Mitochondrial dysfunction associated with ascorbate synthesis in plants.

Mitochondria are the major organelles of energy production; however, active mitochondria can decline their energetic role and show a dysfunctional status. Mitochondrial dysfunction was induced by high non-physiological level of L-galactone-1,4-lactone (L-GalL), the precursor of ascorbate (AsA), in plant mitochondria. The dysfunction induced by L-GalL was associated with the fault in the mitochondrial electron partition and reactive oxygen species (ROS) over-production. Using mitochondria from RNAi-plant lines harbouring silenced L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activity, it was demonstrated that such dysfunction is dependent on this enzyme activity. The capacity of alternative respiration was strongly decreased by L-GalL, probably mediated by redox-inactivation of the alternative oxidase (AOX) enzyme. Although, alternative respiration was shown to be the key factor that helps support AsA synthesis in dysfunctional mitochondria. Experiments with respiratory inhibitors showed that ROS formation and mitochondrial dysfunction were more associated with the decline in the activities of COX (cytochrome oxidase) and particularly AOX than with the lower activities of respiratory complexes I and III. The application of high L-GalL concentrations induced proteomic changes that indicated alterations in proteins related to oxidative stress and energetic status. However, supra-optimal L-GalL concentration was not deleterious for plants. Instead, the L-GalLDH activity could be positive. Indeed, it was found that wild type plants performed better growth than L-GalLDH-RNAi plants in response to high non-physiological L-GalL concentrations.

High light stress induces H2O2 production and accelerates fruit ripening in tomato.

Increased synthesis of H2O2 is observed during the initiation of fruit ripening. However, its association with plant cell processes triggering the maturation of fruit has not yet been demonstrated. The aim of this work is to investigate whether H2O2 participates in the tomato ripening process and particularly through its association with the ethylene signaling pathway. The experiments were carried out with two ethyl methanesulfonate mutant lines of Micro-Tom tomato deficient in GDP-L-galactose phosphorylase activity and displaying lower ascorbic acid content than the corresponding parental genotype (i.e. wild type). Plants were subjected to a high irradiance (HI) treatment to stimulate H2O2 synthesis. HI treatment enhanced H2O2 production and reduced the timing of fruit ripening in both mutants and wild-type fruits. These results could be linked to an increase of the expression of H2O2-related genes and changes in the expression of ethylene-related genes. The fruit H2O2 production increased or decreased after applying the treatments that induced ethylene synthesis or blocked its action, respectively. The results presented in this work give an evidence of the association of redox and hormonal components during fruit ripening in which H2O2 participates downstream in the events regulated by ethylene.

Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.

MAIN CONCLUSION: Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-L-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-L-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production.

Nitric oxide as a key component in hormone-regulated processes.

Nitric oxide (NO) is a small gaseous molecule, with a free radical nature that allows it to participate in a wide spectrum of biologically important reactions. NO is an endogenous product in plants, where different biosynthetic pathways have been proposed. First known in animals as a signaling molecule in cardiovascular and nervous systems, it has turned up to be an essential component for a wide variety of hormone-regulated processes in plants. Adaptation of plants to a changing environment involves a panoply of processes, which include the control of CO2 fixation and water loss through stomatal closure, rearrangements of root architecture as well as growth restriction. The regulation of these processes requires the concerted action of several phytohormones, as well as the participation of the ubiquitous molecule NO. This review analyzes the role of NO in relation to the signaling pathways involved in stomatal movement, plant growth and senescence, in the frame of its interaction with abscisic acid, auxins, gibberellins, and ethylene.

L-galactono-1,4-lactone dehydrogenase (GLDH) forms part of three subcomplexes of mitochondrial complex I in Arabidopsis thaliana.

L-galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.

Consumo de tabaco, alcohol, drogas y su relación con los intentos de suicidio en población asistida en la guardia de un hospital general polivalente / Smoking, Alcohol and Drugs Consumption, and their relationships with suicide attempts in a population assisted in the Ward of a general polyvalent hospital

El problema del consumo de tabaco, alcohol y drogas y los intentos de suicidio son dos afecciones de autodestrucción, la primera con una intencionalidad tal vez inconciente y la otra voluntariamente ejecutada.Objetivo: analizar el consumo de sustancias psicoactivas en pacientes que ingresan en la guardia de un Hospital General polivalente con el diagnóstico de intento de suicidio y definir el perfil de la conducta suicidaMaterial y métodos: se realizaron dos fichas epidemiológicas administradas por profesionales de la guardia y por el Servicio de Psiquiatría del Hospital Posadas, contenían datos generales sobre el intento de suicidio y frecuencia del consumo de sustancias inmediatamente antes del intento de suicidio y en el mes anterior, a 200 pacientes ingresados a la guardia entre junio y diciembre de 2009.Resultados: se encontró una asociación entre el consumo de alcohol, marihuana y cocaína en los varones, y de alcohol y sedantes en las mujeres.

The issue of smoking, alcohol and drugs consumption, as well as suicide attempts constitute two self-destructive affections. The first one is related with an intentionality which is probably unconscious, and the second one, with an intentionality deliberately executed. Purpose: to analyze the consumption of psychoactive substances in patients who are admitted to the ward of a General, polyvalent Hospital with a suicide attempt diagnosis, and to define the profile of the suicidal behavior. Material and methods: two epidemiologic forms administered by professionals from the ward and by the Psychiatry Service of Hospital Posadas, containing general information on the suicide attempt and the frequency of consumption of substances immediately before the suicide attempt and in the previous month, to 200 patients admitted to the ward between June and December 2009. Results: a relationship was found between alcohol, marihuana and cocaine consumption in men and between alcohol and sedatives in women.

Trastornos psicopatológicos e intentos de suicidio asistidos en la guardia de un hospital polivalente del conurbano bonaerense / Pychopathological disorders and suicide attempts assited in the ward of a plyclinic in the suburbs of the province of Buenos Aires

La conducta suicida es una situación de múltiples aristas en la que intervienen varios factores y que se enmarca dentro de las conductas agresivas. Este estudio fue efectuado a fin de acercarse a la comprensión de este angustiante cuadro. Objetivo: estudiar la caracterización de la conducta suicida en pacientes de ambos sexos concurrentes a la Guardia del Hospital "Dr. Alejandro Posadas" con motivo de un intento de suicidio. Material y Métodos: fueron examinados en la Guardia y en el Servicio de Psicopatología 230 personas por intento de suicidio, 200 ingresaron al estudio y 30 no pudieron hacerlo por no hallarse en condiciones. Se utilizaron tres formularios, de consentimiento informado, de la Guardia del Hospital y un tercero para ser llenado por el psiquiatra del Servicio. Se realizó una base de datos y el análisis estadístico utilizando el SPSS V 11. Resultados: se encontró una vinculación entre aquellos que realizaron un intento de suicidio y los diagnósticos de trastornos de personalidad borderline, histérica y los trastornos de ansiedad, bipolar y esquizofrenia. Facilitar el acceso a los pacientes a fin de realizar un diagnóstico y tratamiento precoces es una forma de evitar la conducta agresiva del intento de suicidio.(AU)

Suicide behavior is a disorder of multiple rough edges in which serveral factors are involved, which is framed within aggressive behaviors. This study was performed in order to obtain an insight into this distressing condition. Objective: To study the characterization of suicidal behavior in patients of both sexes attending the Ward of Hospital "Dr. Alejandro Posadas" due to a suicide attempt. Material and Methods: 230 people were examined in the Ward and in the Psychopathological Service due to a suicide attempt, 200 entered the study and 30 could not do so for not being eligible. Three informed consent forms were used, two belonging to the Hospitals Ward and the third one to be completed by the Services Psychiatrist. A database was created, as well as the statistical analysis, by means of the software apllication, SPSS V 11. Results: a connection was found betweeen those patients who committed suicide and the diagnosos of borderline, histrionic personality disorders, and anxiety, bipolar and schizophrenia disorders. Facilitating access to patients in order to perform an early diagnosis and treatment is a way of avoiding the aggressive bahavior relative to the suicide attempt.(AU)

Trastornos psicopatológicos e intentos de suicidio asistidos en la guardia de un hospital polivalente del conurbano bonaerense / Pychopathological disorders and suicide attempts assited in the ward of a plyclinic in the suburbs of the province of Buenos Aires

La conducta suicida es una situación de múltiples aristas en la que intervienen varios factores y que se enmarca dentro de las conductas agresivas. Este estudio fue efectuado a fin de acercarse a la comprensión de este angustiante cuadro. Objetivo: estudiar la caracterización de la conducta suicida en pacientes de ambos sexos concurrentes a la Guardia del Hospital "Dr. Alejandro Posadas" con motivo de un intento de suicidio. Material y Métodos: fueron examinados en la Guardia y en el Servicio de Psicopatología 230 personas por intento de suicidio, 200 ingresaron al estudio y 30 no pudieron hacerlo por no hallarse en condiciones. Se utilizaron tres formularios, de consentimiento informado, de la Guardia del Hospital y un tercero para ser llenado por el psiquiatra del Servicio. Se realizó una base de datos y el análisis estadístico utilizando el SPSS V 11. Resultados: se encontró una vinculación entre aquellos que realizaron un intento de suicidio y los diagnósticos de trastornos de personalidad borderline, histérica y los trastornos de ansiedad, bipolar y esquizofrenia. Facilitar el acceso a los pacientes a fin de realizar un diagnóstico y tratamiento precoces es una forma de evitar la conducta agresiva del intento de suicidio.

Suicide behavior is a disorder of multiple rough edges in which serveral factors are involved, which is framed within aggressive behaviors. This study was performed in order to obtain an insight into this distressing condition. Objective: To study the characterization of suicidal behavior in patients of both sexes attending the Ward of Hospital "Dr. Alejandro Posadas" due to a suicide attempt. Material and Methods: 230 people were examined in the Ward and in the Psychopathological Service due to a suicide attempt, 200 entered the study and 30 could not do so for not being eligible. Three informed consent forms were used, two belonging to the Hospital's Ward and the third one to be completed by the Service's Psychiatrist. A database was created, as well as the statistical analysis, by means of the software apllication, SPSS V 11. Results: a connection was found betweeen those patients who committed suicide and the diagnosos of borderline, histrionic personality disorders, and anxiety, bipolar and schizophrenia disorders. Facilitating access to patients in order to perform an early diagnosis and treatment is a way of avoiding the aggressive bahavior relative to the suicide attempt.

Control of ascorbic acid synthesis and accumulation and glutathione by the incident light red/far red ratio in Phaseolus vulgaris leaves.

The effects of red/far red (R/FR) ratios on leaf ascorbate (AA) and glutathione (GSH) accumulation were examined in common bean (Phaseolus vulgaris L.). Growth under low R/FR ratios resulted in a "shade" phenotype and much lower leaf AA and GSH contents than high (R/FR) ratios. Photosynthesis rates were unaffected by changes in the R/FR ratio but leaf respiration rates, pyridine nucleotide pools and antioxidant enzyme activities were decreased under the low R/FR regime. The GSH pool changed slowly in response to altered R/FR ratios but leaf ascorbate acclimated over a single photoperiod. We conclude that light quality signals, particularly R/FR ratios, are important regulators of antioxidant synthesis and accumulation. These acclimatory changes are an early response to changing light environment.

Vacuolar cysteine proteases of wheat (Triticum aestivum L.) are common to leaf senescence induced by different factors.

Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.

Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins.

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 +/- 0.2 nmol min(-1) mg(-1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 +/- 0.04 nmol min(-1) mg(-1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 +/- 0.04 nmol min(-1) mg(-1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(omega)-nitro-l-Arg methyl ester hydrochloride and N(omega)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 mum S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mum peroxynitrite (ONOO(-)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(-) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(-) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves.

The effects of growth irradiance and respiration on ascorbic acid (AA) synthesis and accumulation were studied in the leaves of wild-type and transformed Arabidopsis thaliana with modified amounts of the mitochondrial alternative oxidase (AOX) protein. Plants were grown under low (LL; 50 micromol photons m(-2) s(-1)), intermediate (IL; 100 micromol photons m(-2) s(-1)), or high (HL; 250 micromol photons m(-2) s(-1)) light. Increasing growth irradiance progressively elevated leaf AA content and hence the values of dark-induced disappearance of leaf AA, which were 11, 55, and 89 nmol AA lost g(-1) fresh weight h(-1), from LL-, IL-, and HL-grown leaves, respectively. When HL leaves were supplied with L-galactone-1,4-lactone (L-GalL; the precursor of AA), they accumulated twice as much AA and had double the maximal L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activities of LL leaves. Growth under HL enhanced dehydroascorbate reductase and monodehydroascorbate reductase activities. Leaf respiration rates were highest in the HL leaves, which also had higher amounts of cytochrome c and cytochrome c oxidase (CCO) activities, as well as enhanced capacity of the AOX and CCO electron transport pathways. Leaves of the AOX-overexpressing lines accumulated more AA than wild-type or antisense leaves, particularly at HL. Intact mitochondria from AOX-overexpressing lines had higher AA synthesis capacities than those from the wild-type or antisense lines even though they had similar L-GalLDH activities. AOX antisense lines had more cytochrome c protein than wild-type or AOX-overexpressing lines. It is concluded that regardless of limitations on L-GalL synthesis by regulation of early steps in the AA synthesis pathway, the regulation of L-GalLDH activity via the interaction of light and respiratory controls is a crucial determinant of the overall ability of leaves to produce and accumulate AA.

Up-regulation of the mitochondrial alternative oxidase pathway enhances photosynthetic electron transport under drought conditions.

The aim of this study was to explore the role of the mitochondrial alternative oxidase (AOX) in the protection of photosynthesis during drought in wheat leaves. The relative water contents of water-replete and drought-exposed wheat plants were 97.2+/-0.3 and 75+/-2, respectively. Drought increased the amount of leaf AOX protein and also enhanced the rate of AOX-dependent O(2) uptake by the respiratory electron transport chain. The amount of the reduced, active form of the AOX protein was specifically increased by drought. The AOX inhibitor salicylhydroxamic acid (1 mM; SHAM) inhibited 70% of AOX activity in vivo in both water-replete and drought-exposed plants. Plants treated with SHAM were then exposed to low (100), high (350), or excess light (800 mumol photons m(-2) s(-1)) for 90 min. SHAM did not modify chlorophyll a fluorescence quenching parameters in water-replete controls after any of these treatments. However, while the maximal quantum yield of photosystem II (PSII) electron transport (F(v)/F(m)) was not affected by SHAM, the immediate quantum yield of PSII electron transport (Phi(PSII)) and photochemical quenching (qP) were gradually reduced by increasing irradiance in SHAM-treated drought-exposed plants, the decrease being most pronounced at the highest irradiance. Non-photochemical quenching (NPQ) reached near maximum levels in plants subjected to drought at high irradiance. However, a combination of drought and low light caused an intermediate increase in NPQ, which attained higher values when AOX was inhibited. Taken together, these results show that up-regulation of the respiratory AOX pathway protects the photosynthetic electron transport chain from the harmful effects of excess light.

Thylakoid-bound ascorbate peroxidase mutant exhibits impaired electron transport and photosynthetic activity.

In chloroplasts, stromal and thylakoid-bound ascorbate peroxidases (tAPX) play a major role in the removal of H(2)O(2) produced during photosynthesis. Here, we report that hexaploid wheat (Triticum aestivum) expresses three homeologous tAPX genes (TaAPX-6A, TaAPX-6B, and TaAPX-6D) mapping on group-6 chromosomes. The tAPX activity of a mutant line lacking TaAPX-6B was 40% lower than that of the wild type. When grown at high-light intensity photosystem II electron transfer, photosynthetic activity and biomass accumulation were significantly reduced in this mutant, suggesting that tAPX activity is essential for photosynthesis. Despite the reduced tAPX activity, mutant plants did not exhibit oxidative damage probably due to the reduced photochemical activity. This might be the result of a compensating mechanism to prevent oxidative damage having as a consequence a decrease in growth of the tAPX mutant plants.