## 3 Discussion  
By cloning the missing genes from the MEP pathway in _C. roseus_, we provide a comprehensive overview of the pathway organization in planta, which may potentially impact its involvement in the synthesis of highly valuable MIAs. As reported for many plants synthesizing specialized metabolites, we first established that at least two isoforms of clade II DXS coexist, namely DXS2.1 and DXS2.2, in the Madagascar periwinkle (Figure S1). Interestingly, these two isoforms display slightly distinct expression profiles in _C. roseus_ organs. DXS2.1 exhibits a coordinated expression with other MEP genes, including IDI, to fulfill IPP and DMAPP to both primary and secondary metabolisms (Figure 3; [30; 34]). By contrast, DXS2.2 is mainly expressed in flower buds and much less expressed in roots compared to other MEP genes. This could potentially be linked to a broader involvement of this isoform in the leaf MIA metabolism and the dedicated MIA metabolism taking place in reproductive organs as described previously for a specific isoform of tabersonine 16-hydroxylase [49]. In young leaves, RNA in situ hybridization confirmed that internal phloem associated parenchyma (IPAP) is the main site of expression of MEP pathway genes (Figure 4). Albeit we cannot exclude a lower expression in other leaf tissues, as described with the immunolabeling of HDS and confirmed for DXR (Figure 10; [35]), this result definitively confirms that IPAP is a key tissue in both IPP synthesis and MIA precursor production. The latter thus begins with a DXS-catalyzed reaction up to the antepenultimate step of the MSI pathway catalyzed by 7-deoxyloganic acid hydroxylase and class II cytochrome P450 reductase to form loganic acid [19; 50], thus encompassing 16 successive enzymatic steps. While transporters allowing import of loganic acid  
Figure 11: Transient transformation of _C. roseus_ young leaf using a decreasing amount of the DXR-YFP expressing construct. Young _C. roseus_ leaves were transiently transformed with decreasing amounts of the plasmid expressing DXR fused to the N-terminus of YFP as indicated at the bottom of each picture. The fluorescence emitted by YFP-fusions proteins was superimposed to cell morphology acquired using differential interference contrast. Arrowhead and star indicate punctuated and diffuse localization in plastids, respectively. (**A**) 800 ng; (**B**) 400 ng; (**C**) 150 ng. Bar: 10 \(\upmu\)m.  
into the epidermis have been identified, transporters ensuring its export from IPAP have yet to be discovered [51]. The identification of such transporters remains of importance since transport is a key element in the control of the whole MIA metabolism. At the subcellular level, we showed that all MEP pathway enzymes are targeted to plastids including stroma and stromules, as previously established for HDS and IDI [34; 35]. A schematic representation of this distribution is given in Figure 12. While DXS2.1, DXS2.2 and DXR were initially reported to display a punctuated pattern of localization in plastids, fusion/deletion experiments, DXR immunolabeling in planta and cell transformations with decreasing amounts of DNA, confirmed that this result was artifactual and probably resulted from artificially high protein expression levels (Figures 5, 7 and 9-11). These increased amounts of proteins may have impeded the protein folding capacities of DXS and DXR resulting in protein aggregation and formation of the punctuated fluorescence signal. Although partial colocalization with the plastoglobulin markers has been observed (Figure 9), this localization could not be observed with TEM. Additionally, no MEP pathway enzyme has so far been detected in the plastoglobulin proteome, suggesting that early MEP pathway enzymes, while expressed in close proximity to plastoglobulin due to their high density in plastids, are not in fact localized to these structures [52].