Aberrant Localizations of Prolamins in the PDIL1;1-Knockout esp2 Endosperm.
(A) Immunoblot analyses of extracts from mature seeds with indicated antibodies. Total seed proteins were extracted under reducing conditions from untransformed wild-type (+/+) control (lane 1), PDL1;1-knockout (−/−) esp2 (lane 2), esp2 expressing both spGFP-crP10 and spDsRed-cpP13 (lanes 3), and esp2 expressing both spGFP-crP10 and spDsRed-cpP13 under the background of PDIL2;3-knockdown (KD) (lane 4).
(B) Immunoblot analyses of seed proteins. Seed proteins were fractionated into the supernatant (S) and pellet (P) fractions by centrifugation under nonreducing conditions (see Methods for details). pGT, proglutelins; Glb, α-globulin; RA17, α-amylase inhibitor.
(C) Confocal fluorescence images of the outer region of endosperm (10 DAF) expressing both spGFP-crP10 and spDsRed-cpP13 in the background of esp2 (pdil1;1). Arrowheads indicate PB-I–like particles containing GFP-crP10. Note that DsRed-cpP13 did not accumulate in these PB-I-like particles. The fluorescence signals of GFP-crP10 (left panel) and DsRed-cpP13 (middle panel) were converted to green and red, respectively, and merged (right panel). Bars = 5 μm.
(D) Confocal fluorescence images of the PDIL2;3-knockdown (KD) endosperm cells (10 DAF) expressing both spGFP-crP10 and spDsRed-cpP13 in the background of esp2 (pdil1;1). The fluorescence signals of GFP-crP10 (left panel) and DsRed-cpP13 (middle panel) were converted to green and red, respectively, and merged (right panel). Bars = 5 μm.