Exogenic ROS Sensors

Two types of exogenous probe molecules are typically employed for monitoring of ROS: spin traps, which interact with ROS giving rise to EPR-detectable species (Hideg et al., 1994, 2011; Zulfugarov et al., 2011), and fluorophores, which change their emission properties due to interaction with ROS (vide infra).

The use of fluorophores offers a most promising tool because it permits the application of recently developed advanced techniques of time- and space-resolved fluorescence microscopy for in vivo studies (see Shim et al., 2012; Schmitt et al., 2013, 2014a, and references therein).

Two different approaches can be used: a) addition of exogenous fluorescence probes, which penetrate into the cell and change their fluorescence properties due to reaction with ROS, and b) expression of ROS- sensitive fluorescent proteins, mostly variants of the green fluorescent protein (GFP), which act as real-time redox reporters for the use in intact cyanobacteria, algae and higher plants. The latter ones are separately described in chapter 3.2.3 (Schmitt et al., 2014a).

Table 3 gives an overview on exogenous fluorophores that typically change their optical properties due to interaction with ROS and additionally mentions the most important properties as ROS specificity, localizability and typical application schemes as well as corresponding references for successful applications.

Most of the mentioned ROS fluorophores are not truly specific to certain ROS. However, as exogenous dyes typically respond in a certain oxidative potential range, appropriate mixtures can permit assays that are selective in a certain range of oxidative potentials (e.g. when both dyes show fluorescence or only one dye shows fluorescence, but the other not). These assays are more selective than those utilizing just a single dye.

Permeability across membranes is necessary at most for the applicability of exogenous ROS-sensing fluorophores (Table 3). Generally, the water/octanol partition coefficient could be utilized to quantify membrane permeability of the probes. For a quantitative analysis, it is necessary to know the reaction mechanism in detail, as well as possible interfering side effects and the cellular localization of these dyes. A good overview on detailed chemical reaction schemes is given by (Mattila et al., 2015).

Table 3. Compilation of ROS-sensitive exogenous fluorescence probes.

Compound/reference

Specificity

Further information/localizability

CM-H2DCFDA (Dixit and Cyr, 2003)

Unspecific

Permeates into animal cells, requires the presence of cellular esterases. Not easily applicable in plants

Singlet oxygen sensor green (SOSG) (Flors et al., 2006)

Highly specific to singlet oxygen

Successfully used for detection of 'AgO2 in

A. thaliana leaves

  • 3,3'-diaminobenzidine
  • (DAB)
  • (Thordal-Christensen et al., 1997; Fryer et al., 2002)

Specific to H2O2 in presence of peroxidase (and other heme-containing proteins)

Generates a dark brown precipitate which reports the presence and distribution of hydrogen peroxide in plant cells. Permeates into plant cells.

Aminophenyl fluorescein (APF)

APF is a cell permeable indicator that can be used to detect hydroxyl radicals (HO), peroxynitrite (ONOO) and hypochlorite (OCl) production in cells. Shows limited photooxidation.

hydroxyphenyl fluorescein (HPF)

Specific to hydroxyl radical and peroxynitrite. Minor sensitivity to other ROS. HPF is cell permeable.

nitroblue tetrazolium (NBT) (Maly et al., 1989; Thordal-Christensen et al., 1997)

Specific to superoxide and with slightly reduced reactivity to hydrogen peroxide

Proxyl fluorescamine (Cohn et al., 2008)

Specific to hydroxyl radicals and superoxide Complementary use as spin trap

Hydroethidine (dihydroethidium) (Gomes et al., 2005)

Unspecific

Binds specifically to DNA, marking the nucleus

DPPP (diphenyl-1- pyrenylphosphine) (Gomes et al., 2005)

Unspecific

Lipophilic, detects ROS in lipids, blood plasma, tissues and food

MCLA (2-methyl-6-(4- methoxyphenyl)-3,7- dihydroimidazo[1, 2-a] pyrazin-3-one, hydrochloride) (Godrant et al., 2009)

Specific to superoxide or singlet oxygen

Trans-1-(2'-Methoxyvi-

nyl)Pyrene

Highly specific to singlet oxygen

Some of these ROS probes can be tuned regarding their properties inside the cell by enzymatic reactions. For instance, the comer- cially available 2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester (H2DCF-DA), a fluorescein-based dye, which is virtually non-fluorescent in the reduced state, becomes fluorescent after oxidation and concomitant splitting of the acetate groups by cellular esterases as 2',7'-dichloro- fluorescein (DCF). H2DCF-DA is widely used in (nonphotosynthetic) animal cells.

Increase of DCF fluorescence due to ROS production upon exposure of Chinese hamster ovary

Figure 56. Increase of DCF fluorescence due to ROS production upon exposure of Chinese hamster ovary (CHO) cells to 440-480 nm light. The image shows the ROS content by intensity of the emission of DCF in three different cells.

Figure 56 illustrates the application of CM-H2DCFDA in monitoring the development of ROS production upon exposure of CHO cells to 440480 nm light in phosphate buffered saline (PBS). After staining the cells with CM-H2DCFDA, the fluorescence of the indicator strongly rises upon illumination of the cells with light of 440 nm - 480 nm wavelengths due to the light-induced production of ROS and subsequent photooxidation in presence of oxidative compounds. It can be seen that after 6 sec, the lower cell has a higher cytosolic redox potential (higher fluorescence yield) than the upper two cells that show a less intense luminescence. On the other hand, these cells exhibit white “dots” indicating “hot spots” of accumulated DCF and/or higher local ROS activity (Schmitt et al., 2014a).

DCF was used to monitor the time course of the ROS production (determined from DCF fluorescence) between WT and the hy2 and hy3 mutants of A.thaliana (Kreslavski et al., 2013b) as it was shown that preillumination of the leaves of A.thaliana with red light reduced the inhibitory effect of UV radiation on the PSII activity in WT but not in the hy3 and hy2 mutants, which are lacking the phytochrome apoprotein and chromophore, respectively. These findings suggested that the active form of a phytochrome is involved in the protection of the PA against UV-A.

However, as Figure 57 indicates no significant kinetic differences of ROS production between WT and both mutants were observed.

CM-H2DCFDA is sensitive to ROS only in the living cell environment (in vivo) which enables the generation of dyes not only sensitive to ROS but also indicating that the ROS are produced inside the cell. Such studies are necessary especially to avoid side effects due to generation of ROS by the applied dyes, monitoring of ROS outside the cells in solution due to unspecific localisation and/or photooxidation of the dye by illumination (Vitali, 2011; for a detailed description see also Dixit and Cyr, 2003).

Dependence of the green fluorescence of DCF increasing with time of continuous irradiation with UV-A

Figure 57. Dependence of the green fluorescence of DCF increasing with time of continuous irradiation with UV-A (360/40 nm). The DCF signal indicates the production of ROS in leaves of WT (black squares) and two phytochrome deficient mutants hy2 (red circles) and hy3 mutant (blue triangles) during illumination.

DCF can be used in plant cells of A.thaliana leaves for measuring the ROS production (mainly H2O2) upon illumination with UV-A. Figure 58 shows the highly fluorescent DCF after incubation of leaves of A. tha- liana to PBS containing 500 pM H2DCFDA. The leaves were exposed to H2DCFDA solution for 1-2 h before starting the UV-A irradiation experiments. It can be seen that after irradiation with 360 nm UV-A light at an intensity of 250 W/m2 for 10 minutes areas which contained microscopic damages exhibit strong DCF emission (Figure 58, left panel) while the Chl a emission at 680 nm appears reduced in the same areas (middle panel) due to photobleaching. The simultaneous reduction of the Chl a fluorescence together with enhanced DCF fluorescence becomes evident in the overlay image (Figure 58, right panel) where Chl a emission is recolored in red and DCF in green. DCF studies together with observation of Chl a bleaching show the interaction of ROS and Chl a (Kreslavski et al., 2013b)

Fluorescence of a section of a 26-d-ol

Figure 58. Fluorescence of a section of a 26-d-old A.thaliana leaf. The fluorescence was emitted from 2',7'-dichlorofluorescein (DCF) (excited at Am = 470 nm) after irradiation of the leaf with UV-A (360 nm; I - 250 Wm-2) registered at 530 nm (left panel) in comparison to the Chl a fluorescence at 680 nm (middle panel). The overlay shows both (right panel) after recoloration.

Incubation of A.thaliana leaves in a PBS buffer with a final apparent concentration of the polyaromatic hydrocarbon (PAH) naphthalene (Naph) of 100 mg l-1 showed severe damage of the cell membrane upon illumination with UV-A as shown in Figure 59. Similar results were obtained for pea leaves (Kreslavski et al., 2014b). UV-radiation in combination with toxic compounds like PAHs lead to generation and accumulation of ROS as visible after staining with DCF.

DCF fluorescence in leaves of A. thaliana incubated with Naph during illumination with UV-A

Figure 59. DCF fluorescence in leaves of A. thaliana incubated with Naph during illumination with UV-A.

Figure 60 clearly shows the accumulation of ROS in the cell membrane while the red Chl fluorescence is emitted from inside the chloroplasts (see Figure 61).

Localization of the green fluorescence of DCF in the cell membrane after illumination with UV light

Figure 60. Localization of the green fluorescence of DCF in the cell membrane after illumination with UV light.

Localization of the green fluorescence of DCF in the cell membrane after illumination with UV light (right side) in contrast to fluorescence emitted from Chl at 680 nm (left side)

Figure 61. Localization of the green fluorescence of DCF in the cell membrane after illumination with UV light (right side) in contrast to fluorescence emitted from Chl at 680 nm (left side).

In leaves of A. thaliana treated with Naph, ROS waves with a temporal frequency of 20 minutes and a “wavelength” of several hundreds of micrometers were observed (Figure 62). Such a behavior is in line with wave-like closure and opening of stomata as observed in green plants under stress conditions.

In pea leaves the reduction of PSII activity at the presence of Naph is accompanied by transient generation of H2O2 as well as swelling of thylakoids and distortion of cell plasma membranes (Kreslavski et al., 2014b). It could be shown that Naph-treated leaves of Arabidopsis thaliana show enhanced DCF fluorescence in the cell membrane. The comparison of short term and long term exposure to different PAHs revealed that at short term exposure, the PAHs with high water solubility lead to the strongest reduction of PS II activity while after long term exposure the effect of PAHs with low water solubility is stronger.

Temporal intensity variation of the DCF fluorescence emitted from a single cell of A.thaliana after incubating the leaves with Naph and illuminating with UV-A over 45 minutes

Figure 62. Temporal intensity variation of the DCF fluorescence emitted from a single cell of A.thaliana after incubating the leaves with Naph and illuminating with UV-A over 45 minutes.

Figure 63 illustrates the use of DanePy (Hideg et al., 1998, 2000), see Table 4 in imaging ROS production in Arabidopsis thaliana leaves. Figure 63 upper panel illustrates an area of the leaf tip exposed to a photosynthetic photon flux density (PPFD) of 600 pmol m-2 s-1 for 60 min. The white oval in frame A shows the illuminated area, frames B and C show the images of fluorescence emission from the DanePy before and after high light treatment, respectively. Fluorescence quenching occurs within the area of the leaf tip exposed to the high light. This fluorescence quenching in frame C reflects the formation of non-fluorescent DanePyO due to reaction of 1AgO2 with DanePy.

Figure 63, lower panel shows ROS sensing in A.thaliana with nitroblue tetrazolium (NBT) which is sensitive to H2O2 (Maly et al., 1989; Thordal- Christensen et al., 1997; see Table 3). In that case the top half of the leaf was exposed to 600 pmol m-2 s-1 for 60 min. The purple coloration indicates the formation of insoluble formazan deposits due to reaction of NBT with superoxide.

Top panel

Figure 63. Top panel: Fluorescence emission from an Arabidopsis thaliana leaf which was infiltrated with 40 mM dansyl-2,2,5,5,-tetramethyl-2,5-dihydro-1H- pyrrole (DanePy). Bottom panel: Image of an Arabidopsis thaliana leaf infiltrated with 6 mM nitroblue tetrazolium (NBT) (adapted from Fryer et al., 2002). Image reproduced with permission.

As DanePy is detectable by fluorescence quenching due to ROS formation it is more often used as a typical spin trap which is highly selective for singlet oxygen.

 
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