In this article, we summarize current protocols for the application of this technique to different plant tissues and include a
detailed protocol on the identification and quantification of phytoene by HPLC analysis. Detectable amounts of phytoene accumulate within a few hours in herbicide-treated photosynthetically
active leaves, equivalent to the level of carotenoid biosynthesis.
Moreover, the pathway activity is dependent on light intensity
and increases significantly after exposure to high light conditions
[5]. We also include a protocol on the treatment of Arabidopsis
seedlings with norflurazon which allows determination of pathway
flux alterations in photomorphogenic mutants [20, 21]. Finally, we
include a method for determining the carotenogenic pathway flux
in callus generated from germinating Arabidopsis seedlings
[10, 22]. Remarkably, pathway flux determined matched with
expected pathway alterations, for example, in mutants or upon
overexpression of enzymes positioned upstream to phytoene formation. This supports the notion that this system as a useful tool
for basic pathway research. We recently identified a number of
apocarotenoid-degradation products in callus, corroborating the
strong impact of oxidative destruction on carotenoid stability.
The carotenoid extraction protocol includes α-tocopheryl acetate as internal standard to correct for extraction errors. Quantification of phytoene is performed without requiring purified
phytoene as authentic standard compound. The amount of phytoene in each sample is calculated using a response factor of the
diode array detector for β-carotene after applying a conversion
factor obtained from molecular extinction coefficients of
β-carotene and phytoene.
2 Materials
2.1 Carotenoid
Pathway Flux
in Leaves
1. 4-week-old Arabidopsis plants grown in soil under long-day
conditions.
2. Small petri dishes (100 mm diameter).
3. Two solutions with 10 μM and 70 μM norflurazon in water.
For this, prepare a 100 mM norflurazon stock solution in
isopropanol and further dilute in water. Norflurazon stocks
can be stored at 4
C for several years.
4. Forceps.
5. Phytochamber or light source with 100 μmol photons m
À2 s
À2 .
2.2 Norflurazon
Treatment Arabidopsis
Seedlings
1. Arabidopsis seeds.
2. Sterilization solution (6% (v/v) sodium hypochlorite, 0.02%
(v/v) Triton X-100), sterile water.
3. Tabletop centrifuge.
Quantification of Carotenoid Pathway Flux
281
detailed protocol on the identification and quantification of phytoene by HPLC analysis. Detectable amounts of phytoene accumulate within a few hours in herbicide-treated photosynthetically
active leaves, equivalent to the level of carotenoid biosynthesis.
Moreover, the pathway activity is dependent on light intensity
and increases significantly after exposure to high light conditions
[5]. We also include a protocol on the treatment of Arabidopsis
seedlings with norflurazon which allows determination of pathway
flux alterations in photomorphogenic mutants [20, 21]. Finally, we
include a method for determining the carotenogenic pathway flux
in callus generated from germinating Arabidopsis seedlings
[10, 22]. Remarkably, pathway flux determined matched with
expected pathway alterations, for example, in mutants or upon
overexpression of enzymes positioned upstream to phytoene formation. This supports the notion that this system as a useful tool
for basic pathway research. We recently identified a number of
apocarotenoid-degradation products in callus, corroborating the
strong impact of oxidative destruction on carotenoid stability.
The carotenoid extraction protocol includes α-tocopheryl acetate as internal standard to correct for extraction errors. Quantification of phytoene is performed without requiring purified
phytoene as authentic standard compound. The amount of phytoene in each sample is calculated using a response factor of the
diode array detector for β-carotene after applying a conversion
factor obtained from molecular extinction coefficients of
β-carotene and phytoene.
2 Materials
2.1 Carotenoid
Pathway Flux
in Leaves
1. 4-week-old Arabidopsis plants grown in soil under long-day
conditions.
2. Small petri dishes (100 mm diameter).
3. Two solutions with 10 μM and 70 μM norflurazon in water.
For this, prepare a 100 mM norflurazon stock solution in
isopropanol and further dilute in water. Norflurazon stocks
can be stored at 4
C for several years.
4. Forceps.
5. Phytochamber or light source with 100 μmol photons m
À2 s
À2 .
2.2 Norflurazon
Treatment Arabidopsis
Seedlings
1. Arabidopsis seeds.
2. Sterilization solution (6% (v/v) sodium hypochlorite, 0.02%
(v/v) Triton X-100), sterile water.
3. Tabletop centrifuge.
Quantification of Carotenoid Pathway Flux
281