to 200 mg of roots, and 10–15 mg of any carotenoid-rich tissues
(e.g., dried leaf tissues and tomato fruits) that can be extractable in
a single step with 0.5–1 mL extraction buffer (see Note 2). While
collecting fresh tissues, it is recommended the excessive moisture
to be removed from tissues by blotting onto a clean lint-free paper,
thereby enhancing accuracy in determination of the fresh weight
between independent samples. Immediately transfer samples into
liquid nitrogen avoiding exposure to high temperatures and/or
light (see Note 5). Samples should be stored at À80
C until
pigment extraction. In this work, we performed metabolic analysis
using Arabidopsis cotyledon tissues collected from 7-day-old darkgrown wild-type (WT) and mutant lines ZETA-CAROTENE
ISOMERASE (z-iso; zic1-3, Salk_136385) and CAROTENOID
ISOMERASE (crtiso; ccr2.1) [36]. Additionally, 14-day-old darkgrown tomato CAROTENOID ISOMERASE mutant (crtiso; tangerine
mic ) [37] is used to confirm peak retention times of identified
carotenoids.
3.2 Carotenoid
Extraction
The complete extraction of carotenoids from tissues enhances the
accuracy of quantification. A single-step extraction method works
effectively for most tissue types (e.g., leaves, roots, etiolated tissues,
and fruits). Extraction under an ambient lighting condition is
preferable to limit carotenoid photoisomerisation. The carotenoid
extraction standard operating procedure is as follows;
1. Weigh plant tissues and place in a 1.5 or 2 mL microcentrifuge
tube (flat-bottom tubes are recommended) with 2 stainless
steel beads (3 mm diameter). Seal the tubes and snap freeze
in liquid N 2 . Prepare 3–5 biological replicates per treatment
(see Note 2).
2. Prepare the cell lysis system for mechanical grinding of samples
by adjusting to 20 Hertz frequency for 2 min of shaking (this
ensures complete grinding) (see Note 2).
3. Place frozen tubes containing plant material into prechilled
(À80
C) cell lysis adaptors, balance as necessary, and begin
tissue grinding (see Note 3).
4. After grinding, return tubes containing the ground tissue back
to liquid N 2 (see Note 6).
5. For pigment extraction, transfer tubes from liquid N 2 to ice
and add 500 μL extraction buffer and vortex vigorously for
1 min.
6. Add an equal volume (500 μL) of water and invert tubes gently
2–3 times. Avoid vigorous shaking, which can emulsify the
extract and hinder phase separation.
7. Centrifuge the tubes for 5 min at 15,000 rpm (~21,100 Â g) at
4
C.
Profiling Carotenoids in Plants Using HPLC
149
(e.g., dried leaf tissues and tomato fruits) that can be extractable in
a single step with 0.5–1 mL extraction buffer (see Note 2). While
collecting fresh tissues, it is recommended the excessive moisture
to be removed from tissues by blotting onto a clean lint-free paper,
thereby enhancing accuracy in determination of the fresh weight
between independent samples. Immediately transfer samples into
liquid nitrogen avoiding exposure to high temperatures and/or
light (see Note 5). Samples should be stored at À80
C until
pigment extraction. In this work, we performed metabolic analysis
using Arabidopsis cotyledon tissues collected from 7-day-old darkgrown wild-type (WT) and mutant lines ZETA-CAROTENE
ISOMERASE (z-iso; zic1-3, Salk_136385) and CAROTENOID
ISOMERASE (crtiso; ccr2.1) [36]. Additionally, 14-day-old darkgrown tomato CAROTENOID ISOMERASE mutant (crtiso; tangerine
mic ) [37] is used to confirm peak retention times of identified
carotenoids.
3.2 Carotenoid
Extraction
The complete extraction of carotenoids from tissues enhances the
accuracy of quantification. A single-step extraction method works
effectively for most tissue types (e.g., leaves, roots, etiolated tissues,
and fruits). Extraction under an ambient lighting condition is
preferable to limit carotenoid photoisomerisation. The carotenoid
extraction standard operating procedure is as follows;
1. Weigh plant tissues and place in a 1.5 or 2 mL microcentrifuge
tube (flat-bottom tubes are recommended) with 2 stainless
steel beads (3 mm diameter). Seal the tubes and snap freeze
in liquid N 2 . Prepare 3–5 biological replicates per treatment
(see Note 2).
2. Prepare the cell lysis system for mechanical grinding of samples
by adjusting to 20 Hertz frequency for 2 min of shaking (this
ensures complete grinding) (see Note 2).
3. Place frozen tubes containing plant material into prechilled
(À80
C) cell lysis adaptors, balance as necessary, and begin
tissue grinding (see Note 3).
4. After grinding, return tubes containing the ground tissue back
to liquid N 2 (see Note 6).
5. For pigment extraction, transfer tubes from liquid N 2 to ice
and add 500 μL extraction buffer and vortex vigorously for
1 min.
6. Add an equal volume (500 μL) of water and invert tubes gently
2–3 times. Avoid vigorous shaking, which can emulsify the
extract and hinder phase separation.
7. Centrifuge the tubes for 5 min at 15,000 rpm (~21,100 Â g) at
4
C.
Profiling Carotenoids in Plants Using HPLC
149