Investigating FLOE1 and FLOE2 granule biophysical function in vitro.
EMSL Project ID
60682
Abstract
Plant seeds undergo a programmed desiccation and rehydration, but the molecular mechanism underpinning cellular survival during these developmental stages has remained elusive. FLOE1 is expressed during these key seed desiccation and germination events and regulates germination under low water stress conditions. FLOE1 is a prion-like domain containing protein, which is expressed in seeds and forms phase separated biomolecular condensates in the presence of water. Both FLOE1 and the related FLOE2 proteins form phase separated granules that co-localize but do not mix together. FLOE2 is found through the plant kingdom, and the FLOE2 proteins are homologous to a truncated versions of FLOE1 (FLOE1∆DS and the FLOE1 splice variant FLOE1.2). FLOE1∆DS forms more solid and larger aggregates that FLOE1, and FLOE1∆DS is constitutively localized to larger cytoplasmic condensates, while FLOE1 granules dissolve in low osmotic conditions. Expressing the FLOE1∆DS mutant increases seed germinating under very limited water conditions, suggesting the change in germination rate is tied to organizational/structural changes in the FLOE1 granules. We hypothesize that the formation and organization of FLOE1 granules serves as a critical water sensing mechanism during plant seed cell dehydration and rehydration.We propose expressing FLOE1, FLOE2, along with the splice variant FLOE1.2, using the cell-free protein synthesis platform at EMSL. Our primary goal is to determine if the cell-free protein synthesis of FLOE1 and FLOE2 in vitro can serve as an advantageous alternative to conventional methods of generating FLOE samples, which suffer from a low yield and a lack of co-expression potential for hetero-systems. If in vitro expression of FLOE1 and FLOE2 proteins work and they form granules in vitro, our future aims would be to use these samples for more thorough native mass spectrometry, high-resolution cryo-electron tomography or microED applications to understand the atomic structure of the FLOE1 and FLOE2 proteins, and gain further insight into the molecular mechanism of FLOE1 and FLOE2 granule formation. Together, uncovering the regulatory mechanism behind the FLOE family is critical for engineer plants to be resilient against water limitation under climate change.
Project Details
Project type
Limited Scope
Start Date
2023-02-28
End Date
2023-05-24
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members