Whether the interaction between CO and COP1 also occurred in vivo in plant cells was tested using fluorescent resonance energy transfer (FRET).
Microprojectile bombardment was used to co-express cyan fluorescent protein (CFP):COP1 and yellow fluorescent protein (YFP):CO in leaf epidermal cells of Arabidopsis.
CFP:COP1 and YFP:CO colocalized to the nucleus and also colocalized in speckles within the nucleus (Figure 4A and B).
Physical interaction of CFP:COP1 and YFP:CO was tested by measuring FRET using photoacceptor bleaching, as previously described (Wenkel et al, 2006) (Figure 4C and D).
Quantification of FRET signals demonstrated that FRET occurred between YFP:CO and CFP:COP1 both in the nucleus and specifically in nuclear speckles (Figure 4C and D).
In control experiments using YFP and CFP, YFP:CO and CFP or YFP and CFP:COP1 FRET was detected at significantly lower levels (Figure 4C).
These experiments demonstrate that YFP:CO and CFP:COP1 colocalize and physically interact in the nuclei of plant cells.
The COP1 full-length cDNA was isolated by RT–PCR and produced as entry clone through BP reaction of Gateway system from Invitrogen.
Then, the entry clone was utilized for the construction of destination vectors for plant transformation, FRET experiments and in vitro-binding assay.
All plasmids for plant transformation were introduced into Agrobacterium strain GV3101 (pMP90RK) and transformed into WT Columbia, cop1–4 or SUC2:CO (An et al, 2004) plants by the floral dip method (Clough and Bent, 1998).
(C) Quantification of FRET in vivo between CFP:CO and YFP:COP1.
YFP:CO detected as an increase in CFP fluorescence after photobleaching of YFP.
Quantification of FRET efficiencies after acceptor photobleaching measured in nuclei and nuclear speckles.
Data are mean±s.d.
of 10–20 cells from three separate experiments.
(D) Visualization of increase in CFP fluorescence after YFP photobleaching.
Left-hand panel, cells expressing CFP:COP1 and YFP, which exerts an effect as a negative control.
Right-hand panel, cells expressing CFP:COP1 and YFP:CO.
Scale bar: 6 μm in (A) and 8 μm in (D).Fluorescence resonance energy transfer (FRET) pairs GFP/DsRed were analysed using a confocal laser-scanning microscope (Zeiss LSM510 META).
FRET measurements of DsRed emission with zero contribution from GFP, was accomplished as described by Erickson et al.
(2003) using the following settings: excitation at 488 nm and emission filters, BP 505–530 nm for GFP and BP 600–637 nm for DsRed.
(C–F) FRET detection in tobacco leaf epidermal cells co-expressing GFP:AtEBP and ACBP4:DsRed; (C) differential interference contrast image of D-F; (D) green channel shows GFP:AtEBP; (E) red channel shows FRET signal of ACBP4:DsRed; (F) co-localization of two signals is indicated by a yellow colour in merged images of (D) and (E).
In FRET analysis, in cells co-expressing GFP:AtEBP and ACBP4:DsRed, not only GFP:AtEBP green fluorescence (Fig.
2D) but also ACBP4:DsRed red fluorescence (Fig.
2E), which overlapped with the GFP signals (Fig.
2F), were detected, indicating that FRET occurred between GFP:AtEBP and ACBP4:DsRed.