Indoleacetic Acid Oxidation Research Articles

Oxidase-like manganese oxide nanoparticles: a mechanism of organic acids/aldehydes as electron acceptors and potential application in cancer therapy.

Identifying the underlying catalytic mechanisms of synthetic nanocatalysts or nanozymes is important in directing their design and applications. Herein, we revisited the oxidation process of 4,4'-diamino-3,3',5,5'-tetramethylbiphenyl (TMB) by Mn3O4 nanoparticles and revealed that it adopted an organic acid/aldehyde-triggered catalytic mechanism at a weakly acidic or neutral pH, which is O2-independent and inhibited by the pre-addition of H2O2. Importantly, similar organic acid/aldehyde-mediated oxidation was applied to other substrates of peroxidase in the presence of nanoparticulate or commercially available MnO2 and Mn2O3 but not MnO. The selective oxidation of TMB by Mn3O4 over MnO was further supported by density functional theory calculations. Moreover, Mn3O4 nanoparticles enabled the oxidation of indole 3-acetic acid, a substrate that can generate cytotoxic singlet oxygen upon single-electron transfer oxidation, displaying potential in nanocatalytic tumor therapy. Overall, we revealed a general catalytic mechanism of manganese oxides towards the oxidation of peroxidase substrates, which could boost the design and various applications of these manganese-based nanoparticles.

Dual-Responsive Enzyme-Polysaccharide Conjugate as a Nanocarrier System for Enzyme Prodrug Therapy.

Biopolymer-based drug delivery systems have gained considerable attention in the field of nanomedicine. In this study, a protein-polysaccharide conjugate was synthesized by covalent conjugation of the enzyme horseradish peroxidase (HRP) with acetalated dextran (AcDex) via a thiol exchange reaction. The resulting bioconjugate shows a dual-responsive behavior in acidic and reductive environments to achieve a controlled release of drugs. The self-assembly of this amphiphilic HRP-AcDex conjugate allows the encapsulation of prodrug indole-3-acetic acid (IAA) into the hydrophobic polysaccharide core. Under slightly acidic conditions, the acetalated polysaccharide reverts to its native hydrophilic form, which triggers the disassembly of micellar nanoparticles and the release of the encapsulated prodrug. The conjugated HRP further activates the prodrug by oxidation of IAA into cytotoxic radicals, which leads to cellular apoptosis. The results indicate that the HRP-AcDex conjugate in combination with IAA has great potential to be used as a novel enzyme prodrug therapy for cancer treatment.

ABA-responsive AREB1/ABI3-1/ABI5 cascade regulates IAA oxidase gene SlDAO2 to inhibit hypocotyl elongation in tomato.

Hypocotyl elongation is dramatically influenced by environmental factors and phytohormones. Indole-3-acetic acid (IAA) plays a prominent role in hypocotyl elongation, whereas abscisic acid (ABA) is regarded as an inhibitor through repressing IAA synthesis and signalling. However, the regulatory role of ABA in local IAA deactivation remains largely uncharacterized. In this study, we confirmed the antagonistic interplay of ABA and IAA during the hypocotyl elongation of tomato (Solanum lycopersicum) seedlings. We identified an IAA oxidase enzyme DIOXYGENASE FOR AUXIN OXIDATION2 (SlDAO2), and its expression was induced by both external and internal ABA signals in tomato hypocotyls. Moreover, the overexpression of SlDAO2 led to a reduced sensitivity to IAA, and the knockout of SlDAO2 alleviated the inhibitory effect of ABA on hypocotyl elongation. Furthermore, an ABA-responsive regulatory SlAREB1/SlABI3-1/SlABI5 cascade was identified to act upstream of SlDAO2 and to precisely control its expression. SlAREB1 directly bound to the ABRE present in the SlDAO2 promoter to activate SlDAO2 expression, and SlABI3-1 enhanced while SlABI5 inhibited the activation ability of SlAREB1 by directly interacting with SlAREB1. Our findings revealed that ABA might induce local IAA oxidation and deactivation via SlDAO2 to modulate IAA homoeostasis and thereby repress hypocotyl elongation in tomato.

Different regulation of auxin homeostasis would be a possible mechanism conferring quinclorac resistance in Echinochloa crusgalli var. zelayensis

AbstractDifferences in ethylene biosynthesis and cyanide detoxification have been reported to be mechanisms of quinclorac resistance in Echinochloa crusgalli var. zelayensis. Resistant phenotypes could be a consequence of the altered endogenous indole acetic acid (IAA) homeostasis induced by the herbicide. In this study, we determined the IAA content and expression levels of auxin homeostasis‐related genes in susceptible and resistant biotypes of E. crusgalli var. zelayensis after quinclorac treatment. The results showed that the IAA content of JNNX‐S (susceptible biotype) was significantly higher than that of SSXB‐R (resistant biotype) after treatment with 50 μM quinclorac. To better understand this rise in IAA, the expression profiles of seven genes (one for auxin synthesis, five for IAA conjugation and one for IAA oxidation) and the biochemical activities of two oxidases involved in IAA homeostasis were measured. The expression of EcYUCCA10 was significantly higher in JNNX‐S than in SSXB‐R. The expression levels of the EcGH3s were significantly lower in JNNX‐S than in SSXB‐R. These expression profiles were consistent with the elevation of IAA levels in the susceptible biotype. In contrast, EcUGT and EcDAO were induced in each biotype, but a smaller increase was observed in SSXB‐R than in JNNX‐S. The enzymatic activities of IAA oxidases and peroxidases were higher in SSXB‐R than in JNNX‐S 24 h after treatment. It was inferred that altered expression of specific genes involved in IAA synthesis, conjugation and oxidation resulted in less IAA being induced in the resistant biotype, resulting in a lower ethylene burst and the associated quinclorac resistance. These results suggest novel layers of complexity in the mechanism of quinclorac resistance.

A Novel Electrochemical Sensor Based on Modified Carbon Paste Electrode with ZnO Nanorods for the Voltammetric Determination of Indole‐3‐acetic Acid in Plant Seed Extracts

AbstractDetection of indole‐3‐acetic acid (IAA), as a phytohormone, is important for precision farming, plant phenotyping, and crop management. Herein, IAA was detected in bean and wheat plant seeds extractions using zinc oxide nanorods/carbon paste electrode (ZnO NRs/CPE). ZnO NRs/CPE showed excellent electrocatalytic activity, high sensitivity, and selectivity toward the oxidation of IAA. The linearity range was from 30.×10−8 to 5.0×10−6 M (r2=0.996, n=10), with a detection limit of 1.7×10−8 M. Moreover, ZnO NRs/CPE exhibited high reproducibility, with a standard deviation of 1.0 % for six successive measurements of IAA.

Interaction of indole-3-acetic acid with horseradish peroxidase as a potential anticancer agent: from docking to molecular dynamics simulation

The oxidation process, catalyzed by the peroxidase enzymes, occurs in all domains of life to detoxify the hydrogen peroxide toxicity. The most well-known, applicable and vastly studied member of the peroxidases family is horseradish peroxidase (HRP), especially the isoenzyme C (HRP C). HRP (primarily HRP C) is commercially available and applicable in biotechnology and diagnosis. Recently, a novel application of HRP has been introduced in cancer therapy as the combination of HRP with indole-3-acetic acid (IAA). The anticancer activity of HRP/IAA complex is through oxidation of IAA by HRP in hypoxic tumor condition, which leads to apoptosis and cancerous cell death. However, the molecular interaction of HRP/IAA has not been elucidated. Identifying the interaction of IAA with HRP would provide a better insight into its function and applications. In this study, molecular docking and molecular dynamics (MD) simulation were applied to determine the molecular interaction of the IAA/HRP complex. The docking study represented that IAA bound at the ‘exposed’ heme edge of the HRP enzyme, and the IAA entrance to the enzyme was situated at the carboxymethyl side-chain of the selected structure. Our computational results showed the HRP/IAA complex structure stability. While hydrogen bond formation with ARG38 and HIS42 stabilized the substrate, hydrophobic interactions with Phe68, Gly69, Leu138, Pro139, Pro141 and Phe179 contributed to IAA/HRP complex stability. The results can help to better understand peroxidase enzyme activity and would pave the way for future development of new therapeutics with improved anticancer efficacy. Communicated by Ramaswamy H. Sarma

Fluazifop-P-butyl induced ROS generation with IAA (indole-3-acetic acid) oxidation in Acanthospermum hispidum D.C.

Acanthospermum hispidum D.C. was particularly susceptible to fluazifop-P-butyl, an aryloxyphenoxypropionate herbicide, and the primary action site for the herbicide was shoot apical meristem, which is also the main site of indole-3-acetic acid (IAA) biosynthesis and action. Membrane lipid peroxidation caused by increasing levels of reactive oxygen species (ROS) was considered as an action mechanism of fluazifop-P-butyl in A. hispidum. To further clarify the ROS inducing mechanism of fluazifop-P-butyl in the plant, the interactions between fluazifop-P-butyl and auxin compounds IAA or 2,4-dichlorophenoxyacetic acid (2,4-D) were studied. Haloxyfop-P-methyl, an AOPP herbicide which is inactive on A. hispidum, was used for comparison. The results showed that the growth inhibition and malondialdehyde or H2O2 increases induced by fluazifop-P-butyl on A. hispidum were reversed by IAA or 2,4-D. The IAA content was decreased but the contents of three IAA oxidation metabolites, indole-3-methanol, indole-3-aldehyde and indole-3-carboxylic acid were increased by fluazifop-P-butyl in A. hispidum, but not by haloxyfop-P-methyl. The growth of A. hispidum was not inhibited by three IAA oxidative compounds. Moreover, the activities of IAA oxidase and peroxidase were increased by fluazifop-P-butyl but not by haloxyfop-P-methyl, and the increase was reversed by IAA or 2,4-D. We suggest that there is an antagonistic effect between fluazifop-P-butyl and IAA or 2,4-D, and the IAA oxidation may be involved in the action mechanism of fluazifop-P-butyl in A. hispidum.

ACCERBATIN, a small molecule at the intersection of auxin and reactive oxygen species homeostasis with herbicidal properties.

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides.

Auxin homeostasis: the DAO of catabolism.

Nearly all programmed and plastic plant growth responses are at least partially regulated by auxins, such as indole-3-acetic acid (IAA). Although vectorial, long distance auxin transport is essential to its regulatory function, all auxin responses are ultimately localized in individual target cells. As a consequence, cellular auxin concentrations are tightly regulated via coordinated biosynthesis, transport, conjugation, and oxidation. The primary auxin oxidative product across species is 2-oxindole-3-acetic acid (oxIAA), followed by glucose and amino acid conjugation to oxIAA. Recently, the enzymes catalyzing the oxidative reaction were characterized in Arabidopsis thaliana. DIOXYGENASE OF AUXIN OXIDATION (DAO) comprises a small subfamily of the 2-oxoglutarate and Fe(II) [2-OG Fe(II)] dependent dioxygenase superfamily. Biochemical and genetic studies have revealed critical physiological functions of DAO during plant growth and development. Thus far, DAO has been identified in three species by homology. Here, we review historical and recent studies and discuss future perspectives regarding DAO and IAA oxidation.

DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana

Tight homeostatic regulation of the phytohormone auxin [indole-3-acetic acid (IAA)] is essential to plant growth. Auxin biosynthetic pathways and the processes that inactivate auxin by conjugation to amino acids and sugars have been thoroughly characterized. However, the enzyme that catalyzes oxidation of IAA to its primary catabolite 2-oxindole-3-acetic acid (oxIAA) remains uncharacterized. Here, we show that DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) catalyzes formation of oxIAA in vitro and in vivo and that this mechanism regulates auxin homeostasis and plant growth. Null dao1-1 mutants contain 95% less oxIAA compared with wild type, and complementation of dao1 restores wild-type oxIAA levels, indicating that DAO1 is the primary IAA oxidase in seedlings. Furthermore, dao1 loss of function plants have altered morphology, including larger cotyledons, increased lateral root density, delayed sepal opening, elongated pistils, and reduced fertility in the primary inflorescence stem. These phenotypes are tightly correlated with DAO1 spatiotemporal expression patterns as shown by DAO1pro:β-glucuronidase (GUS) activity and DAO1pro:YFP-DAO1 signals, and transformation with DAO1pro:YFP-DAO1 complemented the mutant phenotypes. The dominant dao1-2D mutant has increased oxIAA levels and decreased stature with shorter leaves and inflorescence stems, thus supporting DAO1 IAA oxidase function in vivo. A second isoform, DAO2, is very weakly expressed in seedling root apices. Together, these data confirm that IAA oxidation by DAO1 is the principal auxin catabolic process in Arabidopsis and that localized IAA oxidation plays a role in plant morphogenesis.

An Novel Electrochemical Sensor for Indole-3-Acetic Acid Detection Based on the Peroxidase-like Activity of Hemin/Boron Nitride Composite Nanocomposite

An amperometric sensor for the detection of indole-3-acetic acid (IAA) at low potential was firstly established based on the hemin/boron nitride (hemin/BN) nanocomposite. The hemin/BN nanocomposite was prepared by a simple and facile hydrothermal method. It exhibited peroxidase-like activity for the catalytic oxidation of IAA in the presence of oxygen. The consumption of oxygen has a linear relationship with the concentration of IAA in the range from 0.5 uM to 0.08 mM with a detection limit of 0.1 uM (S/N=1/3). This sensor was unaffected by many interfering substances, and was stable over time. The work broadened the application of hemin/rGO in electrochemical sensor areas and provided a new method for IAA detection.

Low potential detection of indole-3-acetic acid based on the peroxidase-like activity of hemin/reduced graphene oxide nanocomposite

An amperometric sensor was firstly established for the detection of indole-3-acetic acid (IAA) at low potential based on the hemin/reduced graphene oxide (hemin/rGO) composite. The hemin/rGO nanocomposite was prepared by a simple and facile hydrothermal method without using any reducing agent. It exhibited peroxidase-like activity for the catalytic oxidation of IAA in the presence of oxygen. The consumption of oxygen has a linear relationship with the concentration of IAA in the range from 0.1 to 43μM and from 43 to 183μM. The detection limit was down to 0.074μM. This sensor was unaffected by many interfering substances and stable over time. Such work broadened the application of hemin/rGO and provided a new method for IAA detection.

Water-dispersed carboxymethyl cellulose-montmorillonite-single walled carbon nanotube composite with enhanced sensing performance for simultaneous voltammetric determination of two trace phytohormones

A simple and sensitive method for the simultaneous voltammetric detection of two trace phytohormones both indole-3-acetic acid (IAA) and salicylic acid (SA) was successfully realized using composite film modified glassy carbon electrode (GCE) with enhanced sensing performance. Carboxymethyl cellulose (CMC), a water-soluble biopolymer with good film-forming ability and adhesive property was selected as the disperser, stabilizer, and binder for improving the water-dispersibility of single-walled carbon nanotube (SWCNT) with excellent electrocatalytic ability and montmorillonite (MMT) with high effective surface area and surface adsorption capacity, and enhancing the water stability and adhesion between the coating and substrate electrode. CMC-SWCNT-MMT/GCE displayed enhanced electrode stability in water, effective surface area, adhesive property and adsorption capacity, and good electrocatalytic activity for the oxidation of both IAA and SA, which could simultaneously detected two trace phytohormones with high sensitivity, low detection limit, and good sensing stability. Satisfactory results indicated the water-dispersed CMC-SWCNT-MMT with enhanced sensing performance provided a promising platform for the electrochemical application of high-performance composite film-modified electrode.

One-step co-electrodeposition of graphene oxide doped poly(hydroxymethylated-3,4-ethylenedioxythiophene) film and its electrochemical studies of indole-3-acetic acid

Abstract A novel graphene oxide (GO) doped poly(hydroxymethylated-3,4-ethylenedioxythiophene) (PEDOTM) film has been achieved via one-step co-electrodeposition and utilized for electrochemical studies of indole-3-acetic acid (IAA). The incorporation of GO into PEDOTM film facilitated the electrocatalytic activity and exhibited a favorable interaction between the PEDOTM/GO film and the phytohormone during the oxidation of IAA. Under optimized conditions, differential pulse voltammetry and square wave voltammetry were used for the quantitative analysis of IAA, respectively, each exhibiting a wide linearity range from 0.6 μmol L −1 to 10 μmol L −1 and 0.05 μmol L −1 to 40 μmol L −1 , good sensitivity with a low detection limit of 0.087 μmol L −1 and 0.033 μmol L −1 , respectively, as well as good stability. With the notable advantages of a green, sensitive method, expeditious response and facile operation, the as-prepared PEDOTM/GO organic-inorganic composite film provides a promising platform for electrochemical studies of IAA.

Effect of Temperature on Guaiacol Peroxidase of <i>Pyrus communis</i>

Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase, POD) is one of the key enzymes controlling plant growth, differentiation and development. The enzyme participates in construction, rigidification and eventual lignification of cell walls, biosynthesis of ethylene from 1-aminocyclopropane-1-carboxylic acid and H2O2, regulation of auxin level through auxin catabolism, protection of tissue from damage and infection by pathogenic microorganisms, the oxidation of indoleacetic acid. For peroxidase activity in wild pears extract one pH optimum was observed at 6.5 that probably belong to atleast one isoenzyme. Activity of peroxidase in presence of guaiacol and H2O2 was optimum after incubation at 40 °C. Maximum activity of peroxidase is 300% .Activity increased to 240%, 300%, 70% and 10% after 60 minute incubation at 30, 40, 45 and 60 °C for peroxidase. Incubation at high temperature (70 °C) was accompanied with decrease of activity to 10% peroxidase activity.

Effect of Temperature on Guaiacol Peroxidase of <i>Pyrus communis</i>

Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase, POD) is one of the key enzymes controlling plant growth, differentiation and development. The enzyme participates in construction, rigidification and eventual lignification of cell walls, biosynthesis of ethylene from 1-aminocyclopropane-1-carboxylic acid and H2O2, regulation of auxin level through auxin catabolism, protection of tissue from damage and infection by pathogenic microorganisms, the oxidation of indoleacetic acid. For peroxidase activity in wild pears extract one pH optimum was observed at 6.5 that probably belong to atleast one isoenzyme. Activity of peroxidase in presence of guaiacol and H2O2 was optimum after incubation at 40 °C. Maximum activity of peroxidase is 300% .Activity increased to 240%, 300%, 70% and 10% after 60 minute incubation at 30, 40, 45 and 60 °C for peroxidase. Incubation at high temperature (70 °C) was accompanied with decrease of activity to 10% peroxidase activity.

Evidence of oxidative attenuation of auxin signalling

Indole-3-acetic acid (IAA) is the principle auxin in Arabidopsis and is synthesized primarily in meristems and nodes. Auxin is transported to distal parts of the plant in response to developmental programming or environmental stimuli to activate cell-specific responses. As with any signalling event, the signal must be attenuated to allow the system to reset. Local auxin accumulations are thus reduced by conjugation or catabolism when downstream responses have reached their optima. In most cell types, localized auxin accumulation increases both reactive oxygen species (ROS) and an irreversible catabolic product 2-oxindole-3-acid acid (oxIAA). oxIAA is inactive and does not induce expression of the auxin-responsive reporters DR5 or 2XD0. Here it is shown that oxIAA is not transported from cell to cell, although it appears to be a substrate for the ATP-binding cassette subfamily G (ABCG) transporters that are positioned primarily on the outer lateral surface of the root epidermis. However, oxIAA and oxIAA-Glc levels are higher in ABCB mutants that accumulate auxin due to defective cellular export. Auxin-induced ROS production appears to be at least partially mediated by the NAD(P)H oxidase RbohD. oxIAA levels are higher in mutants that lack ROS-scavenging flavonoids (tt4) and are lower in mutants that accumulate excess flavonols (tt3). These data suggest a model where IAA signalling is attenuated by IAA catabolism to oxIAA. Flavonoids appear to buffer ROS accumulations that occur with localized increases in IAA. This buffering of IAA oxidation would explain some growth responses observed in flavonoid-deficient mutants that cannot be explained by their established role in partially inhibiting auxin transport.

Biochemical activities of extracts from Hypericum perforatum L. 3rd Communication: modulation of peroxidase activity as a simple method for standardization.

Alcoholic extracts from the herb "St. John's wort" (Hypericum perforatum L.) are widely used to counteract depressive situations, where the question on the mainly active principle is still under discussion. Thus, standardization of the drug on the basis of dry matter has been chosen instead of the popular leading component, hypericin. Inhibition of myeloperoxidase-catalyzed dimerization of enkephalins by Hypericum extracts has recently been reported. This method is based on the separation and quantification of enkephalin dimers by HPLC. In order to simplify this assay myeloperoxidase could be substituted by the cheaper horseradish peroxidase and the enkephalins by the amino acid tyrosine without loss of significance. In this communication we represent a more rapid photometric method based on peroxidase-catalyzed indole acetic acid oxidation suitable for quick, simple and economic drug standardization.

Change in antioxidant and lignifying enzyme activities in rubbing tomato ( Solanum lycopersicum ) internodes

In tomato plant, rubbing applied to a young internode inhibits elongation of the rubbed internode and its neighbouring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL) and peroxidases (POD), 24 h after rubbing of theforth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Lignin synthesis in tomato plant measured 14 days after mechanical stress application was significantly stimulated in the rubbed internodes (n°4) as compared with the control. Fourier transform infrared (FTIR) analyses revealed that lignin synthesized in response to mechanical elicitation displayed a distinct structure, substantially enriched in syringyl (S) units, as compared to constitutive lignin. Taken together, our results suggest that the decrease in rubbed internode length is as a result of IAA oxidation, increases in enzyme activities (PAL and POD) and cell wall rigidification induced by lignification process. Key words: Mechanical stimulation, Solanum lycopersicum, indole-3-acetic acid (IAA), Fourier transform infrared (FTIR), lignin.

A disposable electrochemical sensor for the determination of indole-3-acetic acid based on poly(safranine T)-reduced graphene oxide nanocomposite

A disposable electrochemical sensor for the determination of indole-3-acetic acid (IAA) based on nanocomposites of reduced graphene oxide (rGO) and poly(safranine T) (PST) was reported. The sensor was prepared by coating a rGO film on a pre-anodized graphite electrode (AGE) through dipping–drying and electrodepositing a uniform PST layer on the rGO film. Scanning electron microscopic (SEM) and infrared spectroscopic (IR) characterizations indicated that PST–rGO formed a rough and crumpled composite film on AGE, which exhibited high sensitive response for the oxidation of IAA with 147–fold enhancement of the current signal compared with bare AGE. The voltammetric current has a good linear relationship with IAA concentration in the range 1.0×10−7–7.0×10−6M, with a low detection limit of 5.0×10−8M. This sensor has been applied to the determination of IAA in the extract samples of several plant leaves and the recoveries varied in the range of 97.71–103.43%.