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Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple
通过含有pH敏感指示剂溴甲酚紫的凝胶评估根部pH值的变化   

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PLOS Genetics
Oct 2017

Abstract

The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol purple, treated with the peptide and photographed after 30 min. Herein the protocol is optimized for evaluation of exogenous treatment, described in detail and expected results are presented.

Keywords: pH indicator (pH指示剂), Alkalinization (碱化), RALF (RALF), Bromocresol purple (溴甲酚紫)

Background

Proton transport is induced by a myriad of signals and is used by plants to coordinate growth, defense and development. Some plant hormone peptides can affect proton transport, causing a strong alkalinization of the extracellular medium (Felix and Boller, 1995; Pearce et al., 2001a). The 5 kDa peptide hormone Rapid Alkalinization Factor (RALF), after being secreted, binds to its receptor FERONIA, and causes the phosphorylation of the plasma membrane H+-Adenosine triphosphatase 2, inhibiting proton transport and alkalinizing the extracellular media (Pearce et al., 2001b; Haruta et al., 2014).

Growth media containing the pH indicator bromocresol purple is an effective method to visualize alkalinization or acidification in the media around the roots. This method has been used previously to show that NaRALF is required for regulating root hair extracellular pH (Wu et al., 2007), and that roots of plants overexpressing AtRALF23 have reduced capacity to acidify the rhizosphere (Srivastava et al., 2009). Growth medium with the pH indicator bromocresol purple was also used by Masachis et al. (2016) to demonstrate that RALF homologs produced by fungal pathogens induced alkalinization of media around the roots of tomato plants.

We have optimized the pH indicator bromocresol purple protocol and using the improved protocol we were able to visualize the alkalinization effect around Arabidopsis roots after AtRALF1 treatment in wild type and mutant seedlings (Dressano et al., 2017). Here we demonstrate how this assay can provide qualitative information on the extracellular pH that surrounds Arabidopsis roots.

Materials and Reagents

  1. Biological material
    1. Arabidopsis thaliana seeds and seedlings (Ecotype Columbia, Col-0)
    2. In-house produced 6xHis AtRALF1 recombinant peptide 

  2. Chemicals and materials for seed sterilization and growth
    1. Square Petri dish with Grid 100 mm W x 15 mm H, sterile (Electron Microscopy Sciences, catalog number: 70691 )
    2. Clear plastic wrap
    3. Graduated cylinder 1,000, 100 and 10 ml (Uniglas)
    4. 1,000 µl filter tips (NEST Scientific, catalog number: NPT1000-B-B )
    5. 200 µl filter tips (NEST Scientific, catalog number: NPT0200-B-Y )
    6. 10 µl filter tips (NEST Scientific, catalog number: 301001 )
    7. Gellan Gum Powder (Culture Gel TM Type I- BioTech Grade) (PhytoTechnology Laboratories, catalog number: G434 )
    8. Murashige & Skoog Basal Salt Mixture (PhytoTechnology Laboratories, catalog number: M524 )
    9. Sterile distilled water
    10. Sodium hypochlorite solution
    11. Sodium hypochlorite (NaClO) solution 50% (v/v) (see Recipes)

  3. Chemicals and materials for gel containing the pH-sensitive indicator bromocresol purple
    1. Graduated cylinder 100 ml (Uniglas)
    2. Petri dish 150 x 20 mm sterile
    3. Bromocresol purple free acid reagent Grade (C2H16Br2O5S, AMRESCO, catalog number: 0531-25G )
    4. Calcium sulfate dihydrate (CaSO4·2H2O, Merck, catalog number: 102161 )
    5. Potassium hydroxide (KOH)
    6. Hydrochloric acid (HCl)
    7. Agarose RA (Biotechnology Grade, AMRESCO, catalog number: N605-500G )
    8. Sterile distilled water

Equipment

  1. Weighing balance (RADWAG Wagi Elektroniczne, model: AS 220/C/2 )
  2. Magnetic stirrer (Fisher Scientific)
  3. pH meter (Thermo Orion, PerpHecT LongR meter, model: 350 )
  4. Pipettes (Nichipet EX, P1000, P200, P10)
  5. Beaker 1,000 ml (PHOX, Boro 3.3)
  6. Beaker 250 ml (APRX, Boro 3.3)
  7. Autoclave (Sercon, model: HS 1-0101 )
  8. Laminar flow hood (Pachane, model: PA 440 )
  9. Growth chamber/Controlled environment room
  10. Erlenmeyer (PIREX)
  11. Microwave (BRASTEMP, catalog number: BMJ38ARANA )
  12. Tweezers
  13. Digital camera (Cyber-shot, Sony, model: DSC-H300 )

Procedure

  1. Seed sterilization and germination
    To analyze the root media alkalinization, seedlings were grown on plates containing half-strength MS medium (see Recipes) for 9 days.
    1. Surface sterilize A. thaliana seeds in Eppendorf tubes using 1 ml sodium hypochlorite solution 50% (v/v) for 10 min followed by 5 washes using 1 ml sterile Milli-Q water each. Seeds are washed with the aid of a micropipette (1,000 µl) (Figures 1A).
    2. Stratify the sterilized seeds for 72 h at 4 °C in water and then drop in a line using a micropipette (10 µl) on a square plate dish containing half-strength MS medium (Figures 1B-1D).
    3. Seal the plates containing the seeds with clear plastic wrap.
    4. Incubate the plates vertically in a controlled environmental growth chamber at 22 ± 2 °C, 16 h light (150 μmol m-2 sec-1) and 8 h dark for 9 days (Figure 1E).


      Figure 1. Seed sterilization and seedling growth. A. A. thaliana seeds during the surface sterilization procedure with sodium hypochlorite solution 50% (v/v); B. Pipette tip (which has been cut and then autoclaved) containing stratified seeds; C. Stratified seeds being disposed in half-strength MS medium; D. Seeds aligned on a square Petri dish containing half-strength MS medium; F. Arabidopsis seedlings (9 days old) grown in the growth chamber.

  2. Gel containing the pH-sensitive indicator bromocresol purple
    1. Prepare gel containing the pH-sensitive indicator bromocresol purple
      1. Add 0.006% (w/v) of bromocresol purple free acid reagent grade (60 mg L-1); 1 mM of CaSO4 (172 mg L-1) in sterile distilled water. Adjust pH to 5.7 using KOH or HCl. Pour the solution into an Erlenmeyer.
      2. Add agarose (15 g L-1) into the Erlenmeyer and heat in the microwave until completely dissolved.
    2. Pour the melted gel into Petri dishes (150 x 20 mm), 50 ml per dish (approx. 0.4 cm thick), with the aid of a graduated cylinder (100 ml). The agarose gel is allowed to cool until solidified. (Figure 2).


      Figure 2. pH-Sensitive indicator bromocresol purple in gel. A. Bromocresol purple; B. Calcium sulfate (CaSO4); C. Agarose; D. Bromocresol purple solution in sterile distilled water pH 5.7; E. The solution is heated in a microwave until agarose is completely dissolved; F. Gel containing bromocresol purple completely dissolved and ready to be poured. G. Gel being poured in a Petri dish. H. Plate containing the solidified gel and ready to receive the seedlings (top view). I. Lateral view of the plate shown in H. Gel should be about 0.4 cm thick and a dark yellow color is expected to allow detection of pH differences.

    3. Gently transfer groups of seedlings (6-8) at once onto each Petri dish containing the pH indicator bromocresol purple (pH 5.7) with tweezers (Figures 3A-3B; see Video 1).

      Video 1. Seedlings being transferred to Petri dish containing the pH indicator bromocresol purple

    4. Once placed in the gel pH-sensitive indicator bromocresol purple, only the roots of the seedling are treated with 10 µl AtRALF1 (10 μM solution) or water (10 µl as a control) (see Video 2).

      Video 2. Roots of Arabidopsis seedlings treated with AtRALF1 solution

    5. Capture images with a digital camera (Sony, Cyber-shot) 30 min after treatment.

Data analysis

  1. The data produced is qualitative, highly reproducible but seedlings have to be manipulated carefully. Wounds that may be unintentionally inflicted by handling itself are a signal that causes proton fluxes and may cause alkalinization.
  2. After 30 min, AtRALF1-treated seedlings show a purple color around the roots while water-treated control seedlings develop no color (Figures 3C-3D).
  3. Photographic record: the best results that we have obtained are using a white background and a source of white light from behind the plate. Good photographic records are the result of good equipment, photographer skills and light setup. Reducing the distance between the plate and the white background increases the detection of the color change in the gel. An example of this effect is shown in Figure 3F.


    Figure 3. Evaluation of root pH change on a gel containing bromocresol purple. A. Arabidopsis seedlings being selected to be transferred onto a gel containing the pH indicator. B. Seedlings being placed on top of the gel. C. Pair of 6-8 seedlings before AtRALF1 or water treatment. D. The same pair shown in C, 30 min after treatment. E. Pair of 6-8 seedlings before AtRALF1 or water treatment. F. The same pair shown in E, 30 min after treatment. Image was taken after reducing the distance between the plate and the white background. Scale bars = 1 cm.

Notes

  1. In-house produced 6xHis AtRALF1 recombinant peptide was obtained as described by do Canto et al. (2014).
  2. In our experience, the most critical points for reproducibility are: the thickness of the gel (0.4 cm) that facilitates imaging; the time of incubation with the peptide (30 min); maintaining intact (roots should not be touched/injured with forceps or in any way during handling); and a small volume of peptide solution for root treatment (10 μl).
  3. Recombinant AtRALF1 (7,631 g/mol) is dissolved in distilled water (7.631 μg/100 μl). The final concentration of the solution is 10 μM.

Recipes

  1. Half-strength MS medium
    Note: Weigh all the chemicals used in weighing balance (± 0.0001). Adjust the pH of MS medium using a magnetic stirrer and a pH meter.
    1. Add 2.215 g L-1 MS salts without sucrose and vitamins (PhytoTechnology Laboratories), 10.0 g sucrose to 800 ml ddH2O, adjust pH to 5.7 using KOH
    2. Add Gellan Gum Powder (6 g L-1, PhytoTechnology Laboratories) and add up to 1 L with dH2O
    3. Autoclave for 15 min at 121 °C
    4. After autoclaving, pour the half-strength MS medium in square Petri dishes, 30 ml per plate, with the aid of a graduated cylinder (100 ml) in a laminar flow hood
  2. Gel containing bromocresol purple (pH 5.7)
    0.006% (w/v) bromocresol purple (free acid reagent grade) (60 mg L-1)
    1 mM of CaSO4 (172 mg L-1)
    Sterile distilled water
    Agarose (15 g L-1)
  3. Sodium hypochlorite (NaClO) solution 50% (v/v)
    50 ml of sodium hypochlorite solution
    50 ml of sterile distilled water

Acknowledgments

This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). DSM is a research fellow of CNPq. This protocol was adapted from Wu et al. (2007).
The authors declare that they do not have any conflicts of interest or competing interests.

References

  1. Do Canto, A. M., Ceciliato, P. H. O., Ribeiro, B., Ortiz Morea, F. A., Franco Garcia, A. A., Silva-Filho, M. C. and Moura, D. S. (2014). Biological activity of nine recombinant AtRALF peptides: implications for their perception and function in Arabidopsis. Plant Physiol Biochem 75: 45-54.
  2. Dressano, K, Ceciliato, P. H. O., Silva, A. L., Guerrero-Abad, J. C., Bergonci, T., Ortiz-Morea, F. A., Bürger, M., Silva-Filho, M. C. and Moura, D. S. (2017). BAK1 is involved in AtRALF1-induced inhibition of root cell expansion. PLoS Genet 13 (10): e1007053.
  3. Felix, G. and Boller, T. (1995). Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells. Plant J 7: 381-389.
  4. Haruta, M., Sabat, G., Stecker, K., Minkoff, B. B. and Sussman, M. R. (2014). A peptide hormone and its receptor protein kinase regulate plant cell expansion. Science 343(6169): 408-411.
  5. Masachis, S., Segorbe, D., Turra, D., Leon-Ruiz, M., Furst, U., El Ghalid, M., Leonard, G., Lopez-Berges, M. S., Richards, T. A., Felix, G. and Di Pietro, A. (2016). A fungal pathogen secretes plant alkalinizing peptides to increase infection. Nat Microbiol 1(6): 16043.
  6. Pearce, G., Moura, D. S., Stratmann, J. and Ryan, C. A. (2001a). Production of multiple plant hormones from a single polyprotein precursor. Nature 411(6839): 817-820.
  7. Pearce, G., Moura, D. S., Stratmann, J. and Ryan, C. A., Jr. (2001b). RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development. Proc Natl Acad Sci U S A 98(22): 12843-12847.
  8. Srivastava, R., Liu, J. X., Guo, H., Yin, Y. and Howell, S. H. (2009). Regulation and processing of a plant peptide hormone, AtRALF23, in Arabidopsis. Plant J 59(6): 930-939.
  9. Wu, J., Kurten, E. L., Monshausen, G., Hummel, G. M., Gilroy, S. and Baldwin, I. T. (2007). NaRALF, a peptide signal essential for the regulation of root hair tip apoplastic pH in Nicotiana attenuata, is required for root hair development and plant growth in native soils. Plant J 52(5): 877-890.

简介

快速碱化因子(RALF)是一种植物激素肽,可抑制质子转运,导致细胞外培养基碱化。 为了检测由根细胞中的RALF肽引发的碱化反应,将拟南芥幼苗仔细转移至含有pH敏感指示剂溴甲酚紫紫的凝胶中,用肽处理并在30分钟后拍照。 在此,该方案针对外源性治疗的评估进行了优化,详细描述并呈现预期结果。

【背景】质子运输是由无数的信号引起的,并被植物用来协调增长,防御和发展。一些植物激素肽可以影响质子转运,引起细胞外基质强碱性化(Felix和Boller,1995; Pearce等人,2001a)。 5kDa肽激素快速碱化因子(RALF)在分泌后与其受体FERONIA结合,并导致质膜H + - 腺苷三磷酸酶2的磷酸化,抑制质子转运并碱化细胞外培养基(Pearce等人,2001b; Haruta等人,2014)。

含有pH指示剂溴甲酚紫的生长培养基是在根周围的培养基中观察碱化或酸化的有效方法。先前已经使用该方法来表明NaRALF是调节根毛细胞外pH所需的(Wu等人,2007),并且过表达AtRALF23的植物的根具有降低的将根际酸化的能力(Srivastava ,2009)。 Masachis等人也使用pH指示剂溴甲酚紫紫色的生长培养基。 (2016)证明由真菌病原体产生的RALF同源物诱导了番茄植物根周围的培养基的碱化。

我们已经优化了pH指示剂溴甲酚紫方案,并且使用改进的方案,我们能够在AtRALF1处理后在野生型和突变体幼苗中观察到拟南芥根部周围的碱化作用(Dressano等人, ,2017)。在这里,我们展示了这种分析方法如何提供围绕拟南芥根部的细胞外pH值的定性信息。

关键字:pH指示剂, 碱化, RALF, 溴甲酚紫

材料和试剂

  1. 生物材料
    1. 拟南芥种子和幼苗(Ecotype Columbia,Col-0)
    2. 内部生产的6xHis AtRALF1重组多肽 

  2. 用于种子灭菌和生长的化学品和材料
    1. 方格培养皿,格栅100毫米宽×15毫米高,无菌(Electron Microscopy Sciences,目录号:70691)
    2. 透明塑料包装
    3. 量筒1000,100和10毫升(Uniglas)
    4. 1,000μl过滤嘴(NEST Scientific,目录号:NPT1000-B-B)
    5. 200μl过滤嘴(NEST Scientific,目录号:NPT0200-B-Y)
    6. 10μl过滤嘴(NEST Scientific,目录号:301001)
    7. 结冷胶粉(Culture Gel Gel TM I型 - 生物技术等级)(PhytoTechnology Laboratories,目录号:G434)
    8. Murashige& Skoog Basal盐混合物(PhytoTechnology Laboratories,目录号:M524)
    9. 无菌蒸馏水
    10. 次氯酸钠溶液
    11. 次氯酸钠(NaClO)溶液50%(v / v)(见食谱)

  3. 含有pH敏感指示剂溴甲酚紫的凝胶的化学品和材料
    1. 量筒100毫升(Uniglas)
    2. 培养皿150 x 20毫米无菌
    3. 溴甲酚紫游离酸试剂级(C 2 H 16 Br 2 O 5 S,AMRESCO,目录号: 0531-25G)
    4. 硫酸钙二水合物(CaSO 4·2H 2 O,Merck,目录号:102161)
    5. 氢氧化钾(KOH)
    6. 盐酸(HCl)
    7. 琼脂糖RA(生物技术级,AMRESCO,目录号:N605-500G)
    8. 无菌蒸馏水

设备

  1. 称重天平(RADWAG Wagi Elektroniczne,型号:AS 220 / C / 2)
  2. 磁力搅拌器(Fisher Scientific)
  3. pH计(Thermo Orion,PerpHecT LongR计,型号:350)
  4. 移液器(Nichipet EX,P1000,P200,P10)
  5. 烧杯1,000毫升(PHOX,Boro 3.3)
  6. 烧杯250毫升(APRX,Boro 3.3)
  7. 高压灭菌器(Sercon,型号:HS 1-0101)
  8. 层流罩(Pachane,型号:PA 440)
  9. 生长室/受控环境室
  10. 埃伦迈耶(PIREX)
  11. 微波炉(BRASTEMP,目录号:: BMJ38ARANA)
  12. 镊子
  13. 数码相机(Cyber-shot,Sony,型号:DSC-H300)

程序

  1. 种子灭菌和发芽
    为了分析根培养基碱化,将幼苗在含有半强度MS培养基(参见食谱)的平板上生长9天。
    1. 使用1ml次氯酸钠溶液50%(v / v)10分钟在Eppendorf管中对拟南芥种子进行表面消毒,然后分别使用1ml无菌Milli-Q水进行5次洗涤。种子用微量移液器(1,000μl)洗涤(图1A)。
    2. 将灭菌的种子在4°C的水中分层72小时,然后使用微量移液管(10μL)在含有半强度MS培养基的方盘平皿上滴加(图1B-1D)。

    3. 使用透明塑料包装密封包含种子的盘子。
    4. 在22±2℃,16小时光照(150μmolm -2 s -1)和8小时黑暗的情况下,将受控环境生长室中的板竖直孵育9天天(图1E)。


      图1.种子灭菌和幼苗生长。 :一种。 甲。用次氯酸钠溶液50%(v / v)进行表面灭菌过程中的拟南芥种子; B.含有分层种子的移液管尖(已经切割然后高压灭菌); C.分层种子置于半强度MS培养基中; D.在含有半强度MS培养基的方形培养皿上对齐种子; F.生长在生长室中的拟南芥幼苗(9日龄)。

  2. 含有pH敏感指示剂溴甲酚紫的凝胶
    1. 准备含有pH敏感指示剂溴甲酚紫的凝胶
      1. 加入0.006%(w / v)溴甲酚紫游离酸试剂级(60g L-1);在无菌蒸馏水中加入1mM CaSO 4(172g L -1)。使用KOH或HCl调节pH至5.7。将溶液倒入锥形瓶中。
      2. 将琼脂糖(15g L -1)加入锥形瓶中并在微波炉中加热直至完全溶解。
    2. 借助于量筒(100ml),将熔化的凝胶倒入培养皿(150×20mm),每皿50ml(约0.4cm厚)。使琼脂糖凝胶冷却直至凝固。 (图2)。


      图2.凝胶中pH敏感指示剂溴甲酚紫。A.溴甲酚紫; B.硫酸钙(CaSO4·4); C.琼脂糖; D.溴甲酚紫溶液,无菌蒸馏水pH 5.7; E.溶液在微波中加热直至琼脂糖完全溶解; F.含有溴甲酚紫的凝胶完全溶解并准备倒入。 G.凝胶被倒入培养皿中。 H.含有固化凝胶并准备接种幼苗的平板(顶视图)。 I.在H中显示的板的横向视图。凝胶应该约0.4厘米厚,预计深黄色可以检测到pH差异。

    3. 用镊子轻轻转移幼苗组(6-8)至含有pH指示剂溴甲酚紫(pH 5.7)的每个培养皿中(图3A-3B;参见视频1)。

      视频1

    4. 一旦放入凝胶pH敏感指示剂溴甲酚紫,只用10μlAtRALF1(10μM溶液)或水(10μl作为对照)处理幼苗的根部(见视频2)。

      视频2

    5. 使用数码相机(Sony,Cyber-shot)在处理后30分钟拍摄图像。

数据分析

  1. 产生的数据是定性的,可重复性很高,但幼苗必须仔细操作。通过处理本身可能会无意中造成的创伤是一个信号,可能会导致质子通量并可能导致碱化。
  2. 30分钟后,AtRALF1处理的幼苗在根部周围呈现紫色,而水处理的对照幼苗不发色(图3C-3D)。
  3. 摄影记录:我们获得的最佳结果是使用白色背景和来自背后的白光源。良好的摄影记录是良好的设备,摄影师技巧和灯光设置的结果。减少平板和白色背景之间的距离会增加凝胶中颜色变化的检测。图3F显示了这种效应的一个例子。


    图3.含有溴甲酚紫的凝胶的根部pH变化评估。 :一种。选择拟南芥幼苗转移到含有pH指示剂的凝胶上。 B.将幼苗放在凝胶上。 C.在AtRALF1或水处理前对6-8幼苗。 D.在C中显示的同一对,处理后30分钟。 E.在AtRALF1或水处理前6-8对幼苗。 F.处理后30分钟E中显示的相同对。图像是在减少板和白色背景之间的距离后拍摄的。比例尺= 1厘米。

笔记

  1. 如do Canto等人所述获得自制产生的6xHis AtRALF1重组肽。 (2014)。
  2. 根据我们的经验,最重要的重现点是:便于成像的凝胶厚度(0.4厘米);与肽孵育的时间(30分钟);保持完整(在处理过程中不应用镊子或任何方式触摸/伤害根部);和少量用于根处理的肽溶液(10μl)。
  3. 将重组AtRALF1(7,631g / mol)溶于蒸馏水(7.631μg/100μl)中。该解决方案的最终浓度是10μM。

食谱

  1. 半强度MS培养基
    注意:称量称量天平中使用的所有化学物质(±0.0001)。使用磁力搅拌器和pH计调整MS培养基的pH值。
    1. 将不含蔗糖和维生素的2.215g L -1 MS盐(PhytoTechnology Laboratories),10.0g蔗糖加入到800ml ddH 2 O中,使用KOH调节pH至5.7, >
    2. 加入结冷胶粉(6g L -1,PhytoTechnology Laboratories)并用dH 2 O加入至1L。

    3. 在121°C高压灭菌15分钟
    4. 高压灭菌后,将半强度MS培养基倒入正方形Petri培养皿中,每个培养皿30ml,借助量筒(100ml)在层流罩中进行。
  2. 含溴甲酚紫的凝胶(pH 5.7)
    0.006%(w / v)溴甲酚紫(游离酸试剂级)(60g L -1)
    1毫摩尔CaSO 4(172克L -1)
    无菌蒸馏水
    琼脂糖(15克L -1)
  3. 次氯酸钠(NaClO)溶液50%(v / v)
    50毫升次氯酸钠溶液

    50毫升无菌蒸馏水

致谢

本研究得到Fundaçãode AmparoàPesquisa do Estado deSãoPaulo(FAPESP)的支持。帝斯曼是CNPq的研究员。该协议改编自Wu等人(2007)。
作者声明他们没有任何利益冲突或利益冲突。

参考

  1. Do Canto,A. M.,Ceciliato,P. H. O.,Ribeiro,B.,Ortiz Morea,F. A.,Franco Garcia,A. A.,Silva-Filho,M. C.和Moura,D. S.(2014)。 9种重组AtRALF肽的生物学活性:它们在拟南芥中的感知和功能的意义 EM>。 Plant Physiol Biochem 75:45-54。
  2. Dressano,K,Ceciliato,P.H.O.,Silva,A.L.,Guerrero-Abad,J.C.,Bergonci,T.,Ortiz-Morea,F.A.,Bürger,M.,Silva-Filho,M.C和Moura,D.S。(2017)。 BAK1参与AtRALF1诱导的根细胞扩增抑制。 Genet 13(10):e1007053。
  3. Felix,G。和Boller,T。(1995)。 Systemin在Lycopersicon中诱导快速离子通量和乙烯生物合成peruvianum 细胞。 Plant J 7:381-389。
  4. Haruta,M.,Sabat,G.,Stecker,K.,Minkoff,B. B.和Sussman,M. R.(2014)。 肽类激素及其受体蛋白激酶调节植物细胞扩增。 Science 343(6169):408-411。
  5. Masachis,S.,Segorbe,D.,Turra,D.,Leon-Ruiz,M.,Furst,U.,El Ghalid,M.,Leonard,G.,Lopez-Berges,MS,Richards,TA,Felix, G.和Di Pietro,A。(2016)。 真菌病原体分泌植物碱化肽以增加感染。 Nat Microbiol 1(6):16043.
  6. Pearce,G.,Moura,D. S.,Stratmann,J.和Ryan,C. A.(2001a)。 从单一多蛋白前体制备多种植物激素。 Nature 411(6839):817-820。
  7. Pearce,G.,Moura,D.S.,Stratmann,J。和Ryan,C.A.,Jr.(2001b)。 RALF是植物中5 kDa无处不在的多肽,可抑制根的生长和发育
    美国国家科学院院刊98(22):12843-12847。
  8. Srivastava,R.,Liu,J.X.,Guo,H.,Yin,Y。和Howell,S.H。(2009)。 在拟南芥中调控和加工植物肽激素AtRALF23 。 Plant J 59(6):930-939。
  9. Wu,J.,Kurten,E.L。,Monshausen,G.,Hummel,G.M.,Gilroy,S.and Baldwin,I.T。(2007)。 NaRALF,一种肽信号,用于调节根尖毛发质外pH值,是本地土壤根毛发育和植物生长所必需的。 Plant J 52(5):877-890。
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引用:Silva, A. L., Dressano, K., Ceciliato, P. H., Guerrero-Abad, J. and Moura, D. S. (2018). Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple. Bio-protocol 8(7): e2796. DOI: 10.21769/BioProtoc.2796.
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