3. We found that light intensity directly affects carotenoid pathway flux in detached leaves. For standard experiments, use
100 μmol photons m
À2 s
À2 which corresponds to standard
growth chamber conditions.
4. If higher number of seeds needs to be sterilized, up to 55 mg
seeds can be treated at once with the same amount of sterilization solution and resuspended in accordingly increased final
medium.
5. This low concentrated agar-agar solution is viscous and can
easily be pipetted. Arabidopsis seeds resuspended in this solution do not settle and distribute equally after mixing which
allows to distribute them evenly on several metal grids.
6. If higher number of seeds need to be sterilized, up to 55 mg
seeds can be treated at once with the same procedure and
resuspended in equivalently increased final medium.
7. Seeds should not settle, see Note 5.
8. A convenient way to do this is to place both plates in a row
(fresh plate in front), lift the filter paper with sterile forceps and
drag it over to the front plate touching the fringe of the rear
petri dish.
9. Grinding to a fine powder is essential as carotenoids will not be
quantitatively extracted from a sample if grinding is omitted or
not sufficient. Grinding lyophilized plant material is less effective than grinding fresh frozen material. Aluminum foil envelopes should not be closed too tight to allow water to sublimate
during lyophilization. Avoid thawing of powder throughout
the whole process.
10. Addition of basic Tris neutralizes acids in samples to prevent
acid-catalyzed furanoid rearrangement of epoxy groups [24].
11. While slight volume errors during extraction process are tolerable, addition of exact and identical volumes of internal standard is crucial. In dispenser-type pipettes (e.g., Eppendorf
multipette) piston is in direct contact with solvent, in contrast
to standard pipettes in which piston is filled with air. Absence of
vapor pressure in dispenser-type pipettes avoids dripping of
solvents and results in exact and precise pipetting volumes.
Use these pipettes for addition of internal and external
standards.
12. These chromatographic conditions are only sufficient for separation and quantification of α-tocopheryl acetate and phytoene
in the presence of other carotenoids and chlorophylls. If quantification of other carotenoids in the presence of chlorophylls is
required, chromatography can be performed on a 250 mm
C30 reverse phase column, for instance as described in Hoa
et al. [25]. If quantification of other carotenoids in the absence
Quantification of Carotenoid Pathway Flux
289
100 μmol photons m
À2 s
À2 which corresponds to standard
growth chamber conditions.
4. If higher number of seeds needs to be sterilized, up to 55 mg
seeds can be treated at once with the same amount of sterilization solution and resuspended in accordingly increased final
medium.
5. This low concentrated agar-agar solution is viscous and can
easily be pipetted. Arabidopsis seeds resuspended in this solution do not settle and distribute equally after mixing which
allows to distribute them evenly on several metal grids.
6. If higher number of seeds need to be sterilized, up to 55 mg
seeds can be treated at once with the same procedure and
resuspended in equivalently increased final medium.
7. Seeds should not settle, see Note 5.
8. A convenient way to do this is to place both plates in a row
(fresh plate in front), lift the filter paper with sterile forceps and
drag it over to the front plate touching the fringe of the rear
petri dish.
9. Grinding to a fine powder is essential as carotenoids will not be
quantitatively extracted from a sample if grinding is omitted or
not sufficient. Grinding lyophilized plant material is less effective than grinding fresh frozen material. Aluminum foil envelopes should not be closed too tight to allow water to sublimate
during lyophilization. Avoid thawing of powder throughout
the whole process.
10. Addition of basic Tris neutralizes acids in samples to prevent
acid-catalyzed furanoid rearrangement of epoxy groups [24].
11. While slight volume errors during extraction process are tolerable, addition of exact and identical volumes of internal standard is crucial. In dispenser-type pipettes (e.g., Eppendorf
multipette) piston is in direct contact with solvent, in contrast
to standard pipettes in which piston is filled with air. Absence of
vapor pressure in dispenser-type pipettes avoids dripping of
solvents and results in exact and precise pipetting volumes.
Use these pipettes for addition of internal and external
standards.
12. These chromatographic conditions are only sufficient for separation and quantification of α-tocopheryl acetate and phytoene
in the presence of other carotenoids and chlorophylls. If quantification of other carotenoids in the presence of chlorophylls is
required, chromatography can be performed on a 250 mm
C30 reverse phase column, for instance as described in Hoa
et al. [25]. If quantification of other carotenoids in the absence
Quantification of Carotenoid Pathway Flux
289