José Antonio Hernández

Evidence for the Presence of the Ascorbate-Glutathione Cycle in Mitochondria and Peroxisomes of Pea Leaves

The presence of the enzymes of the ascorbate-glutathione cycle was investigated in mitochondria and peroxisomes purified from pea (Pisum sativum L.) leaves. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were present in mitochondria and peroxisomes, as well as in the antioxidants ascorbate and glutathione. The activity of the ascorbate-glutathione cycle enzymes was higher in mitochondria than in peroxisomes, except for APX, which was more active in peroxisomes than in mitochondria. Intact mitochondria and peroxisomes had no latent APX activity, and this remained in the membrane fraction after solubilization assays with 0.2 M KCl. Monodehydroascorbate reductase was highly latent in intact mitochondria and peroxisomes and was membrane-bound, suggesting that the electron acceptor and donor sites of this redox protein are not on the external side of the mitochondrial and peroxisomal membranes. Dehydroascorbate reductase was found mainly in the soluble peroxisomal and mitochondrial fractions. Glutathione reductase had a high latency in mitochondria and peroxisomes and was present in the soluble fractions of both organelles. In intact peroxisomes and mitochondria, the presence of reduced ascorbate and glutathione and the oxidized forms of ascorbate and glutathione were demonstrated by high-performance liquid chromatography analysis. The ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H2O2 generated in both plant organelles.

Salt-induced oxidative stress in chloroplasts of pea plants

Abstract The possible involvement of activated oxygen species in the mechanism of damage by NaCl strees was studied in chloroplasts from leaves of two cultivars of pea ( Pisum sativum L.) with differential sensitivity to NaCl. Intact organelles were purified by centrifugation in density-gradients of Percoll. In chloroplasts from tolerant plants, NaCl stress produced a significant increase of CuZn-SOD II and ascorbate peroxidase activities as well as in ascorbate content, while in those from sensitive plants NaCl produced increases in the H 2 O 2 content and lipid peroxidation and no changes were observed in the enzymatic activities. Chlorophyll content significantly decreased in chloroplasts from sensitive plants and chloroplast integrity was lower in sensitive than in tolerant plants. Electron microscopy showed that the thylakoidal structure of chloroplast was notably disorganized in the NaCl-treated leaves. In purified chloroplasts, an increase in the number and size of plastoglobuli was produced by NaCl in chloroplasts from tolerant plants and to a lesser extent, in chloroplasts from sensitive plants. The relative starch content only decreased in chloroplasts from tolerant plants by NaCl-treatment. Results obtained suggest that in the cellular toxicity of NaCl in pea plants, superoxide- and H 2 O 2 -mediated oxidative damage in chloroplasts may play an important role.

Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation

Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol VBMCP/VBAD induction plus autologous stem cell transplantation [ASCT]). MRD status by MFC was determined at day 100 after ASCT. Progression-free survival (PFS; median 71 vs 37 months, P < .001) and overall survival (OS; median not reached vs 89 months, P = .002) were longer in patients who were MRD negative versus MRD positive at day 100 after ASCT. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation-negative complete response after ASCT. Moreover, MRD(-) immunofixation-negative (IFx(-)) patients and MRD(-) IFx(+) patients had significantly longer PFS than MRD(+) IFx(-) patients. Multivariate analysis identified MRD status by MFC at day 100 after ASCT as the most important independent prognostic factor for PFS (HR = 3.64, P = .002) and OS (HR = 2.02, P = .02). Our findings demonstrate the clinical importance of MRD evaluation by MFC, and illustrate the need for further refinement of MM re-sponse criteria. This trial is registered at http://clinicaltrials.gov under identifier NCT00560053.

Differential response of antioxidative enzymes of chloroplasts and mitochondria to long-term NaCl stress of pea plants.

In this work the activity of superoxide dismutase (SOD) and the enzymes of the ascorbate-glutathione (ASC-GSH) cycle were investigated in chloroplasts and mitochondria from leaves of Pisum sativum L. cv. Puget after 15 days treatment with 0-130 mM NaCl. The main chloroplastic SOD activity was due to CuZn-SOD II, which was increased significantly (about 1.7-fold) by NaCl, although during severe NaCl stress (110-130 mM) chloroplastic Fe-SOD exhibited a stronger enhancement in its activity (about 3.5-fold). A sudden induction in chloroplastic APX, DHAR and GR was also caused by NaCl (70-110 mM), but not by the highest salt concentration (130 mM), at which GR and DHAR activities were similar to the control values and APX decreased. In addition, the H2O2 concentration and lipid peroxidation of membranes increased significantly, 3.5- and 7-fold, respectively, in chloroplasts under severe NaCl stress. In purified mitochondria DHAR and GR were significantly induced only at 90 and 130 mM NaCl, respectively, although DHAR activity was below control values in the highest NaCl concentrations. APX and MDHAR activities started their response to salt in mild NaCl conditions (70 mM) and increased significantly with the severity of the stress. Mn-SOD was induced only under severe NaCl concentrations. The mitochondrial H2O2 and lipid peroxidation were increased at the highest NaCl concentration although to a lesser extent (about 2-2.5-fold) than in chloroplasts, whereas the increase in carbonyl protein contents was higher in mitochondria. The results suggest that the degree of enhanced tolerance to NaCl seems to require the induction of specific isoforms, depending on the different organelles.

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

Plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungi modify alleviation biochemical mechanisms in water-stressed plants

This study examined the effect of inoculation with the plant-growth-promoting rhizobacterium (PGPR) Pseudomonas mendocina Palleroni, alone or in combination with an arbuscular mycorrhizal (AM) fungus, Glomus intraradices (Schenk & Smith) or Glomus mosseae (Nicol & Gerd.) Gerd. & Trappe, on antioxidant enzyme activities (superoxide dismutase, catalase and total peroxidase activities), phosphatase and nitrate reductase activities and solute accumulation in leaves of Lactuca sativa L. cv. Tafalla affected by three different levels of water stress. At moderate drought, bacterial inoculation and mycorrhizal inoculation with G. intraradices, alone or in combination, stimulated significantly nitrate reductase activity. At severe drought, fertilisation and P. mendocina inoculation, alone or in combination with either of the selected AM fungi, increased significantly phosphatase activity in lettuce roots and proline accumulation in leaves. Total peroxidase (POX) and catalase (CAT) activities increased in response to drought, whereas superoxide dismutase activity decreased. Inorganic fertilisation and both combined treatments of PGPR and AM fungus showed the highest values of leaf POX activity under severe drought. The highest CAT activity was recorded in the fertilised plants followed by the P. mendocina-inoculated plants grown under severe stress conditions. These results support the potential use of a PGPR as an inoculant to alleviate the oxidative damage produced under water stress.

Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to plum pox virus

In this work, a recombinant plum pox virus (PPV, Sharka) encoding green fluorescent protein is used to study its effect on antioxidant enzymes and protein expression at the subcellular level in pea plants (cv. Alaska). PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13–15 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 d post-inoculation) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level. Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased generation of reactive oxygen species in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.

High‐sensitive immunophenotyping and DNA ploidy studies for the investigation of minimal residual disease in multiple myeloma

Sensitive techniques for monitoring minimal residual disease (MRD) in multiple myeloma (MM) are needed to evaluate the effectiveness of new intensive treatment strategies. The aim of the present study was to explore the applicability and sensitivity of flow cytometry immunophenotyping and DNA ploidy studies for the investigation of residual myelomatous plasma cells (PC) in MM patients. Bone marrow (BM) samples from 61 untreated MM patients were immunophenotypically analysed with a panel of 21 monoclonal antibodies, using a high‐sensitive method based on a two‐step acquisition procedure through a SSC/CD38+++‐CD138+‘live‐gate’. Overall, in 87% of MM cases, PC displayed an aberrant phenotype at diagnosis. The most important aberrant criteria were: antigen over‐expression of CD56 (62%), CD28 (16%) and CD33 (6%) and asynchronous expression of CD117 (28%), sIg (21%) and CD20 (10%). DNA aneuploidy was found in 62% of cases. The simultaneous use of these two techniques allowed the detection of aberrant/aneuploid PC in 95% of the cases. Based on dilutional experiments, the detection limit of both techniques ranged from 10−4 to 10−5. In 29 stem cells harvests and 19 BM samples obtained 3 months after autologous transplantation, we have investigated the presence of residual myelomatous PC; they were detected in 44% of the stem cell collections and in 61% of the BM samples obtained after transplant. The percentage of pathological PC did not significantly change during the days of harvest. In summary, the present study shows that the combined use of immunophenotyping and DNA ploidy studies is a suitable approach for MRD investigation in MM patients based on their applicability (95% of cases) and sensitivity (up to 10−5).

Induction of Several Antioxidant Enzymes in the Selection of a Salt-Tolerant Cell Line of Pisum sativum

Summary Using the in vitro culture technique, we selected a cell line of Pisum sativum cv. Challis adapted to 85.5 mM NaCl. The possible relationships between the activity of enzymes related to oxygen metabolism and the salt adaptation of pea calli were analysed. The induction of two new Cu,Zn-SOD isozymes in salt-resistant calli was observed. Peroxidase activity was greatly increased in selected calli and catalase did not show a significant variation. The activity changes observed are discussed in terms of their possible relevance to pea calli adaptation to salt-stress.

Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings

Hydrogen peroxide (H(2)O(2)) increased the germination percentage of pea seeds, as well as the growth of seedlings in a concentration-dependent manner. The effect of H(2)O(2) on seedling growth was removed by incubation with 10 microm ABA. The H(2)O(2)-pretreatment produced an increase in ascorbate peroxidase (APX), peroxidase (POX) and ascorbate oxidase (AAO). The increases in these ascorbate-oxidizing enzymes correlated with the increase in the growth of the pea seedlings as well as with the decrease in the redox state of ascorbate. Moreover, the increase in APX activity was due to increases in the transcript levels of cytosolic and stromal APX (cytAPX, stAPX). The proteomic analysis showed that H(2)O(2) induced proteins related to plant signalling and development, cell elongation and division, and cell cycle control. A strong correlation between the effect of H(2)O(2) on plant growth and the decreases in ABA and zeatin riboside (ZR) was observed. The results suggest an interaction among the redox state and plant hormones, orchestrated by H(2)O(2), in the induction of proteins related to plant signalling and development during the early growth of pea seedlings.