of chlorophylls is required, separation on a 150 mm C30
reverse phase column is suitable (e.g., YMC carotenoid
(150 mm  3 mm, 5 μm) with solvent system A: methanol–
tert-Butyl methyl ether (1:1, v/v) and B: methanol–tert-Butyl
methyl ether–water (5:1:1, v/v/v), 0.75 ml min
À1 ; 40
C;
increase linearly from 0% A to 100% A within 20 min, maintain
final conditions for 4 min).
13. Dissolve some β-carotene crystals in 20 μl chloroform in a
15 ml tube and add PE up to 10 ml. Determine concentration
photometrically at λ max (β-carotene) ¼ 450 nm by Beer’s law
usingthemolarextinctioncoefficientε134,000LÂmol
À1
Âcm
À1
[23] and dilute accordingly to obtain a 5 pmol/μl solution in PE.
14. If enough care was taken during extraction and sample preparation, the TocAc correction factor ranges between 0.75 and
1.0.
15. Phytoene and α-tocopheryl acetate are quantified at 285 nm
using the deuterium lamp (D2) signals, whereas all other carotenoids, such as β-carotene for calibration, are quantified
using the Tungsten lamp (W) signal. For reliable phytoene
quantification with the method described, it is necessary to
monitor sensitivity of both lamps over time. That is,
β-carotene calibration to evaluate W lamp sensitivity should
be repeated in regular intervals. Similarly, deuterium lamp
(D2) sensitivity should be evaluated by monitoring the
α-tocopheryl acetate peak area at 285 nm in external standard
samples in repeated experiments, that is, signals should remain
fairly constant over a longer period of time. In this context,
lamp lifetime as recommended by the manufacturer should not
be exceeded.
Acknowledgments
This work was supported in part by the HarvestPlus research consortium (grant 2014H6320.FRE) to R.W. J.K. was funded by
Grant WE 4731/3-1 from the Deutsche Forschungsgemeinschaft.
The authors thank the present and former members of the laboratory of Prof. Peter Beyer for their contributions to establishing this
method.
References
1. Schweiggert RM, Mezger D, Schimpf F et al
(2012) Influence of chromoplast morphology
on carotenoid bioaccessibility of carrot,
mango, papaya, and tomato. Food Chem
135:2736–2742
2. Li L, Yuan H (2013) Chromoplast biogenesis
and carotenoid accumulation. Arch Biochem
Biophys 539:102–109
3. Sun T, Yuan H, Cao H et al (2017) Carotenoid
metabolism in plants: the role of plastids. Mol
Plant 11:58–74
290
Julian Koschmieder and Ralf Welsch
reverse phase column is suitable (e.g., YMC carotenoid
(150 mm  3 mm, 5 μm) with solvent system A: methanol–
tert-Butyl methyl ether (1:1, v/v) and B: methanol–tert-Butyl
methyl ether–water (5:1:1, v/v/v), 0.75 ml min
À1 ; 40
C;
increase linearly from 0% A to 100% A within 20 min, maintain
final conditions for 4 min).
13. Dissolve some β-carotene crystals in 20 μl chloroform in a
15 ml tube and add PE up to 10 ml. Determine concentration
photometrically at λ max (β-carotene) ¼ 450 nm by Beer’s law
usingthemolarextinctioncoefficientε134,000LÂmol
À1
Âcm
À1
[23] and dilute accordingly to obtain a 5 pmol/μl solution in PE.
14. If enough care was taken during extraction and sample preparation, the TocAc correction factor ranges between 0.75 and
1.0.
15. Phytoene and α-tocopheryl acetate are quantified at 285 nm
using the deuterium lamp (D2) signals, whereas all other carotenoids, such as β-carotene for calibration, are quantified
using the Tungsten lamp (W) signal. For reliable phytoene
quantification with the method described, it is necessary to
monitor sensitivity of both lamps over time. That is,
β-carotene calibration to evaluate W lamp sensitivity should
be repeated in regular intervals. Similarly, deuterium lamp
(D2) sensitivity should be evaluated by monitoring the
α-tocopheryl acetate peak area at 285 nm in external standard
samples in repeated experiments, that is, signals should remain
fairly constant over a longer period of time. In this context,
lamp lifetime as recommended by the manufacturer should not
be exceeded.
Acknowledgments
This work was supported in part by the HarvestPlus research consortium (grant 2014H6320.FRE) to R.W. J.K. was funded by
Grant WE 4731/3-1 from the Deutsche Forschungsgemeinschaft.
The authors thank the present and former members of the laboratory of Prof. Peter Beyer for their contributions to establishing this
method.
References
1. Schweiggert RM, Mezger D, Schimpf F et al
(2012) Influence of chromoplast morphology
on carotenoid bioaccessibility of carrot,
mango, papaya, and tomato. Food Chem
135:2736–2742
2. Li L, Yuan H (2013) Chromoplast biogenesis
and carotenoid accumulation. Arch Biochem
Biophys 539:102–109
3. Sun T, Yuan H, Cao H et al (2017) Carotenoid
metabolism in plants: the role of plastids. Mol
Plant 11:58–74
290
Julian Koschmieder and Ralf Welsch