Natalia V. Bykova

Identification of 14 new phosphoproteins involved in important plant mitochondrial processes

Protein phosphorylation is a very important posttranslational modification the role of which is practically unexplored in mitochondria. Using two‐dimensional gel electrophoresis followed by mass spectrometry, 14 new phosphoproteins are identified in potato tuber mitochondria, all household proteins also present in mammalian and fungal mitochondria. Seven of the new phosphoproteins are involved in the tricarboxylic acid cycle or associated reactions, four are subunits of respiratory complexes and involved in electron transport, ATP synthesis and protein processing, two are heat shock proteins and one is involved in defence against oxidative stress. These findings open up entirely new possibilities for the regulation and signal integration of mitochondrial processes.

INVOLVEMENT OF CYANIDE-RESISTANT AND ROTENONE-INSENSITIVE PATHWAYS OF MITOCHONDRIAL ELECTRON TRANSPORT DURING OXIDATION OF GLYCINE IN HIGHER PLANTS

Metabolism of glycine in isolated mitochondria and protoplasts was investigated in photosynthetic, etiolated (barley and pea leaves) and fat‐storing (maize scutellum) tissues using methods of [1‐14C]glycine incorporation and counting of 14CO2 evolved, oxymetric measurement of glycine oxidation and rapid fractionation of protoplasts incubated in photorespiratory conditions with consequent determination of ATP/ADP ratios in different cell compartments. The involvement of different paths of electron transport in mitochondria during operation of glycine decarboxylase complex (GDC) was tested in different conditions, using aminoacetonitrile (AAN), the inhibitor of glycine oxidation in mitochondria, rotenone, the inhibitor of Complex I of mitochondrial electron transport, and inhibitors of cytochrome oxidase and alternative oxidase. It was shown that glycine has a preference to other substrates oxidized in mitochondria only in photosynthetic tissue where succinate and malate even stimulated its oxidation. Rotenone had no or small effect on glycine oxidation, whereas the role of cyanide‐resistant path increased in the presence of ATP. Glycine oxidation increased ATP/ADP ratio in cytosol of barley protoplasts incubated in the presence of CO2, but not in the CO2‐free medium indicating that in conditions of high photorespiratory flux oxidation of NADH formed in the GDC reaction passes via the non‐coupled paths. Activity of GDC in fat‐storing tissue correlated with the activity of glyoxylate‐cycle enzymes, glycine oxidation did not reveal preference to other substrates and the involvement of paths non‐connected with proton translocation was not pronounced. It is suggested that the preference of glycine to other substrates oxidized in mitochondria is achieved in photosynthetic tissue by switching to rotenone‐insensitive intramitochrondrial NADH oxidation and by increasing of alternative oxidase involvement in the presence of glycine.

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.

Origins and metabolism of formate in higher plants

Formate, a simple one-carbon compound, is readily metabolized in plant tissues. In greening potato tubers, similar to some procaryotes, formate is directly synthesized via a ferredoxin-dependent fixation of CO2, serving as the main precursor for carbon skeletons in biosynthetic pathways. In other plant species and tissues, formate appears as a side-product of photorespiration and of fermentation pathways, but possibly also as a product of direct CO2 reduction in chloroplasts. Formate metabolism is closely related to serine synthesis and to all subsequent reactions originating from serine. Formate may have a role in biosynthesis of numerous compounds, in energetic metabolism and in si,signal transduction pathways related to stress response. This review summarizes the current state of formate research, physiological/biochemical and molecular aspects

Metabolic response of potato plants to an antisense reduction of the P-protein of glycine decarboxylase

Abstract. Potato (Solanum tuberosum L. cv. Desiré) plants with reduced amounts of P-protein, one of the subunits of glycine decarboxylase (GDC), have been generated by introduction of an antisense transgene. Two transgenic lines, containing about 60–70% less P-protein in the leaves compared to wild-type potato, were analysed in more detail. The reduction in P-protein amount led to a decrease in the ability of leaf mitochondria to decarboxylate glycine. Photosynthetic and growth rates were reduced but the plants were viable under ambient air and produced tubers. Glycine concentrations within the leaves were elevated up to about 100-fold during illumination. Effects on other amino acids and on sucrose and hexoses were minor. Nearly all of the glycine accumulated during the day was metabolised during the following night. The data suggest that the GDC operates far below substrate saturation under normal conditions thus allowing a flexible and fast response to changes in the environment.

Interaction between photorespiration and respiration in transgenic potato plants with antisense reduction in glycine decarboxylase.

Potato (Solanum tuberosum L. cv. Désirée) plants with an antisense reduction in the P-protein of the glycine decarboxylase complex (GDC) were used to study the interaction between respiration and photorespiration. Mitochondria isolated from transgenic plants had a decreased capacity for glycine oxidation and glycine accumulated in the leaves. Malate consumption increased in leaves of GDC deficient plants and the capacity for malate and NADH oxidation increased in isolated mitochondria. A lower level of alternative oxidase protein and decreased partitioning of electrons to the alternative pathway was found in these plants. The adenylate status was altered in protoplasts from transgenic plants, most notably the chloroplastic ATP/ADP ratio increased. The lower capacity for photorespiration in leaves of GDC deficient plants was compensated for by increased respiratory decarboxylations in the light. This is interpreted as a decreased light suppression of the tricarboxylic acid cycle in GDC deficient plants in comparison to wild-type plants. The results support the view that respiratory decarboxylations in the light are restricted at the level of the pyruvate dehydrogenase complex and/or isocitrate dehydrogenase and that this effect is likely to be mediated by mitochondrial photorespiratory products.

Genomic analysis of MAP kinase cascades inArabidopsis defense responses

The process of phosphorylation and dephosphorylation is a common mechanism of signal transduction in plants, connecting the perception of extracellular signals with the final responses to those signals. This paper will concentrate on the mitogen-activated protein (MAP) kinase pathway, one of the main phosphorylation pathways that plants use in biotic and abiotic stress resistance. It is a cascade consisting of several classes of kinases, each having a different role in signal integration and divergence. The cascade is regulated by various mechanisms, including not only transcriptional and translational regulations but also post-transcriptional regulations and protein-protein interactions. Recent detailed analysis of certain specific MAP kinase pathways has revealed the specificity of the kinases in the cascade, signal transduction patterns, identity of pathway targets, and the complexity of the cascade. Strategies in the study of phosphorylation pathways are discussed, and approaches integrating various genomics and proteomics technologies are suggested.

Dihydrolipoamide dehydrogenase from porcine heart catalyzes NADH-dependent scavenging of nitric oxide

Dihydrolipoamide dehydrogenase (DLDH; EC 1.8.1.4) from porcine heart is capable of using nitric oxide (NO) as an electron acceptor, with NADH as the electron donor, forming nitrate in the reaction. NADPH was not effective as an electron donor. The reaction had a pH optimum near 6 and was not inhibited by cyanide or diphenyleneiodonium ions. The K m for NADH was 10 μM, while that for NO was 0.5 μM. The rate of NO conversion was comparable to the rate of lipoamide conversion (200 μmol min−1 mg−1 protein at pH 6). Cytochrome c or myoglobin were poor electron acceptors by themselves but, in the presence of methylene blue, DLDH had an activity of 5–7 μmol min−1 mg−1 protein with these substrates, indicating that DLDH can act also as a methemoglobin reductase. While the K m of DLDH for NO is relatively low, it is in the physiological range of NO levels encountered in the tissue. The enzyme may, therefore, have a significant role in modifying NO levels under specific cell conditions.

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.

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.