Figure 7.

PDIL2;3 Does Not Perform the Functions of PDIL1;1 in the Endosperm.

(A) Confocal fluorescence images of the Rhodamine-stained endosperm cells of the wild type, esp2 (pdil1;1), and esp2 expressing spGFP-PDIL1;1 (esp2+PDIL1;1) or spGFP-PDIL2;3 (esp2+PDIL2;3). Typical PB-I (arrowheads) and PB-II (arrows) are indicated. Bars = 5 μm.

(B) Relative rRNase-refolding activities of recombinant PDIL1;1 variants. The arrowheads in the top panel indicate the positions of redox active sites of PDIL1;1: Cys69-X-X-Cys72 (a domain) and Cys414-X-X-Cys417 (a′ domain). The Cys residues in the active sites were substituted with Ala residues: Ala69-X-X-Ala72 (a_m domain) and Ala414-X-X-Ala417 (a′_m domain). The wild type (abb′a′) and three mutants (a_mbb′a′, abb′a′_m, and a_mbb′a′_m) were purified, and their refolding activities were compared. The relative activities are presented as means with standard deviation of three replicates.

(C) Complementation analysis of the esp2 endosperm with wild-type spGFP-PDIL1;1, spGFP-PDIL2;3, or mutated spGFP-PDIL1;1 at the redox active sites. The total proteins extracted from the mature seeds were separated by SDS-PAGE, stained with Coomassie blue (panel CBB) or subjected to immunoblot analysis with antiglutelin antibody (panel pGT). Immunoblot with anti-GFP antibody detected GFP-PDIL1;1 (predicted molecular mass = 84 kD) and GFP-PDIL2;3 (predicted molecular mass = 76 kD) (panel GFP). Seed proteins extracted from untransformed wild-type control (lane 1, WT), PDIL2;3 knockdown in the wild-type background with respect to PDIL1;1 (lane 2, PDIL2;3-KD), and esp2 (lane 3) were also analyzed as controls. pGT, proglutelins; αGT, glutelin acidic subunits; βGT, glutelin basic subunits; crP10, Cys-rich 10-kD prolamin.