Farnesyl pyrophosphate fpp

Barley seeds (Hordeum vulgare L. cultivar. Steffi, Saatzucht Ackermann & Co, Irlbach, Germany) were grown for 4.5 days at 23°C. Intact etioplasts were isolated and the concentration of plastids determined by counting [19 (link), 34 (link)]. For phototransformation of Pchlide, and synthesis of Chlide and Chl, etioplasts were preincubated in the absence or presence of GGPP (1,35 mM, Sigma-Aldrich, St. Louis, USA). For esterification of GGPP (1,35 mM) and Farnesyl pyrophosphate (FPP) (1,3 mM) (Sigma-Aldrich, St. Louis, USA), etioplasts were investigated in the presence of NADPH (0,5 mM Sigma-Aldrich, St. Louis, USA) for 1 min on ice. Etioplasts were transferred to dry ice and either maintained in the dark (D) or illuminated (L) for 2 s with white light via a light guide (10 mEm^-2s^-1, Schott, DE). For comparison of Chl synthesis in light and darkness, etioplast were illuminated with white light (50 E/m²s) or supplemented with 0,77 nmol of Zn-pheophorbide a (Zn-pheide) in darkness in the presence or absence of GGPP for 20 min at 25°C. Pigment concentrations were determined according to Helfrich [37 (link)].\

An in vitro enzyme assay for TPS activity was performed in a final volume of 500 μl of reaction buffer [25mM HEPES, pH 7.4, 100mM KCl, 7.5mM MgCl₂, 5% (v/v) glycerol, 5mM dithiothreitol], with about 20 μg of recombinant protein and 10 µg of either farnesyl pyrophosphate (FPP) or geranyl pyrophosphate (GPP) (Sigma-Aldrich). The reaction mixtures were mixed gently and carefully overlaid with 250 µl of hexane (Sigma-Aldrich) to trap volatile products. The tube was then sealed with Parafilm and incubated at 30 °C for 2h, followed by 1min of vortexing. After centrifugation at 1200g at 4 °C for 30min, the hexane upper layer was transferred into a 2ml glass vial for GC-MS analysis (see below). As a negative control, heat-inactivated recombinant protein was added to the enzyme assay.

Alendronate was obtained from Merck Pharmaceuticals (Whitehouse Station, NJ, USA). Alizarin Red S, thiazolyl blue tetrazolium bromide (MTT), Percoll, L-ascorbic acid-2-phosphate, beta-glycerolphosphate, sodium bicarbonate, dexamethasone, N-acetyl-L-cysteine (NAC), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Dulbecco's Modified Eagle's Medium (DMEM), ascorbic acid, non-essential amino acids, penicillin/streptomycin, fetal bovine serum (FBS), keratinocyte-serum free medium (SFM), epidermal growth factor-bovine pituitary factor (EGF-BPE) and trypsin/EDTA were purchased from Gibco-BRL (Rockville, MD, USA). Dimethyl sulfoxide (DMSO) was purchased from Amresco LLC (Solon, OH, USA).