采用离子色谱法测定水稻根有机酸分泌

Chun-quan Zhu; Xiao-fang  Zhu; Ren-fang  Shen

doi:10.21769/BioProtoc.2141

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Peer-reviewed

Rice Root Organic Acid Efflux Measurement by Using Ion Chromatography

采用离子色谱法测定水稻根有机酸分泌

Chun-quan Zhu

Xiao-fang Zhu

Ren-fang Shen email

发布: 2017年02月20日第7卷第4期 DOI: 10.21769/BioProtoc.2141 浏览次数: 8568

评审: Arsalan DaudiWan-Jun ZhangAnonymous reviewer(s)

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Cover of Plant Physiology, featuring study using the protocol.

Jun 2016

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Abstract

Organic acids secreted from plant roots play important roles in various biological processes including nutrient acquisition, metal detoxification, and pathogen attraction. The secretion of organic acids may be affected by various conditions such as plant growth stage, nutrient deficiency, and abiotic stress. For example, when white lupin (Lupinus albus L.) is exposed to phosphorus (P)-deficient conditions, the secretion of citrate acid from its proteoid roots significantly increases (Neumann et al., 1999). This protocol describes a method for the collection and measurement of the efflux of organic acids (oxalate, malate, and citrate) from the roots of rice cultivar Nipponbare (‘Nip’) under different nitrogen forms (NH₄⁺ and NO₃^-), together with different P supply (+P and -P) conditions.

Keywords: Organic acids (有机酸)

Rice (水稻)

Collection (采集)

Measurement (测定)

Ion chromatography (离子色谱法)

Background

In addition to enzymatic methods (Delhaize et al., 1993) and high-performance liquid chromatography (HPLC) (Chen et al., 2013), ion chromatography is another widely used method for the determination of organic acids, which has previously been employed to detect the significant increase in oxalate content in taro root exudates during Al³⁺ stress (Ma and Miyasaka, 1998). Compared to ion chromatography, alternative methods have their own defects. For example, enzymatic methods require the use of enzymes that can easily undergo denaturation. Moreover, it is difficult to distinguish oxalate acid from Cl- peaks by HPLC. Here, we describe a method for analyzing organic acids secreted by rice roots using ion chromatography. This method could be used in the analysis of organic acids that are secreted by other hydroponically cultivated plants.

Materials and Reagents

0.2 µm syringe filter (Beyotime, catalog number: FF252 )
Rice seedlings
Nutrient solution
Amerlite IR-120B resin (H⁺ form) (Alfa Aesar, catalog number: L14285 )
Dowex 1 x 8 resin (100-200 mesh, formate form)
Distilled water
Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
Formic acid (HCOOH) (Sigma-Aldrich, catalog number: 695076 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: 223506 )
Oxalate (Sigma-Aldrich, catalog number: 75688 )
Malate (Sigma-Aldrich, catalog number: M8304 )
Citrate (Sigma-Aldrich, catalog number: 251275 )
1 N HCl (see Recipes)
2 N HCl (see Recipes)
2 M HCOOH (see Recipes)
4 N NaOH (see Recipes)
0.5 mM CaCl₂ (see Recipes)

Equipment

1.25 L black plastic pot (plastic pot must be opaque, if required, wrap it in black tape)
pH meter (Mettler Toledo, model: S40 / SG78 / SG23 / SG68 )
Cation exchange column (16 mm x 14 cm) (Bio-Rad Laboratories, model: 732-1010 )
Rotary evaporator (IKA, model: RV10 )
Ion chromatography (Thermo Fisher Scientific, Dionex, model: ICS 3000 System )
IonPac AS11 anion-exchange analytical column (4 x 250 mm)
Guard column (4 x 50 mm)