Salicylic acid changes the properties of extracellular peroxidase activity secreted from wounded wheat (Triticum aestivum L.) roots

Summary.  Wheat (Triticum aestivum L.) roots released proteins showing peroxidase activity in the apoplastic solution in response to wound stress. Preincubation of excised roots with 1 mM salicylic acid at pH 7.0 enhanced the guaiacol peroxidase activity of the extracellular solution (so-called extracellular peroxidase). The soluble enzymes were partially purified by precipitation with ammonium sulfate followed by size exclusion and ion exchange chromatography. Despite an increase in the total activity of secreted peroxidase induced by pretreatment of excised roots with salicylic acid, the specific activity of the partially purified protein was significantly lower compared to that of the control. Purification of the corresponding proteins by ion exchange chromatography indicates that several isoforms of peroxidase occurred in both control and salicylic acid-treated samples. The activities of the extracellular peroxidases secreted by the salicylic acid-treated roots responded differently to calcium and lectins compared with those from untreated roots. Taken together, our data suggest that salicylic acid changes the isoforms of peroxidase secreted by wounded wheat roots. Received June 10, 2002; accepted September 24, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.     

Inhibition of catalase activity by oxidative stress and its relationship to salicylic acid accumulation in plants

The decrease in catalase activity and its relationship to change in salicylic acid content were investigated in rice, wheat, and cucumber seedlings exposed to oxidative stresses. A decrease in chlorophyll fluorescence (F/Fm), measured as an indicator of the oxidative stress, and a drop in catalase activity were observed following treatment with NaCl in all plant seedlings tested . Furthermore, such decreases in F/Fm and catalase activity were also observed under low temperature conditions in both rice cultivars, whereas the degrees of decrease were dependent on their low temperature tolerance . Although the content of salicylic acid increased in rice seedlings stressed by NaCl treatment, it was inversely correlated with the decrease in the catalase activity . Such a relationship between the decrease in catalase activity and increase in salicylic acid content was confirmed with paraquat treatment of the rice seedlings . These results suggested that the fall in catalase activity is a phenomenon occurring in many plant species under oxidative stress and is related to the accumulation of salicylic acid in oxidatively-stressed plants.     

水杨酸对黄瓜叶片抗氧化剂酶系的调节作用

分析了水杨酸（ＳＡ）对黄瓜（ＣｕｃｕｍｉｓｓａｔｉｖｕｓＬ．）叶片抗氧化剂酶系活性及活性氧水平的调节作用。不同浓度的ＳＡ（０．５ｍｍｏｌ／Ｌ、１ｍｍｏｌ／Ｌ、２．５ｍｍｏｌ／Ｌ、５ｍｍｏｌ／Ｌ）均能显著地提高被处理叶片超氧化物歧化酶（ＳＯＤ）和过氧化物酶（ＰＯＤ）活性,而且还能诱导同株的非处理叶片中ＳＯＤ和ＰＯＤ活性增加。用１ｍｍｏｌ／ＬＳＡ处理第一片真叶,在处理后６～７２ｈ,ＰＯＤ活性增加了２２％～６７％,同株非处理的第二片真叶ＰＯＤ活性增加了１４％～８６％,但是,在ＳＡ处理后３ｈ之前以及处理９６ｈ之后,ＰＯＤ活性没有变化。ＳＡ能够显著降低超氧物阴离子含量和提高过氧化氢水平,但它对过氧化氢酶（ＣＡＴ）活性的抑制作用很弱,表明ＳＡ提高体内过氧化氢含量的原因主要是通过提高ＳＯＤ活性而不是抑制ＣＡＴ活性。同工酶分析表明,ＳＡ不能诱导新的ＳＯＤ同工酶,但可以诱导新的ＰＯＤ同工酶。     

Determination of aspirin and salicylic acid in transdermal perfusates

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of aspirin and salicylic acid in transdermal perfusates. The compounds were separated on a C₈ Nucleosil column (5 μm, 250×4.6 mm) using a mobile phase containing a mixture of water–acetonitrile–orthophosphoric acid (650:350:2, v/v/v) and a flow-rate of 1 ml/min. The transdermal samples were in phosphate-buffered saline (PBS) and could be injected directly onto the HPLC system. The method was reproducible with inter-day R.S.D. values of no greater than 3.46 and 2.60% for aspirin and salicylic acid, respectively. The method was linear over the concentration range 0.2–5.0 μg/ml and had a limit of detection of 0.05 μg/ml for both compounds. For certain samples, it was necessary to ensure that no transmembrane leakage of the aspirin prodrugs had occurred. In these cases, a gradient was introduced by increasing the acetonitrile content of the mobile phase after the salicylic acid had eluted. The method has been applied to the determination of aspirin and salicylic acid in PBS following in vitro application of the compounds to mouse skin samples.     

A geometrical model of dermal capillary clearance

A new microscopic model is developed to describe the dermal capillary clearance process of skin permeants. The physiological structure is represented in terms of a doubly periodic array of absorbing capillaries. Convection-dominated transport in the blood flow within the capillaries is coupled with interstitial diffusion, the latter process being quantified via a slender-body-theory approach. Convection across the capillary wall and in the interstitial phase is treated as a perturbation which may be added to the diffusive transport. The model accounts for the finite permeability of the capillary wall as well as for the geometry of the capillary array, based on realistic values of physiological parameters. Calculated dermal concentration profiles for permeants having the size and lipophilicity of salicylic acid and glucose illustrate the power and general applicability of the model. Furthermore, validation of the model with published in vivo experimental results pertaining to human skin permeation of hydrocortisone is presented. The model offers the possibility for in-depth theoretical understanding and prediction of subsurface drug distribution in the human skin following topical application, as well as rates of capillary clearance into the systemic circulation. A simpler approach that treats the capillary bed as a homogeneously absorbing zone is also employed. The latter may be used in conjunction with the capillary exchange model to estimate measurable dermal transport and clearance parameters in a straightforward manner.     

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.     

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.     

AtPTR3, a wound-induced peptide transporter needed for defence against virulent bacterial pathogens in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mutation in the wound-induced peptide transporter gene AtPTR3 (At5g46050) of Arabidopsis thaliana has been shown to affect germination on media containing a high salt concentration. The heterologous expression in yeast was utilized to verify that the AtPTR3 protein transports di-and tripeptides. The T-DNA insert in the Atptr3-1 mutant in the Arabidopsis ecotype C24 revealed two T-DNA copies, the whole vector sequence, and the gus marker gene inserted in the second intron of the AtPTR3 gene. An almost identical insertion site was found in the Atptr3-2 mutant of the Col-0 ecotype. The AtPTR3 expression was shown to be regulated by several signalling compounds, most clearly by salicylic acid (SA), but also methyl jasmonate (MeJA) and abscisic acid. Real-time PCR experiments suggested that the wound-induction of the AtPTR3 gene was abolished in the SA and JA signalling mutants. The Atptr3 mutant plants had increased susceptibility to virulent pathogenic bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tomato, and produced more reactive oxygen species when grown on media containing paraquat or rose bengal. Public microarray data suggest that the AtPTR3 expression was induced by Pseudomonas elicitors and by avirulent P. syringae pathovars and type III secretion mutants. This was verified experimentally for the hrpA mutant with real-time PCR. These results suggest that AtPTR3 is one of the defence-related genes whose expression is reduced by virulent bacterium by type III dependent fashion. Our results suggest that AtPTR3 protects the plant against biotic and abiotic stresses.