拟南芥根分泌酸性磷酸酶活性分析

Liangsheng  Wang; Dong  Liu

doi:10.21769/BioProtoc.2202

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Peer-reviewed

Analyses of Root-secreted Acid Phosphatase Activity in Arabidopsis

拟南芥根分泌酸性磷酸酶活性分析

LW Liangsheng Wang email

Dong Liu

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2202 浏览次数: 13144

评审: Marisa RosaNing LiuAnonymous reviewer(s)

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Abstract

Induction and secretion of acid phosphatase (APase) is a universal adaptive response of higher plants to low-phosphate stress (Tran et al., 2010). The intracellular APases are likely involved in the remobilization and recycling of phosphate (Pi) from intracellular Pi reserves, whereas the extracellular or secreted APases are believed to release Pi from organophosphate compounds in the rhizosphere. The phosphate starvation-induced secreted APases can be released into the rhizosphere or retained on root surfaces (root-associated APases). In this article, we describe the protocols for analyzing root-secreted APase activity in the model plant Arabidopsis thaliana (Arabidopsis). In Arabidopsis, the activity of both root-associated APases and APases that are released into the rhizosphere can be quantified based on their ability to cleave a synthesized substrate, para-nitrophenyl-phosphate (pNPP), which releases a yellow product, para-nitrophenol (pNP) (Wang et al., 2011 and 2104). The root-associated APase activity can also be directly visualized by applying a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), to the root surface (Lloyd et al., 2001; Tomscha et al., 2004; Wang et al., 2011 and 2014) whereas the isozymes of APases that are released into rhizosphere can be profiled using an in-gel assay (Trull and Deikman, 1998; Tomscha et al., 2004; Wang et al., 2011 and 2014). The protocol for analysis of intracellular APase activity in Arabidopsis has been previously described (Vicki and William, 2013).

Keywords: Arabidopsis thaliana (拟南芥)

Phosphate starvation (磷酸盐饥饿)

Secretion (分泌)

Acid phosphatase (酸性磷酸酶)

Phosphatase activity (磷酸酶活性)

Isozyme (同工酶)

Histochemical staining (组织化学染色)

Quantitative analysis (定量分析)

Background

Phosphate (Pi) is the major form of phosphorus that plants take up through their root systems, and Pi levels in most soils are low, resulting in Pi starvation. To cope with this nutritional stress, plants trigger an array of adaptive responses that increase their survival and growth. Induction and secretion of APases is a hallmark Pi starvation response that has been documented in a variety of plant species (Tran et al., 2010), and root-secreted APase activity has been widely used as a diagnostic tool to evaluate the magnitude of plant responses to Pi starvation. In this article, we provide three protocols for assay of root-secreted APase activity. An assay using pNPP as a substrate is the most commonly used method to quantify APase activity. A BCIP staining assay provides a simple, one-step method for the histochemical detection of APase activity on the root surface. Neither of these methods, however, can reveal the number of APase isoforms that contribute to the observed activity. A third method (in-gel assay), which combines electrophoresis, protein renaturation, and conversion of a substrate into a red-brown product, can reveal the diverse compositions of APase isoforms in different samples, and can therefore provide additional information about root-secreted APases.

Materials and Reagents

9-cm-diameter Petri dish
Surgical blade (Swann Morton Surgical Scalpel Blade No. 20)
1.7 ml and 2 ml Eppendorf tubes
Dialysis tubing (Sigma-Aldrich, catalog number: D9777 )
50 ml Falcon tube
Whatman #1 filter paper (GE Healthcare, catalog number: 1001-125 )
Pipette tips
Arabidopsis seeds
Triton X-100 (Sigma-Aldrich, catalog number: X100 )
30% acrylamide/Bis solution (Solarbio, catalog number: A1010 )
TEMED (Sigma-Aldrich, catalog number: T22500 )
Agar (Sigma-Aldrich, catalog number: A1296 )
Bovine serum albumin (BSA)*
95% ethanol*
Ammonium persulfate (NH₄)₂S₂O₈*
Sodium hypochlorite solution (NaClO)*
Sodium hydroxide (NaOH)*
Sodium acetate (NaAc)*
Phosphoric acid (H₃PO₄)*
Hydrochloric acid (HCl)*
Ammonium nitrate (NH₄NO₃)*
Potassium nitrate (KNO₃)*
Calcium chloride (CaCl₂)*
Magnesium sulfate (MgSO₄)*
Potassium dihydrogen phosphate (KH₂PO₄)*
Potassium sulfate (K₂SO₄)*
Potassiu iodide (KI)*
Orthoboric acid (H₃BO₃)*
Manganese sulfate (MnSO₄)*
Zinc sulfate (ZnSO₄)*
Sodium molybdate (Na₂MoO₄)*
Cobalt(II) chloride (CoCl₂)*
Copper sulfate (CuSO₄)*
Fe-EDTA*
Myo Inositol*
Nicotinic acid*
Pyridoxine HCl*
Thiamine HCl*
Glycine*
Sucrose*
Bromophenol blue*
Coomassie Brilliant Blue G-250*
MES (AMRESCO, catalog number: E169 )
β-naphthyl acid phosphate (Sigma-Aldrich, catalog number: N7375 )
Fast Black K (Sigma-Aldrich, catalog number: F7253 )
pNPP (Sigma-Aldrich, catalog number: P4744 )
BCIP (Gold Bio, catalog number: B-500-10 )
Tris base (NOVON SCIENTIFIC, catalog number: ZZ02531 )
DTT (Sigma-Aldrich, catalog number: DTT-RO )
SDS (AMRESCO, catalog number: 0227 )
DMSO (AMRESCO, catalog number: 0231 )
PMSF (Sigma-Aldrich, catalog number: 52332 )
Liquid nitrogen
Murashige and Skoog (MS) medium (P+ and P- MS medium) (see Recipes, Murashige and Skoog, 1962)
APase activity profiling detection buffer, pH 4.9 (see Recipes)
Reaction buffer, pH 4.9 (see Recipes)
pNPP solution (1.0 mg/ml) (see Recipes)
BCIP stock solution (100x) (see Recipes)
Bradford solution (see Recipes)
5x protein loading buffer (see Recipes)

*Note: Similar reagents from any qualified company are suitable for this experiment.

Equipment

Pipettes
Forceps (Fisher Scientific, catalog number: 22-327379 )
Spectrophotometer (WPA, Biowave)
250 ml Erlenmeyer (conical) flask
Orbital shaker (Qilinbeier, model: TS-1 )
Freeze dryer (Beijing Songyuanhuaxing, model: LGJ-10 )
Microfuge (Eppendorf, model: 5415 D )
Microwave oven
Small mortar and pestle
Water bath (Shanghai Yiheng, model: DK-8D )**
Analytical balance (Mettler Toledo, model: PB153-L)**
pH meter (Mettler Toledo, model: FE20 )**
Mini-PROTEAN^® Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories, model: Mini-PROTEAN^® Tetra Vertical Electrophoresis Cell , catalog number: 1658004)