Fig. 2.

Confirmation of enzyme activity in melon DXS and HDR genes. (A) Complementation of a dxs- E. coli mutant with melon DXS isoforms. The dxs– strain can grow normally in the presence of 1mM mevalonate but requires transformation with a plasmid encoding a functional DXS gene for viability on mevalonate-free media. Picea abies DXS1 (PaDXS1) is included as a positive control (Phillips et al., 2007) while melon 1-deoxyxyulose 5-phosphate reductoisomerase (CmDXR) was used as a negative control. (B) Crude lysates of E. coli overnight cultures expressing CmDXS proteins were used in DXS in vitro reactions with pyruvate, glyceraldehyde 3-phosphate, and all necessary co-factors. DXP was analysed by LC-MS/MS. The non-transformed control (NT) indicates endogenous DXS levels. (C) complementation of the hdr E. coli mutant MG1655 ara<>ispH (McAteer et al., 2001) with melon HDR isoforms. The LB-carbenicillin-kanamycin plate on the left also contains 0.2% arabinose, which induces expression of the endogenous HDR gene in this mutant strain. The plate on the right contains the same antibiotics and 0.2% glucose, which represses endogenous HDR expression. Under these conditions, the strain is viable only when transformed with a plasmid encoding a functional HDR gene.