Gregorio Barba-Espín

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.

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.

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.

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.

Characterization of the antioxidant system during the vegetative development of pea plants

The antioxidative system was studied during the development of pea plants. The reduced glutathione (GSH) content was higher in shoots than in roots, but a greater redox state of glutathione existed in roots compared with shoots, at least after 7 d of growth. The 3-d-old seedlings showed the highest content of oxidised ascorbate (DHA), which correlated with the ascorbate oxidase (AAO) activity. Also, the roots exhibited higher DHA content than shoots, correlated with their higher AAO activity. The activities of antioxidant enzymes were much higher in shoots than in roots. Ascorbate peroxidase (APX) activity decreased during the progression of growth in both shoots and roots, whereas peroxidase (POX) activity strongly increased in roots, reflecting a correlation between POX activity and the enhancement of growth. Catalase activity from shoots reached values nearly 3 or 4-fold higher than in roots. The monodehydroascorbate reductase (MDHAR) activity was higher in young seedlings than in more mature tissues, and in roots a decrease in MDHAR was noticed at the 11th day. No dehydroascorbate reductase (DHAR) was detected in roots from the pea plants and DHAR values detected in seedlings and in shoots were much lower than those of MDHAR. In shoots, GR decreased with the progression of growth, whereas in roots an increase was seen on the 9th and 11th days. Finally, superoxide dismutase (SOD) activity increased in shoots during the progression of growth, but specific SOD activity was higher in roots than in shoots.

Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants

The possible involvement of polyamines in the salt stress adaptation was investigated in grapevine (Vitis vinifera L.) plantlets focusing on photosynthesis and oxidative metabolism. Salt stress resulted in the deterioration of plant growth and photosynthesis, and treatment of plantlets with methylglyoxal-bis(guanylhydrazone) (MGBG), a S-adenosylmethionine decarboxylase (SAMDC) inhibitor, enhanced the salt stress effect. A decrease in PSII quantum yield (Fv/Fm), effective PSII quantum yield (Y(II)) and coefficient of photochemical quenching (qP) as well as increases in non-photochemical quenching (NPQ) and its coefficient (qN) was observed by these treatments. Salt and/or MGBG treatments also triggered an increase in lipid peroxidation and reactive oxygen species (ROS) accumulation as well as an increase of superoxide dismutase (SOD) and peroxidase (POX) activities, but not ascorbate peroxidase (APX) activity. Salt stress also resulted in an accumulation of oxidized ascorbate (DHA) and a decrease in reduced glutathione. MGBG alone or in combination with salt stress increased monodehydroascorbate reductase (MDHAR), SOD and POX activities and surprisingly no accumulation of DHA was noticed following treatment with MGBG. These salt-induced responses correlated with the maintaining of high level of free and conjugated spermidine and spermine, whereas a reduction of agmatine and putrescine levels was observed, which seemed to be amplified by the MGBG treatment. These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.

Role of H2O2 in pea seed germination

The imbibition of pea seeds with hydrogen peroxide (H2O2) increased the germination as well as the seedling growth, producing an invigoration of the seeds. We propose that H2O2 could acts as signaling molecule in the beginning of seed germination involving specific changes at proteomic, transcriptomic and hormonal levels. These findings have practical implication in the context of seed priming technologies to invigorate low vigour seeds.

Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

Overlapping responses of the aleurone layer proteome to tunicamycin and heat shock identify components of the plant protein secretory machinery. The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion.