prtocolos sod

7/24/2019 Prtocolos Sod

1/8


2/8

218

response to oxidative stress [13]. The steady state ROS level andthe rates by which ROS can be trapped by dyes in tissues reflect thebalance of ROS production and ROS detoxification.

To detect ROS in plant cells and organs various in situ and invitro methods have been developed. For in situ pattern analysis

preference has been given to histochemical staining [1416] and,for quantification, to ROS-trapping by fluorophors [17, 18].

Here, we present a method for quantitative analysis of ROSaccumulation in leaves based on histochemical ROS staining. ROSlevels are quantified with the help of digital image analysis. Ourmethod is cheap, fast, and easy. Special preparation and illumina-tion of stained plant material enable visualization of ROS productionsites not only in the external cell layer but also in the inner parts ofthese organs. Therefore, the method permits determination ofROS-accumulating leaf areas as well as quantification of ROS accu-

mulation within particular leaf areas. The method has been optimizedwith respect to quantification. It can be used for both, detection ofO2 by NBT staining and the analysis of H2O2 accumulation byDAB staining, with minimal variation.

2 Materials

All solutions are prepared using distilled water and analytical gradereagents (seeNote 1). All reagents and solutions are stored at 4 C

(unless indicated otherwise). For disposal of waste material care-fully follow your waste disposal regulations (remember that sodiumazide is acutely toxic).

1. 10 Phosphate-buffered saline (PBS) stock solution: Dissolve80 g of NaCl, 2 g of KCl, 14.4 g of Na2HPO4, and 2.4 g ofKH2PO4in 800 mL of distilled water. Mix and adjust pH to7.4. Adjust volume with distilled water to 1 L. Sterilize byautoclaving and store at 4 C.

2. 1 PBS: To obtain 1 L of 1 PBS dilute 100 mL of 10 PBS

with 900 mL of sterile water. Store at 4 C.3. 10 mM sodium azide: Dissolve 0.65 g of sodium azide (NaN3)

in 950 mL of 1 PBS. Adjust volume to 1 L. Caution! Sodiumazide is acutely toxic. Do not prepare more solution than youreally need.

4. 1 mg/mL NBT staining solution: Dissolve 1 g of nitro bluetetrazolium chloride (NBT) in 1 L of 1 PBS (seeSubheading 2,item 1for recipe). Store in the darkness at 4 C.

5. 1 mg/mL DAB staining solution: Dissolve 1 g of 3,3-diami-

nobenzidine (DAB) in 1 L of 1 PBS (seeSubheading 2, item1for recipe). Store in the darkness at 4 C.

6. Sample containers (bottles/beakers): Prepare an appropriatenumber of sample containers (each sample should be placed in

Ilona Juszczak and Margarete Baier
http://-/?-http://-/?-http://-/?-http://-/?-


3/8

219

a separate sample container; the size of sample container dependson the size of stained plant tissue). Fill them with NBT or DABstaining solution for NBT and DAB staining, respectively.

7. Desiccators and vacuum pump: The number of desiccatorsneeded depends on the number of samples. The same desicca-tors can be used for both NBT and DAB stainings.

8. De-staining solution: Mix 100 mL of 100 % acetic acid,100 mL of glycerol, and 300 mL of 96 % ethanol. Right beforestarting the de-staining procedure, heat the mixture in a waterbath up to 6080 C.

9. Photos: You can take photos using either a camera with a highresolution or a light microscope with an integrated camera. Ifnot using a light microscope, an additional light source mightbe helpful, which lights up your samples from the bottom

(e.g., White-light plate, INTAS, Germany). For quantificationit is absolutely essential that the light is evenly applied and theobject is not shaded or shading.

10. Quantification: Download and install the freeware packageImageJ on your computer (http://rsbweb.nih.gov/ij/down-load.html) [19].

3 Methods

All staining reactions are light sensitive. Therefore, the whole stain-ing procedure should be carried out in darkness or at very low lightintensities. Samples subjected to staining (e.g., leaves, seedlings)should be treated with care. Injuries promote ROS formation andlead to false-positive results.

1. Place samples (e.g., detached leaves, whole seedlings, or rosettes)immediately after the harvest in 10 mM sodium azide. Theazide inhibits superoxide dismutases and heme-type peroxidasesat the active sites. The azide solution should fully cover the plant

material. Put the sample containers to the desiccator and applya vacuum (see Note 2) to remove air from the intercellularspaces and to flood the tissue with the azide solution. Incubatethe plant material for 10 min under vacuum. Afterwards, slowlyrelease the vacuum. Take the sample containers out of the desic-cator. Discard the sodium azide solution.

2. Fill the sample containers (containing your samples) with NBTstaining solution, place them back into the desiccator andapply a vacuum (seeNote 3) to remove the oxygen in the des-iccator and to protect the NBT from auto-oxidation.

3. Keep the samples under vacuum for 624 h (see Note 4).Release the vacuum and subject the samples to the de-stainingprocedure (Subheading 3.3).

3.1 NBT Staining

for O2 Detection

O2- and H2O2Quantification in Leaves
http://rsbweb.nih.gov/ij/download.htmlhttp://rsbweb.nih.gov/ij/download.htmlhttp://rsbweb.nih.gov/ij/download.htmlhttp://-/?-http://-/?-http://rsbweb.nih.gov/ij/download.htmlhttp://rsbweb.nih.gov/ij/download.html


4/8

220

1. Place the samples in containers filled with DAB stainingsolution immediately after the harvest. Put them into a desic-cator and apply a vacuum (seeNote 3).

2. Incubate the samples in the DAB solution for 1024 h indarkness until they are optimally stained.

3. For clearing of the background from chlorophylls and otherpigments, continue with (Subheading 3.3).

1. Discard the staining solutions.

2. Fill the sample containers (containing samples) with hot de-staining solution, which will remove chlorophylls, carotenoids,and other plant pigments, but stabilize the dye. Incubate thesamples in the de-staining solution until the chlorophyll iscompletely removed (seeNote 5). Non-stained tissue (parts)

should be as white as possible. Due to the high glycerol con-tent the samples get soft and luminescent. Caution! Acetic acidhas a pungent smell (especially when hot). Perform de-stainingunder fume hood!

1. Take the samples out of the de-staining solution (be carefulnot to destroy the sample with forceps while taking out).

2. Remove the traces of de-staining solution by placing thesamples on a paper towel.

3. Place the samples under the microscope or on the externallight device.

4. Illuminate the samples with white light from the bottom andtake photos (seeNote 6).

5. Using your favorite software (e.g., Adobe Photoshop or thefreeware GIMP) subject the photos to digital processing (e.g.,background subtraction, contrast improvement, color correction).For taking photos from NBT-stained plants, decrease the yel-low background to its minimum. For DAB-stained samplesremove the yellow and the blue colors to minimize background

effects (seeNote 7). The photos should be of similar quality tothose in Fig. 1after such processing.

1. Open the photo, which you want to analyze, with ImageJ.

2. Change the color scale of the photo to a grey-scale. To do that,choose from ImageJ main menu Image (Fig. 2a) and fromthe sub-menus Type and 32-bit. Store the grey picture ina new file.

3. To segment the grey picture into features of interest (areastained stronger than a defined threshold) and background,adjust the threshold for the intensity of grey color. FromImageJ main menu use the following options: Image

3.2 DAB Staining

for H2O2Detection

3.3 De-staining

Procedure

3.4 Photographing

of Stained Samples

3.5 Quantification

of Staining Results

http://-/?-http://-/?-


5/8

221

Fig. 1Arabidopsis thalianaleaves (middle-age and young) stained with NBT (a, b) and DAB (c, d). The samples

were harvested before (a, c) and after (b, d) 14 days of cold treatment [8]

Fig. 2 ImageJ main menu (a), Threshold window (b), and Results window (c) with the most important options

marked with red squares(Color figure online)



6/8

222

AdjustThreshold (Fig. 2a). In the Threshold win-dow (Fig. 2b) move the lower slider to the set threshold level.Make sure that only the stained area is marked in red (Fig. 3c).

4. Surround the area of the first leaf using the Freehandselection tool (Fig. 2a).

5. Choose Measure from the ImageJ main menu Analyze.

6. To choose the type of measurement, here in [%] of stained leafarea, use the following options in the Results window:ResultsSet measurementsArea fraction (Fig. 2c). Theresults are shown in [%] as %Area (Fig. 2c).

7. To determine the staining intensity within defined leaf areas takethe grey picture. Select the area of interest using the rectangularor oval selection tool. Measure the staining intensity by choosingMeasure from the ImageJ main menu Analyze. The type ofthe measurement should be set as Mean grey value (from theResults window choose: ResultsSet measurementsMean

grey value). The result is shown as mean of the intensities withinthe selected area.

8. Repeat steps 47to quantify the staining in further leaves.

Fig. 3 Quantification workflow. (a) Original picture; (b) Picture in grey(32-bit) scale; (c) Picture before threshold

setting, in which the stained area is marked in red; (d) Picture after threshold setting with the area of the first

leaf surrounded by the red line(using Freehand selection tool)



7/8

223

4 Notes

1. Use of freshly prepared staining solutions improves the qualityof staining. PBS can be prepared in advance and stored at 4 C.

2. To enable the comparison between samples (e.g., control andstress treatment), put all of them to the same desiccator andincubate them for the same time. It ensures that the samestrength of vacuum is applied to all of them.

3. To improve penetration of the staining solution into your sam-ple, apply and release the vacuum at least three times (each for5 min) prior to the incubation.

4. Longer staining periods usually give better results, but can alsoresult in over-staining. Therefore, carefully optimize the dura-tion of staining.

5. To speed up de-staining, the incubation can be performed in athermoblock (approximately 1520 min at 60 C). It is recom-mended to change the de-staining solution at least twice foroptimal de-staining of young tissues and three times for olderleaves, which are more difficult to de-stain.

6. Avoid any light from the side. If possible use a soft ring lightfor taking the photos.

7. Take care that all digital modifications are identical for all sam-ples treated in parallel. If samples should be compared which

are harvested on different days, prepare extra samples for com-parison of staining intensities and de-staining efficiency andmaintain half of them in the dark in the staining solution andhalf in the de-staining solution until the next set of plants isprepared.

References

1. Thomashow MF (1999) Plant cold acclima-tion: freezing tolerance genes and regulatory

mechanisms. Annu Rev Plant Physiol PlantMol Biol 50:571599

2. Heidervand L, Amiri LM (2010) What hap-pens in plant molecular responses to coldstress? Acta Physiol Plant 32:419431

3. Juszczak I, Rudnik R, Pietzenuk B, Baier M(2012) Natural genetic variation in the expres-sion regulation of the chloroplast antioxidantsystem amongArabidopsis thalianaaccessions.Physiol Plant 146:5370

4. Torres MA, Dangl JL, Jones JD (2002)Arabidopsis gp91phox homologues AtrbohD

and AtrbohF are required for accumulation of

reactive oxygen intermediates in the plantdefense response. Proc Natl Acad Sci U S A

99:5175225. Bolwell GP, Bindschedler LV, Blee KA, Butt

VS, Davies DR, Gardner SL, Gerrish C,Minibayeva F (2002) The apoplastic oxidativeburst in response to biotic stress in plants: athree-component system. J Exp Bot53:13671376

6. Bindschedler LV, Dewdney J, Blee KA, StoneJM, Asai T, Plotnikov J, Denoux C, Hayes T,Gerrish C, Davies DR, Ausubel FM, BolwellGP (2006) Peroxidase-dependent apoplasticoxidative burst in Arabidopsis required for

pathogen resistance. Plant J 47:851863



8/8

224

7. Mehler AH (1951) Studies on reactivities ofilluminated chloroplasts. I. Mechanism of thereduction of oxygen and other Hill reagents.

Acta Biochem Biophys 33:6577

8. Mittler R (2002) Oxidative stress, antioxidantsand stress tolerance. Trends Plant Sci 7:

4054109. Apel K, Hirt H (2004) Reactive oxygen species:

metabolism, oxidative stress, and signal trans-duction. Annu Rev Plant Biol 55:373399

10. Mittler R, Vanderauwera S, Gollery M, VanBreusegem F (2004) Reactive oxygen gene net-

work of plants. Trends Plant Sci 9:490498

11. Baier M, Dietz KJ (1997) The plant 2-Cysperoxiredoxin BAS1 is a nuclear-encodedchloroplast protein: its expressional regulation,phylogenetic origin, and implications for itsspecific physiological function in plants. Plant J

12:17919012. Baier M, Pitsch NT, Mellenthin M, Guo W

(2010) Regulation of genes encoding antioxi-dant enzymes in comparison to regulation ofthe extra-plastidic antioxidant defense system.In: Anjum NA, Chan MT, Umar S (eds)

Ascorbate-glutathione pathway and stress tol-erance in plants. Springer, Dordrecht,Heidelberg, London, New York

13. Foyer CH, Shigeoka S (2011) Understandingoxidative stress and antioxidant functions to

enhance photosynthesis. Plant Physiol 155:93100

14. Vanacker H, Carver TL, Foyer CH (2000) EarlyH2O2accumulation in mesophyll cells leads toinduction of glutathione during the hyper-sensi-tive response in the barley-powdery mildew

interaction. Plant Physiol 123:1289130015. Kawai-Yamada M, Ohori Y, Uchimiya H

(2004) Dissection of Arabidopsis Bax inhibi-tor-1 suppressing Bax-, hydrogen peroxide-,and salicylic acid-induced cell death. Plant Cell16:2132

16. Bournonville CF, Diaz-Ricci JC (2010)Quantitative determination of superoxide inplant leaves using a modified NBT stainingmethod. Phytochem Anal 22:268271

17. Hideg E, Barta C, Kalai T, Vass I, Hideg K,Asada K (2002) Detection of singlet oxygen

and superoxide with fluorescent sensors inleaves under stress by photoinhibitionor UV radiation. Plant Cell Physiol 43:11541164

18. Fryer MJ, Oxborough K, Mullineaux PM,Baker NR (2002) Imaging of photo-oxidativestress responses in leaves. J Exp Bot53:12491254

19. Schneider CA, Rasband WS, Eliceiri KW(2012) NIH Image to ImageJ: 25 years ofimage analysis. Nat Methods 9:671675


prtocolos sod

Documents