Competence for Elicitation of H2O2 in Hypocotyls of Cucumber Is Induced by Breaching the Cuticle and Is Enhanced by Salicylic Acid

To study H2O2 production, the epidermal surfaces of hypocotyl segments from etiolated seedlings of cucumber (Cucumis sativus L.) were gently abraded. Freshly abraded segments were not constitutively competent for rapid H2O2 elicitation. This capacity developed subsequent to abrasion in a time-dependent process that was greatly enhanced in segments exhibiting an acquired resistance to penetration of their epidermal cell walls by Colletotrichum lagenarium, because of root pretreatment of the respective seedlings with 2,6-dichloroisonicotinic acid. When this compound or salicylic acid was applied to abraded segments, it also greatly enhanced the induction of competence for H2O2 elicitation. This process was fully inhibited by 5 [mu]M cycloheximide or 200 [mu]M puromycin, suggesting a requirement for translational protein synthesis. Both a crude elicitor preparation and a partially purified oligoglucan mixture from Phytophthora sojae also induced, in addition to H2O2 production, a refractory state, which explains the transient nature of H2O2 elicitation. Taken together, these results suggest that the cucumber hypocotyl epidermis becomes conditioned for competence to produce H2O2 in response to elicitors by a stimulus resulting from breaching the cuticle and/or cutting segments. This conditioning process is associated with protein synthesis and is greatly enhanced when substances able to induce systemic acquired resistance are present in the tissue.     

Pretreatment of Parsley Suspension Cultures with Salicylic Acid Enhances Spontaneous and Elicited Production of H2O2

Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to study the regulation of extracellular H2O2. After resuspension, the washed cells regulated the H2O2 concentration spontaneously to a constant level that was greatly increased when the cultures were pretreated for 1 d with salicylic acid (SA). The H2O2 level was further increased on addition of a fungal elicitor preparation, macromolecular chitosan, the sterol-binding polyene macrolide amphotericin B, the G protein-activating peptide mastoparan, or La3+. In all cases, this induced H2O2 burst was also greatly enhanced in cell suspensions pretreated with SA. Both the spontaneous and the induced H2O2 production were decreased by the protein kinase inhibitor K-252a. It is suggested that production of extracellular H2O2 occurs by an endogenously controlled plasma membrane enzyme complex that requires continuous phosphorylation for function and whose activity is increased by pretreatment of the cells with SA. This system can also receive various external stimuli, including those resulting from binding of fungal elicitor. SA can induce acquired resistance against pathogens. The conditioning of the parsley suspension culture by SA represents, therefore, a model for the long-term regulation of apoplastic H2O2 concentration by this signal substance, as suggested previously for the wound hormone methyl jasmonate.     

Cutin Monomers and Surface Wax Constituents Elicit H2O2 in Conditioned Cucumber Hypocotyl Segments and Enhance the Activity of Other H2O2 Elicitors

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface and cut segments were conditioned to develop competence for H₂O₂ elicitation. Alkaline hydrolysates of cutin from cucumber, tomato, and apple elicited H₂O₂ in such conditioned segments. The most active constituent of cucumber cutin was identified as dodecan-1-ol, a novel cutin monomer capable of forming hydrophobic terminal chains. Additionally, the cutin hydrolysates enhanced the activity of a fungal H₂O₂ elicitor, similar to cucumber surface wax, which contained newly identified alkan-1,3-diols. The specificity of elicitor and enhancement activity was further elaborated using some pure model compounds. Certain saturated hydroxy fatty acids were potent H₂O₂ elicitors as well as enhancers. Some unsaturated epoxy and hydroxy fatty acids were also excellent H₂O₂ elicitors but inhibited the fungal elicitor activity. Short-chain alkanols exhibited good elicitor and enhancer activity, whereas longer-chain alkan-1-ols were barely active. The enhancement effect was also observed for H₂O₂ elicitation by ergosterol and chitosan. The physiological significance of these observations might be that once the cuticle is degraded by fungal cutinase, the cutin monomers may act as H₂O₂ elicitors. Corrosion of cutin may also bring surface wax constituents in contact with protoplasts and enhance elicitation.     

Induction of competence for elicitation of defense responses in cucumber hypocotyls requires proteasome activity

The epidermal cells of hypocotyls from etiolated cucumber seedlings are not constitutively competent for elicitation of the rapid H2O2 defense response. However, elicitor competence developed while conditioning the surface-abraded seedlings by rotating them in buffer for 4 h. Competence development was greatly potentiated by inducers of systemic acquired resistance and suppressed by specific inhibitors of proteasome activity, clastolactacystin beta-lactone (LAC) and carboxybenzoyl-L-leucyl-L-leucyl-L-leucinal (LLL). In the freshly abraded seedlings, chitinase gene activation became evident approximately 4 h after elicitor addition. Accumulation of chitinase mRNA was enhanced upon conditioning prior to elicitation and was inhibited by LAC and LLL, indicating that the process which leads to H2O2 elicitation competence is also superimposed on the elicitation of chitinase mRNA. LAC and LLL caused an accumulation of ubiquitin-conjugated proteins and enhanced the expression of a proteasome alpha-subunit, suggesting that proteasome activity was specifically inhibited and that the effect observed on gene expression was not due to impaired gene induction in general. Together, our results suggest that the ubiquitin-proteasome system may play a crucial role in a process which switches the signaling pathway for diverse plant defense responses into a functional state, as is known for many basic cellular processes in both animals and yeast.     

Hydrogen peroxide from the oxidative burst is not involved in the induction of taxol biosynthesis in Taxus chinensis cells

In cell suspension cultures of Taxus chinensis, 40 mg/l fungal elicitor from Aspergillus niger and 20 microM HgCl2 elicited 5.7 and 3.6 mg/l taxol, which was a 9-fold and 5-fold increase vs. compared with the control, respectively. The fungal elicitor induced hydrogen peroxide (H2O2) accumulation but HgCl2 did not, indicating that H2O2 was not necessary for enhancement of taxol induced by elicitor. Compared with the treatment with fungal elicitor alone, exogenous catalase, ascorbic acid, diphenylene iodonium and superoxide dismutase induced a 0.45, 0.4, 0.7 and 1.4-fold H2O2, but elicited taxol production, which was 0.98, 1.2, 1.1 and 0.9-fold, respectively, vs. non-treated cells Elicitor-induced taxol production was not accorded with the amount of H2O2 production.     

Defense Responses in Infected and Elicited Cucumber (Cucumis sativus L.) Hypocotyl Segments Exhibiting Acquired Resistance

Segments from dark-grown cucumber (Cucumis sativus L.) hypocotyls were used to study defense reactions occurring upon fungal infection and induced by elicitors in the same tissue. The segments were rendered resistant to infection by Colletotrichum lagenarium either by growing the seedlings in the presence of dichloroisonicotinic acid (DCIA) or by preincubation of the cut segments with DCIA, salicylic acid (SA), or 5-chlorosalicylic acid (5CSA). This resistance appears to be due mainly to inhibition of fungal penetration into epidermal cells. In the resistant hypocotyl segments, the fungus induced, at the time of attempted penetration, an increased deposition of phenolics, which were visualized by autofluorescence. These phenolics were located mainly in the epidermal cell wall around and in the emerging papillae below appressoria and were quantified either as lignin-like polymers by the thioglycolic acid method or as 4-OH-benzaldehyde, 4-OH-benzoic, or 4-coumaric acid liberated upon treatment with alkali at room temperature. Pretreatment with DCIA, SA, and 5CSA induced little chitinase activity, but this activity greatly increased in resistant tissues upon subsequent infection. These observations indicate that resistance is associated with an improved perception of the pathogen stimulus resulting in the enhanced induction of diverse defense reactions. When the cut segments were pretreated with DCIA, SA, or 5CSA and then split and incubated with chitosan fragments, the deposition of cell wall phenolics was also enhanced. These pretreated and split segments also exhibited an increase in the rapid production of activated oxygen species induced by an elicitor preparation from Phytophthora megasperma f. sp. Glya. Pretreatment of the segments with methyl jasmonate neither induced resistance nor enhanced induction of cell wall phenolics upon fungal infection, although we observed in the corresponding split segments some increase in chitosan-induced cell wall phenolics and in elicitor-induced rapid production of activated oxygen species.     

Involvement of nitric oxide in elicitor-induced defense responses and secondary metabolism of Taxus chinensis cells

This work was to characterize the generation of nitric oxide (NO) in Taxus chinensis cells induced by a fungal elicitor extracted from Fusarium oxysporum mycelium and the signal role of NO in the elicitation of plant defense responses and secondary metabolite accumulation. The fungal elicitor at 10-100 microg/ml (carbohydrate equivalent) induced a rapid and dose-dependent NO production in the Taxus cell culture, which exhibited a biphasic time course, reaching the first plateau within 1 h and the second within 12 h of elicitor treatment. The NO donor sodium nitroprusside potentiated elicitor-induced H2O2 production and cell death but had little influence on elicitor-induced membrane K+ efflux and H+ influx (medium alkalinization). NO inhibitors Nomega-nitro-L-arginine and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide partially blocked the elicitor-induced H2O2 production and membrane ion fluxes. Moreover, the NO inhibitors suppressed elicitor-induced activation of phenylalanine ammonium-lyase and accumulation of diterpenoid taxanes (paclitaxel and baccatin III). These results suggest that NO plays a signal role in the elicitor-induced responses and secondary metabolism activities in the Taxus cells.     

Chitinase in cucumber hypocotyls is induced by germinating fungal spores and by fungal elicitor in synergism with inducers of acquired resistance

After root pretreatment with 2,6-dichloroisonicotinic acid (DCIA or INA), hypocotyls of etiolated cucumber seedlings acquired resistance to infection by Colletotrichum lagenarium caused by the failure of the fungus to penetrate epidermal cell walls. The hypocotyls contained only low levels of class III chitinase and its mRNA prior to infection. This pathogenesis-related (PR) gene was expressed strongly upon infection but only in resistant hypocotyls and soon after germination of the fungal spores. Chitinase was also induced early by an albino mutant strain of C. lagenarium that can barely penetrate the epidermis. Thus, early recognition of the fungus implies signal compounds able to pass, or being generated in, the hydrophobic epidermal surface. As the apoplastic chitinase accumulates timely at the site of a subsequent attack, it may contribute to disease resistance. The mechanism behind the enhanced responsiveness of epidermal cells was studied by gently abrading the cuticle of susceptible hypocotyls to allow permeation of a water-soluble polymeric fungal elicitor. Induction of chitinase occurred only when the elicitor was applied simultaneously with a resistance inducer such as DCIA, salicylic acid (SA) or a benzothiadiazole (BTH). In addition, long-term root pretreatment with DCIA conditioned the hypocotyls for enhanced elicitor responses. These results demonstrate that the above inducers of acquired resistance can affect expression of the cucumber chitinase gene not only as direct inducers. They can also act synergistically with fungal elicitors and, in addition, condition the hypocotyls in a developmental manner for potentiated elicitation.     

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.     

Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. Using elicitation and precursor feeding

The effect of biotic elicitors (yeast extract, chitosan), signaling molecule (salicylic acid), and polyamines (putrescine and spermidine) was studied with respect to isoflavones accumulation in hairy root cultures of Psoralea corylifolia L. Untreated hairy roots (control) accumulated 1.55% dry wt of daidzein and 0.19% dry wt of genistein. In precursor feeding experiment, phenylalanine at 2 mM concentration led to 1.3 fold higher production of daidzein (1.91% dry wt) and genistein (0.27% dry wt). In biotic elicitors, chitosan (2 mg/L) was found to be the most efficient elicitor to induce daidzein (2.78% dry wt) and genistein (0.279% dry wt) levels in hairy roots. Salicylic acid at 1 mM concentration stimulated the maximum accumulation of daidzein (2.2% dry wt) and genistein (0.228% dry wt) 2 days after elicitation. In case of polyamines, putrescine (50 mM) resulted in highest accumulation of daidzein (3.01% dry wt) and genistein (0.227% dry wt) after 5 days of addition. Present results indicated the effectiveness of elicitation and precursor feeding on isoflavones accumulation in hairy roots of P. corylifolia. This is the first report of elicitation on isoflavones production by hairy roots of P. corylifolia.     

Early Responses in Methyl Jasmonate-Preconditioned Cells toward Pathogen-Derived Elicitors

Early, signal transduction-related responses in cultured tobacco cells due to methyl jasmonate (MeJa), a cell-wall-derived elicitor from Phytophthora nicotianae and chitosan, were investigated. MeJa was an effective inducer of lipid peroxidation and lipoxygenase (LOX) activity with maximum levels reached within 2 h and 4–8 h, respectively. Chitosan and the elicitor induced a transient increase (1–4 h) in lipid peroxidation. Conditioning with MeJA, followed by secondary elicitation, led to a significant increase in malondialdehyde concentration after 1 h. Chitosan and the elicitor induced transient activation of LOX with maximal values between 8 and 12 h, with preconditioning resulting in a rapid increase in LOX activity at 4 h post elicitation. MeJA did not effect phosphoprotein accumulation but conditioning led to the potentiation and differential induction of phosphoproteins due to chitosan and elicitor. The results indicate that cells are sensitized by the exposure to MeJa to respond more intensely and rapidly toward secondary elicitation by fungal pathogen derived elicitors.     

茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞中紫杉醇含量的影响

比较了茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞几个抗病相关指标（POD、CAT活力、H2O2含量）及紫杉醇含量的影响,3种信号分子的组合对POD、CAT、H2O2及紫杉醇含量的影响是不一致的,MJ单独添加,MJ与SA联合作用以及MJ与F5联合作用都可使POD活力增加,且12h后H2O2含量均升高,约在48h达到高峰,为对照的2倍左右,但72h后,MJ单独添加和MJ与SA联合作用组中H2O2含量变化不大,F5与MJ联合作用则使H2O2含量持续比对照高。MJ单独添加使CAT酶活在144h后才较对照低,F5、SA的加入都可使CAT酶活下降,SA的作用更显著。说明三者的诱导途径并不完全一样,以SA和MJ联合添加对紫杉醇合成的促进作用最大,含量达到细胞干重的0．04％。     

Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor

Effect of chitosan elicitor on growth and phenylethanoid glycosides (PeGs) accumulation in Cistanche deserticola cell suspension cultures was investigated. PeGs accumulation was dramatically improved by addition of selected chitosan at optimal elicitation conditions. Furthermore, a strategy of repeated addition of the chitosan elicitor for enhancing PeGs accumulation was developed. The chitosan elicitor of 10 mg l(-1)-medium repeatedly added on days 15 and 17 improved PeGs accumulation further, and the final PeGs production in the treated cell cultures of C. deserticola reached 364.6 mg l(-1), which was 3.4-fold higher than that of the control without elicitation. The increase of PeGs accumulation in C. deserticola cell suspension cultures was related to the increase of phenylalanine ammonium lyase activity stimulated by the chitosan elicitor.     

Early changes in membrane permeability, production of oxidative burst and modification of PAL activity induced by ergosterol in cotyledons of Mimosa pudica

Ergosterol (a fungal membrane component) was shown to induce transient influx of protons and membrane hyperpolarization in cotyledonary cells of Mimosa pudica L. By contrast, chitosan (a fungal wall component with known elicitor properties) triggered membrane depolarization. In the processes induced by ergosterol, a specific desensitization was observed, since cells did not react to a second ergosterol application but did respond to a chitosan treatment. This comparative study correspondingly shows that ergosterol and chitosan were perceived in a distinct manner by plant cells. Generation of O2*-, visualized by infiltration with nitroblue tetrazolium, was displayed in organs treated with ergosterol and chitosan. This AOS production was preceded by an increase in activity of NADPH oxidase measured in protein extracts of treated cotyledons. In all the previously described processes, cholesterol had no effect, thereby indicating that ergosterol specifically induced these physiological changes known to participate in the reaction chain activated by characteristic elicitors. Contrary to chitosan, ergosterol did not greatly activate secondary metabolism as shown by the small change in content of free phenolics and by the low modification in activity of PAL, the key enzyme of this metabolic pathway. Therefore, future studies have to clarify the signalling cascade triggered by ergosterol recognition.     

Some common signal transduction events are not necessary for the elicitor-induced accumulation of silymarin in cell cultures of Silybum marianum

A variety of pharmacological effectors of signal transduction pathways were used to investigate the elicitor-activated sequence of cellular responses by which yeast extract (YE) or methyljasmonate (MeJA) enhanced production of silymarin in cell cultures of Silybum marianum. As we recently showed that inhibition of external and internal calcium fluxes significantly increased flavonolignan production in S. marianum cultures, we examined whether calcium mediates signaling events leading to enhancement of silymarin production upon YE or MeJA elicitation. Pre-treatment of cultures with calcium chelators, calcium blockers or intracellular antagonists enhanced the elicitor effect of YE or MeJA. The increase of intracellular-free Ca(2+) level also promoted the elicitor effect, suggesting that an external source of calcium or alterations in internal calcium fluxes were not required for the elicitation to occur. Activation of phosphorylation/dephosphorylation cascades did not appear to mediate the elicitation mechanism; the increase in silymarin induced by elicitation was not suppressed by inhibitors of protein phosphatases or by protein kinase inhibitors. No H(2)O(2) generation was detected at any time after elicitation. Also, diphenyleneiodonium, a potent inhibitor of NAD(P)H-oxidase, did not block silymarin production in elicited cultures. From these results, we conclude that S. marianum cell cultures do not appear to employ conserved signaling components in the transduction of the elicitor signal to downstream responses such as silymarin production.     

大丽轮枝菌毒素与拟南芥互作反应中SA、NO与H2O2信号的相关性

本文研究了大丽轮枝菌毒素（VD-toxin)与拟南芥互作反应中外源SA、NO供体、NO合酶抑制剂等对拟南芥幼苗H2O2含量的影响,并对H2O2的积累部位进行了DAB组化染色检测。大丽轮枝菌毒素、外源SA、NO供体处理拟南芥幼苗均能诱导H2O2的积累,NO供体的诱导作用最强;NO合酶抑制剂处理则未表现出H2O2含量的增强;H2O2的积累部位主要在叶片的表皮毛和维管束组织。结果表明,在大丽轮枝菌毒素与拟南芥互作反应中,H2O2可能作为信号分子参与了SA和NO调控的拟南芥防卫反应,NO信号与H2O2信号间的关系可能更密切。     

A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor

We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 microM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 microM), had little effect, but the phosphatase inhibitor, calyculin A (1 microM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 microM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA.     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Salicylic acid-induced inactivation of creatine kinase in the presence of lactoperoxidase and H2O2

To clarify one mechanism of aspirin-induced gastric mucosal damage, inactivation of creatine kinase (CK) by salicylic acid that is easily produced from aspirin in vivo was examined in the presence of lactoperoxidase (LPO) and H2O2 (LPO-H2O2). Salicylic acid inactivated CK (rabbit muscle) during its interaction with LPO-H2O2. CK activity in gastric mucosal homogenate decreased dependent on the concentration of salicylic acid in the presence of LPO-H2O2. Oxygen radical scavengers did not prevent the inactivation of CK. Direct detection of free radicals of salicylic acid by electron spin resonance was unsuccessful. However, glutathionyl radicals were formed during the interaction of salicylic acid with LPO-H2O2 in the presence of reduced glutathione and 5,5-dimethyl-1-pyrroline oxide as a spin trap agent. Among salicylic acid-related drugs, salsalate, but not aspirin and ethenzamide, inactivated CK, indicating the phenolic hydroxyl group is oxidized by LPO-H2O2. During oxidation of salicylic acid by LPO-H2O2, the sulfhydryl group in CK markedly decreased, and salicylic acid bound to CK. These results indicate that CK was inactivated through loss of the sulfhydryl group and binding of salicylic acid.