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Aug 2016

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Analyses of Root-secreted Acid Phosphatase Activity in Arabidopsis
拟南芥根分泌酸性磷酸酶活性分析   

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Abstract

Induction and secretion of acid phosphatase (APase) is a universal adaptive response of higher plants to low-phosphate stress (Tran et al., 2010). The intracellular APases are likely involved in the remobilization and recycling of phosphate (Pi) from intracellular Pi reserves, whereas the extracellular or secreted APases are believed to release Pi from organophosphate compounds in the rhizosphere. The phosphate starvation-induced secreted APases can be released into the rhizosphere or retained on root surfaces (root-associated APases). In this article, we describe the protocols for analyzing root-secreted APase activity in the model plant Arabidopsis thaliana (Arabidopsis). In Arabidopsis, the activity of both root-associated APases and APases that are released into the rhizosphere can be quantified based on their ability to cleave a synthesized substrate, para-nitrophenyl-phosphate (pNPP), which releases a yellow product, para-nitrophenol (pNP) (Wang et al., 2011 and 2104). The root-associated APase activity can also be directly visualized by applying a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), to the root surface (Lloyd et al., 2001; Tomscha et al., 2004; Wang et al., 2011 and 2014) whereas the isozymes of APases that are released into rhizosphere can be profiled using an in-gel assay (Trull and Deikman, 1998; Tomscha et al., 2004; Wang et al., 2011 and 2014). The protocol for analysis of intracellular APase activity in Arabidopsis has been previously described (Vicki and William, 2013).

Keywords: Arabidopsis thaliana (拟南芥), Phosphate starvation (磷酸盐饥饿), Secretion (分泌), Acid phosphatase (酸性磷酸酶), Phosphatase activity (磷酸酶活性), Isozyme (同工酶), Histochemical staining (组织化学染色), Quantitative analysis (定量分析)

Background

Phosphate (Pi) is the major form of phosphorus that plants take up through their root systems, and Pi levels in most soils are low, resulting in Pi starvation. To cope with this nutritional stress, plants trigger an array of adaptive responses that increase their survival and growth. Induction and secretion of APases is a hallmark Pi starvation response that has been documented in a variety of plant species (Tran et al., 2010), and root-secreted APase activity has been widely used as a diagnostic tool to evaluate the magnitude of plant responses to Pi starvation. In this article, we provide three protocols for assay of root-secreted APase activity. An assay using pNPP as a substrate is the most commonly used method to quantify APase activity. A BCIP staining assay provides a simple, one-step method for the histochemical detection of APase activity on the root surface. Neither of these methods, however, can reveal the number of APase isoforms that contribute to the observed activity. A third method (in-gel assay), which combines electrophoresis, protein renaturation, and conversion of a substrate into a red-brown product, can reveal the diverse compositions of APase isoforms in different samples, and can therefore provide additional information about root-secreted APases.

Materials and Reagents

  1. 9-cm-diameter Petri dish
  2. Surgical blade (Swann Morton Surgical Scalpel Blade No. 20)
  3. 1.7 ml and 2 ml Eppendorf tubes
  4. Dialysis tubing (Sigma-Aldrich, catalog number: D9777 )
  5. 50 ml Falcon tube
  6. Whatman #1 filter paper (GE Healthcare, catalog number: 1001-125 )
  7. Pipette tips  
  8. Arabidopsis seeds
  9. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  10. 30% acrylamide/Bis solution (Solarbio, catalog number: A1010 )
  11. TEMED (Sigma-Aldrich, catalog number: T22500 )
  12. Agar (Sigma-Aldrich, catalog number: A1296 )
  13. Bovine serum albumin (BSA)*
  14. 95% ethanol*
  15. Ammonium persulfate (NH4)2S2O8*
  16. Sodium hypochlorite solution (NaClO)*
  17. Sodium hydroxide (NaOH)*
  18. Sodium acetate (NaAc)*
  19. Phosphoric acid (H3PO4)*
  20. Hydrochloric acid (HCl)*
  21. Ammonium nitrate (NH4NO3)*
  22. Potassium nitrate (KNO3)*
  23. Calcium chloride (CaCl2)*
  24. Magnesium sulfate (MgSO4)*
  25. Potassium dihydrogen phosphate (KH2PO4)*
  26. Potassium sulfate (K2SO4)*
  27. Potassiu iodide (KI)*
  28. Orthoboric acid (H3BO3)*
  29. Manganese sulfate (MnSO4)*
  30. Zinc sulfate (ZnSO4)*
  31. Sodium molybdate (Na2MoO4)*
  32. Cobalt(II) chloride (CoCl2)*
  33. Copper sulfate (CuSO4)*
  34. Fe-EDTA*
  35. Myo Inositol*
  36. Nicotinic acid*
  37. Pyridoxine HCl*
  38. Thiamine HCl*
  39. Glycine*
  40. Sucrose*
  41. Bromophenol blue*
  42. Coomassie Brilliant Blue G-250*
  43. MES (AMRESCO, catalog number: E169 )
  44. β-naphthyl acid phosphate (Sigma-Aldrich, catalog number: N7375 )
  45. Fast Black K (Sigma-Aldrich, catalog number: F7253 )
  46. pNPP (Sigma-Aldrich, catalog number: P4744 )
  47. BCIP (Gold Bio, catalog number: B-500-10 )
  48. Tris base (NOVON SCIENTIFIC, catalog number: ZZ02531 )
  49. DTT (Sigma-Aldrich, catalog number: DTT-RO )
  50. SDS (AMRESCO, catalog number: 0227 )
  51. DMSO (AMRESCO, catalog number: 0231 )
  52. PMSF (Sigma-Aldrich, catalog number: 52332 )
  53. Liquid nitrogen
  54. Murashige and Skoog (MS) medium (P+ and P- MS medium) (see Recipes, Murashige and Skoog, 1962)
  55. APase activity profiling detection buffer, pH 4.9 (see Recipes)
  56. Reaction buffer, pH 4.9 (see Recipes)
  57. pNPP solution (1.0 mg/ml) (see Recipes)
  58. BCIP stock solution (100x) (see Recipes)
  59. Bradford solution (see Recipes)
  60. 5x protein loading buffer (see Recipes)

*Note: Similar reagents from any qualified company are suitable for this experiment.

Equipment

  1. Pipettes    
  2. Forceps (Fisher Scientific, catalog number: 22-327379 )
  3. Spectrophotometer (WPA, Biowave)
  4. 250 ml Erlenmeyer (conical) flask
  5. Orbital shaker (Qilinbeier, model: TS-1 )
  6. Freeze dryer (Beijing Songyuanhuaxing, model: LGJ-10 )
  7. Microfuge (Eppendorf, model: 5415 D )
  8. Microwave oven
  9. Small mortar and pestle
  10. Water bath (Shanghai Yiheng, model: DK-8D )**
  11. Analytical balance (Mettler Toledo, model: PB153-L)**
  12. pH meter (Mettler Toledo, model: FE20 )**
  13. Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories, model: Mini-PROTEAN® Tetra Vertical Electrophoresis Cell , catalog number: 1658004)

**Note: Similar equipment from any qualified company is suitable for this experiment.

Software

  1. SPSS Statistics

Procedure

  1. Quantification of root-associated APase activity and the activity of APases released into the growth media
    1. Quantification of root-associated APase activity
      1. Arabidopsis seeds are suspended with 5% sodium hypochlorite solution/0.1% Triton X-100 and incubated for 10 min at room temperature with occasionally inverting for surface-sterilization.
      2. Remove the liquid and wash the seeds with sterilized ddH2O for 4 times.
      3. Place 20-30 surface-sterilized Arabidopsis seeds horizontally on the upper part of each 9-cm-diameter Petri dish containing full-strength or half-strength Murashige and Skoog (MS) medium (P+ or P- medium). For each biological sample, use three replicates.
      4. Keep the Petri dishes at 4 °C for 2 days for stratification.
      5. Place the Petri dishes vertically in a controlled environment growth room and allow the seedlings to grow for 6-7 days under a 16-h light/8-h dark regime (90 µmol photons m-2 sec-1) at 22-23 °C.
      6. Excise roots with a razor blade and measure root length. The average root length of the Arabidopsis seedlings grown on P+ medium is 4.0-5.5 cm and that grown on P- medium is 2.5-4.0 cm.
      7. Immerse two roots with forceps into 650 µl of pre-warmed (37 °C) reaction buffer in a 1.7 ml Eppendorf tube.
      8. Quickly add 50 µl of pNPP solution.
      9. Incubate at 37 °C for 1-4 h.
      10. Add 100 µl of 0.4 N NaOH and shake vigorously by hand to stop the reaction.
      11. Remove the roots with forceps and measure the absorbance at 410 nm (A410) in the remaining solution with a spectrophotometer.
      12. Calculate the root-associated APase activity as A410/cm root/h (Figure 1A).


        Figure 1. Analysis of APase activity secreted by Arabidopsis seedlings of WT and various single, double, and triple pap (purple acid phosphatase) mutants grown in or on P+ or P- medium. Root-associated APase activities (A) and APase activities in the medium (B) were determined with pNPP as the substrate. Values are means with SE. A one-way ANOVA analysis was carried out for the whole data set, and post hoc comparisons were conducted using the SPSS Tukey HSD test at P < 0.05. Within each of the four plots, values with different letters are significantly different. Adapted from Wang et al. (2014).

    2. Concentrating the proteins in the liquid growth medium
      1. Transfer about 150 9-day-old seedlings that were grown vertically on P+ solid medium with autoclaved forceps to a 250 ml autoclaved conical flask containing 100 ml of P+ or P- liquid medium.
      2. Place the flasks on an orbital shaker (40 rpm/min) and allow the seedlings to grow for 6-7 days under a 16-h light/8-h dark regime (90 µmol photons m-2 sec-1) at 22-23 °C.
      3. Remove all of the seedlings from the flasks and freeze the liquid medium at -20 °C.
      4. Put the medium in the holder of a pre-cooled freeze dryer, cover the lid, and lyophilize the medium for 3-4 days.
      5. Thoroughly wash the bottom of the flask using 5-6 ml of ddH2O and transfer the washing solution to dialysis tubing.
      6. Dialyze the sample for 24 h at 4 °C against 4 L of pre-cooled ddH2O and change the ddH2O every 6 h.
      7. Transfer the dialyzed sample to a 2 ml tube and centrifuge for 5 min at 1,500 x g.
      8. Transfer the supernatant to a 50 ml Falcon tube and freeze the liquid at -20 °C, then lyophilize for 2 days as described in step A2d.
      9. Dissolve the lyophilized sample in 200 µl of ddH2O.
      10. Measure the protein concentration using the Bradford method as described in step A3.
      11. The concentrated, secreted proteins can be used immediately or can be stored at -80 °C for further analysis.
    3. Determination of protein concentration
      1. Pipette 0, 2, 4, 8, 12, 16, or 20 μl of 1 μg/μl BSA into 1.7 ml Eppendorf tubes and add ddH2O to a final volume of 20 μl.
      2. Add 980 μl of Bradford solution to each tube and mix well.
      3. Incubate at room temperature for 5 min.
      4. Measure the absorbance at 595 nm (A595) with a spectrophotometer.
      5. Make a Bradford standard curve by plotting BSA content and A595.
      6. Add 20 μl of concentrated secreted proteins to 980 μl of Bradford solution.
      7. Mix well and incubate at room temperature for 5 min.
      8. Measure the A595 with a spectrophotometer.
      9. Compare the resulting A595 value with the Bradford standard curve to determine the protein concentration of various samples.
    4. Quantification of APase activity in the growth medium
      1. Add 10 µl of concentrated proteins (about 2.5 µg) collected from the liquid growth medium to 590 µl of reaction buffer in a 1.7 ml Eppendorf tube. For each sample, make a least three replicates.
      2. Add 80 μl of pNPP solution to the reaction mixture.
      3. Incubate at 37 °C for 1 h.
      4. Add 120 μl of 0.4 N NaOH to terminate the reaction.
      5. Measure the A410 with a spectrophotometer.
      6. Calculate the APase activity in the growth medium as A410/mg protein/min (Figure 1B).

  2. Analysis of root-associated APase activity using histochemical staining
    1. Grow seedlings vertically on P+ or P- MS medium for 6-7 days as described in steps A1a-A1e.
    2. Add 100 ml of ddH2O and 0.5 g agar to a 250 ml conical flask, gently shake the flask, and then heat the flask in a microwave oven until the agar is completely dissolved.
    3. Add 1 ml of the BCIP stock solution (100x) and shake briefly by hand.
    4. Keep the BCIP/agar solution at room temperature or use flowing water to cool the solution for a few minutes to about 40 °C.
    5. Keep the BCIP/agar solution in a 40 °C water bath.
    6. Uniformly overlay about 5 ml of the BCIP/agar solution on one row of the Arabidopsis roots and incubate at room temperature for about 24 h (Figure 2).


      Figure 2. A demonstration of how to overlay the BCIP/agar solution on the root surface of Arabidopsis seedlings grown on Petri dish

    7. The roots of Arabidopsis grown on P- medium will be stained blue, while these grown on P+ medium will remain unstained (Figure 3).


      Figure 3. Histochemical staining of root-associated APase activity. Upper row: The morphologies of 7-day-old Arabidopsis seedlings grown on MS P+ and P- medium without BCIP staining. Bottom row: Seedlings whose roots were stained with a 0.01% BCIP/0.5% agar solution for 24 h. In the lower row, root-associated APase activity (as indicated by the blue color) was detected on roots of seedlings grown on P- medium (bottom right) but not on roots of seedlings grown on P+ medium (bottom left).

    8. APase histochemical staining is greatly affected by the BCIP concentration. A high concentration of BCIP (0.08%-0.16%) can cause over-staining, which may mask differences in the level of secreted APase activity (Figure 4).


      Figure 4. The effect of different concentrations of BCIP on staining of root-associated APase activity of 7-day-old Arabidopsis seedlings grown on MS P- medium. The concentrations of BCIP used are indicated at the left bottom corner. nop1-1 and nop1-2 are Arabidopsis mutants that have mutations in the AtPAP10 gene. With the increase in BCIP concentration, the activities of other root-associated APases, mainly AtPAP12 and AtPAP26, are also detected. Adapted from Wang et al. (2011).

  3. Analysis of profiles of APase isoforms released into the growth medium (in-gel assay)
    The isozyme profile of APases released into the growth medium is visualized by separating protein samples on SDS-PAGE (denaturing gel) or Native-PAGE (non-denaturing gel), which are then stained with the chromogenic substrates β-NAP and fast black K. For Native-PAGE analysis, omit SDS from the gel and running buffer.
    Prepare a 10% separating gel and a 5% stacking gel (SDS-PAGE) according to the following Table 1.

    Table 1. Recipes for separating gel and stacking gel
    Stock solution
    5% stacking gel (5 ml)
    10% separating gel (10 ml)
    30% Acrylamide/Bis solution
    0.83 ml
    3.3 ml
    1.5 M Tris-HCl, pH 8.8
    -
    2.5 ml
    1 M Tris-HCl, pH 6.8
    0.63 ml
    -
    10% SDS
    0.05 ml
    0.1 ml
    10% (NH4)2S2O8
    0.05 ml
    0.1 ml
    TEMED
    0.005 ml
    0.004 ml
    ddH2O
    3.40 ml
    4.0 ml

    1. Set up the SDS-PAGE gel running system according to the Bio-Rad instructions (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf).
    2. Add 1x protein running buffer that was pre-cooled to 4 °C.
    3. Thoroughly mix 2.5 μg of secreted proteins with 1/5 volume of 5x protein loading buffer and load the sample on the SDS-PAGE gel.
      Note: Do not heat the sample.
    4. Separate proteins at 60 V for 30 min followed by 120 V for 150 min (both at 4 °C).
    5. Carefully transfer the gel to pre-cooled (4 °C) ddH2O in a container and gently shake for 10 min to remove SDS from the gel.
    6. Change pre-cooled (4 °C) ddH2O and repeat 4 times.
    7. Add 50 ml of reaction buffer and shake gently for 15 min at 4 °C.
    8. Change reaction buffer and repeat 4 times.
    9. Add 50 ml of APase activity profiling detection buffer and incubate at 37 °C for 1-3 h with gentle agitation. The isozyme profile of APases is visualized as the red-brown bands on the gel (Figure 5).


      Figure 5. Profiles of APase activity in the growth medium. A 2.5-μg quantity of secreted protein was separated by SDS-PAGE (A) or Native-PAGE (B) and stained with 0.3 mg/ml β-NAP and 0.5 mg/ml Fast Black K. Protein molecular markers are on the left side of the gel in (A). The numbers on the right of each panel indicate different APase isoforms. Adapted from Wang et al. (2014).

Notes

  1. The temperature of BCIP/agar solution is important for detecting root-associated APase activity. If the solution is too hot (above 60 °C), it will damage Arabidopsis roots and inactivate the APase on the root surface.
  2. Removal of SDS is critical for obtaining a clear profile of APase isozymes.

Recipes

  1. Murashige and Skoog (MS) medium (P+ and P- MS medium)
    P+ MS medium is the full-strength MS medium (Murashige and Skoog, 1962). The recipe for full-strength MS medium is shown below. For P- MS medium, the 1.25 mM KH2PO4 in P+ MS medium is replaced with 1.25 mM K2SO4. Autoclave P+ and P- medium at 121 °C for 10 min, and store the medium at room temperature
    NH4NO3
    20.62 mM
    CuSO4
    0.1 µM
    KNO3
    18.79 mM
    Fe-EDTA
    100 µM
    CaCl2
    2.99 mM
    Myo inositol
    0.55 µM
    MgSO4
    1.5 mM
    Nicotinic acid
    4.1 µM
    KH2PO4
    1.25 mM
    Pyridoxine HCl
    2.4 µM
    K2SO4
    0.625 mM
    Thiamine HCl
    0.3 µM
    KI
    5 µM
    Glycine
    30 µM
    H3BO3
    100 µM


    MnSO4
    92.5 µM
    Sucrose
    10 g/L
    ZnSO4
    30 µM
    MES
    5.1 g/L
    NaMoO4
    1 µM
    pH
    5.8
    CoCl2
    0.1 µM
    Agar
    10 g/L

  2. APase activity profiling detection buffer, pH 4.9 (freshly prepared)
    0.3 mg/ml β-naphtyl acid phosphate
    0.5 mg/ml Fast Black K
    10 mM MgCl2
    50 mM NaAc
  3. Reaction buffer, pH 4.9
    10 mM MgCl2
    50 mM NaAc
    Store at 4 °C
  4. pNPP solution (1.0 mg/ml)
    A 10-mg quantity of pNPP is dissolved in 10 ml of reaction buffer
    Store aliquots at -20 °C
  5. BCIP stock solution (100x)
    The concentration of the BCIP stock solution is 10 g/L, and that of the working solution is 0.1 g/L
    To prepare the BCIP stock solution:
    1. Dissolve 0.1 g of BCIP powder in 8 ml of ddH2O and then add approximately 14 drops of 1 N NaOH until the BCIP power is totally dissolved
    2. Add ddH2O to a total volume of 10 ml and then filter the solution to remove un-dissolved BCIP particles
    3. Store aliquots at -20 °C
  6. Bradford solution
    Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol
    Add 100 ml of 85% H3PO4 and then add ddH2O to 1,000 ml
    Pass through a Whatman #1 filter paper, and store the filtrate at 4 °C in a brown bottle
  7. 5x protein loading buffer
    250 mM Tris-HCl, pH 6.8
    10 mM DTT
    10% SDS
    30% (v/v) glycerol
    0.05% (w/v) bromophenol blue
    Store the buffer at room temperature

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant Nos. 31370290 and 30670170).

References

  1. Lloyd, J. C., Zakhleniuk, O. V. and Raines, C. A. (2001). Identification of mutants in phosphorus metabolism. Annals Applied Biol 138: 111-115.
  2. Murashige, T and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497.
  3. Tomscha, J. L., Trull, M. C., Deikman, J., Lynch, J. P. and Guiltinan, M. J. (2004). Phosphatase under-producer mutants have altered phosphorus relations. Plant Physiol 135(1): 334-345.
  4. Tran, H. T., Hurley, B. A. and Plaxton, W. C. (2010). Feeding hungry plants: The role of purple acid phosphatases in phosphate nutrition. Plant Sci 179: 14-27.
  5. Trull, M. C. and Deikman, J. (1998). An Arabidopsis mutant missing one acid phosphatase isoform. Planta 206(4): 544-550.
  6. Vicki, K. and William, P. (2013). Protein extraction, acid phosphatase activity assays, and determination of soluble protein concentration. Bio-protocol e889.
  7. Wang, L., Li, Z., Qian, W., Guo, W., Gao, X., Huang, L., Wang, H., Zhu, H., Wu, J. W., Wang, D. and Liu, D. (2011). The Arabidopsis purple acid phosphatase AtPAP10 is predominantly associated with the root surface and plays an important role in plant tolerance to phosphate limitation. Plant Physiol 157(3): 1283-1299.
  8. Wang, L., Lu, S., Zhang, Y., Li, Z., Du, X. and Liu, D. (2014). Comparative genetic analysis of Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation. J Integr Plant Biol 56(3): 299-314.

简介

酸性磷酸酶(APase)的诱导和分泌是高等植物对低磷酸盐胁迫的普遍适应性反应(Tran et al。,2010)。细胞内APase可能参与磷酸盐(Pi)从细胞内Pi储备的再利用和再循环,而细胞外或分泌的APase被认为从根际中的有机磷酸盐化合物中释放出Pi。磷酸盐饥饿诱导的分泌的APase可以释放到根际中或保留在根表面(根相关的APase)上。在本文中,我们描述了在拟南芥(Arabidopsis thaliana)(拟南芥)中分析根分泌的APase活性的方案。在拟南芥中,释放到根际的根系相关APase和APase的活性可以基于它们切割合成底物,释放黄色产物的对硝基苯基磷酸(pNPP)的能力来定量,对硝基苯酚(pNP)(Wang等,2011和2104)。根系相关的APase活性也可以通过将显色底物5-溴-4-氯-3-吲哚基 - 磷酸酯(BCIP)施用到根表面来直接显现(Lloyd等人,2001; Tomscha et al。 ,2004; Wang等人,2011和2014),而释放到根际中的APase的同功酶可以使用凝胶内测定法(Trull和Deikman,1998; Tomscha等人,2004; Wang等人, 2011年和2014年)。之前已经描述了用于分析拟南芥细胞内APase活性的方案(Vicki和William,2013)。
【背景】磷酸盐(Pi)是植物通过根系发生磷的主要形式,大多数土壤中的Pi水平较低,导致Pi饿。为了应对这种营养压力,植物引发了一系列适应性反应,增加了其生存和增长。 APase的诱导和分泌是各种植物物种中记载的标志性的Pi饥饿反应(Tran et al。,2010),根分泌的APase活性已被广泛用作评估植物大小的诊断工具对Pi饿的反应。在本文中,我们提供三种方法来测定根分泌的APase活性。使用pNPP作为底物的测定法是用于量化APase活性的最常用的方法。 BCIP染色测定提供了用于组织化学检测根表面上APase活性的简单的一步法。然而,这些方法都不能揭示有助于观察到的活性的APase同工型的数量。将电泳,蛋白质复性和底物转化成红棕色产品的第三种方法(凝胶内测定)可以揭示不同样品中APase异构体的不同组成,因此可以提供关于根 - 分泌的APase。

关键字:拟南芥, 磷酸盐饥饿, 分泌, 酸性磷酸酶, 磷酸酶活性, 同工酶, 组织化学染色, 定量分析

材料和试剂

  1. 9厘米直径的培养皿
  2. 外科刀片(Swann Morton手术刀片20号)
  3. 1.7ml和2ml Eppendorf管
  4. 透析管(Sigma-Aldrich,目录号:D9777)
  5. 50ml Falcon管
  6. Whatman#1滤纸(GE Healthcare,目录号:1001-125)
  7. 移液器提示
  8. 拟南芥种子
  9. Triton X-100(Sigma-Aldrich,目录号:X100)
  10. 30%丙烯酰胺/Bis溶液(Solarbio,目录号:A1010)
  11. TEMED(Sigma-Aldrich,目录号:T22500)
  12. 琼脂(Sigma-Aldrich,目录号:A1296)
  13. 牛血清白蛋白(BSA)*
  14. 95%乙醇*
  15. 过硫酸铵(NH 4)2 S 2 O 8 *
  16. 次氯酸钠溶液(NaClO)*
  17. 氢氧化钠(NaOH)*
  18. 醋酸钠(NaAc)*
  19. 磷酸(H 3 3 PO 4)*
  20. 盐酸(HCl)*
  21. 硝酸铵(NH 4 NO 3)*
  22. 硝酸钾(KNO 3 )*
  23. 氯化钙(CaCl 2)*
  24. 硫酸镁(MgSO 4)*
  25. 磷酸二氢钾(KH 2 PO 4)*
  26. 硫酸钾(K 2 O 4 SO 4)*
  27. 碘化钾(KI)*
  28. 正硼酸(H 3 3 BO 3)*
  29. 硫酸锰(MnSO 4)*
  30. 硫酸锌(ZnSO 4)*
  31. 钼酸钠(Na 2 MoO 4)*
  32. 氯化钴(II)(CoCl 2)*
  33. 硫酸铜(CuSO 4 )*
  34. Fe-EDTA *
  35. Myo肌醇*
  36. 烟酸*
  37. 盐酸吡哆醇*
  38. 硫胺素HCl *
  39. 大豆*
  40. 蔗糖*
  41. 溴酚蓝*
  42. 考马斯亮蓝G-250 *
  43. MES(AMRESCO,目录号:E169)
  44. β-萘酸磷酸酯(Sigma-Aldrich,目录号:N7375)
  45. Fast Black K(Sigma-Aldrich,目录号:F7253)
  46. pNPP(Sigma-Aldrich,目录号:P4744)
  47. BCIP(Gold Bio,目录号:B-500-10)
  48. Tris碱(NOVON SCIENTIFIC,目录号:ZZ02531)
  49. DTT(Sigma-Aldrich,目录号:DTT-RO)
  50. SDS(AMRESCO,目录号:0227)
  51. DMSO(AMRESCO,目录号:0231)
  52. PMSF(Sigma-Aldrich,目录号:52332)
  53. 液氮
  54. Murashige和Skoog(MS)培养基(P +和P- MS培养基)(参见食谱,Murashige和Skoog,1962)
  55. APase活动分析检测缓冲液,pH 4.9(参见食谱)
  56. 反应缓冲液,pH 4.9(参见食谱)
  57. pNPP溶液(1.0mg/ml)(参见食谱)
  58. BCIP库存解决方案(100x)(见配方)
  59. 布拉德福德解决方案(见配方)
  60. 5x蛋白质加载缓冲液(参见食谱)

注意:来自任何合格公司的类似试剂都适用于本实验。

设备

  1. 移液器
  2. 镊子(Fisher Scientific,目录号:22-327379)
  3. 分光光度计(WPA,Biowave)
  4. 250毫升锥形瓶(锥形瓶)
  5. 轨道摇床(Qilinbeier,型号:TS-1)
  6. 冷冻干燥机(北京松原华兴,型号:LGJ-10)
  7. Microfuge(Eppendorf,型号:5415 D)
  8. 微波炉
  9. 小砂浆和杵
  10. 水浴(上海益恒,型号:DK-8D)**
  11. 分析天平(Mettler Toledo,型号:PB153-L)**
  12. pH计(Mettler Toledo,型号:FE20)**
  13. Mini-PROTEAN ® Tetra Vertical Electrophoresis Cell(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Vertical Electrophoresis Cell,目录号:1658004)

注意:任何合格公司的类似设备都适合本实验。

软件

  1. SPSS统计

程序

  1. 根系相关APase活性的量化和释放到生长培养基中的APase的活性
    1. 根相关APase活性的量化
      1. 将种子用5%次氯酸钠溶液/0.1%Triton X-100悬浮,并在室温下温育10分钟,偶尔倒置进行表面灭菌。
      2. 取出液体,用灭菌的ddH 2 O洗涤种子4次
      3. 将含有全强度或半强度Murashige和Skoog(MS)培养基的每个9cm直径的培养皿的上部水平放置20-30个表面灭菌的拟南芥种子(P +或P-中)。对于每个生物样本,使用三个重复
      4. 保持培养皿在4°C 2天分层
      5. 将培养皿垂直放置在受控的环境生长室中,并允许幼苗在16小时光/8小时黑暗方案(90μmol光子 sec < sup> -1 )在22-23℃
      6. 用剃须刀切割根并测量根长度。在P +培养基上生长的拟南芥幼苗的平均根长度为4.0-5.5厘米,在P-培养基上生长的幼苗的长度为2.5-4.0厘米。
      7. 将两根镊子浸入650微升预热(37℃)的1.7ml Eppendorf管中的反应缓冲液中。
      8. 快速加入50μlpNPP溶液。
      9. 在37°C孵育1-4小时。
      10. 加入100μl0.4 N NaOH,用手摇动停止反应。
      11. 用镊子去除根部,并用分光光度计测量剩余溶液中410 nm处的吸光度(A <410>)。
      12. 计算与根相关的APase活动为A 410//root/h(图1A)。


        图1.由拟南芥(WT)和各种单,双和三重(或)紫色磷酸酶分泌的APase活性的分析)突变体在P +或P-培养基中或培养基中生长。使用pNPP作为底物测定培养基(B)中的根相关APase活性(A)和APase活性。价值观是SE的手段。对整个数据集进行单因素方差分析,使用SPSS Tukey HSD检验进行比较,比较 0.05。在四个地块的每一个中,具有不同字母的值显着不同。改编自Wang等人。 (2014)。

    2. 将蛋白质浓缩在液体生长培养基中
      1. 转移大约150只9日龄的幼苗,在P +固体培养基上用高压灭菌镊子垂直生长至含有100ml P +或P-液体培养基的250ml高压灭菌锥形瓶中。
      2. 将烧瓶放置在轨道振荡器(40rpm/min)上,并允许幼苗在16小时光/8小时黑暗方案(90μmol光子)下生长6-7天。 sec -1 )在22-23°C
      3. 从烧瓶中取出所有幼苗,并将液体介质冷冻至-20°C
      4. 将介质放在预冷冻式冷冻干燥机的支架上,盖上盖子,并将介质冻干3-4天。
      5. 使用5-6ml的ddH 2 O彻底清洗烧瓶的底部,并将洗涤液转移到透析管上。
      6. 在4℃下对样品进行24小时透析4升预冷ddH 2 O,每6小时更换ddH 2 O。
      7. 将透析的样品转移到2 ml管中,并以1,500 x g离心5分钟。
      8. 将上清液转移到50ml Falcon管中,并将液体冷冻至-20℃,然后如步骤A2d所述将其冻干2天。
      9. 将冻干的样品溶解于200μl的ddH 2 O。
      10. 使用如步骤A3所述的Bradford方法测量蛋白质浓度。
      11. 浓缩的分泌蛋白质可以立即使用,也可以储存在-80℃进行进一步分析。
    3. 蛋白质浓度的测定
      1. 移取0,2,4,8,12,16或20μl1μg/μlBSA至1.7ml Eppendorf管中,加入ddH 2 O至终体积为20μl。 >
      2. 向每个管中加入980μlBradford溶液,并充分混匀
      3. 在室温下孵育5分钟。
      4. 用分光光度计测量595nm处的吸光度(A 595 )
      5. 通过绘制BSA内容和A 595 制作Bradford标准曲线。
      6. 向980μlBradford溶液中加入20μl浓缩分泌的蛋白质。
      7. 混匀,室温孵育5分钟
      8. 用分光光度计测量A 595。
      9. 将得到的A 595值与Bradford标准曲线进行比较,以确定各种样品的蛋白质浓度。
    4. 生长培养基中APase活性的定量
      1. 将从液体生长培养基中收集的10μl浓缩的蛋白质(约2.5μg)加入到在1.7ml Eppendorf管中的590μl反应缓冲液中。对于每个样品,至少进行三次重复。
      2. 向反应混合物中加入80μl的pNPP溶液
      3. 在37℃孵育1小时。
      4. 加入120μl0.4 N NaOH终止反应。
      5. 用分光光度计测量A <410>
      6. 计算生长培养基中的APase活性为Aβ410/mg蛋白/分钟(图1B)。

  2. 使用组织化学染色分析根相关APase活性
    1. 在P +或P- MS培养基上垂直栽培幼苗6-7天,如步骤A1a-A1e所述。
    2. 向250ml锥形瓶中加入100ml ddH 2 O和0.5g琼脂,轻轻摇动烧瓶,然后在微波炉中加热烧瓶直到琼脂完全溶解。
    3. 加入1ml BCIP储备液(100x),用手短暂摇匀。
    4. 将BCIP /琼脂溶液保持在室温或使用流动的水将溶液冷却几分钟至约40℃。
    5. 将BCIP /琼脂溶液保存在40°C水浴中
    6. 在一排拟南芥根部上均匀地覆盖约5ml BCIP /琼脂溶液,并在室温下孵育约24小时(图2)。


      图2.关于如何将BCIP /琼脂溶液覆盖在培养皿上生长的拟南芥幼苗的根表面的示范

    7. 在P-培养基上生长的拟南芥的根将被染成蓝色,而在P +培养基上生长的这些根将保持未染色(图3)。


      图3.根相关APase活性的组织化学染色上排:在没有BCIP染色的MS P +和P-培养基上生长的7天龄拟南幼苗的形态。底行:其根用0.01%BCIP/0.5%琼脂溶液染色24小时的幼苗。在下排中,在P培养基(右下方)生长但不在P +培养基上生长的幼苗(左下)的幼苗的根上检测到根相关的APase活性(如蓝色所示)。
    8. APase组织化学染色受BCIP浓度的影响很大。高浓度的BCIP(0.08%-0.16%)可引起过度染色,这可能掩盖分泌的APase活性水平的差异(图4)。


      图4.不同浓度的BCIP对在MS-P培养基上生长的7天龄拟南芥幼苗的根相关APase活性的染色的影响使用的BCIP在左下角显示。 nop1-1 和 nop1-2 是在AtPAP10基因中具有突变的拟南芥突变体。随着BCIP浓度的增加,其他根系相关APase(主要是AtPAP12和AtPAP26)的活性也被检测到。改编自Wang等人。 (2011)。

  3. 分析释放到生长培养基中的APase同种型(凝胶测定)
    通过在SDS-PAGE(变性凝胶)或Native-PAGE(非变性凝胶)上分离蛋白质样品,观察释放到生长培养基中的APase的同功酶谱,然后用显色底物β-NAP和快速黑色K对于Native-PAGE分析,从凝胶和运行缓冲液中省去SDS 根据下表1制备10%分离凝胶和5%堆积凝胶(SDS-PAGE)
    表1.用于分离凝胶和堆叠凝胶的配方
    库存解决方案
    <5>堆积凝胶(5 ml)
    10%分离凝胶(10 ml)
    30%丙烯酰胺/双溶液
    0.83 ml
    3.3 ml
    1.5M Tris-HCl,pH8.8
    -
    2.5 ml
    1M Tris-HCl,pH6.8。
    0.63 ml
    -
    10%SDS
    0.05 ml
    0.1 ml
    10%(NH 4)2 2 2
    0.05 ml
    0.1 ml
    TEMED
    0.005 ml
    0.004 ml
    ddH 2 O
    3.40 ml
    4.0 ml

    1. 根据Bio-Rad说明设置SDS-PAGE凝胶运行系统( http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf )。
    2. 加入1x预冷却至4℃的蛋白质运行缓冲液
    3. 彻底混合2.5μg分泌蛋白与1/5体积的5x蛋白加载缓冲液,并将样品载于SDS-PAGE凝胶上。
      注意:不要加热样品。
    4. 在60V下分离蛋白质30分钟,然后120V,150分钟(均为4℃)
    5. 小心地将凝胶转移到容器中的预冷却(4℃)ddH 2 O,并轻轻摇动10分钟以从凝胶中除去SDS。
    6. 更换预冷(4℃)ddH 2 O并重复4次。
    7. 加入50ml反应缓冲液,并在4℃下轻轻摇动15分钟。
    8. 更换反应缓冲液并重复4次
    9. 加入50毫升APase活性分析检测缓冲液,并在37℃下温和搅拌孵育1-3小时。 APase的同功酶谱可视化为凝胶上的红棕色带(图5)

      图5.生长培养基中APase活性的谱。通过SDS-PAGE(A)或天然PAGE(B)分离2.5μg量的分泌蛋白,并用0.3mg/ml β-NAP和0.5mg/ml Fast Black K.蛋白质分子标记位于(A)中凝胶的左侧。每个面板右侧的数字表示不同的APase同种型。改编自Wang等人。 (2014)。

笔记

  1. BCIP /琼脂溶液的温度对于检测根相关的APase活性很重要。如果溶液太热(高于60°C),则会损害拟南芥根系,并使根表面上的APase失活。
  2. 去除SDS对于获得APase同功酶的清晰概况至关重要。

食谱

  1. Murashige和Skoog(MS)培养基(P +和P- MS培养基)
    P + MS培养基是全强度MS培养基(Murashige and Skoog,1962)。全强度MS培养基的配方如下所示。对于P-MS培养基,将P + MS培养基中的1.25mM KH 2 PO 4 N 4替换为1.25mM K 2 SO 4 。在121℃高压灭菌P +和P-培养基10分钟,并将培养基储存在室温下
    NH 4< 3>< 3>
    20.62  mM
    CuSO 4
    0.1μM
    KNO 3
    18.79 mM
    Fe-EDTA
    100μM
    CaCl 2
    2.99  mM
    Myo肌醇
    0.55μM
    MgSO 4
    1.5 mM
    烟酸
    4.1μM
    KH 2 PO 4
    1.25 mM
    吡哆醇盐酸盐
    2.4μM
    K> SO SO SO SO>
    0.625 mM
    盐酸硫胺素
    0.3μM
    KI
    5μM
    大豆
    30μM
    3 <3> 3
    100μM


    MnSO 4
    92.5μM
    蔗糖
    10克/升
    ZnSO 4
    30μM
    MES
    5.1 g/L
    NaMoO 4
    1μM
    pH
    5.8
    CoCl 2
    0.1μM
    琼脂
    10克/升

  2. APase活性分析检测缓冲液,pH 4.9(新鲜制备)
    0.3mg/mlβ-萘酸磷酸盐
    0.5 mg/ml Fast Black K
    10mM MgCl 2
    50 mM NaAc
  3. 反应缓冲液,pH 4.9
    10mM MgCl 2
    50 mM NaAc
    储存于4°C
  4. pNPP溶液(1.0mg/ml)
    将10mg量的pNPP溶解在10ml的反应缓冲液
    中 在-20°C储存等分试样
  5. BCIP库存解决方案(100x)
    BCIP储液的浓度为10g/L,工作液的浓度为0.1g/L 准备BCIP库存解决方案:
    1. 将0.1g BCIP粉末溶于8ml ddH 2 O中,然后加入约14滴1N NaOH直至BCIP功率完全溶解。
    2. 加入ddH 2 O至总体积为10ml,然后过滤溶液以除去未溶解的BCIP颗粒
    3. 在-20°C储存等分试样
  6. 布拉德福德解决方案
    将100毫克考马斯亮蓝G-250溶于50ml 95%乙醇中 加入100ml 85%H 3 PO 4,然后加入ddH 2 O至1,000ml
    通过Whatman#1滤纸,并将滤液在4℃下储存在棕色瓶中
  7. 5x蛋白加载缓冲液
    250mM Tris-HCl,pH6.8
    10 mM DTT
    10%SDS
    30%(v/v)甘油 0.05%(w/v)溴酚蓝
    将缓冲液储存在室温下

致谢

这项工作得到了中国国家自然科学基金(授权号31370290和30670170)的支持。

参考文献

  1. Lloyd,JC,Zakhleniuk,OV和Raines,CA(2001)。  鉴定磷代谢中的突变体。 Annals Applied Biol 138:111-115。
  2. Murashige,T和Skoog,F.(1962)。  用于烟草组织培养物的快速生长和生物测定的修订培养基。生物学植物化学 15(3):473-497。
  3. Tomscha,JL,Trull,MC,Deikman,J.,Lynch,JP和Guiltinan,MJ(2004)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih。磷酸酶生物碱突变体具有改变的磷关系。植物生理学135(1):334-345。
  4. Tran,HT,Hurley,BA和Plaxton,WC(2010)。 
  5. Trull,MC和Deikman,J.(1998)。  一个拟南芥突变体缺失一个酸性磷酸酶同种型。植物 206(4):544-550。
  6. Vicki,K.和William,P。(2013)。蛋白质提取,酸性磷酸酶活性测定和可溶性蛋白质浓度的测定。生物方案 e889。
  7. 王丽丽,李,,,钱,吴国强,高尧,黄,,王,,朱,,吴,,Wang and and Liu ,D.(2011)。拟南芥紫色磷酸酶AtPAP10主要与根表面相关,并且在磷酸盐限制的植物耐受性中起重要作用。植物生理学157(3):1283-1299。
  8. Wang,L.,Lu,S.,Zhang,Y.,Li,Z.,Du,X. and Liu,D。(2014)。拟南芥紫色酸性磷酸酶的比较遗传分析AtPAP10,AtPAP12和AtPAP26为其在植物中的作用提供了新的见解适应磷酸盐剥夺。 J Integr Plant Biol 56(3):299-314。
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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. Wang, L. and Liu, D. (2017). Analyses of Root-secreted Acid Phosphatase Activity in Arabidopsis. Bio-protocol 7(7): e2202. DOI: 10.21769/BioProtoc.2202.
  2. Wang, L., Li, Z., Qian, W., Guo, W., Gao, X., Huang, L., Wang, H., Zhu, H., Wu, J. W., Wang, D. and Liu, D. (2011). The Arabidopsis purple acid phosphatase AtPAP10 is predominantly associated with the root surface and plays an important role in plant tolerance to phosphate limitation. Plant Physiol 157(3): 1283-1299.
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