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Radiolabeled compound uptake in plants

Plant culture

  1. Sow surface sterilized seeds on solid MS/2 1% sucrose
  2. Grow for 7 days in 16/8 h light/dark
  3. Transfer 4-6 plants in a well of a 12-well plate containing 3 ml of liquid MS/2 1% sucrose
  4. Grow for 4 more days in the same light regime as above, with gentle shaking (~40 rpm)

Uptake Experiment 

  1. Transfer the plants into a 24-well plate containing 1 ml uptake medium (most often MS/2 1 % sucrose)
  2. Place the plate in the same conditions of the uptake experiment, and let the plant adapt for 1-2 h (I shake the plant at 300 rpm on an Eppendorf thermomixer)
  3. Add 100 μl 11X radiolabeled compound. I use 2 μl radiolabeled compound and the desired amount of could compound for 1 well
  4. Let the plant take up for the desired amount of time under shaking as above
  5. Take the plants out of the wells and transfer them to the vacuum filtration manifold (e.g. # EQU-FM-10X20 from DHI Laboratory Products, Denmark)
  6. Wash three times with 0.2 mM CaSO4

Exfullex experiment (Optional)

  1. Transfer the washed plants into 1 ml of efflux solution (generally MS/2 1% sucrose)
  2. Let the plant excrete the compound for the desired time under shaking as above
  3. Collect the medium with a pipette

Plant sample preparation for counting 

  1. Put the plants in empty scintillation vials
  2. Dry out at 70C for 2 - 3 h
  3. Weigh the dried samples
  4. Add 1 ml 5% NaClO and leave O/N at RT in a closed vial
  5. Open the vial under the fume hood for > 1 h
  6. Add 5 ml Ultima Gold XR (Perkin Elmer), mix well by inversion (10 times)
  7. Leave 1 h in the dark
  8. Count in a LSC (I do 4 min)

Protocol by Guillaume Pilot. July 2009

 

Thin Layer Chromatography of amino acids

Sample preparation 

  1. Dry your sample (medium or plant extract) in a speed vac
  2. Solubilize the dried pellet in 70% ethanol

Running the TLC

  1. Load you samples 1 μl by 1μl on the silica plate, wait for the drop to be dry before loading the next one
  2. Develop by dipping the bottom of the plate in a butanol-1:acetic acid:water (3:1:1) mix, in a TLC chamber
  3. Close the chamber and wait for the solvent to migrate in the silica for 2 h
  4. Dry the plate under the fume hood

Revelation of the amino acid

  1. Spay the plate with a solution of 0.3% ninhydrine in ethanol
  2. Bake for 20 min at 120C
  3. Scan

Protocol by Guillaume Pilot. July 2009

 

Xylem and Phloem sap collection from Arabidopsis

Xylem Sap collection

  1. Sow plants and keep in high humidity for 1 week (this will lengthen the hypocotyl)
  2. Grow plants until they start bolting (short day growth would increase the size of the plants)
  3. Water thoroughly 1-2 days before the collection
  4. Cut the hypocotyl below the rosette with a razor blade
  5. Discard the first drop of xylem sap
  6. Collect the upcoming drops with a P2 pipette


Note: the amount of exudate collected shoudl be between 10-30 ul with 5-100 plant

Phloem Sap collection

Solutions: 10 mM EDTA pH7 or 8.5

  1. Cut an Arabidopsis plant below the rosette
  2. Cut out the leaves to be sampled at the base of the petiole in a watch-glass containing 10 mM EDTA
  3. Rinse the petiole of the leaves in 5 mM EDTA
  4. Immerse the petiole in 400 µl of 5 mM EDTA in a microtube
  5. Incubate for 6-8 in the dark, in 100% RH
  6. Remove the leaves and analyze the solution


Protocol by Guillaume Pilot - July 2009
 

Tobacco leaf infiltration

Agrobacteria preparation

Infiltration solution:
MgCl2 10 mM
Acetosyringone 100 µM (3,5-dimethoxy-4-hydroxy-acetophenone)

  1. Grow a 2 ml-culture of agrobacteria with all the selective antibiotics until it reaches the plateau (over-night for GV3101)
  2. Transfer 1.5 ml of the culture in an eppendorf tube
  3. Spin 3 min @ 3300 g
  4. Resuspend the pellet in 1 ml the infiltration solution by pipetting up and down
  5. Repeat this washing step
  6. Take the OD of a 1/10 dilution
  7. Dilute an aliquot of the culture in the infiltration solution so that the OD of each of the strains is 0.05

Infiltration into tobacco leaves

  1. Take 5-6 week-old Nicotiana benthaminana or Nicotiana tabacum
  2. Scratch the leaf with a razor blade
  3. With a 1 ml syringe, infiltrate the agro solution onto the lower epidermis at the level of the scratch

Protocol by Guillaume Pilot - July 2009

 

Arabidopsis callus generation and culture

Media

MS medium (1 L)

  1. MS salts 4.3 g
  2. Sucrose 20 g
  3. MES 1 g
  4. KOH pH 5.7
  5. Agar 8 g

Autoclave

Vitamins 1000 X (10 ml)

  1. myo-inositol 1 g
  2. nicotinic acid 10 mg
  3. thiamin HCl 100 mg
  4. pyridoxine HCl 10 mg
  5. glycine 40 mg
  6. Filter sterilize and store a -20°C

Add to 50 ml cooled medium:

                                                            Induction                              Propagation
1 mg/ml (4.5 mM) 2,4-D                 50 µl              4.5 µM            25 µl                  2.25 µM
0.5 mg/ml (2.3 mM) kinetin              5 µl             0.23 µM           10 µl                  0.46 µM
Vitamins                                        50 µl             1 X                  50 µl                 1 X
 

Procedure

  1. Sow seeds on induction medium, grow @ 22 °C (wounding increases callus formation rate)
  2. Transfer to propagation medium after 4 weeks
  3. Transfer to fresh plates every 3 weeks

Protocol by Guillaume Pilot - July 2009

 

Amino acid analysis by ninhydrin

Solutions

Solution A

  1. Propionic acid 25 ml
  2. 10 N NaOH 21 ml
  3. H2O 4 ml
  4. 2-ethoxy-ethanol 50 ml
  5. Ninhydrine 2 g

Solution B
        1 mg/ml Ascorbic acid in H2O

Sample preparation

  1. Extract around 100 mg with 500µl 80% EtOH
  2. Place 20 min @ 80°C (vortex @ 1400 rpm if possible)
  3. Centrifuge 3 min @ max speed
  4. Transfer the supernatant in a fresh eppi, and keep on ice
  5. Resuspend the pellet in 500 µl 80% EtOH
  6. Place 20 min @ 80°C (vortex @ 1400 rpm if possible)
  7. Centrifuge 3 min @ max speed
  8. Pool the supernatant with the previous one
  9. Dry in a speed vac

Reaction

  1. Resuspend in 250 µl 20 % EtOH
  2. Centrifuge 5 min @ max speed
  3. Take 20-40 µl of the supernatant
  4. Complete to 200 µl with H2O
  5. Add 200 µl solution A
  6. Add 100 µl solution B
  7. Place for 15 min @ 95°C
  8. Cool 5 min on ice
  9. Add 500 µl 60% EtOH
  10. Take OD570, and use 20% EtOH as blank (should stay brown and not turn violet)

Standard

0 to 120 nmol glycine

Protocol by Guillaume Pilot - July 2009

 

GUS staining

Pre-treatment (Optional, good for leaves of soil grown plants)

  1. Soak tissue for 10 min. in heptane. Some tissues might require longer treatment
  2. But be careful not to dehydrate it too much. Handle heptane in the hood
  3. Heptane might be reused (and reduces waste)
  4. Remove heptane as much as possible. Allow the sample to dry from 2-5 min
  5. Watch the process; do not allow the tissue to dry

Solution

  1. Phosphate buffer (50 mM, pH 7.2) 50 ml
  2. 0.5 M NaPi buffer pH 7.2 5 ml
  3. 25% Triton 100 ul
  4. H2O to make 50 ml
  5. Note.Phosphate buffer, dissolve 4.83 g NaH2PO4 H2O, and 12.78 g Na2HPO4 in 250 ml H2O

Sampling

        Cut plant tissues in 1 ml phosphate buffer

Prefixing

Add 500 µl of the 3x prefixing solution:

  1. Phosphate buffer 9 ml
  2. Formaldehyde 36 % 1.25 ml (4.5%)

Vacuum 10 min, incubate @ root temperature 20-30 more minutes

Washing

3 times 5 min in phosphate buffer + Ferro- and Ferricyanide at the same concentration as below

Reaction

  1. Phosphate buffer 10 ml
  2. K ferrocyanide 50 mM 100 ul
  3. K ferricyanide 50 mM 100 ul
  4. X-Gluc 100 mM (52 mg/ml) 100 ul
  5. vacuum 15 min
  6. Place at 37°C for the desired amount of time

Fixing

Fixer (50 ml)

  1. 100 mM phosphate buffer pH 7.2 25 ml (50 mM)
  2. 36% formaldehyde 2.8 ml (2%)
  3. 25% glutaraldehyde 2 ml (1%)
  4. SDS 10 % 100 ul
  5. H2O to make 50 ml

vacuum 15 min and place for 24 h @ 4°C

Dehydratation/destaining

  1. 2x 70% EtOH 15min
  2. 4-5x 100% EtOH 30 min + overnight

Protocol by Guillaume Pilot - July 2009

 

Arabidopsis suspension cells - culture and media

Media

For 1L:

  1. 4.4 g MS salts
  2. 2 % glucose
  3. 0.2 µM Kinetin (4.6 mM stock- in 150 ul 1 M KOH add 10 ml H2O); stored at 4°C
  4. 2.5 µM 1-Naphtaleneacetic acid (25 mM stock prepared in 70% EtOH); at -20 °C
  5. pH to 5.8 with 1 M KOH

Culture

Cells are subcultured every 7 days. The cells are grown under constant light at 22 °C, constantly rotating at 120 rpm

Protocol by Frommer Lab