Biochemical study of the effect of stress conditions on the mandelonitrile-associated salicylic acid biosynthesis in peach.

Salicylic acid (SA) plays a central role in plant responses to environmental stresses. In a recent study, we suggested a third pathway for SA biosynthesis from mandelonitrile (MD) in peach plants. This pathway is an alternative to the phenylalanine ammonia-lyase pathway and links SA biosynthesis and cyanogenesis. In the present work, using biochemical approaches, we studied the effect of salt stress and Plum pox virus (PPV) infection on this proposed SA biosynthetic pathway from MD. Peach plants were submitted to salt stress and Plum pox virus (PPV) infection. We studied the levels of SA and its intermediates/precursors (phenylalanine, MD, amygdalin and benzoic acid) in in vitro shoots. Moreover, in peach seedlings, we analysed the content of H2 O2 -related enzymes, SA and the stress-related hormones abscisic acid and jasmonic acid. We showed that the contribution of this SA biosynthetic pathway from MD to the total SA pool does not seem to be important under the stress conditions assayed. Nevertheless, MD treatment not only affected the SA content, but also had a pleiotropic effect on abscisic acid and jasmonic acid levels. Furthermore, MD modulates the antioxidative metabolism via SA-dependent or -independent redox-related signalling pathways. Even though the proposed SA biosynthetic pathway seems to be functional under stress conditions, MD, and hence cyanogenic glycosides, may be operating more broadly than by influencing SA pathways and signalling. Thus, the physiological function of the proposed SA biosynthetic pathway remains to be elucidated.

The effect of mandelonitrile, a recently described salicylic acid precursor, on peach plant response against abiotic and biotic stresses.

In a previous work, we observed that mandelonitrile (MD), which controls cyanogenic glycoside turnover, is involved in salicylic acid (SA) biosynthesis in peach plants. In order to gain knowledge about the possible roles of this SA biosynthetic pathway, this current study looks at the effect of MD and phenylalanine (Phe; MD precursor) treatments on peach plant performance from an agronomic point of view. Abiotic (2 g·l-1 NaCl) and biotic (Plum pox virus, PPV) stresses were assayed. We recorded the following chlorophyll fluorescence parameters: quantum yield of photochemical energy conversion in PSII [Y(II)], photochemical quenching (qP) and quantum yield of regulated non-photochemical energy loss in PSII and its coefficient [Y(NPQ) and qN]. In addition, considering that environmental stresses lead to nutritional disorders, we determined the soluble K+ , Ca2+ , Na+ and Cl- concentrations in NaCl-stressed seedlings. In PPV-infected seedlings, we recorded the Ca2+ level, which has been suggested to play critical roles in regulating SA-related plant defence responses against pathogens. The MD treatment lessened the effect of both stresses on plant development. In addition, an increase in non-photochemical quenching parameters was observed in MD-treated seedlings, suggesting a safer dissipation of excess energy under stress conditions. In NaCl-stressed peach seedlings both treatments stimulated the accumulation of phytotoxic ions in roots, whereas in PPV-infected seedlings MD increased Ca2+ content. Our results suggest that MD and Phe influence the response of peach seedlings to the deleterious effects of salt and PPV infection stresses.

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.

Benzothiadiazole and l-2-oxothiazolidine-4-carboxylic acid reduce the severity of Sharka symptoms in pea leaves: effect on antioxidative metabolism at the subcellular level.

The effect of treatment with benzothiadiazole (BTH) or l-2-oxothiazolidine-4-carboxylic acid (OTC), and their interaction with Plum pox virus (PPV) infection, on antioxidative metabolism of pea plants was studied at the subcellular level. PPV infection produced a 20% reduction in plant growth. Pre-treatment of pea plants with OTC or BTH afforded partial protection against PPV infection, measured as the percentage of leaves showing symptoms, but neither BTH nor OTC significantly reduced the virus content. PPV infection caused oxidative stress, as monitored by increases in lipid peroxidation and protein oxidation in soluble and chloroplastic fractions. In leaves of non-infected plants, OTC increased the content of reduced glutathione (GSH) and total glutathione; accordingly, an increase in the redox state of glutathione was observed. An increase in oxidized glutathione (GSSG) was found in symptomatic leaves from infected plants. A similar increase in GSSG was also observed in asymptomatic leaves from infected, untreated plants. However, no changes in GSSG occurred in asymptomatic leaves from infected plants treated with BTH and OTC and, accordingly, a higher redox state of GSH was recorded in those leaves, which could have had a role in the reduction of symptoms, as observed in asymptomatic leaves from infected plants treated with BTH or OTC. Treatment with BTH or OTC had some effect on antioxidant enzymes in soluble and chloroplastic fractions from infected pea leaves. An increase in antioxidative mechanisms, such as GSH-related enzymes (DHAR, GR and G6PDH), as well as APX and POX, at the subcellular level was observed, which could play a role in reducing the severity of cellular damage induced by Sharka in pea leaves.

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.

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-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (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. It is concluded 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 plants.