## 4 Material and Methods
### RNA In Situ Hybridization  
The protocol used for the detection of the MEP pathway transcripts in _C. roseus_ leaves was previously described [32; 60]. Full-length cDNAs of DXS2.2, CMS, CMK and HDR amplified with the specific corresponding primers (Table S1) and cloned in pSC-A amp/kan (Agilent Technologies) were used for the synthesis of sense and antisense digoxigenin-labeled RNA probes. For DXS2.1, DXR, MECS and HDS, the previously described plasmids were used for the transcription of riboprobes [32; 41]. Briefly, RNA hybridization was performed at room temperature, unless otherwise stated. After rehydration, _C. roseus_ leaf sections were treated with proteinase K (5 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) in 100 mM Tris-HCl and 50 mM EDTA (pH 8.0)) for 30 min at 37 \({}^{\circ}\)C, and rinsed twice with TBS150 (10 mM Tris-HCl, 150 mM NaCl (pH 7.5)), followed by blocking of proteinase K with glycine (2 mg\(\cdot\)mL\({}^{-1}\) in TBS150) for 2 min, and by two rinses in TBS150. After postfixation with 3.7% formaldehyde in PBS during 20 min and two washing steps in TBS150 (5 min), sections were acetylated with acetic anhydride (0.25% in 0.1 M triethanolamine-HCl (pH 8.0)) for 10 min, washed with TBS150, dehydrated in an ethanol series and air-dried. To detect RNA, 120 \(\upmu\)L of the hybridization mix (200 ng\(\cdot\)mL\({}^{-1}\) partially hydrolyzed digoxigenin-labeled RNA transcripts, 40% formamide, 10% dextran sulfate, 1 \(\upmu\)g\(\cdot\)mL\({}^{-1}\)oproteinised yeast total RNA, 0.3 M NaCl, 0.01 M Tris-HCl (pH 6.8), 10 mM sodium phosphate (pH 6.8), 5 mm EDTA and 40 units-mL\({}^{-1}\) RNAsin ribonuclease inhibitor) were dispersed on the sections before mounting under cover slips to limit evaporation. Hybridization was performed during 16 h at 50 \({}^{\circ}\)C in an atmosphere containing 50% formamide. Cover slips were removed from slides through soaking in 2 \(\times\) SSC at 37 \({}^{\circ}\)C (1 \(\times\) SSC is 0.15 M NaCl and 15 mM sodium citrate). Before detection, slides were treated with RNase A (50 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) in 10 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA (pH 7.5)) for 30 min at 37 \({}^{\circ}\)C followed by washing under gentle agitation in 2 \(\times\) SSC for 1 h, 1 \(\times\) SSC for 1 h and in 0.1 \(\times\) SSC for 1 h at 65 \({}^{\circ}\)C. To detect hybridized transcripts, slides were washed in Tween TBS (TTBS) (0.1 M Tris-HCl (pH 8.0), 0.15 M NaCl and 0.3% Triton X-100) for 10 min and blocked with 2% BSA in TTBS for 30 min. Sheep antidigoxigenin Fab fragments-alkaline phosphatase conjugate (Roche) at a 1:200 dilution in a solution of 1% BSA in TTBS was dispensed on the sections and mounted under cover slips. After incubation for 2 h, unbound conjugates were washed two times for 15 min with TTBS and two times for 10 min with AP buffer (0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl and 10 mM MgCl\({}_{2}\)). For color development, slides were immerged in 175 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 350 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) nitro blue tetrazolium chloride in AP buffer for 1-16 h. After development, slides were washed in water, dried and mounted in immersion oil under cover slips.