The Starch Granule Surface: Technological and Biological Implications of Puroindoline and Host-pathogen Interactions

Abstract

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.