expression and plastid import, the fluorescence is visualized by
confocal microscopy. For testing interactions between two carotenoid enzymes, each construct encodes an enzyme, whose
C-terminus is fused in frame to the N-terminus of one of two halves
of a fluorescent protein, such as Yellow Fluorescent Protein (YFP).
If the enzymes interact in vivo, the full structure of the fluorescent
protein is restored and can be visualized by the known technique of
bimolecular fluorescence [11]. The use of protoplasts for transient
expression of fluorescently tagged carotenoid enzymes reveals
enzyme location and potential interactions in vivo. Detailed
below is the methodology, with careful explanations and cautionary
notes, used to examine carotenoid enzyme localization and
enzyme–enzyme interactions by creation of test plasmids that are
introduced into freshly isolated protoplasts that are then visualized
by fluorescence microscopy.
2 Materials
Distilled water is recommended. All manipulations for the isolation
and transformation of protoplasts should be carried out at room
temperature. The procedure is not sterile, no flow hood is required
and all manipulations with protoplasts can be performed on a lab
bench. Transformed protoplasts survive no longer than 24–48 h,
thus fluorescence should be examined the day after transformation.
Here, a C4 plant, Zea mays (maize), is the choice for protoplast
preparation, since most carotenoid biosynthetic enzymes studied in
our laboratory were of maize origin. In maize, etiolated leaves give
protoplasts with the best transformation efficiency of 80–90%.
However, if the carotenoid enzymes to be tested are thought to
interact with a developed thylakoid membrane, green leaves should
be used for protoplasts preparation. If a C3 plant is preferred as a
source of protoplasts, then Vigna unguiculata subsp. unguiculata
(black-eyed peas) is recommended, however, transformation efficiency in protoplasts isolated from these leaves is lower and peas do
not grow well as etiolated. The methodology detailed here, with
extensive tips and notes, is based on the protocols used to examine
localization and interaction of carotenoid enzymes [6, 8–10].
1. Cellulase from Trichoderma viridae or Trichoderma
sp. (preferably plant cell culture-tested).
2. Pectinase from Rhizopus sp.
3. Reagents #1 and #2 can be substituted with Onozuka R10
(cellulase+pectinase mix).
4. BSA (bovine serum albumin).
5. Mannitol.
6. MES (2-(N-morpholino)ethanesulfonic acid) CaCl 2 .
Elucidating Carotenoid Enzyme Localization and Interactions
225
confocal microscopy. For testing interactions between two carotenoid enzymes, each construct encodes an enzyme, whose
C-terminus is fused in frame to the N-terminus of one of two halves
of a fluorescent protein, such as Yellow Fluorescent Protein (YFP).
If the enzymes interact in vivo, the full structure of the fluorescent
protein is restored and can be visualized by the known technique of
bimolecular fluorescence [11]. The use of protoplasts for transient
expression of fluorescently tagged carotenoid enzymes reveals
enzyme location and potential interactions in vivo. Detailed
below is the methodology, with careful explanations and cautionary
notes, used to examine carotenoid enzyme localization and
enzyme–enzyme interactions by creation of test plasmids that are
introduced into freshly isolated protoplasts that are then visualized
by fluorescence microscopy.
2 Materials
Distilled water is recommended. All manipulations for the isolation
and transformation of protoplasts should be carried out at room
temperature. The procedure is not sterile, no flow hood is required
and all manipulations with protoplasts can be performed on a lab
bench. Transformed protoplasts survive no longer than 24–48 h,
thus fluorescence should be examined the day after transformation.
Here, a C4 plant, Zea mays (maize), is the choice for protoplast
preparation, since most carotenoid biosynthetic enzymes studied in
our laboratory were of maize origin. In maize, etiolated leaves give
protoplasts with the best transformation efficiency of 80–90%.
However, if the carotenoid enzymes to be tested are thought to
interact with a developed thylakoid membrane, green leaves should
be used for protoplasts preparation. If a C3 plant is preferred as a
source of protoplasts, then Vigna unguiculata subsp. unguiculata
(black-eyed peas) is recommended, however, transformation efficiency in protoplasts isolated from these leaves is lower and peas do
not grow well as etiolated. The methodology detailed here, with
extensive tips and notes, is based on the protocols used to examine
localization and interaction of carotenoid enzymes [6, 8–10].
1. Cellulase from Trichoderma viridae or Trichoderma
sp. (preferably plant cell culture-tested).
2. Pectinase from Rhizopus sp.
3. Reagents #1 and #2 can be substituted with Onozuka R10
(cellulase+pectinase mix).
4. BSA (bovine serum albumin).
5. Mannitol.
6. MES (2-(N-morpholino)ethanesulfonic acid) CaCl 2 .
Elucidating Carotenoid Enzyme Localization and Interactions
225