Fig. 4.

GFP-ICL driven from the ICL promoter reports native ICL localization and levels during seedling development. (A) GFP fluorescence in hypocotyls of light-grown seedlings expressing ICLp-GFP-ICL or 35S-GFPSKL constructs at 4–8 days after sowing. Peroxisomal ICLp-GFP-ICL fluorescence diminishes after 5 days in wild-type (lines K325 and K345), whereas fluorescence persists for at least 8 days in pex4–1 pex22–1 (lines K5 and K23). GFP fluorescence from 35S-GFPSKL is constitutive. Exposure times in each row were normalized to the 4-day-old seedling. Bar, 20 μm. Insets are confocal optical slices showing individual peroxisomes in hypocotyl cells; inset bar, 5 μm. (B) Immunoblot of protein extracts (32 cotyledons per lane) from wild-type and pex4–1 pex22–1 plants expressing ICLp-GFP-ICL. Blots were probed with α-ICL and α-HSC70 antibodies. The α-ICL antibodies detect GFP-ICL (absent in untransformed wild-type, right lane), native ICL, and a cross-reacting band marked with an asterisk. Positions of molecular weight markers (in kDa) are indicated on the left.