Reactive oxygen species (ROS), including superoxide (O₂•⁻) and hydrogen peroxide (H ₂ O ₂ ), 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 H ₂ O ₂ standards ( Figure 1B, complete). Omission of HRP from the assay solution eliminated the detectable fluorescence ( Figure 1B, −HRP), indicating that H ₂ O ₂ 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.

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.

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.