Data on detection of singlet oxygen, hydroxyl radical and organic radical in Arabidopsis thaliana

This article contains data related to the research article entitled, “Organic radical imaging in plants: Focus on protein radicals” (Kumar et al., 2018). The data presented herein focus on reactive oxygen species (ROS) and organic radical formed within photosynthetic tissues of Arabidopsis thaliana during high light stress and includes (1) Confocal laser scanning microscopic images using 3′-p-(hydroxyphenyl) fluorescein (HPF) as specific probe for the detection of hydroxyl radical (HO•); (2) Confocal laser scanning microscopic images using Singlet Oxygen Sensor Green (SOSG) as a specific probe for the detection of singlet oxygen (1O2) and; (3) Electron paramagnetic resonance (EPR) spectroscopy using spin traps for the detection of organic radical.


a b s t r a c t
This article contains data related to the research article entitled, "Organic radical imaging in plants: Focus on protein radicals" (Kumar et al., 2018). The data presented herein focus on reactive oxygen species (ROS) and organic radical formed within photosynthetic tissues of Arabidopsis thaliana during high light stress and includes (1) Confocal laser scanning microscopic images using 3 0 -p-(hydroxyphenyl) fluorescein (HPF) as specific probe for the detection of hydroxyl radical (HO ); (2) Confocal laser scanning microscopic images using Singlet Oxygen Sensor Green (SOSG) as a specific probe for the detection of singlet oxygen ( 1 O 2 ) and; (3) Electron paramagnetic resonance (EPR) spectroscopy using spin traps for the detection of organic radical. &

Value of the data
We suggest organic radicals and other biomolecules oxidized by HO and 1 O 2 as significant members of the signaling pathways in high-light stressed plants.
We illustrate the localization of hydroxyl radical (HO ) and singlet oxygen ( 1 O 2 ) within Arabidopsis leaves as well as detection of organic radicals in thylakoid membranes by techniques which might be of interest to the community of plant redox biology and ROS-mediated signaling.
Apart from malondialdehyde (MDA), organic radicals/ biomolecules oxidized by HO and 1 O 2 have not been reported as major regulators of signaling in stressed plants. However, our current data article can be useful as it deals with the current aspect and hypothesizes the phenomenon.
We present histochemical staining followed by confocal laser scanning microscopy which can be used as an imaging tool for HO , 1 O 2 localization and spin-trap based EPR spectroscopy for qualitative analysis of organic radical.

Data
Data presented herein bring together microscopic techniques used to visualize the formation of ROS (HO and 1 O 2 ) within Arabidopsis leaves in the first stage and detection of organic radical formation in the second stage of stress-induced protein oxidation as suggested in [1]. Plants were subjected to high white light stress (1500 mmol photons m À 2 s À 1 ) at low temperature (8°C) for 13 h, i.e. conditions chosen so as to reach oxidative stress in photosystem II but concurrently to avoid chlorophyll degradation. Immediately after the light-induced stress, the leaves were cut into pieces and incubated with specific fluorescent probes in dark/RL for 30 min to gain confocal images from spongy mesophyll cells, omitting 3 lines of cells along the injured edges. To avoid photosensitization of SOSG [2], an RL source (λ Z 600 nm) has been used during histochemical staining, recently with both fluorescent probes. Organic radicals formation were determined using EPR spin trapping spectroscopy in thylakoid membranes isolated from pre-illuminated Arabidopsis leaves and illuminated by a high white light (30 min/5 min, as specified).

Plant samples -Arabidopsis thaliana
Seeds of A. thaliana wild-type Columbia-0 was purchased from the Nottingham Arabidopsis Stock Centre (NASC), U.K. The seeds were soaked for 4 days (at 4°C) followed by potting with a peat substrate (Klasmann, Potgrond H). The plants were grown for 5-6 weeks in a walk-in type growth chamber Fytoscope FS-WI-HY (Photon Systems Instruments, Drásov, Czech Republic) under following conditions: photoperiod of 8/16 h light/dark (100 μmol photons m À 2 s À 1 ); temperature of 22/20°C light/dark and a relative humidity of 60%. For high light treatment, illumination at 1500 mmol photons m À 2 s À 1 at low temperature (8°C) for 13 h was achieved using AlgaeTron AG 230 (Photon Systems Instruments, Drásov, Czech Republic).

Histochemical staining for microscopy
Leaf pieces of size 5 Â 5 mm were excised from Arabidopsis leaf blade on a glass slide wetted with HEPES buffer (pH 7.5) and infiltrated with fluorescent probes (either 10 μM HPF or 50 μM SOSG) in a syringe. Subsequently, the leaf pieces together with probe solution were transferred into 1.5 ml Eppendorf tube and incubated for 30 min either in dark or exposed to high red light. The illumination was performed utilizing a LED source with a light guide CL6000 LED Zeiss (Carl Zeiss Microscopy GmbH, Jena, Germany). The exposure was achieved using a long-pass edge interference filter (λ Z 600 nm) (Andover Corporation, Salem, NH, USA). Fig. 2A shows a schematic representation of the experimental procedure for details refer to references [2,3].

Confocal laser scanning microscopy
Following the staining procedure, the leaf pieces were transferred into a fresh HEPES buffer (pH 7.5) on a slide and visualized by confocal laser scanning microscopy (Fluorview 1000 unit attached to IX80 microscope; Olympus Czech Group, Prague, Czech Republic). The excitation of HPF and SOSG was performed by a 488 nm line of an argon laser and the emission was detected by a 505-525 nm filter, respectively. The excitation/emission parameters utilized is slightly different as compared to excitation/emission maxima mentioned in datasheet (please refer to legend to Fig. 1) due to variation of the instrument used in our study. The laser intensities for Figs. 2-4 were set as described in our previous study [2].

Thylakoid membrane isolation
Thylakoid membranes were prepared according to the protocol of Casazza and co-workers [4] as briefly described below.
Leaves were harvested from 5-6 weeks old Arabidopsis plants. The leaves (5-10 g) were floated in dark on ice cold water for 5-10 min and then blotted. All glassware's were pre-cooled before use and all further steps were performed at 4°C under dark condition.       NaHCO 3 (10 mM), HEPES (20 mM, pH 7.6), 0.5% (w/v) fatty acid-free BSA] followed by a last-step centrifugation (2600g, 3 min at 4°C).
The pellet was dissolved in a small volume (0.5-1 ml) of the re-suspension buffer; chlorophyll concentration was calculated from the absorbance of an 80% (v/v) acetone extract measured at 645 and 663 nm [5].