Fig 1.
(A) Microscopy images of WT protonema cells grown in for 10 days (i) and WT gametophytes grown for 6 weeks (ii). Stained with BODIPY (green) and overlaid with chloroplast autofluorescence (red), showing the presence of cytosolic LDs. (B) (i) Image depicts 3D projection of surface rendering Z-stack image of a portion of a cell stained with BODIPY and ER-Tracker™ Red dye was used. and overlaid with red chloroplast autofluorescence (ii) BODIPY stained LDs in green overlaid with red chloroplast autofluorescence after removing the ER tracker stained Z-stack image. The right-hand side shows a model for the localization of LD-associated proteins. Seipin oligomers localize in the ER at the ER-LD contact site. Lipid droplet associated proteins mainly fold as a helical protein and covers the LD surface without disturbing the LD membrane. Oleosin proteins penetrate the LD membrane into the LD matrix with a long conserved hydrophobic hairpin structure.
Fig 2.
Co-localization of PpOLE1 to LDs of P. patens.
(A) PpOLE1 was visualized by the fluorescence of the C-terminal tagged Venus and LDs were visualized by LipidTOX Red stain (artificial yellow color). (B) 3D projection of surface rendering Z-stacks of LipidTOX Red stained LDs (artificial yellow color) were merged with Venus for the depiction of LD co-localization)).
Fig 3.
Co-localization of AtLDAP1 to P. patens LDs.
A) Processed confocal Z-stacks of Venus tagged AtLDAP1 (yellow), LipidTOX red-stained LD (green) and subsequent overlap of the two micrographs (scale = 10 μm). B) 3D projection of surface rendering Z-stack image of the stained LDs (green) with chloroplast auto-fluorescent (red) and subsequent overlap of Venus localization of AtLDAP1.
Table 1.
LD size of different P. patens lines (based on biological triplicates).
Fig 4.
Localization of PpSeipin325 in P. patens.
A) PpSeipin325 was tagged at the C-terminal with Venus protein (yellow) and imaged for the localization using confocal microscopy. LDs were stained with LipidTOX red (artificial green color) and merged with the Venus expression. Merged image depicts that the majority of LDs (green) are following the expression pattern of PpSeipin325 (yellow). (B) (i)3D projection of surface rendering Z-stack PpSeipin325 (yellow) with LDs (green). (ii) PpSeipin 325 expression pattern (yellow) after removing the LD Z-stack image. Arrows indicated the holes where the LDs (green) are emerging (scale = 10 μm).
Table 2.
Patchoulol production of three weeks old modified P. patens cell lines (biological triplicates).
Fig 5.
Ectopic expression of LD-associated proteins and PTS promoted LD occurrence.
Representative confocal Z-stack images of LDs in gametophyte leaves of different P. patens lines. Green color shows the BODIPY stained LDs and the red shows the chloroplast autofluorescence. Images were collected at the same laser intensity and magnification (scale = 10 μm).
Fig 6.
Log (x+) normalized heatmap of the different P. patens lines and the wildtype P. patens with a 256-fold up (red) or down (blue) regulation of gene expression in one or more P. patens lines compared to the wild type.
For some this also show that there is little to no change in one or more P. patens lines, but a 256-fold change in just one P. patens line. The annotation of the contig numbers is seen in Table 3.
Table 3.
List of contigs shown in Fig 3.