## 2 Results
### MEP Pathway Enzymes Are Targeted to Plastids  
The subcellular localization of _C. roseus_ MEP pathway enzymes was then analyzed through a fluorescent protein (FP) imaging procedure recently developed for _C. roseus_ cell cultures [47; 48]. The full-length sequence of each enzyme was fused to the N-terminal end of green FP (GFP) to express a MEP enzyme-GFP fusion maintaining accessibility of the predicted TP. The resulting proteins were transiently expressed in _C. roseus_ chlorophyll-containing cells for imaging. Depending on enzymes, two different patterns of fluorescence were observed (Figure 5). From CMS to HDR, all enzyme fusions displayed a GFP fluorescence signal colocalizing with the autofluorescence of chlorophylls, demonstrating that these enzymes were targeted to plastids (Figure 5D1-H4). The signal appeared diffused into the stroma, as well as the stromules as previously reported for HDS and IDI [34; 36]. In  
Figure 4: Coexpression of the MEP pathway genes in internal phloem associated parenchyma (IPAP) cells of _C. roseus_ young leaves. Paraffin-embedded serial sections of young leaves were hybridized with digoxigenin-labeled transcripts, which were subsequently localized with anti-digoxigenin-alkaline phosphatase conjugates followed by nitro blue tetrazolium chloride (NBT)/5-bromo 4-chloro-3-indolyl phosphate (BCIP) color development. The antisense (AS) probes used for RNA labeling and control hybridization with sense (S) RNA probes are mentioned in the figure. (**A**) DXS2.1 AS; (**B**) DXS2.1 S; (**C**) DXS2.2 AS; (**D**) DXS2.2 S; (**E**) DXR AS; (**F**) DXR S; (**G**) CMS AS; (**H**) CMS S; (**I**) CMK AS; (**J**) CMK S; (**K**) MECS AS; (**L**) MECS S; (**M**) HDS AS; (**N**) HDS S; (**O**) HDR AS; (**P**) HDR S. Bars: 100 \(\upmu\)m.  
contrast, the two first steps of the MEP pathway, including DXS2.1, DXS2.2 and DXR, displayed a GFP fluorescence signal in punctuated aggregates within plastids (Figure 5A1-C4). Although diffuse fluorescence signals were also observed in plastid stroma of a few numbers of cells, the punctuated aggregates were detected in around 95% of the observed cells for both DXS and DXR. To determine whether this punctuated localization depended on the cell type used for localization, we also performed biolistic-mediated transient transformations of cells from detached _C. roseus_ leaves. In these cells, while HDS-Yellow FP (YFP) exhibited a diffuse signal in plastids, both DXS isoforms and DXR YFP fusions still displayed a punctuated fluorescence signal in plastids (Figure 6A-D). Such a protein distribution observed in different cell types was intriguing and raised the question of possible subplastitial targeting of DXS and DXR.  
Figure 5: Subcellular localization of the MEP pathway enzymes in _C. roseus_ chlorophyll-containing cells. _C. roseus_ cells (CR6 cell line) were transiently transformed with plasmids expressing MEP pathway enzymes fused to the N-terminus of GFP as indicated in the first column. The fluorescence emitted by GFP-fused proteins (**A1**–**H1**) was compared to chlorophyll autofluorescence (**A2**–**H2**). Colocalization of the two signals appeared in yellow while merging the two individual (green/red) color images (**A3**–**H3**). Cell morphology was observed with differential interference contrast (DIC, **A4**–**H4**). Bar: 10 \(\upmu\)m.