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. 2023 Jul 6;120(29):e2304870120. doi: 10.1073/pnas.2304870120

Fig. 6.

Fig. 6.

Overexpressed mNeonGreen–MIEL1 decreases oleosin levels, slows TAG utilization, enlarges lipid droplets, and localizes to peroxisomes. (A) Diagram of constitutively expressed fluorescent reporters marking peroxisomal lumen (tdTomato–PTS1) and MIEL1 (mNeonGreen–HA–MIEL1). (B) Relative mRNA levels in 2-d-old seedlings were measured via RT-qPCR and statistically analyzed as in the legend of Fig. 4. (C) Immunoblot of seedlings without a reporter (WT), segregating for mNeonGreen–HA, or homozygous for mNeonGreen–HA–MIEL1 probed with the indicated antibodies. (D) Immunoblot probed with the indicated antibodies. (E) TLC of extracted lipids. The graph shows relative TAG content in three biological replicates (including the one shown) normalized to the WT signal at 0 d and statistically analyzed as in the legend of Fig. 1. (F) Confocal images (maximum-intensity projections of twelve 1-µm slices) of hypocotyl epidermal cells showing tdTomato–PTS1 (magenta) and mNeonGreen–HA (green) at the indicated seedling ages. Neutral lipids were stained with MDH (cyan) in 2-d-old seedlings. (G) Magnification of boxed region in 7-d image from panel F. Plot shows tdTomato–PTS1 (magenta) and mNeonGreen–HA (green) fluorescence intensity across multiple peroxisomes (orange arrow). (H) As in panel F, but with mNeonGreen–HA–MIEL1. (I) Magnification of boxed region in 7-d image from panel H. Plot shows tdTomato–PTS1 (magenta) and mNeonGreen–HA–MIEL1 (green) fluorescence intensity across multiple peroxisomes (orange arrow). In panels C–E, 0-d samples were stratified seeds; other samples were seedlings harvested at the indicated times following sowing.