FIGURE 2. Role of CPT3 in Dol biosynthesis – studies in planta and functional assay in yeast.
A, B Relative expression of CPT3 and content of dolichols (Dol-15 to Dol-17) in the leaves of 4-week-old Arabidopsis plants, measured for wild-type Col-0, four independent RNAi lines targeting CPT3 (RNAi-1, -12, -14 and -23) and aCPT3 -overexpressing line (OE-7). The results are means (±SD) of three independent experiments. Asterisks indicate statistically significant differences between WT and mutant plants (* 0.01<P<0.05, ** 0.001<P<0.01, *** P<0.001, Student’s t-test).
C Changes in the levels of CPT3 mRNA (black curves) and in the content of Dols and Prens (respectively, orange and green bars) in the tissues of three Arabidopsis accessions: Col-0, Or-0, and Stw-0, during the plant life-span. Transcript levels and lipid content were estimated in Arabidopsis seedlings (1-3 weeks, bright colors) and leaves (4-6 weeks, deep colors), shown are means ± SD (n=3). Please note that the content of Prens is rescaled (0.01 multiples are presented) due to their high cellular level.
D Co-localization of fluorescence signals of CPT3-GFP (green) and ER-CFP (compartmental marker, blue) upon transient expression inNicotiana benthamiana leaves. Bar: 10 μm.
E Analysis of CPT3 and Lew1 protein–protein interaction using split yellow fluorescent protein (YFP) BiFC assay in tobacco leaf cells. Shown is the co-localization of fluorescence signals from the CPT1/Lew1 complex (green) and from the compartmental marker ER-CFP (blue). Bar: 10 μm. See Materials and methods.
F Analysis of CPT3 and Lew1 interaction using yeast two-hybrid system.
G, H Polyisoprenoid profiles and glycosylation status of CPY analyzed for rer2 Δ transformed with empty vector, CPT3 ,LEW1, and CPT3/LEW1. Representative HPLC/UV chromatograms are shown. The positions of mature CPY (mCPY) and its hypoglycosylated glycoforms (lacking between one and four N -linked glycans, -1 to -4) are indicated. See Materials and Methods.
FIGURE 3. Manhattan plot of genome-wide associations for phytosterols, plastoquinone, and dolichols. The dotted horizontal lines indicate a significance level of 0.05 after Bonferroni correction for multiple testing (see Materials and Methods). Manhattan plot of genome-wide association results for polyprenols, chlorophylls and tocopherols are shown in Figure S5.