Pedro Díaz-Vivancos

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.

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.

Understanding the role of H2O2 during pea seed germination: a combined proteomic and hormone profiling approach

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H(2)O(2)) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mm H(2)O(2) on several oxidative features such as protein carbonylation, endogenous H(2)O(2) and lipid peroxidation levels. We report that H(2)O(2) treatment of the pea seeds increased their endogenous H(2)O(2) content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H(2)O(2) in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H(2)O(2) , whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H(2)O(2) in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels.

A different role for hydrogen peroxide and the antioxidative system under short and long salt stress in Brassica oleracea roots

Salinity affects normal growth and development of plants depending on their capacity to overcome the induced stress. The present study was focused on the response and regulation of the antioxidant defence system in Brassica oleracea roots under short and long salt treatments. The function and the implications of hydrogen peroxide as a stressor or as a signalling molecule were also studied. Two different zones were analysed—the elongation and differentiation zone and the fully differentiated root zone—in order to broaden the knowledge of the different effects of salt stress in root. In general, an accumulation of hydrogen peroxide was observed in both zones at the highest (80 mM NaCl) concentration. A higher accumulation of hydrogen peroxide was observed in the stele of salt-treated roots. At the subcellular level, mitochondria accumulated hydrogen peroxide in salt-treated roots. The results confirm a drastic decrease in the antioxidant enzymes catalase, ascorbate peroxidase, and peroxidases under short salt treatments. However, catalase and peroxidase activities were recovered under long salt stress treatments. The two antioxidant molecules analysed, ascorbate and glutathione, showed a different trend during salt treatments. Ascorbate was progressively accumulated and its redox state maintained, but glutathione was highly accumulated at 24 h of salt treatment, but then its concentration and redox state progressively decreased. Concomitantly, the antioxidant enzymes involved in ascorbate and glutathione regeneration were modified under salt stress treatments. In conclusion, the increase in ascorbate levels and the maintenance of the redox state seem to be critical for root growth and development under salt stress.

Glutathione – linking cell proliferation to oxidative stress

SIGNIFICANCE The multifaceted functions of reduced glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) continue to fascinate plants and animal scientists, not least because of the dynamic relationships between GSH and reactive oxygen species (ROS) that underpin reduction/oxidation (redox) regulation and signalling. Here we consider the respective roles of ROS and GSH in the regulation of plant growth, with a particular focus on regulation of the plant cell cycle. Glutathione is discussed not only as a crucial low molecular weight redox buffer that shields nuclear processes against oxidative challenge but also a flexible regulator of genetic and epigenetic functions. RECENT ADVANCES The intracellular compartmentalization of GSH during the cell cycle is remarkably consistent in plants and animals. Moreover, measurements of in vivo glutathione redox potentials reveal that the cellular environment is much more reducing than predicted from GSH/GSSG ratios measured in tissue extracts. The redox potential of the cytosol and nuclei of non-dividing plant cells is about -300 mV. This relatively low redox potential maintained even in cells experiencing oxidative stress by a number of mechanisms including vacuolar sequestration of GSSG. We propose that regulated ROS production linked to glutathione-mediated signalling events are the hallmark of viable cells within a changing and challenging environment. CRITICAL ISSUES The concept that the cell cycle in animals is subject to redox controls is well established but little is known about how ROS and GSH regulate this process in plants. However, it is increasingly likely that redox controls exist in plants, although possibly through different pathways. Moreover, redox-regulated proteins that function in cell cycle checkpoints remain to be identified in plants. While GSH-responsive genes have now been identified, the mechanisms that mediate and regulate protein glutathionylation in plants remain poorly defined. FUTURE DIRECTIONS The nuclear GSH pool provides an appropriate redox environment for essential nuclear functions. Future work will focus on how this essential thiol interacts with the nuclear thioredoxin system and nitric oxide to regulate genetic and epigenetic mechanisms. The characterization of redox-regulated cell cycle proteins in plants, and the elucidation of mechanisms that facilitate GSH accumulation in the nucleus are keep steps to unravelling the complexities of nuclear redox controls.

Low glutathione regulates gene expression and the redox potentials of the nucleus and cytosol in Arabidopsis thaliana

Reduced glutathione (GSH) is considered to exert a strong influence on cellular redox homeostasis and to regulate gene expression, but these processes remain poorly characterized. Severe GSH depletion specifically inhibited root meristem development, while low root GSH levels decreased lateral root densities. The redox potential of the nucleus and cytosol of Arabidopsis thaliana roots determined using roGFP probes was between -300 and -320 mV. Growth in the presence of the GSH-synthesis inhibitor buthionine sulfoximine (BSO) increased the nuclear and cytosolic redox potentials to approximately -260 mV. GSH-responsive genes including transcription factors (SPATULA, MYB15, MYB75), proteins involved in cell division, redox regulation (glutaredoxinS17, thioredoxins, ACHT5 and TH8) and auxin signalling (HECATE), were identified in the GSH-deficient root meristemless 1-1 (rml1-1) mutant, and in other GSH-synthesis mutants (rax1-1, cad2-1, pad2-1) as well as in the wild type following the addition of BSO. Inhibition of auxin transport had no effect on organ GSH levels, but exogenous auxin decreased the root GSH pool. We conclude that GSH depletion significantly increases the redox potentials of the nucleus and cytosol, and causes arrest of the cell cycle in roots but not shoots, with accompanying transcript changes linked to altered hormone responses, but not oxidative stress.

Salicylic acid negatively affects the response to salt stress in pea plants

We studied the effect of salicylic acid (SA) treatment on the response of pea plants to salinity. Sodium chloride (NaCl)-induced damage to leaves was increased by SA, which was correlated with a reduction in plant growth. The content of reduced ascorbate and glutathione in leaves of salt-treated plants increased in response to SA, although accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxide also occurred in leaves of salt-exposed plants treated with SA. In the absence of NaCl, SA increased ascorbate peroxidase (APX; 100 μm) and glutathione-S transferase (GST; 50 μm) activities and increased catalase (CAT) activity in a concentration-dependent manner. Salinity decreased glutathione reductase (GR) activity, but increased GST and CAT activity. In salt-stressed plants, SA also produced changes in antioxidative enzymes: 100 μm SA decreased APX but increased GST. Finally, a concentration-dependent increase in superoxide dismutase (SOD) activity was induced by SA treatment in salt-stressed plants. Induction of PR-1b was observed in NaCl-stressed plants treated with SA. The treatment with SA, as well as the interaction between salinity and SA treatment, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mm NaCl, but was statistically higher in the absence than in the presence of SA. Overall, the results show that SA treatment negatively affected the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidant metabolism. The data also show that SA treatment could enhance the resistance of salt-stressed plants to possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression.

Elucidating hormonal/ROS networks during seed germination: insights and perspectives

While authors have traditionally emphasized the deleterious effects of reactive oxygen species (ROS) on seed biology, their role as signaling molecules during seed dormancy alleviation and germination is now the focus of many studies around the world. Over the last few years, studies using “-omics” technologies together with physiological and biochemical approaches have revealed that seed germination is a very complex process that depends on multiple biochemical and molecular variables. The pivotal role of phytohormones in promoting germination now appears to be interdependent with ROS metabolism, involving mitogen-activated protein kinase cascade activation, gene expression and post-translational protein modifications. This review is, thus, an attempt to summarize the new discoveries involving ROS and seed germination. The study of these interactions may supply markers of seed quality that might eventually be used in breeding programs to improve crop yields.

Effect of arbuscular mycorrhizae and induced drought stress on antioxidant enzyme and nitrate reductase activities in Juniperus oxycedrus L. grown in a composted sewage sludge-amended semi-arid soil

We studied the influence of inoculation with a mixture of three exotic arbuscular mycorrhizal (AM) fungi, Glomus intraradices Schenck & Smith, Glomus deserticola Trappe, Bloss. & Menge and Glomus mosseae (Nicol & Gerd.) Gerd. & Trappe, and the addition of composted sewage sludge (SS) on the activities of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and total peroxidase (POX) and of shoot and root nitrate reductase (NR, EC 1.6.6.1) in Juniperus oxycedrus L. seedlings, an evergreen shrub, grown in a non-sterile soil under well-watered and drought-stress conditions. Both the inoculation with exotic AM fungi and the addition of composted SS stimulated significantly growth and the N and P contents in shoot tissues of J. oxycedrus with respect to the plants neither inoculated nor treated with composted SS that were either well-watered or droughted. Under drought-stress conditions, only inoculation with exotic AM fungi increased shoot and root NR activity (about 188% and 38%, respectively, with respect to the plants neither inoculated nor treated with composted SS). Drought increased the POX and SOD activities in both shoots of J. oxycedrus seedlings inoculated with exotic AM fungi and grown with composted SS, but the increase was less than in the plants neither inoculated nor treated with SS. Both the plants inoculated with exotic AM fungi and the plants grown with composted SS developed additional mechanisms to avoid oxidative damage produced under water-shortage conditions.

Antioxidant enzyme activities and hormonal status in response to Cd stress in the wetland halophyte Kosteletzkya virginica under saline conditions

Salt marshes constitute major sinks for heavy metal accumulation but the precise impact of salinity on heavy metal toxicity for halophyte plant species remains largely unknown. Young seedlings of Kosteletzkya virginica were exposed during 3 weeks in nutrient solution to Cd 5 µM in the presence or absence of 50 mM NaCl. Cadmium (Cd) reduced growth and shoot water content and had major detrimental effect on maximum quantum efficiency (F(v) /F(m) ), effective quantum yield of photosystem II (Y(II)) and electron transport rates (ETRs). Cd induced an oxidative stress in relation to an increase in O(2) (•-) and H(2) O(2) concentration and lead to a decrease in endogenous glutathione (GSH) and α-tocopherol in the leaves. Cd not only increased leaf zeatin and zeatin riboside concentration but also increased the senescing compounds 1-aminocyclopropane-1-carboxylic acid (ACC) and abscisic acid (ABA). Salinity reduced Cd accumulation already after 1 week of stress but was unable to restore shoot growth and thus did not induce any dilution effect. Salinity delayed the Cd-induced leaf senescence: NaCl reduced the deleterious impact of Cd on photosynthesis apparatus through an improvement of F(v) /F(m) , Y(II) and ETR. Salt reduced oxidative stress in Cd-treated plants through an increase in GSH, α-tocopherol and ascorbic acid synthesis and an increase in glutathione reductase (EC 1.6.4.2) activity. Additional salt reduced ACC and ABA accumulation in Cd+NaCl-treated leaves comparing to Cd alone. It is concluded that salinity affords efficient protection against Cd to the halophyte species K. virginica, in relation to an improved management of oxidative stress and hormonal status.