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Extraction of Apoplastic Wash Fluids and Leaf Petiole Exudates from Leaves of Arabidopsis thaliana

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The Plant Journal
Feb 2015



The long-distance translocation of metabolites and mineral elements is crucial for plant growth and reproduction. In most cases, source-to-sink translocation of metabolites and minerals requires their passage through the apoplast, irrespective whether they are transported via the xylem or the phloem. This apoplast-mediated pathway is of particular importance during plant senescence, when photoassimilates as well as organic, inorganic or chelated forms of nutrients are translocated from leaves to fruits or seeds. Recent genetic and physiological studies revealed the involvement of numerous membrane transporters mediating phloem loading of amino acids, sugars, urea or mineral elements. To evaluate the contribution of individual transporters to xylem unloading or phloem loading, the collection of apoplastic fluids and of phloem sap is essential. Here, we describe a method for the extraction of apoplastic fluids and the collection of leaf petiole exudates from Arabidopsis leaves, the latter representing an approximation to the real composition of the phloem sap.

Materials and Reagents

  1. Paper towel
  2. 50 ml Falcon tube (Tubes, 50 ml, PP) (Greiner Bio-One GmbH, catalog number: 227 261 )
  3. 1.5 ml tube (Safe-Lock tubes 1.5 ml) (Eppendorf, catalog number: 0030 120.086 )
  4. For extraction of apoplastic fluids
    1. Arabidopsis leaves (4 g fresh weight for one repetition)
      Note: Healthy leaves should be used, preferentially from hydroponic or soil culture.
    2. Autoclaved distilled water
    3. Ice
  5. For extraction of petiole exudates
    1. Arabidopsis leaves (2-3 leaves for one repetition)
    2. Ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15576-028 )
    3. Potassium hydroxide (KOH) (Carl Roth GmbH + Co., catalog number: P747 )
    4. 10 mM EDTA solution (see Recipes)


  1. Glass beaker, 600 ml (Kavalierglass of North America, Simax, catalog number: 1632417010600 )
  2. Vacuum desiccator
  3. Vacuum pump with air gauge (KNF Neuberger GmbH, model: N022AT.18 )
  4. Centrifuge for 50 ml tubes (Centrifuge 5810R) Eppendorf, catalog number: 5810 000.017 )
  5. Growth chamber for plant cultivation (Percival Scientific, model: CU-41L4X )


  1. Extraction of apoplastic wash fluids
    1. At least 4 g of fresh leaves, corresponding to approx. 60-70 leaves with an average blade length of 3-4 cm, are collected from intact Arabidopsis plants. Notably, these leaves should be intact. While very young leaves have smaller intercellular space yielding less apoplastic fluid, old leaves may contaminate the apoplastic fluid with cytosolic constituents whenever membrane integrity decreases; thus fully expanded leaves are ideal. Leaves are divided into 3 technical replicates of approx. 1.3 g each and transferred to 50 ml Falcon tubes before weighing and recording fresh biomass per sample. Each sample is then washed with approx. 40 ml ice-cold autoclaved distilled water for two or three times (Figure 1A).
    2. Wet leaves of one sample are incubated in an open Falcon tube filled with 40 ml of ice-cold distilled water and placed into a vacuum desiccator. The intercellular space of the leaves is then infiltrated with water by applying a vacuum of -80 hPa five times for 2 min each time using a vacuum pump (Figure 1B). To facilitate the removal of air bubbles from the leaves, tubes are gently shaken after the vacuum has been released. In the desiccator, tubes are kept on ice to decrease the risk of intracellular components leaching to the apoplastic fluid e.g., whenever the plasma membrane of leaf cells is damaged or may have become leaky.
    3. All water is removed from the tube, and the leaf tissues are wiped dry with paper towels (Figure 1C). Successfully infiltrated parts of the leaves usually become transparent (Figure 1D). If large parts of the leaf areas are found not to be infiltrated, vacuum infiltration is performed again to fill also the remaining intercellular space with water. Finally, all water droplets on the surface of the leaves are carefully removed.
    4. The leaves are transferred to a fresh 50 ml Falcon tube, and the tube is centrifuged at 100 x g and 4 °C for 20 min. To better separate the leaves from the apoplastic wash fluid, leaves can be rolled in saran wrap in a way that the roll is opened towards the bottom, allowing the apoplastic fluid to drain out during centrifugation (Figure 1E-F).
    5. Leaves are removed from the tube, and the collected apoplastic wash fluid is transferred to a fresh and preweighed 1.5 ml tube. The volume of the fluid is then measured by weighing. In general, 10-50 μl of apoplastic fluid can be collected by centrifugation.
    6. To test for possible intracellular contaminants in the apoplastic wash fluid, the activity of cellular marker enzymes like malate dehydrogenase or hexosephosphate isomerase can be measured within the fluid (Lohaus et al., 2001).
    7. The collected apoplastic wash fluid is then ready for the analysis of mineral elements by ICP-MS (Eggert and von Wirén, 2013) or amino acids and other compounds (Bohner et al., 2015). The apoplastic fluid can be stored at -20 °C for several weeks, depending on the stability of target compounds.

      Figure 1. Extraction of leaf apoplastic wash fluid. A. Collection of fresh leaf material. B. Incubation of immersed leaf samples under vacuum pressure. C. Leaves are wiped dry to remove water and droplets from the leaf surface. D. Successfully infiltrated leaves become transparent. Left image shows leaves after infiltration, while in the right image the infiltrated leaf tissue is marked in red. E. Leaves after being wrapped in Saran. The Saran wrap is not closed at the bottom to allow apoplastic fluid passing through. F. Placement of Saran-wrapped leaves in a centrifuge tube.

  2. Collection of leaf petiole exudates
    1. Intact leaves are detached from plants close to their base (Figure 2A) and weighed for fresh weight determination. Plants may be cultivated on soil or in hydroponics under controlled conditions. However, any growth condition is fine as long as healthy, intact and physiologically active leaves are given. These leaves are immersed in EDTA solution, and the base of their petioles is cut again to avoid embolism (Figure 2B). Droplets on the leaf surface are removed by paper towel.
    2. Petioles of 2-3 leaves are rapidly transferred to a 2.0 ml tube filled with 1.8 ml EDTA solution. If the petioles are short, the volume of the EDTA solution can be increased to 1.9 ml (Figure 2C). EDTA is used as exudate collection medium since it chelates calcium and thereby prevents re-sealing of sieve tubes (King and Zeevaart, 1974). Since the chelating activity of EDTA ceases at acidic conditions, the pH of the EDTA solution is adjusted to 8.5 with 5 M KOH.
    3. These leaves are kept for 6 h in a glass container in a growth chamber e.g., at 22°C and 80-90% humidity and a light intensity of 200-280 μmol m-2 s-1 to ensure ongoing leaf photosynthesis. High humidity is required to suppress leaf transpiration and uptake of EDTA solution through the leaf petiole. In general, the rate of exudation from leaf petioles is maintained for a period of 1-4 h and reaches a plateau after 6 h (King and Zeevaart, 1974).
    4. The leaves are removed, and the leaf petiole exudates are eventually concentrated using a vacuum concentrator or speed vac at 4 °C before re-suspension in a smaller volume of water, methanol or any other type of solvent depending on the subsequent analysis. For any type of metabolite analysis exudates should be stored at ≤ -20 °C. After the collection of leaf exudates, the leaves are dried in the oven at 55 °C until constant weight, and the dry weight of the leaf tissue can be determined by weighing. Leaf petiole exudation can be related either to fresh or dry weight basis.

      Figure 2. Collection of leaf petiole exudates. A. Leaves are detached from plants at the base of the leaf petiole. B. The leaf petiole is cut again when the petiole is immersed in 10 mM EDTA. C. Petioles of 2-3 leaves are immersed to 10 mM EDTA in a 2 ml tube and kept under light condition for 6 h.


  1. 10 mM EDTA solution
    Dissolve 3.72 g of EDTA in 800 ml of distilled water
    Adjust pH to 8.5 with 5 M KOH
    Add distilled water to 1,000 ml


This protocol was adapted from previously published studies by King and Zeevaart (1974), Corbesier et al. (2001), Lohaus et al. (2001), Kojima et al. (2007) and Carvalhais et al. (2011), and was performed by Bohner et al. (2015). This work was financially supported by Deutsche Forschungsgemeinschaft DFG Research Unit FOR948 (W1728/14). We also thank Lisa Unrath for excellent technical assistance.


  1. Bohner, A., Kojima, S., Hajirezaei, M., Melzer, M. and von Wirén, N. (2015). Urea retranslocation from senescing Arabidopsis leaves is promoted by DUR3-mediated urea retrieval from leaf apoplast. Plant J 81: 377-387.
  2. Carvalhais, L. C., Dennis, P. G., Fedoseyenko, D., Hajirezaei, M. R., Borriss, R. and von Wirén, N. (2013). Root exudation of sugars, amino acids, and organic acids by maize as affected by nitrogen, phosphorus, potassium, and iron deficiency. J Plant Nutr Soil Sci 176: 3-11.
  3. Corbesier, L., Havelange, A., Lejeune, P., Bernier, G. and Périlleux, C. (2001). N content of phloem and xylem exudates during the transition to flowering in Sinapis alba and Arabidopsis thaliana. Plant Cell Environ 24: 367-375.
  4. King, R. W. and Zeevaart, J. A. (1974). Enhancement of phloem exudation from cut petioles by chelating agents. Plant Physiol 53: 96-103.
  5. Kojima, S., Bohner, A., Gassert, B., Yuan, L. and von Wirén, N. (2007). AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots. Plant J 52: 30-40.
  6. Lohaus, G., Pennewiss, K., Sattelmacher, B., Hussmann, M. and Muehling, K.H. (2001). Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species. Physiol Plant 111(4): 457-465.




  1. 毛巾
  2. 将50ml falcon管(Tubes,50ml,PP)(Greiner Bio-One GmbH,目录号:227 261)
  3. 1.5ml管(Safe-Lock管1.5ml)(Eppendorf,目录号:0030120.086)
  4. 用于萃取非塑性流体
    1. 拟南芥叶(一次重复4g鲜重)
    2. 高压蒸馏水
  5. 用于提取叶柄渗出液
    1. 拟南芥叶(2-3个叶重复一次)
    2. 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,Invitrogen TM,目录号:15576-028)
    3. 氢氧化钾(KOH)(Carl Roth GmbH + Co.,目录号:P747)
    4. 10 mM EDTA溶液(见配方)


  1. 玻璃烧杯,600ml(Kavalierglass of North America,Simax,目录号:1632417010600)
  2. 真空干燥器
  3. 带气量计的真空泵(KNF Neuberger GmbH,型号:N022AT.18)
  4. 离心机用于50ml管(Centrifuge 5810R)Eppendorf,目录号:5810 000.017)
  5. 用于植物栽培的生长室(Percival Scientific,型号:CU-41L4X)


  1. 外质洗涤液的提取
    1. 至少4g的新鲜叶,对应于约。 60-70叶 ?平均叶片长度为3-4cm,从完整的拟南芥植物中收集。值得注意的是,这些叶片应该是完整的。虽然很 幼叶具有较小的细胞间隙,产生较少的外体 流体,老叶可能污染具有胞浆的质外体液 当膜完整性降低时,从而完全展开 叶是理想的。叶被分为3个技术重复 约。每个1.3g并转移到50ml falcon管中,然后称重 ?并记录每个样品的新鲜生物量。然后用每个样品洗涤 ?约。 40毫升冰冷的高压灭菌蒸馏水2或3 次(图1A)。
    2. 将一个样品的湿叶在37℃下孵育 打开的falcon管充满40毫升冰冷的蒸馏水和 置于真空干燥器中。叶的细胞间空间 然后通过施加-80hPa的真空使其渗透水 每次2分钟,使用真空泵(图1B)。方便 ?从叶子中除去气泡,轻轻摇动试管 在真空释放后。在干燥器中,保持管 ?冰降低细胞内组分浸出的风险 只要叶细胞的质膜是,就可以使用非质外体液 损坏或可能已泄漏
    3. 所有的水从中除去 管,并用纸巾擦干叶组织(图1C)。 成功渗透的叶子的部分通常变得透明 (图1D)。如果发现叶面积的大部分不是 渗透,再次进行真空渗透以填充 保留与水的细胞间隙。最后,所有的水滴 ?小心地除去叶子的表面
    4. 叶子是 转移到新鲜的50ml falcon管中,并将管离心 ?100×g /孔和4℃20分钟。为了更好地分离叶子 外部清洗流体,叶子可以卷在saran包裹在一种方式 辊朝向底部打开,允许非塑性流体到达 离心期间排出(图1E-F)
    5. 叶被去除 ?,并且收集的外质性洗涤液被转移 到新鲜和预称重的1.5ml管。然后流体的体积 通过称重测量。一般来说,10-50微升的质外体液可以 通过离心收集
    6. 测试可能的细胞内 外质洗涤液中的污染物,细胞的活性 标记酶如苹果酸脱氢酶或己糖磷酸异构酶 可以在流体内测量(Lohaus et al。,,2001)
    7. 的 收集的外质洗涤液然后准备用于分析 矿物元素(ICPert)(Eggert和vonWirén,2013)或氨基酸 和其他化合物(Bohner等人,2015)。外质液可以是 储存在-20°C几个星期,取决于目标的稳定性 ?化合物

      图1.叶外质性洗涤液的提取。 新鲜的叶子材料的汇集。 B.浸没叶的孵育 样品在真空压力下。 C.擦干叶子以除去水 和来自叶表面的液滴。 D.成功渗透叶 变得透明。左图显示浸润后的叶子,而in ?右侧图像中,浸润的叶组织标记为红色。 E.叶子 ?后被包裹在萨兰。 Saran包没有关闭 底部以允许质外体流体通过。 F.放置 Saran包裹的叶子在离心管。

  2. 叶柄渗出物的收集
    1. 完整叶片从接近其基部的植物分离(图2A) 并称重以测定鲜重。可以栽培植物 土壤或在受控条件下的水培。但是,任何增长 条件是良好的,只要健康,完整和生理活性 叶子。将这些叶子浸入EDTA溶液中, 再次切除其叶柄的基部以避免栓塞(图2B)。 用纸巾除去叶表面上的液滴。
    2. 叶柄 ?的2-3片叶快速转移到装有1.8的2.0ml管中 ml EDTA溶液。如果叶柄短,EDTA的体积 溶液可以增加至1.9ml(图2C)。 EDTA用作渗出物 ?收集介质,因为其螯合钙并因此防止 重新密封筛管(King和Zeevaart,1974)。由于螯合 ?EDTA的活性在酸性条件下停止,EDTA的pH 溶液用5M KOH调节至8.5
    3. 这些叶子保留 在生长室中的玻璃容器中例如6小时。在22℃和80-90% ?湿度和200-280μmolm -2 s -1的光强度 -1 以确保 正在进行的叶片光合作用。需要高湿度来抑制叶 蒸腾和EDTA溶液通过叶柄的吸收。在 一般来说,叶柄的渗出速率保持为a 周期1-4 h,6 h后达到平台(King和Zeevaart, 1974)。
    4. 除去叶子,叶柄叶分泌物 最后使用真空浓缩器或4℃的真空速度浓缩 ?然后再悬浮于较小体积的水,甲醇或任何其中 其他类型的溶剂,取决于随后的分析。任何类型 ?的代谢物分析渗出物应储存在≤-20°C。之后 ?收集叶子渗出物,将叶子在55℃的烘箱中干燥 直到恒重,并且可以确定叶组织的干重 通过称重确定。叶柄叶片渗出可能与之相关 新鲜或干重

      图2.叶柄的收集 A.叶片与叶片底部的植物分离 叶柄。 B.当叶柄浸入时,再次切叶叶柄 在10mM EDTA中。将2-3片叶的叶柄浸入10mM EDTA中 ?2ml管中,并在光照条件下保持6小时。


  1. 10mM EDTA溶液
    将3.72g EDTA溶于800ml蒸馏水中
    用5 M KOH将pH调节至8.5 加蒸馏水至1000 ml


该方案改编自King和Zeevaart(1974),Corbesier等人(2001),Lohaus等人(2001),Kojima等人的先前发表的研究。 et al。(2007)和Carvalhais等人(2011),并且由Bohner等人(2015)进行。这项工作得到了德国Forschungsgemeinschaft DFG研究单位FOR948(W1728/14)的资助。我们还感谢Lisa Unrath的优秀技术援助。


  1. Bohner,A.,Kojima,S.,Hajirezaei,M.,Melzer,M。和vonWirén,N.(2015)。 通过DUR3介导的尿素回收促进拟南芥叶片中的尿素再转化from leaf apoplast。 Plant J 81:377-387。
  2. Carvalhais,L.C.,Dennis,P.G.,Fedoseyenko,D.,Hajirezaei,M.R.,Borriss,R.and vonWirén,N.(2013)。 受氮,磷影响的玉米的糖,氨基酸和有机酸的根系渗出,钾和铁缺乏。 176植物营养土壤科学176:3-11。
  3. Corbesier,L.,Havelange,A.,Lejeune,P.,Bernier,G。和Périlleux,C。(2001)。 韧皮部和木质部的N含量在向开花过渡期间渗出 Sinapis alba 和拟南芥 植物细胞环境 24:367-375。
  4. King,R.W。和Zeevaart,J.A。(1974)。 通过螯合剂增强切根叶片的韧皮部渗出。植物生理 53:96-103。
  5. Kojima,S.,Bohner,A.,Gassert,B.,Yuan,L.and vonWirén,N。(2007)。 AtDUR3代表高亲和力尿素转运穿过氮缺乏质膜的主要转运蛋白> Arabidopsis 根。植物J 52:30-40
  6. Lohaus,G.,Pennewiss,K.,Sattelmacher,B.,Hussmann,M。和Muehling,K.H。 (2001)。 渗透离心技术是否适用于分离外质液?对不同植物物种的临界评价。 生理植物 111(4):457-465。
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引用:Araya, T., Bohner, A. and Wirén, N. v. (2015). Extraction of Apoplastic Wash Fluids and Leaf Petiole Exudates from Leaves of Arabidopsis thaliana. Bio-protocol 5(24): e1691. DOI: 10.21769/BioProtoc.1691.