Mitochondria in photosynthetic cells: Coordinating redox control and energy balance.

In photosynthetic tissues in the light, the function of energy production is associated primarily with chloroplasts, while mitochondrial metabolism adjusts to balance ATP supply, regulate the reduction level of pyridine nucleotides, and optimize major metabolic fluxes. The tricarboxylic acid cycle in the light transforms into a noncyclic open structure (hemicycle) maintained primarily by the influx of malate and the export of citrate to the cytosol. The exchange of malate and citrate forms the basis of feeding redox energy from the chloroplast into the cytosolic pathways. This supports the level of NADPH in different compartments, contributes to the biosynthesis of amino acids, and drives secondary metabolism via a supply of substrates for 2-oxoglutarate-dependent dioxygenase and for cytochrome P450-catalyzed monooxygenase reactions. This results in the maintenance of redox and energy balance in photosynthetic plant cells and in the formation of numerous bioactive compounds specific to any particular plant species. The noncoupled mitochondrial respiration operates in coordination with the malate and citrate valves and supports intensive fluxes of respiration and photorespiration. The metabolic system of plants has features associated with the remarkable metabolic plasticity of mitochondria that permit the use of energy accumulated during photosynthesis in a way that all anabolic and catabolic pathways become optimized and coordinated.

Matrix Redox Physiology Governs the Regulation of Plant Mitochondrial Metabolism through Posttranslational Protein Modifications.

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.

A decade of plant proteomics and mass spectrometry: translation of technical advancements to food security and safety issues.

Tremendous progress in plant proteomics driven by mass spectrometry (MS) techniques has been made since 2000 when few proteomics reports were published and plant proteomics was in its infancy. These achievements include the refinement of existing techniques and the search for new techniques to address food security, safety, and health issues. It is projected that in 2050, the world's population will reach 9-12 billion people demanding a food production increase of 34-70% (FAO, 2009) from today's food production. Provision of food in a sustainable and environmentally committed manner for such a demand without threatening natural resources, requires that agricultural production increases significantly and that postharvest handling and food manufacturing systems become more efficient requiring lower energy expenditure, a decrease in postharvest losses, less waste generation and food with longer shelf life. There is also a need to look for alternative protein sources to animal based (i.e., plant based) to be able to fulfill the increase in protein demands by 2050. Thus, plant biology has a critical role to play as a science capable of addressing such challenges. In this review, we discuss proteomics especially MS, as a platform, being utilized in plant biology research for the past 10 years having the potential to expedite the process of understanding plant biology for human benefits. The increasing application of proteomics technologies in food security, analysis, and safety is emphasized in this review. But, we are aware that no unique approach/technology is capable to address the global food issues. Proteomics-generated information/resources must be integrated and correlated with other omics-based approaches, information, and conventional programs to ensure sufficient food and resources for human development now and in the future.

Modulating protein function through reversible oxidation: Redox-mediated processes in plants revealed through proteomics.

It has been clearly demonstrated that plants redox control can be exerted over virtually every cellular metabolic pathway affecting metabolic homeostasis and energy balance. Therefore, a tight link exists between cellular/compartmental steady-state redox level and cellular metabolism. Proteomics offers a powerful new way to characterize the response and regulation of protein oxidation in different cell types and in relation to cellular metabolism. Compelling evidence revealed in proteomics studies suggests the integration of the redox network with other cellular signaling pathways such as Ca(2+) and/or protein phosphorylation, jasmonic, salicylic, abscisic acids, ethylene, and other phytohormones. Here we review progress in using the various proteomics techniques and approaches to answer biological questions arising from redox signaling and from changes in redox status of the cell. The focus is on reversible redox protein modifications and on three main processes, namely oxidative and nitrosative stress, defense against pathogens, cellular redox response and regulation, drawing on examples from plant redox proteomics studies.

Diversity of transgene integration and gene-editing events in wheat (Triticum aestivum L.) transgenic plants generated using Agrobacterium-mediated transformation.

Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype-dependency. Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) chimeric gene. Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low- and high-copy-number integration events were recovered in the T0 population with various levels of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome. A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary screening of transgene quality and GE events in the T0 population of plants regenerated through the co-delivery of GRF-GIF can allow for the propagation of the best candidates for further phenotypic analysis.

Proteomics and plant disease: advances in combating a major threat to the global food supply.

The study of plant disease and immunity is benefiting tremendously from proteomics. Parallel streams of research from model systems, from pathogens in vitro and from the relevant pathogen-crop interactions themselves have begun to reveal a model of how plants succumb to invading pathogens and how they defend themselves without the benefit of a circulating immune system. In this review, we discuss the contribution of proteomics to these advances, drawing mainly on examples from crop-fungus interactions, from Arabidopsis-bacteria interactions, from elicitor-based model systems and from pathogen studies, to highlight also the important contribution of non-crop systems to advancing crop protection.

Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control.

Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox-sensitive proteome upon wheat seed dormancy release. Using thiol-specific fluorescent labelling, solubility-based protein fractionation, 2-D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non-dormant, abscisic, or gibberellic acid-treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox-, stress-, and pathogen-responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non-dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage.

Structural and functional properties of class 1 plant hemoglobins.

Nonsymbiotic class 1 plant hemoglobins are induced under hypoxia. Structurally they are protein dimers consisting of two identical subunits, each containing heme iron in a weak hexacoordinate state. The weak hexacoordination of heme-iron binding to the distal histidine results in an extremely high avidity to oxygen, with a dissociation constant in the nanomolar range. This low dissociation constant is due to rapid oxygen binding resulting in protein conformational changes that slow dissociation from the heme site. Class 1 hemoglobins are characterized by an increased rate of Fe³(+) reduction which is likely mediated by cysteine residue. This cysteine can form a reversible covalent bond between two monomers as shown by mass spectrometry analysis and, in addition to its structural role, prevents the molecule from autoxidation. The structural properties of class 1 hemoglobins allow them to serve as soluble electron transport proteins in the enzymatic system scavenging nitric oxide produced in low oxygen via reduction of nitrite. During oxygenation of nitric oxide to nitrate, oxidized ferric hemoglobin is formed (methemoglobin), which can be reduced by an associated reductase. The identified candidate for this reduction is monodehydroascorbate reductase. It is suggested that hemoglobin functions as a terminal electron acceptor during the hypoxic turnover of nitrogen, the process aided by its extremely high affinity for oxygen.

Metabolic Changes in Seed Embryos of Hypoxia-Tolerant Rice and Hypoxia-Sensitive Barley at the Onset of Germination.

Rice (Oryza sativa L.) and barley (Hordeum vulgare L.) are the cereal species differing in tolerance to oxygen deficiency. To understand metabolic differences determining the sensitivity to low oxygen, we germinated rice and barley seeds and studied changes in the levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), activities of the enzymes involved in their scavenging, and measured cell damage parameters. The results show that alcohol dehydrogenase activity was higher in rice than in barley embryos providing efficient anaerobic fermentation. Nitric oxide (NO) levels were also higher in rice embryos indicating higher NO turnover. Both fermentation and NO turnover can explain higher ATP/ADP ratio values in rice embryos as compared to barley. Rice embryos were characterized by higher activity of S-nitrosoglutathione reductase than in barley and a higher level of free thiols in proteins. The activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase) in imbibed embryos were higher in rice than in barley, which corresponded to the reduced levels of ROS, malonic dialdehyde and electrolyte leakage. The observed differences in metabolic changes in embryos of the two cereal species differing in tolerance to hypoxia can partly explain the adaptation of rice to low oxygen environments.

The phosphoproteome of Fusarium graminearum at the onset of nitrogen starvation.

Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEFxSDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO(2) enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12 h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum.

Anoxic nitric oxide cycling in plants: participating reactions and possible mechanisms.

At sufficiently low oxygen concentrations, hemeproteins are deoxygenated and become capable of reducing nitrite to nitric oxide (NO), in a reversal of the reaction in which NO is converted to nitrate or nitrite by oxygenated hemeproteins. The maximum rates of NO production depend on the oxygen avidity. The hemeproteins with the highest avidity, such as hexacoordinate hemoglobins, retain oxygen even under anoxic conditions resulting in their being extremely effective NO scavengers but essentially incapable of producing NO. Deoxyhemeprotein-related NO production can be observed in mitochondria (at the levels of cytochrome c oxidase, cytochrome c, complex III and possibly other sites), in plasma membrane, cytosol, endoplasmic reticulum and peroxisomes. In mitochondria, the use of nitrite as an alternative electron acceptor can contribute to a limited rate of ATP synthesis. Non-heme metal-containing proteins such as nitrate reductase and xanthine oxidase can also be involved in NO production. This will result in a strong anoxic redox flux of nitrogen through the hemoglobin-NO cycle involving nitrate reductase, nitrite: NO reductase, and NO dioxygenase. In normoxic conditions, NO is produced in very low quantities, mainly for signaling purposes and this nitrogen cycling is inoperative.

Roles for Plant Mitochondrial Alternative Oxidase Under Normoxia, Hypoxia, and Reoxygenation Conditions.

Alternative oxidase (AOX) is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain (ETC) that has a lower affinity for oxygen than does cytochrome (cyt) oxidase. To investigate the role(s) of AOX under different oxygen conditions, wild-type (WT) Nicotiana tabacum plants were compared with AOX knockdown and overexpression plants under normoxia, hypoxia (near-anoxia), and during a reoxygenation period following hypoxia. Paradoxically, under all the conditions tested, the AOX amount across plant lines correlated positively with leaf energy status (ATP/ADP ratio). Under normoxia, AOX was important to maintain respiratory carbon flow, to prevent the mitochondrial generation of superoxide and nitric oxide (NO), to control lipid peroxidation and protein S-nitrosylation, and possibly to reduce the inhibition of cyt oxidase by NO. Under hypoxia, AOX was again important in preventing superoxide generation and lipid peroxidation, but now contributed positively to NO amount. This may indicate an ability of AOX to generate NO under hypoxia, similar to the nitrite reductase activity of cyt oxidase under hypoxia. Alternatively, it may indicate that AOX activity simply reduces the amount of superoxide scavenging of NO, by reducing the availability of superoxide. The amount of inactivation of mitochondrial aconitase during hypoxia was also dependent upon AOX amount, perhaps through its effects on NO amount, and this influenced carbon flow under hypoxia. Finally, AOX was particularly important in preventing nitro-oxidative stress during the reoxygenation period, thereby contributing positively to the recovery of energy status following hypoxia. Overall, the results suggest that AOX plays a beneficial role in low oxygen metabolism, despite its lower affinity for oxygen than cytochrome oxidase.

Methods for functional proteomic analyses.

The term 'Proteomics' was introduced in 1997 to describe a growing interest in the study of the proteome - the expressed protein set of an organism. As this new discipline evolved, it quickly became obvious that proteomics would be a very complex and ambitious undertaking, perhaps even more so than genomics, which had engendered it. New techniques for both the separation and analysis/identification of proteins were emerging or being refined, and these facilitated the development of this new field. Many proteomics experiments are now routine in some laboratories. In this chapter we describe a typical proteomics experiment, using examples from our laboratory: the separation of complex mixtures of proteins by 2-dimensional electrophoresis and subsequent identification of a protein spot by mass spectrometry with two commonly used instruments: MALDI-QqTOF and ESI-ion trap.

Role of organic acids in the integration of cellular redox metabolism and mediation of redox signalling in photosynthetic tissues of higher plants.

Organic acids play a crucial role in numerous metabolic processes accompanied by transfer of electrons and protons and linked to the reduction/oxidation of major redox couples in plant cells, such as NAD, NADP, glutathione, and ascorbate. Fluxes through the pathways metabolizing organic acids modulate redox states in cell compartments, contribute to generation of reactive oxygen and nitrogen species, and mediate signal transduction processes. Organic acid metabolism not only functions to equilibrate the redox potential in plant cells but also to transfer redox equivalents between cell compartments supporting various metabolic processes. The most important role in this transfer belongs to different forms of malate dehydrogenase interconverting malate and oxaloacetate or forming pyruvate (malic enzymes). During photosynthesis malate serves as a major form of transfer of redox equivalents from chloroplasts to the cytosol and other compartments via the malate valve. On the other hand, mitochondria, via alterations of their redox potential, become a source of citrate that can be transported to the cytosol and support biosynthesis of amino acids. Citrate is also an important retrograde signalling compound that regulates transcription of several genes including those encoding the alternative oxidase. The alternative oxidase, which is activated by increased redox potential and by pyruvate, is, in turn, important for the maintenance of redox potential in mitochondria. The roles of organic acids in establishing redox equilibrium, supporting ionic gradients on membranes, acidification of the extracellular medium, and regulation of production of reactive oxygen and nitrogen species are discussed.

Arabidopsis thaliana alcohol dehydrogenase is differently affected by several redox modifications.

In plant cells, many stresses, including low oxygen availability, result in a higher production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can lead to redox-dependent post-translational modification of proteins Cys residues. Here, we studied the effect of different redox modifications on alcohol dehydrogenase (ADH) from Arabidopsis thaliana. ADH catalyzes the last step of the ethanol fermentation pathway used by plants to cope with energy deficiency during hypoxic stress. Arabidopsis suspension cell cultures showed decreased ADH activity upon exposure to H2O2, but not to the thiol oxidizing agent diamide. We purified recombinant ADH and observed a significant decrease in the enzyme activity by treatments with H2O2 and diethylamine NONOate (DEA/NO). Treatments leading to the formation of a disulfide bond between ADH and glutathione (protein S-glutathionylation) had no negative effect on the enzyme activity. LC-MS/MS analysis showed that Cys47 and Cys243 could make a stable disulfide bond with glutathione, suggesting redox sensitivity of these residues. Mutation of ADH Cys47 to Ser caused an almost complete loss of the enzyme activity while the Cys243 to Ser mutant had increased specific activity. Incubation of ADH with NAD+ or NADH prevented inhibition of the enzyme by H2O2 or DEA/NO. These results suggest that binding of ADH with its cofactors may limit availability of Cys residues to redox modifications. Our study demonstrates that ADH from A. thaliana is subject to different redox modifications. Implications of ADH sensitivity to ROS and RNS during hypoxic stress conditions are discussed.

Phosphoproteomic profiling of wheat callus labelled in vivo.

Callus is an important intermediate tissue for the propagation of plants through tissue culture, as it can form new shoots, and hence plants, through somatic embryogenesis, a process whose underlying biochemical mechanisms are poorly understood. The involvement of kinases suggests that signalling through phosphorylation cascades may play an important role. In this report methods for labelling wheat (Triticum aestivum L.) callus phosphoproteins in vivo through the uptake of [(32)P]orthophosphate and subsequently revealing the phosphoproteome of this tissue by two-dimensional electrophoresis and autoradiography are described. Labelled proteins from 2D gels were identified by tandem mass spectrometry analysis using the 'Mascot' search engine, and by de novo sequencing in combination with BLAST and MS-BLAST, using a cross-species protein identification approach. Eight putative phosphoproteins from 10 spots were identified, confirming the potential utility of this method; three of these have not previously shown to be phosphorylated. Furthermore, the phosphorylation site for one of the proteins was identified. Protein synthesis functions were tentatively assigned to five of the phosphoproteins, one reactive oxygen species (ROS)-detoxifying enzyme ascorbate peroxidase, and two could not be identified reliably from current database matches.

Cytosolic Triosephosphate Isomerase from Arabidopsis thaliana Is Reversibly Modified by Glutathione on Cysteines 127 and 218.

In plant cells, an increase in cellular oxidants can have multiple effects, including the promotion of mixed disulfide bonds between glutathione and some proteins (S-glutathionylation). The present study focuses on the cytosolic isoform of the glycolytic enzyme triosephosphate isomerase (cTPI) from Arabidopsis thaliana and its reversible modification by glutathione. We used purified recombinant cTPI to demonstrate the enzyme sensitivity to inhibition by N-ethylmaleimide, hydrogen peroxide and diamide. Treatment of cTPI with diamide in the presence of reduced glutathione (GSH) led to a virtually complete inhibition of its enzymatic activity by S-glutathionylation. Recombinant cTPI was also sensitive to the oxidized form of glutathione (GSSG) in the micromolar range. Activity of cTPI was restored after reversion of S-glutathionylation by two purified recombinant A. thaliana cytosolic glutaredoxins (GRXs). GRXs-mediated deglutathionylation of cTPI was dependent on a GSH-regenerating system. Analysis of cTPI by mass spectrometry after S-glutathionylation by GSSG revealed that two Cys residues (Cys127 and Cys218) were modified by glutathione. The role of these two residues was assessed using site-directed mutagenesis. Mutation of Cys127 and Cys218 to Ser separately or together caused different levels of decrease in enzyme activity, loss of stability, as well as alteration of intrinsic fluorescence, underlining the importance of these Cys residues in protein conformation. Comparison of wild-type and mutant proteins modified with biotinyl glutathione ethyl ester (BioGEE) showed partial binding with single mutants and total loss of binding with the double mutant, demonstrating that both Cys residues were significantly S-glutathionylated. cTPI modification with BioGEE was reversed using DTT. Our study provides the first identification of the amino acid residues involved in cTPI S-glutathionylation and supports the hypothesis that this reversible modification could be part of an oxidative stress response pathway.

Nitric Oxide and Reactive Oxygen Species Mediate Metabolic Changes in Barley Seed Embryo during Germination.

The levels of nitric oxide (NO) and reactive oxygen species (ROS), ATP/ADP ratios, reduction levels of ascorbate and glutathione, expression of the genes encoding proteins involved in metabolism of NO and activities of the enzymes involved in fermentation and in metabolism of NO and ROS were studied in the embryos of germinating seeds of two barley (Hordeum vulgare L.) cultivars differing in dormancy level. The level of NO production continuously increased after imbibition while the level of nitrosylated SH-groups in proteins increased. This corresponded to the decrease of free SH-groups in proteins. At early stage of germination (0-48 h post imbibition) the genes encoding class 1 phytoglobin (the protein scavenging NO) and S-nitrosoglutathione reductase (scavenging S-nitrosoglutathione) were markedly expressed. More dormant cultivar exhibited lower ATP/ADP and ascorbate/dehydroascorbate ratios and lower lactate and alcohol dehydrogenase activities, while the production of NO and nitrosylation of proteins was higher as compared to the non-dormant cultivar. The obtained data indicate that at the onset of germination NO is actively generated causing nitrosylation of SH-groups and a switch from respiration to fermentation. After radicle protrusion the metabolism changes in a more reducing type as recorded by ratio of reduced and oxidized glutathione and ascorbate. The turnover of NO by the scavenging systems (phytoglobin, S-nitrosoglutathione reductase and interaction with ROS) might contribute to the maintenance of redox and energy balance of germinating seeds and lead to alleviation of dormancy.

Glyoxylate cycle and metabolism of organic acids in the scutellum of barley seeds during germination.

During the developmental processes from dry seeds to seedling establishment, the glyoxylate cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the glyoxylate cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the glyoxylate cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis.