Polyclonal antibody preparation and immunolocalization of maize (Zea mays) seed protein EMB564

Maize protein EMB564 is a member of group 1 LEA (late embryogenesis abundant) proteins. Currently, there is accumulating information available concerning the biological functions and subcellular localization of group 1 LEA proteins. We recently found that EMB564 protein is associated with maize seed viability. In order to elucidate the function of EMB564, its tissue and subcellular localization in mature seeds was analyzed by immunochemical analysis. A peptide corresponding to a 20-mer consensus (TVVPGGTGGKSVEAQEHLAE) of EMB564 was chemically synthesized and used to immunize rabbits for polyclonal antibody preparation. This antibody reacted strongly with EMB564. Immunoblot analysis revealed that EMB564 existed in highest abundance in the plumule, followed by the radicle and scutellum in the maize embryo. Immunoelectron microscopy demonstrated the preferential nuclear localization of EMB564, associated with chromatin, in the embryonic cells. This subcellular localization is consistent with the previous bioinformatic prediction. This is the first immunoelectron microscopy evidence of subcellular localization of the group 1 LEA proteins. This work would help to understand the mechanism underlying the function of the EMB564 in seed vigor maintenance.