Hydrogen peroxide (H₂O₂) is a key reactive oxygen species generated in plant chloroplasts under high light. In this study, we evaluated the performance of a commercial fluorometric H₂O₂ assay kit by testing its applicability to Arabidopsis thaliana leaf extracts. Fluorescence was detected even in the absence of the enzyme horseradish peroxidase (HRP), indicating non-specific Amplite® Red oxidation by endogenous compounds. This response was further enhanced under high-light conditions. These findings demonstrate that the H2O2 assay kit can generate false-positive signals and lacks sufficient specificity for reliable H₂O₂ quantification in plant tissues.
","acknowledgements":"We thank Dr. N. Sato for excellent technical assistance.
","authors":[{"affiliations":["Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"i2487290@cc.kyoto-su.ac.jp","firstName":"Akira","lastName":" Fukuwatari","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University","Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences","Center for Plant Sciences"],"credit":["conceptualization","methodology","supervision","validation","writing_originalDraft","writing_reviewEditing"],"email":"motohas@cc.kyoto-su.ac.jp","firstName":"Ken","lastName":"Motohashi","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-8414-2836"}],"awards":[],"conflictsOfInterest":null,"dataTable":null,"extendedData":[],"funding":"This work was supported by JSPS KAKENHI Grant Number 16K07409 and by a grant from the Center for Plant Sciences, Kyoto Sangyo University.
","image":{"url":"https://portal.micropublication.org/uploads/2cefac80f9a3e44e4d31c0c07bee6342.jpg"},"imageCaption":"A, Schematic illustration of the detection mechanism of the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence).
B, Standard curve generated from hydrogen peroxide standards (0, 12.5, 25, 50, 125, 250, 500, and 1,000 pmol per 50 µL). Data represent mean ± standard deviation (SD) (n = 3).
C, Fluorescence measurements of Arabidopsis leaf extracts (approximately 40 mg fresh weight) under the conditions defined below. D and L represent dark and light conditions, respectively. Data represent mean ± SD (n = 3).
Definitions for panels B and C: “complete” = Amplite® Red + HRP; “−HRP” = HRP omitted; “−HRP, −Amplite® Red” = both HRP and Amplite® Red omitted.
","imageTitle":"Quantification of hydrogen peroxide in Arabidopsis thaliana leaf tissues using a fluorometric assay kit
","methods":"Plant materials and growth conditions ----- Arabidopsis thaliana ecotype Columbia-0 (Col-0) seeds were surface-sterilized in 5% (v/v) sodium hypochlorite for 5 minutes, and sown on Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with 1.2% (w/v) agar and 1% (w/v) sucrose. Plants were grown in petri dishes under continuous light (50 μmol photons m⁻² s⁻¹) at 23°C for 21 days in a growth chamber.
Sample preparation for hydrogen peroxide detection in Arabidopsis leaves----- For dark treatment, plants were incubated in complete darkness for 12 hours. For light treatment, plants were exposed to high light intensity (1,000 μmol photons m−2 s−1) for 1 hour to induce hydrogen peroxide production. After treatment, leaf tissues (approximately 40 mg) were harvested and immediately frozen in liquid nitrogen in 2 mL loop screw cap tube containing a 7-mm zirconia bead (1392-200-SS-LP-C, WATSON, Tokyo, Japan). Tissue samples were homogenized using a bead homogenizer (Fisherbrand Bead Mill24, Fisher Scientific, Waltham, MA, USA) at speed (S) = 2.10 ms-1 for one 15-s cycle.
Quantification of hydrogen peroxide in Arabidopsis leaf samples ----- Hydrogen peroxide levels were quantified using the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest, Pleasanton, CA, USA). Assay buffer (1,500 µL) was added to homogenized samples, followed by centrifugation at 20,000 × g for 10 minutes. The resulting supernatants (50 µL) were transferred to assay solutions containing Amplite® Red with or without HRP. Fluorescence was measured in a 96-well black plate (#3915, Corning) using a fluorescence spectrometer (BioTek Synergy HTX, Agilent, Santa Clara, CA, USA). Excitation was set at 528 nm (20-nm bandwidth), and emission was detected at 590 nm (20-nm bandwidth).
","reagents":"Hydrogen peroxide detection assay kit ----- The Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest) was used for quantifying hydrogen peroxide in Arabidopsis leaf samples. The kit includes Amplite® Red, 3% hydrogen peroxide, assay buffer, and HRP.
","patternDescription":"Reactive oxygen species (ROS), including superoxide (O₂⁻) and hydrogen peroxide (H2O2), are generated during the light reactions of photosynthesis. Excess ROS can damage photosynthetic components, including Photosystem I, thereby reducing the efficiency of photosynthetic electron transport (Vilyanen and Kozuleva, 2025). ROS are traditionally visualized in plant leaves using nitro blue tetrazolium staining for superoxide (Jabs et al., 1996; Lee et al., 2002; Okegawa and Motohashi, 2015) and 3,3'-diaminobenzidine (DAB) staining for hydrogen peroxide (Bindschedler et al., 2006; Rojas et al., 2012; Okegawa and Motohashi, 2015). However, although these methods enable spatial visualization in leaves, they require multiple procedural steps and are not readily quantitative. Fluorescence-based assays are widely used for hydrogen peroxide detection in non-plant systems (Mori et al., 1991; Von Rein and Hirschelmann, 1991; Zhang et al., 2021). Commercial kits are also readily available for this purpose, and hydrogen peroxide was detected by horseradish peroxidase (HRP)-catalyzed oxidation of fluorometric indicators (Zhou et al., 1997). In this study, we aimed to evaluate the applicability of a commercially available fluorometric hydrogen peroxide quantification kit to plant leaf samples. Because H₂O₂ levels in leaves are relatively low, we selected kits utilizing highly sensitive fluorescence-based detection methods.
Amplite® Red undergoes horseradish peroxidase (HRP)-catalyzed oxidation in the presence of hydrogen peroxide, generating fluorescence with an emission maximum at ~585 nm (Figure 1A). We used this fluorescence to quantify hydrogen peroxide using a fluorescence spectrometer. To quantify hydrogen peroxide in leaf tissues, we generated a standard curve using 0 – 1 nmol hydrogen peroxide standard according to the kit protocol (Figure1B, complete). Omission of HRP from the assay solution eliminated the detectable fluorescence (Figure1B, −HRP), indicating that hydrogen peroxide alone does not oxidize Amplite® Red.
Thereafter, we attempted to determine hydrogen peroxide accumulation in Arabidopsis leaves. In land plants, high-light conditions promote ROS production by the excess reducing power generated through photosynthetic electron transport in chloroplasts; thus, ROS accumulation is not observed under dark conditions (Okegawa and Motohashi, 2015). To measure hydrogen peroxide in lysates prepared from Arabidopsis leaf tissues, samples were collected from plants exposed to either dark or high light conditions. Fluorescence from oxidized Amplite® Red was observed under both dark and high light conditions; however, the high light-treated samples exhibited approximately 2.7-fold higher intensity than the dark-treated samples (Figure 1C, dark and light (complete)). Furthermore, no fluoresce signals were detected in the leaf lysates in the absence of both HRP and Amplite® Red (Figure 1C, dark and light (−HRP, −Amplite® Red)).
Subsequently, we attempted to quantify hydrogen peroxide in the dark and light-treated samples using a detection system lacking HRP (Figure 1C, dark and light (−HRP)). In this system, the Amplite® Red is not directly oxidized by hydrogen peroxide without HRP (Figure 1B, −HRP); therefore, no fluorescence was expected. However, fluorescence intensities comparable to those in the complete system were observed, suggesting that Amplite® Red was oxidized independently of the intended enzymatic reaction, possibly by endogenous leaf components.
Notably, this fluorescence intensity was lower in dark-treated samples and higher in light-treated samples, suggesting that this HRP-independent,
non-specific oxidation of Amplite® Red is enhanced under high-light conditions. The increased fluorescence in light-treated samples suggests that certain endogenous oxidizing substances accumulate in Arabidopsis leaf tissues upon illumination. Plants contain abundant glutathione (Rahantaniaina et al., 2013; Maruta et al., 2024). Reduced glutathione (GSH) is listed as an inhibitor in the assay kit documentation, and oxidized glutathione (GSSG) or other light-induced reactive metabolites may have contributed to the direct oxidation of Amplite® Red.
In conclusion, these results indicate that endogenous oxidants—particularly those whose level increase upon light exposure—can interfere with fluorescence-based hydrogen peroxide assays and generate false-positive signals in plant samples. Therefore, without additional purification or validation steps, the Amplite® Red assay kit is not suitable for reliable quantification of hydrogen peroxide in Arabidopsis leaf samples. These results emphasize that additional validation is required when applying fluorometric H₂O₂ assays to plant tissues, and that improved methods with higher specificity for plant redox conditions will be important for accurate ROS quantification.
","references":[{"reference":"Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C, Davies DR, Ausubel FM, Bolwell GP (2006) Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47: 851-863
","pubmedId":"","doi":""},{"reference":"Jabs T, Dietrich RA, Dangl JL (1996) Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 273: 1853-1856
","pubmedId":"","doi":""},{"reference":"Lee BH, Lee H, Xiong L, Zhu JK (2002) A mitochondrial complex I defect impairs cold-regulated nuclear gene expression. Plant Cell 14: 1235-1251
","pubmedId":"","doi":""},{"reference":"Maruta T, Tanaka Y, Yamamoto K, Ishida T, Hamada A, Ishikawa T (2024) Evolutionary insights into strategy shifts for the safe and effective accumulation of ascorbate in plants. J Exp Bot 75: 2664-2681
","pubmedId":"","doi":""},{"reference":"Mori I, Fujita Y, Toyoda M, Kato K, Yoshida N, Akagi M (1991) Fluorimetric determination of hydrogen peroxide by use of the fluorescence reaction between N-(4'-hydroxyphenyl)-N-(4-methylquinolinyl)amine and cobalt(II) in the presence of trimethylstearylammonium chloride. Talanta 38: 683-686
","pubmedId":"","doi":""},{"reference":"Okegawa Y, Motohashi K (2015) Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo. Plant J 84: 900-913
","pubmedId":"","doi":""},{"reference":"Rahantaniaina MS, Tuzet A, Mhamdi A, Noctor G (2013) Missing links in understanding redox signaling via thiol/disulfide modulation: how is glutathione oxidized in plants? Front Plant Sci 4: 477
","pubmedId":"","doi":""},{"reference":"Rojas CM, Senthil-Kumar M, Wang K, Ryu CM, Kaundal A, Mysore KS (2012) Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis. Plant Cell 24: 336-352
","pubmedId":"","doi":""},{"reference":"Vilyanen DV, Kozuleva MA (2025) Photosynthetic Control and Its Role in Protection of Photosystem I against Photoinhibition. Biochemistry (Mosc) 90: 840-859
","pubmedId":"","doi":""},{"reference":"Von Rein C, Hirschelmann R (1991) Comparison of a fluorimetric and a colorimetric method for the determination of hydrogen peroxide production by rat pleural macrophages. J Pharmacol Methods 26: 61-65
","pubmedId":"","doi":""},{"reference":"Zhang GQ, Li YS, Liu WP, Gao XF (2021) A fluorimetric and colorimetric dual-signal sensor for hydrogen peroxide and glucose based on the intrinsic peroxidase-like activity of cobalt and nitrogen co-doped carbon dots and inner filter effect. Anal Methods 13: 3196-3204
","pubmedId":"","doi":""},{"reference":"Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253: 162-168
","pubmedId":"","doi":""}],"title":"The Amplite® fluorometric H₂O₂ assay kit does not accurately quantify hydrogen peroxide in Arabidopsis leaf tissue
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Leonore Reiser"},"openAcknowledgement":false,"submitted":"1771972776439"}]},{"id":"85b1eb9c-f6e3-4d2f-9054-368de33d42fd","decision":"revise","abstract":"Hydrogen peroxide (H₂O₂) is a key reactive oxygen species generated in plant chloroplasts under high light. In this study, we evaluated the performance of a commercial fluorometric H₂O₂ assay kit by testing its applicability to Arabidopsis thaliana leaf extracts. Fluorescence was detected even in the absence of the enzyme horseradish peroxidase (HRP), indicating non-specific Amplite® Red oxidation by endogenous compounds. This response was further enhanced under high-light conditions. These findings demonstrate that the H2O2 assay kit can generate false-positive signals and lacks sufficient specificity for reliable H₂O₂ quantification in plant tissues.
","acknowledgements":"We thank Dr. N. Sato for excellent technical assistance.
","authors":[{"affiliations":["Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"i2487290@cc.kyoto-su.ac.jp","firstName":"Akira","lastName":" Fukuwatari","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University","Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences","Center for Plant Sciences"],"credit":["conceptualization","methodology","supervision","validation","writing_originalDraft","writing_reviewEditing"],"email":"motohas@cc.kyoto-su.ac.jp","firstName":"Ken","lastName":"Motohashi","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-8414-2836"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This work was supported by JSPS KAKENHI Grant Number 16K07409 and by a grant from the Center for Plant Sciences, Kyoto Sangyo University.
","image":{"url":"https://portal.micropublication.org/uploads/10367545f16f85125ebfa78f19082c61.jpg"},"imageCaption":"(A) Schematic illustration of the detection mechanism of the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence). (B) Standard curve generated from hydrogen peroxide standards (0, 12.5, 25, 50, 125, 250, 500, and 1,000 pmol per 50 µL). Data represent mean ± standard deviation (SD) (n = 3). (C) Fluorescence measurements of Arabidopsis leaf extracts (approximately 40 mg fresh weight) under the conditions defined below. D and L represent dark and light conditions, respectively. Data represent mean ± SD (n = 3). Definitions for panels B and C: “complete” = Amplite® Red + HRP; “−HRP” = HRP omitted; “−HRP, −Amplite® Red” = both HRP and Amplite® Red omitted.
","imageTitle":"Quantification of hydrogen peroxide in Arabidopsis thaliana leaf tissues using a fluorometric assay kit
","methods":"Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) seeds were surface-sterilized in 5% (v/v) sodium hypochlorite for 5 minutes, and sown on Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with 1.2% (w/v) agar and 1% (w/v) sucrose. Plants were grown in petri dishes under continuous light (50 μmol photons m⁻² s⁻¹) at 23°C for 21 days in a growth chamber.
Sample preparation for hydrogen peroxide detection in Arabidopsis leaves
For dark treatment, plants were incubated in complete darkness for 12 hours. For light treatment, plants were exposed to high light intensity (1,000 μmol photons m−2 s−1) for 1 hour to induce hydrogen peroxide production. After treatment, leaf tissues (approximately 40 mg) were harvested and immediately frozen in liquid nitrogen in 2 mL loop screw cap tube containing a 7-mm zirconia bead (1392-200-SS-LP-C, WATSON, Tokyo, Japan). Tissue samples were homogenized using a bead homogenizer (Fisherbrand Bead Mill24, Fisher Scientific, Waltham, MA, USA) at speed (S) = 2.10 ms-1 for one 15-s cycle.
Quantification of hydrogen peroxide in Arabidopsis leaf samples
Hydrogen peroxide levels were quantified using the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest, Pleasanton, CA, USA). Assay buffer (1,500 µL) was added to homogenized samples, followed by centrifugation at 20,000 × g for 10 minutes. The resulting supernatants (50 µL) were transferred to assay solutions containing Amplite® Red with or without HRP. Fluorescence was measured in a 96-well black plate (#3915, Corning) using a fluorescence spectrometer (BioTek Synergy HTX, Agilent, Santa Clara, CA, USA). Excitation was set at 528 nm (20-nm bandwidth), and emission was detected at 590 nm (20-nm bandwidth).
","reagents":"Hydrogen peroxide detection assay kit
The Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest) was used for quantifying hydrogen peroxide in Arabidopsis leaf samples. The kit includes Amplite® Red, 3% hydrogen peroxide, assay buffer, and HRP.
","patternDescription":"Reactive oxygen species (ROS), including superoxide (O₂⁻) and hydrogen peroxide (H2O2), are generated during the light reactions of photosynthesis. Excess ROS can damage photosynthetic components, including Photosystem I, thereby reducing the efficiency of photosynthetic electron transport (Vilyanen and Kozuleva, 2025). ROS are traditionally visualized in plant leaves using nitro blue tetrazolium staining for superoxide (Jabs et al., 1996; Lee et al., 2002; Okegawa and Motohashi, 2015) and 3,3'-diaminobenzidine (DAB) staining for hydrogen peroxide (Bindschedler et al., 2006; Rojas et al., 2012; Okegawa and Motohashi, 2015). However, although these methods enable spatial visualization in leaves, they require multiple procedural steps and are not readily quantitative. Fluorescence-based assays are widely used for hydrogen peroxide detection in non-plant systems (Mori et al., 1991; Von Rein and Hirschelmann, 1991; Zhang et al., 2021). Commercial kits are also readily available for this purpose, and hydrogen peroxide was detected by horseradish peroxidase (HRP)-catalyzed oxidation of fluorometric indicators (Zhou et al., 1997). In this study, we aimed to evaluate the applicability of a commercially available fluorometric hydrogen peroxide quantification kit to plant leaf samples. Because H₂O₂ levels in leaves are relatively low, we selected kits utilizing highly sensitive fluorescence-based detection methods.
Amplite® Red undergoes horseradish peroxidase (HRP)-catalyzed oxidation in the presence of hydrogen peroxide, generating fluorescence with an emission maximum at ~585 nm (Figure 1A). We used this fluorescence to quantify hydrogen peroxide using a fluorescence spectrometer. To quantify hydrogen peroxide in leaf tissues, we generated a standard curve using 0 – 1 nmol hydrogen peroxide standard according to the kit protocol (Figure1B, complete). Omission of HRP from the assay solution eliminated the detectable fluorescence (Figure1B, −HRP), indicating that hydrogen peroxide alone does not oxidize Amplite® Red.
Thereafter, we attempted to determine hydrogen peroxide accumulation in Arabidopsis leaves. In land plants, high-light conditions promote ROS production by the excess reducing power generated through photosynthetic electron transport in chloroplasts; thus, ROS accumulation is not observed under dark conditions (Okegawa and Motohashi, 2015). To measure hydrogen peroxide in lysates prepared from Arabidopsis leaf tissues, samples were collected from plants exposed to either dark or high light conditions. Fluorescence from oxidized Amplite® Red was observed under both dark and high light conditions; however, the high light-treated samples exhibited approximately 2.7-fold higher intensity than the dark-treated samples (Figure 1C, dark and light (complete)). Furthermore, no fluoresce signals were detected in the leaf lysates in the absence of both HRP and Amplite® Red (Figure 1C, dark and light (−HRP, −Amplite® Red)).
Subsequently, we attempted to quantify hydrogen peroxide in the dark and light-treated samples using a detection system lacking HRP (Figure 1C, dark and light (−HRP)). In this system, the Amplite® Red is not directly oxidized by hydrogen peroxide without HRP (Figure 1B, −HRP); therefore, no fluorescence was expected. However, fluorescence intensities comparable to those in the complete system were observed, suggesting that Amplite® Red was oxidized independently of the intended enzymatic reaction, possibly by endogenous leaf components.
Notably, this fluorescence intensity was lower in dark-treated samples and higher in light-treated samples, suggesting that this HRP-independent, non-specific oxidation of Amplite® Red is enhanced under high-light conditions. The increased fluorescence in light-treated samples suggests that certain endogenous oxidizing substances accumulate in Arabidopsis leaf tissues upon illumination. Plants contain abundant glutathione (Rahantaniaina et al., 2013; Maruta et al., 2024). Reduced glutathione (GSH) is listed as an inhibitor in the assay kit documentation, and oxidized glutathione (GSSG) or other light-induced reactive metabolites may have contributed to the direct oxidation of Amplite® Red.
In conclusion, these results indicate that endogenous oxidants—particularly those whose level increase upon light exposure—can interfere with fluorescence-based hydrogen peroxide assays and generate false-positive signals in plant samples. Therefore, without additional purification or validation steps, the Amplite® Red assay kit is not suitable for reliable quantification of hydrogen peroxide in Arabidopsis leaf samples. These results emphasize that additional validation is required when applying fluorometric H₂O₂ assays to plant tissues, and that improved methods with higher specificity for plant redox conditions will be important for accurate ROS quantification.
","references":[{"reference":"Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, et al., Paul Bolwell. 2006. Peroxidase‐dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. The Plant Journal 47: 851-863.
","pubmedId":"","doi":"DOI: 10.1111/j.1365-313X.2006.02837.x"},{"reference":"Jabs T, Dietrich RA, Dangl JL. 1996. Initiation of Runaway Cell Death in an\n Arabidopsis\n Mutant by Extracellular Superoxide. Science 273: 1853-1856.
","pubmedId":"","doi":"DOI: 10.1126/science.273.5283.1853"},{"reference":"Lee Bh, Lee H, Xiong L, Zhu JK. 2002. A Mitochondrial Complex I Defect Impairs Cold-Regulated Nuclear Gene Expression. The Plant Cell 14: 1235-1251.
","pubmedId":"","doi":"DOI: 10.1105/tpc.010433"},{"reference":"Maruta T, Tanaka Y, Yamamoto K, Ishida T, Hamada A, Ishikawa T. 2024. Evolutionary insights into strategy shifts for the safe and effective accumulation of ascorbate in plants. Journal of Experimental Botany 75: 2664-2681.
","pubmedId":"","doi":"DOI: 10.1093/jxb/erae062"},{"reference":"Mori I, Fujita Y, Toyoda M, Kato K, Yoshida N, Akagi M. 1991. Fluorimetric determination of hydrogen peroxide by use of the fluorescence reaction between N-(4′-hydroxyphenyl)-N-(4-methylquinolinyl)amine and cobalt(II) in the presence of trimethylstearylammonium chloride. Talanta 38: 683-686.
","pubmedId":"","doi":"DOI: 10.1016/0039-9140(91)80157-u"},{"reference":"Okegawa Y, Motohashi K. 2015. Chloroplastic thioredoxin\n m\n functions as a major regulator of Calvin cycle enzymes during photosynthesis\n in vivo. The Plant Journal 84: 900-913.
","pubmedId":"","doi":"DOI: 10.1111/tpj.13049"},{"reference":"Rahantaniaina MS, Tuzet Ae, Mhamdi A, Noctor G. 2013. Missing links in understanding redox signaling via thiol/disulfide modulation: how is glutathione oxidized in plants?. Frontiers in Plant Science 4: 10.3389/fpls.2013.00477.
","pubmedId":"","doi":"DOI: 10.3389/fpls.2013.00477"},{"reference":"Rojas CM, Senthil-Kumar M, Wang K, Ryu CM, Kaundal A, Mysore KS. 2012. Glycolate Oxidase Modulates Reactive Oxygen Species–Mediated Signal Transduction during Nonhost Resistance in Nicotiana benthamiana and Arabidopsis\n . The Plant Cell 24: 336-352.
","pubmedId":"","doi":"DOI: 10.1105/tpc.111.093245"},{"reference":"Vilyanen DV, Kozuleva MA. 2025. Photosynthetic Control and Its Role in Protection of Photosystem I against Photoinhibition. Biochemistry (Moscow) 90: 840-859.
","pubmedId":"","doi":"DOI: 10.1134/S0006297925601121"},{"reference":"Von Rein C, Hirschelmann R. 1991. Comparison of a fluorimetric and a colorimetric method for the determination of hydrogen peroxide production by rat pleural macrophages. Journal of Pharmacological Methods 26: 61-65.
","pubmedId":"","doi":"DOI: 10.1016/0160-5402(91)90054-9"},{"reference":"Zhang GQ, Li Ys, Liu WP, Gao XF. 2021. A fluorimetric and colorimetric dual-signal sensor for hydrogen peroxide and glucose based on the intrinsic peroxidase-like activity of cobalt and nitrogen co-doped carbon dots and inner filter effect. Analytical Methods 13: 3196-3204.
","pubmedId":"","doi":"DOI: 10.1039/d1ay00781e"},{"reference":"Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP. 1997. A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases. Analytical Biochemistry 253: 162-168.
","pubmedId":"","doi":"DOI: 10.1006/abio.1997.2391"}],"title":"The Amplite® fluorometric H₂O₂ assay kit does not accurately quantify hydrogen peroxide in Arabidopsis leaf tissue
","reviews":[{"reviewer":{"displayName":"Kyaw Aung"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"788f85da-b2ee-4c3b-9c23-10fa82925954","decision":"revise","abstract":"Hydrogen peroxide (H₂O₂) is a key reactive oxygen species in plants. We evaluated a commercial Amplite® fluorometric H₂O₂ assay in Arabidopsis thaliana leaf extracts. The extracts produced strong fluorescence without exogenous horseradish peroxidase (HRP), with higher signals after high-light than dark treatment. However, spiking the extracts with 5–15 µM H₂O₂ produced no concentration-dependent increase either with or without HRP, whereas the same additions increased fluorescence in assay buffer. Thus, the assay did not reliably quantify H₂O₂ in Arabidopsis leaf extracts under the conditions tested.
","acknowledgements":"We thank Dr. N. Sato for excellent technical assistance.
","authors":[{"affiliations":["Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"fafudu0@gmail.com","firstName":"Akira","lastName":" Fukuwatari","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"g2356970@cc.kyoto-su.ac.jp","firstName":"Yuto","lastName":"Nawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University","Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences","Center for Plant Sciences"],"credit":["conceptualization","methodology","supervision","validation","writing_originalDraft","writing_reviewEditing"],"email":"motohas@cc.kyoto-su.ac.jp","firstName":"Ken","lastName":"Motohashi","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-8414-2836"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by JSPS KAKENHI Grant Number 16K07409 and by a grant from the Center for Plant Sciences, Kyoto Sangyo University.
","image":{"url":"https://portal.micropublication.org/uploads/ff237d6ea535fcb0f97ef1ce57b82730.jpg"},"imageCaption":"(A) Schematic illustration of the detection mechanism of the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence). (B) Standard curve generated using hydrogen peroxide standards (0, 12.5, 25, 50, 125, 250, 500, 1,000 and 1,500 pmol per 50 µL). (C) Fluorescence measurements of Arabidopsis leaf extracts prepared from approximately 40 mg fresh weight under the assay conditions defined below. (D–F) H₂O₂ spike-recovery analysis in assay buffer and Arabidopsis leaf extracts. Exogenous H₂O₂ was added at final concentrations of 0, 5, 10, and 15 µM to assay buffer containing Amplite® Red and HRP but no leaf extract (D), to Arabidopsis leaf extracts under complete assay conditions (E), or to Arabidopsis leaf extracts under assay conditions lacking HRP (F). D and L indicate extracts prepared from dark-treated and high-light-treated plants, respectively, in panels C, E and F. Data represent the mean ± SD. Data in panels B and D were obtained from three independent assay replicates, whereas data in panels C, E, and F were obtained from three biological replicates. “complete” indicates reactions containing Amplite® Red and HRP; “−HRP” indicates reactions without HRP; and “−HRP, −Amplite® Red” indicates reactions without either HRP or Amplite® Red. Panel B and panels D–F were obtained in separate assay runs; panels D–F were measured in parallel using the same instrument settings.
","imageTitle":"Evaluation of hydrogen peroxide detection and spike recovery in Arabidopsis thaliana leaf extracts using the Amplite® fluorometric assay
","methods":"Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) seeds were surface-sterilized in 5% (v/v) sodium hypochlorite for 5 minutes, and sown on Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with 1.2% (w/v) agar and 1% (w/v) sucrose. Plants were grown in Petri dishes under continuous light (50 μmol photons m⁻² s⁻¹) at 23 °C for 21 days in a growth chamber.
Sample preparation for hydrogen peroxide detection in Arabidopsis leaves
For dark treatment, plants were incubated in complete darkness for 12 hours. For high-light treatment, plants were exposed to high light intensity (1,000 μmol photons m−2 s−1) for 1 hour to induce hydrogen peroxide production. After treatment, leaves (approximately 40 mg) were harvested and immediately frozen in liquid nitrogen in 2-mL loop screw-cap tube containing a 7-mm zirconia bead (1392-200-SS-LP-C, WATSON, Tokyo, Japan). Tissue samples were homogenized using a bead homogenizer (Fisherbrand Bead Mill24, Fisher Scientific, Waltham, MA, USA) at speed (S) = 2.10 m s-1 for one 15-s cycle.
Fluorometric hydrogen peroxide assay of Arabidopsis leaf extracts
Fluorescence responses were measured using the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest, Pleasanton, CA, USA). Assay buffer (1,500 µL) was added to homogenized samples, followed by centrifugation at 20,000 × g for 10 minutes. The resulting supernatants (50 µL) were transferred to assay solutions containing Amplite® Red with or without HRP. After incubation for 30 min at room temperature protected from light, fluorescence was measured in a 96-well black plate (#3915, Corning) using a fluorescence microplate reader (BioTek Synergy HTX, Agilent, Santa Clara, CA, USA). Excitation was set at 528 nm (20-nm bandwidth), and emission was detected at 590 nm (20-nm bandwidth).
For the H₂O₂ spike-recovery analysis, H₂O₂ was added to 50-µL aliquots of the leaf extracts to give final assay concentrations of 0, 5, 10, and 15 µM. The spiked extracts were then assayed with Amplite® Red in the presence or absence of HRP as described above. Control reactions contained assay buffer instead of leaf extract. The control and leaf-extract reactions shown in Figure 1D–F were analyzed in parallel under the same measurement conditions.
Use of artificial intelligence
OpenAI Codex (GPT-5.6 Sol) was used during manuscript revision for English-language editing and consistency checks among the text, figure, and figure legend. It was not used to generate experimental data or perform primary data analysis. All scientific content and interpretations were reviewed and approved by the authors.
","reagents":"Hydrogen peroxide detection assay kit
The Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest Cat. No. 11501) was used for fluorometric analysis of hydrogen peroxide in Arabidopsis leaf extracts. The kit includes Amplite® Red, 3% hydrogen peroxide, assay buffer, HRP, and DMSO.
","patternDescription":"Reactive oxygen species (ROS), including superoxide (O₂•⁻) and hydrogen peroxide (H2O2), are generated during the light reactions of photosynthesis. Excess ROS can damage photosynthetic components, including Photosystem I, thereby reducing the efficiency of photosynthetic electron transport (Vilyanen and Kozuleva, 2025). ROS are traditionally visualized in plant leaves using nitro blue tetrazolium staining for superoxide (Jabs et al., 1996; Lee et al., 2002; Okegawa and Motohashi, 2015) and 3,3′-diaminobenzidine (DAB) staining for hydrogen peroxide (Bindschedler et al., 2006; Rojas et al., 2012; Okegawa and Motohashi, 2015). However, although these methods enable spatial visualization in leaves, they require multiple procedural steps and are not readily quantitative. Fluorescence-based assays are widely used for hydrogen peroxide detection in non-plant systems (Mori et al., 1991; Von Rein and Hirschelmann, 1991; Zhang et al., 2021). Commercial kits are also readily available for this purpose, and hydrogen peroxide is detected by horseradish peroxidase (HRP)-catalyzed oxidation of fluorometric indicators (Zhou et al., 1997). Amplite® Red (CAS: 119171-73-2) is chemically identical to Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Amplex Red has been used without exogenous HRP for semiquantitative visualization of ROS in living Arabidopsis root hairs (Kubenova et al., 2023), showing that the addition of exogenous HRP is not always required to observe Amplex Red fluorescence in intact Arabidopsis tissues. Thus, fluorescence detected without added HRP does not, by itself, establish whether the signal quantitatively reflects H₂O₂ concentration. Because H₂O₂ levels in leaves are relatively low, we selected a highly sensitive commercial fluorometric assay and evaluated both its applicability to Arabidopsis leaf extracts and the quantitative relationship between H₂O₂ concentration and fluorescence by H₂O₂ spike-recovery analysis.
Amplite® Red undergoes horseradish peroxidase (HRP)-catalyzed oxidation in the presence of hydrogen peroxide, generating fluorescence with an emission maximum at ~585 nm (Figure 1A). We assessed whether this fluorescence could be used to quantify hydrogen peroxide using a fluorescence microplate reader. We first generated a standard curve using 0–1.5 nmol hydrogen peroxide standards according to the kit protocol (Figure 1B, complete). Omission of HRP from the assay solution eliminated the detectable fluorescence (Figure 1B, −HRP), indicating that H2O2 alone did not produce detectable fluorescence under the conditions tested.
We next examined the fluorescence response of Arabidopsis leaf extracts. In land plants, high-light conditions promote ROS production by the excess reducing power generated through photosynthetic electron transport in chloroplasts; high-light treatment was therefore used to induce ROS production relative to dark-treated plants (Okegawa and Motohashi, 2015). Leaf extracts were prepared from plants exposed to either dark or high-light conditions. Amplite® Red-dependent fluorescence was observed under both dark and high-light conditions; however, the high-light-treated samples exhibited approximately 1.4-fold higher intensity than the dark-treated samples (Figure 1C, dark and light (complete)). Furthermore, no fluorescence signals were detected in the leaf extracts in the absence of both HRP and Amplite® Red (Figure 1C, dark and light (−HRP, −Amplite® Red)).
Subsequently, we examined the fluorescence response of the leaf extracts in the absence of exogenous HRP (Figure 1C, −HRP). Although H₂O₂ alone did not generate detectable Amplite® Red fluorescence in assay buffer without HRP (Figure 1B), the leaf extracts produced strong fluorescence even when HRP was omitted. The fluorescence intensities were comparable to those obtained under the complete assay conditions. These results demonstrate that exogenous HRP was not required for the generation of Amplite® Red-dependent fluorescence in the leaf extracts. However, fluorescence in the absence of exogenous HRP does not establish whether the signal quantitatively reflects H₂O₂ concentration, because endogenous peroxidases or other components of the leaf extract may support probe oxidation. We therefore evaluated the quantitative validity of the assay by H₂O₂ spike-recovery analysis.
To determine whether the fluorescence observed in Arabidopsis leaf extracts quantitatively reflected H₂O₂, known amounts of H₂O₂ were added to the extracts. Although the same amounts of H₂O₂ produced the expected increases in fluorescence in assay buffer (Figure 1D), no corresponding increase was observed in the leaf extracts (Figures 1E and 1F). The markedly different responses to added H₂O₂ in assay buffer and leaf extracts indicate a sample-matrix effect; that is, components of the leaf extract altered the relationship between added H₂O₂ and fluorescence. Possible explanations include rapid removal of added H₂O₂, inhibition or quenching of the Amplite® Red reaction by components of the leaf extract, and strong probe-derived fluorescence that is not quantitatively related to H₂O₂ concentration. The present experiments do not distinguish among these possibilities. An Amplex Red-based method has previously been used to quantify H₂O₂ in tissue extracts from several plant species, including Arabidopsis, after optimization of the extraction conditions and evaluation of tissue-dependent loss of added H₂O₂ (Chakraborty et al., 2016). Thus, the present results do not indicate that Amplex Red is intrinsically unsuitable for plant samples. Our conclusion is limited to the conditions tested here: fluorescence from Arabidopsis leaf extracts could not be reliably converted to H₂O₂ concentrations using a standard curve prepared in assay buffer.
In conclusion, Arabidopsis leaf extracts produced substantial Amplite® Red fluorescence both with and without exogenous HRP. However, fluorescence did not increase in proportion to the amount of H₂O₂ added to the extracts, whereas a clear concentration-dependent response was obtained in assay buffer. Thus, irrespective of the mechanism responsible for probe oxidation or loss of the added H₂O₂, the buffer-based standard curve could not be used to quantify H₂O₂ in the leaf extracts. These results show that the Amplite® fluorometric assay does not provide reliable quantitative measurements of H₂O₂ in Arabidopsis leaf extracts under the conditions tested.
","references":[{"reference":"Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, et al., Paul Bolwell. 2006. Peroxidase‐dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. The Plant Journal 47: 851-863.
","pubmedId":"","doi":"DOI: 10.1111/j.1365-313X.2006.02837.x"},{"reference":"Chakraborty S, Hill AL, Shirsekar G, Afzal AJ, Wang GL, Mackey D, Bonello P. 2016. Quantification of hydrogen peroxide in plant tissues using Amplex Red. Methods 109: 105-113.
","pubmedId":"27476009","doi":"10.1016/j.ymeth.2016.07.016"},{"reference":"Jabs T, Dietrich RA, Dangl JL. 1996. Initiation of Runaway Cell Death in an\n Arabidopsis\n Mutant by Extracellular Superoxide. Science 273: 1853-1856.
","pubmedId":"","doi":"DOI: 10.1126/science.273.5283.1853"},{"reference":"Kuběnová L, Haberland J, Dvořák P, Šamaj J, Ovečka M. 2023. Spatiotemporal distribution of reactive oxygen species production, delivery, and use in Arabidopsis root hairs. Plant Physiology 193: 2337-2360.
","pubmedId":"37666000","doi":"10.1093/plphys/kiad484"},{"reference":"Lee Bh, Lee H, Xiong L, Zhu JK. 2002. A Mitochondrial Complex I Defect Impairs Cold-Regulated Nuclear Gene Expression. The Plant Cell 14: 1235-1251.
","pubmedId":"","doi":"DOI: 10.1105/tpc.010433"},{"reference":"Mori I, Fujita Y, Toyoda M, Kato K, Yoshida N, Akagi M. 1991. Fluorimetric determination of hydrogen peroxide by use of the fluorescence reaction between N-(4′-hydroxyphenyl)-N-(4-methylquinolinyl)amine and cobalt(II) in the presence of trimethylstearylammonium chloride. Talanta 38: 683-686.
","pubmedId":"","doi":"DOI: 10.1016/0039-9140(91)80157-u"},{"reference":"Okegawa Y, Motohashi K. 2015. Chloroplastic thioredoxin\n m\n functions as a major regulator of Calvin cycle enzymes during photosynthesis\n in vivo. The Plant Journal 84: 900-913.
","pubmedId":"","doi":"DOI: 10.1111/tpj.13049"},{"reference":"Rojas CM, Senthil-Kumar M, Wang K, Ryu CM, Kaundal A, Mysore KS. 2012. Glycolate Oxidase Modulates Reactive Oxygen Species–Mediated Signal Transduction during Nonhost Resistance in Nicotiana benthamiana and Arabidopsis\n . The Plant Cell 24: 336-352.
","pubmedId":"","doi":"DOI: 10.1105/tpc.111.093245"},{"reference":"Vilyanen DV, Kozuleva MA. 2025. Photosynthetic Control and Its Role in Protection of Photosystem I against Photoinhibition. Biochemistry (Moscow) 90: 840-859.
","pubmedId":"","doi":"DOI: 10.1134/S0006297925601121"},{"reference":"Von Rein C, Hirschelmann R. 1991. Comparison of a fluorimetric and a colorimetric method for the determination of hydrogen peroxide production by rat pleural macrophages. Journal of Pharmacological Methods 26: 61-65.
","pubmedId":"","doi":"DOI: 10.1016/0160-5402(91)90054-9"},{"reference":"Zhang GQ, Li Ys, Liu WP, Gao XF. 2021. A fluorimetric and colorimetric dual-signal sensor for hydrogen peroxide and glucose based on the intrinsic peroxidase-like activity of cobalt and nitrogen co-doped carbon dots and inner filter effect. Analytical Methods 13: 3196-3204.
","pubmedId":"","doi":"DOI: 10.1039/d1ay00781e"},{"reference":"Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP. 1997. A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases. Analytical Biochemistry 253: 162-168.
","pubmedId":"","doi":"DOI: 10.1006/abio.1997.2391"}],"title":"Matrix effects in Arabidopsis leaf extracts limit H₂O₂ quantification with the Amplite® fluorometric assay
","reviews":[],"curatorReviews":[]},{"id":"80d5a0c1-fd36-44e0-8ba1-bd9ba4db2ef3","decision":"accept","abstract":"Hydrogen peroxide (H₂O₂) is a key reactive oxygen species in plants. We evaluated a commercial Amplite® fluorometric H₂O₂ assay in Arabidopsis thaliana leaf extracts. The extracts produced strong Amplite® Red-dependent fluorescence without exogenous horseradish peroxidase (HRP), and fluorescence signals were higher following high-light treatment than following dark treatment. However, this difference could not be quantitatively attributed to H2O2: adding 5–15 µM H₂O₂ produced no concentration-dependent increase with or without HRP, whereas the same additions increased fluorescence in assay buffer. Thus, under the conditions examined, the fluorescence observed in Arabidopsis leaf extracts did not permit reliable H2O2 quantification.
","acknowledgements":"We thank Dr. N. Sato for excellent technical assistance.
","authors":[{"affiliations":["Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"fafudu0@gmail.com","firstName":"Akira","lastName":" Fukuwatari","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"g2356970@cc.kyoto-su.ac.jp","firstName":"Yuto","lastName":"Nawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University","Kyoto Sangyo University"],"departments":["Department of Frontier Life Sciences","Center for Plant Sciences"],"credit":["conceptualization","methodology","supervision","validation","writing_originalDraft","writing_reviewEditing"],"email":"motohas@cc.kyoto-su.ac.jp","firstName":"Ken","lastName":"Motohashi","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-8414-2836"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by JSPS KAKENHI Grant Number 16K07409 and by a grant from the Center for Plant Sciences, Kyoto Sangyo University.
","image":{"url":"https://portal.micropublication.org/uploads/3f97739218e69340ee5994f65735d438.jpg"},"imageCaption":"(A) Schematic illustration of the detection mechanism of the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence). (B) Standard curve generated using hydrogen peroxide standards (0, 12.5, 25, 50, 125, 250, 500, 1,000 and 1,500 pmol per 50 µL). (C) Fluorescence measurements of Arabidopsis leaf extracts prepared from approximately 40 mg fresh weight under the assay conditions defined below. (D–F) H₂O₂ spike-recovery analysis in assay buffer and Arabidopsis leaf extracts. Exogenous H₂O₂ was added at final concentrations of 0, 5, 10, and 15 µM to assay buffer containing Amplite® Red and HRP but no leaf extract (D), to Arabidopsis leaf extracts under complete assay conditions (E), or to Arabidopsis leaf extracts under assay conditions lacking HRP (F). D and L indicate extracts prepared from dark-treated and high-light-treated plants, respectively, in panels C, E and F. Data represent the mean ± SD. Data in panels B and D were obtained from three independent assay replicates, whereas data in panels C, E, and F were obtained from three biological replicates. “complete” indicates reactions containing Amplite® Red and HRP; “−HRP” indicates reactions without HRP; and “−HRP, −Amplite® Red” indicates reactions without either HRP or Amplite® Red. Panel B and panels D–F were obtained in separate assay runs; panels D–F were measured in parallel using the same instrument settings.
","imageTitle":"Evaluation of hydrogen peroxide detection and spike recovery in Arabidopsis thaliana leaf extracts using the Amplite® fluorometric assay
","methods":"Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) seeds were surface-sterilized in 5% (v/v) sodium hypochlorite for 5 minutes, and sown on Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with 1.2% (w/v) agar and 1% (w/v) sucrose. Plants were grown in Petri dishes under continuous light (50 μmol photons m⁻² s⁻¹) at 23 °C for 21 days in a growth chamber.
Preparation of Arabidopsis leaf extracts
For dark treatment, plants were incubated in complete darkness for 12 hours. For high-light treatment, plants were exposed to high light intensity (1,000 μmol photons m−2 s−1) for 1 hour as a physiological treatment expected to increase ROS production. After treatment, leaves (approximately 40 mg fresh weight) were harvested and immediately frozen in liquid nitrogen in a 2-mL screw-cap tube with a loop cap containing a 7-mm zirconia bead (1392-200-SS-LP-C, WATSON, Tokyo, Japan). Tissue samples were homogenized using a bead homogenizer (Fisherbrand Bead Mill24, Fisher Scientific, Waltham, MA, USA) at speed (S) = 2.10 m s-1 for one 15-s cycle.
Amplite® fluorometric assay of Arabidopsis leaf extracts
Fluorescence responses were measured using the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest, Pleasanton, CA, USA). Assay buffer (1,500 µL) was added to homogenized samples, followed by centrifugation at 20,000 × g for 10 minutes. Assay reactions were prepared using the manufacturer-recommended reaction volumes: 50 µL of each supernatant was mixed with 50 µL of working solution containing Amplite® Red with or without HRP, giving a final assay volume of 100 µL per well. After incubation for 30 min at room temperature while protected from light, fluorescence was measured in a 96-well black plate (#3915, Corning) using a fluorescence microplate reader (BioTek Synergy HTX, Agilent, Santa Clara, CA, USA). Excitation was set at 528 nm (20-nm bandwidth), and emission was detected at 590 nm (20-nm bandwidth).
To examine the fluorescence response to added H₂O₂, H₂O₂ was added to 50-µL aliquots of the leaf extracts to give final assay concentrations of 0, 5, 10, and 15 µM. These samples were then assayed with Amplite® Red in the presence or absence of HRP as described above. Control reactions contained assay buffer instead of leaf extract. The control and leaf-extract reactions shown in Figure 1D–F were analyzed in parallel under the same measurement conditions.
Use of artificial intelligence
OpenAI Codex (GPT-5.6 Sol) was used solely for English-language refinement and consistency checks during manuscript revision. It was not used to draft the original manuscript, generate experimental data, perform primary data analysis, or determine scientific conclusions. All AI-assisted changes were reviewed and approved by the authors.
","reagents":"Hydrogen peroxide detection assay kit
The Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest Cat. No. 11501) was used for fluorometric measurements in Arabidopsis leaf extracts. The kit includes Amplite® Red, 3% hydrogen peroxide, assay buffer, HRP, and DMSO.
","patternDescription":"Reactive oxygen species (ROS), including superoxide (O₂•⁻) and hydrogen peroxide (H2O2), are generated during the light reactions of photosynthesis. Excess ROS can damage photosynthetic components, including Photosystem I, thereby reducing the efficiency of photosynthetic electron transport (Vilyanen and Kozuleva, 2025). ROS are traditionally visualized in plant leaves using nitro blue tetrazolium staining for superoxide (Jabs et al., 1996; Lee et al., 2002; Okegawa and Motohashi, 2015) and 3,3′-diaminobenzidine (DAB) staining for hydrogen peroxide (Bindschedler et al., 2006; Rojas et al., 2012; Okegawa and Motohashi, 2015). However, although these methods enable spatial visualization in leaves, they require multiple procedural steps and are not readily quantitative. Fluorescence-based assays are widely used for hydrogen peroxide detection in non-plant systems (Mori et al., 1991; Von Rein and Hirschelmann, 1991; Zhang et al., 2021). Commercial kits are readily available for fluorescence-based H₂O₂ detection. Because H₂O₂ levels in leaves are relatively low, we selected a highly sensitive Amplite® fluorometric H₂O₂ assay. The assay is based on HRP-catalyzed oxidation of Amplite® Red in the presence of H₂O₂ (Zhou et al., 1997). Amplite® Red (CAS: 119171-73-2) is chemically identical to Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Amplex Red has been used without exogenous HRP for semiquantitative visualization of ROS in living Arabidopsis root hairs (Kubenova et al., 2023), indicating that probe fluorescence can be generated in intact plant tissues without added HRP. However, the observation of such fluorescence does not, by itself, establish either that the signal originates from H₂O₂ or that it quantitatively reflects H₂O₂ concentration. We therefore evaluated whether the fluorescence measured with the Amplite® assay in Arabidopsis leaf extracts could be used for H₂O₂ quantification by determining whether it increased in response to known additions of H₂O₂.
Amplite® Red undergoes horseradish peroxidase (HRP)-catalyzed oxidation in the presence of hydrogen peroxide, generating fluorescence with an emission maximum at ~585 nm (Figure 1A). We assessed whether this fluorescence could be used to quantify hydrogen peroxide using a fluorescence microplate reader. We first generated a standard curve in assay buffer using 0–1.5 nmol H2O2 standards (Figure 1B, complete). Omission of HRP from the assay solution eliminated the detectable fluorescence (Figure 1B, −HRP), indicating that H2O2 alone did not produce detectable fluorescence under the conditions tested.
We next examined the fluorescence response of Arabidopsis leaf extracts. In land plants, high-light conditions promote ROS production by the excess reducing power generated through photosynthetic electron transport in chloroplasts; high-light treatment was therefore used as a physiological treatment expected to increase ROS production relative to dark treatment (Okegawa and Motohashi, 2015). Leaf extracts were prepared from plants exposed to either dark or high-light conditions. Amplite® Red-dependent fluorescence was observed under both dark and high-light conditions; however, the high-light-treated samples exhibited approximately 1.4-fold higher intensity than the dark-treated samples (Figure 1C, dark and light (complete)). Furthermore, no fluorescence signals were detected in the leaf extracts in the absence of both HRP and Amplite® Red (Figure 1C, dark and light (−HRP, −Amplite® Red)). However, this treatment-associated difference in fluorescence did not establish a quantitative difference in H₂O₂ concentration.
Subsequently, we examined the fluorescence response of the leaf extracts in the absence of exogenous HRP (Figure 1C, −HRP). Although H₂O₂ alone did not generate detectable Amplite® Red fluorescence in assay buffer without HRP (Figure 1B), the leaf extracts produced strong fluorescence even when HRP was omitted. The fluorescence intensities were comparable to those obtained under the complete assay conditions. These results demonstrate that exogenous HRP was not required for the generation of Amplite® Red-dependent fluorescence in the leaf extracts. However, this finding does not establish that the observed fluorescence originated from H₂O₂ or quantitatively reflected H₂O₂ concentration, because endogenous peroxidases or other components of the leaf extract may support probe oxidation. We therefore tested whether the leaf-extract fluorescence increased in response to known additions of H₂O₂.
To determine whether the fluorescence observed in Arabidopsis leaf extracts quantitatively reflected H₂O₂, known amounts of H₂O₂ were added to the extracts. Although the same amounts of H₂O₂ produced the expected increases in fluorescence in assay buffer (Figure 1D), no corresponding increase was observed in the leaf extracts either with or without exogenous HRP (Figures 1E and 1F). Thus, the relationship between H₂O₂ concentration and fluorescence established in assay buffer could not be applied to the leaf extracts. Moreover, the present results do not establish that the fluorescence observed in the leaf extracts originated from H₂O₂. Possible explanations include rapid removal of added H₂O₂ by endogenous H₂O₂-scavenging activities, inhibition or quenching of the Amplite® Red reaction by components of the leaf extract, and strong probe-derived fluorescence that is not quantitatively related to H₂O₂ concentration. The present experiments do not distinguish among these possibilities. An Amplex Red-based method has previously been used to quantify H₂O₂ in tissue extracts from several plant species, including Arabidopsis, after optimization of the extraction conditions and evaluation of tissue-dependent loss of added H₂O₂ (Chakraborty et al., 2016). Thus, the present results do not indicate that Amplex Red is intrinsically unsuitable for plant samples. Our conclusion is limited to the conditions tested here: fluorescence from Arabidopsis leaf extracts could not be reliably converted to H₂O₂ concentrations using a standard curve prepared in assay buffer.
In conclusion, Arabidopsis leaf extracts produced substantial Amplite® Red fluorescence both with and without exogenous HRP. However, fluorescence did not increase in proportion to the amount of H₂O₂ added to the extracts, whereas a clear concentration-dependent response was obtained in assay buffer. Thus, irrespective of the mechanism responsible for probe oxidation or loss of the added H₂O₂, the buffer-based standard curve could not be used to quantify H₂O₂ in the leaf extracts. Therefore, neither the observed fluorescence nor the difference between high-light- and dark-treated samples could be quantitatively attributed to H₂O₂. These results show that the Amplite® fluorometric assay does not provide reliable quantitative measurements of H₂O₂ in Arabidopsis leaf extracts under the conditions tested.
","references":[{"reference":"Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, et al., Paul Bolwell. 2006. Peroxidase‐dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. The Plant Journal 47: 851-863.
","pubmedId":"","doi":"DOI: 10.1111/j.1365-313X.2006.02837.x"},{"reference":"Chakraborty S, Hill AL, Shirsekar G, Afzal AJ, Wang GL, Mackey D, Bonello P. 2016. Quantification of hydrogen peroxide in plant tissues using Amplex Red. Methods 109: 105-113.
","pubmedId":"27476009","doi":"10.1016/j.ymeth.2016.07.016"},{"reference":"Jabs T, Dietrich RA, Dangl JL. 1996. Initiation of Runaway Cell Death in an\n Arabidopsis\n Mutant by Extracellular Superoxide. Science 273: 1853-1856.
","pubmedId":"","doi":"DOI: 10.1126/science.273.5283.1853"},{"reference":"Kuběnová L, Haberland J, Dvořák P, Šamaj J, Ovečka M. 2023. Spatiotemporal distribution of reactive oxygen species production, delivery, and use in Arabidopsis root hairs. Plant Physiology 193: 2337-2360.
","pubmedId":"37666000","doi":"10.1093/plphys/kiad484"},{"reference":"Lee Bh, Lee H, Xiong L, Zhu JK. 2002. A Mitochondrial Complex I Defect Impairs Cold-Regulated Nuclear Gene Expression. The Plant Cell 14: 1235-1251.
","pubmedId":"","doi":"DOI: 10.1105/tpc.010433"},{"reference":"Mori I, Fujita Y, Toyoda M, Kato K, Yoshida N, Akagi M. 1991. Fluorimetric determination of hydrogen peroxide by use of the fluorescence reaction between N-(4′-hydroxyphenyl)-N-(4-methylquinolinyl)amine and cobalt(II) in the presence of trimethylstearylammonium chloride. Talanta 38: 683-686.
","pubmedId":"","doi":"DOI: 10.1016/0039-9140(91)80157-u"},{"reference":"Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15: 473-497
","pubmedId":"","doi":""},{"reference":"Okegawa Y, Motohashi K. 2015. Chloroplastic thioredoxin\n m\n functions as a major regulator of Calvin cycle enzymes during photosynthesis\n in vivo. The Plant Journal 84: 900-913.
","pubmedId":"","doi":"DOI: 10.1111/tpj.13049"},{"reference":"Rojas CM, Senthil-Kumar M, Wang K, Ryu CM, Kaundal A, Mysore KS. 2012. Glycolate Oxidase Modulates Reactive Oxygen Species–Mediated Signal Transduction during Nonhost Resistance in Nicotiana benthamiana and Arabidopsis\n . The Plant Cell 24: 336-352.
","pubmedId":"","doi":"DOI: 10.1105/tpc.111.093245"},{"reference":"Vilyanen DV, Kozuleva MA. 2025. Photosynthetic Control and Its Role in Protection of Photosystem I against Photoinhibition. Biochemistry (Moscow) 90: 840-859.
","pubmedId":"","doi":"DOI: 10.1134/S0006297925601121"},{"reference":"Von Rein C, Hirschelmann R. 1991. Comparison of a fluorimetric and a colorimetric method for the determination of hydrogen peroxide production by rat pleural macrophages. Journal of Pharmacological Methods 26: 61-65.
","pubmedId":"","doi":"DOI: 10.1016/0160-5402(91)90054-9"},{"reference":"Zhang GQ, Li Ys, Liu WP, Gao XF. 2021. A fluorimetric and colorimetric dual-signal sensor for hydrogen peroxide and glucose based on the intrinsic peroxidase-like activity of cobalt and nitrogen co-doped carbon dots and inner filter effect. Analytical Methods 13: 3196-3204.
","pubmedId":"","doi":"DOI: 10.1039/d1ay00781e"},{"reference":"Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP. 1997. A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases. Analytical Biochemistry 253: 162-168.
","pubmedId":"","doi":"DOI: 10.1006/abio.1997.2391"}],"title":"The Amplite® fluorometric assay does not reliably quantify H₂O₂ in Arabidopsis leaf extracts
","reviews":[],"curatorReviews":[]},{"id":"dce2ebdc-6fbb-434e-a069-b2c216863c80","decision":"publish","abstract":"Hydrogen peroxide (H₂O₂) is a key reactive oxygen species in plants. We evaluated a commercial Amplite® fluorometric H₂O₂ assay in Arabidopsis thaliana leaf extracts. The extracts produced strong Amplite® Red-dependent fluorescence without exogenous horseradish peroxidase (HRP), and fluorescence signals were higher following high-light treatment than following dark treatment. However, this difference could not be quantitatively attributed to H2O2: adding 5–15 µM H₂O₂ produced no concentration-dependent increase with or without HRP, whereas the same additions increased fluorescence in assay buffer. Thus, under the conditions examined, the fluorescence observed in Arabidopsis leaf extracts did not permit reliable H2O2 quantification.
","acknowledgements":"We thank Dr. N. Sato for excellent technical assistance.
","authors":[{"affiliations":["Kyoto Sangyo University, Kyoto, Japan"],"departments":["Department of Frontier Life Sciences, Faculty of Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"fafudu0@gmail.com","firstName":"Akira","lastName":" Fukuwatari","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University, Kyoto, Japan"],"departments":["Department of Frontier Life Sciences, Faculty of Life Sciences"],"credit":["investigation","methodology","dataCuration","writing_reviewEditing"],"email":"g2356970@cc.kyoto-su.ac.jp","firstName":"Yuto","lastName":"Nawa","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Kyoto Sangyo University, Kyoto, Japan","Kyoto Sangyo University, Kyoto, Japan"],"departments":["Department of Frontier Life Sciences, Faculty of Life Sciences","Center for Plant Sciences"],"credit":["conceptualization","methodology","supervision","validation","writing_originalDraft","writing_reviewEditing"],"email":"motohas@cc.kyoto-su.ac.jp","firstName":"Ken","lastName":"Motohashi","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-8414-2836"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by JSPS KAKENHI Grant Number 16K07409 and by a grant from the Center for Plant Sciences, Kyoto Sangyo University.
","image":{"url":"https://portal.micropublication.org/uploads/3f97739218e69340ee5994f65735d438.jpg"},"imageCaption":"(A) Schematic illustration of the detection mechanism of the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence). (B) Standard curve generated using hydrogen peroxide standards (0, 12.5, 25, 50, 125, 250, 500, 1,000 and 1,500 pmol per 50 µL). (C) Fluorescence measurements of Arabidopsis leaf extracts prepared from approximately 40 mg fresh weight under the assay conditions defined below. (D–F) Fluorescence responses to added H₂O₂ in assay buffer and Arabidopsis leaf extracts. Exogenous H₂O₂ was added at final concentrations of 0, 5, 10, and 15 µM to assay buffer containing Amplite® Red and HRP but no leaf extract (D), to Arabidopsis leaf extracts under complete assay conditions (E), or to Arabidopsis leaf extracts under assay conditions lacking HRP (F). D and L indicate extracts prepared from dark-treated and high-light-treated plants, respectively, in panels C, E and F. Data represent the mean ± SD. Data in panels B and D were obtained from three independent assay replicates, whereas data in panels C, E, and F were obtained from three biological replicates. “complete” indicates reactions containing Amplite® Red and HRP; “−HRP” indicates reactions without HRP; and “−HRP, −Amplite® Red” indicates reactions without either HRP or Amplite® Red. Panel B and panels D–F were obtained in separate assay runs; panels D–F were measured in parallel using the same instrument settings.
","imageTitle":"Fluorescence responses of the Amplite® assay to H₂O₂ in assay buffer and Arabidopsis thaliana leaf extracts
","methods":"Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia-0 (Col-0) seeds were surface-sterilized in 5% (v/v) sodium hypochlorite for 5 minutes, and sown on Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented with 1.2% (w/v) agar and 1% (w/v) sucrose. Plants were grown in Petri dishes under continuous light (50 μmol photons m⁻² s⁻¹) at 23 °C for 21 days in a growth chamber.
Preparation of Arabidopsis leaf extracts
For dark treatment, plants were incubated in complete darkness for 12 hours. For high-light treatment, plants were exposed to high light intensity (1,000 μmol photons m−2 s−1) for 1 hour as a physiological treatment expected to increase ROS production. After treatment, leaves (approximately 40 mg fresh weight) were harvested and immediately frozen in liquid nitrogen in a 2-mL screw-cap tube with a loop cap containing a 7-mm zirconia bead (1392-200-SS-LP-C, WATSON, Tokyo, Japan). Tissue samples were homogenized using a bead homogenizer (Fisherbrand Bead Mill24, Fisher Scientific, Waltham, MA, USA) at speed (S) = 2.10 m s-1 for one 15-s cycle.
Amplite® fluorometric assay of Arabidopsis leaf extracts
Fluorescence responses were measured using the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest, Pleasanton, CA, USA). Assay buffer (1,500 µL) was added to homogenized samples, followed by centrifugation at 20,000 × g for 10 minutes. Assay reactions were prepared using the manufacturer-recommended reaction volumes: 50 µL of each supernatant was mixed with 50 µL of working solution containing Amplite® Red with or without HRP, giving a final assay volume of 100 µL per well. After incubation for 30 min at room temperature while protected from light, fluorescence was measured in a 96-well black plate (#3915, Corning) using a fluorescence microplate reader (BioTek Synergy HTX, Agilent, Santa Clara, CA, USA). Excitation was set at 528 nm (20-nm bandwidth), and emission was detected at 590 nm (20-nm bandwidth).
To examine the fluorescence response to added H₂O₂, H₂O₂ was added to 50-µL aliquots of the leaf extracts to give final assay concentrations of 0, 5, 10, and 15 µM. These samples were then assayed with Amplite® Red in the presence or absence of HRP as described above. Control reactions contained assay buffer instead of leaf extract. The control and leaf-extract reactions shown in Figure 1D–F were analyzed in parallel under the same measurement conditions.
Use of artificial intelligence
OpenAI Codex (GPT-5.6 Sol) was used solely for English-language refinement and consistency checks during manuscript revision. It was not used to draft the original manuscript, generate experimental data, perform primary data analysis, or determine scientific conclusions. All AI-assisted changes were reviewed and approved by the authors.
","reagents":"Hydrogen peroxide detection assay kit
The Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Red fluorescence; AAT Bioquest Cat. No. 11501) was used for fluorometric measurements in Arabidopsis leaf extracts. The kit includes Amplite® Red, 3% hydrogen peroxide, assay buffer, HRP, and DMSO.
","patternDescription":"Reactive oxygen species (ROS), including superoxide (O₂•⁻) and hydrogen peroxide (H2O2), are generated during the light reactions of photosynthesis. Excess ROS can damage photosynthetic components, including Photosystem I, thereby reducing the efficiency of photosynthetic electron transport (Vilyanen and Kozuleva, 2025). ROS are traditionally visualized in plant leaves using nitro blue tetrazolium staining for superoxide (Jabs et al., 1996; Lee et al., 2002; Okegawa and Motohashi, 2015) and 3,3′-diaminobenzidine (DAB) staining for hydrogen peroxide (Bindschedler et al., 2006; Rojas et al., 2012; Okegawa and Motohashi, 2015). However, although these methods enable spatial visualization in leaves, they require multiple procedural steps and are not readily quantitative. Fluorescence-based assays are widely used for hydrogen peroxide detection in non-plant systems (Mori et al., 1991; Von Rein and Hirschelmann, 1991; Zhang et al., 2021). Commercial kits are readily available for fluorescence-based H₂O₂ detection. Because H₂O₂ levels in leaves are relatively low, we selected a highly sensitive Amplite® fluorometric H₂O₂ assay. The assay is based on HRP-catalyzed oxidation of Amplite® Red in the presence of H₂O₂ (Zhou et al., 1997). Amplite® Red (CAS: 119171-73-2) is chemically identical to Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Amplex Red has been used without exogenous HRP for semiquantitative visualization of ROS in living Arabidopsis root hairs (Kubenova et al., 2023), indicating that probe fluorescence can be generated in intact plant tissues without added HRP. However, the observation of such fluorescence does not, by itself, establish either that the signal originates from H₂O₂ or that it quantitatively reflects H₂O₂ concentration. We therefore evaluated whether the fluorescence measured with the Amplite® assay in Arabidopsis leaf extracts could be used for H₂O₂ quantification by determining whether it increased in response to known additions of H₂O₂.
Amplite® Red undergoes horseradish peroxidase (HRP)-catalyzed oxidation in the presence of hydrogen peroxide, generating fluorescence with an emission maximum at ~585 nm (Figure 1A). We assessed whether this fluorescence could be used to quantify hydrogen peroxide using a fluorescence microplate reader. We first generated a standard curve in assay buffer using 0–1.5 nmol H2O2 standards (Figure 1B, complete). Omission of HRP from the assay solution eliminated the detectable fluorescence (Figure 1B, −HRP), indicating that H2O2 alone did not produce detectable fluorescence under the conditions tested.
We next examined the fluorescence response of Arabidopsis leaf extracts. In land plants, high-light conditions promote ROS production by the excess reducing power generated through photosynthetic electron transport in chloroplasts; high-light treatment was therefore used as a physiological treatment expected to increase ROS production relative to dark treatment (Okegawa and Motohashi, 2015). Leaf extracts were prepared from plants exposed to either dark or high-light conditions. Amplite® Red-dependent fluorescence was observed under both dark and high-light conditions; however, the high-light-treated samples exhibited approximately 1.4-fold higher intensity than the dark-treated samples (Figure 1C, dark and light (complete)). Furthermore, no fluorescence signals were detected in the leaf extracts in the absence of both HRP and Amplite® Red (Figure 1C, dark and light (−HRP, −Amplite® Red)). However, this treatment-associated difference in fluorescence did not establish a quantitative difference in H₂O₂ concentration.
Subsequently, we examined the fluorescence response of the leaf extracts in the absence of exogenous HRP (Figure 1C, −HRP). Although H₂O₂ alone did not generate detectable Amplite® Red fluorescence in assay buffer without HRP (Figure 1B), the leaf extracts produced strong fluorescence even when HRP was omitted. The fluorescence intensities were comparable to those obtained under the complete assay conditions. These results demonstrate that exogenous HRP was not required for the generation of Amplite® Red-dependent fluorescence in the leaf extracts. However, this finding does not establish that the observed fluorescence originated from H₂O₂ or quantitatively reflected H₂O₂ concentration, because endogenous peroxidases or other components of the leaf extract may support probe oxidation. We therefore tested whether the leaf-extract fluorescence increased in response to known additions of H₂O₂.
To determine whether the fluorescence observed in Arabidopsis leaf extracts quantitatively reflected H₂O₂, known amounts of H₂O₂ were added to the extracts. Although the same amounts of H₂O₂ produced the expected increases in fluorescence in assay buffer (Figure 1D), no corresponding increase was observed in the leaf extracts either with or without exogenous HRP (Figures 1E and 1F). Thus, the relationship between H₂O₂ concentration and fluorescence established in assay buffer could not be applied to the leaf extracts. Moreover, the present results do not establish that the fluorescence observed in the leaf extracts originated from H₂O₂. Possible explanations include rapid removal of added H₂O₂ by endogenous H₂O₂-scavenging activities, inhibition or quenching of the Amplite® Red reaction by components of the leaf extract, and strong probe-derived fluorescence that is not quantitatively related to H₂O₂ concentration. The present experiments do not distinguish among these possibilities. An Amplex Red-based method has previously been used to quantify H₂O₂ in tissue extracts from several plant species, including Arabidopsis, after optimization of the extraction conditions and evaluation of tissue-dependent loss of added H₂O₂ (Chakraborty et al., 2016). Thus, the present results do not indicate that Amplex Red is intrinsically unsuitable for plant samples. Our conclusion is limited to the conditions tested here: fluorescence from Arabidopsis leaf extracts could not be reliably converted to H₂O₂ concentrations using a standard curve prepared in assay buffer.
In conclusion, Arabidopsis leaf extracts produced substantial Amplite® Red fluorescence both with and without exogenous HRP. However, fluorescence did not increase in proportion to the amount of H₂O₂ added to the extracts, whereas a clear concentration-dependent response was obtained in assay buffer. Thus, irrespective of the mechanism responsible for probe oxidation or loss of the added H₂O₂, the buffer-based standard curve could not be used to quantify H₂O₂ in the leaf extracts. Therefore, neither the observed fluorescence nor the difference between high-light- and dark-treated samples could be quantitatively attributed to H₂O₂. These results show that the Amplite® fluorometric assay does not provide reliable quantitative measurements of H₂O₂ in Arabidopsis leaf extracts under the conditions tested.
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