Rhizosphere Acidification Assay

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Plant survival depends on the ability of root systems to establish themselves in locations where water and nutrients are available for uptake and translocation (Hawes et al., 2003). Rhizosphere influences crop productivity by mediating efficient nutrient transformation, acquisition, and use (Shen et al., 2013). Rhizosphere acidification is a central mechanism for plant mineral nutrition since it contributes to nutrient solubility and the proton motive force (pmf). This pmf is generated by the plasma membrane H+-ATPases (Miller and Smith, 1996; Forde, 2000) in root epidermal and cortical cells, and is coupled to active nutrient acquisition (e.g., N, K, P). Roots are able to acidify the rhizosphere by up to two pH units compared to the surrounding bulk soil mainly through the release of protons, but also bicarbonate, organic acids and CO2. Here we present an easy and inexpensive protocol to quantify protons released to the media by the root system-a method successfully used in our recently published work (Pizzio et al., 2015).

Keywords: Root(根), Arabidopsis(拟南芥), Rhyzosphere(rhyzosphere), Nutrient-use-efficiency(养分利用效率), Acidification(酸化)

Materials and Reagents

  1. Arabidopsis thaliana seeds (Col-0)
  2. Eppendorf tubes (1.5 ml)
  3. Commercial Bleach
  4. Pipettes and tips (1 and 5 ml)
  5. Silicon caps (or aluminum foil)
  6. Square Petri dishes
  7. Plastic wrap
  8. Spatula and forceps
  9. Flasks (200 ml)
  10. Plastic wrap
  11. Glass culture tubes
  12. Tween-20 (Sigma-Aldrich, catalog number: P-1379 )
  13. Murashige and Skoog medium (MS) (PhytoTechnology Laboratories®, catalog number: M524 )
  14. Sucrose (VWR International, catalog number: BDH-0308 )
  15. Potassium Hydroxide (KOH) (Thermo Fisher Scientific, catalog number: P-250 )
  16. Agar-agar (Sigma-Aldrich, catalog number: A-1296 )
  17. Sterile distilled water
  18. MES hydrate (Sigma-Aldrich, catalog number: M-8250 )
  19. Seed sterilization solution (see Recipes)
  20. MS solid (see Recipes)
  21. MS liquid (see Recipes)
  22. Assay solution (see Recipes)


  1. Autoclave
  2. Rotary shaker
  3. Flow hood
  4. Fridge (4 °C)
  5. Plant growth chamber (Conviron, catalog number: ATC26 )
  6. Magnetic stirrer and stirring bars
  7. pH meter (Beckman Coulter)
    Note: pH probe should be capable of measuring pH in samples with volumes <= 3 ml.
  8. Balance


  1. Seed sterilization
    1. Put 5 mg (200-300 seeds) of Arabidopsis seeds into an Eppendorf tube and add 750 µl sterilization solution.
    2. Vortex for 15 min at room temperature.
    3. Decant the sterilization solution (with sterile tips) under flow hood and add 750 µl sterile water.
    4. Vortex briefly.
    5. Repeat steps c and d 3 times.
    6. Stratify the seeds in 750 µl sterile water at 4 °C for 2 d in the dark.

  2. Plating seeds (under flow hood)
    1. Sow seeds (about 10) on sterile solid MS plates. Keep a density of approximately one seed per cm2. Crowded plates produce unhealthy seedlings.
    2. Close and seal plates with plastic wrap. Use only one layer of plastic wrap. More than one layer prevents gaseous exchange and healthy plant growth.
    3. Incubate plates in a vertical position in a growth chamber at 21 °C with a 12-h-light/12-h-dark cycle (150 μmol m-2 s-1) (Forde, 2000). Vertical growth reduces root stress and prevents agar transfer when transplanting seedlings.
    4. Grow seedlings for 5-7 d.

  3. Plant growth in liquid media
    1. Prepare 200 ml flasks with 4 ml liquid MS each.
    2. Close flasks with a “silicon” cap (or with aluminum foil).
    3. Autoclave flasks at 121 °C for 20 min.
    4. Let flasks cool in flow hood.
    5. Transfer 10 seedlings into each flask. Put seedlings in the bottom with the help of sterile spatula and forceps. Be sure that the seedlings stay in contact with the media.
    6. Close flasks with a layer of plastic wrap. Transparent plastic wrap favors transmission of light, promotes uniform plant growth, and prevents media evaporation.
    7. Grow plants on a rotary shaker at 50 rpm in chamber at 21 °C with a 12-h-light/12-h-dark cycle (150 μmol m-2 s-1) for 2 weeks.
    8. Pay attention: usually seedlings consume all the liquid media before the 2-week period. In this case, add 4 ml sterile liquid MS.

  4. Acidification assay
    1. Empty flasks and wash roots with the assay solution (5 ml) for 5 min.
    2. Empty flasks again and add fresh assay solution (3 ml).
    3. Prepare a control flask with assay solution (3 ml), but without any plants.
    4. Close flasks with plastic (transparent) foil and keep them in the growth chamber for 6 h.
    5. Transfer all the 3 ml of assay solution from flasks to a glass tube and proceed to pH measurements.
    6. Take out plants from flasks and check the number of plants in each flask.
    7. Dry plants with paper towels, cut roots from shoots, and immediately weigh to avoid plant dehydration, and the concomitant weight loss. Root and shoot weights are important values because we can either calculate protons released per gram of root or seedling.

  5. Calculation
    1. Use pH values from each flask to compute [H+] (mole/L) in sample and control using the equation pH=-log [H+].
    2. Calculate the number of H+ moles in 3 ml of experimental and control samples [mole/ 3 ml].
    3. Compute crude H+ released to the medium by subtracting the average values of H+control from H+sample. A typical experiment requires from 3 to 5 independent replicates.
    4. Relativize average number of H+ moles to either plant number, or to root or plant fresh weight. The units are going to be either mole/plant or mole/g. These relativized values allow us to calculate an average from several flasks and also to compare flasks with different plant numbers or plant lines.

Representative data

Figure 1. Protons released from the roots during day and night hours by Col-0 (empty bars) and transgenic plants overexpressing the type I H+-PPase AVP1 (AVP1-1; Pizzio et al., 2015) (black bars) plants grown in liquid media (mean ± SE; n= 6 pools of 10 plants per line, per time of day, per trial; two independent trials)


  1. Seed sterilization solution
    30% (v/v) commercial bleach
    0.05% (v/v) Tween-20
  2. Murashige and Skoog (MS) solid
    One-half-strength MS
    1% [w/v] Sucrose
    pH 5.7 with KOH
    1% [w/v] agar
  3. MS liquid
    One-half-strength MS+
    1% [w/v] Sucrose (pH 5.7 with KOH)
  4. Assay solution
    One-quarter-strength MS
    2 mM MES buffer (pH 6.8 with KOH). Low MES concentration assures similar starting pH for all plants.


RAG, GAP, and KR were supported by the National Science Foundation (grant no. IOS-1122148). This protocol was adapted from Pizzio et al. (2015).


  1. Forde, B. G. (2000). Nitrate transporters in plants: structure, function and regulation. Biochim Biophys Acta 1465(1-2): 219-235.
  2. Hawes, M. C., Bengough, G., Cassab, G. and Ponce, G. (2003). Root caps and rhizosphere. J Plant Growth Regul 21: 352-367.
  3. Miller, A. J. and Smith, S. J. (1996). Nitrate transport and compartmentation in cereal root cells. Journal of Experimental Botany, Vol. 47, No. 300, pp. 843-854.
  4. Pizzio, G. A., Paez-Valencia, J., Khadilkar, A. S., Regmi, K., Patron-Soberano, A., Zhang, S., Sanchez-Lares, J., Furstenau, T., Li, J., Sanchez-Gomez, C., Valencia-Mayoral, P., Yadav, U. P., Ayre, B. G. and Gaxiola, R. A. (2015). Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning. Plant Physiol 167(4): 1541-1553.
  5. Shen, J., Li, C., Mi, G., Li, L., Yuan, L., Jiang, R. and Zhang, F. (2013). Maximizing root/rhizosphere efficiency to improve crop productivity and nutrient use efficiency in intensive agriculture of China. J Exp Bot 64(5): 1181-1192.


植物生存取决于根系统在水和营养物可用于摄取和易位的位置建立自己的能力(Hawes等人,2003)。根际土壤通过介导有效的营养物转化,获得和使用来影响作物生产力(Shen等人,2013)。根际酸化是植物矿物营养的中心机制,因为它有助于营养物溶解度和质子动力(pmf)。该pmf由根表皮和皮层细胞中的质膜H sup + -ATPases(Miller和Smith,1996; Forde,2000)产生,并且与活性营养物获得(例如,/em> N,K,P)。与周围的主体土壤相比,根部能够使根际酸化多达两个pH单位,主要通过质子的释放,但是还有碳酸氢盐,有机酸和CO 2。在这里,我们提出了一个简单和便宜的协议,量化由根系统释放到媒体的质子 - 这种方法在我们最近出版的作品中成功地使用(Pizzio等人,2015)。

关键字:根, 拟南芥, rhyzosphere, 养分利用效率, 酸化


  1. 拟南芥种子(Col-0)
  2. Eppendorf管(1.5ml)中
  3. 商业污点
  4. 移液器和吸头(1和5 ml)
  5. 硅帽(或铝箔)
  6. 方形培养皿
  7. 塑料包装
  8. 小铲和镊子
  9. 烧瓶(200ml)
  10. 塑料包装
  11. 玻璃培养管
  12. Tween-20(Sigma-Aldrich,目录号:P-1379)
  13. Murashige和Skoog培养基(MS)( Phyto Technology Laboratories ,目录号:M524)
  14. 蔗糖(VWR International,目录号:BDH-0308)
  15. 氢氧化钾(KOH)(Thermo Fisher Scientific,目录号:P-250)
  16. 琼脂(Sigma-Aldrich,目录号:A-1296)
  17. 无菌蒸馏水
  18. MES水合物(Sigma-Aldrich,目录号:M-8250)
  19. 种子灭菌溶液(见配方)
  20. MS实体(参见配方)
  21. MS液体(参见配方)
  22. 测定解决方案(参见配方)


  1. 高压灭菌器
  2. 旋转振动器
  3. 流量罩
  4. 冰箱(4°C)
  5. 植物生长室(Conviron,目录号:ATC26)
  6. 磁力搅拌器和搅拌棒
  7. pH计(Beckman Coulter)
    注意:pH探针应该能够测量体积<= 3ml的样品的pH。
  8. 余额


  1. 种子灭菌
    1. 将5mg(200-300粒种子)拟南芥种子放入Eppendorf管中,加入750μl灭菌溶液。
    2. 在室温下涡旋15分钟
    3. 在流动罩下倒出灭菌溶液(无菌尖端),加入750μl无菌水
    4. 短暂涡旋。
    5. 重复步骤c和d 3次。
    6. 将种子在750μl无菌水中在4℃在黑暗中分层2天。

  2. 电镀种子(流动罩下)
    1. 在无菌固体MS平板上播种种子(约10)。保持密度 大约每平方厘米1个种子。拥挤的板材产生不健康 苗。
    2. 关闭和密封板用塑料套。只使用一个 塑料包装层。多于一层防止气体交换 ?健康植物生长
    3. 孵育板在垂直位置 ?生长室中在21℃下以12小时光/12小时暗循环(150μmolm -2 -2s -1 s -1)(Forde,2000)。垂直生长减少根部压力并防止 移植苗时的琼脂转移
    4. 生长幼苗5-7 d。

  3. 液体培养基中的植物生长
    1. 分别用4ml液体MS制备200ml烧瓶
    2. 关闭带"硅"帽(或铝箔)的瓶子。
    3. 高压灭菌瓶在121℃下20分钟
    4. 让烧瓶在流动罩中冷却。
    5. 转移10幼苗到每个烧瓶。把幼苗放在底部 在无菌刮铲和镊子的帮助下。确保幼苗 ?保持与媒体接触。
    6. 关闭有一层的烧瓶 保鲜膜。透明塑料包装有利于透光, 促进植物均匀生长,防止培养基蒸发
    7. 在旋转振荡器上在50rpm的室中在21℃下用a生长植物 12小时光/12小时暗循环(150μmolm -2 -2s -1 s -1))2周。
    8. 工资 注意:通常苗前消耗所有液体培养基 2周。在这种情况下,加入4 ml无菌液体MS。

  4. 酸化测定
    1. 空的烧瓶和洗涤根与测定溶液(5ml)5分钟
    2. 再次倒空烧瓶并加入新鲜的测定溶液(3ml)
    3. 用测定溶液(3ml)制备对照烧瓶,但不含任何植物
    4. 用塑料(透明)箔封闭烧瓶,并将它们保持在生长室中6小时
    5. 将所有3ml的测定溶液从烧瓶中转移到玻璃管中,并进行pH测量
    6. 从烧瓶中取出植物,并检查每个烧瓶中的植物数量
    7. 用纸巾干燥植物,从枝条切根,和 立即称重,避免植物脱水,并伴有重量 ?失利。根和茎的重量是重要的值,因为我们可以 ?计算每克根或幼苗释放的质子。

  5. 计算
    1. 使用来自每个烧瓶的pH值,使用方程pH = -log [H sup + ]计算样品和对照中的[H sup +](mole/>
    2. 计算3ml实验和对照样品中的H sup +摩尔数[摩尔/3ml]。
    3. 通过减去平均值来计算粗H + sup + +释放到培养基 来自H + sample 的H + 典型的实验需要3 到5个独立的重复
    4. 将相对于植物数或根或植物鲜重的H 摩尔的平均数相对。的 单位将是摩尔/植物或摩尔/g。这些相对化 值允许我们从几个烧瓶计算平均值 比较具有不同植物数量或植物品系的烧瓶。


图1.通过Col-0(空条)和过量表达IH i + -/- >顶层AVP1(AVP1-1)的转基因植物在白天和夜晚从根部释放的质子, (平均值±SE; n = 6个池,每行10个植物,每次每个时间,每次试验)的植物(黑色条)(黑色条)两次独立试验)


  1. 种子灭菌溶液
  2. Murashige和Skoog(MS)固体
    1%[w/v]蔗糖 pH 5.7用KOH
  3. MS液体
    半强度MS +
  4. 测定溶液
    2mM MES缓冲液(pH6.8,KOH)。低MES浓度保证了所有植物的类似的起始pH




  1. Forde,B.G。(2000)。 植物中的硝酸盐转运蛋白:结构,功能和调节。 Biochim Biophys Acta 1465(1-2):219-235。
  2. Hawes,M.C.,Bengough,G.,Cassab,G。和Ponce,G。(2003)。 根冠和根际。 J植物生长调节 21:352-367。
  3. Miller,A.J。和Smith,S.J。(1996)。 谷类根细胞中的硝酸盐转运和分隔。实验植物学杂志,Vol。 47,No. 300,pp。843-854。
  4. Pizzio,GA,Paez-Valencia,J.,Khadilkar,AS,Regmi,K.,Patron-Soberano,A.,Zhang,S.,Sanchez-Lares,J.,Furstenau,T.,Li,J.,Sanchez -Gomez,C.,Valencia-Mayoral,P.,Yadav,UP,Ayre,BG和Gaxiola,RA(2015)。 拟南芥 I型质子泵浦焦磷酸酶在韧皮部中强烈表达,其中是焦磷酸盐代谢和光合分配所必需的。植物生理学167(4):1541-1553。
  5. Shen,J.,Li,C.,Mi,G.,Li,L.,Yuan,L.,Jiang,R。和Zhang,F。 最大限度地提高根/根际效率,以提高中国集约化农业的作物生产力和养分利用效率。 a> J Exp Bot 64(5):1181-1192。
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Copyright: © 2015 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. Pizzio, G. A., Regmi, K. and Gaxiola, R. (2015). Rhizosphere Acidification Assay. Bio-protocol 5(23): e1676. DOI: 10.21769/BioProtoc.1676.
  2. Pizzio, G. A., Paez-Valencia, J., Khadilkar, A. S., Regmi, K., Patron-Soberano, A., Zhang, S., Sanchez-Lares, J., Furstenau, T., Li, J., Sanchez-Gomez, C., Valencia-Mayoral, P., Yadav, U. P., Ayre, B. G. and Gaxiola, R. A. (2015). Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning. Plant Physiol 167(4): 1541-1553.

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