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Measurement of Net High-affinity Urea Uptake in Maize Plants
玉米植株高亲和力转运系统吸收尿素的测定方法   

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Abstract

Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only in a few plant species highlighting the importance of a urea transporter in roots, DUR3 (Kojima et al., 2007; Wang et al., 2012; Zanin et al., 2014a). Regarding maize plants, a crop that needs a large amount of urea fertilizer, the capability to take up urea via an inducible and high-affinity transport system has been recently characterized (Zanin et al., 2014a; Zanin et al., 2014b). Here, we described a small-scale protocol suitable for the measurement of urea net high-affinity uptake in roots of intact maize plants.

Keywords: Transport(运输), Nitrogen(氮), Root(根), Corn(玉米), Method(方法)

Materials and Reagents

  1. Maize seeds (Zea mays L., cv. PR33T56, Pioneer Hi-bred Italia S.p.A., Parma, Italy)
  2. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60370 )
  3. Calcium sulphate (CaSO4) (Sigma-Aldrich, catalog number: 12090 )
  4. Urea [CO(NH2)2] (Sigma-Aldrich, catalog number: 15604 )
  5. Diacetylmonoxime [CH3C(=NOH)COCH3] (Sigma-Aldrich, catalog number: 31550 )
  6. Thiosemicarbazide (NH2CSNHNH2) (Sigma-Aldrich, catalog number: 89050 )
  7. Sulphuric acid (H2SO4) (Sigma-Aldrich, catalog number: 320501 )
  8. Ortho-phosphoric acid (H3PO4) (Sigma-Aldrich, catalog number: W290017 )
  9. Ferric chloride hexahydrate (FeCl3) (Sigma-Aldrich, catalog number: 12319 )
  10. Sterile deionized water
  11. KCl (Sigma-Aldrich, catalog number: P9541 )
  12. CaSO4 (Sigma-Aldrich, catalog number: 12090)
  13. MgSO4 (Sigma-Aldrich, catalog number: 746452 )
  14. KH2PO4 (Sigma-Aldrich, catalog number: P9791 )
  15. NaFe-EDTA (Sigma-Aldrich, catalog number: 03650 )
  16. H3BO3 (Sigma-Aldrich, catalog number: B7901 )
  17. MnSO4 (Sigma-Aldrich, catalog number: 221287 )
  18. ZnSO4 (Sigma-Aldrich, catalog number: Z1001 )
  19. CuSO4 (Sigma-Aldrich, catalog number: C3036 )
  20. Na2MoO4 (Sigma-Aldrich, catalog number: M1651 )
  21. Nutrient solution (see Recipes)
  22. Urea solution stock (see Recipes)
  23. Mixed colour reagent (see Recipes)
  24. Mixed acid reagent (see Recipes)
  25. Colour development reagent (see Recipes)

Equipment

  1. Growth chamber and hydroponic growing system (including plastic net, plastic pots)
  2. pH meter (Jenway, model: 3510 )
  3. Plastic box (15 x 10 cm; H 4 cm; Figure 1A, alternatively you can use the bottom of the pipette tips, Sigma-Aldrich, catalog number: P5161 )
  4. 0.2 ml 96-well plate (AB ANALITICA Advanced Biomedicine, catalog number: B50601 ; Figure 1B)
  5. Clear 96-well microplate with flat bottoms (STARLAB, catalog number: S1837-9600 ; Figure 1C)
  6. Sealing tapes, optically clear (SARSTEDT AG, catalog number: 95.1994 )
  7. Thermocycler (Eppendorf, model: Mastercycler® personal )
  8. Orbital shaker (Janke & Kunkel IKA-Labortechnik, model: KS 501D )
  9. Spectrophotometric multiwell plate reader (TECAN, model: GENios Microplate Reader )
  10. Timer
  11. Pipettes (Eppendorf, model: 0.5-10 μl, 20-200 μl, 100-1,000 μl, Eppendorf Reference® 2 ) and tips
  12. 1.5 ml plastic tubes (Eppendorf, catalog number: 0030 125.150 )
  13. Absorbent paper (Sigma-Aldrich, catalog number: Z270849 )


    Figure 1. Plastic equipment. A. plastic box; B. 0.2 ml 96-well plate; C. Clear 96-well microplate.

Procedure

  1. Maize growth conditions
    1. Germinate maize seeds on a plastic net placed at the surface of an aerated 0.5 mM CaSO4 solution in a growth chamber at 25 °C in the dark.
    2. After 3 days, transfer the seedlings into an aerated hydroponic system containing 0.5 mM CaSO4 under controlled climatic conditions: day/night photoperiod, 16/8 h; light intensity, 220 µmol m-2s-1; temperature (day/night) 25/20 °C; relative humidity 70 to 80%.
    3. After 2 days (5-day-old) plants were transferred for 4 hours in a N-free nutrient solution containing (µM): KCl 5; CaSO4 500; MgSO4 100; KH2PO4 175; NaFe-EDTA 20; H3BO3 2.5; MnSO4 0.2; ZnSO4 0.2; CuSO4 0.05; Na2MoO4 0.05. N is supplied in form of 1 mM CO(NH2)2 (urea-treated plants); or as control, plants are exposed to a N-free nutrient solution (control-plants). The pH of solution is adjusted to pH 6.0 with potassium hydroxide (KOH).

  2. Root uptake of urea and collection of samples
    1. Gently remove intact plants (six urea-treated plants and six control plants) from hydroponic system and rinse plant roots for 10 sec in 500 ml of calcium sulphate solution (500 µM CaSO4), repeat this step twice (Figure 2A).
    2. On an absorbent paper, absorb the excess of calcium sulphate solution from maize plants taking care to not damage roots (Figure 2B).
    3. For each measurement, fill three plastic boxes with 40 ml of urea solution (8 ml of 1 mM urea solution stock and 32 ml of 500 µM calcium sulphate, final urea concentration 200 µM) and place the boxes on the orbital shaker (speed 90 rpm).
    4. Set the timer to count up 10 min.
    5. In each plastic box, place two intact plants submerging the roots in the urea solution (avoid to submerge the seed of the plants). Starting the timer (T = 0 min), collect 60 µl of the urea solution in a 96-well plate (Figure 2C-E).
    6. Net uptake is measured as urea depletion from the solution per unit of time, removing samples of solution (60 µl) for urea determination every 2 min for 10 min, span time during which uptake had a linear trend. Thus, continue to collect the urea solution from each plastic box every two minutes from the start point, at T = 2, 4, 6, 8 and 10 min.
    7. At the end of the harvesting time, turn off the orbital shaker. In each plastic box, cut and dry roots on absorbent paper; then weigh the maize roots of two plants (Figure 2F).


      Figure 2. Procedures to collect the samples. A. Rinse the maize roots; B. dry maize roots on absorbent paper; C. Prepare plants on an orbital shaker; D. Two plants of maize are transferred in each plastic box, with roots submerged in the urea solution; E. during the experimental time span of 10 min, collect 60 µl of urea solution every two minutes; F. at the end of the experiment, cut and weight the roots.

  3. Urea standards
    1. In 1.5 ml plastic tubes, dilute 1 mM urea solution stock as indicated in Table 1 to create the standard curve.

      Table 1. Urea standards
      Urea solution stock
      (1 mM)
      Calcium sulphate
      (500 µM)
      Final urea concentration
      0 µl
      1000 µl
      0 µM
      100 µl
      900 µl
      100 µM
      120 µl
      880 µl
      120 µM
      140 µl
      860 µl
      140 µM
      160 µl
      840 µl
      160 µM
      180 µl
      820 µl
      180 µM
      200 µl
      800 µl
      200 µM
      250 µl
      750 µl
      250 µM

    2. Transfer 60 µl of the standards into separate wells of the 96-well plate.

  4. Urea determination by assay colorimetric reaction
    1. Prepare fresh the colour development reagent by mixing 25 ml of mixed acid reagent with 25 ml of mixed colour reagent.
    2. Add 120 µl of colour development reagent to each well of 96-well plate containing the samples or the standards.
    3. Seal the plate with a sealing tape.
    4. Incubate for 15 min at 99 °C (lid temperature: 105 °C) in a thermocycler.
    5. Cool the samples for 5 min on ice.
    6. Remove the sealing tape from the plate and transfer 160 µl of all samples in a clear 96-well microplate with flat bottoms.
      Note: To avoid to loss material, de-pressurize the wells with a needle before removing the sealing tape from the plate.
    7. Measure the absorbance at 540 nm using a microtiter plate reader (Figure 3).


      Figure 3. An example of output data from a microtiter plate reader at 540 nm. In blue and red are highlighted standards and samples, respectively.

    8. The capacity of maize roots to take up urea was determined measuring urea depletion from the solution during the time span of 10 minutes. Net-uptake rates of urea were expressed as µmol urea/g root fresh weight (FW)/h. An example of calculation is provided in Supplemental file 1.

Notes

After the harvesting, it is possible to freeze the samples at -20 °C and in the following day proceed the samples.
The urea was determined by diacetylmonoxime and thiosemicarbazide colorimetric assay modified from Killingsbaeck (1975) and Mérigout et al. (2008). In order to analyze a great numbers of samples, the colorimetric reaction was performed using 96-well microplates and the volumes of reagents were optimized. Kojima et al. (2007) used the same colorimetric reaction to determined urea accumulation in Arabidopsis tissues. The authors pointed out that the ureides allantoin, ornithine, arginine, and uric acid, did not interfere with urea determinations, although other ureides were not tested.

Recipes

  1. Nutrient solution
    KCl 5 µM
    CaSO4 500 µM
    MgSO4 100 µM
    KH2PO4 175 µM
    NaFe-EDTA 20 µM
    H3BO3 2.5 µM
    MnSO4 0.2 µM
    ZnSO4 0.2 µM
    CuSO4 0.05 µM
    Na2MoO4 0.05 µM
  2. Urea solution stock
    1 mM urea
    500 µM calcium sulphate
  3. Mixed colour reagent
    7% (v/v) 0.2 M diacetylmonoxime
    7% (v/v) 0.05 M thiosemicarbazide
  4. Mixed acid reagent
    20% (v/v) sulphuric acid
    9% (v/v) ortho-phosphoric acid
    0.06% (v/v) 74 mM ferric chloride hexahydrate
  5. Colour development reagent
    50% (v/v) mixed colour reagent
    50% (v/v) mixed acid reagent

Acknowledgments

The procedures for the root-uptake measurements were adapted from previous studies on nitrate uptake (Rizzardo et al., 2012). The work was supported by a grant from the Italian autonomous region of Friuli Venezia Giulia and the Italian Ministry of University and Research.

References

  1. Kojima, S., Bohner, A., Gassert, B., Yuan, L. and von Wiren, N. (2007). AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots. Plant J 52(1): 30-40.
  2. Merigout, P., Lelandais, M., Bitton, F., Renou, J. P., Briand, X., Meyer, C. and Daniel-Vedele, F. (2008). Physiological and transcriptomic aspects of urea uptake and assimilation in Arabidopsis plants. Plant Physiol 147(3): 1225-1238.
  3. Rizzardo, C., Tomasi, N., Monte, R., Varanini, Z., Nocito, F. F., Cesco, S. and Pinton, R. (2012). Cadmium inhibits the induction of high-affinity nitrate uptake in maize (Zea mays L.) roots. Planta 236(6): 1701-1712.
  4. Wang, W. H., Kohler, B., Cao, F. Q., Liu, G. W., Gong, Y. Y., Sheng, S., Song, Q. C., Cheng, X. Y., Garnett, T., Okamoto, M., Qin, R., Mueller-Roeber, B., Tester, M. and Liu, L. H. (2012). Rice DUR3 mediates high-affinity urea transport and plays an effective role in improvement of urea acquisition and utilization when expressed in Arabidopsis. New Phytol 193(2): 432-444.
  5. Zanin, L., Tomasi, N., Wirdnam, C., Meier, S., Komarova, N. Y., Mimmo, T., Cesco, S., Rentsch, D. and Pinton, R. (2014a). Isolation and functional characterization of a high affinity urea transporter from roots of Zea mays. BMC Plant Biol 14: 222.
  6. Zanin, L., Zamboni, A., Monte, R., Tomasi, N., Varanini, Z., Cesco, S. and Pinton, R. (2015). Transcriptomic analysis highlights reciprocal interactions of urea and nitrate for nitrogen acquisition by maize roots. Plant Cell Physiol 56(3): 532-548.

简介

尽管其作为氮肥的广泛使用,但是作为直接可及的作物植物氮源的脲的作用仍然知之甚少。 迄今为止,仅在少数植物物种中研究了尿素获取的生理学和分子学方面,突出了根中的尿素转运蛋白DUR3的重要性(Kojima等人,2007; et al。,2012; Zanin et al。,2014a)。 关于玉米植物,需要大量尿素肥料的作物,通过诱导型和高亲和力转运系统摄取尿素的能力最近已被表征(Zanin等人,2014a; Zanin et al。,2014b)。 在这里,我们描述了一个小规模的协议,适合测量尿素净高亲和力摄取完好的玉米植物的根。

关键字:运输, 氮, 根, 玉米, 方法

材料和试剂

  1. 玉米种子( Zea mays L.,cv.PR33T56,Pioneer Hi-bred Italia S.p.A.,Parma,Italy)
  2. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:60370)
  3. 硫酸钙(CaSO 4)(Sigma-Aldrich,目录号:12090)
  4. 脲[CO(NH 2)2](Sigma-Aldrich,目录号:15604)
  5. 二乙酰基肟[CH 3 C(= NOH)COCH 3](Sigma-Aldrich,目录号:31550)
  6. 氨基硫脲(NH 2 2 CSNHNH 2)(Sigma-Aldrich,目录号:89050)
  7. 硫酸(H 2 SO 4)(Sigma-Aldrich,目录号:320501)
  8. 正磷酸(H 3 PO 4)(Sigma-Aldrich,目录号:W290017)
  9. 氯化铁六水合物(FeCl 3)(Sigma-Aldrich,目录号:12319)
  10. 无菌去离子水
  11. KCl(Sigma-Aldrich,目录号:P9541)
  12. CaSO 4(Sigma-Aldrich,目录号:12090)
  13. MgSO 4(Sigma-Aldrich,目录号:746452)
  14. KH sub 2 PO 4(Sigma-Aldrich,目录号:P9791)
  15. NaFe-EDTA(Sigma-Aldrich,目录号:03650)
  16. H sub 3 BO 3(Sigma-Aldrich,目录号:B7901)
  17. MnSO 4(Sigma-Aldrich,目录号:221287)
  18. ZnSO 4(Sigma-Aldrich,目录号:Z1001)
  19. CuSO 4(Sigma-Aldrich,目录号:C3036)
  20. Na 2 MoO 4(Sigma-Aldrich,目录号:M1651)
  21. 营养液(见配方)
  22. 尿素溶液(见配方)
  23. 混合颜色试剂(见配方)
  24. 混合酸试剂(见配方)
  25. 显色试剂(见配方)

设备

  1. 生长室和水培生长系统(包括塑料网,塑料盆)
  2. pH计(Jenway,型号:3510)
  3. 塑料盒(15 x 10 cm; H 4 cm;图1A,或者您可以使用移液管吸头的底部,Sigma-Aldrich,目录号:P5161)
  4. 0.2ml 96孔板(AB ANALITICA Advanced Biomedicine,目录号:B50601;图1B)
  5. 澄清96孔平底微孔板(STARLAB,目录号:S1837-9600;图1C)
  6. 密封胶带,光学透明(SARSTEDT AG,目录号:95.1994)
  7. 热循环仪(Eppendorf,型号:Mastercycler 个人)
  8. 轨道振动器(Janke& Kunkel IKA-Labortechnik,型号:KS 501D)
  9. 分光光度多孔板读数器(TECAN,型号:GENios Microplate Reader)
  10. 计时器
  11. 移液管(Eppendorf,型号:0.5-10μl,20-200μl,100-1,000μl,Eppendorf Reference 2)和提示
  12. 1.5ml塑料管(Eppendorf,目录号:0030125.150)
  13. 吸收纸(Sigma-Aldrich,目录号:Z270849)


    图1.塑料设备。 A.塑料盒; B. 0.2ml 96孔板; C.清除96孔微孔板

程序

  1. 玉米生长条件
    1. 发芽在塑料网的玉米种子被安置在表面的 充气的0.5mM CaSO 4溶液在生长室中在25℃在黑暗中。
    2. 3天后,将幼苗转移到充气的水培中 系统在受控气候条件下含有0.5mM CaSO 4: 日/夜光周期,16/8h;光强度,220μmolm -2 s -1 s -1。 温度(日/夜)25/20°C;相对湿度70〜80%。
    3. 2天后(5日龄)植物在无N的条件下转移4小时  营养液含有(μM):KCl 5; CaSO 4 500; MgSO 4 4 100; KH 2 PO 4 4 175; NaFe-EDTA 20; H sub 3 BO 3 3 2.5; MnSO 4 <0.2; ZnSO 4 <0.2; CuSO 4 <0.05; Na 2 MoO 4 4。 N以1mM CO(NH 2)2(脲处理的植物)的形式提供;要么 作为对照,将植物暴露于无N营养液 (对照植物)。用水将溶液的pH调节至6.0 氢氧化钾(KOH)。

  2. 根部吸收尿素和收集样品
    1. 轻轻地删除完好的植物(六个尿素处理的植物和六个控制 植物)和水培系统,冲洗植物根在500秒10秒 ml硫酸钙溶液(500μMCaSO 4),重复该步骤两次 (图2A)。
    2. 在吸收纸上,吸收过量的钙 硫酸盐溶液从玉米植物注意不损害根 (图2B)。
    3. 对于每个测量,填充三个塑料盒 将40ml尿素溶液(8ml的1mM尿素溶液储备液和32ml 500μM硫酸钙,最终尿素浓度200μM),并将其放置 (速度90rpm)。
    4. 将计时器设置为向上计数10分钟。
    5. 在每个塑料盒中,放置两个完整的植物淹没根   尿素溶液(避免浸没植物的种子)。 开始 定时器(T = 0min),在96孔中收集60μl尿素溶液 板(图2C-E)
    6. 净摄取量测量为尿素消耗 从溶液每单位时间,移除溶液样品(60μl)   用于尿素测定每2分钟10分钟,跨度时间期间 摄取具有线性趋势。 因此,继续收集尿素溶液 从每个塑料盒每两分钟从起点,在T = 2, 4,6,8和10分钟。
    7. 在收获时间结束时,关闭 轨道振动器。 在每个塑料盒中,切根和干根吸收 纸; 然后称重两株植物的玉米根(图2F)

      图 2.收集样品的程序。 A.冲洗玉米根; B.干燥 玉米根在吸收纸上; C.在轨道摇床上准备植物; D.两个玉米植物在每个具有根的塑料盒中转移 浸没在尿素溶液中; E.在实验时间跨度 10 min,每两分钟收集60μl尿素溶液; F.结束 的实验,切割和重量的根。

  3. 尿素标准
    1. 在1.5ml塑料管中,稀释1mM尿素溶液原液,如表1所示,以产生标准曲线
      表1.尿素标准
      尿素溶液储液
      (1 mM)
      硫酸钙
      (500μM)
      最终尿素浓度
      0微升
      1000微升
      0μM
      100微升
      900μl
      100μM
      120微升
      880μl
      120μM
      140微升
      860微升
      140μM
      160微升
      840微升
      160μM
      180微升
      820微升
      180μM
      200μl
      800μl
      200μM
      250微升
      750微升
      250μM

    2. 将60μl的标准品转移到96孔板的单独的孔中。

  4. 通过测定比色反应测定尿素
    1. 通过混合25ml混合酸试剂和25ml混合颜色试剂制备新鲜的显色试剂
    2. 向含有样品或标准品的96孔板的每个孔中加入120μl显色试剂
    3. 用密封带密封板。
    4. 在热循环仪中在99℃下孵育15分钟(盖温度:105℃)
    5. 在冰上冷却样品5分钟。
    6. 从板上取下密封胶带,将160μl的所有样品转移到一个带有平底的透明96孔微孔板中。
      注意:为了避免材料损失,在从板上取下密封胶带前,用针头对孔进行减压。
    7. 使用微量滴定板读数器测量540nm处的吸光度(图3)

      图3.来自微量滴定板读数器的输出数据的示例 540 nm。蓝色和红色是突出显示的标准品和样品, 分别
    8. 玉米根吸收尿素的能力 测定尿素从溶液中的消耗 时间跨度为10分钟。尿素的净吸收率表示为μmol  尿素/g根鲜重(FW)/h。提供了计算的示例 在补充文件1 中。

笔记

收获后,可以在-20℃下冷冻样品,并在第二天进行样品。
通过由Killingsbaeck(1975)和Mérigout等人(2008)修改的二乙酰基肟和氨基硫脲比色测定法测定尿素​​。为了分析大量样品,使用96孔微量培养板进行比色反应,并优化试剂的体积。 Kojima等人(2007)使用相同的比色反应来确定拟南芥组织中的尿素累积。作者指出,脲脲尿囊素,鸟氨酸,精氨酸和尿酸,不干扰尿素测定,虽然没有测试其他脲类。

食谱

  1. 营养液
    KCl 5μM
    CaSO 4 <500μM
    MgSO 44μM100μM KH <2> PO 175微米。
    NaFe-EDTA20μM
    H <3> BO <3> 2.5μM
    MnSO 4 <0.2μM ZnSO 4 <0.2μM CuSO 40.05μM Na MoO 40.05μM
  2. 尿素溶液仓
    1 mM尿素
    500μM硫酸钙
  3. 混合试剂
    7%(v/v)0.2M二乙酰基单肟
    7%(v/v)0.05M硫代氨基脲
  4. 混合酸试剂
    20%(v/v)硫酸 9%(v/v)原磷酸 0.06%(v/v)74mM氯化铁六水合物
  5. 显色试剂
    50%(v/v)混色试剂
    50%(v/v)混合酸试剂

致谢

根吸收测量的程序改变自先前关于硝酸盐吸收的研究(Rizzardo等人,2012)。这项工作得到意大利自治区Friuli Venezia Giulia和意大利大学和研究部的资助。

参考文献

  1. Kojima,S.,Bohner,A.,Gassert,B.,Yuan,L。和von Wiren,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(1):30-40。
  2. Merigout,P.,Lelandais,M.,Bitton,F.,Renou,J.P.,Briand,X.,Meyer,C.and Daniel-Vedele,F。(2008)。 拟南芥植物中尿素摄取和同化的生理和转录组学方面。/a> Plant Physiol 147(3):1225-1238。
  3. Rizzardo,C.,Tomasi,N.,Monte,R.,Varanini,Z.,Nocito,F.F.,Cesco,S.and Pinton,R。(2012)。 镉抑制玉米中高亲和力硝酸盐摄入的诱导( Zea mays L.)根。植物 236(6):1701-1712。
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引用:Zanin, L., Tomasi, N. and Pinton, R. (2015). Measurement of Net High-affinity Urea Uptake in Maize Plants. Bio-protocol 5(11): e1490. DOI: 10.21769/BioProtoc.1490.
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