Formation of the Azodication (ABTS²⁺) from ABTS [2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulphonate)] in Sterile Plant Cultures: Root–Exuded Oxidoreductases Contribute to Rhizosphere Priming

Abstract

Rhizosphere priming by terrestrial plants comprises increased or repressed efflux of CO₂ and N from soil organic matter (SOM), decaying under the impact of temperature, moisture, and the composition of rhizodeposits. Contemporarily, increases in water solubility vs. losses in molecular size, aromaticity, and the content in phenolic OH groups denote the degradation of SOM in planted soil. Root peroxidases (POs) and ‘polyphenoloxidases’ are surmised to contribute to these effects, however, final evidence for this is lacking. Therefore, seedlings of white mustard, alfalfa, and oilseed rape with wide spans in PO release were grown in hydroponic cultures at variable levels of Cu/Fe/Mn as Fenton metals, but also under P and Fe starvation to stimulate the release of carboxylic acids that form catalytic Mn³⁺ chelants from Mn²⁺ and MnO₂. The shortage in active oxygen as a cosubstrate of POs delayed the immediate oxidation of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) supplements to the green ABTS^•+ by PO/H₂O₂, the possible formation of Mn³⁺ via PO catalyzed aryloxy radicals from root–released phenolics, and of HO^• by metal cations in H₂O₂ dependent Fenton–like reactions. Enhanced by exuded and external malate, O₂ independent MnO₂ supplements in some treatments formed ABTS^•+ spontaneously. The culture fluids then turned red in all treatments within 24–60 h by the formation of azodication (ABTS²⁺) derivatives in a second plant initiated oxidation step that is known to be catalyzed by substrate radicals. It is concluded that plants initiate oxidative activities that contribute to rhizosphere priming in an environment of oxidoreductase and carboxylate exudates, the indicated presence of mediating substrate radicals, and the cations and (hydr)oxides of transition metals. Pathways of H₂O₂ production upon the degradation of carboxylates and by the POs themselves are indicated.

1. Introduction

Soil organic matter with an estimated amount of 1.6 × 10¹⁸ g C [1] is a major player in the terrestrial carbon cycle. Its formation from plant residues by soil fauna and microbiota is overlaid from the rhizosphere priming effect [2]. Poorly defined rhizosphere factors of herbs inhibit [3,4], but mainly stimulate, the mineralization of C (by 27–245%) and N compounds (by 36–62%) [5,6,7] to accelerate the biotic degradation of arable land [8].

Part of soil formation processes is the degradation of the recalcitrant aromatic structures of lignin and humic substances (HS). It is catalyzed by the joint action of dehydrogenases, oxygenases and oxidoreductases such as the laccases and peroxidases of plants, white–rot, soft–rot, brown–rot fungi and bacteria [9,10,11,12]. The enzymes operate in concert with low–molecular weight (MW) carboxylic acids, (reactive) oxygen species, transition metal cations, and small organic radical molecules of substrate or metabolic origin with redox mediator properties [13,14,15,16]. They mediate electron abstractions from substrates whose electron withholding capacity (E⁰) surpasses the redox potential of the enzyme itself.

Initial one–electron oxidations of the dominating non-phenolic lignin units presuppose redox potentials of E⁰ > 1.5 V taken vs. normal hydrogen electrodes (NHE) [17]. This catalytic potential is afforded by lignin peroxidases and versatile peroxidases of certain wood-decaying basidiomycete fungi (E⁰ = 1.4–1.5 V). The enzymes use H₂O₂ as an electron acceptor, see [9,15,18,19,20] for reviews. Manganese dependent peroxidases of basidiomycetous wood-decay and ground fungi complete their redox cycle by the oxidation of two Mn²⁺ cations to the abiotic oxidant Mn³⁺ (E⁰ = 0.8–0.9 V for Mn³⁺/malonate or oxalate complexes) [20,21]. The cation performs one-electron abstractions (H⁺/e⁻) from phenolic substrate molecules or phenolic lignin structures [22,23] upon its reduction to Mn²⁺. It is enzymatically reoxidized to Mn³⁺ by electron transfer to H₂O₂. Laccases of basidiomycetes (E⁰ up to 0.79 V) catalyze one-electron abstractions from hydroxyl groups of four phenolic molecules (up to 0.81 V) [24] to convert the electron acceptor O₂ to 2 H₂O. They initiate oxidations, decarboxylations, demethylations, and demethoxylations from phenolic acids (derivatives) and phenolic lignin moieties. Resulting aryloxy radicals polymerize or depolymerize to low-MW mediators to initiate spontaneous C_α-C_β bond cleavages, C_α oxidations, and alkyl-aryl and aromatic ring cleavages in phenolic lignin model compounds [25,26]. Laccase/synthetic mediator systems (E⁰ up to 1.5 V) oxidize the lignin model surrogate veratryl alcohol (E⁰ = 1.4 V) and nonphenolic structures of lignin model compounds [27,28,29].

The Mn-independent class (iii) plant peroxidases (PO; EC 1.11.1.7) have comparable substrate spectra as actors in rhizosphere priming. Their redox potential of E⁰ = 0.89–0.95 V [18] surpasses that of fungal laccases and especially of plant laccases (E⁰ = 0.4 V) and monophenol monooxygenases (MMO, EC 1.14.18.1, E⁰ = 0.26 V) [15,30]. Plant POs catalyze one-electron oxidations of phenolics and humic substances [31] using H₂O₂ as electron acceptor according to the redox cycle.

Two phenolic molecules + H₂O₂→Two aryloxy radicals + two H₂O

(1)

Plant released phenolics acting as aryloxy radicals mediate the formation of Mn³⁺ (0.8–0.9 V) [15,32] and a partial oxidation of veratryl alcohol from maize roots (1.4 V) [30]. They expand the substrate range at least in part to that of fungal POs.

In several plant tissues, more than 30 PO and several MMO [33] and laccase isoforms [34] can be found. In plant roots, the bulk of the PO is localized in the primary cell wall but also in membranes, organelles, vacuoles, and the cytoplasm [33]. Class (iii) peroxidases could thus be leached from roots of cotton, wheat, cress, tomato, water hyacinth, French bean, rice [33,35] and other plants [15,30,36]. Taken with the chromogen guaiacol at pH 7.0, alfalfa plants but not oilseed rape surpassed the root exuded PO activities of white mustard 167–182 times both in axenic and natural soil cultures [15,37]. Contemporarily, sterile root effluents of herbs contained little, if any, H₂O₂, or its precursor O₂^•–, as the co-substrates of PO. Soil-grown alfalfa plants (40 d old) contained 42 mg kg⁻¹ DW of peroxide in the shoot, and traces in root tissue and in aseptic root exudates [38].

With its content of up to 30% phenolic units [39], the humus polymer matches with the substrate spectrum of laccases, peroxidases, and abiotically generated oxidants such as hydroxyl radical (HO^•) that is reduced to water upon H⁺/e^- abstractions from organics (E⁰ = 1.8–2.7 V) [40]. According to Haber and Weiss [41], HO^• as the ultimate oxidant is presumably formed by multivalent transition metals (M) such as Ce, Co, Cr, Cu, Fe, La, Mn, Ni, Ru, V, and others. They serve as electron donors upon H₂O₂ consume in a Fenton-like reaction [42,43,44]. Clay and oxide minerals such as MnO₂ can also serve as catalysts in Fenton-like reactions [45].

(Mⁿ)⁺ + H₂O₂→(Mⁿ⁺¹)⁺ + HO^• + HO⁻

(2)

Soil humic substances (HS) and their alkali-extractable humic acids (HA, apparent molecular size 10–>300 kDa) and fulvic acids (FA, 0.1–9 kDa) are predominantly formed, and immediately released, from senescent plant tissues [46]. Humic acids are mainly composed of (C-substituted) aromatics and alkyl-C (aliphatic structures) whereas fulvic acids represent randomly composed polysaccharides and little aromatics [47,48]. Their reactive dissociable groups are mainly carboxyl (-COOH), phenolic, enolic, and alcoholic hydroxyl (-OH), methoxyl (-OCH₃), sulphydryl (-SH), amino (-NH₂), and imino (=NH) type. Their protons (H⁺) are exchangeable with metal cations and are preferred targets of oxidative enzymes [49,50,51]. The mean molecular size and the degree of aromaticity of soil derived HS were reduced by the fungal manganese peroxidase/Mn³⁺ couple, by the Mn-independent plant peroxidase, and the abiotic oxidants H₂O₂ and Mn(III)acetate in vitro. The hydrolytic β-glucosidase caused discoloration. Fungal tyrosinases and laccases had little, if any, effect [31]. Beside active catalysts as above, those of whole fungal and bacterial cultures, too, greatly reduced the chemical reactivity of soil HS to tetrazotized o-dianisidine [31]. This indicator of hydroxylated aromatics [52] reveals the oxidative depletion of OH groups by apparent H⁺/e⁻ abstractions.

The immediate impact of root released POs on soil forming processes evokes little attention. Their ecological role is poorly understood [15]. It is suggested that the presence of POs and MMOs released by potted herbs and soil microbiota could add to an accelerated efflux of nitrogen upon an expected oxidative degradation of their HS matrices [7,53]. Wood-decay fungi growing on sterile, HS amended media released MMOs, laccases, and manganese peroxidases. Within three weeks, the concentrations of HS decreased by 29–54%, their molecular size by 53–59%, and the loss in hydroxyl groups (-OH) amounted 89–99.6%. This indicated shifts to the aromatic-poor fulvic-acids pool [31] and mineralization by the MnP/Mn³⁺ system [54]. A natural forest soil C_org 15–20% incubated with a set of plants for 56 d contained POs beside traces of laccase and MMO. Relative to unplanted controls and without H₂O₂ amendments, the water solubility of HS rose to 115–156%. The concentrations of their phenolic OH groups dropped by 53–79% (r = 0.815, p ≥ 0.67) in PO releasing plants, and by 28–34% in plants with no extractable PO exudates, to copy the reactions obtained in fungal cultures [37].

The evidence for an apparent contribution of plant released oxidoreductases to rhizosphere priming as above is nevertheless indirect. In the present study, seedlings of the strategy-I plants white mustard, alfalfa, and oilseed rape were therefore cultivated in sterile hydroponic cultures by exclusion of enzyme releasing microorganisms. The plants were grown at variable levels of Fe and Mn as Fenton metals with potential HO^• generation. In addition, Fe and P starvation was observed to promote the release of carboxylic acids that form with Mn³⁺ chelates from MnO₂ supplements another abiotic catalyst [55]. The grown-up seedlings were amended with the susceptible and colorless chromogen 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS). It turns green upon its one-electron oxidation to the cation radical ABTS^•+ (E⁰ = 0.68 V), and red after a second oxidation step from ABTS^•+ to the azodication ABTS²⁺ (ABTS^•+ to ABTS²⁺, E⁰ = 1.09 V) (Figure 1) [56]. In a second trial, Fenton metals acting as micronutrients were exposed to H₂O₂ or O₂^•–. Their ability was tested to oxidize ABTS by the formation of hydroxyl radical (HO^•) even at the physiological pH conditions of the plant cultures. The goal was to use the unambiguous color reactions of ABTS for the definitive proof of the joint, and almost inseparable, oxidative activities afforded:

• by released oxidoreductases of gnotobiotic plants in the absence of H₂O₂ or O₂^•– supplements,
• by contributions of transition metal cations in Fenton-like reactions with active oxygen species,
• and by Mn³⁺ catalyst derived from MnO₂ by root-released and external malate.

2. Materials and Methods

2.1. Surface Sterilization of Seeds for Gnotobiotic Flask Cultures

Commercially available seeds (N. L. Chrestensen, Erfurt, Germany) were immersed in deionized water amended with the anionic detergent Fit (Fit GmbH, Zittau, Germany) for 2–6 h to remove adhering bubbles and make the kernels sink to the beaker’s bottom. Those drifting above were removed. The remaining submerged seeds of white mustard (Sinapis alba L.) were surface-sterilized to 100% in 15% NaOCl (13% active chlorine) for 30 min and washed once in autoclaved water prior to the transfer into sterile 100-mL Erlenmeyer flasks for germination. The pre-submerged seeds of oilseed rape (Brassica napus L.) were surface-sterilized at a rate of around 67% in 30% NaOCl for 210 min and washed once prior to germination. Pre-submerged seeds of alfalfa (Medicago sativa L.) were surface-sterilized up to 100% in 30% H₂O₂ diluted to one-third for 20–30 min and used without washing. The treatment was accompanied by foam production incited by the kernels’ surface peroxidase enzyme.

2.2. Hydroponic Flask Cultures

Cotton stoppered 100-mL Erlenmeyer flasks with 2.5 mL deionized water were autoclaved at 121 °C for 30 min. They were equipped with 1 cm³ surface-sterilized seeds under air-controlled conditions, weighed, and incubated in the daylight at room temperature in a tilted position to avoid submergence of the kernels by the water resource. After 6–7 days, the emerging germlings were raised with a sterile nutrient solution adapted from Fries [57] and composed of (L⁻¹) 1 g KNO₃, 5 g KH₂PO₄, 0.01 g CaCl₂, 0.1 g NaCl, 0.01 g FeSO₄·7H₂O, 2 mg MnSO₄·H₂O, 0.5 mg ZnSO₄·H₂O, and 0.5 mg CuSO₄·5H₂O (Merck, Darmstadt), pH 4.5. In the early growth stages, KH₂PO₄, FeSO₄ and/or MnSO₄ supplements were omitted. Within 3–4 weeks of incubation, the plants reached 45–65 mm (white mustard, oilseed rape) and 20–30 mm (alfalfa), respectively, in height and developed secondary leaves. At the outset of testing, the free liquid held at 2 mL was filled up to around 8.5 mL with the sterile nutrient solution modified in the P, Fe, and Mn content. Thereby, the mode of P or P/Fe(II) starvation was retained to increase the root exudation of malate/malonate [55]. Applications of Mn(II) were also withheld that could lead to the formation of the abiotic Mn³⁺ catalyst [58] by plant PO/root phenolic mediator systems [32,38]. Supplements of 50 mg autoclaved pyrolusite (MnO₂, Merck) in oxide activated or in the more passive powdered stage were used to serve as active-oxygen independent abiotic catalysts [45]. They could also serve as sources of Mn³⁺ liberated by root-exuded malate/malonate chelants or by external malate input [55]. The role of oxidoreductase enzymes in ABTS transformations was illustrated with the application of Pyricularia oryzae laccase that, unlike plant PO, acts in the absence of active oxygen species such as O₂^•– and H₂O₂ and uses O₂ as electron acceptor [9,20]. Sterile-filtered high-performance liquid chromatography (HPLC)--grade ABTS (0.45 μm; Fluka) dissolved in a nutrient solution aliquot was applied one week later. The pH values of the resulting culture fluids rose from initial 4.4–4.7 to the later 5.1 in alfalfa and to 6.2 in white mustard. The final plant biomass ranged 0.38 to 0.5 g DW per flask. The sterility of the cultures was carefully supervised with the transfer of fluid aliquots to Standard I bacterial agar (Merck, Darmstadt).

2.3. Enzymatic and Chemical Tests

The activity of Mn-independent peroxidase (EC 1.11.1.7) in plant culture fluids war recorded as increase in A₄₃₆ with 0.33 mL of the culture filtrate, 0.33 mL of 1.8 mM guaiacol, and 0.33 mL of 13.2 mM H₂O₂ prepared in 0.13 M potassium phosphate buffer pH 6.0 (ε₄₃₆ = 6400 M⁻¹ cm⁻¹ for the resulting tetraguaiacol and other products) [59]. Enzyme reactions were followed at least in triplicate for 4 to 10 (to 30) min using UV-VIS spectrophotometry (Helios Beta, Unicam UV-VIS, Cambridge, UK). For the determination of plant laccase, H₂O₂ amendments were replaced by water. Monophenol monooxygenase (EC1.14.18.1) at pH 6.0 was measured as increase in A₄₇₅ with 0.1 mL of culture filtrate and 0.9 mL of 15.6 mM DL-DOPA, 3,4-dihydroxyphenylalanine, prepared in 0.1 M potassium phosphate buffer (ε₄₇₅ = 3600 M⁻¹ cm⁻¹ for the resulting dopachrome) [60].

For the qualitative detection of root released phenolics, culture fluid aliquots were boiled up for 10 s to inactivate enzymes. Then 1-mL aliquots were amended with 0.1 mL of a freshly prepared aqueous solution of 4.21 mM Fast Blue B salt (Acros). Increases in absorbance were recorded over the initial 10 s at 530 nm in quadruplicate and expressed as an increase in absorbance min⁻¹ [52,61]. Peroxides in culture fluids were indicated with Merckoquant peroxide test strips (Merck). Non-oxidized remnants of ABTS in the fluids of plant cultures with azodication formation were determined in 1-mL reaction mixtures of 0.1 M potassium phosphate buffer pH 4.5 containing 0.1 mL of horseradish peroxidase (HRP, 0.3 mg 10 mL⁻¹; Merck) and 0.1 mL of the culture filtrate. The reaction was started with the application of 0.1 mL H₂O₂ (45 mg 10 mL⁻¹) and followed at A₄₂₀.

2.4. Oxidation of ABTS by Abiotic Catalysts

Triplicate samples of 4 mg from the transition metal compounds, CoCl₂·6H₂O, CuSO₄·5H₂O, FeSO₄·7H₂O, MnSO₄·H₂O, and Pb(CH₃CO₂)₂·3H₂O (Merck) were weighed into 2 mL plastic microtubes. They were dissolved in 1.5 mL bideionized water or 0.15 M KH₂PO₄ buffer pH 4.5 and amended with 0.1 mL of aqueous 1.2 mM ABTS. Active oxygen species were added in the form of H₂O₂ (11 mg) and the superoxide anion radical (O₂^•–) derived from 1.5–2 mg of the alkaline KO₂. The reaction mixtures were maintained in the physiological pH range of (2.3) 3.8 to >6.0 or acidified to pH 1.5–2 with H₂SO₄. UV-VIS spectrophotometry was used to quantify the formation of the green ABTS^•+ solution at λ = 420 nm [59] and the red ABTS²⁺ moieties by scanning spectrophotometry. The long-term presence of active oxygen was controlled with O₂^•– test strips (Merck).

2.5. Extraction of Aliphatic Carboxylic Acids for HPLC Examination

Carboxylic acids in culture fluids were extracted twice for 3 and 1 h, respectively, with 1.5 volumes of diethylether. Ether extracts were pooled and evaporated to a near-dryness (to prevent fatty acids from being blown out). Residues were resuspended in 1 mL of 0.005 M H₂SO₄ in bideionized water. Acids were quantified by HPLC using a Shimadzu SCL-10A model with a SPD-M10Avp diode array detector (Shimadzu Corp., Kyoto, Japan), and a Chrompack Organic acids column 300 × 6.5 mm (Varian Australia Pty Ltd., Mulgrave, Australia) under isocratic conditions. The mobile phase (0.6 mL min⁻¹) consisted of 0.005 M H₂SO₄ in bideionized water. Working conditions included 10 μL of sample injection, running time 20 min, column temperature 40 °C, and ultraviolet (UV) detection from 210 nm to 500 nm. Calibration (considering the losses to ether extraction) and establishment of a library to compare spectral profiles were performed with 23 individual HPLC-grade samples of mono- to tricarboxylic acids (Merck). Several culture fluids were acidified with 0.2-% formic acid, centrifuged, and re-examined by HPLC (Agilent 1100; Agilent Technologies, Waldbronn, Germany) equipped with an Esquire 6000 ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany).

2.6. Actual Mineral Concentrations of White Mustard Culture Fluids

Appreciating the role of Cu, Fe, Mn, and P in the assays, aliquots of the final 8.5 mL of spent white mustard culture fluids were drawn from those five-week-old cultures that had not been amended with the respective minerals of interest (compare Section 2.2). The detected minor mineral pools were rated as seed-borne and liberated by leaching and root exudation. Samples of culture fluids were acidified to pH 2 with HNO₃, passed through 0.45 μm membrane filters, and analyzed by inductively coupled plasma mass spectrometry (ICP-MS; Thermo, X series).

2.7. Examination of ABTS²⁺ Derivative by Liquid Chromatography

Non-purified culture fluid of the red ABTS²⁺ derivative generated with white mustard was centrifuged at 14,000 g for 5 min, passed through a 0.45 μm membrane filter and analyzed by liquid chromatography with mass spectrometry detection (LC-MS) and pneumatically assisted atmospheric pressure ionization (API). The Perkin Elmer Sciex API 16 S with Series 200 pump and autosampler was run with 5.2 KV ionization voltage, 5 µL sample injection, and a flow of 0.25 mL min⁻¹ under isocratic conditions. The eluent was composed of 10% methanol, 85% AFFA A, and 5% AFFA B.

AFFA A: 50 mM formic acid, 2 mM NH₄⁺HCOO^- in water.

AFFA B: 50 mM formic acid, 2 mM NH₄⁺HCOO^- in 95% acetonitrile.

2.8. Data Processing

In the case of numerical data, SPSS 8.0 software (Chicago, IL, USA) was used to calculate standard deviations (SD) of quadruplicate results, linear correlations, and to perform one-way analyses of variance. To determine the positions of the comparatively flat peaks obtained by spectrophotometric scans of ABTS solutions in the A₃₄₀ to A₈₀₀ range, absorbance values of the curves were determined for 5-nm intervals. Peaks were accepted where the differences between neighboring intervals dropped to zero. Most ABTS transformation data represent maximum values of four replicates.

3. Results and Discussion

3.1. Oxidation of ABTS by Gnotobiotic Plant Cultures

The three to four weeks old plants of the 100-mL microcosms raised aseptically in the absence of external P (from KH₂PO₄), Fe(II), or Mn(II) supplements had used seed internal resources to cover their mineral demand. They leached the Fenton metals Cu, Fe, and Mn at ppb amounts into the final 8.5 mL of free liquid (

Table 1. Oxidation of ABTS to ABTS^•+ (A₄₂₀, temporary maximum values) and ABTS²⁺ derivatives (A₅₂₀ to A₅₆₁ ± SD) by sterile hydroponic cultures of white mustard (treatments 1a to 4b), oilseed rape, and alfalfa in the alternating presence of phosphate and transition metals leached from seeds (maximum Leach values) or added up with the nutrient solution (sum, in mg L⁻¹). Letters a and b denote two independent replicates.

Minerals, Treatment	P of KH2PO4	Cu(II)	Fe(II)	Mn(II)	Mn(IV)O2 c	Malate/Malon-Ate, mg L−1	A420 of ABTS•+	A520–A561of ABTS2+	Single Absorbance Peaks
1a	Leach 263 Sum 1697 (+)	0.050 0.177 (+)	0.049 3.349 (+)	0.013 0.013 (−)	0 0 (−)	0 0 (−)	BD, below detection	0.014 ± 0.007 up to 0.189	A552–A560
1b							BD	0.044 ± 0.014	A542–A551
2a	263 (−)	0.177 (+)	3.349 (+)	55 (+)	690 (+)	7.7 (−)	0.780 (7 h)	0.108 ± 0.019	A546–A561
2b							0.214	0.114 ± 0.038	A527–A550
3a	263 (−)	0.177 (+)	3.349 (+)	55 (+)	690 (+)	679	0.999 (7 h)	0.071 ± 0.030	A542–A546
3b							0.416 (7 h)	0.057 ± 0.010	A520–A548
4b	1697 (+)	0.177 (+)	0.049 (-)	0.013 (−)	0 (−)	0 (−)	BD	0.024 ± 0.006	A520–A550
4b Lacc							0.311	0.444 ± 0.060	A515–A551
Rape	Leach rate, not determined (−)	>0.127 (+)	>3.30 (+)	>55 (+)	690 (+)	0 (−)	1.347 (30 h)	0.076 ± 0.013	A548
Alfalfa							BD	0.248 ± 0.127	A543

Time span denoting the drop of A₄₂₀ absorbance values to (near) zero by reduction of the cation radical, followed by its partial transformation to ABTS²⁺ derivatives within ≤ 60 h. Unmarked absorbance values indicate stability of the green ABTS^•+ solution for > 6 d of observation; (+), Minerals of the nutrient solution increased the pool of those leached from seeds; (−), in the current treatment, the modified nutrient solution did not contain these compounds; ^c MnO₂ oxide activated (Fluka) was applied to 2a, 3a, oilseed rape, and alfalfa. The less active MnO₂ powder (Merck) was applied to 2b and 3b.

) to complicate interpretations of their role in the catalytic system by their ubiquitous background presence. Root released peroxidases converted guaiacol (mM min⁻¹, 20 °C) at 0.202 ± 0.069 in white mustard, at 0.376 ± 0.04 in oilseed rape, and at 11.23 ± 1.72 in alfalfa (n = 15). Traces of laccases and MMO were negligible. Active oxygen and root exudate of phenolics ranged below the detection limit. Concentrations (mg L⁻¹) of malate/malonate released by white mustard roots amounted zero in the presence of P and Fe supplements, 7.7 under P starvation, and 24 under P and Fe starvation. Both carboxylates were not found in cultures of oilseed rape and alfalfa.

With the contemporary exposure to the chromogenic ABTS and some of its potential oxidants, the plant cultures did not immediately form notable amounts of the green ABTS^•+ solutions absorbing at λ = 420 nm (A₄₂₀; Table 1). The apparent shortage in active oxygen species hid the minor oxidative activities of the root-released PO, the possible formation of Mn³⁺ oxidant by PO-catalyzed aryloxy radicals, or the production of HO^• with the aid of Cu, Fe, and Mn cations in H₂O₂ dependent Fenton-like reactions [42]. However, not only laccase (treatment 4b, Table 1), the Mn(II)/Mn(IV) couple, too, oxidized ABTS spontaneously to the green cation radical. Thereby, the oxide activated MnO₂ reached higher A₄₂₀ values than its powdered surrogate, supported by malate supplements. The green solutions bleached within 7–30 h to gradually express red color tones after 24–60 h in all treatments of white mustard (1a–4b) and in oilseed rape. Alfalfa plants known to surpass root exuded PO activities of white mustard up to 167–182 times both in axenic and natural soil cultures [15,37] formed red derivatives peaking at A₅₄₃ in a superior quantity without a notable phase of preceding A₄₂₀ intermediates (Figure 2). The position of absorbance peaks in red culture fluids point to the presence of ABTS²⁺ in the A₅₁₅ to A₅₂₀ range [56]. Modifications of the azo dication in solutions peak at A₅₄₂ to A₅₆₁ [62]. Heat-killed plant control cultures devoid of MnO₂ and PO activity did not oxidize ABTS.

Upon the formation of the red derivatives, both active oxygen and PO proved to be the limiting factors. Adding H₂O₂ and HRP to culture fluid aliquots resulted in spontaneous and intense reddenings. This includes that root-derived phenolics converted to aryloxy radicals by PO/active oxygen and/or by Mn³⁺ must have been present in excess.

3.2. Liquid Chromatography-Mass Spectrometry (LC-MS) Examination of a Non-Purified ABTS Derivative from White Mustard

Under the ionizing conditions of the weak acids in the eluent, the red ABTS derivative yielded major peaks at m/z 452.6 > 490.4 > 485.3. Among the numerous minor substances, m/z 514.1 to 514.2 represented the ABTS dianion molecule completed by two protons at the former NH₄ binding sites.

A derivative appearing at m/z 486.3 points to the replacement of two ethyl (C₂H₅) by methyl groups (CH₃) (Figure 3a,b).

Comparable disintegrations of the red ABTS²⁺ related product were also documented in preliminary Fourier-transform infrared spectrometry (FT-IR) analyses [62]. The lilac precipitates of a completely by K₂O₈S₂ oxidized monoazo compound of functional ABTS showed major stretching vibrations at 1506.6, 1472.8, and 1441.4 cm⁻¹. The range of (1555) 1525–1410 cm⁻¹ is thereby associated with the presence of the -N=N- double bonds in the majority of azo dyes [63,64,65,66].

A soluble compound generated by fresh beech wood chips in the mode as above then showed the azo group related band at 1473.77 cm⁻¹ in an environment of a general structural decay. Signals from P-O bonds around 859 cm⁻¹ dominated [67].

3.3. Contributions of Abiotic Catalysts to the Oxidation of ABTS

The rate of ABTS oxidation by Fenton-like catalysts at pH 1.5–2 surpassed that in the physiological pH range of 3.8–6.0 drastically (

Table 2. Abiotic oxidation of ABTS to the green cation radical (ABTS^•+) and to red ABTS²⁺ products with absorbance peaks in the A₄₈₂ to A₆₀₁ range by active oxygen species alone or in combination with transition metal catalysts at physiological or suboptimal pH. Digits, temporary maximum absorbances A₄₂₀ of ABTS^•+ solutions.

Catalyst and Solvent	pH (2.3) 3.8–>6.0		pH 1.5–2.0
	Water	KH2PO4 Buffer	Water	KH2PO4 Buffer
O2•− from KO2	0 a	0	1.602 (6 days)	0.330
H2O2	0	0	1.604 (<4–5 h)	0.560
MnSO4/KO2	0	0	Insoluble red product A482/529 b	1.165
MnSO4/H2O2	0	0	1.636 (2.5–3 h)	0.620 (3 days)
FeSO4/KO2	0	0	0	0
FeSO4/H2O2	0	0	0	0.395 (4 days)
CuSO4/KO2	0.252	0.650	1.276	0.744
CuSO4/H2O2	0.816	0.506	1.115	0.511
NiSO4/KO2	0	0.021	1.748	0.738
NiSO4/H2O2	0	0	1.804 (4 days)	0.180
Pb(CH3CO2)2/KO2	0	0.064	Insoluble red product A553/601 b	0.813
Pb(CH3CO2)2/H2O2	0.762	0	0.996	0.581
CoCl2/KO2	0.022	0.376	Insoluble red product A514/596 b	0.950
CoCl2/H2O2	0.384	Soluble red product A514 b,c	1.750 (1.5 h) c	1.182 (28 h) c

Time span denoting the drop of A₄₂₀ absorbance values to (near) zero by reduction of the cation radical to the original ABTS. Unmarked absorbance values indicate stability of the green ABTS^•+ solution for >6 d of observation. ^a No notable oxidative reaction; ^b Position of the absorbance peaks of the red ABTS²⁺-related products; ^c The red solution of CoCl₂ peaking at A_511/512 may account for, or contribute to, the soluble red products of A₅₁₄ that finally replace the green ABTS^•+ stage.

). Moreover, the absence of KH₂PO₄ in the reaction mixture yielded higher A₄₂₀ values and enabled the immediate production of red and insoluble ABTS²⁺ products generated by two-electron oxidation (Figure 4 and Figure 5). They are generally formed at pH around 2.0 in the presence of excess oxidant [16]. The insoluble compounds comproportionated to functional ABTS^•+ by electron exchange with subsequently applied ABTS. Red compounds were not formed in the presence of buffer.

The insoluble red products generated by Co, Mn, and Pb catalysts in concert with KO₂ (O₂^•–) displayed up to two absorbance peaks in the ABTS²⁺-relevant 514-600 nm range (Figure 4 and Figure 5). They corresponded with the insoluble ABTS²⁺ compound formed by excess applications of K₂O₈S₂ oxidant at pH 2.15 with absorbance peaks at λ = 556 and 603 nm [16,62]. The soluble red product formed in three of four CoCl₂/H₂O₂ treatments peaking at A₅₁₄ could be pretended, to the greatest extent, by the red color of the CoCl₂ itself with its single peak at A₅₁₁ to A₅₁₂.

In general, abiotic contributions to the initial oxidation of ABTS at physiological pH could be expected from Co and Cu compounds in cooperation with O₂^•– and H₂O₂ both in water and KH₂PO₄ solution, and of Pb/H₂O₂ in water (Table 2). Combinations of Mn, Fe, and Ni alone with active oxygen were virtually inactive. Contemporarily, pH 1.5–2 conditions denoted by excess protons made the single active oxygen compounds O₂^•– and H₂O₂ as well as all metal/active oxygen combinations but those of Fe potent one-electron oxidants of ABTS. The reactions were somewhat less intense in the presence of KH₂PO₄ where white phosphate precipitates suggest binding of the transition metals. Low-pH disintegration of the ABTS itself did not seem to occur as its respective ABTS^•+ lambda scans did not differ from those taken at physiological pH (Figure 4).

3.4. Interaction of Catalysts

The formation of red azodication derivatives from ABTS by aseptic plants is first evidence for their ability to initiate oxidative (chain) reactions with rhizosphere priming effects. As recently indicated, the stable and soluble azodication derivatives consist of ABTS²⁺ molecules as a minority among lower-MW moieties with -N=N- double bonds (Figure 1 and Figure 3a,b). They were generated by incubating PO-bearing sapwood chips of trees with ABTS and H₂O₂ in buffer solution. The catalysis of the initial green oxidation stage of ABTS^•+ was ascribed to the PO/H₂O₂ system. The second oxidation step to ABTS²⁺ only preceded with timber-released mediator molecules converted to radicals by ABTS^•+ and possibly by the PO/H₂O₂ couple, too [62].

In this study, the outcome in red azodication derivatives was higher with PO-rich plants such as alfalfa and with supplements of fungal laccase and mineral catalysts such as MnO₂ that are independent of active oxygen species (Table 1). The spontaneous intense reddening of culture fluid aliquots amended with H₂O₂ and HRP showed that lacks in active oxygen and PO were the main limiting factors in a system containing enough root-exuded molecules that could act as catalysis-mediating aryloxy radicals.

The redox potential E⁰ = 0.68 V for the oxidation of ABTS to ABTS^•+ is in the range of plant PO (E⁰ = 0.89–0.95 V) [18] and Mn³⁺ (0.8–0.9V) [20,21] formed from Mn(II) by PO-catalyzed aryloxy radicals [15,32]. Accordingly, Mn(II) starvation in the treatments 1a, 1b, and 4b led to the lowest outcome in azodication production (Table 1) but possibly, too, in a drop of enzyme release that is stimulated by mineral fertilizing [37]. Contributions of root-exuded laccase (E⁰ = 0.4 V) and MMO (E⁰ = 0.26 V) traces as active oxygen independent enzymes may solely depend on proper redox mediator systems to overcome their deficit in redox potential. The oxidation of ABTS by pyrolusite (MnO₂) as a member of oxygen neutral clay minerals [45] may obey the rule upon the formation of manganite, with Mn being in the three-valent stage as a consequence of a transient H⁺/e^- incorporation [68,69].

Mn(IV)O₂ + ABTS + H⁺→Mn(III)OOH + ABTS^•+

(3)

Pyrolusite in plant-soil systems was mainly regarded as a source of Mn³⁺ released from the mineral by root-exuded carboxylate chelants [55]. Actually, P and Fe(II) starvation increased malate/malonate release and may have contributed, as underpinned by further exogenous malate supply, to higher ABTS transformation rates. It is known that Mn³⁺ initiated, too, the degradation of malonate to formate at least in the presence of MnP and yielded O₂^•–, H₂O₂, and acetate radical (COOH-CH₂^•) catalysts [70]. In reactions of O₂^•– with metal cations, Mn³⁺ or HO^• [71] are formed:

O₂^•− + Mn²⁺ + 2 H⁺→Mn³⁺ + H₂O₂

(4)

O₂^•− + Fe²⁺ + 2 H⁺→Fe³⁺ + H₂O₂

(5)

O₂^•− + Fe³⁺→O₂ + Fe²⁺

(6)

Fe²⁺ + H₂O₂→Fe³⁺ + HO⁻ + HO^• (Fenton reaction)

(7)

O₂^•− + H₂O₂→HO⁻ + HO^• + O₂ (Haber-Weiss reaction)

(8)

The transition metals Co, Cu, Fe, Mn, Ni, (and Pb) released from the plants and supplied, in part, with the nutrient solution formed therefore the ultimate oxidant HO^• (E⁰ = 1.8–2.7 V) in a Fenton–like reaction (see Equation (2)) [40,41] both with O₂^•– and H₂O₂. As indicated by the conversion of ABTS, the high rate of hydroxyl radical formation at pH 1.5–2 dropped to a moderate level at the physiological pH of the plant cultures in the reaction with Co, Cu, and Pb, whereas Fe and Mn failed to cooperate (Table 2).

Contemplating the treatments 1a and 4b with Cu(II) as the only efficient Fenton metal (Table 1), it is concluded that plants initiate oxidative activities in the rhizosphere by the release of peroxidases, carboxylates, and occasional traces of active oxygen species at least in alfalfa [38]. Azodication derivatives in the culture fluids confirm the presence of root–released phenolic molecules, too, that had been oxidized to aryloxy radicals with the respective redox mediator potential [62]. Their formation could mainly be ascribed to the PO/active–oxygen couple but also to the by–production of Mn³⁺ with the aid of the radicals in turn. In the absence of external H₂O₂, POs generate it by themselves from electron donors such as the intracellular NAD(P)H, indole-3-acetic acid, thiols (glutathione), and carboxylates [15,33,70]. Oxidative contributions of HO^• generated by Cu(I)/Cu(II) in a Fenton–like reaction with O₂^•– and H₂O₂ may be less efficient. Cu(I) mainly reacts with O₂ without the production of the hydroxyl radical [42]. In contrast, active oxygen independent catalysts such as pyrolusite, laccase, and Mn³⁺/malate complexes like the excessive release of alfalfa PO increased the efficacy of ABTS oxidation. The results encourage further studies into the control of micropollutants such as agrochemicals, pharmaceuticals, and endocrine disrupting compounds in soil by PO releasing crops.