The Aqueous Extract of Brassica oleracea L. Exerts Phytotoxicity by Modulating H2O2 and O2− Levels, Antioxidant Enzyme Activity and Phytohormone Levels

Allelopathic interactions between plants serve as powerful tools for weed control. Despite the increasing understanding of the allelopathic mechanisms between different plant species, the inhibitory effects of B. oleracea on weed growth remain poorly understood. In this study, we conducted experiments to demonstrate that B. oleracea extract can suppress the germination of Panicum miliaceum L.varruderale Kit. seeds as well as of the roots, shoots and hypocotyl elongation of P. miliaceum seedlings. Furthermore, we observed that B. oleracea extract reduced the levels of hydrogen peroxide and superoxide anion in the roots while increasing the activities of catalase and ascorbate peroxidase. In the shoots, B. oleracea extract enhanced the activities of superoxide dismutase and peroxidase. Moreover, the use of the extract led to an increase in the content of phytohormones (indole-3-acetic acid, indole-3-acetaldehyde, methyl indole-3-acetate, N6-isoPentenyladenosine, dihydrozeatin-7-glucoside, abscisic acid and abscisic acid glucose ester) in P. miliaceum seedlings. Interestingly, the aqueous extract contained auxins and their analogs, which inhibited the germination and growth of P. miliaceum. This may contribute to the mechanism of the B. oleracea-extract-induced suppression of P. miliaceum growth.


Introduction
Weeds pose a significant challenge to crop production, as they compete with crops for essential resources such as soil nutrients and water [1,2].To address this issue, chemical herbicides have been widely used and have introduced significant benefits to agriculture [3].However, these herbicides also come with several disadvantages, including the development of weed resistance [4,5].Consequently, there is an urgent need to improve weed control methods in a more sustainable manner.One promising approach is allelopathy, a biological phenomenon involving chemical interactions between plants [6,7].Allelopathy has the potential to be an effective and environmentally friendly tool for the weed management of field crops [8].By harnessing the natural chemical compounds produced by certain plants, it is possible to suppress the growth and development of weeds [9,10].
The Brassicaceae family holds significant potential for the exertion of allelopathic effects between weeds and other crops [11].Incorporating the residues of B. oleracea, a Brassicaceae vegetable, into the soil can significantly reduce weed biomass and weed species diversity [12].However, the specific effects of B. oleracea on the physiochemistry of weeds are not yet fully understood.Reactive oxygen species (ROS) play crucial roles in various biological processes within plant growth [13][14][15].Variations in the accumulation of superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ) in root tips have a significant impact on root growth and differentiation [16][17][18].
Antioxidant systems in cell membranes and other organelles are closely related to ROS and help prevent oxidative damage by regulating the production and elimination of ROS [19].This includes antioxidant defense enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT) [20].Further investigation is needed to understand the role of the antioxidant enzyme system in the exertion of allelopathic effects.
Allelochemicals can modulate plant growth and development by influencing phytohormones and cell division [21].Some allelochemicals can reduce ethylene and auxin levels in plants, thereby inhibiting the growth and development of recipient plants [22].Understanding the physiological and biochemical mechanisms involved, such as the role of ROS and antioxidant defense enzymes, as well as the modulation of phytohormones by allelochemicals, is crucial for comprehending the full extent of allelopathic effects.
Currently, limited knowledge is available regarding the effects of allelochemicals released by B. oleracea on the physiological and biochemical aspects of P. miliaceum, a grass weed species.Therefore, this study investigates the influence of B. oleracea on the growth parameters (shoot length, root length, hypocotyl length and germination rate), antioxidant enzyme activities (CAT, APX, POD and SOD) and phytohormone levels (auxins, cytokinins and abscisic acid) of P. miliaceum.Additionally, the components of the B. oleracea extracts are identified.These findings may provide valuable insights into the weed control of B. oleracea and offer useful parameters for its management.

The Phytotoxicity of Aqueous Extract on P. miliaceum
During the observation of P. miliaceum seed germination, a strong inhibitory effect was noted as a result of an aqueous extract concentration of 0.28 g/mL, resulting in an 85% inhibition rate.Upon the addition of the aqueous extract, both the shoot and root growth of P. miliaceum seedlings were significantly suppressed, and the inhibitory effect increased with increasing concentrations of the extract.The strongest inhibition was observed on the roots when the concentration reached 0.28 g/mL.By calculating the EC 50 , it was found that the roots were the most sensitive to the aqueous extract, with an EC 50 value of 0.08013 g/mL, indicating the strongest inhibitory effect (Figure 1).

The Effect of Aqueous Extract on H2O2 and O2 − Content in P. miliaceum Seedlings
The aqueous extract treatment resulted in a reduction in H2O2 and O2 − content in the roots of P. miliaceum seedlings (Figure 2a,b).The H2O2 content in the roots of P. miliaceum treated with the aqueous extract decreased to 2.65 ± 0.03 µmol/mL.Similarly, the O2 − content in the roots of the seedlings treated with the aqueous extract decreased significantly to 0.28 ± 0.04 µmol/mL compared to that of the control.However, there were no significant changes in the levels of H2O2 and O2 − in the shoots (Figure 2c,d).The aqueous extract treatment resulted in a reduction in H 2 O 2 and O 2 − content in the roots of P. miliaceum seedlings (Figure 2a,b).The H 2 O 2 content in the roots of P. miliaceum treated with the aqueous extract decreased to 2.65 ± 0.03 µmol/mL.Similarly, the O 2 − content in the roots of the seedlings treated with the aqueous extract decreased significantly to 0.28 ± 0.04 µmol/mL compared to that of the control.However, there were no significant changes in the levels of H 2 O 2 and O 2 − in the shoots (Figure 2c,d).

Effect of Aqueous Extract on Antioxidant Enzyme Activity
Under the influence of the aqueous extract, the brightness of the CAT and APX bands isolated from the shoots of P. miliaceum on native gels was greater than that of the control.The activities of CAT and APX were enhanced in the shoots.In the roots, the brightness of

Effect of Aqueous Extract on Antioxidant Enzyme Activity
Under the influence of the aqueous extract, the brightness of the CAT and APX bands isolated from the shoots of P. miliaceum on native gels was greater than that of the control.The activities of CAT and APX were enhanced in the shoots.In the roots, the brightness of CAT and PPO bands on the gel was higher than that of the control, indicating an increase in their activities.The activity of PPO showed different changes in the shoots and roots under the influence of the aqueous extract.However, the aqueous extract did not affect the activity of SOD (Figure 3).

The Effect of Aqueous Extract on Phytohormone Levels in P. miliaceum Seedlings
The treatment with the aqueous extract of B. oleracea resulted in significant increases in the contents of several phytohormones.The content of indole-3-carboxylic acid (ICA) increased by 34.8 ng/g.Additionally, there were significant increases in the contents of indole-3-carboxaldehyde (ICAID) and methyl indole-3-acetate (ME-IAA), with changes of 290.67 ng/g and 7.08 ng/g, respectively.Among the cytokinins, only the contents of dihydrozeatin-7-glucoside (DHZ7G) and N6-isoPentenyladenosine (IPR) showed significant increases.Moreover, the contents of abscisic acid (ABA) and abscisic acid glucosyl ester (ABA-GE) also increased significantly under the treatment with B. oleracea extract, with contents of 6.97 ng/g and 35.99 ng/g, respectively (Figure 4).

The Effect of Aqueous Extract on Phytohormone Levels in P. miliaceum Seedlings
The treatment with the aqueous extract of B. oleracea resulted in significant increases in the contents of several phytohormones.The content of indole-3-carboxylic acid (ICA) increased by 34.8 ng/g.Additionally, there were significant increases in the contents of indole-3-carboxaldehyde (ICAID) and methyl indole-3-acetate (ME-IAA), with changes of 290.67 ng/g and 7.08 ng/g, respectively.Among the cytokinins, only the contents of dihydrozeatin-7-glucoside (DHZ7G) and N6-isoPentenyladenosine (IPR) showed significant increases.Moreover, the contents of abscisic acid (ABA) and abscisic acid glucosyl ester (ABA-GE) also increased significantly under the treatment with B. oleracea extract, with contents of 6.97 ng/g and 35.99 ng/g, respectively (Figure 4).

The Effects of Auxins on the Growth of P. miliaceum
We conducted experiments using ICA, ICAID and ME-IAA on the growth and development of P. miliaceum and calculated the EC50 values.We found that indole compounds had significant effects on the growth and development of P. miliaceum.In terms of the seed germination rate, ICAID showed the strongest inhibitory effect.For shoot elongation, ME-IAA had the lowest EC50 value, indicating the strongest inhibitory effect on

The Effects of Auxins on the Growth of P. miliaceum
We conducted experiments using ICA, ICAID and ME-IAA on the growth and development of P. miliaceum and calculated the EC 50 values.We found that indole compounds had significant effects on the growth and development of P. miliaceum.In terms of the seed germination rate, ICAID showed the strongest inhibitory effect.For shoot elongation, ME-IAA had the lowest EC 50 value, indicating the strongest inhibitory effect on shoots.Regarding root elongation, ICAID exhibited the most potent inhibitory effect (Figure 5).

Composition Analysis of B. oleracea Extract
After conducting a comparative analysis of the characteristic ion peaks using mass spectrometry, indole alkaloids were identified in the aqueous extract of B. oleracea.Among these, indole-3-acetic acid acts as an auxin, while indole-3-aldehyde, indole-3-acetonitrile and indole-3-carboxylic acid serve as analogs of auxin.Furthermore, the aqueous extract also contained other indole alkaloids, such as indole-3-acrylic acid and methoxyindoleacetic acid (Table 1).After we demonstrated the inhibitory effect of ICA on P. miliaceum, we also found the presence of ICA in the extract, indicating that ICA was one of the crucial molecules responsible for the phytotoxicity induced by B. oleracea extract.

Composition Analysis of B. oleracea Extract
After conducting a comparative analysis of the characteristic ion peaks using mass spectrometry, indole alkaloids were identified in the aqueous extract of B. oleracea.Among these, indole-3-acetic acid acts as an auxin, while indole-3-aldehyde, indole-3-acetonitrile and indole-3-carboxylic acid serve as analogs of auxin.Furthermore, the aqueous extract also contained other indole alkaloids, such as indole-3-acrylic acid and methoxyindoleacetic acid (Table 1).After we demonstrated the inhibitory effect of ICA on P. miliaceum, we also found the presence of ICA in the extract, indicating that ICA was one of the crucial molecules responsible for the phytotoxicity induced by B. oleracea extract.

Discussion
This study indicated that the aqueous extract obtained from B. oleracea inhibited the growth of P. miliaceum.The roots of P. miliaceum seedlings were the most sensitive to the aqueous extract, experiencing the strongest inhibitory effect.This was achieved through multiple mechanisms, including a reduction in reactive oxygen species (ROS) levels, an increase in the activity of antioxidant defense enzymes, and the modulation of phytohormone levels.ROS play a vital role as signaling molecules in regulating root growth [23,24].Studies have demonstrated that spermidine and brassinosteroids can reduce the overall growth of maize seedlings by increasing oxidative damage through ROS production [25].Additionally, the copper-mediated inhibition of primary root elongation is believed to occur via increasing H 2 O 2 levels [26].These findings suggest that ROS may serve as common signaling molecules for root growth across different plant species.However, our research findings indicated that the aqueous extract reduced the levels of H 2 O 2 and O 2 − .Therefore, we concluded that H 2 O 2 and O 2 − , as signaling molecules, generated different responses under various stress conditions.The treatment with the aqueous extract resulted in increased activities of POD, SOD, CAT and APX, which collectively contributed to a reduction in ROS accumulation in the roots.By enhancing the activities of these antioxidant defense enzymes, the extract treatment effectively mitigated ROS levels in root tissues [27].B. oleracea extract significantly enhances the activity of CAT and PPO in P. miliaceum roots.However, in the shoots, treatment with B. oleracea extract altered the activity of CAT and APX, indicating that the activity of PPO and APX is organ-specific.Nevertheless, CAT activity was increased in both the roots and shoots, leading us to believe that CAT played a significant role in the phytotoxicity exerted by B. oleracea extract.Artemisia absinthium and Psidium guajava extracts increase the activity of CAT and APX in Parthenium hysterophorus leaves [28].Nevertheless, further research is needed to understand the specific interplay between ROS clearance and antioxidant enzymes in allelopathic interactions.
Phytohormones play a crucial role in regulating seedling growth [29].In this study, the content of the methyl ester of indole-3-acetic acid (ME-IAA) increased under treatment with the aqueous extract compared to that in the control.ME-IAA is an inactivated form of indole-3-acetic acid (IAA), while ME-IAA is believed to serve as a storage form that can be converted into IAA and utilized for homeostatic regulation in plants [30,31].Meanwhile, our study also demonstrated that ME-IAA exerted a strong inhibitory effect on P. miliaceum growth.However, it neither exhibited a growth-promoting effect at low concentrations nor a growth-inhibitory effect at high concentrations, as is observed with auxin in plants [32,33].Our research has revealed that P. miliaceum seedlings are influenced by the aqueous extract, which leads to an increase in the content of auxin.Interestingly, the aqueous extract also contains auxin.Exogenous auxin has been found to reduce H 2 O 2 levels, thereby affecting the growth of plant seedlings.There exists an antagonistic relationship between H 2 O 2 and auxin distribution.Therefore, we believe that the addition of exogenous auxin from B. oleracea results in the absorption of excessive auxin by P. miliaceum, leading to an antagonistic interaction with reactive oxygen species in the roots, thereby co-regulating the growth of P. miliaceum seedlings.However, the proportions and content of the phytohormones in B. oleracea extract need further determination, since these also accelerated the application of B. oleracea extract in field weed management.
Both ICA and ICAID are natural products, and ICAID has been demonstrated to play a significant role in the apical dominance of pea seedlings by acting as an inhibitor of lateral shoot growth.In addition, ICAID, as an inhibitor of TIR1, has promising prospects for development as a herbicide, [34,35], including during ethylene and ABA production in germinating seeds under allelopathic stress [36].ABI4 is an AP2/ERF-type transcription factor that regulates the ABA signaling pathway during seed germination and post-germination growth [37].ABI4 regulates the expression of genes involved in ROS production and the response to ROS clearance [38].We believe that the increase in the content of abscisic acid activates the antioxidant system in P. miliaceum seedlings, thereby reducing the level of ROS.For example, ABI4 responds to salt stress in Arabidopsis by regulating ascorbate synthesis and ROS accumulation [39].As a precursor of ABA, ABA-GE levels increase with elevated levels of ABA.Previous studies have suggested that the increase in endogenous ABA observed in plants may be partially attributed to the breakdown of endogenous ABA-GE [40,41].
Allelochemicals are bioactive mediators of allelopathy, and they are non-nutritional compounds formed as secondary metabolites during various physiological processes in plants [42].Allelopathic effects typically arise from the combined action of different compounds.There is no single plant toxin exclusively responsible for exerting allelopathic effects [43].Therefore, the phytotoxicity of B. oleracea extract on P. miliaceum was the result of the combined action of multiple allelochemicals.
In summary, B. oleracea exerts phytotoxic effects on P. miliaceum by reducing the germination of seeds, and inhibits root, shoot and embryo growth in P. miliaceum seedlings.Specific biochemical parameters associated with this allelopathic effect include a decrease in H 2 O 2 and O 2 − levels in the roots, as well as the induction of CAT and POD activity in the roots.Additionally, B. oleracea extract contains auxin and its analogs.This plant extract, which has the potential to control and manage weeds, warrants further research.If B. oleracea extract contains a variety of auxin analogs in abundant quantities and is used to regulate weed growth, it could contribute to weed management.

Preparation of the Aqueous Extract of B. oleracea
Briefly, 280 g of fresh B. oleracea leaves was weighed and ground into a powder using liquid nitrogen in a mortar.The powder was transferred into a 1 L conical flask, to which 1000 mL of sterile water was added.The flask was shaken with a shaker at 4 • C for 48 h.The resulting solution was filtered through four layers of cheesecloth and the filtrate was centrifuged at 4000 rpm for 20 min [44].The supernatant was collected and a mother solution with a concentration of 0.28 g/mL was obtained.The mother solution was diluted with sterile water to obtain extraction solutions with concentrations of 0.01 g/mL, Plants 2023, 12, 3086 9 of 13 0.03 g/mL, 0.05 g/mL, 0.07 g/mL, 0.14 g/mL and 0.28 g/mL.The prepared extraction solutions were then stored in a refrigerator at 4 • C for future use.

Germination and Growth Conditions of P. miliaceum
The growth inhibition of P. miliaceum was assessed using a medium composed of MS (2.16 g/L Murashige and Skoog basal medium, 8 g/L sucrose and 8 g/L agar).When the MS medium had nearly solidified, different concentrations of the sterilized extract were added, while the control group received an equal volume of sterile water.After sterilization, the P. miliaceum seeds were added to the culture bottles.P. miliaceum was cultivated at 23 • C under long day conditions (16/8 h light/dark) with cool-white fluorescence bulbs serving as the light source.The aqueous extract of B. oleracea was added to the medium before culturing the P. miliaceum [45].The medium of the control treatment contained the same amount of water.After seven days, the germination rate, root length, shoot length and hypocotyl length of the P. miliaceum seeds were measured.

Determination of H 2 O 2 and O 2
− Levels P. miliaceum seedlings were placed in a staining solution mixed with 10 mM Tris-HCl (pH 7.6) in an amount of 9 mL + DAB (3, 3-diaminobenzidine) + 2 mL of MES and vacuumed with a vacuum pump to allow the dye to enter the plant.They were stained in the dark for 24 h and then discolored with 95% alcohol and photographed; 0.5 g of NBT was dissolved in a 0.1 M phosphoric acid buffer to prepare 10 mL of the solution [46].The ROS content was measured using assay kits (Suzhou Grace Biotechnology Co., Ltd.; Suzhou, China).

Assay of Antioxidant Enzyme Activity through Native-PAGE Profiling
Non-denatured polyacrylamide gel electrophoresis was performed on a vertical slab at 4 • C using 0.01 M Tris-glycine (pH 8.3) as an electrode buffer with 12% running and 3.5% stacking gel.Using a Mini-PROTEAN ® Tetra electrophoresis tank (Bio-Rad Laboratories Co., Ltd., Hercules, CA, USA) for electrophoresis.An enzyme sample corresponding to 30 mg of protein mixed with glycerol was layered on top of the stacking gel and electrophoresis was performed at a current of 25 mA per slab.After electrophoresis, the gels were washed with distilled water.The gel was incubated in 10 mM potassium phosphate buffer (pH 6.0) containing 20 mM guaiacol and 0.01% H 2 O 2 for 15 min, and the POD isozymes were observed as orange-brown bands.After incubating them in 2.5 mmol/L NBT for 30 min, the SOD isozymes were observed on the gels as colored bands.After immersing the gels in 1.17 × 10 −6 mol/L riboflavin for 20 min, they were transferred to Petri dishes and irradiated with fluorescent lamps.With the exception of the areas where SOD was localized, light exposure resulted in the production of a purple tint of insoluble formazan throughout the gel.The gels were incubated at room temperature in 0.1 M potassium phosphate buffer (pH 6.4) containing 4 mM ascorbate and 4 mM H 2 O 2 for 15 min to localize the APX isozymes.Then, the gels were washed with distilled water and stained with 0.125 N HCl solution containing 0.1% ferrocyanide and 0.1% ferrichloride (w/v).Colorless bands against a Prussian blue backdrop were used to identify the APX isozymes.The CAT isozymes were visualized by soaking the gels in potassium phosphate buffer (pH 7.0) followed by incubating them in the same buffer containing 5 mM H 2 O 2 .After incubation for 10 min, the gels were rinsed with water and stained in a reactive mixture containing 2% (w/v) potassium ferricyanide and 2% ferric chloride (v/v).The isozymes were shown as colorless bands against a deep-blue background [47,48].
4.5.The Detection of Phytohormone Levels in P. miliaceum P. miliaceum samples were ground to a powder using liquid nitrogen and extracted with 1 mL of methanol for subsequent LC-MS (AB 6500 + QTRAP ® , Agilent Technologies Co., Ltd., Beijing, China) analysis.The liquid chromatography conditions were as follows: the column used was from Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 100 mm × 2.1 mm Plants 2023, 12, 3086 10 of 13 i.d.); the mobile phase consisted of water and acetonitrile; the quantification of phytohormones was performed based on the retention time and peak shape of the standard compounds; each peak area represented the relative content of the corresponding plant hormone; standard solutions with concentrations of 0.01 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL were prepared; the concentration versus peak area standard curve was calculated using the standard solutions; the peak areas of the P. miliaceum samples were then substituted into the standard curve equation to calculate the absolute content of phytohormones [49].

Identification of Secondary Metabolites in Aqueous Extract of B. oleracea
The B. oleracea leaf extract was spun for 10 s to ensure thorough mixing.Briefly, 200 µL of the mixed sample was taken and transferred into a 2 mL EP tube.Then, 200 µL of 70% methanol was added and the sample was vortexed for 3 min before being centrifuged at 12,000 rpm at 4 • C for 10 min.The supernatant was filtered through an organic-based microporous membrane (0.22 µm) into a liquid phase injection vial and analyzed using LC-MS/MS.The liquid phase conditions were as follows: column, Agilent SB-C18 (1.8 µm, 2.1 mm × 100 mm); mobile phase A, ultrapure water (with 0.1% formic acid to ensure peak sharpness); mobile phase B, acetonitrile (0.1% formic acid); gradient elution, 0.00 min B at 5%, with an increase in the B proportion to 95% within 9.00 min, which was maintained for 1 min, and a decrease in the B proportion to 5%, from 10.00 to 11.10 min, which was equilibrated at 5% until the 14 min time point; flow rate, 0.35 mL/min; column temperature, 40 • C; injection volume, 4 µL.The main mass spectrometry conditions included the following ESI source operating parameters: ion source, turbo spray; source temperature, 550 • C; ion spray voltage (IS), 5500 V (positive ion mode)/-4500 V (negative ion mode); gas 1 (GS1), gas 2 (GS2) and curtain gas (CUR) set at 50, 60 and 25.0 psi, respectively; collision-induced dissociation (CID) parameters set to high.Instrument tuning and mass calibration were performed using 10 and 100 µmol/L of polyethylene glycol solutions for the QQQ and LIT modes, respectively.A QQQ scan was performed in the MRM mode with the collision gas (nitrogen) set to medium.The DP and CE for each MRM ion pair were optimized through further DP and CE optimization.A specific set of MRM ion pairs was monitored for each metabolite eluted during each period.The obtained mass spectrometry information was matched with that in the metabolite public database HMDB (http://www.hmdb.ca/accessed on 20 June 2023) to obtain secondary metabolite information [50].

Statistical Analysis
The data were determined using the F-test or Levene's test.Statistical significance was evaluated using a two-tailed t-test (for all two-group comparisons).The data are presented as mean ± standard error (SE) and a p-value of < 0.05 was considered statistically significant with * p < 0.05 and ** p < 0.01.

Conclusions
Our research on the allelopathic effect of B. oleracea on weeds indicates that B. oleracea aqueous extract can effectively inhibit the growth of P. miliaceum, making it a promising choice for sustainable weed management.Our study has revealed that the allelopathic activity of B. oleracea extract on P. miliaceum seedlings is mediated through the modulation of hydrogen peroxide, superoxide anions and phytohormone levels.Given the escalating

Figure 1 .
Figure 1.Effects of aqueous extract on the germination rate, root length, shoot length and hypocotyl length of P. miliaceum.(a) Effects of aqueous extract on the germination of P. miliaceum.(b) Effects of aqueous extract on the shoot of P. miliaceum.(c) Effects of aqueous extract on the root of P. miliaceum.(d) Effects of aqueous extract on the hypocotyl of P. miliaceum.2.2.The Effect of Aqueous Extract on H 2 O 2 and O 2 − Content in P. miliaceum Seedlings

Figure 2 .
Figure 2. Effects of aqueous extract on H2O2 and O2 − accumulation in the roots and shoots of P. miliaceum.(a,b) Quantification of NBT and DAB staining.The blue staining represents O2 − , while the red staining represents H2O2.(c,d) Content of O2 − and H2O2 (** p < 0.01).Each bar graph is the mean ± standard error.

Figure 2 .
Figure 2. Effects of aqueous extract on H 2 O 2 and O 2 − accumulation in the roots and shoots of P. miliaceum.(a,b) Quantification of NBT and DAB staining.The blue staining represents O 2 − , while the red staining represents H 2 O 2 .(c,d) Content of O 2 − and H 2 O 2 (** p < 0.01).Each bar graph is the mean ± standard error.

Figure 3 .
Figure 3. Isozyme profile of CAT, APX, PPO and SOD from the shoots and roots.

Figure 3 .
Figure 3. Isozyme profile of CAT, APX, PPO and SOD from the shoots and roots.

Figure 5 .
Figure 5.The effects of indole-3-carboxaldehyde (ICA), methyl indole-3-acetate (ME-IAA) and indole-3-carboxylic acid (ICAID) on the growth and development of P. miliaceum.(a) The effect of ICA on P. miliaceum germination.(b) The effect of ME-IAA on P. miliaceum germination.(c) The effect of ICAID on P. miliaceum germination.(d) The effect of ICA on P. miliaceum shoot length.(e) The effect of ME-IAA on P. miliaceum shoot length.(f) The effect of ICAID on P. miliaceum shoot length.(g) The effect of ICA on P. miliaceum root length.(h) The effect of ME-IAA on P. miliaceum root length.(i) The effect of ICAID on P. miliaceum root length.

Figure 5 .
Figure 5.The effects of indole-3-carboxaldehyde (ICA), methyl indole-3-acetate (ME-IAA) and indole-3-carboxylic acid (ICAID) on the growth and development of P. miliaceum.(a) The effect of ICA on P. miliaceum germination.(b) The effect of ME-IAA on P. miliaceum germination.(c) The effect of ICAID on P. miliaceum germination.(d) The effect of ICA on P. miliaceum shoot length.(e) The effect of ME-IAA on P. miliaceum shoot length.(f) The effect of ICAID on P. miliaceum shoot length.(g) The effect of ICA on P. miliaceum root length.(h) The effect of ME-IAA on P. miliaceum root length.(i) The effect of ICAID on P. miliaceum root length.

Table 1 .
Secondary metabolites in the aqueous extract of B. oleracea.