transporters are tissue-specific, development-specific, or stress-response specific, but this is a relatively unexplored area in proteomics that merits much more attention.

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

Seed vigor is a complex property that determines the seed's potential for rapid uniform emergence and subsequent growth. However, the mechanism for change in seed vigor is poorly understood. The seeds of poplar (Populus × Canadensis Moench), which are short-lived, were stored at 30 °C and 75 ± 5% relative humidity for different periods of time (0-90 days) to obtain different vigor seeds (from 95 to 0% germination). With decreasing seed vigor, the temperature range of seed germination became narrower; the respiration rate of the seeds decreased markedly, while the relative electrolyte leakage increased markedly, both levelling off after 45 days. A total of 81 protein spots showed a significant change in abundance (≥ 1.5-fold, P < 0.05) when comparing the proteomes among seeds with different vigor. Of the identified 65 proteins, most belonged to the groups involved in metabolism (23%), protein synthesis and destination (22%), energy (18%), cell defense and rescue (17%), and storage protein (15%). These proteins accounted for 95% of all the identified proteins. During seed aging, 53 and 6 identified proteins consistently increased and decreased in abundance, respectively, and they were associated with metabolism (22%), protein synthesis and destination (22%), energy (19%), cell defense and rescue (19%), storage proteins (15%), and cell growth and structure (3%). These data show that the decrease in seed vigor (aging) is an energy-dependent process, which requires protein synthesis and degradation as well as cellular defense and rescue.

Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(P)H) and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified.

Amplitude- and frequency-modulated waves of Ca2+ ions transmit information inside cells. Reactive Oxygen Species, specifically hydrogen peroxide, have been proposed a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view of the physical and biochemical conditions in plant cells. As model system, we use a H2O2 signal originating at the plasma membrane and spreading through the cytosol.We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits on such signals as regards signal origin, frequency, amplitude, and distance. Then we establish the impact of ROS-removing enzymes on the ability of H2O2 to transmit signals. Finally, we consider to what extent cytoplasmic streaming distorts signals. This modelling allows us to predict the conditions under which diffusion-mediated signalling is possible.We show that purely diffusive transmission of intracellular information by H2O2 over a distance of 1 µm (typical distance between organelles, which may function as relay stations) is possible at frequencies well above 1 Hz, which is the highest frequency observed experimentally. This allows both frequency and amplitude modulation of the signal. Signalling over a distance of 10 µm (typical distance between the plasma membrane and the nucleus) may be possible, but requires high signal amplitudes or, equivalently, a very low detection threshold. Furthermore, at this longer distance a high level of enzymatic degradation is required make signalling at frequencies above 0.1 Hz possible. In either case, cytoplasmic streaming does not seriously disturb signals. We conclude that although purely diffusion-mediated signalling without relaying stations is theoretically possible, it is unlikely to work in practice, since it requires a much faster enzymatic degradation and a much lower cellular background concentration of H2O2 than observed experimentally.

Mitochondria function as hubs of plant metabolism. Oxidative phosphorylation produces ATP, but it is also a central high-capacity electron sink required by many metabolic pathways that must be flexibly coordinated and integrated. Here, we review the crucial roles of redox-associated posttranslational protein modifications (PTMs) in mitochondrial metabolic regulation. We discuss several major concepts. First, the major redox couples in the mitochondrial matrix (NAD, NADP, thioredoxin, glutathione, and ascorbate) are in kinetic steady state rather than thermodynamic equilibrium. Second, targeted proteomics have produced long lists of proteins potentially regulated by Cys oxidation/thioredoxin, Met-SO formation, phosphorylation, or Lys acetylation, but we currently only understand the functional importance of a few of these PTMs. Some site modifications may represent molecular noise caused by spurious reactions. Third, different PTMs on the same protein or on different proteins in the same metabolic pathway can interact to fine-tune metabolic regulation. Fourth, PTMs take part in the repair of stress-induced damage (e.g. by reducing Met and Cys oxidation products) as well as adjusting metabolic functions in response to environmental variation, such as changes in light irradiance or oxygen availability. Finally, PTMs form a multidimensional regulatory system that provides the speed and flexibility needed for mitochondrial coordination far beyond that provided by changes in nuclear gene expression alone. [ABSTRACT FROM AUTHOR]

Seed ageing is a process that results in a delayed germination, a decreased germination percentage, and finally a total loss of seed viability. However, the mechanism of seed ageing is poorly understood. In the present study, Yliangyou 2 hybrid rice (Oryza sativa L.) seeds were artificially aged at 100% relative humidity and 40°C, and the effect of artificial ageing on germination, germination time course and the change in protein profiles of embryo and endosperm was studied to understand the molecular mechanism behind seed ageing. With an increasing duration of artificial ageing, the germination percentage and germination rate of hybrid rice seeds decreased. By comparing the protein profiles from the seeds aged for 0, 10 and 25 d, a total of 91 and 100 protein spots were found to show a significant change of more than 2-fold (P

Mutations in the gene encoding the enzyme tafazzin, TAZ, cause Barth syndrome (BTHS). Individuals with this X-linked multisystem disorder present cardiomyopathy (often dilated), skeletal muscle weakness, neutropenia, growth retardation and 3-methylglutaconic aciduria. Biopsies of the heart, liver and skeletal muscle of patients have revealed mitochondrial malformations and dysfunctions. It is the purpose of this review to summarize recent results of studies on various animal or cell models of Barth syndrome, which have characterized biochemically the strong cellular defects associated with TAZ mutations. Tafazzin is a mitochondrial phospholipid-lysophospholipid transacylase that shuttles acyl groups between phospholipids and regulates the remodeling of cardiolipin (CL), a unique inner mitochondrial membrane phospholipid dimer consisting of two phosphatidyl residues linked by a glycerol bridge. After their biosynthesis, the acyl chains of CLs may be modified in remodeling processes involving up to three different enzymes. Their characteristic acyl chain composition depends on the function of tafazzin, although the enzyme itself surprisingly lacks acyl specificity. CLs are crucial for correct mitochondrial structure and function. In addition to their function in the basic mitochondrial function of ATP production, CLs play essential roles in cardiac function, apoptosis, autophagy, cell cycle regulation and Fe-S cluster biosynthesis. Recent developments in tafazzin research have provided strong insights into the link between mitochondrial dysfunction and the production of reactive oxygen species (ROS). An important tool has been the generation of BTHS-specific induced pluripotent stem cells (iPSCs) from BTHS patients. In a complementary approach, disease-specific mutations have been introduced into wild-type iPSC lines enabling direct comparison with isogenic controls. iPSC-derived cardiomyocytes were then characterized using biochemical and classical bioenergetic approaches. The cells are tested in a ‘‘heart-on-chip’’ assay to model the pathophysiology in vitro, to characterize the underlying mechanism of BTHS deriving from TAZ mutations, mitochondrial deficiencies and ROS production and leading to tissue defects, and to evaluate potential therapies with the use of mitochondrially targeted antioxidants.