Polimorfismo enzimático de peroxidasas en hipocotilos de altramuz blanco / Alfonso Ros Barceló ; Dirección Muñoz Girón.

Tesis - Universidad de Murcia.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TM 3274

The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees

The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H2O2 levels in lignifying tissues, whose capacity to produce and to accumulate H2O2 is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H2O2 in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O2 to remove ASC, it could regulate O2 availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H2O2 production in lignifying tissues) at substrate level, by controlling the tension of O2. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.This work was supported by grants from the MCYT (HI2001-0064 and BOS2002-03550). LVGR hold a FPI fellowship from the MCYT.Peer reviewe

Oxidative stress induced by long-term plum pox virus infection in peach (Prunus persica)

10 pages, 4 figures, 4 tables.In this study, the effect of long-term plum pox virus (PPV) infection on the response of certain antioxidant enzymes at the subcellular level was studied in peach plants (Prunus persica (L.) Batch) (cv. GF305), which are characterized by great susceptibility to the virus. In infected plants, a decrease in the efficiency of excitation energy capture by PSII (Fv'/Fm') was observed, which was accompanied by a decrease in non-photochemical quenching (NPQ). p-Hydroxy-mercury benzoic acid (pHMB)-insensitive ascorbate peroxidase (APX) activity (class III peroxidase) was detected in both chloroplast and soluble fractions. In soluble fractions from inoculated peaches, a significant increase in pHMB-sensitive APX activity and a significant decrease in superoxide dismutase (SOD) activity were observed. These changes were correlated with the observations in isolated chloroplasts, where an increase in both pHMB-sensitive and pHMB-insensitive APX activities was observed, whereas significant decreases in SOD, monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activities were produced. According to these results, as a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and protein oxidation, was produced in peach leaves, which was monitored by the diaminobenzidine (DAB) peroxidase-coupled H2O2 probe. PPV infection produced an alteration in chloroplast ultrastructure, giving rise to dilated thylakoid membranes. PPV-infected peach leaves showed a decreased amount of starch in chloroplasts from palisade parenchyma, as well as an increase in the number and size of plastoglobuli, in relation to control plants. The results suggest that long-term PPV infection produces an oxidative stress, and that an antioxidative metabolism imbalance may be related to the progress of PPV infection and symptoms in peach plants.This research was supported by grant AGL 2002-02115 from the CICYT (Spanish Ministry of Science and Technology).Peer reviewe

Long-term plum pox virus infection produces an oxidative stress in a susceptible apricot, Prunus armeniaca, cultivar but not in a resistant cultivar

The effect of plum pox virus (PPV) infection on the response of some antioxidant enzymes was studied in two apricot cultivars, which behaved differently against PPV infection: cultivar Real Fino (susceptible) and cultivar Stark Early Orange (cv. SEO, resistant). In the susceptible cultivar, PPV produced a decrease in ΦPSII, F'v/F'm and Qp. PPV infection produced a drop in p-hydroxy mercury benzoic acid (pHMB)-sensitive ascorbate peroxidase, dehydroascorbate reductase and peroxidase in the soluble fraction from susceptible plants, whereas in the resistant apricot cultivar, pHMB-insensitive ascorbate peroxidase, monodehydroascorbate reductase, glutathione reductase and superoxide dismutase increased. However, catalase decreased in the soluble fractions from both infected cultivars. Long-term PPV infection also produced a decrease in the chloroplastic ascorbate–glutathione cycle enzymes only in the susceptible plants. As a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and in protein oxidation, was produced only in the leaves from the susceptible cultivar which was also monitored by the diaminobenzidine peroxidase-coupled H2O2 probe. The loss of ΦPSII, indicative of activated oxygen species production, and the decrease in the levels of antioxidant enzymes in chloroplasts from susceptible plants could be responsible for the chlorosis symptoms observed. The results suggest that the higher antioxidant capacity showed by cv. SEO could be a consequence of a systemic acquired resistance induced by PPV penetration in stem tissue at the graft site and could be related, among other factors, to their resistance to PPV.This research was supported by grant AGL 2002-02115 from the CICYT (Spanish Ministry of Education and Science).Peer reviewe

The apoplastic antioxidant system in Prunus: Response to long-term Plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to Plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxy mercury benzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (Class I APX) and pHMB-insensitive APX (Class III APX), superoxide dismutase (SOD), peroxidases (POX), NADH-POX and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in Class I APX, POX, NADH-POX and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in Class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. We conclude that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plantsThis work has been supported by grant AGL-2002-02115 from the CICYT (Comisión Interministerial de Ciencia y Tecnología) of Spain and grant 23BIO2005-04-6444 from the Comunidad de Murcia (Spain). P.M. Periago thanks the Ministerio de Ciencia y Tecnología for her contract in the Programme “Ramón y Cajal”.Peer reviewe