Figure 4

Protein accumulation and synthesis in RLSB/RLSB and rlsb-1/rlsb-2 double mutant sibling plants. For each panel, proteins were isolated from lower (lower third) or upper (upper third) regions of the first or second emerging leaves (4 – 6 cm in length) from each genotype. Panel A: Equalized amounts of total proteins (soluble plus membrane) isolated from these regions were loaded and separated by SDS-PAGE, and silver stained. The positions of LSU and SSU (solid circles), unidentified proteins reduced in mutant leaf base (solid triangles), and unidentified proteins increased in mutant leaf base (open triangles) are indicated. Panel B: Soluble protein accumulation in lower leaf regions of RLSB/RLSB, heterozygote RLSB/rlsb-1, and mutant maize rlsb-1/rlsb-2 plants. Positions of the Rubisco LSU, as well as three other soluble proteins affected in the mutant, are indicated (arrows). Note that in this panel, the LSU band is overlapping (and slightly below) another unidentified protein, which somewhat obscures its reduction in the double mutant plants. Panel C, top: In vivo synthesis of total soluble proteins in lower and upper leaf regions. Protein extracts were prepared from leaf disks labeled with ³⁵S-met/cys for one hour, as described in Methods. Equalized amounts of labeled extract were separated by SDS-PAGE and visualized using a phosphorimager. Position of the LSU is indicated. Panel C middle: Immunoprecipitation of LSU from the ³⁵S-labeled extracts. LSU protein was immunoprecipitated from equalized amounts of labeled plant extract, separated by SDS-PAGE, visualized and quantitated using a phosphorimager. Panel C, bottom: Immunoblot showing accumulation of RLSB and PEPCase (as a loading control) proteins in lower and upper regions of RLSB/RLSB and rlsb-1/rlsb-2 maize leaves. Equalized amounts of total (soluble plus membrane) proteins from RLSB/RLSB and rlsb-1/rlsb-2 leaves were separated by SDS-PAGE and analyzed by immunostaining using the antisera indicted, as described in Methods.