Root Mitochondria Research Articles

Combined Action of Melatonin and Water Deficiency on Growth and MDA Content of Hypocotyls and Roots of Lupine and Respiration of Mitochondria Isolated from These Organs

Melatonin is known to improve the tolerance of plants to adverse abiotic factors (AAF), including dehydration. Nevertheless, details of the action of this phytohormone are not sufficiently comprehended. In the current study, the effects of melatonin on the growth, water status, and content of malonic dialdehyde were evaluated in etiolated seedlings of narrow-leaved lupine (Lupinus angustifolius L.) subjected to dehydration. The respiration of mitochondria isolated from different organs of these plants was also analyzed. Here, the 4-day-old seedlings have been deprived of water for 1 day. This entailed the increased water deficit up to 22% in the roots and 6% in the hypocotyls. Such desiccation of the seedlings retarded the growth of their hypocotyls but increased that of the roots by 13% in comparison with the undesiccated control. Both root and hypocotyl tissues accumulated MDA under these conditions. The dehydration of the seedlings negatively affected the respiration of the mitochondria isolated from these organs. The rate of oxidation of the substrates, especially malate, was diminished at the metabolic State 3 mainly due to an inhibition of the cytochrome respiration pathway (CP). For example, upon oxidation of malate, the activity of CP decreased 1.8 times in the mitochondria from hypocotyls and four to five times in those from roots. Pretreatment of the seedlings with 0.1 µM melatonin prevented the dehydration-induced rise in the MDA level in the hypocotyls but, by contrast, stimulated this parameter by 27% in the roots. Under drought conditions combined with exogenous melatonin, the growth of hypocotyls was close to that in the undesiccated control, while the root growth was lower than that in the control. In the seedlings normally supplied with water, treatment with melatonin did not significantly influence the respiration of the mitochondria prepared from the hypocotyls or roots. However, under water shortage, melatonin completely blocked the inhibition of a substrate oxidation in the hypocotyl mitochondria, chiefly because of sustaining the CP activity. As a result, exogenous melatonin increased the rate of malate oxidation by 87% and succinate oxidation by 26% in metabolic state 3 in the mitochondria from the hypocotyls. Application of this hormone to the plants only partially reversed the dehydration-induced inhibition of the oxidative phosphorilation in the root mitochondria. In this case, the oxidation of respiratory substrates, especially succinate, and activity of CP were still inhibited. Possible causes of the differences between the hypocotyl and root cells of the lupine seedling in their responses to melatonin and water deficit, namely, an extent of oxidative stress and functions of mitochondria, are discussed.

The Effects of Mg Deficiency on Activities and Isoenzyme Patterns of Antioxidant Enzymes in Cotton Roots

Magnesium (Mg) is an essential nutrient that takes vital roles in biochemical processes related to energy metabolism, regulation of enzyme activity, nucleic acid biosynthesis and carbohydrate metabolism. Mg deficiency in arable lands is an emerging problem. Loss of balance in different metabolic pathways due to Mg deficiency causes increased production of reactive oxygen species (ROS) such as O2.-, H2O2 and HO. in the roots. The aim of this work was to elucidate the antioxidant response of roots of cotton (Gossypium hirsutum) to Mg deficiency. For this aim, antioxidant enzyme activities were measured and changes in their isoenzymes were determined. In addition, NADPH oxidase (NOX) activity was investigated to determine the changes in ROS signaling. Moreover, H2O2 and lipid peroxidation levels were also measured as an indicator of oxidative damage. Findings of this work indicate that Mg deficiency does not cause oxidative load in roots of Gossypium hirsutum plants that overwhelms cellular antioxidant defence system, as evident by H2O2 and TBARS levels. However, to adjust to the new redox status, root cells drastically downregulate NOX dependent ROS production and induced total superoxide dismutase activity. These findings indicate that site of O2.- production is mitochondria in roots. Although, catalase and ascorbate peroxidase activities were decreased with Mg deficiency induced peroxidase activity seems to limit H2O2 accumulation. These adaptive responses and changes in antioxidant defence were sufficient to prevent excess ROS accumulation and occurrence of oxidative damage in roots of Gossypium hirsutum plants under Mg deficiency

Effects of three phenolic compounds on mitochondrial function and root vigor of cotton (Gossypium hirsutum L.) seedling roots

Continuous cropping of cotton causes accumulation of allelochemicals in soil that results in substantial crop yield and quality losses. To elucidate the physiological mechanism of the effects of allelochemicals on cotton root growth, and solve the problem of continuous cropping obstacles, hydroponics experiments were carried out to study the effects of three allelochemicals (p-hydroxybenzoic acid (PHBA), phloroglucinol, and ferulic acid) at different concentrations (0.8, 4.0, and 20.0 mmol L−1) on the production of reactive oxygen species, antioxidant enzyme activities, and mitochondrial function of cotton seedling roots. All three phenolic compounds suppressed cotton root growth, decreased the activity of antioxidant enzymes (superoxide dismutase, catalase and peroxidase) and H+-ATPase in root mitochondria, and increased generation of O2− and the content of H2O2. They also increased the degree of openness of mitochondria permeability transition pores, and decreased the membrane fluidity of mitochondria, and the ratio of cytochrome (Cyt) c/a, thus resulting in the damage of mitochondrial structure and overall function of the root system. Ferulic acid at 20.0 mmol L−1 inhibited cotton root growth more than the other treatments. Above all, all three kinds of allelochemicals inhibited antioxidant enzyme activity and mitochondrial function in cotton seedling roots, and the inhibition depended on the dose of phenolic compounds. Compared to PHBA and phloroglucinol, ferulic acid was a stronger inhibitor of cotton seedling root growth.

Effects of p-hydroxybenzoic acid and phloroglucinol on mitochondria function and root growth in cotton (Gossypium hirsutum L.) seedling roots.

With early-maturing cotton cultivar CCRI-50 widely grown in China as experimental material, water culture experiment was conducted to study the effects of p-hydroxybenzoic acid and phloroglucinol with different concentrations (0.8, 4.0, and 20.0 mmol·L-1) on generation rate of reactive oxygen, changes of antioxidant enzyme activities and mitochondria function of cotton roots. Results showed that p-hydroxybenzoic acid and phloroglucinol treatments inhibited the cotton root growth, reduced SOD, POD, CAT and H+-ATPase activities in root mitochondria, increased the generation rate of O2-· and H2O2 content. In addition, they also increased the opening of mitochondrial permeability transition pores (MPTP), decreased the membrane fluidity and cytochrome c/a (Cyt c/a). Difference of mitochondria function between p-hydroxybenzoic acid and phloroglucinol treatments was minor at concentration of 0.8 mmol·L-1, while the inhibition to root growth and mitochondria function under treatment of p-hydroxybenzoic acid at concentration of 4.0 and 20.0 mmol·L-1 was stronger than that of phloroglucinol. Above all, p-hydroxybenzoic acid and phloroglucinol inhibited antioxidant enzyme activity and mitochondrial function in cotton seedling roots, and the inhibition depended on dose of phenolic acids. The inhibition to root growth and mitochondria function between p-hydroxybenzoic acid and phloroglucinol treatment was different, and p-hydroxybenzoic acid had stronger inhibition than phloroglucinol at the concentration more than 4.0 mmol·L-1.

Changes in the OXPHOS system in leaf and root mitochondria of Arabidopsis thaliana subjected to long-term sulphur deficiency

Long-term sulphur (S) deficiency in Arabidopsis thaliana affects the functioning of the mitochondrial oxidative phosphorylation system (OXPHOS) via alteration of the multisubunit NADH-ubiquinone oxidoreductase (Complex I; EC 1.6.5.3), which contains several iron–sulphur clusters. Densitometric analysis of bands of respiratory chain complexes after one-dimensional blue-native polyacrylamide gel electrophoresis (BN-PAGE) showed that levels and in-gel capacities of Complex I in leaf and root mitochondria were lower than those of the control. Two-dimensional BN/SDS-PAGE showed lower abundance of all Complex I subunits, but the qualitative structural composition (subunit expression and mobility) did not change. In mitochondria of S-deficient A. thaliana, impairment of Complex I could be compensated to some extent by additional type II NADH dehydrogenases that do not contain iron–sulphur clusters. The level and capacity of external NADH dehydrogenases in leaf and root mitochondria was higher under S deficiency, but that of internal NADH dehydrogenases did not differ from the control. The amount of COXII (mitochondrial-encoded subunit of cytochrome c oxidase in Complex IV; EC 1.9.3.1) and the capacity of Complex IV were lower under S deficiency, but levels of alternative oxidase, a bypass to Complex IV, did not change. We discuss S deficiency in A. thaliana in relation to the assembly and stability of Complex I and to a bypass of Complex I by external type II NADH dehydrogenases.

Anatomical changes to protect organelle integrity account for tolerance to alkali and salt stresses in Melilotus officinalis

The adaptation mechanism of Melilotus officinalis was studied to understand this species’ mechanisms. Seedlings of Melilotus officinalis were treated with different NaHCO3 levels, with the control plants being grown without NaHCO3. Anatomical structure of three organs and ultrastruture of two organelles widespread were observed by making paraffin and ultrathin sections. The results showed that NaHCO3 treatments significantly affected vascular system of Melilotus officinalis. Vascular tissue throughout the roots, stems and leaflets underwent changes. Particularly, the diameter of xylem vessels declined significantly, which greatly limited the transportation and transverse diffusion of water; the cross section of stems was changed from near quadrangle to irregular shape, which increased the surface area of the stems and furthered the ability of photosynthesis; epidermis cell walls cutinization occurred and the diameter of xylem vessel decreased greatly, which reduced water loss through stomata and non-stomata cells. In spite of these dramatic morphological changes, organelles in cells, both chloroplast in stems and leaflets and mitochondria in roots, had minor changes. Particularly, thylakoid in chloroplast and cristae in mitochondria still had intact membrane system and sharp-edged laminated structure. Vascular tissue, the cross section of stems, cell walls of epidermis and mesophyll cell all underwent changes in Melilotus officinalis under NaHCO3 stress. While organelles in cells had minor changes. Maybe structural integrity of organelle was protected by active morphological change of tissues and cells in Melilotus officinalis.

Mitochondrial ferritin is a functional iron-storage protein in cucumber (Cucumis sativus) roots

In plants, intracellular Fe trafficking must satisfy chloroplasts' and mitochondrial demands for Fe without allowing its accumulation in the organelles in dangerous redox-active forms. Protein ferritin is involved in such homeostatic control, however its functional role in mitochondria, differently from its role in chloroplasts, is still matter of debate. To test ferritin functionality as a 24-mer Fe-storage complex in mitochondria, cucumber seedlings were grown under different conditions of Fe supply (excess, control, deficiency) and mitochondria were purified from the roots. A ferritin monomer of around 25 KDa was detected by SDS-PAGE in Fe-excess root mitochondria, corresponding to the annotated Csa5M215130/XP_004163524 protein: such a monomer is barely detectable in the control mitochondria and not at all in the Fe-deficient ones. Correspondingly, the ferritin 24-mer complex is abundant in root mitochondria from Fe-excess plants and it stores Fe as Fe(III): such a complex is also detectable, though to a much smaller extent, in control mitochondria, but not in Fe-deficient ones. Cucumber ferritin Csa5M215130/XP_004163524 is therefore a functional Fe(III)-store in root mitochondria and its abundance is dependent on the Fe nutritional status of the plant.

Comprehensive Analysis of Mitochondria in Roots and Hypocotyls of Soybean under Flooding Stress using Proteomics and Metabolomics Techniques

Flooding is a serious problem for soybeans because it reduces growth and grain yield. Proteomic and metabolomic techniques were used to examine whether mitochondrial function is altered in soybeans by flooding stress. Mitochondrial fractions were purified from the roots and hypocotyls of 4-day-old soybean seedlings that had been flooded for 2 days. Mitochondrial matrix and membrane proteins were separated by two-dimensional polyacrylamide gel electrophoresis and blue-native polyacrylamide gel electrophoresis, respectively. Differentially expressed proteins and metabolites were identified using mass spectrometry. Proteins and metabolites related to the tricarboxylic acid cycle and γ-amino butyrate shunt were up-regulated by flooding stress, while inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were down-regulated. The amounts of NADH and NAD were increased; however, ATP was significantly decreased by flooding stress. These results suggest that flooding directly impairs electron transport chains, although NADH production increases in the mitochondria through the tricarboxylic acid cycle.

Respiratory activity, energy and redox status in sulphur-deficient bean plants

Sulphur (S) is an essential nutrient that due to its chemistry plays important roles in many metabolic processes. S-deficient bean plants ( Phaseolus vulgaris L. cv. Złota saxa) showed decreased sulphate concentrations and sulphur to nitrogen ratios in the leaves and roots, less chloroplastic pigments and lower dry matter production. Phenotypic effects of S deficiency appeared as depressed shoot growth, paling and curling of the youngest leaves, chlorotic and/or necrotic spots on the leaf surface. Our results show that S deficiency changes mitochondrial function, cellular energy status and redox homeostasis. ATP production in bean leaf and root mitochondria was lower as the result of decreased activity of Complex I. Increased activities of internal NADH dehydrogenases (ND in) may at least partially compensate for Complex I impairment. External NADH dehydrogenases (ND ex) activities, as well as protein level and capacity of alternative oxidase (AOX), did not change in S-deficient bean plants. Total ATP concentration severely decreased in leaf and root tissues. Pyridine nucleotide level was changed in S-deficient bean plants, NAD(H) pool became more reduced in leaf and root tissues whereas NADP(H) pool was more oxidized in the leaves. Our findings indicate that flexible function of plant mitochondrial respiratory chain could be an important target during adaptations to S deficiency.

Activities of antioxidant enzymes in mitochondria of growing and dormant sugar beet roots

In mitochondria isolated from growing (70–85 days) and dormant (stored for 8–12 weeks) sugar beet (Beta vulgaris L.) roots, activities of superoxide dismutase (SOD) and enzymes of the ascorbate-glutathione cycle were determined. The activity of SOD, the enzyme involved in superoxide detoxification, was much higher in mitochondria of the growing root, whereas activities of ascorbate peroxidase (APO) and glutathione reductase (GR), key enzymes of the ascorbate-glutathione cycle involved in the hydrogen peroxide degradation, increased substantially in mitochondria of dormant storage roots. Catalase (CAT) activity was detected in the fraction of root mitochondria purified in the sucrose density gradient, which activity was inhibited by cyanide by 85–90% and much weaker, by aminotriazol (by 30–35%). Submitochondrial localization of APO and CAT was analyzed using proteinase K. It was established that a substrate-binding APO center is localized on the external side of the inner membrane, whereas CAT is localized in the mitochondrial matrix. A possible role of mitochondria as ROS (hydrogen peroxide) acceptors in the cells of storage parenchyma of the stored root is discussed.

Combining proteomics of root and shoot mitochondria and transcript analysis to define constitutive and variable components in plant mitochondria

Mitochondria undertake respiration in plant cells, but through metabolic plasticity utilize differ proportions of substrates and deliver different proportions of products to cellular metabolic and biosynthetic pathways. In Arabidopsis the mitochondrial proteome from shoots and cell culture have been reported, but there has been little information on mitochondria in roots. We compare the root mitochondrial proteome with mitochondria isolated from photosynthetic shoots to define the role of protein abundance in these differences. The major differences observed were in the abundance and/or activities of enzymes in the TCA cycle and the mitochondrial enzymes involved in photorespiration. Metabolic pathways linked to TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation. Comparisons to microarray analysis of these same tissues showed a positive correlation between mRNA and mitochondrial protein abundance, but still ample evidence for the role of post-transcriptional processes in defining mitochondrial composition. Broader comparisons of transcript abundances for mitochondrial components across Arabidopsis tissues provided additional evidence for specialization of plant mitochondria, and clustering of these data in functional groups showed the constitutive vs variably expressed components of plant mitochondria.

New insights into the mitochondrial nitric oxide production pathways

Considerable evidence has appeared over the past few years that nitric oxide (NO) is an important anoxic metabolite and a potent signal molecule in plants. Several pathways operative in different cell compartments, lead to NO production. Mitochondria, being a major NO producing compartment, can generate it by either nitrite reduction occurring at nearly anoxic conditions or by the oxidative route via nitric oxide synthase (NOS). Recently we compared both pathways by ozone collision chemiluminescence and by DAF fluorescence. We found that nitrite reduction to NO is associated with the mitochondrial membrane fraction but not with the matrix. In case of the nitric oxide synthase pathway, an L-arginine dependent fluorescence was detected but its response to NOS inhibitors and substrates was untypical. Therefore the existence of NOS or NOS-like activity in barley root mitochondria is very doubtful. We also found that mitochondria scavenge NO. In addition, we found indirect evidence that mitochondria are able to convert NO to gaseous intermediates like NO2 and N2O.

Production and Scavenging of Nitric Oxide by Barley Root Mitochondria

We examined whether root mitochondria and mitochondrial membranes produce nitric oxide (NO) exclusively by reduction of nitrite or also via a nitric oxide synthase (NOS), and to what extent direct NO measurements could become undetectable due to NO oxidation. Chemiluminescence detection of NO in the gas phase was used to monitor NO emission from suspensions (i.e. direct chemiluminescence). For comparison, diaminofluorescein (DAF) and diaminorhodamine (DAR) were used as NO indicators. NO oxidation to nitrite and nitrate was quantified after reduction of nitrite + nitrate to NO by vandium (III) with subsequent chemiluminescence detection (i.e. indirect chemiluminescence). Nitrite and NADH consumption were also measured. Anaerobic nitrite-dependent NO emission was exclusively associated with the membrane fraction, without participation of matrix components. Rates of nitrite and NADH consumption matched, whereas the rate of NO emission was lower. In air, mitochondria apparently produced no nitrite-dependent NO, and no NOS activity was detected by direct or indirect chemiluminescence. In contrast, with DAF-2 or DAR-4M, an l-arginine-dependent fluorescence increase took place. However, the response of this apparent low NOS activity to inhibitors, substrates and cofactors was untypical when compared with commercial inducible NOS (iNOS), and the existence of NOS in root mitochondria is therefore doubtful. In a solution of commercial iNOS, about two-thirds of the NO (measured as NADPH consumption) were oxidized to nitrite + nitrate. Addition of mitochondria to iNOS decreased the apparent NO emission, but without a concomitant increase in nitrite + nitrate formation. Thus, mitochondria appeared to accelerate oxidation of NO to volatile intermediates.

Methylene blue increases the tolerance of tomato plants to abiotic stresses

SummaryEnvironmental stresses such as high temperature and high salinity are known to cause oxidative stress in plants by enhancing the generation of reactive oxygen species (ROS), which have a strong impact on plant development. Mitochondria constitute one of the major sources of ROS in roots. Recently, it has been shown in mammalian systems that methylene blue (MB) at very low concentrations can attenuate mitochondrial ageing by scavenging ROS. In the present study, we tested whether MB could be used to protect tomato (Lycopersicon esculentum L.) seedlings against the harmful effects of high salinity and high root temperature. Tomato seedlings were grown hydroponically and were subjected to two abiotic stresses: high salinity (150 mM NaCl) in the nutrient medium, or high root temperature (35ºC) for 14 d. These stress treatments were applied with or without the addition of 10–8 M MB. The results showed that, under normal conditions, MB enhanced root and shoot growth, increased root respiration, decreased root H2O2 and malonyldialdehyde (MDA) concentrations, and enhanced root superoxide dismutase (SOD) activity. Both high salinity and high root temperature stress impaired root and shoot growth, and caused an enhancement of root oxidative stress. MB had a significant protective effect against both abiotic stresses and restored the levels of oxidative stress-related components (i.e., H2O2 and MDA) in root and leaf tissues almost to normal levels. It is hypothesised that MB could confer its protective effect not only through interactions with root mitochondria, but also via additional cellular targets in the root.

BN-PAGE analysis of the respiratory chain complexes in mitochondria of cucumber MSC16 mutant

Rearrangements of mitochondrial DNA in MSC16 mutant of cucumber (Cucumis sativus L.) affect mitochondrial functioning due to the alteration mainly of Complex I resulting in several metabolic changes. One-dimensional Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) and densitometric measurements showed that the level and in-gel capacity of Complex I were lower in MSC16 leaf and root mitochondria as compared to wild-type (WT). The level and capacity of supercomplex I+III(2) were always lower in leaf but not in MSC16 root mitochondria. Two-dimensional BN/SDS-PAGE indicated that the band abundance for most of the subunits of Complex I was lower in MSC16 leaf and root mitochondria. Supercomplex I+III(2) level was only altered in MSC16 leaf mitochondria as measured after 2D BN/SDS-PAGE. No differences in the qualitative composition of the subunits of Complex I and supercomplex I+III(2) between MSC16 and WT mitochondria were observed. In MSC16 mitochondria Complex I impairment could be compensated to some extent by additional respiratory chain NADH dehydrogenases. A higher capacity and level of NDB-1 protein of external NADH dehydrogenase was observed in MSC16 leaf and root mitochondria as compared to WT. The level of COX II, mitochondrial-encoded subunit of Complex IV, was higher in MSC16 leaf and root mitochondria. However, the capacity of Complex IV was slightly higher only in MSC16 leaf mitochondria. The levels of complexes: III(2) and V and Complex V capacity did not differ in mitochondria between genotypes. An abundance of the subunits of respiratory complexes is one of the key factors determining not only their structure and functional stability but also a formation of the supercomplexes. We discuss here mitochondrial genome rearrangements in MSC16 mutant in a relation to assembly and/or stability (the lower level and capacity) of Complex I and supercomplex I+III(2).

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

Nitric oxide (NO) has emerged as a central signaling molecule in plants and animals. However, the long search for a plant NO synthase (NOS) enzyme has only encountered false leads. The first works describing a pathogen-induced NOS-like plant protein were soon retracted. New hope came from the identification of NOS1, an Arabidopsis thaliana protein with an atypical NOS activity that was found to be targeted to mitochondria in roots. Although concerns about the NO-producing activity of this protein were raised (causing the renaming of the protein to NO-associated 1), compelling data on its biological role were missing until recently. Strong evidence is now available that this protein functions as a GTPase that is actually targeted to plastids, where it might be required for ribosome function. These and other results support the argument that the defective NO production in loss-of-function mutants is an indirect effect of interfering with normal plastid functions and that plastids play an important role in regulating NO levels in plant cells.

Organophosphorous plant growth regulator Melaphen: Resistance of plant and animal cells to stress factors

The addition of the organophosphorous plant growth regulator Melaphen (4 × 10−12 M) to the incubation medium increases the maximum rate of oxidation of NAD-dependent substrates in rat liver and sugar beet root mitochondria. In addition, Melaphen stimulates electron transport during oxidation of succinate by rat liver mitochondria, but has no effect on the rate of this substrate oxidation in sugar beet root mitochondria. In storage organs of plants, the rate of oxidation of NAD-dependent substrates by mitochondria is relatively low. By stimulating the activity of NAD-dependent dehydrogenases, Melaphen stimulates energy metabolism in the cells and manifests adaptogenic activity by accelerating the germination of seeds. Melaphen does not influence the fluorescence of lipid peroxidation (LPO) products in mitochondria non-exposed to stress, but decreases 1.5–2 fold the LPO fluorescence in rat liver mitochondria exposed to cold stress and artificially “aged” sugar beet root mitochondria. Besides, Melaphen increases the rate of electron transport in a terminal site of respiratory chains of plant and animal mitochondria and decreases LPO. The data obtained testify to antistress activity of Melaphen.

Arabidopsis Nitric Oxide Synthase1 Is Targeted to Mitochondria and Protects against Oxidative Damage and Dark-Induced Senescence

The Arabidopsis thaliana protein nitric oxide synthase1 (NOS1) is needed for nitric oxide (NO) synthesis and signaling during defense responses, hormonal signaling, and flowering. The cellular localization of NOS1 was examined because it is predicted to be a mitochondrial protein. NOS1-green fluorescent protein fusions were localized by confocal microscopy to mitochondria in roots. Isolated mitochondria from leaves of wild-type plants supported Arg-stimulated NO synthesis that could be inhibited by NOS inhibitors and quenched by a NO scavenger; this NOS activity is absent in mitochondria isolated from nos1 mutant plants. Because mitochondria are a source of reactive oxygen species (ROS), which participate in senescence and programmed cell death, these parameters were examined in the nos1 mutant. Dark-induced senescence of detached leaves and intact plants progressed more rapidly in the mutant compared with the wild type. Hydrogen peroxide, superoxide anion, oxidized lipid, and oxidized protein levels were all higher in the mutant. These results demonstrate that NOS1 is a mitochondrial NOS that reduces ROS levels, mitigates oxidative damage, and acts as an antisenescence agent.

In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ

At oxygen concentrations of < or =1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that, in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR-containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it has been observed with Arabidopsis, barley, pea, and tobacco. A specific role for nitrite to NO reduction in roots under anoxia is discussed.

Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants

5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria. 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria. Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides. These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA. The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms. Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence. Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms. Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process. Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria.