植物多重应激下脂质过氧化作用的希夫试剂定性分析：组织化学方法

Jay Prakash  Awasthi; Bedabrata  Saha; Bhaben  Chowardhara; Sanjenbam Sanjibia Devi; Pankaj  Borgohain; Sanjib Kumar  Panda

doi:10.21769/BioProtoc.2807

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Peer-reviewed

Qualitative Analysis of Lipid Peroxidation in Plants under Multiple Stress Through Schiff’s Reagent: A Histochemical Approach

植物多重应激下脂质过氧化作用的希夫试剂定性分析：组织化学方法

Jay Prakash Awasthi

Bedabrata Saha

Bhaben Chowardhara

Sanjenbam Sanjibia Devi

Pankaj Borgohain

SP Sanjib Kumar Panda email

发布: 2018年04月20日第8卷第8期 DOI: 10.21769/BioProtoc.2807 浏览次数: 14933

评审: Samik BhattacharyaVenkatasalam ShanmugabalajiAnonymous reviewer(s)

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Abstract

Lipid peroxidation is a physiological indicator of both biotic and abiotic stress responses, hence is often used as a biomarker to assess stress-induced cell damage or death. Here we demonstrate an easy, quick and cheap staining method to assess lipid peroxidation in plant tissues. In this methodology, Schiff’s reagent, is used to assay for membrane degradation. Histochemical detection of lipid peroxidation is performed in this protocol. In brief, Schiff’s reagent detects aldehydes that originate from lipid peroxides in stressful condition. Schiff’s reagent is prepared and applied to plants tissue. After the reaction, plant tissue samples are rinsed with a sulfite solution to retain the staining color. From this analysis, qualitative visualization of lipid peroxidation in plant tissue is observed in the form of magenta coloration. This reagent is useful for visualization of stress induced lipid peroxidation in plants. In this protocol, Indica rice root, Assam tea root and Indian mustard seedlings are used for demonstration.

Keywords: Schiff’s reagent (希夫试剂)

Rice (水稻)

Tea (茶叶)

Mustard (芥末)

Membrane degradation (膜降解)

Lipid peroxidation (脂质过氧化)

Background

Oxidative stress induced by both biotic and abiotic stress factors leads to lipid peroxidation. The peroxidation of unsaturated lipids in membrane is the most apparent symptom of oxidative stress. Lipid peroxidation is a deleterious process in plants, which affects membrane properties, causes protein degradation and limits the capacity of ionic transport, ultimately triggering the cell death process (Yamamoto et al., 2001). Malondialdehyde (MDA) content, which is a byproduct of lipid peroxidation process was found to be enhanced in rice (Ma et al., 2012; Awasthi et al., 2017), Salvinia (Mandal et al., 2013), pea (Yamamoto et al., 2001), Soyabean (Cakmak and Horst, 1991; Du et al., 2010), Indian mustard (Saha et al., 2016) on exposure to stress. The reactive oxygen species (ROS) associated with oxidative stress acts on membrane lipids to decrease membrane stability. Thus making the study of lipid peroxidation an important parameter and our protocol offers a very rapid method for the study. Schiff’s reagent reacts with aldehyde functional group of MDA to give the magenta coloration, thus acting as a key determinant of lipid peroxidation in plant tissues.

Materials and Reagents

Glass Petri plates of 10 cm diameter (Scott Duran)
Absorbent cotton
Whatman 1 filter paper
Razor blade (Glassvan, No. 23, Scalpel blade)
15 ml disposable centrifuge tubes (Tarsons)
Slide
Needle (Dispovan, sterile needle, 0.45 x 13 mm)
Brush (Camel, Size 0)
Gas mask
Plant tissue (Leaf, root or whole seedling) collected freshly after stress treatment
Distilled water
Mercuric chloride (HgCl₂) (SRL Sisco Research Laboratories, catalog number: 25699 )
Schiff’s reagents (HiMedia Laboratories, catalog number: S074-500ML )
Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O) (HiMedia Laboratories, catalog number: GRM3903-500G )
Potassium nitrate (KNO₃) (HiMedia Laboratories, catalog number: GRM1401-500G )
Potassium phosphate monobasic (KH₂PO₄) (HiMedia Laboratories, catalog number: RM3943-500G )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (HiMedia Laboratories, catalog number: RM684-5KG )
Boric acid (H₃BO₃) (HiMedia Laboratories, catalog number: MB007-1KG )
Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (HiMedia Laboratories, catalog number: RM685-500G )
Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (HiMedia Laboratories, catalog number: PCT0118-1KG )
Sodium molybdate dihydrate (Na₂MoO₄·2H₂O) (HiMedia Laboratories, catalog number: GRM415-100G )
Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (HiMedia Laboratories, catalog number: RM630-500G )
Ferric chloride (FeCl₃) (HiMedia Laboratories, catalog number: RM1178-1KG )
EDTA (HiMedia Laboratories, catalog number: RM678-100G )
Ammonium sulfate ((NH₄)₂SO₄) (HiMedia Laboratories, catalog number: MB004-250G )
Potassium sulfate (K₂SO₄) (HiMedia Laboratories, catalog number: GRM404-500G )
Manganese sulfate monohydrate (MnSO₄·H₂O) (SRL Sisco Research Laboratories, catalog number: 1347151 , 500G)
Sodium Meta-bisulphate (Na₂S₂O₅) (Sigma-Aldrich, catalog number: 255556-100G )
Calcium chloride dihydrate (CaCl₂·2H₂O) (HiMedia Laboratories, catalog number: PCT0004-500G )
Aluminum chloride (AlCl₃) (Merck, catalog number: 8010810100 )
Cadmium chloride (CdCl₂) (Thermo Fisher Scientific, catalog number: Q17584 )
Zinc chloride (ZnCl₂) (Thermo Fisher Scientific, catalog number: Q28785 )
Ethanol (Diluent for DNA Extraction, HiMedia Laboratories, catalog number: MB228-500ML )
Glycerol (Sigma-Aldrich, catalog number: V800196-500ML )
Acetic acid (Thermo Fisher Scientific, catalog number: Q21057 )
Hydrochloric acid (HCl) (HiMedia Laboratories, catalog number: AS004-2.5L )
Schiff’s reagent staining solution (see Recipe 1)
Hoagland solution (see Recipe 2)
Modified nutrient solution (see Recipe 3)
Treatment solutions (AlCl₃, CdCl₂, ZnCl₂) (see Recipe 4)
Sulfite solution (see Recipe 5)
Bleaching solution (see Recipe 6)
Storage solution (see Recipe 7)

Equipment

Pipettes (Gilson, PIPETMAN, 2-20,20-200 and 100-1,000 µl)
pH meter (pH Tutor, Eutech Instruments)
Magnetic stirrer cum hot plate (Tarsons)
Weighing balance (Sartorius, 0.1 mg-220 g)
Water bath (Equitron unstirred water bath, Medica Instruments)
Light microscope (Olympus, 10x objective)
Digital camera (Nikon, model: COOLPIX P100 , 10.3 megapixel, 26x zoom)

Procedure

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Awasthi, J. P., Saha, B., Chowardhara, B., Devi, S. S., Borgohain, P. and Panda, S. K. (2018). Qualitative Analysis of Lipid Peroxidation in Plants under Multiple Stress Through Schiff’s Reagent: A Histochemical Approach. Bio-protocol 8(8): e2807. DOI: 10.21769/BioProtoc.2807.