局部和系统性铁过量对拟南芥侧根起始影响的测定

Guangjie   Li; Lin Zhang; Weiming Shi

doi:10.21769/BioProtoc.2387

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Peer-reviewed

Determination of the Effects of Local and Systemic Iron Excess on Lateral Root Initiation in Arabidopsis thaliana

局部和系统性铁过量对拟南芥侧根起始影响的测定

GL Guangjie Li

LZ Lin Zhang

Weiming Shi email

发布: 2017年07月05日第7卷第13期 DOI: 10.21769/BioProtoc.2387 浏览次数: 10716

评审: Marisa RosaManjula MummadisettiAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

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Dec 2015

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Abstract

Root system architecture depends on nutrient availability. A symptom of iron (Fe) toxicity in plants is stunted root growth, yet little is known about the effects of excess Fe on lateral root (LR) development. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of Arabidopsis thaliana root systems to Fe by testing homogeneous supply and localized Fe supply treatment.

Keywords: Arabidopsis (拟南芥)

Fe toxicity (铁毒性)

Lateral root (侧根)

Localized iron supply (局部铁供应)

Homogeneous iron supply (均质铁供应)

Background

A localized supply of nutrients regulates lateral root elongation and/or lateral root density in Arabidopsis thaliana. Lateral root formation is affected by nutrients at different developmental stages (e.g., initiation versus elongation) and in a nutrient-specific manner (Li et al., 2011; Giehl et al., 2012). Iron toxicity as a syndrome in plants is typically associated with an excessive amount of the ferrous form (the Fe²⁺ ion) in the soil (Vigani, 2012). Iron toxicity symptoms vary with cultivars and are characterized by a reddish-brown, purple bronzing, yellow, or orange discoloration of the lower leaves and a stunted root growth. The presence of the Fe²⁺ ion is increased by low pH and anoxic conditions, and there is an increasing presence in vertically lower strata (Becker and Asch, 2005; Li et al., 2016). We describe a detailed pipeline used for localized iron supply in Arabidopsis grown in vitro which we validated in Arabidopsis (Li et al., 2015a; 2015b and 2016).

Materials and Reagents

Eppendorf tubes (1.5 ml)
Sterile tips
Plastic wrap (Bemis, catalog number: PM996 )
130 x 130 x 12 mm square plastic plates (self-made, see Figure 1)
130 x 13 x 2 mm glass strip (self-made, see Figure 1)

Figure 1. Square plastic plates used for treatment
Arabidopsis thaliana seeds (DR5:GUS lines, Col-0 background)
Distilled water
Ethanol (Sinopharm Chemical Reagent, catalog number: 80176961 )
Sodium hypochlorite (NaClO) (Sinopharm Chemical Reagent, catalog number: 80010428 )
Sodium dodecyl sulfate, sodium salt (SDS) (Sinopharm Chemical Reagent, catalog number: 30166428 )
Potassium phosphate monobasic (KH₂PO₄) (Sinopharm Chemical Reagent, catalog number: 10017618 )
Sodium nitrate (NaNO₃) (Sinopharm Chemical Reagent, catalog number: 10019918 )
Magnesium sulfate (MgSO₄) (Sinopharm Chemical Reagent, catalog number: 10013018 )
Calcium chloride (CaCl₂) (Sinopharm Chemical Reagent, catalog number: 20011160 )
Ferrous sulfate (FeSO₄·7H₂O) (Sinopharm Chemical Reagent, catalog number: 10012116 )
Ethylene diamine tetraacetic acid (EDTA) (Sinopharm Chemical Reagent, catalog number: 10009717 )
Boric acid (H₃BO₃) (Sinopharm Chemical Reagent, catalog number: 10004818 )
Manganese sulfate (MnSO₄) (Sinopharm Chemical Reagent, catalog number: LB2208102 )
Zinc chloride (ZnCl₂) (Sinopharm Chemical Reagent, catalog number: 10023828 )
Copper sulfate (CuSO₄) (Sinopharm Chemical Reagent, catalog number: 10008216 )
Sodium molybdate (Na₂MoO₄) (Sinopharm Chemical Reagent, catalog number: 10019818 )
Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
MES hydrate (Sigma-Aldrich, catalog number: M8250 )
Agar-agar (Sigma-Aldrich, catalog number: A7002 )
Seed sterilization solution (see Recipes)
Normal growth medium (see Recipes)
Fe-supplemented medium and the control medium (see Recipes)
Control medium (see Recipes)

Equipment

Glass bottles (Schott Duran glass bottle, 500 ml capacity or higher)
Incubation chamber (23 ± 1 °C, under fluorescent lamps at 100 μmol/m²/sec, 16-h-light/8-h-dark)
pH meter (Mettler-Toledo International, model: FE20K )
Autoclave (TOMY DIGITAL BIOLOGY, model: SX-500 )
Refrigerator (Haier, model: BCD-648WDBE )
Flow hood (Suzhou Antai Air-tech, model: SW-CJ-1F(D) )
Vortex
Tweezers

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Li, G., Zhang, L. and Shi, W. (2017). Determination of the Effects of Local and Systemic Iron Excess on Lateral Root Initiation in Arabidopsis thaliana. Bio-protocol 7(13): e2387. DOI: 10.21769/BioProtoc.2387.
Li, G., Song, H., Li, B., Kronzucker, H. J. and Shi, W. (2015). Auxin Resistant1 and PIN-FORMED2 Protect Lateral Root Formation in Arabidopsis under Iron Stress. Plant Physiol 169(4): 2608-2623.