Tissue Printing and Dual Excitation Flow Cytometry for Oxidative Stress—New Tools for Reactive Oxygen Species Research in Seed Biology

The intracellular homeostasis of reactive oxygen species (ROS) and especially of superoxide anion and hydrogen peroxide participate in signaling cascades which dictate developmental processes and reactions to stresses. ROS are also biological molecules that play important roles in seed dormancy and germination. Because of their rapid reactivity, short half-life and low concentration, ROS are difficult to measure directly with high accuracy and precision. In presented work tissue printing method with image analysis and dual excitation flow cytometry (FCM) were developed for rapid detection and localization of O2•− and H2O2 in different part of seed. Tissue printing and FCM detection of ROS showed that germination of wild oat seeds was associated with the accumulation of O2•− and H2O2 in embryo (coleorhiza, radicle and scutellum), aleurone layer and coat. To verify if printing and FCM signals were specified, the detection of O2•− and H2O2 in seeds incubated in presence of O2•− generation inhibitor (DPI) or H2O2 scavenger (CAT) were examined. All results were a high level of agreement among the level of ROS derived from presented procedures with the ones created from spectrophotometric measured data. In view of the data obtained, tissue printing with image analysis and FCM are recommended as a simple and fast methods, which could help researchers to detection and level determination of ROS in the external and inner parts of the seeds.


Introduction
Reactive Oxygen Species (ROS) have long been known to be a damaging compound for biomolecules [1] and also signaling intermediates [2]. ROS are by-product of aerobic metabolism under natural conditions and plants have evolved an array of self-protective defensive tools to counteract loss of redox homeostasis of the cell [3,4]. Detoxification of ROS by antioxidative defense system comprising of both non-enzymatic and enzymatic antioxidants is paramount for the maintenance of physiological level of ROS and function of ROS as a signal molecules in a number of cellular processes.
Several works in the last decade strongly convey the role of ROS at all stages of seed life from embryogenesis to germination [5]. Out of several ROS, superoxide anion (O 2 •− ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (HO•) have been implicated in dormancy releasing and germination [6][7][8]. ROS accumulation occurs both in quiescent dry seeds during after-ripening (AR) and metabolically active seeds [9,10]. ROS participate in dormancy release during AR through the direct oxidation of a subset of biomolecules, such as nucleic acid, especially mRNA [11]. During seed imbibition, their metabolic activity vary dramatically in most of the cell organelles like glyoxysomes, peroxisomes and mitochondria, results in ROS accumulation [12,13]. Likewise, the enzymatic activity of NADPH oxidase, which transfer electrons across the plasma membrane from cytoplasmic NADPH to molecular oxygen, is an important source of O 2 •− in the plasma membrane [14]. Bailly et al. [5] proposed that the Timing of generation, diffusion and degradation of various types of ROS within various cellular compartments leads to that ROS at different concentrations would play distinctly different roles therein, thus eventually leading to different fates of cells. Therefore, it has become very important to determine the location and concentration of ROS. Because ROS arise from various mechanisms, various qualitative and quantitative techniques are currently available. Furthermore, due to instability, short half-life and mutual interference of most oxygen radicals, ROS are not an easy object to analyze. Protocols for ROS detection and determination of its concentration in cell are available, like spectrophotometry [17], histochemical staining for stereomicroscopy and light microscopy [18,19], fluorescence [20,21], chemiluminescence [22], high-performance liquid chromatography (HPLC) [23], fluorescent protein-based redox probe [24], electron spin resonance (ESR) [25] and electrochemical biosensors [26]. However, each technique has its advantages and disadvantages, so it is recommended to use more than one method to test ROS in cells, trying to rule out specific drawbacks of single techniques [27]. Tissue printing onto membranes is a simple and rapid method employed to study the localization of nucleic acid, proteins and metabolites from freshly cut surface of a bisected tissue. Seed tissue imprints on nitrocellulose membranes were described by Reference [28] for localization of extensin in soybean seed coat. The KI/starch-or DAB-mediated tissue printing has been proposed for direct detection of H 2 O 2 production in seedlings [29][30][31] and fruits [32]. A procedure based on the reduction of nitro blue tetrazolium (NBT) by O 2 •− , visualized by the formation of the dark blue formazan and oxidation of 3,3 -diaminobenzidine (DAB) by H 2 O 2 , visualized by the formation of the yellow complex, have recently been used for a localization of ROS in whole seeds or embryos isolated from seeds [33][34][35][36][37][38][39][40]. However, there is no evidence of studies using NBT-or DAB-mediated tissue printing for ROS analysis in seeds. Detection and histochemical localization of ROS provides information about in situ distribution and accumulation of ROS in different part of tissue or organ. An important objective for analysis of stained tissue or tissue imprinting signal is to statistically compare staining intensity for a particular component that has been located in the analyzed object. The stained area can be detected and quantification analyzed with the methods of freeware or commercial applications for image analysis.
The cost effective answer for quantitative analysis is ImageJ, image processing program inspired by NIH (National Institutes of Health) Image (https://imagej.nih.gov/ij/index.html). Image analysis with ImageJ is rapid and simply method, which proceeds as follows: acquisition of an image, preprocessing of the image for facilitating further processing, selection of pixels of interest and extraction of characteristic features. The quantitative analysis of O 2 •− and H 2 O 2 level based on histochemical staining was used for whole leaves [41][42][43]. However, there is no evidence of studies using method of quantification with the help of digital image analysis of histochemical staining and tissue printing results for ROS level estimation in seed. Fluorescence is one of the excellent technique for visualize ROS due to the sensitivity and selectivity offered by fluorescent probe [44]. Detection of ROS by fluorescence involves the oxidation of the fluorescent probe itself but the probe being a stable molecule in the reduced state with fluorescence properties. Two specific dyes are available to measure intracellular ROS using fluorescence microscopy or standard fluorometer. Dihydroethidium (DHE) can detect intracellular

In Situ Localization of O2 •− and H2O2 by Histochemical Staining
To verify the tissue printing results, the detection and localization of O2 •− and H2O2 were performed by histochemical NBT or DAB staining in the seeds imbibed in water, DPI or CAT. The experiment was first carried out using whole seeds but the staining was not completed; ROS were accumulating on the surface of coleorhiza of seeds when the coat was ruptured ( Figure S2a-d). However, stained longitudinally bisected half caryopses showed accumulation of O2 •-and H2O2 in the coleorhiza, radicle, scutellum, aleurone layer and coat ( Figure S3). The obtained data confirmed the results of tissue printing experiment ( Figure 1). ROS were produced both on the outer and inner side of the cover. Furthermore, the signal intensity of stained longitudinally bisected half seeds was stronger than printing signal of half seeds. The tissue printing results shown, that ROS signal, not related with mechanical tissue damage, can be registered only when the tissue printing was done in less than 2 min after the cut was done. Probably, if the histochemical staining from NBT (last 10 min) or DAB (last up to 90 min) has been used to detect ROS in half seeds, resulted with very strong signal which might be related to mechanical damage. Therefore, the embryos were isolated from dry and imbibed seeds before NBT or DAB staining and it was found that embryos were stained ( Figure 2).

In Situ Localization of O 2 •− and H 2 O 2 by Histochemical Staining
To verify the tissue printing results, the detection and localization of O 2 •− and H 2 O 2 were performed by histochemical NBT or DAB staining in the seeds imbibed in water, DPI or CAT. The experiment was first carried out using whole seeds but the staining was not completed; ROS were accumulating on the surface of coleorhiza of seeds when the coat was ruptured ( Figure S2a-d).
However, stained longitudinally bisected half caryopses showed accumulation of O 2 •and H 2 O 2 in the coleorhiza, radicle, scutellum, aleurone layer and coat ( Figure S3). The obtained data confirmed the results of tissue printing experiment ( Figure 1). ROS were produced both on the outer and inner side of the cover. Furthermore, the signal intensity of stained longitudinally bisected half seeds was stronger than printing signal of half seeds. The tissue printing results shown, that ROS signal, not related with mechanical tissue damage, can be registered only when the tissue printing was done in less than 2 min after the cut was done. Probably, if the histochemical staining from NBT (last 10 min) or DAB (last up to 90 min) has been used to detect ROS in half seeds, resulted with very strong signal which might be related to mechanical damage. Therefore, the embryos were isolated from dry and imbibed seeds before NBT or DAB staining and it was found that embryos were stained ( Figure 2). In the embryo isolated from dry seeds accumulation of ROS was not detected ( Figure 2). When seeds were incubated in presence of water for 8 h, embryo showed a significant accumulation of O 2 •− and H 2 O 2 in coleorhiza and scutellum or in coleorhiza, respectively, as compared to the same parts of the dry embryo. The intensity of the signal increased during imbibition time and was more evident after 16 h. However, when the seeds were incubated in presence of DPI or CAT, intensity of staining was fully decreased. In the embryo isolated from dry seeds accumulation of ROS was not detected ( Figure 2). When seeds were incubated in presence of water for 8 h, embryo showed a significant accumulation of O2 •− and H2O2 in coleorhiza and scutellum or in coleorhiza, respectively, as compared to the same parts of the dry embryo. The intensity of the signal increased during imbibition time and was more evident after 16 h. However, when the seeds were incubated in presence of DPI or CAT, intensity of staining was fully decreased.

O2 •− and H2O2 Quantification by ImageJ
Digital image analysis package ImageJ was applied for quantification of the intensity of stained area in tissue printing from seeds and histochemical stained embryo. The quantification by the measure of staining intensity of printing signal showed that after incubation of seeds in presence of water for 8 h, the content of O2 •− was increased in embryo (

O 2 •− and H 2 O 2 Quantification by ImageJ
Digital image analysis package ImageJ was applied for quantification of the intensity of stained area in tissue printing from seeds and histochemical stained embryo. The quantification by the measure of staining intensity of printing signal showed that after incubation of seeds in presence of water for was about 1.5-fold higher. Prolongation of incubation for up to 16 h progressively increased the intensity of the signal; the signal was ca. 1.8-fold higher in embryo, coleorhiza, radicle and aleurone layer from 16 h-imbibed seeds than signal from the dry seeds. Furthermore, in this time point, intensity of the staining in scutellum was only 1.3-fold higher than in scutellum from dry seeds ( Figure 4d). The signal of H2O2 was not detected when the seeds were incubated in presence of CAT for 8 and 16 h.  representing shades of gray. Vertical bars indicate ± SD. One-way ANOVA with the Duncan's post hoc test was used to determine the significance of differences. Mean values with different letters (a-c) are significantly different (p < 0.05, n = 5). The NBT histostains demonstrate that during incubation of seeds in presence of water for 8 h, content of O2 •− was increased in coleorhiza and scutellum 1.5 times in comparison to the same parts of embryo from dry seeds (Figure 5a,b). During further incubation up to 16 h, O2 •− showed about 2.5 and 3 times higher level in coleorhiza and scutellum, respectively, than in coleorhiza and scutellum from dry embryo. The signal of O2 •− was fully suppressed when the seeds were incubated in presence The NBT histostains demonstrate that during incubation of seeds in presence of water for 8 h, content of O 2 •− was increased in coleorhiza and scutellum 1.5 times in comparison to the same parts of embryo from dry seeds (Figure 5a  of DPI for 8 and 16 h. After 8 h of imbibition in water, the amount of H2O2 was ca. 1.7 times higher in coleorhiza than in coleorhiza from dry embryo and remained constant up to 16 h (Figure 5c). The signal of H2O2 was fully suppressed when the seeds were incubated in presence of CAT for 8 and 16 h.    Fluorescence-based signal for H 2 O 2 was also detected in different part of seed by flow cytometry (FCM), when seeds were stained using 6-carboxy-2 ,7 -dichlorodihydrofluorescein diacetate di(acetoxymethyl ester) (CDCDHFDA-AM). The fluorescence signal was more strongly in embryo (Figure 7a

O2 •− and H2O2 Quantification by Spectrophotometric Method
To test the precision of tissue printing method with ImageJ and FCM to ROS estimation, intensity of tissue printing signal and stained embryo signal to results obtained using the UV-VIS method were compared. In agreement with higher accumulation of O2 •− estimated by tissue printing (Figures 1 and  2) and FCM (Figure 6

O 2 •− and H 2 O 2 Quantification by Spectrophotometric Method
To test the precision of tissue printing method with ImageJ and FCM to ROS estimation, intensity of tissue printing signal and stained embryo signal to results obtained using the UV-VIS method were compared. In agreement with higher accumulation of O 2 •− estimated by tissue printing (Figures 1 and 2) and FCM (Figure 6  content of H2O2 was about 1.6-fold higher than content of H2O2 in the same parts of dry seeds. Prolongation of incubation up to 16 h increased the content of H2O2; in this time point, H2O2 showed about 1.7-2.2 times higher level than in dry seeds. Furthermore, the level of H2O2 in scutellum was not significantly changed throughout the whole period of imbibition (Figure 9d). The content of H2O2 was decreased when the seeds were incubated in presence of CAT.

Discussion
Seed germination is a critical developmental step regulated by multiple plant endogenous signals, such as phytohormones, reactive oxygen species (ROS) or reactive nitrogen species (RNS) [49][50][51]. ROS are efficiently interlinked with the environmental condition and major hormonal regulators, such as gibberellin, abscisic acid and ethylene, which are associated with seed dormancy and germination [52][53][54]. A number of studies have shown that ROS promote the germination of several seeds, both dormant and non-dormant [6][7][8], including Avena fatua [36,55].
Endogenously produced ROS acts as a cell signaling molecules and as a redox potential regulators. However, despite their positive role, their accumulation in the cells leads to oxidative stress. Therefore, understanding the role of ROS as cellular messenger and response for the stress, requires its precise localization, quantification and global dynamics of ROS in different parts of the seed. Due to short half-life and high reactivity, the detection of ROS in the cell has always been very

Discussion
Seed germination is a critical developmental step regulated by multiple plant endogenous signals, such as phytohormones, reactive oxygen species (ROS) or reactive nitrogen species (RNS) [49][50][51]. ROS are efficiently interlinked with the environmental condition and major hormonal regulators, such as gibberellin, abscisic acid and ethylene, which are associated with seed dormancy and germination [52][53][54]. A number of studies have shown that ROS promote the germination of several seeds, both dormant and non-dormant [6][7][8], including Avena fatua [36,55].
Endogenously produced ROS acts as a cell signaling molecules and as a redox potential regulators. However, despite their positive role, their accumulation in the cells leads to oxidative stress. Therefore, understanding the role of ROS as cellular messenger and response for the stress, requires its precise localization, quantification and global dynamics of ROS in different parts of the seed. Due to short half-life and high reactivity, the detection of ROS in the cell has always been very challenging.
During past decades, a variety of methods for detection and quantification of ROS, including its reactive intermediates, have been applied.
Tissue printing method has become an important tool for visualization and localization of different molecules in plant tissue [56]. In this study, it has been used to detect and determine the localization of ROS in non-dormant Avena fatua L. seeds during imbibition. One of known techniques of detection of O 2 •− is histochemical staining based on the reduction of nitro blue tetrazolium by O 2 • , visualized by the formation of the dark blue formazan [18]. Employment of this reaction together with the tissue printing method, made the detection and localization of O 2 •− in plant tissue possible. According to our knowledge, the detection and localization of O 2 •− using the NBT-mediated tissue printing in plant material is unavailable. In our protocol, O 2 •− was localized in embryo, coleorhiza, radicle, scutellum, aleurone layer and coat during seeds imbibition up to 16 h (Figures 1 and 2). However, when the seeds were incubated in presence of DPI, intensity of staining was completely decreased. Until now, the tissue printing for H 2 O 2 detection and localization was used only for seedlings of Glycine max, Pisum sativum, Phaseolus vulgaris, Helianthus annuus, Cucumis sativus and Solanum tuberosum [29]. It is worth noting, that aforementioned procedure was based on the oxidation of KI to I 2 by H 2 O 2 . Used nitrocellulose membranes were presoaked with starch and KI, reagent mixture for the histochemical assay of H 2 O 2 [57]. The DAB-mediated tissue printing, has been proposed for direct detection of H 2 O 2 production also for seedlings of Zea mays [30] and Lycopersicon esculentum [31], as well as fruits of L. esculentum [32]. Usage of this method allowed for identification of H 2 O 2 signals in coleorhiza and radicle of embryo, aleurone layer and coat during seeds imbibition up to 16 h (Figures 1 and 4). Nonetheless, when the seeds were incubated in presence of CAT, intensity of staining was entirely decreased. ROS accumulation in coleorhiza and radicle can be associated with elongation growth and cell wall loosening. Previously, ROS have been demonstrated to play a role in radicle elongation in Lepidium sativum [58] and Lactuca sativa [33]. Taken as a whole, presented results show that NBT-or DAB-mediated tissue printing for O 2

•−
and H 2 O 2 , respectively, are a very rapid and specific methods which can be used for detection and visualization of ROS in seeds. Rapidity of this protocol (within 10 s, see Materials and Methods for details) can completely avoid the interference of wound-induced ROS. Usage of image processing software for further analysis of received results, is fundamental and allow to extract very useful information from images. Results analysis can be done using open source image analysis tools, such as CellProfiler [59], Fiji/ImageJ [60] or Icy [61] can be used. ImageJ, a Java-based application, currently very popular in human science, thanks to its versatility can also be used as a tool for the quantification of histological results [62]. In the field of plant and seed science, ImageJ provides a way to measure many parameters, such as length, width, area or shape [63] and has become necessary tool for plant physiology, such as leaf disease or leaf color changes [64].
There are several example studies where the image processing method was used for the quantitative estimate of ROS accumulation in leaves. ImageJ has been used for determination of O 2 •− and H 2 O 2 level in leaves [41][42][43]65] and roots [66]. According to available data, O 2 •− and H 2 O 2 in seeds are visualized by staining with NBT or DAB, respectively. The data interpretation was based on subjective visual estimation and provided only qualitative results. Stained tissue can be digitalized and opened in Fiji/ImageJ for quantification of blue (NBT for O 2 •− ) or yellow (DAB for H 2 O 2 ) color intensity (in the negative, corresponding to the lighter tones of gray). In this work, aforementioned method has been applied for NBT-or DAB-mediated tissue printing of whole seeds and histochemical stained embryo, as well as used to detect the spatial distribution of the NBT or DAB intensity at different parts of seeds. As a result, an image with NBT or DAB only staining is generated and the average intensity of its pixels can be quantified after the selection of specific ROI (Region of Interest). In digital image analysis, the pixel intensity values for color range from 0 to 255, wherein, 0 represents the darkest shade of the color and 255 represent the lightest shade of the color as a standard. Combining tissue printing and image processing with Fiji/ImageJ software, it was possible to obtain a relative ROS concentration based on the pixel intensity resulting from the NBT or DAB signal (Figures 1-5). Obtained results demonstrate that the Fiji/ImageJ is a promising tool for the quantification of ROS in different part of seeds. Oxidative stress can be studied using a large variety of methodological approaches, including fluorescence: microscopy or classical fluorimeter. Several fluorescent dyes have been tested so far. They allowed to obtain information on the localization of ROS and thus the dynamics of oxidative stress in plants exposed to unfavorable environmental conditions. In present study, small-molecule fluorescent ROS dyes (DHE and CDCDHFDA-AM) and dual excitation flow cytometry, which can provide information at the single cell level, have been used for ROS quantification in different parts of seeds. The fluorescence signal showed that during imbibition of seeds up to 16 h, the content of O 2  (Figure 7f). To confirm that FCM measures the ROS level, caryopses were treated with DPI or CAT. The application of DPI or CAT, fully reduced of fluorescence signal intensity.
In plant physiology, the fluorescence probes DHE and CDCDHFDA-AM are perhaps the most frequently used when studying ROS signaling and oxidative stress in plant cells and green algae using fluorescence microscopy and fluorometry [67]. To our knowledge, the present study is the first application of dual excitation FCM with DHE and CDCDHFDA-AM to determination of O 2 •and H 2 O 2 level in seeds. The results obtained demonstrate that FCM is a promising tool for the precise localization and quantification of ROS in different part of seeds.
To validate results obtained using tissue printing with image analysis and FCM methods for ROS analysis in seed material, UV-VIS method was used to measure O 2 •− and H 2 O 2 levels. In this study, it was proved a high correlation between mean gray value in tissue printing signal, FCM signal and the biochemically-quantified amount of O 2 •− and H 2 O 2 (Figures 8 and 9). In agreement with higher accumulation of O 2 •− estimated by tissue printing (Figures 1-3) and FCM (Figures 6 and 7),

Seed Material
Avena fatua (wild oat) spikelets, which contained 2-3 florets covered with glumes, were collected in July 2010 near Szczecin (Poland). After collection, the florets (a single caryopsis, covered by the lemma and palea) were dried at room temperature (RT) for 7 days to a constant moisture content (ca. 12%). To obtain non-dormant caryopses (seeds), dormant florets were stored dry under ambient relative humidity for up to 4 months in darkness at 25 • C.
In all the experiments, seeds (25 in each of 5 replicates) were incubated at 20 • C in the dark, in Petri dishes (ø 6 cm) on one layer of filter paper (Whatman No. 1) moistened with 1.5 mL of distilled water or with solutions: diphenyleneiodonium (DPI) (10 −4 M) (Merck, Darmstadt, Germany) or catalase (CAT) (1000 U) (Merck, Darmstadt, Germany) for 8 or 16 h. After treatment whole seeds and embryo, coleorhiza, radicle, coat (testa + pericarp) and aleurone layer isolated from caryopses were used for analysis.

Localization of O 2
•− and H 2 O 2 using tissue printing was determined according to Liu et al. [31] with some modification. After incubation whole seeds were placed on Petri dish and cut using razor blade. To obtain a high-quality cut surface without damaging tissue, each edge of the razor blade was used several times. Longitudinally bisected half seed was immediately used for tissue printing. Nitrocellulose blotting membrane (Amersham™ Protran™ 0.2 µm NC, Amersham, UK) was soaked in 6 mM NBT (Merck, Darmstadt, Germany) (in 10 mM Tris-HCl, pH 7.4) for O 2 •− or 2 mg mL −1 DAB-HCl solution (pH 3.8) (Merck, Darmstadt, Germany) for H 2 O 2 localization and then air-dried at 25 • C for 30 min in darkness. The soaked membrane was placed on three layers of filter paper (Whatman No 1) (Merck, Darmstadt, Germany). The half seed was placed with its cut surface down on membrane, covered with parafilm and pressed for 10 s. Then, the half seed or embryo was carefully removed with the forceps. After 5 min at RT, when reaction between ROS and NBT or DAB was completed, membranes were photographed (Canon EOS 500, Tokyo, Japan). All the images were saved as TIFF files, with 3072 × 2304 pixel resolution and 24 bits RGB color depth (pixel transformation factor = 1, no scaling of result images). The image analysis was conducted using the ImageJ (version 1.51s/Java 1.6, LOCI, University of Wisconsin, U.S.). NBT or DAB stained membranes were converted to gray scale for analysis. Staining can be assessed by setting a 'threshold' using the thresholding tool. However, to limit the ROS determination to the defined parts of the seeds or embryos, specific ROI (Region of Interest; using one of the drawing tools) was analyzed. Minimum and maximum threshold values were established to remove background staining and a mean gray value for the plaques was then calculated. In digital image analysis, the pixel intensity values for color range from 0 to 255, wherein, 0 represents the darkest shade of the color and 255 represent the lightest shade of the color as a standard. The data are presented as a mean gray value of the total plaque area to give a relative staining density for each sample [68]. The dried membranes can be kept at room temperature without losing signals or the original resolution of printed images for several months. To verify whether there is a production of wound-induced ROS under experimental conditions, tissue printing was performed at 0, 1, 2 and 4 min after cutting.

In Situ Localization and Level of O 2 •− and H 2 O 2 and Content Determination by ImageJ
Generation of ROS in situ was detected by monitoring the reduction of nitro blue tetrazolium (NBT) as described by Beyer and Fridovich [18] or polymerization of 3,3 -diaminobenzidine (DAB) according to Thordal-Christensen et al. [19]. After incubation embryos were isolated from seeds and stained for 10 min in darkness in 6 mM NBT (in 10 mM Tris-HCl, pH 7.4) or 90 min in darkness in 1 mg ml-1 DAB containing 0.05% (v/v) Tween-20 and 10 mM Na 2  The stained samples were transferred to 2 mL of the isolation buffer, pH 7.4, containing 45 mM MgCl 2 , 30 mM sodium citrate, 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) and 0.1% (v/v) Triton X-100 [69] and using a razor blade, were chopped for 2 min. Subsequently, the suspension was passed through a 50 µm nylon mesh. The labelled cells were analyzed using flow cytometer (Partec, Germany) with an air-cooled 20 mV argon-ion laser and HBO mercury arc lamp. In the flow chamber, each cell crosses a region of fluorescence excitation. There, the green-or red-fluorescence emissions are excited consecutively with blue light at 488 nm and UV and recorded by photomultiplier tubes. The flow cytometer was equipped with dichroic mirror with the edge at 420 nm (TK420), a full mirror (FM) and a long pass filter with the edge at 515 nm (OG515). The final gated cell populations contained 20,000 cells and signals were recorded on a histogram by logarithmic amplifiers. The histograms present the fluorescence intensity (log; Geo Mean) on the x-axis and cell count on the y-axis in gated population of cells. The relative O 2 •− and H 2 O 2 level was expressed as the mean fluorescence intensity (percentage of the control).

Determination of O 2 •− and H 2 O 2 Content by Spectrophotometric Method
Extracellular O 2 •− production was estimated using the method developed by Misra and Fridovich [17]. After incubation, the embryos, coleorhiza, radicle, scutellum, coat (testa + pericarp) and aleurone layer were isolated from seeds. Sample were ground and homogenized by pestle and mortar in cold 50 mM Tris-HCl pH 7.5 with 2% (w/v) PVPP (fresh weight: buffer, 1:10, w/v). The homogenate was centrifuged for 20 min at 14,000× g at 4 • C and then the supernatant was immediately used. The reaction mixture was composed of 0.05 mL of 50 mM Tris-HCl pH 7.5, 0.05 mL of 60 mM epinephrine (in 0.5 M HCl) and 0.05 mL of supernatant. The oxidation of epinephrine to adrenochrome was measured in reaction mixture at 480 nm for 2 min. In each test, oxidation of adrenaline was carried out in the reaction mixture (without extract). The epinephrine extinction coefficient was ε = 4.02 mM −1 cm −1 . The results were expressed as relative unit corresponds to the rate of epinephrine oxidation in extracts of sample from dry caryopses calculated as µmol min −1 g −1 FW). Extracellular H 2 O 2 content was determined according to Velikova et al. [70]. After incubation, the embryos, coleorhiza, radicle, scutellum, coat (testa + pericarp) and aleurone layer were isolated from seeds. Sample were ground and homogenized by pestle and mortar in cold 1% (w/v) trichloroacetic acid (TCA) (fresh weight:TCA, 1:10, w/v). After 20 min of centrifugation at 14,000× g at 4 • C, the resulting supernatant was immediately used for spectrophotometric analysis. The content of H 2 O 2 was measured in reaction mixture (0.5 mL of 10 mM potassium phosphate buffer pH 7.0, 1 mL of 1 M KI in 10 mM potassium phosphate buffer, pH 7.0 and 0.5 mL of the supernatant) at 390 nm after incubation at 25 • C for 60 min. A standard curve was prepared by using the H 2 O 2 standard. The results were expressed as nmol H 2 O 2 g −1 FW.

Data Analysis
All the experiments were carried out in five biological replicates and the results are expressed as mean ± SD. The means were analyzed for significance using one-way analysis of variance, ANOVA (Statistica for Windows version 13.0, Stat-Soft Inc., Tulsa, OK, USA). Data were checked for normality and homogeneity of variance and met these criteria. Duncan's multiple range test was used to test for significance of differences (p ≤ 0.05). Every experiment was repeated three times and the results presented correspond to a representative single experiment.

Conclusions
ROS are hot topics in seed biology because they function both as agents of damage and mediators of cellular signals. This small molecules are now known to play an important role in seed dormancy and germination, stress responses and environmental interactions. Understanding of their functions needs knowledge about the level of ROS, especially superoxide anion and hydrogen peroxide. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signaling molecule to various biological processes.
The quantitative method for ROS determination based on tissue printing with image analysis and flow cytometry are simple and fast. This methods can be used for determination of ROS accumulation in the external and inner parts of the seeds. Finally, all steps of tissue printing protocol was done within 10 s which avoided interference of ROS resulting of tissue damage. Image analysis applied to NBT-or DAB-mediated tissue printing and FCM provides a very efficient means of quantifies ROS level in seed sample. It has been practically proven, by comparison of received results with the UV-VIS results, that developed methods provide repeatable, accurate and reliable results. Furthermore, the use of image analysis methods avoids the effects of human subjectivity. These methods have the potential to significantly enhance both precision and reproducibility and making quantitative methods more readily accessible to most plant laboratories.

Acknowledgments:
The author would like to thank Jan Kępczyński for critical remarks on some parts of the manuscript.

Conflicts of Interest:
The author declares no conflict of interest.