Figure 1.
Simplified sterol biosynthetic pathway in Arabidopsis and yeast.
Sterol biosynthesis starts preferentially with cycloartenol in plants and lanosterol in animals and fungi. Main biosynthetic steps and sterols accumulating in the mutant lines considered in this study are indicated in colors: red, ste1; green, dwarf5; blue, dim1. The major observed enzymatic activity of the YFP-fused enzymes generated is also indicated by the same color code. LAS1 = lanosterol synthase, CAS1 = cycloartenol synthase. The dashed arrows are indicating more than one enzymatic step. Accurate sterol nomenclature can be found at IUPAC http://www.iupac.org/
Table 1.
Sterol composition of wild type (W303) and mutant (erg3 and erg4) yeast strains expressing the corresponding YFP-fused proteins compared to the non-transformed strains.
Figure 2.
Genetic complementation of ste1-1, dwarf5-2 and dim sterol biosynthetic mutants expressing STE1-YFP, DWARF5-YFP, and DIM-YFP cDNAs, respectively.
GC-FID chromatograms of steryl acetates are shown. (A) dwarf5-2 mutant; (B) dwarf5-2/DWARF5-YFP partially complemented mutant; (C) dwarf5-2/DWARF5-YFP fully complemented mutant. (D) dim mutant; (E), dim/DIM-YFP partially complemented mutant; (F) dim/DIM-YFP fully complemented mutant. (G) ste1-1 mutant; (H), ste1-1/STE1-YFP partially complemented mutant; (I) ste1-1/STE1-YFP fully complemented mutant. Sterol peaks identified by their retention time and confirmed by GC-MS (prominent mass fragments not shown here) are: 1, cholesterol; 2, Δ5,7-cholesterol; 3, Δ7-cholesterol; 4, campesterol; 5, Δ7-campesterol; 6, Δ5,7-campesterol; 7, Δ5,7-stigmasterol; 8, Δ8-sitosterol; 9, Δ5,7-sitosterol; 10, sitosterol; 11, isofucosterol; 12, Δ7-sitosterol; 13, 24-methylene cholesterol; 14, stigmasterol; 15, Δ7-avenasterol. Full complementation of dwarf5-2, dim and ste1-1 results in the accumulation of sitosterol (10) instead of Δ5,7-sitosterol (9), isofucosterol (11) and Δ7-sitosterol (12), respectively. The relevant peaks in each complementation are labelled in bold in the relevant panels.
Table 2.
Sterol composition of wild type and dwarf5-2 (d5), dim (d1) and ste1-1 (s1) Arabidopsis mutant plants.
Figure 3.
Phenotype of dwarf5-2, dim and ste1-1 mutants complemented with DWARF5-YFP, DIM-YFP and STE1-YFP, respectively.
(A, D, G) dwarf5-2, dim and ste1-1 (sterol profiles given in Figure 2). (B, E, H) dwarf5-2, dim and ste1.1 partial complemented. (C, F, I) dwarf5-2, dim and ste1-1fully complemented. (J) Wild-type (sterol profile given in Supplemental Figure 2).
Figure 4.
Subcellular localization of DWARF5-YFP protein in Arabidopsis dwarf5-2::DWARF5-YFP plants.
(A) Confocal images of leaves showing protein distribution in the ER and (D) to the periphery of the cell (yellow arrow). (B) Chlorophyll autofluorescence. (E) In red is shown the chlorophyll autofluorescence combined with the FM4-64 fluorescence localized to the PM. The overlay images show (C) the complete separation of red and yellow signal and (F) the co-localization of DWARF5-YFP and FM4-64 indicating the PM association of DWARF5. Scale bars = 25 µm.
Figure 5.
Subcellular localization of DIM-YFP protein in Arabidopsis dim::DIM-YFP plants.
(A) The DIM-YFP signal is localized to structures surrounding the nuclei resembling ER (white arrow) and to the cell periphery (yellow arrow). (B) In red is shown the chlorophyll autofluorescence combined with the FM4-64 fluorescence localized to the PM. (C) The overlay image shows the co-localization of DIM-YFP and FM4-64 suggesting the PM association of DIM. Scale bars = 50 µm.
Figure 6.
Subcellular localization of STE1-YFP protein in Arabidopsis ste1-1::STE1-YFP plants (panel A to D) and Nile Red staining of ste1-1 mutants (panel E to H).
(A) STE1-YFP localization to structures resembling ER (white arrow) and LPs (yellow arrows). (B) and (F) chlorophyll autofluorescence. (C) and (G) LPs stained with Nile Red (yellow arrows). (D) Overlay image of (A), (B) and (C) showing the overlap of the Nile Red and YFP signal (yellow arrow) and the ER localization (white arrow) of STE1-YFP. (E) YFP signal absent in ste1-1 mutant. (H) Overlay image of (E), (F) and (G) showing the distribution of LPs in cell of ste1-1 plant. Scale bars = 10 µm.
Figure 7.
Subcellular localization of STE1-YFP protein in Arabidopsis ste1-1::STE1-YFP plants.
(A) STE1-YFP fluorescence signal in leaf vascular tissue. (B) Chlorophyll autofluorescence. (C) Overlay image of (A) and (B). Scale bars = 250 µm.