514 nm line of an argon laser is used to excite the fluorescence
of YFP, and the 543 nm laser is used to excite RFP. Chlorophyll
autofluorescence is detected between 664 and 696 nm, GFP
fluorescence is detected between 500 and 539 nm, YFP
between 524 and 575 nm, and RFP between 600 and
650 nm. One of the advantages of the Leica system is the ability
to perform wavelength scanning (lambda scan) to obtain the
emission spectrum of the excited molecules. The analyzed
emission spectra should be identical to the spectra of fluorescent molecules as described in the reference database (included
in the Leica LAS AF software) to exclude potential false signals
caused by multiple autofluorescent components of plant cells.
Typical localization results can be seen for test carotenoid
enzymes such as PSY from maize and Arabidopsis, and
β-carotene hydroxylase 4 (HYD4) from maize (see Fig. 2).
The red chlorophyll autofluorescence provides a good internal
marker for plastid identification and therefore it is useful to
merge the red fluorescence signal with the fluorescent tag
(GFP, YFP, or RFP). Note that the PSY enzymes from maize
and Arabidopsis do not localize in the same place, as previously
reported [6]. Figure 3 shows typical results for protein-protein
interactions, as evidenced by the restored fluorescence seen
when coexpressing BiFC fusions of the P450 carotene hydroxylases from maize, CYP97C and CYP97A (see Fig. 3a)
[10]. BiFC fusion pairs using the nonheme diiron enzyme
HYD4 also restores YFP fluorescence, evidence for HYD4
forming a dimeric complex (Fig. 3b); this complex localization
is consistent with the GFP-tagged localization in Fig. 2b.
Figure 3c shows restored fluorescence indicating interaction
for a known homodimeric plastid protein, ChrD (carotenoidassociated protein D), which is commonly used as a positive
control for BiFC experiments. Figure 3d shows no restoration
of fluorescence, indicating that while CYP97C can interact
with the P450 carotene β-ring hydroxylase CYP97A, it cannot
interact with the nonheme diiron enzyme β-ring
hydroxylase HYD4.
4 Notes
1. Any maize variety can be used, preferably with a known genotype and/or one for which there is known sequence information such as the B73 cultivar. Maize mutants defective in
carotenoid biosynthesis may be useful as long as the phenotype
does not affect leaf photosynthesis [13]. Many maize genetic
stocks can be obtained from the Maize Genetic Cooperation
Stock Center (University of Illinois; http://maizecoop.cropsci.
uiuc.edu) which is part of the National Plant Germplasm
Elucidating Carotenoid Enzyme Localization and Interactions
231
of YFP, and the 543 nm laser is used to excite RFP. Chlorophyll
autofluorescence is detected between 664 and 696 nm, GFP
fluorescence is detected between 500 and 539 nm, YFP
between 524 and 575 nm, and RFP between 600 and
650 nm. One of the advantages of the Leica system is the ability
to perform wavelength scanning (lambda scan) to obtain the
emission spectrum of the excited molecules. The analyzed
emission spectra should be identical to the spectra of fluorescent molecules as described in the reference database (included
in the Leica LAS AF software) to exclude potential false signals
caused by multiple autofluorescent components of plant cells.
Typical localization results can be seen for test carotenoid
enzymes such as PSY from maize and Arabidopsis, and
β-carotene hydroxylase 4 (HYD4) from maize (see Fig. 2).
The red chlorophyll autofluorescence provides a good internal
marker for plastid identification and therefore it is useful to
merge the red fluorescence signal with the fluorescent tag
(GFP, YFP, or RFP). Note that the PSY enzymes from maize
and Arabidopsis do not localize in the same place, as previously
reported [6]. Figure 3 shows typical results for protein-protein
interactions, as evidenced by the restored fluorescence seen
when coexpressing BiFC fusions of the P450 carotene hydroxylases from maize, CYP97C and CYP97A (see Fig. 3a)
[10]. BiFC fusion pairs using the nonheme diiron enzyme
HYD4 also restores YFP fluorescence, evidence for HYD4
forming a dimeric complex (Fig. 3b); this complex localization
is consistent with the GFP-tagged localization in Fig. 2b.
Figure 3c shows restored fluorescence indicating interaction
for a known homodimeric plastid protein, ChrD (carotenoidassociated protein D), which is commonly used as a positive
control for BiFC experiments. Figure 3d shows no restoration
of fluorescence, indicating that while CYP97C can interact
with the P450 carotene β-ring hydroxylase CYP97A, it cannot
interact with the nonheme diiron enzyme β-ring
hydroxylase HYD4.
4 Notes
1. Any maize variety can be used, preferably with a known genotype and/or one for which there is known sequence information such as the B73 cultivar. Maize mutants defective in
carotenoid biosynthesis may be useful as long as the phenotype
does not affect leaf photosynthesis [13]. Many maize genetic
stocks can be obtained from the Maize Genetic Cooperation
Stock Center (University of Illinois; http://maizecoop.cropsci.
uiuc.edu) which is part of the National Plant Germplasm
Elucidating Carotenoid Enzyme Localization and Interactions
231