Characterization of <i>fps2-1</i> mutant lines harbouring <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> genes.

<p>(A) The expression of <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> was investigated using total RNA from 12-day-old seedlings of Arabidopsis wild-type, <i>fps2-1</i> and the indicated lines of the <i>fps2-1</i> mutant harbouring <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> chimeric genes. PCR products were electrophoresed in a 1% agarose gel. The size in bp of the amplified cDNA fragments corresponding to <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> (1088 bp) and <i>PP2A</i> genes (307 bp) is indicated on the right. Numbers on the left indicate the sizes in bp of DNA markers shown in lane M. (B) Western blot analysis of total FPS protein in 16,000 <i>g</i> extracts from seeds of plant lines indicated above (upper panel). The lower panel shows the Coomassie blue-stained electrophoretic protein patterns in the 35 to 50 kDa range of extracts used for FPS protein level determinations. Images show the results of one representative experiment. (C) FPS activity in the 16,000 <i>g</i> protein extracts used for Western blot analysis. FPS activity in mutants is expressed relative to that in the wild-type, which was assigned a value of 100. The mean values and SE were calculated from three independent experiments.</p

Expression in <i>E. coli</i> and purification of recombinant FPS1S and FPS2 proteins.

<p>(A) Total protein extracts from <i>E. coli</i> cells harbouring either pGEX-3X-NotI-FPS1 or pGEX-3X-NotI-FPS2 before (lanes 1 and 3) and after induction (lanes 2 and 4) of GST-FPS1S and GST-FPS2 expression with 0.4 mM IPTG for 6 hours at 22°C. (B) Soluble protein extracts of IPTG-induced <i>E. coli</i> cells harbouring either pGEX-3X-NotI-FPS1 (lane 1) or pGEX-3X-NotI-FPS2 (lane 2), and purified native FPS1S (lane 3) and FPS2 (lane 4) protein preparations after Glutathione-Sepharose 4B affinity column chromatography, proteolytic digestion with Factor Xa and protease removal. Arrows indicate the position of GST-FPS protein fusions and purified native FPS proteins. Molecular masses of standards (M) are indicated in kDa.</p

Thermal stability of FPS1S and FPS2 enzyme activity.

<p>(A) Activity of purified FPS1S (open symbols) and FPS2 (closed symbols) was measured after incubation at either 37°C (squares) or 45°C (circles) for the indicated time periods. (B) FPS activity in 16,000 <i>g</i> protein extracts from <i>fps1-1</i> (FPS2 activity, closed circles) and <i>fps2-1</i> (FPS1 activity, open circles) mutants was determined after incubation at 45°C for the indicated times. In both cases enzyme activities are expressed relative to the FPS activity values at time 0 min and the mean values and SE were calculated from three independent experiments. (C) Differential scanning fluorimetry (DSF) results plotted as change in fluorescence emission intensity (normalized to unity at its maximum) with increasing temperature (20–80°C). The FPS1S and FPS2 curves correspond to 6 µM enzyme.</p

Homology modelling of FPS1S and FPS2 proteins and in silico <i>DD</i>G calculations.

<p>(A) Ribbon representation of the dimeric and monomeric structures of human FPS (PDB 2F7M), which was used to template the threading of Arabidopsis FPS1S and FPS2. The active site cleft is labelled and a bound phosphate ion is shown in sticks. (B) Sequence substitutions between FPS1S (top) and FPS2 (bottom) were mapped onto the ribbon structure (left monomer) or the molecular surface (right monomer) of the homology modeled dimers. Chemical character is colour coded as follows: red, acidic (Asp, Glu); blue, basic (Arg, Lys, His); green, polar (Ser, Thr, Asn, Gln, Tyr); orange, apolar (Met, Phe, Pro, Trp, Val, Leu, Ile, Ala). (C) Histogram of <i>DD</i>G (kcal/mol) upon single-site substitution calculated using Rosetta <i>DD</i>G application (top) or CC/PBSA (bottom). Mutations predicted to occur with a decrease in <i>DD</i>G are coloured green and those expected to increase <i>DD</i>G are coloured pink. Horizontal dashed lines at –1 to +1 kcal/mol bound the neutral area where <i>DD</i>G is supposed to contribute little to the overall stabilization or destabilization of the mutated protein.</p

Effect of pH and NaCl on FPS1S and FPS2 enzyme activity.

<p>(A) FPS activity of purified FPS1S and FPS2 was determined at pH values ranging from 5.5 to 9.0 as described under Materials and Methods. Enzyme activities are expressed relative to the maximal activity values for FPS1S and FPS2. (B) Effect of NaCl on FPS1S and FPS2 enzyme activity. FPS activity of purified FPS1S and FPS2 was determined at the indicated NaCl concentrations. Enzyme activities are expressed relative to the activity values for FPS1S and FPS2 measured without NaCl. The mean values and SE were calculated from three independent experiments.</p

Reversion of <i>fps2-1</i> phenotypes by expressing FPS1S under control of the <i>FPS2</i> promoter.

<p>Sitosterol (A) and HMGR activity (B) levels in seeds from Arabidopsis wild-type, <i>fps2-1</i> and the indicated lines of the <i>fps2-1</i> mutant harbouring <i>FPS2p::FPS1S-mutdis</i> and <i>FPS2p::FPS1S</i> chimeric genes. Values are expressed relative to the wild-type values. Data represent the mean values and SE of three independent experiments. (C) Reversion of mevastatin hypersensitivity. Representative seedlings of the same lines were grown for 12 days under long-day conditions (16 h light/8 h dark) on MS plates supplemented with 1 µM mevastatin.</p

Steady-state kinetic constants for FPS1S and FPS2.

<p>The <i>K</i>_m mean values and SE were calculated from three independent experiments.</p><p>k_cat values were calculated for the dimeric form of the enzyme.</p

Schematic representation of chimeric genes <i>FPS2p::FPS1-mutdis</i> (A), <i>FPS2p::FPS1S</i> (B), <i>FPS1mutdisp::FPS2</i> (C) and <i>FPS1p::FPS2</i> (D).

<p>Dark gray boxes represent <i>FPS2</i> genomic sequences whereas sequences corresponding to <i>FPS1</i> gene are represented by light gray boxes. In all constructs the 3′-untranslated regions of both <i>FPS</i> genes is represented by an empty box. The position of the translational start codons is indicated as ATG and that of stop codons is indicated as TAG. In the <i>FPS2p::FPS1S-mutdis</i> (A) and <i>FPS1mutdisp::FPS2</i> (C) constructs, the 5′-most ATG codon in exon 1 of the <i>FPS1</i> gene was mutated to an ATC codon (encoding Ile). Arrows indicate the transcription start sites. In the <i>FPS1</i> gene, position +1 was assigned to the most internal transcription start site.</p

Reversion of <i>fps2-1</i> phenotypes by expressing FPS2 under the control of the <i>FPS1</i> promoter.

<p>Sitosterol (A) and HMGR activity (B) levels in seeds from Arabidopsis wild-type, <i>fps2-1</i> and the indicated lines of the <i>fps2-1</i> mutant harbouring <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> chimeric genes. Values are expressed relative to the wild-type values. Data represent the mean values and SE of three independent experiments. (C) Reversion of mevastatin hypersensitivity. Representative seedlings of the same lines were grown for 12 days under long-day conditions (16 h light/8 h dark) on MS plates supplemented with 1 µM mevastatin.</p

Subcellular localization of GFP-tagged FPS1S and FPS2 proteins.

<p>Confocal laser scanning micrographs showing the distribution of fluorescence in agroinfiltrated <i>N. benthamiana</i> epidermal cells transiently co-expressing FPS1S-GFP (A), GFP-FPS1S (E), FPS2-GFP (I) or GFP-FPS2 (M) with the peroxisome protein marker CFP-SKL (B, F, J and N). Co-localization evaluation of GFP-tagged FPS proteins with the peroxisome protein marker (C, G, K and O). Differential interference contrast (DIC) images showing the morphology of transformed cells (D, H, L and P). Scale bars = 20 µm.</p