## 2 Results
### Punctuated Localization of DXS and DXR is Likely Caused by Protein Overexpression  
To identify a potential plastid subcompartment hosting DXS2.1-, DXS2.2- and DXR-YFP fusions, we performed cell transformations with distinct markers targeted to thylakoids (CAB-CFP; Figure 9A,B), the inner membrane of plastid envelope (TIC-CFP; Figure 9C,D) and plastoglobulin (CCD4-CFP; Figure 9E,F). While thylakoid and inner membrane markers exhibited circular fluorescent signals in plastids, the plastoglobulin marker displayed a punctuated signal similar to that of DXS2.1, DXS2.2 and DXR fusion proteins. However, cotransformations of cells with plasmids expressing this marker (CCD4-CFP) or DXS2.1, DXS2.2, DXR, HDS-YFP did not reveal a perfect superimposition of the two fluorescent signals, preventing us to firmly conclude on a potential targeting of MEP pathway enzyme to plastoglobulin (Figure 9G-Z). Figure 9: Coexpressions of DXS2.1-YFP, DXS2.2-YFP and DXR-YFP fusions with different plastid markers. Chlorophyll-containing _C. roseus_ cells were transiently transformed with plasmids expressing the CFP-marker of thylakoid (CAB-CFP, **A,B**), the inner membrane of chloroplast envelope (TIC-CFP, **C,D**) and plastoglobulin (Carotenoid Cleavage Dioxygenase (CCD) 4-CFP, **E,F**) or cotransformed with plasmids expressing the CCD4-CFP marker and the DXS2.1-YFP fusion protein (**G–J**). Chlorophyll-free _C. roseus_ cells were transiently transformed with plasmids expressing the CCD4-CFP marker and the DXS2.1-YFP (**K–N**), DXS2.2-YFP (**O–R**), DXR-YFP (**S–V**) and HDS-YFP (**W–Z**) fusion proteins. Colocalization of the two signals appeared in yellow by merging the two individual (green/red) color images (**I**,**M**,**Q**,**U**,**Y**). Cell morphology is observed with differential interference contrast (DIC, **B**,**D**,**F**,**J**,**N**,**R**,**V**,**Z**). Bar: 10 μm. This result prompted us to analyze subcellular localization in native conditions by performing anti-DXR immunogold labeling on cells of _C. roseus_ young leaves and transmission electron microscopy (TEM) observation. First, we noticed that the immunogold labeling was only barely detectable in plastids of palissadic parenchyma while a pronounced signal was observed in plastids of IPAP cells (Figure 10A-D), thus confirming our previous results of RNA in situ hybridization (Figure 4). Figure 10: Immunogold labeling of DXR in the stroma of plastids from _C. roseus_ leaf cells. **(A)** Morphology of _C. roseus_ young leaves observed on ultrathin cross-sections highlighting epidermis (ep), palissadic parenchyma (pp), spongy parenchyma (sp), xylem (x), phloem (ph) and internal phloem associated parenchyma (* or ipap). **(B)** Intermediate magnification highlighting plastids from pp and ipap. **(C)** A very low abundant—but specific—labeling was observed in the stroma of palissadic parenchyma plastids neighboring ipap cells. **(D,E)** Typical dense labeling was specifically observed in the stroma of ipap plastids devoid of plastoglobulin-like structures but including stromule budding (s). cp, chloroplast; cw, cell wall; sg, starch granule. Interestingly, a homogenous immunogold labeling was observed in the stroma of all observed plastids that never displayed plastoglobulin-like structures (Figure 10D,E) but could show some stromule budding including labeling (Figure 10E). Such an absence of the aggregated localization observed through the GFP imaging procedure strongly suggested that it might result from the procedure of protein expression in cells and notably the strong gene overexpression caused by the use of the CaMV355 promoter. To test this hypothesis, additional transformations of the construct expressing the DXR-YFP fusion were performed in cells of _C. roseus_ young leaves using a decreasing amount of transforming plasmid DNA.