Puroindolines co-localize to the starch granule surface and increase seed bound polar lipid content

Endosperm texture in wheat is controlled by the Pina and Pinb genes that comprise the Hardness (Ha) locus. Studies have shown that soft and hard varieties differ in the amount of starch bound polar lipids but have not addressed whether PINs are directly involved and whether the presence of one particular PIN affects seed polar lipid levels and cellular localization. Here, we determined the effect of overexpressing PINA or PINB on seed bound polar lipids and PIN localization. F₃ recombinants homozygous for either a Pina or Pinb null Ha locus with or lacking a transgenically added Pina or Pinb were analyzed for grain hardness, PIN abundance, and seed bound polar lipid levels. Overexpressed PINs resulted in reduced hardness, increased starch bound PINs, and increased seed bound polar lipids. Addition of PINA to the PINA nulls or PINB to the PINB nulls resulted in higher bound polar lipid levels than the addition of the alternative PIN. Both PINs localized to the starch surface in the presence or absence of the other protein. Our results indicate that PIN overexpression results in reduced endosperm texture and increased seed bound polar lipids and that PINs independently localize to the surface of starch granules.