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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   

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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

  1. 0.2 µm syringe filter (Beyotime, catalog number: FF252 )
  2. Rice seedlings
  3. Nutrient solution
  4. Amerlite IR-120B resin (H+ form) (Alfa Aesar, catalog number: L14285 )
  5. Dowex 1 x 8 resin (100-200 mesh, formate form)
  6. Distilled water
  7. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148 )
  8. Formic acid (HCOOH) (Sigma-Aldrich, catalog number: 695076 )
  9. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  10. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: 223506 )
  11. Oxalate (Sigma-Aldrich, catalog number: 75688 )
  12. Malate (Sigma-Aldrich, catalog number: M8304 )
  13. Citrate (Sigma-Aldrich, catalog number: 251275 )
  14. 1 N HCl (see Recipes)
  15. 2 N HCl (see Recipes)
  16. 2 M HCOOH (see Recipes)
  17. 4 N NaOH (see Recipes)
  18. 0.5 mM CaCl2 (see Recipes)

Equipment

  1. 1.25 L black plastic pot (plastic pot must be opaque, if required, wrap it in black tape)
  2. pH meter (Mettler Toledo, model: S40 / SG78 / SG23 / SG68 )
  3. Cation exchange column (16 mm x 14 cm) (Bio-Rad Laboratories, model: 732-1010 )
  4. Rotary evaporator (IKA, model: RV10 )
  5. Ion chromatography (Thermo Fisher Scientific, Dionex, model: ICS 3000 System )
  6. IonPac AS11 anion-exchange analytical column (4 x 250 mm)
  7. Guard column (4 x 50 mm)

Procedure

  1. Cultivate rice seedlings in full strength nutrient solution for 7 d. Afterwards, transplant 10 uniform seedlings to 1.5-L pots. After 6 d of treatments, discard the nutrient solution, and treat these rice seedlings with 0.5 mM CaCl2 (pH 5.5) overnight to collect the organic acids (Figure 1).


    Figure 1. The apparatus for collection of root exudates

  2. After 12 h, collect and weigh the fresh roots of the treated rice seedlings. Pass the root exudates in the 0.5 mM CaCl2 solution through a cation exchange column (16 mm x 14 cm) that contained 5 g of Amerlite IR-120B resin (H+ form) and an anion exchange column filled with 1.5 g of Dowex 1 x 8 resin (100-200 mesh, formate form) (Figure 2). Overall, this process takes approximately about 5.5 h.


    Figure 2. The equipment for retention of the organic acids

  3. Elute the organic acid anions that were retained in the anion exchange resin immediately after the second step using 15 ml of 1 N HCl (Figure 3).


    Figure 3. The equipment for eluting the organic acids

  4. Then, evaporate the eluent by using a rotary evaporator at 40 °C, a rotation speed of 120 rpm, and a vacuum pressure of 35 hPa (Figure 4).


    Figure 4. The equipment used in concentrating the organic acids

  5. Terminate the evaporation process when the eluent solution has completely dried into powder (approximately 30 min later). Then re-dissolve the powder in 1 ml of distilled water and filter the solution through a 0.2 µm syringe filter prior to analysis.
  6. Detect the organic acid anions by ion chromatography (ICS 3000; Dionex) equipped with an IonPac AS11 anion-exchange analytical column (4 x 250 mm) and a guard column (4 x 50 mm). The mobile phase is 1.2 g/L NaOH at a flow rate of 0.6 ml/min. Generate a standard curve by using 1, 5, 10, 30, 50, 80, and 100 mg/L of the organic acid (oxalate, malate, and citrate) stock solution.
  7. Calculate the organic acid efflux expressing it as a function of the root fresh weight. 

Notes:

  1. The recovery rate of organic acids must be > 80%. To calculate the recovery rate, at least two different concentrations of an organic acid (oxalate, malate, and citrate) stock solution were employed. For example, mix 20 μl or 100 μl 100 mg/L of organic acid stock solution with 1 L of distilled water, and repeat the procedure from 1 to 5 (theoretical concentration in distilled water is 2 or 10 μg/ml). Then, repeat step 6 of procedure and calculate the actual organic acids concentration. Finally, calculate the recovery rate using the following equation:
    Recovery rate = (Actual value/Theoretical value) x 100.
  2. To prepare the anion exchange resin, wash the anion exchange resin powder (500 g) in 1 L 4 N NaOH for about 30 min at room temperature. Repeat three times and wash with distilled water to neutral pH. Wash three more times in 1 L 2 M formic acid for about 30 min each and wash again with distilled water to neutral pH.
    Similarly, wash the cation exchange resin (500 g) four times with 0.5 L 4 N NaOH about 1 h at 60 °C each, then rinse it with distilled water to a neutral pH, afterwards, wash it three times in 0.5 L 2 N HCl about 1 h each at room temperature, then rinse it with distilled water to a neutral pH.
    The prepared resins can be stored at 4 °C for at least 1 year.
  3. This method can be easily adapted to other plant species such as Arabidopsis (Zhu et al., 2012). However, the secretion of organic acids is limited for some small plant species. In this case, the recovery rate can be relatively low, and thus the number of plants per pot must be scaled up in order to collect enough organic acids.

Data analysis

  1. Create a standard curve for each organic acid by plotting the peak area vs. the known concentration.
  2. Use the standard curves to calculate the concentrations of each organic acid. This will give the concentration of organic acid present in the eluent. Data can be divided by root fresh weight to determine the amount of organic acids secreted per grams of rice root.
    The concentration of organic acids was calculated using the following formula:
    F= C x V/W
    Where,
    F is the organic acid secreted in roots (μg/g Fw),
    C is the concentration of organic acid (mg/L),
    V is the volume of the organic acid (this is 1 ml in the present study),
    W is the fresh weigh of 10 rice roots in one pot (g Fw)
    Below is an example of a calculation for the concentration of malate. The area of the sample is 0.0612, which was then used in the standard curve equation y = 0.0084x + 0.0056 (Figure 5), which calculated that the concentration of malate is 6.6190 mg/L. The volume of malate in present study was 1 ml, whereas the root fresh weight was 0.6126 g. The obtained value was then used in the above mentioned formula to calculate for the concentration in the roots (10.8048 μg/g Fw).


    Figure 5. Standard curve of malate

Recipes

  1. 1 N HCl
    Add ~88 ml 37% HCl to about 500 ml of distilled water, then adjust the final volume of the solution to 1 L using distilled water
  2. 2 N HCl
    Add ~176 ml 37% HCl to about 500 ml of distilled water, then adjust the final volume of the solution to 1 L using distilled water
  3. 2 M formic acid (HCOOH)
    Add ~78.6 ml 96% HCOOH to about 500 ml of distilled water, then adjust the final volume of the solution to 1 L using distilled water
  4. 4 N NaOH
    Add 166.7 g NaOH to distilled water to a final volume of 1 L
  5. 0.5 mM CaCl2 (pH 5.5)
    1. 0.5 M CaCl2 stock solution
      Dissolve 73.505 g CaCl2·2H2O in distilled water and add distilled water to a final volume of 1 L
    2. 0.5 mM CaCl2
      Dilute 5 ml of 0.5 M CaCl2 stock solution with distilled water to a final volume of 5 L
      Adjust the pH of 0.5 mM CaCl2 solution to 5.5 with 1 N HCl
  6. 100 mg/L organic acid stock solution (oxalate, malate and citrate)
    100 mg oxalate, malate, and citrate together
    Add distilled water to 1 L to make a 100 mg/L stock solution
    Dilute the stock solution to 1, 5, 10, 30, 50, 80, and 100 mg/L to generate a standard curve

Acknowledgments

The methods to collect and concentrate organic acids were modified from Ma et al. (2004), the measurement of organic acids content was modified from Zhu et al. (2015) and developed in the Ren Fang Shen’s Lab-Institute of Soil Science, Chinese Academy of Science. This work was supported by the National Key Basic Research Program of China (No. 2014CB441000), Natural Science Foundation of China (31501825) and the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (Nos. XDB15030302 and XDB15030202).

References

  1. Chen, J., Wan, W. H., Wu, F. H., You, C. Y., Liu, T. W., Dong, X. J., He, J. X. and Zheng, H. L. (2013). Hydrogen sulfide alleviates aluminum toxicity in barley seedlings. Plant Soil 362: 301-318.
  2. Delhaize, E., Ryan, P. R. and Randall, P. J. (1993). Aluminum Tolerance in Wheat (Triticum aestivum L.) (II. Aluminum-stimulated excretion of malic acid from root apices). Plant Physiol 103(3): 695-702.
  3. Ma, J. F., Nagao, S., Sato, K., Ito, H., Furukawa, J. and Takeda, K. (2004). Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley. J Exp Bot 55(401): 1335-1341.
  4. Ma, Z. and Miyasaka, S. C. (1998). Oxalate exudation by taro in response to Al. Plant Physiol 118(3): 861-865.
  5. Neumann, G., Massonneau, A., Martinoia, E. and Römheld, V. (1999). Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta 208(3): 373-382.
  6. Zhu, X. F., Lei, G. J., Jiang, T., Liu, Y., Li, G. X. and Zheng, S. J. (2012). Cell wall polysaccharides are involved in P-deficiency-induced Cd exclusion in Arabidopsis thaliana. Planta 236(4): 989-997.
  7. Zhu, X. F., Wang, Z. W., Wan, J. X., Sun, Y., Wu, Y. R., Li, G. X., Shen, R. F. and Zheng, S. J. (2015). Pectin enhances rice (Oryza sativa) root phosphorus remobilization. J Exp Bot 66(3): 1017-1024.

简介

从植物根系分泌的有机酸在各种生物过程中起重要作用,包括营养获取,金属解毒和病原体吸引。有机酸的分泌可能受植物生长阶段,营养缺乏和非生物胁迫等各种条件的影响。例如,当白羽扇豆(Lupinus albus)暴露于磷(P)缺乏条件时,柠檬酸从其类软骨根分泌显着增加(Neumann等, ,1999)。该方案描述了从不同氮形式的水稻品种Nipponbare('Nip')的根中收集和测量有机酸(草酸盐,苹果酸盐和柠檬酸盐)的流出的方法(NH 4) + 和NO 3 - )以及不同的P电源(+ P和-P)条件。

背景 除了酶法(Delhaize等人,1993)和高效液相色谱(HPLC)(Chen等人,2013),离子色谱是另一个广泛的用于测定有机酸的方法,其先前已被用于检测在Al 3 + 应力期间芋头根系分泌物中草酸盐含量的显着增加(Ma和Miyasaka,1998)。与离子色谱相比,替代方法有其自身的缺陷。例如,酶法需要使用容易经历变性的酶。此外,通过HPLC区分草酸和Cl-峰是困难的。在这里,我们描述了使用离子色谱法分析水稻根系分泌的有机酸的方法。该方法可用于分析其他水培栽培植物分泌的有机酸。

关键字:有机酸, 水稻, 采集, 测定, 离子色谱法

材料和试剂

  1. 0.2μm注射器过滤器(Beyotime,目录号:FF252)
  2. 稻苗
  3. 营养液
  4. Amerlite IR-120B树脂(H + 形式)(Alfa Aesar,目录号:L14285)
  5. Dowex 1 x 8树脂(100-200目,甲酸酯形式)
  6. 蒸馏水
  7. 盐酸(HCl)(Sigma-Aldrich,目录号:258148)
  8. 甲酸(HCOOH)(Sigma-Aldrich,目录号:695076)
  9. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465)
  10. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:223506)
  11. 草酸盐(Sigma-Aldrich,目录号:75688)
  12. 苹果酸(Sigma-Aldrich,目录号:M8304)
  13. 柠檬酸盐(Sigma-Aldrich,目录号:251275)
  14. 1 N HCl(参见食谱)
  15. 2 N HCl(参见食谱)
  16. 2 M HCOOH(见配方)
  17. 4 N NaOH(参见食谱)
  18. 0.5mM CaCl 2(参见食谱)

设备

  1. 1.25L黑色塑料罐(塑料罐必须不透明,如果需要,将其包装成黑色胶带)
  2. pH计(Mettler Toledo,型号:S40/SG78/SG23/SG68)
  3. 阳离子交换柱(16mm×14cm)(Bio-Rad Laboratories,型号:732-1010)
  4. 旋转蒸发器(IKA,型号:RV10)
  5. 离子色谱(Thermo Fisher Scientific,Dionex,型号:ICS 3000系统)
  6. IonPac AS11阴离子交换分析柱(4 x 250 mm)
  7. 护栏(4 x 50毫米)

程序

  1. 培养充分营养液中的水稻幼苗7 d。然后将10只均匀的幼苗移植到1.5升的盆中。 6 d处理后,弃去营养液,用0.5mM CaCl 2(pH5.5)处理这些水稻幼苗过夜,收集有机酸(图1)。

    图1.用于收集根系分泌物的装置

  2. 12小时后,收集和称重处理的水稻幼苗的新鲜根。将根系渗出物通过含有5g Amerlite IR-120B树脂(H +)的阳离子交换柱(16mm×14cm)渗透到0.5mM CaCl 2 /形式)和填充有1.5g Dowex 1×8树脂(100-200目,甲酸盐形式)(图2)的阴离子交换柱。总的来说,这个过程大约需要5.5小时

    图2.保留有机酸的设备

  3. 在第二步后立即用15ml 1N HCl洗脱保留在阴离子交换树脂中的有机酸阴离子(图3)。


    图3.用于洗脱有机酸的设备

  4. 然后,使用40℃旋转蒸发器,120rpm的转速和35hPa的真空压力蒸发洗脱液(图4)。


    图4.用于浓缩有机酸的设备

  5. 当洗脱液完全干燥成粉末(约30分钟后)时,终止蒸发过程。然后将粉末重新溶解在1ml蒸馏水中,然后通过0.2μm注射器过滤器过滤溶液,然后再分析
  6. 通过离子色谱法(ICS 3000; Dionex)检测有机酸阴离子,该离子色谱装备有IonPac AS11阴离子交换分析柱(4×250mm)和保护柱(4×50mm)。流动相为0.6g/min,流速为1.2g/L NaOH。通过使用1,5,10,30,50,80和100 mg/L有机酸(草酸盐,苹果酸盐和柠檬酸盐)储备溶液产生标准曲线。
  7. 计算表达其的有机酸流出量与根鲜重的函数。 

注意:

  1. 有机酸的回收率必须> 80%。为了计算回收率,使用至少两种不同浓度的有机酸(草酸盐,苹果酸盐和柠檬酸盐)储备溶液。例如,将20μl或100μl100 mg/L有机酸储备溶液与1 L蒸馏水混合,重复步骤1至5(蒸馏水中理论浓度为2或10μg/ml)。然后重复步骤6,计算实际有机酸浓度。最后,使用以下公式计算回收率:
    恢复率=(实际值/理论值)x 100.
  2. 为了制备阴离子交换树脂,在室温下将1L 4N NaOH中的阴离子交换树脂粉末(500g)洗涤约30分钟。重复三次,用蒸馏水冲洗至中性pH值。在1L 2 M甲酸中洗涤三次,每次约30分钟,再用蒸馏水洗至中性pH。
    类似地,用0.5L 4N NaOH在60℃下将阳离子交换树脂(500g)洗涤四次约1小时,然后用蒸馏水冲洗至中性pH,然后在0.5℃下洗涤三次L 2 N HCl在室温下每次约1小时,然后用蒸馏水冲洗至中性pH。
    所制备的树脂可以在4℃下储存至少1年。
  3. 这种方法可以容易地适应其他植物物种,如拟南芥(Zhu et al。,2012)。然而,对于一些小的植物物种,有机酸的分泌是有限的。在这种情况下,回收率可以相对较低,因此必须按比例放大每盆的植物数量以便收集足够的有机酸。

数据分析

  1. 通过绘制峰面积与已知浓度来绘制每种有机酸的标准曲线。
  2. 使用标准曲线计算每种有机酸的浓度。这将提供洗脱液中存在的有机酸的浓度。数据可以按根鲜重除以确定每克水稻根分泌的有机酸的量。
    使用以下公式计算有机酸的浓度:
    F = C×V/W
    哪里,
    F是根中分泌的有机酸(μg/g Fw),
    C是有机酸的浓度(mg/L),
    V是有机酸的体积(在本研究中为1ml),
    W是一盆(g Fw)
    的10个水稻根的鲜重 以下是苹果酸浓度计算的一个例子。样品面积为0.0612,然后在标准曲线方程y = 0.0084x + 0.0056(图5)中使用,计算出苹果酸盐的浓度为6.6190 mg/L。本研究中苹果酸盐的体积为1ml,根鲜重为0.6126g。然后将所得值用于上述公式计算根中的浓度(10.8048μg/g Fw)。


    图5.苹果酸的标准曲线

食谱

  1. 1 N HCl
    向约500ml蒸馏水中加入〜88ml 37%的HCl,然后用蒸馏水将溶液的最终体积调节至1L。
  2. 2 N HCl
    向约500ml蒸馏水中加入〜176ml 37%的HCl,然后用蒸馏水将溶液的最终体积调节至1L。
  3. 2 M甲酸(HCOOH)
    向约500ml蒸馏水中加入〜78.6ml 96%HCOOH,然后使用蒸馏水将溶液的最终体积调节至1L。
  4. 4 N NaOH
    向蒸馏水中加入166.7g NaOH至1L的最终体积
  5. 0.5mM CaCl 2(pH5.5)
    1. 0.5M CaCl 2 储备溶液
      在蒸馏水中溶解73.505g CaCl 2·2H 2 O,并加入蒸馏水至1L的最终体积
    2. 0.5mM CaCl 2
      用蒸馏水稀释5ml的0.5M CaCl 2 2储备溶液至最终体积为5升。
      用1N HCl将0.5mM CaCl 2溶液的pH调节至5.5
  6. 100 mg/L有机酸储备溶液(草酸盐,苹果酸盐和柠檬酸盐)
    100毫克草酸盐,苹果酸盐和柠檬酸盐一起
    加入蒸馏水至1升,制成100mg/L储液 将储备溶液稀释至1,5,10,30,50,80和100mg/L以产生标准曲线

致谢

从Ma等人修改了收集和浓缩有机酸的方法。 (2004),有机酸含量的测定由Zhu等人修改。 (2015),并在中国科学院沉芳沉土壤科学实验室研究开发。这项工作得到了中国国家重点基础研究计划(2014CB441000),中国自然科学基金(31501825)和中国科学院"战略重点研究计划"(编号XDB15030302和XDB15030202)的支持。

参考文献

  1. Chen,J.,Wan,WH,Wu,FH,You,CY,Liu,TW,Dong,XJ,He,JX and Zheng,HL(2013)。  硫化氢减轻了大麦幼苗中的铝毒性。植物土壤 362:301-318。
  2. Delhaize,E.,Ryan,PR和Randall,PJ(1993)。  小麦中的耐铝性( L))(II。从根尖诱导的苹果酸的铝刺激排泄物)。植物生理学103 (3):695-702。
  3. Ma,JF,Nagao,S.,Sato,K.,Ito,H.,Furukawa,J.and Takeda,K。(2004)。负责大麦中柠檬酸激活的Al激活分泌的基因的分子图谱.J Exp Bot 55(401):1335-1341。
  4. Ma,Z.和Miyasaka,SC(1998)。  芋头中的草酸盐渗出物响应于Al。植物生理学118(3):861-865。
  5. Neumann,G.,Massonneau,A.,Martinoia,E.和Römheld,V.(1999)。< a class ="ke-insertfile"href ="http://link.springer.com/article/10.1007/s004250050572"target ="_ blank">白羽扇豆类软组织根系发育期间磷缺乏的生理适应性。植物208(3):373-382。
  6. Zhu,XF,Lei,GJ,Jiang,T.,Liu,Y.,Li,GX and Zheng,SJ(2012)。  细胞壁多糖参与拟南芥中P缺陷诱导的Cd排斥。/em> 236(4):989-997。
  7. Zhu,XF,Wang,ZW,Wan,JX,Sun,Y.,Wu,YR,Li,GX,Shen,RF and Zheng,SJ(2015)。  果胶增强水稻(Oryza sativa)根磷再生作用。 Exp Bot 66(3):1017-1024。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhu, C., Zhu, X. and Shen, R. (2017). Rice Root Organic Acid Efflux Measurement by Using Ion Chromatography. Bio-protocol 7(4): e2141. DOI: 10.21769/BioProtoc.2141.
  2. Zhu, C. Q., Zhu, X. F., Hu, A. Y., Wang, C., Wang, B., Dong, X. Y. and Shen, R. F. (2016). Differential Effects of Nitrogen Forms on Cell Wall Phosphorus Remobilization Are Mediated by Nitric Oxide, Pectin Content, and Phosphate Transporter Expression. Plant Physiol 171(2): 1407-1417.
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