Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells

Abstract

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP ). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants

“…The gel was silver stained using an EzStain Silver kit (ATTO) according to the manufacturer's instructions. LC-MS/MS analysis of silver-stained proteins was performed as previously described (Igawa et al, 2017). For MASCOT analysis of each peptide, RNA sequencing of L. longiflorum generative cells was performed (InfoBio).…”