RESUMEN
Mitochondrial complex I (CI) plays a crucial role in oxidising NADH generated by the metabolism (including photorespiration) and thereby participates in the mitochondrial electron transfer chain feeding oxidative phosphorylation that generates ATP. However, CI mutations are not lethal in plants and cause moderate phenotypes, and therefore CI mutants are instrumental to examine consequences of mitochondrial homeostasis disturbance on plant cell metabolisms and signalling. To date, the consequences of CI disruption on the lipidome have not been examined. Yet, in principle, mitochondrial dysfunction should impact on lipid synthesis through chloroplasts (via changes in photorespiration, redox homeostasis, and N metabolism) and the endoplasmic reticulum (ER) (via perturbed mitochondrion-ER crosstalk). Here, we took advantage of lipidomics technology (by LC-MS), phospholipid quantitation by 31P-NMR, and total lipid quantitation to assess the impact of CI disruption on leaf, pollen, and seed lipids using three well-characterised CI mutants: CMSII in N. sylvestris and both ndufs4 and ndufs8 in Arabidopsis. Our results show multiple changes in cellular lipids, including galactolipids (chloroplastic), sphingolipids, and ceramides (synthesised by ER), suggesting that mitochondrial homeostasis is essential for the regulation of whole cellular lipidome via specific signalling pathways. In particular, the observed modifications in phospholipid and sphingolipid/ceramide molecular species suggest that CI activity controls phosphatidic acid-mediated signalling.
Asunto(s)
Arabidopsis , Lipidómica , Arabidopsis/genética , Arabidopsis/metabolismo , Esfingolípidos/metabolismo , Cloroplastos/metabolismo , Ceramidas/metabolismo , Ácidos Fosfatidicos/metabolismoRESUMEN
Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (Rd ) is lacking. Here, we used isotopic methods and metabolomics to investigate the impact of complex I dysfunction on Rd in two respiratory mutants of forest tobacco (Nicotiana sylvestris): cytoplasmic male sterile II (CMSII) and nuclear male sterile 1 (NMS1), previously characterized for complex I disruption. Rd was higher in mutants and the inhibition of leaf respiration by light was lower. Higher Rd values were caused by increased (phosphoenol)pyruvate (PEP) metabolism at the expense of anaplerotic (PEP carboxylase (PEPc) -catalyzed) activity. De novo synthesis of Krebs cycle intermediates in the light was larger in mutants than in the wild-type, although numerically small in all genotypes. Carbon metabolism in mutants involved alternative pathways, such as alanine synthesis, and an increase in amino acid production with the notable exception of aspartate. Our results show that the alteration of NADH re-oxidation activity by complex I does not cause a general inhibition of catabolism, but rather a re-orchestration of fluxes in day respiratory metabolism, leading to an increased CO2 efflux.
Asunto(s)
Dióxido de Carbono/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Análisis de Flujos Metabólicos , Mitocondrias/metabolismo , Nicotiana/citología , Nicotiana/metabolismo , Hojas de la Planta/metabolismo , Isótopos de Carbono , Respiración de la Célula , Descarboxilación , Gases/metabolismo , Metaboloma , Metabolómica , Ácido Pirúvico/metabolismoRESUMEN
Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8 Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Complejo I de Transporte de Electrón/genética , Fotoperiodo , Antioxidantes/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carbono/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Regulación de la Expresión Génica de las Plantas , Luz , Mutación , Nitrógeno/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismoRESUMEN
Cardiolipin is a key phospholipid most specifically found in the membrane of mitochondria in yeasts, plants, and animals. Cardiolipins are essential for the maintenance, the integrity, and the dynamics of mitochondria. In most eukaryotes mitochondria play a central role in the response and adaptation to stress conditions especially through their importance in the control of programmed cell death. To assess the impact of the absence of cardiolipin, knock-down of the expression of cardiolipin synthase, the last enzyme of cardiolipin synthesis pathway in eukaryotes has been performed in yeasts, animals, and plants. These studies showed that cardiolipin is not only important for mitochondrial ultrastructure and for the stability of respiratory complexes, but it is also a key player in the response to stress, the formation of reactive oxygen species, and the execution of programmed cell death.
Asunto(s)
Cardiolipinas/metabolismo , Animales , Apoptosis , Citocromos c/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Mutación , Plantas/genética , Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Estrés Fisiológico , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Levaduras/genética , Levaduras/metabolismoRESUMEN
Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , Proteínas de la Membrana/fisiología , Mitocondrias/ultraestructura , Transferasas (Grupos de Otros Fosfatos Sustitutos)/fisiología , Antioxidantes/metabolismo , Apoptosis , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Cardiolipinas/química , ADN Bacteriano , Luz , Proteínas de la Membrana/genética , Membranas Mitocondriales/química , Mutagénesis Insercional , Protoplastos/enzimología , Plantones/crecimiento & desarrollo , Estrés Fisiológico , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Photosynthetic responses to persisting mild water stress were compared between the wild type (WT) and the respiratory complex I mutant CMSII of Nicotiana sylvestris. In both genotypes, plants kept at 80% leaf-RWC (WT80 and CMSII80) had lower photosynthetic activity and stomatal/mesophyll conductances compared to well-watered controls. While the stomatal conductance and the chloroplastic CO2 molar ratio were similar in WT80 and CMSII80 leaves, net photosynthesis was higher in CMSII80. Carboxylation efficiency was lowest in WT80 leaves both, on the basis of the same internal and chloroplastic CO2 molar ratio. Photosynthetic and fluorescence parameters indicate that WT80 leaves were only affected in the presence of oxygen. Photorespiration, as estimated by electron flux to oxygen, increased slightly in CMSII80 and WT80 leaves in accordance with increased glycerate contents but maximum photorespiration at low chloroplastic CO2 was markedly lowest in WT80 leaves. This suggests that carbon assimilation of WT80 leaves is impaired by limited photorespiratory activity. The results are discussed with respect to a possible pre-acclimation of complex I deficient leaves in CMSII to drive photosynthesis and photorespiration at low CO2 partial pressure.
Asunto(s)
Dióxido de Carbono/metabolismo , Nicotiana/fisiología , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/fisiología , Agua/fisiología , Aclimatación , Carbono/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Deshidratación , Genotipo , Luz , Mitocondrias/metabolismo , Mutación , Oxígeno/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Estomas de Plantas , Transpiración de Plantas , Nicotiana/genética , Nicotiana/efectos de la radiaciónRESUMEN
Ranunculus glacialis leaves were tested for their plastid terminal oxidase (PTOX) content and electron flow to photorespiration and to alternative acceptors. In shade-leaves, the PTOX and NAD(P)H dehydrogenase (NDH) content were markedly lower than in sun-leaves. Carbon assimilation/light and Ci response curves were not different in sun- and shade-leaves, but photosynthetic capacity was the highest in sun-leaves. Based on calculation of the apparent specificity factor of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), the magnitude of alternative electron flow unrelated to carboxylation and oxygenation of Rubisco correlated to the PTOX content in sun-, shade- and growth chamber-leaves. Similarly, fluorescence induction kinetics indicated more complete and more rapid reoxidation of the plastoquinone (PQ) pool in sun- than in shade-leaves. Blocking electron flow to assimilation, photorespiration and the Mehler reaction with appropriate inhibitors showed that sun-leaves were able to maintain higher electron flow and PQ oxidation. The results suggest that PTOX can act as a safety valve in R. glacialis leaves under conditions where incident photon flux density (PFD) exceeds the growth PFD and under conditions where the plastoquinone pool is highly reduced. Such conditions can occur frequently in alpine climates due to rapid light and temperature changes.
Asunto(s)
Oxidorreductasas/fisiología , Proteínas de Plantas/fisiología , Plastidios/enzimología , Ranunculus/enzimología , Oxidorreductasas/metabolismo , Fotosíntesis , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Proteínas de Plantas/metabolismo , Plastidios/metabolismo , Estrés Fisiológico , Luz SolarRESUMEN
The cytoplasmic male-sterile (CMS) mutant of Nicotiana sylvestris which lacks NAD7, one of the subunits of respiratory complex I (NADH: ubiquinone oxidoreductase, EC 1.6.5.3), is characterized by very low (~10 times lower as compared to the wild type plants) emissions of nitric oxide (NO) under hypoxic conditions. The level of the non-symbiotic class 1 hemoglobin, as shown by Western blotting, is increased compared to the wild type plants not only under hypoxia but this protein reveals its marked expression in the CMS mutant even under normoxic conditions. The activity of aconitase (EC 4.2.1.3) is low in the CMS mutant, especially in the mitochondrial compartment, which indicates the suppression of the tricarboxylic acid cycle. The CMS mutant exhibits the severalfold higher activities of alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) under the normoxic conditions as compared to the wild type plants. It is concluded that the lack of functional complex I results in upregulation of the pathways of hypoxic metabolism which include both fermentation of pyruvate and scavenging of NO by the non-symbiotic hemoglobin.
Asunto(s)
Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/metabolismo , Hemoglobinas/metabolismo , Nicotiana/metabolismo , Óxido Nítrico/metabolismo , Proteínas de Plantas/metabolismo , Aconitato Hidratasa/metabolismo , Fermentación , Regulación de la Expresión Génica de las PlantasRESUMEN
To investigate the role of plant mitochondria in drought tolerance, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant, which has low-efficient respiration and photosynthesis, high levels of amino acids and pyridine nucleotides, and increased antioxidant capacity. We show that the delayed decrease in relative water content after water withholding in CMSII, as compared to WT leaves, is due to a lower stomatal conductance. The stomatal index and the abscisic acid (ABA) content were unaffected in well-watered mutant leaves, but the ABA/stomatal conductance relation was altered during drought, indicating that specific factors interact with ABA signalling. Leaf hydraulic conductance was lower in mutant leaves when compared to WT leaves and the role of oxidative aquaporin gating in attaining a maximum stomatal conductance is discussed. In addition, differences in leaf metabolic status between the mutant and the WT might contribute to the low stomatal conductance, as reported for TCA cycle-deficient plants. After withholding watering, TCA cycle derived organic acids declined more in CMSII leaves than in the WT, and ATP content decreased only in the CMSII. Moreover, in contrast to the WT, total free amino acid levels declined whilst soluble protein content increased in CMSII leaves, suggesting an accelerated amino acid remobilisation. We propose that oxidative and metabolic disturbances resulting from remodelled respiration in the absence of Complex I activity could be involved in bringing about the lower stomatal and hydraulic conductances.
Asunto(s)
Sequías , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/metabolismo , Nicotiana/metabolismo , Nicotiana/fisiología , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Estomas de Plantas/metabolismo , Estomas de Plantas/fisiología , Ácido Abscísico/metabolismo , Adenosina Trifosfato/metabolismo , Complejo I de Transporte de Electrón/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Hojas de la Planta/genética , Estomas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/fisiología , Nicotiana/genéticaRESUMEN
Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO(2) and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO(2) and increased N supply both significantly enhanced NAD(+) and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD(+) and NADH compared to the WT, but the differences in NADH were smaller at high CO(2) than in air because high CO(2) increased WT NADH/NAD(+). The CMSII-linked increases in NAD(+) and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD(+) and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD(+) and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD(+) and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent signaling pathways.
Asunto(s)
Carbono/metabolismo , Metaboloma , Mitocondrias/metabolismo , NAD/metabolismo , Nicotiana/metabolismo , Nitrógeno/metabolismo , Aminoácidos/metabolismo , Dióxido de Carbono/metabolismo , Cromatografía Líquida de Alta Presión , Transporte de Electrón , Ambiente , Regulación de la Expresión Génica de las Plantas , Genotipo , Redes y Vías Metabólicas , Modelos Biológicos , Mutación/genética , Hojas de la Planta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nicotiana/genética , Nicotiana/crecimiento & desarrolloRESUMEN
The relationship between the development of photoprotective mechanisms (non-photochemical quenching, NPQ), the generation of the electrochemical proton gradient in the chloroplast and the capacity to assimilate CO(2) was studied in tobacco dark-adapted leaves at the onset of illumination with low light. These conditions induce the generation of a transient NPQ, which relaxes in the light in parallel with the activation of the Calvin cycle. Wild-type plants were compared with a CMSII mitochondrial mutant, which lacks the respiratory complex I and shows a delayed activation of photosynthesis. In the mutant, a slower onset of photosynthesis was mirrored by a decreased capacity to develop NPQ. This correlates with a reduced efficiency to reroute electrons at the PSI reducing side towards cyclic electron flow around PSI and/or other alternative acceptor pools, and with a smaller ability to generate a proton motive force in the light. Altogether, these data illustrate the tight relationship existing between the capacity to evacuate excess electrons accumulated in the intersystem carriers and the capacity to dissipate excess photons during a dark to light transition. These data also underline the essential role of respiration in modulating the photoprotective response in dark-adapted leaves, by poising the cellular redox state.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Transporte de Electrón/fisiología , Luz , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Nicotiana/efectos de la radiación , Adenosina Trifosfato/metabolismo , Dióxido de Carbono/metabolismo , Complejo I de Transporte de Electrón/genética , Fotoquímica , Fotosíntesis , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiaciónRESUMEN
The interaction of photosynthesis and respiration has been studied in vivo under conditions of limited water supply and after consecutive rewatering. The role of the alternative (v(alt)) and cytochrome (v(cyt)) pathways on drought stress-induced suppression of photosynthesis and during photosynthetic recovery was examined in the Nicotiana sylvestris wild type (WT) and the complex I-deficient CMSII mutant. Although photosynthetic traits, including net photosynthesis (A(N)), stomatal (g(s)) and mesophyll conductances (g(m)), as well as respiration (v(cyt) and v(alt)) differed between well-watered CMSII and WT, similar reductions of A(N), g(s), and g(m) were observed during severe drought stress. However, total respiration (V(t)) remained slightly higher in CMSII due to the still increased v(cyt) (to match ATP demand). v(alt) and maximum carboxylation rates remained almost unaltered in both genotypes, while in CMSII, changes in photosynthetic light harvesting (i.e. Chl a/b ratio) were detected. In both genotypes, photosynthesis and respiration were restored after 2 d of rewatering, predominantly limited by a delayed stomatal response. Despite complex I dysfunction and hence altered redox balance, the CMSII mutant seems to be able to adjust its photosynthetic machinery during and after drought stress to reduce photo-oxidation and to maintain the cell redox state and the ATP level.
Asunto(s)
Sequías , Complejo I de Transporte de Electrón/deficiencia , Nicotiana/fisiología , Fotosíntesis/fisiología , Estrés Fisiológico , Agua/fisiología , Western Blotting , Respiración de la Célula , Citocromos/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Gases/metabolismo , Redes y Vías Metabólicas , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Mitocondriales , Mutación/genética , Oxidorreductasas/metabolismo , Pigmentos Biológicos/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Carácter Cuantitativo Heredable , Solubilidad , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
Norflurazon is a bleaching herbicide known to block carotenoid biosynthesis by inhibiting phytoene desaturase activity. Soybean plants were treated with norflurazon, and we examined the effects on the desaturation of lipid molecular species in leaves using ammonium [1-(14)C] oleate labeling. In monogalactosyldiacylglycerol (MGDG), the main chloroplast lipid, a decrease in 18:3/18:3 molecular species and an increase in its precursors 18:2/18:3 and 18:2/18:2 were observed suggesting that the omega(3) FAD7 desaturase activity in planta was inhibited by norflurazon. The in vitro activity of MGDG synthase was also inhibited by 69%. In contrast, the amount of 18:3/18:3 molecular species of phosphatidylcholine (PC) in the extraplastid compartment increased. The observed increase in in vitro lysoPC-acyltransferase activity and activation of desaturation of [1-(14)C] oleate suggest that extraplastid omega(3)FAD3 desaturase was activated. Analysis of the expression of omega(3) FAD3 and omega(3) FAD7 genes in norflurazon treated plants indicate that omega(3) FAD7 and omega(3) FAD3 desaturases are controlled at the post-transcriptional level.
Asunto(s)
Cloroplastos/metabolismo , Glycine max/metabolismo , Herbicidas/farmacología , Metabolismo de los Lípidos , Piridazinas/farmacología , Cloroplastos/química , Cloroplastos/enzimología , Cloroplastos/genética , Ácido Graso Desaturasas/metabolismo , Lípidos/química , Hojas de la Planta/química , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Glycine max/química , Glycine max/enzimología , Glycine max/genéticaRESUMEN
Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.
Asunto(s)
Complejo I de Transporte de Electrón , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/fisiología , Arabidopsis/genética , Membrana Celular/enzimología , Genes de Plantas , Modelos Biológicos , Mutación , Fosforilación Oxidativa , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Oxígeno/metabolismo , Fosforilación , Extractos Vegetales/metabolismo , Hojas de la Planta/metabolismo , Nicotiana/genéticaRESUMEN
Mitochondrial electron transport pathways exert effects on carbon-nitrogen (C/N) relationships. To examine whether mitochondria-N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered 'nitrate-sensing' phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of <1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots.
Asunto(s)
Mitocondrias/metabolismo , Nicotiana/crecimiento & desarrollo , Nitratos/metabolismo , Nitrógeno/metabolismo , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Carbono/metabolismo , Análisis por Conglomerados , ADN de Plantas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Giberelinas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Zeatina/metabolismoRESUMEN
Plant mitochondria contain alternative external NAD(P)H dehydrogenases, which oxidize cytosolic NADH or NADPH and reduce ubiquinone without inherent linkage to proton pumping and ATP production. In potato, St-NDB1 is an external Ca2+-dependent NADPH dehydrogenase. The physiological function of this enzyme was investigated in homozygous Nicotiana sylvestris lines overexpressing St-ndb1 and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leaf mitochondria isolated from the overexpressor lines, higher activity of alternative oxidase (AOX) was detected. However, the AOX induction was substantially weaker than in the complex I-deficient CMSII mutant, previously shown to contain elevated amounts of NAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene were up-regulated in CMSII, but only aox1b showed a response, albeit smaller, in the transgenic lines, indicating differences in AOX activation between the genotypes. As in CMSII, the increase of AOX in the overexpressing lines was not due to a general oxidative stress. The lines overexpressing St-ndb1 had consistently lowered leaf NADPH/NADP+ ratios in the light and variably decreased levels in darkness, but unchanged NADH/NAD+ ratios. CMSII instead had similar NADPH/NADP+ and lower NADH/NAD+ ratios than the wild type. These results demonstrate that St-NDB1 is able to modulate the cellular balance of NADPH and NADP+ at least in the day and that reduction of NADP(H) and NAD(H) is independently controlled. Similar growth rates, chloroplast malate dehydrogenase activation and xanthophyll ratios indicate that the change in reduction does not communicate to the chloroplast, and that the cell tolerates significant changes in NADP(H) reduction without deleterious effects.
Asunto(s)
Mitocondrias/enzimología , NADPH Deshidrogenasa/genética , NADPH Deshidrogenasa/metabolismo , NADP/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Hojas de la Planta/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Oxidación-Reducción , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Especies Reactivas de OxígenoRESUMEN
Nicotiana sylvestris leaves challenged by the bacterial elicitor harpin N(Ea) were used as a model system in which to determine the respective roles of light, oxygen, photosynthesis, and respiration in the programmed cell death response in plants. The appearance of cell death markers, such as membrane damage, nuclear fragmentation, and induction of the stress-responsive element Tnt1, was observed in all conditions. However, the cell death process was delayed in the dark compared with the light, despite a similar accumulation of superoxide and hydrogen peroxide in the chloroplasts. In contrast, harpin-induced cell death was accelerated under very low oxygen (<0.1% O(2)) compared with air. Oxygen deprivation impaired accumulation of chloroplastic reactive oxygen species (ROS) and the induction of cytosolic antioxidant genes in both the light and the dark. It also attenuates the collapse of photosynthetic capacity and the respiratory burst driven by mitochondrial alternative oxidase activity observed in air. Since alternative oxidase is known to limit overreduction of the respiratory chain, these results strongly suggest that mitochondrial ROS accumulate in leaves elicited under low oxygen. We conclude that the harpin-induced cell death does not require ROS accumulation in the apoplast or in the chloroplasts but that mitochondrial ROS could be important in the orchestration of the cell suicide program.
Asunto(s)
Nicotiana/metabolismo , Oxígeno/química , Antioxidantes/química , Dióxido de Carbono/química , Muerte Celular , Núcleo Celular/metabolismo , Electrólitos , Peróxido de Hidrógeno/química , Luz , Modelos Biológicos , Oxígeno/metabolismo , Fotosíntesis , Hojas de la Planta/metabolismo , Fenómenos Fisiológicos de las Plantas , Especies Reactivas de Oxígeno , Superóxidos/químicaRESUMEN
The hypersensitive response has been mostly studied by molecular and biochemical methods after sample destruction. The development of imaging techniques allows the monitoring of physiological changes before any signs of cell death. Here, we follow the early steps of a hypersensitive-like response induced by the bacterial elicitor harpin in Nicotiana sp. We describe cytological modifications after inoculation of the harpin protein, using confocal fluorescence microscopy (CFM) and optical coherence tomography (OCT), an interferometric-based microscopy. The changes detected by CFM occurred 5 h after harpin infiltration and corresponded to a redistribution of the chloroplasts from the upper to the inner regions of the palisade mesophyll cells which could be related to a perturbation in the microtubule network. Using OCT, we were able to detect a decrease in chloroplast backscattered signal as early as 30 min after harpin infiltration. A simple physical model, which accounted for the structure and distribution of thylakoid membranes, suggested that this loss of scattering could be associated with a modification in the refractive index of the thylakoid membranes. Our OCT observations were correlated with a decrease in photosynthesis, emphasizing changes in chloroplast structure as one of the earliest hallmarks of plant hypersensitive cell death.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/farmacología , Cloroplastos/metabolismo , Tomografía de Coherencia Óptica/métodos , Apoptosis/efectos de los fármacos , Cloroplastos/efectos de los fármacos , Microscopía Confocal , Microscopía Fluorescente , Fotosíntesis/efectos de los fármacos , Nicotiana/citología , Nicotiana/efectos de los fármacos , Nicotiana/fisiología , Tomografía de Coherencia Óptica/instrumentaciónRESUMEN
Mitochondria are key players in plant cell redox homeostasis and signalling. Earlier concepts that regarded mitochondria as secondary to chloroplasts as the powerhouses of photosynthetic cells, with roles in cell proliferation, death and ageing described largely by analogy to animal paradigms, have been replaced by the new philosophy of integrated cellular energy and redox metabolism involving mitochondria and chloroplasts. Thanks to oxygenic photosynthesis, plant mitochondria often operate in an oxygen- and carbohydrate-rich environment. This rather unique environment necessitates extensive flexibility in electron transport pathways and associated NAD(P)-linked enzymes. In this review, mitochondrial redox metabolism is discussed in relation to the integrated cellular energy and redox function that controls plant cell biology and fate.
Asunto(s)
Homeostasis , Mitocondrias/metabolismo , Plantas/metabolismo , Apoptosis , Senescencia Celular , Metabolismo Energético , NADP/metabolismo , Oxidación-Reducción , Células Vegetales , Plantas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Transformación GenéticaRESUMEN
Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I-deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin N(Ea) induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.