Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (K MGlc = 140 mM versus K MFrc = 1,375 mM). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H 2 O 2 ) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H 2 O 2 release. The blockage of H 2 O 2 release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F 0 F 1 ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H 2 O 2 release. Inhibition in H 2 O 2 release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H 2 O 2 release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H 2 O 2 release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic respiration (Chance et al., 1979). The mitochondrial electron transport system (ETS) is the major site of ROS production in mammalian and nonphotosynthesizing plant cells (Puntarulo et al., 1991;Halliwell and Gutteridge, 2007). Depending on the mitochondrial respiratory states, a small portion of the consumable oxygen is partially reduced to generate ROS (

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846 -39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H 2 O 2 production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H 2 O 2 generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H 2 O 2 production in a Crsensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both ⌬⌿ m and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.Mitochondrial electron transport chain is the major and continuous source of cellular reactive oxygen species (ROS), 5 which are involved in several conditions, such as apoptosis, ischemia-reperfusion injury, neurodegenerative diseases, and toxicity induced by hyperglycemia (1-5). Electron leakage at the complexes I (6, 7) and III (6, 8 -10) are the main sites for the monoelectronic reduction of oxygen, which results in superoxide (O 2 . ) radical production in the respiratory chain. The rate of mitochondrial ROS production is highly dependent on the mitochondrial membrane potential (⌬⌿ m ) (9, 11) and evidence supporting these observations have long demonstrated (9) that pharmacological uncoupling of oxidative phosphorylation caused a drastic reduction in mitochondrial H 2 O 2 formation. Similarly, activation of oxidative phosphorylation by ADP can also reduce the ⌬ m and ROS formation through activation of F 1 F 0 -ATP synthase complex by using the energy of the ⌬⌿ m to drive ATP synthesis (9, 11). On the other hand, when mitochondrial ADP levels drop, the respiratory rate is reduced, increasing the ⌬⌿ m levels, which ultimately leads to ROS generation. There is a clear link between the increased levels of oxidative stress markers and several neuropathies such as amyotrophic lateral sclerosis, Parkinson and Alzheimer disease and h...

RESUMO-A atividade de peroxidase e concentração de fenóis foi determinada com o objetivo de se avaliar aspectos de compatibilidade entre porta-enxertos e enxertos. As amostras foram processadas e obtidas a partir da casca e lenho dos porta-enxertos de pessegueiros (GF 677, Okinawa, Capdeboscq e Aldrighi) e de ameixeiras (Mirabolano e Marianna), enxertados ou não com as cultivares Diamante, Eldorado e Santa Rosa. Concluiu-se que a atividade de peroxidase e a concentração de fenóis foram relacionadas com união entre enxerto e porta-enxerto, particularmente, em Marianna e Mirabolano, onde a atividade de peroxidase e a concentração de fenóis foram mais elevados. A cultivar Santa Rosa foi compatível tanto com os porta-enxertos de ameixeiras quanto com os de pessegueiros.Palavras-chave: incompatibilidade da enxertia, peroxidase, fenóis, ameixeira. EVALUATION OF THE GRAFT COMPATIBILITY IN Prunus sp.ABSTRACT -The work was accomplished aiming to quantify the peroxidase activity and total phenols, in order to verify the physiological and biochemical processes in grafting of Prunus sp. cultivars. The samples were processed and obtained in bark and wood of the peach rootstocks (GF 677, Okinawa, Capdeboscq and Aldrighi) and plum rootstocks (Mirabolano and Marianna), after they had or not been grafted with the stock Diamante, Eldorado and Santa Rosa. It could be concluded that the peroxidase and the total phenols activity influenced the union between stock and rootstock; after grafting, the incompatibility degree is related with high peroxidase activity and total phenols in the rootstock Marianna and Mirabolano. The Santa Rosa plum graft is as compatible to plum rootstocks as to the peach ones.Key word: graft incompatibility, peroxidase activity, total phenols, Prunus. INTRODUÇÃOA peroxidase e os fenóis são substâncias de grande importância na união entre o enxerto e o porta-enxerto, podendo influenciar nas respostas do processo de enxertia.A peroxidase (PO) está envolvida em muitas das reações metabólicas e processos fisiológicos dos tecidos vegetais. Essa enzima participa da síntese de lignina que é de fundamental importância no desenvolvimento das plantas frutíferas e na compatibilidade da enxertia (Flurkey e Jen, 1978; Musacchi, 1994).A peroxidase é uma enzima cuja especificidade é atuar sobre os peróxidos, principalmente os de hidrogênio, decompondo-os e liberando oxigênio (Gaspar et al., 1982).Embora se reconheça sua importância no metabolismo das frutíferas, pouco se conhece sobre seu papel no crescimento e desenvolvimento das plantas, nas respostas ao estresse (Lagrimini et al., 1990), bem como seu envolvimento no processo de soldadura do enxerto.Um efeito da ação da peroxidase foi observado na combinação incompatível de Prunus avium com Prunus cerasus, onde o aumento da atividade de peroxidase coincidiu com o escurecimento dos tecidos na região enxertada (Schmid & Feucht, 1984;Schmid & Feucht, 1985;Schmid & Freitag, 1988).Os fenóis participam em grande número de processos metabólicos de plantas, sendo que 60% da prod...

RESUMO -Objetivou-se, no presente trabalho, identificar a profundidade de dormência e a velocidade de brotação em gemas de pereira, submetidas a diferentes períodos de frio à temperatura de 4 º C ±1. O experimento foi conduzido na Embrapa-Clima Temperado, em Pelotas, em 1999. Em 1º de junho, foram coletados 50 ramos, na cultivar Carrick, com aproximadamente 30 cm de comprimento. Após, foram divididos em 5 lotes de 10 ramos, sendo 4 mantidos a 4 º C± 1, e um em condições ambiente, constituindo, assim, 5 tratamentos: 0 (Testemunha); 272; 544; 816 e 1088 horas de frio (HF). No final de cada tratamento, os ramos foram divididos em pequenas estacas, contendo apenas uma única gema, sendo, após, armazenados em câmara climática a 25 º C ± 1. Avaliou-se a brotação, considerando-se o estádio de ponta verde. A partir destes dados, calculou-se o tempo médio de brotação (TMB), bem como a percentagem de gemas brotadas, em cada um dos tratamentos. Utilizou-se o índice de velocidade de brotação (IVB), para determinar a eficiência da temperatura na brotação das gemas. A profundidade de dormência, das gemas terminais, diminuiu à medida que se aumentou o período de frio. As gemas axilares não foram influenciadas pelo tempo de exposição ao frio. Com base nos dados do IVB e dos coeficientes angulares, as gemas terminais da cv. Carrick necessitam de 800 horas de frio para completar a brotação, nas condições que foram conduzidos os experimentos.Termos para indexação: Temperatura, dormência, Pyrus communis, tempo médio de brotação, método biológico. EFFECT OF CHILLING ON THE BUD BREAKING OF PEAR CV. CARRICK, IN PELOTAS, RS.ABSTRACT -The objective for this work was to identify the dormancy depth and the bud-sprouting rate of pear trees kept at chilling conditions (4ºC±1) for different periods. The experiment was carried out using buds of twigs of the previous growth season from a pear orchard of the Embrapa Clima Temperado Research Center. The twigs were collected on June 1, 1999. The treatments were five period of chilling: 0 (control); 272; 544; 816; or 1088 hours at 4ºC±1. At the end of each treatment, the twigs were cut into short cuttings containing one lateral or terminal bud each and placed in controlled conditions at 25ºC± 1. The variables evaluated were percentage of bud break and period of time to reach that phase. The mean of time of bud break (MTB) for the terminal buds decreased significantly on the cuttings that received 816 or 1088 chilling hours which were equivalent on MTB. Similarly the bud break rate (time to reach 100% bud break) was significantly shorter on the buds that received 816 or 1088 chilling hours. Therefore the terminal buds of pear cv. Carrick need 800 hours for a satisfactory bud break.Index Terms: Temperature, dormancy, Pyrus communis, mean time to bud break (MTB), biological method. INTRODUÇÃONo Sul do Brasil, existem regiões com condições edafoclimáticas propícias para o cultivo da pereira. Testes realizados com coleções de cultivares mostraram que a cv. Carrick apresenta boa adaptação, constituindo-s...

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min -1 mg PTN -1 . After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min -1 mg PTN -1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein. Correspondence

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The culture of meristems, shoot tips, and axillary buds leads to the method of in vitro multiplication that is easily used and safe to obtain uniform copies with no undesirable variations. This work aimed to propagate five in vitro pear cultivars: Housui, Carrick, Nijisseiki, Packham's Triumph, and Red Bartlett. The work was carried out in the Tissue Culture Laboratory at Embrapa Temperate Climate. The plants were sprayed with benomyl (1.0 mg./L) and agrimicine (2.4 mg/L) in the fields, 2 weeks before the shoots were collected. The shoots were then cut with two buds with no leaves and desinfested with alcohol 70% for 10 s and 1% sodium hypochloride for 20 min, 50 explants, 25 buds, and 25 meristems, were then transferred to test tubes containing MS salts and vitamins, myo-inositol (100.0 mg/L), sucrose (30.0 g/L), agar (6.0 g/L), added to in mg/L: BAP (1.0), GA3 (0.1), and NAA (0.01). Three pear cultivars were used for in vitro multiplication (`Nijisseiki', `Red Bartlett', and `Housui') by using the same basal salt with N reduced to strength, added to (in mg/L): BAP (1.6), NAA (0.16). The material was kept in growth room under 16-h photoperiod, 25 ± 2 °C and 19 μMol·m-2·s-1 of flux radiation. The in vitro contaminations were mainly due to bacteria derived from the bud material (71.5%). Higher oxidation for meristem material was observed for `Carrick' and `Packham`s Triumph'. `Red Bartlett' showed the best results for all the variable studied, although all cultivars in general presented low response.