5. Acetone, petroleum ether–diethyl ether (2:1, v/v), chloroform–methanol (2:1, v/v).
6. α-Tocopheryl acetate (2 mg/ml in acetone).
2.5 Quantitative
HPLC Carotenoid
Analysis
1. β-carotene.
2. Chloroform–methanol (2:1, v/v), petroleum ether.
3. Photometer, 15 ml plastic reaction tubes (see Note 1).
4. C18 column (e.g., Hypersil Gold, 150 Â 2.1 mm i.d., 1.9 μm,
Thermo Scientific, Erlangen, Germany) or equivalent.
3 Methods
3.1 Carotenoid
Pathway Flux
in Leaves
This method is adopted from Ref. [14].
1. Collect 20 rosette leaves from 4-week-old Arabidopsis plants
and place them with the bottom side facing downward in a
small petri dish containing 25 ml of 70 μM norflurazon. Control leaves are placed on water (see Note 2).
2. Incubate for 2 h in the dark at room temperature, for example,
in a cardboard box.
3. Transfer leaves to 25 ml 10 μM norflurazon in small petri dishes
using a forceps, again with the abaxial side in contact with the
liquid.
4. Illuminate for 4 h with 100 μmol photons m
À2 s
À2 (see Note
3).
5. Collect leaves with forceps on paper wipe to remove adhering
liquid. Put harvested leaves in small aluminum foil envelopes,
freeze in liquid nitrogen immediately after harvest and store for
carotenoid extraction (see Subheading 3.4) and HPLC analysis
(Subheading 3.5).
3.2 Norflurazon
Treatment Arabidopsis
Seedlings
This method is adopted from Ref. [20].
1. Weigh 10 mg Arabidopsis seeds per large petri dish (145 mm
diameter) into reaction tube, add 1 ml sterilization solution
and incubate for 10 min with gentle shaking at room temperature (see Note 4).
2. Centrifuge seeds for 2 min at 3000 Â g in a table top centrifuge, remove supernatant under the sterile hood, wash three
times by adding 1 ml of sterile water followed by centrifugation. After the last washing, resuspend seeds in 1 ml 0.4% (w/v)
agar-agar (see Note 5).
3. Distribute seeds onto three metal grids per big petri dish containing Arabidopsis growth agar. Let the plates with lids closed
Quantification of Carotenoid Pathway Flux
283
6. α-Tocopheryl acetate (2 mg/ml in acetone).
2.5 Quantitative
HPLC Carotenoid
Analysis
1. β-carotene.
2. Chloroform–methanol (2:1, v/v), petroleum ether.
3. Photometer, 15 ml plastic reaction tubes (see Note 1).
4. C18 column (e.g., Hypersil Gold, 150 Â 2.1 mm i.d., 1.9 μm,
Thermo Scientific, Erlangen, Germany) or equivalent.
3 Methods
3.1 Carotenoid
Pathway Flux
in Leaves
This method is adopted from Ref. [14].
1. Collect 20 rosette leaves from 4-week-old Arabidopsis plants
and place them with the bottom side facing downward in a
small petri dish containing 25 ml of 70 μM norflurazon. Control leaves are placed on water (see Note 2).
2. Incubate for 2 h in the dark at room temperature, for example,
in a cardboard box.
3. Transfer leaves to 25 ml 10 μM norflurazon in small petri dishes
using a forceps, again with the abaxial side in contact with the
liquid.
4. Illuminate for 4 h with 100 μmol photons m
À2 s
À2 (see Note
3).
5. Collect leaves with forceps on paper wipe to remove adhering
liquid. Put harvested leaves in small aluminum foil envelopes,
freeze in liquid nitrogen immediately after harvest and store for
carotenoid extraction (see Subheading 3.4) and HPLC analysis
(Subheading 3.5).
3.2 Norflurazon
Treatment Arabidopsis
Seedlings
This method is adopted from Ref. [20].
1. Weigh 10 mg Arabidopsis seeds per large petri dish (145 mm
diameter) into reaction tube, add 1 ml sterilization solution
and incubate for 10 min with gentle shaking at room temperature (see Note 4).
2. Centrifuge seeds for 2 min at 3000 Â g in a table top centrifuge, remove supernatant under the sterile hood, wash three
times by adding 1 ml of sterile water followed by centrifugation. After the last washing, resuspend seeds in 1 ml 0.4% (w/v)
agar-agar (see Note 5).
3. Distribute seeds onto three metal grids per big petri dish containing Arabidopsis growth agar. Let the plates with lids closed
Quantification of Carotenoid Pathway Flux
283