## 2 Results
### Punctuated Pattern of Localization of DXS and DXR Requires the Whole Protein Sequence  
To gain insight into the punctuated localization of DXS and DXR, we performed additional fusions with fluorescent proteins. First, to analyze the role of the mature part of these proteins, fusion engaging only the predicted TP (first 100 residues) of DXS2.1, DXS2.2, DXR or HDS (named tpDXS2.1-GFP, tpDXS2.2-GFP, tpDXR-GFP and tpHDS-GFP) were transiently expressed in _C. roseus_ cells. Compared to full-length proteins, deletions of the mature part of DXS, DXS2 and DXR led to the loss of the aggregated pattern of fluorescence and resulted in diffuse patterns of fluorescence within the stroma and stromules (Figure 7A1-C6). By contrast, both tpHDS-GFP, HDS-GFP and HDS-GFP displayed a similar diffuse fluorescent signal in the stroma and stromules of plastids (Figure 7D1-D6), clearly distinct from the signal obtained with the DXS2.1-GFP protein (Figure 7E1-E4). In addition, internal fusions with YFP were also created for both DXR and HDS (tpDXR-YFP-DXR and tpHDS-YFP-HDS) (Figure 8A,B). For each of these constructs, the punctuated (DXR) and diffuse plastid (HDS) localizations were conserved (Figure 8C-J), confirming the requirement of the mature part of DXS2.1, DXS2.2 and DXR to their peculiar localization in plastids.  
Figure 6: Subcellular localization of HDS-YFP, DXS2.1-YFP, DXS2.2-YFP and DXR-YFP fusions in cells of young _C. roseus_ leaf. Young _C. roseus_ leaves were transiently transformed with plasmids expressing HDS (**A**) and both DXS (**B**,**C**) and DXR (**D**) fused to the N-terminus of YFP as indicated on the top of each picture. The fluorescence emitted by YFP-fusion proteins was superimposed to cell morphology acquired using differential interference contrast. Arrowhead and star indicate punctuated and diffuse localization in plastids, respectively. Bar: 10 μm.  
Figure 8: Subcellular localization of DXR and HDS studied using internal YFP protein fusions. _C. roseus_ cells were transiently transformed with plasmids expressing internal YFP fused to split DXR (**A**) and HDS (**B**). The fluorescence emitted by YFP-fused proteins (**C**,**G**) was compared to the fluorescence emitted by the plastid-CFP marker (**D**,**H**). Colocalization of the two signals appeared in yellow by merging the two individual (green/red) color images (**E**,**I**). Cell morphology is observed with differential interference contrast (DIC, **F**,**J**). Bar: 10 \(\upmu\)m.  
Figure 7: Comparison of the subcellular localization of DXS2.1, DXS2.2, DXR and HDS expressed as full-length-GFP fusion proteins or as transit peptide-GFP fusion proteins. _C. roseus_ cells were transiently transformed with plasmids expressing a GFP protein fused to the first 100 residues of the enzyme, encompassing the predicted transit peptide (tp) of DXS2.1 (tpDXS2.1-GFP, **A1–A3**), DXS2.2 (tpDXS2.2-GFP, **B1–B3**), DXR (tpDXR-GFP, **C1–C3**), HDS (tpHDS-GFP, **D1–D3**), or fused to full-length DXS2.1 (DXS2.1-GFP, **A4–A6**), DXS2.2 (DXS2.2-GFP, **B4–B6**), DXR (DXR-GFP, **C4–C6**), HDS (HDS-GFP, **D4–D6**). An additional co-transformation of DXS2.1-YFP (**E1**) and HDS-CFP (**E2**) was performed to highlight differences of localization in plastids as revealed on the merged image (**E3**). Cell morphology was observed with differential interference contrast (DIC, **E4**). Bar: 10 \(\upmu\)m.