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Polyamine and Paraquat Transport Assays in Arabidopsis Seedling and Callus
拟南芥幼苗和愈伤组织中多胺和百草枯转运测定   

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Abstract

Polyamines (PAs) are polycationic compounds found in all living organisms and play crucial roles in growth and survival. We here show the ‘Polyamine and paraquat (PQ) transport assay’ protocol, which can be used to examine the uptake activity of PA/PQ transporters. We have used this protocol to demonstrate that PUT3 in Arabidopsis is a polyamine transporter and is able to take up spermidine and its analog paraquat.

Keywords: Arabidopsis thaliana(拟南芥), Polyamine(多胺), Paraquat(百草枯), Transport(运输), Uptake(摄取)

Background

PAs are involved in gene regulation by interacting with and modulating the functions of anionic macromolecules such as DNA, RNA and proteins. In living cells, PAs’ contents must be regulated to maintain the cellular hemostasis. In higher plants, three major polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm), are present in either free form or conjugated forms with other molecules (Gill and Tuteja, 2010). In yeast, four plasma membrane polyamine transporters, DUR3, SAM3, GAP1 and AGP2 were identified (Uemura et al., 2007). In Arabidopsis, five putative polyamine uptake transporters (PUT1-PUT5) were identified and PUT1-3 have been experimentally validated as polyamine transporters (Mulangi et al., 2012; Li et al., 2013). Our protocol described below has successfully confirmed that PUT3 is an influx transporter for polyamines and paraquat, and PQ/Spd uptake is impaired in the put3 mutant (Shen et al., 2016).

Materials and Reagents

  1. Pipette tips
  2. Plastic Petri dish (VWR, catalog number: 25384-326 )
  3. Parafilm
  4. 1.5 ml Eppendorf centrifuge tube (VWR, catalog number: 20170-355 )
  5. Filter paper
  6. Blue pestle (DWK Life Sciences, Kimble, catalog number: 749521-1500 )
  7. Cuvette (VWR, catalog number: 414004-051 )
  8. Syringe filters, 0.2 µm pore size (VWR, catalog number: 28145-475 )
  9. Syringe
  10. Arabidopsis thaliana ecotype Columbia (Col-0) and mutant line lhr1 (put3)
  11. ScintiVerseTM BD Cocktail (Fisher Scientific, catalog number: SX18-4 )
  12. Clorox Bleach
  13. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  14. (2,4-Dichlorophenoxy) acetic acid sodium salt monohydrate (Sigma-Aldrich, catalog number: D6679 )
  15. Kinetin (Duchefa Biochemie, catalog number: K0905 )
  16. Sodium hydroxide (NaOH)
  17. Murashige & Skoog Basal Salt Mixture (PhytoTechnology Laboratories, catalog number: M524 )
  18. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  19. Agar (Sigma-Aldrich, catalog number: A1296 )
  20. Low melting point agarose (Sigma-Aldrich, catalog number: A9414 )
  21. Methyl viologen dichloride hydrate (Sigma-Aldrich, catalog number: 856177 )
  22. Spermidine (MP Biomedicals, catalog number: 02152068 )
  23. Paraquat-methyl-14C dichloride hydrate (Sigma-Aldrich, catalog number: 313947 )
    Note: This product has been discontinued.
  24. Spermidine trihydrochloride [Terminal Methylenes-3H (N)] (PelkinElmer, catalog number: NET522001MC )
  25. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  26. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  27. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
    Note: This product has been discontinued.
  28. Seed sterilization solution (see Recipes)
  29. 2,000x 2,4-D (9.05 mM) (see Recipes)
  30. 2,000x kinetin (1.86 mM) (see Recipes)
  31. Callus induction solid medium (see Recipes)
  32. Callus induction liquid medium (see Recipes)
  33. ½ MS solid medium (see Recipes)
  34. ½ MS liquid medium (see Recipes)
  35. 200 µM non-14C-labeled PQ solution (see Recipes)
  36. 2.04 mM non-3H-labeled Spd solution (see Recipes)
  37. 40.37 µM non-3H-labeled Spd solution (see Recipes)
  38. Solution 1: 14C-labeled PQ solution (see Recipes)
  39. Solution 2: 14C-labeled PQ solution (see Recipes)
  40. Solution 3: 3H-labeled Spd solution (see Recipes)
  41. Solution 4: 3H-labeled Spd solution (see Recipes)
  42. Solution 5: 3H-labeled Spd solution (see Recipes)
  43. Solution 6: 3H-labeled Spd solution (see Recipes)
  44. 1 M Tris-HCl, pH 7.5 (see Recipes)
  45. 0.5 M EDTA, pH 8.0 (see Recipes)
  46. Crude protein extraction buffer (see Recipes)

Equipment

  1. Pipettes
  2. pH meter
  3. Weighing balance
  4. Laminar flow hood
  5. Stirring bar
  6. Magnetic stirrer (VWR, model: 200 Mini-stirrer )
  7. Vortex (Fisher Scientific, model: Vortex-Genie 2 )
  8. Scintillation counter (Beckman Coulter, model: LS-6500 )
  9. Centrifuge (Beckman Coulter, model: Microfuge® 22R , catalog number: 368831)
  10. Spectrophotometer (Bio-Rad Laboratories, model: SmartSpec Plus, catalog number: 1702525 )
    Note: This product has been discontinued.
  11. Autoclave

Procedure

  1. Preparation of callus cells and plant seedlings
    1. Callus cells
      1. Put the Arabidopsis seeds in seed sterilization solution (see Recipes) for 15-20 min. Discard the sterilization solution and resuspend the seeds with sterilized ddH2O in a laminar flow hood. Let the seeds settle down and discard the solution. Repeat the washing step 5 times.
      2. Sow the surface-sterilized seeds in a row on callus induction medium (see Recipes) in a laminar flow hood, seal the Petri dishes with Parafilm and incubate the Petri dishes at 4 °C in the dark for 2 days.
      3. Place the Petri dishes in a horizontal position and let the seeds germinate and grow under 16 h light/8 h dark cycle (lighting provided by fluorescent bulbs giving an average light intensity of ~150 µmol/m2/sec) at 22 °C for 3 weeks.
      4. Transfer induced callus (Figure 1) to new callus induction medium in a laminar flow hood every 3 weeks until the transport assay (see Note 1).


        Figure 1. 3-week-old induced callus cells and 2-week-old Arabidopsis seedlings. Bars = 1 cm.

    2. Plant seedlings
      1. Put the Arabidopsis seeds in seed sterilization solution for 15-20 min. Discard the sterilization solution and resuspend the seeds with sterilized ddH2O in a laminar flow hood. Let the seeds settle down and discard the solution. Repeat the washing step 5 times.
      2. Sow the surface-sterilized seeds in a row on ½ MS medium (see Recipes) in a laminar flow hood, seal the Petri dishes with Parafilm and incubate the Petri dishes at 4 °C in the dark for 2 days.
      3. Place the Petri dish in a vertical position and let the seeds germinate and grow under 16 h light/8 h dark cycle (lighting provided by fluorescent bulbs giving an average light intensity of ~150 µmol/m2/sec) at 22 °C for 2 weeks (Figure 1).

  2. Generation of standard curve
    1. Standard curve for 14C-labeled PQ
      1. Make a 50x and 500x dilution of 10 µl solution 2 (see Recipes) with ddH2O.
      2. In 1 ml of the ScintiVerseTM BD Cocktail, add 0 µl, 25 µl, 125 µl and 250 µl of 500x diluted solution to make 14C-labeled PQ solutions with 0 nCi, 0.1 nCi, 0.5 nCi, 1 nCi radioactivity, and add 125 µl and 250 µl of 50x diluted solution to make 14C-labeled PQ with 5 nCi and 10 nCi radioactivity, respectively.
      3. Measure the radioactivity by using Beckman LS-6500 scintillation counter to generate a standard curve for 14C-labeled PQ.
      4. Perform 3 sets of this test to ensure accuracy of the standard curve (Figure 2).


        Figure 2. Representative of standard curves for radioactive labeled PQ and Spd

    2. Standard curve for 3H-labeled Spd
      1. Make a 100x dilution of 10 µl solution 3 (see Recipes) with ddH2O.
      2. In 1 ml of the ScintiVerseTM BD Cocktail, add 0 µl, 2.5 µl, 5 µl, 25 µl, 50 µl and 250 µl 100x diluted solution to make 3H-labeled Spd solutions with 0 nCi, 0.5 nCi, 1 nCi, 5 nCi, 10 nCi and 50 nCi radioactivity, respectively.
      3. Measure the radioactivity by using Beckman LS-6500 scintillation counter to generate a standard curve for 3H-labeled Spd.
      4. Perform 3 sets of this test to ensure accuracy of the standard curve (Figure 2).

  3. Transport assays
    1. Transport assays with callus (Table 1)

      Table 1. Designed temperatures and Spd or PQ concentrations used in transport assay of callus cells


      1. Transfer 100 mg fresh callus cells into a 1.5 ml Eppendorf centrifuge tube containing 950 µl liquid callus-inducing medium and vortex for 30 sec.
      2. Pre-treat samples at designed temperature for 30 min to reach equilibrium (Shen et al., 2016).
      3. Add 50 µl solution 2 (final PQ concentration at 10 µM), solution 3 (final Spd concentration at 2 µM) or solution 4 (final Spd concentration at 100 µM) (see Recipes) into the tubes according to the treatment conditions (see Note 2).
      4. Briefly vortex for mixing and return the tubes back to the designed temperature.
      5. Incubate for 1 h, gently invert each tube by hand every 15 min to resuspend the cells and ensure the equilibrium of PQ or Spd in the solution.
      6. Place tubes on ice immediately.
      7. Let the callus cells settle down and remove the PQ- or Spd-containing liquid callus inducing medium by using pipette.
      8. Wash callus cells five times on ice each with 1.5 ml pre-chilled liquid callus inducing medium. For each treatment, three replicate samples should be used at the same time.
    2. Transport assays with seedlings (Table 2)

      Table 2. Designed temperatures and Spd or PQ concentrations used in transport assay of plant seedlings


      1. Transfer 20 seedlings of both wild type Col-0 and put3 mutant line with intact roots onto a filter paper saturated with 5 ml liquid ½ MS medium (see Recipes), and pre-incubate the seedlings at the designed temperature for 30 min (see Note 3).
      2. Mix 250 µl solution 2 (final PQ concentration at 10 µM), solution 5 (final Spd concentration at 2 µM) (see Recipes), or solution 6 (final Spd concentration at 100 µM) (see Recipes) with 4.75 ml ½ MS medium to reach the final concentrations.
      3. Saturate another piece of filter paper with the radioactive solution made in step C2b and pre-incubate it at the designed temperature for 30 min.
      4. Transfer pre-incubated seedlings in the step C2a one by one to the filter paper containing radioactive-labeled PQ or Spd and pre-incubate at the same temperature as stated in the step C2c. Carefully arrange the seedlings to make sure all roots are directly in contact with the filter paper.
      5. Incubate at designed temperature for 1 h.
      6. Cut off roots of the seedlings from each treatment.
      7. Transfer the roots from each treatment into a 1.5 ml Eppendorf centrifuge tube.
      8. Wash the roots 5 times on ice by using 1.5 ml pre-chilled liquid ½ MS medium. For each treatment, three replicate samples should be used at the same time.

  4. Extraction of intracellular contents
    1. Grind washed callus cells or seedling roots by using small blue pestle in 1 ml (for callus cells) or 500 µl (for seedling roots) crude protein extracting buffer (see Recipes) on ice until no clumps remain to release all intracellular contents. The solution should be homogenized to an even mixture that can be pipetted without clogs.
    2. Centrifuge the mixture at 15,500 x g for 20 min at 4 °C.
    3. Transfer supernatant into a new tube for radioactive and protein concentration measurements.
    4. Measure the UV absorbance of 300 µl (for callus cells) or 100 µl (for seedling roots) of the supernatant of each sample by using Bio-Rad SmartSpec Plus Spectrophotometer. The total protein concentration is then calculated based on the UV absorbance using the equation:

      Protein concentration (mg/ml) = (1.55 x A280) - (0.76 x A260)

  5. Radioactive measurement
    1. Add 300 µl (for callus cells) or 100 µl (for seedling roots) of the supernatant of each sample into 1 ml ScintiVerseTM BD Cocktail solution.
    2. Measure the radioactivity of each sample by using Beckman LS-6500 scintillation counter.

Data analysis

The radioactivity (nCi) of each sample was calculated based on the scintillation counter reading (CPM) and the equation of the corresponding standard curve. The total amount of transported PQ/Spd in each sample was then calculated based on the radioactivity and the used nCi per nmol in the transport assay solution. The total protein of each sample was calculated based on the protein concentration and the volume. The PQ/Spd transport rates were presented in the unit of µmole PQ/Spd per gram protein per hour (µmole g-1 h-1). The PQ/Spd transport rate at 0 °C of each sample was considered as non-specific binding of PQ/Spd, and therefore was subtracted from the PQ/Spd transport rates at 25 °C to calculate the adjusted and accurate PQ/Spd transport rates at normal condition. Readers are referred to Shen et al. (2016) for examples of data graphs.

Notes

  1. We maintained the callus cells by transferring fresh and fast-growing callus cells to a new callus induction medium every 3 weeks. By judging the color and growth of the callus cells, it is easy to select the callus cells for transfer. Fresh calli usually show light color whereas relative old calli show dark color.
  2. 2 µM Spd was designed to test if the transporter is a high-affinity PA transporter whereas 100 µM Spd was designed to test if other transporters are also working at high Spd concentrations. In our test, the callus cells and seedlings of the put3 mutant showed approximately 20-fold and 4-fold reduction, respectively, on 2 µM Spd uptake rate compared with the wild type. However, when a higher concentration (100 µM) of Spd was added into the medium, the transport rate was comparable in both put3 mutant and wild type. This might suggest that PUT3 protein is a high-affinity PA transporter and some low-affinity PA transporters may start to work at high PA concentrations.
  3. In the transport assay with seedlings, we used 150 x 15 mm Petri dish and put a filter paper fitting into the Petri dish. Seedlings were arranged relatively separate.

Recipes

  1. Seed sterilization solution
    25% Clorox Bleach
    0.05% Triton X-100
    Store at room temperature
  2. 2,000x 2,4-D (9.05 mM)
    26 mg (2,4-dichlorophenoxy) acetic acid sodium salt monohydrate
    Add ddH2O to 11 ml and sterilize by filtration through a syringe filter (0.2 µm)
    Store at -20 °C
  3. 2,000x kinetin (1.86 mM)
    10 mg kinetin
    1 ml 1 N NaOH
    Add ddH2O to 25 ml and sterilize by filtration through a syringe filter (0.2 µm)
    Store at -20 °C
  4. Callus induction solid medium
    4.33 g/L Murashige & Skoog basal salts
    3% sucrose
    Adjust pH to 5.7 with 0.1 N NaOH and add agar to 0.7% (w/v)
    Autoclave
    Cool medium to ~60 °C
    Add 2,4-D to a final concentration at 4.52 µM
    Add Kinetin to a final concentration at 0.93 µM
    Store at room temperature
  5. Callus induction liquid medium
    4.33 g/L Murashige & Skoog basal salts
    3% sucrose
    Adjust pH with 0.1 N NaOH to 5.7 and autoclave
    Cool medium to ~60 °C
    Add 2,4-D to a final concentration at 4.52 µM
    Add Kinetin to a final concentration at 0.93 µM
    Store at room temperature
  6. ½ MS solid medium
    2.17 g/L Murashige & Skoog basal salts
    1.5% sucrose
    Adjust pH to 5.7 with 0.1 N NaOH and add agar to 1.2% (w/v)
    Autoclave
    Store at room temperature
  7. ½ MS liquid medium
    2.17 g/L Murashige & Skoog basal salts
    1.5% sucrose
    Adjust pH with 0.1 N NaOH to 5.7 and autoclave
    Store at room temperature
  8. 200 µM non-14C-labeled PQ solution
    5.14 mg methyl viologen dichloride hydrate
    100 ml ddH2O
    Store at -20 °C
  9. 2.04 mM non-3H-labeled Spd solution
    3.20 µl spermidine
    10 ml ddH2O
    Store at -20 °C
  10. 40.37 µM non-3H-labeled Spd solution
    198 µl 2.04 mM spermidine
    9.8 ml ddH2O
    Store at -20 °C
  11. Solution 1: 14C-labeled PQ solution (200 µM, 32.3 nCi/nmol, 15.55 ml)
    0.8 mg paraquat-methyl-C14 dichloride
    15.55 ml ddH2O
    Store at -20 °C
  12. Solution 2: 14C-labeled PQ solution (200 µM, 10 nCi/nmol, 1 ml)
    309.6 µl solution 1
    690.4 µl 200 µM non-14C-labeled PQ solution
    Store at -20 °C
  13. Solution 3: 3H-labeled Spd solution (40 µM, 500 nCi/nmol, 1 ml)
    20 µl spermidine trihydrochloride, [terminal methylenes-3H (N)] - solution
    980 µl 40.37 µM non-3H-labeled Spd solution
    Store at -20 °C
  14. Solution 4: 3H-labeled Spd solution (2 mM, 10 nCi/nmol, 1 ml)
    20 µl spermidine trihydrochloride, [terminal methylenes-3H (N)] - solution
    980 µl 2.04 mM non-3H-labeled Spd solution
    Store at -20 °C
  15. Solution 5: 3H-labeled Spd solution (40 µM, 250 nCi/nmol, 1 ml)
    500 µl solution 3
    500 µl 40 µM non-3H-labeled Spd solution
    Store at -20 °C
  16. Solution 6: 3H-labeled Spd solution (2 mM, 5 nCi/nmol, 1 ml)
    500 µl solution 4
    500 µl 2 mM non-3H-labeled Spd solution
    Store at -20 °C
  17. 1 M Tris-HCl, pH 7.5
    121.1 g Trizma® base
    800 ml ddH2O
    Adjust pH to 7.5 with concentrated HCl
    Add ddH2O to 1,000 ml, autoclave
    Store at room temperature
  18. 0.5 M EDTA, pH 8.0
    186.1 g ethylenediaminetetraacetic acid disodium salt dihydrate
    800 ml ddH2O
    Adjust pH to 8.0 with NaOH (about 20 g)
    Add ddH2O to 1,000 ml, autoclave
    Store at room temperature
  19. Crude protein extraction buffer
    50 mM Tris-HCl (pH 7.5)
    0.1 mM EDTA
    1 mM β-mercaptoethanol
    Make fresh solution and store at room temperature

Acknowledgments

This work was partly supported by the US Department of Agriculture National Research Initiative competitive grant 2007-35100-18378 to H.S. and by the National Natural Science Foundation of China (Grant # 31328004) to H.S.

References

  1. Gill, S. S. and Tuteja, N. (2010). Polyamines and abiotic stress tolerance in plants. Plant Signal Behav 5(1): 26-33.
  2. Li, J., Mu, J., Bai, J., Fu, F., Zou, T., An, F., Zhang, J., Jing, H., Wang, Q., Li, Z., Yang, S. and Zuo, J. (2013). Paraquat Resistant1, a Golgi-localized putative transporter protein, is involved in intracellular transport of paraquat. Plant Physiol 162(1): 470-483.
  3. Mulangi, V., Chibucos, M. C., Phuntumart, V. and Morris, P. F. (2012). Kinetic and phylogenetic analysis of plant polyamine uptake transporters. Planta 236(4): 1261-1273.
  4. Shen, Y., Ruan, Q., Chai, H., Yuan, Y., Yang, W., Chen, J., Xin, Z. and Shi, H. (2016). The Arabidopsis polyamine transporter LHR1/PUT3 modulates heat responsive gene expression by enhancing mRNA stability. Plant J 88(6): 1006-1021.
  5. Uemura, T., Kashiwagi, K. and Igarashi, K. (2007). Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae. J Biol Chem 282(10): 7733-7741.

简介

多胺(PA)是在所有生物体中发现的聚阳离子化合物,在生长和生存中起关键作用。 我们在这里展示了“多胺和百草枯(PQ)运输测定”方案,可用于检查PA / PQ转运蛋白的摄取活性。 我们已经使用该协议来证明拟南芥中的PUT3是多胺转运蛋白,并且能够吸收亚精胺及其类似百草枯。
【背景】PA通过与阴离子大分子如DNA,RNA和蛋白质的功能相互作用和调节而参与基因调控。 在活细胞中,必须调节PA的含量以维持细胞止血。 在高等植物中,三种主要的多胺,腐胺(Put),亚精胺(Spd)和精胺(Spm)以游离形式或缀合形式与其他分子一起存在(Gill和Tuteja,2010)。 在酵母中,鉴定了四种质膜多胺转运体DUR3,SAM3,GAP1和AGP2(Uemura等人,2007)。 在拟南芥中,鉴定了5种推定的多胺摄取转运蛋白(PUT1-PUT5),并且PUT1-3已被实验验证为多胺转运蛋白(Mulangi等人,2012; Li et al,2013)。 我们以下所述的方案成功证实了PUT3是多胺和百草枯的流入转运蛋白,并且在Put3突变体(Shen等人,2016)中PQ /Spd吸收受损)。

关键字:拟南芥, 多胺, 百草枯, 运输, 摄取

材料和试剂

  1. 移液器提示
  2. 塑料培养皿(VWR,目录号:25384-326)
  3. 石蜡膜
  4. 1.5ml Eppendorf离心管(VWR,目录号:20170-355)
  5. 滤纸
  6. 蓝杵(DWK Life Sciences,Kimble,目录号:749521-1500)
  7. 比维埃(VWR,目录号:414004-051)
  8. 注射器过滤器,0.2μm孔径(VWR,目录号:28145-475)
  9. 注射器
  10. 拟南芥生态型哥伦比亚(Col-0)和突变体株系lhr1( put3 )
  11. ScintiVerse TM BD Cocktail(Fisher Scientific,目录号:SX18-4)
  12. Clorox Bleach
  13. Triton X-100(Sigma-Aldrich,目录号:T8787)
  14. (2,4-二氯苯氧基)乙酸钠盐一水合物(Sigma-Aldrich,目录号:D6679)
  15. Kinetin(Duchefa Biochemie,目录号:K0905)
  16. 氢氧化钠(NaOH)
  17. Murashige& Skoog基础盐混合物(PhytoTechnology Laboratories,目录号:M524)
  18. 蔗糖(Sigma-Aldrich,目录号:S0389)
  19. 琼脂(Sigma-Aldrich,目录号:A1296)
  20. 低熔点琼脂糖(Sigma-Aldrich,目录号:A9414)
  21. 甲基紫精二氯化物水合物(Sigma-Aldrich,目录号:856177)
  22. 亚精胺(MP Biomedicals,目录号:02152068)
  23. 百草枯 - 甲基 - 二氯化物水合物(Sigma-Aldrich,目录号:313947)
    注意:本产品已停产。
  24. 亚精胺三盐酸盐[甲基亚甲基-3H(N)](PelkinElmer,目录号:NET522001MC)
  25. Trizma ®基质(Sigma-Aldrich,目录号:T1503)
  26. 乙二胺四乙酸二钠盐二水合物(EDTA)(Sigma-Aldrich,目录号:E5134)
  27. 2-巯基乙醇(Sigma-Aldrich,目录号:M7522)
    注意:本产品已停产。
  28. 种子灭菌方案(见食谱)
  29. 2,000x 2,4-D(9.05mM)(参见食谱)
  30. 2,000x激动素(1.86mM)(参见食谱)
  31. 愈伤组织诱导固体培养基(参见食谱)
  32. 愈伤组织诱导液介质(参见食谱)
  33. ½MS固体培养基(参见食谱)
  34. ½MS液体介质(见配方)
  35. 200μM非标准的C标记的PQ溶液(参见食谱)
  36. 2.04mM非标记的H标记的Spd溶液(参见食谱)
  37. 40.37μM非 3 H标记的Spd溶液(参见食谱)
  38. 解决方案1: 14 C标记的PQ溶液(参见食谱)
  39. 解决方案2: 14 C标记的PQ溶液(参见食谱)
  40. 解决方案3: 3 H标记的Spd溶液(参见食谱)
  41. 解决方案4: 3 H标记的Spd溶液(参见食谱)
  42. 解决方案5: 3 H标记的Spd溶液(参见食谱)
  43. 解决方案6: 3 H标记的Spd溶液(参见食谱)
  44. 1M Tris-HCl,pH7.5(参见食谱)
  45. 0.5 M EDTA,pH 8.0(参见食谱)
  46. 粗蛋白提取缓冲液(参见食谱)

设备

  1. 移液器
  2. pH计
  3. 称重平衡
  4. 层流罩
  5. 搅拌酒吧
  6. 磁力搅拌器(VWR,型号:200小型搅拌器)
  7. 涡旋(Fisher Scientific,型号:Vortex-Genie 2)
  8. 闪烁计数器(Beckman Coulter,型号:LS-6500)
  9. 离心机(Beckman Coulter,型号:Microfuge ® 22R,目录号:368831)
  10. 分光光度计(Bio-Rad Laboratories,型号:SmartSpec Plus,目录号:1702525)
    注意:本产品已停产。
  11. 高压灭菌器

程序

  1. 愈伤组织细胞和植物幼苗的制备
    1. 愈伤组织细胞
      1. 将拟南芥种子种子种子灭菌溶液(见食谱)15-20分钟。弃去灭菌溶液,并将灭菌的ddH 2 O的种子重新悬浮在层流罩中。让种子安定下来,丢弃解决方案。重复洗涤步骤5次。
      2. 在层流罩中将表面灭菌的种子连续地播种在愈伤组织诱导培养基(参见食谱)上,用Parafilm密封培养皿,并在4℃下在黑暗中培养培养皿2天。
      3. 将培养皿放置在水平位置,使种子在16小时光/ 8小时黑暗周期内发芽并生长(由荧光灯提供的照明,平均光强度为〜150μmol/ m 2 /秒)在22℃下3周
      4. 将传递诱导的愈伤组织(图1)每3周在层流罩中的新愈伤组织诱导培养基中直至运输测定(见注1)。


        图1. 3周龄的诱导愈伤组织细胞和2周龄 拟南芥 幼苗 1厘米。

    2. 植物幼苗
      1. 将拟南芥种子种子种子灭菌溶液15-20分钟。弃去灭菌溶液,并将灭菌的ddH 2 O的种子重新悬浮在层流罩中。让种子安定下来,丢弃解决方案。重复洗涤步骤5次。
      2. 在层流罩中的½MS培养基(见食谱)上播种表面灭菌的种子,用Parafilm密封培养皿,并在4℃下在黑暗中孵育培养皿2天。
      3. 将培养皿放置在垂直位置,让种子在16小时光/ 8小时黑暗周期内发芽并生长(由荧光灯提供的照明,平均光强度为〜150μmol/ m 2 /秒)在22℃下2周(图1)

  2. 生成标准曲线
    1. 标准曲线 14 C标记的PQ
      1. 用ddH 2 O进行50x和500x稀释的10μl溶液2(参见食谱)。
      2. 在1ml的ScintiVerse TM BD Cocktail中,加入0μl,25μl,125μl和250μl500x稀释的溶液,以制备具有0〜 nCi,0.1nCi,0.5nCi,1nCi放射性,并加入125μl和250μl50x稀释的溶液,分别制备具有5nCi和10nCi放射性的 C标记的PQ。 >
      3. 使用Beckman LS-6500闪烁计数器测量放射性,以生成标准曲线。 C标记的PQ。
      4. 执行3套本次测试以确保标准曲线的准确性(图2)。


        图2.放射性标记PQ和Spd的标准曲线的代表

    2. 标准曲线 3 H标记Spd
      1. 用ddH 2 O进行100倍稀释10μl溶液3(参见食谱)。
      2. 在1ml的ScintiVerse TM BD Cocktail中,加入0μl,2.5μl,5μl,25μl,50μl和250μl100x稀释溶液,使得<分别具有0 nCi,0.5 nCi,1 nCi,5 nCi,10 nCi和50 nCi放射性的标记Spd解。
      3. 使用Beckman LS-6500闪烁计数器测量放射性,以产生 3 H标记Spd的标准曲线。
      4. 执行3套本次测试以确保标准曲线的准确性(图2)。

  3. 运输测定
    1. 运输测试与愈伤组织(表1)

      表1.在愈伤组织细胞运输测定中使用的设计温度和Spd或PQ浓度


      1. 将100毫克新鲜的愈伤组织细胞转移到含有950微升液体愈伤组织诱导培养基的1.5ml Eppendorf离心管中,并旋涡30秒。
      2. 在设计温度下预处理样品30分钟以达到平衡(Shen等人,2016)。
      3. 根据处理条件将50μl溶液2(10μM的最终PQ浓度),溶液3(2μM的最终Spd浓度)或溶液4(最终Spd浓度在100μM)(参见食谱)加入管中(见附注2)
      4. 短暂地涡流混合并将管返回设计温度。
      5. 孵育1小时,每15分钟用手轻轻倒转每个管,以重新悬浮细胞,并确保溶液中PQ或Spd的平衡。
      6. 将管立即放在冰上。
      7. 让愈伤组织沉淀下来,用移液管去除含有PQ或含Spd的液体愈伤组织诱导培养基。
      8. 在冰上洗涤愈伤组织细胞5次,每次1.5ml预冷液体愈伤组织诱导培养基。对于每个处理,应同时使用三个重复样品。
    2. 带幼苗的运输测定(表2)

      表2.植物幼苗运输测定中使用的设计温度和Spd或PQ浓度


      1. 将具有完整根的野生型Col-0和put3突变体系的20个幼苗转移到用5ml液体½MS培养基饱和的滤纸上(参见食谱),并将设计的幼苗预孵育温度30分钟(见注3)。
      2. 混合250μl溶液2(10μM的最终PQ浓度),溶液5(最终Spd浓度为2μM)(参见食谱)或溶液6(最终Spd浓度为100μM)(参见食谱)与4.75ml 1/2 MS培养基达到最终浓度。
      3. 用步骤C2b中制备的放射性溶液饱和另一张滤纸,并在设计温度下预孵育30分钟。
      4. 将步骤C2a中的预孵育的幼苗逐个转移到含有放射性标记的PQ或Spd的滤纸上,并在与步骤C2c中所述的相同温度下预孵育。小心安排苗木,确保所有根部都直接与滤纸接触。
      5. 在设计温度下孵育1小时。
      6. 每次处理切断幼苗根部。
      7. 将根从每个处理转移到1.5ml Eppendorf离心管中
      8. 使用1.5ml预冷液体½MS培养基在冰上洗涤根5次。对于每个处理,应同时使用三个重复样品。

  4. 细胞内含量的提取
    1. 通过在冰上使用1ml(用于愈伤组织细胞)或500μl(用于幼苗根)粗蛋白质提取缓冲液(参见食谱)的小蓝杵研磨洗涤的愈伤组织细胞或幼苗根,直至不存在团块以释放所有细胞内内容物。该溶液应均质化成均匀的混合物,可以移液而无堵塞。
    2. 在15℃下以15,500 x g离心混合物20分钟。
    3. 将上清液转移到新的管中进行放射性和蛋白质浓度测量
    4. 使用Bio-Rad SmartSpec Plus分光光度计测量每个样品的上清液的UV吸收值为300μl(对于愈伤组织细胞)或100μl(用于幼苗根)。然后使用以下等式,基于UV吸光度计算总蛋白浓度:

      蛋白质浓度(mg / ml)=(1.55×A280) - (0.76×A260)

  5. 放射性测量
    1. 向每个样品的上清液中加入300μl(用于愈伤组织细胞)或100μl(作为幼苗根),加入1ml ScintiVerse BD Cocktail溶液中。
    2. 使用Beckman LS-6500闪烁计数器测量每个样品的放射性

数据分析

基于闪烁计数器读数(CPM)和相应标准曲线的方程计算每个样品的放射性(nCi)。然后基于运输测定溶液中的放射性和每nmol使用的nCi计算每个样品中运输的PQ / Spd的总量。基于蛋白质浓度和体积计算每个样品的总蛋白。 PQ / Spd传输速率以每小时每克蛋白质的μmolePQ / Spd单位(μmoleg 1 h -1)表示。每个样品0℃的PQ / Spd传输速率被认为是PQ / Spd的非特异性结合,因此从25℃的PQ / Spd传输速率中减去,以计算调整和准确的PQ / Spd传输正常情况下的率。读者参考Shen <等等。(2016),了解数据图表的例子。

笔记

  1. 我们通过每3周将新鲜和快速生长的愈伤组织细胞转移到新的愈伤组织诱导培养基来维持愈伤组织细胞。通过判断愈伤组织细胞的颜色和生长,容易选择愈伤组织细胞进行转移。新鲜的愈伤组织通常显示浅色,而相对老的愈伤组织显示深色。
  2. 2μMSpd被设计用于测试转运蛋白是否是高亲和力PA转运蛋白,而100μMSpd被设计用于测试其他转运蛋白是否也以高Spd浓度工作。在我们的测试中,与野生型相比,在2μMSpd摄取速率下,put3突变体的愈伤组织细胞和幼苗分别显示约20倍和4倍的降低。然而,当将较高浓度(100μM)的Spd加入到培养基中时,在put3突变体和野生型之间的转运率是相当的。这可能表明PUT3蛋白是高亲和力PA转运蛋白,一些低亲和力PA转运蛋白可能开始在高PA浓度下工作。
  3. 在带有幼苗的运输测定中,我们使用150 x 15 mm培养皿,并将滤纸配件放入培养皿中。幼苗相对分开排列。

食谱

  1. 种子灭菌解决方案
    25%Clorox Bleach
    0.05%Triton X-100
    在室温下存放
  2. 2,000x 2,4-D(9.05mM)
    26mg(2,4-二氯苯氧基)乙酸钠盐一水合物
    将ddH 2 O加入到11ml中并通过注射器过滤器(0.2μm)过滤灭菌
    储存于-20°C
  3. 2,000x激动素(1.86 mM)
    10mg激动素
    1毫升1N NaOH 将ddH 2 O加入25ml,并通过注射器过滤器(0.2μm)过滤灭菌
    储存于-20°C
  4. 愈伤组织诱导固体培养基
    4.33 g / L Murashige&amp; Skoog基础盐
    3%蔗糖
    用0.1N NaOH将pH调节至5.7,并加入琼脂至0.7%(w / v) 高压灭菌器
    冷却至〜60°C
    添加2,4-D至最终浓度为4.52μM
    将Kinetin添加至0.93μM的终浓度
    在室温下存放
  5. 愈伤组织诱导液体培养基
    4.33 g / L Murashige&amp; Skoog基础盐
    3%蔗糖
    用0.1N NaOH调节pH至5.7,高压釜
    冷却至〜60°C
    添加2,4-D至最终浓度为4.52μM
    将Kinetin添加至0.93μM的终浓度
    在室温下存放
  6. ½MS固体培养基
    2.17 g / L Murashige&amp; Skoog基础盐
    1.5%蔗糖
    用0.1N NaOH将pH调节至5.7,并加入琼脂至1.2%(w / v) 高压灭菌器
    在室温下存放
  7. ½MS液体介质
    2.17 g / L Murashige&amp; Skoog基础盐
    1.5%蔗糖
    用0.1N NaOH调节pH至5.7,高压釜
    在室温下存放
  8. 200μM非 14 C标记的PQ溶液
    5.14毫克甲基紫精二氯化物水合物
    100ml ddH 2 O 储存于-20°C
  9. 2.04mM非标记的H标记的Spd溶液
    3.20微克亚精胺
    10ml ddH 2 O O
    储存于-20°C
  10. 40.37μM非 3 H标记的Spd溶液
    198μl2.04mM亚精胺
    9.8毫升ddH 2 O - / - 储存于-20°C
  11. 溶液1:C标记的PQ溶液(200μM,32.3nCi / nmol,15.55ml)
    0.8毫克百草枯 - 甲基-C 14二氯化物
    15.55ml ddH 2 O
    储存于-20°C
  12. 溶液2:C标记的PQ溶液(200μM,10nCi / nmol,1ml)
    309.6μl溶液1
    690.4μl200μM非标准的C标记的PQ溶液
    储存于-20°C
  13. 溶液3: 3 H标记的Spd溶液(40μM,500ntCi / nmol,1ml)
    20μl亚精胺三盐酸盐,[末端亚甲基 - 3 H(N)] - 溶液
    980μl40.37μM非 3 H标记的Spd溶液
    储存于-20°C
  14. 溶液4: 3 H标记的Spd溶液(2mM,10nCi / nmol,1ml)
    20μl亚精胺三盐酸盐,[末端亚甲基 - 3 H(N)] - 溶液
    980μl2.04mM非标记H标记的Spd溶液
    储存于-20°C
  15. 溶液5:H标记的Spd溶液(40μM,250ntCi / nmol,1ml)
    500μl溶液3
    500μl40μM非标记的H标记的Spd溶液
    储存于-20°C
  16. 溶液6: 3 H标记的Spd溶液(2mM,5ntCi / nmol,1ml)
    500μl溶液4
    500μl2mM非标准H标记的Spd溶液
    储存于-20°C
  17. 1M Tris-HCl,pH7.5
    121.1g Trizma ® base
    800毫升ddH 2 O
    用浓HCl调节pH至7.5 将ddH 2 O加入到1000ml高压釜中 在室温下存放
  18. 0.5 M EDTA,pH 8.0
    186.1g乙二胺四乙酸二钠盐二水合物
    800毫升ddH 2 O
    用NaOH调节pH至8.0(约20g) 将ddH 2 O加入到1000ml高压釜中 在室温下存放
  19. 粗蛋白提取缓冲液
    50mM Tris-HCl(pH7.5)
    0.1 mM EDTA
    1mMβ-巯基乙醇
    在室温下制作新鲜的溶液并储存

致谢

这项工作得到了美国农业部国家研究计划竞争力补助金2007-35100-18378美国部分支持。并由中国国家自然科学基金(批准号:31328004)提交给美国

参考

  1. Gill,SS和Tuteja,N。(2010)。植物中的多胺和非生物胁迫耐受性。植物信号行为 5(1):26-33。
  2. Li,J.,Mu,J.,Bai,J.,Fu,F.,Zou,T.,An,F.,Zhang,J.,Jing,H.,Wang,Q.,Li, Yang,S.and Zuo,J。(2013)。&nbsp; 百草枯抗性1,一种高尔基体定位的推定转运蛋白参与百草枯的细胞内运输。植物生理学162(1):470-483。
  3. Mulangi,V.,Chibucos,MC,Phuntumart,V.and Morris,PF(2012)。&nbsp; 植物多胺摄取转运蛋白的动力学和系统发育分析。植物 236(4):1261-1273。
  4. Shen,Y.,Ruan,Q.,Chai,H.,Yuan,Y.,Yang,W.,Chen,J.,Xin,Z.和Shi,H。(2016)。拟南芥多胺转运蛋白LHR1 / PUT3调节热响应基因表达通过增强mRNA的稳定性。植物J 88(6):1006-1021。
  5. Uemura,T.,Kashiwagi,K。和Igarashi,K。(2007)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/17218313”目标=“_ blank”>酿酒酵母中DUR3和SAM3的多胺吸收。生物化学282(10):7733-7741。
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引用:Chai, H., Shen, Y. and Shi, H. (2017). Polyamine and Paraquat Transport Assays in Arabidopsis Seedling and Callus. Bio-protocol 7(15): e2421. DOI: 10.21769/BioProtoc.2421.
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