Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice
Fig 4
RUPO localizes to the plasma membrane and cytoplasmic vesicles of the pollen tube.
(A-F) Subcellular localization of the 35S::RUPOΔC-GFP fusion protein in onion epidermal cells. (A) Single confocal section and (B) bright-field image of the cell bombarded with 35S::RUPOΔC-GFP plasmid. (C,D) The same cell as in (A,B) was treated with 0.8 M mannitol to induce plasmolysis. (E) Single confocal section and (F) bright-field image of the cell bombarded with 35S::GFP. (G,H,I) Subcellular localization of the Ubi::RUPO-GFP fusion protein in lily pollen tubes. (G,H) RUPO or (I) GFP alone driven by ubiquitin promoter was transiently expressed in lily pollen tubes. Scale bars, 50 μm in (A) to (G), 5 μm in (H,I). (J-K) RUPO was enriched in membrane fraction. Homogenate from mature pollen grains was centrifuged at 10,000×g, and resultant supernatant (S10) was further centrifuged at 100,000×g to separate into supernatant (S100) and membrane fraction (P100). Proteins in each fraction were resolved by SDS-PAGE and probed with anti-RUPO antibody. 30 μg soluble protein (S10, S100) and 10 μg membrane protein (P100) were loaded. (J) Western blot image, (K) Protein loading control image stained with Coomassie brilliant blue. (L) Crude membrane vesicles were fractionized on a discontinuous sucrose density gradient, and proteins in individual fractions were probed with antibodies against RUPO, ER marker protein SMT1 and PM marker protein H+-ATPase.