Characterization of proteins influencing the nutritional qualityof maize (Zea mays L.) endosperm

Elongation factor 1A is one of the lysine-rich proteins increased in o2 mutants, and its concentration is highly predictive of the protein-bound lysine content of the endosperm. Understanding the biological basis of this relationship could help to explain the mechanisms of lysine accumulation in the endosperm, providing new insights for developing maize genotypes with better nutritional quality. Three different eEF1A isoforms were purified from developing endosperm and investigated in their accumulation, structural and functional activities. The accumulation of the isoforms appears to be developmentally regulated and independent of the o2 mutation. The purified proteins differed in their ability to bind F-actin in vitro, suggesting they are functionally distinct. The isoform that binds actin most effectively was the most predominant in high eEF1A genotypes, which may be related to enhanced cytoskeleton formation, and therefore increased synthesis of cytoskeleton-associated proteins in these genotypes. Tandem mass spectrometry revealed each isoform is composed of the four same gene products, which are modified post-translationally by methylation and phosphorylation. The chemical differences that account for their different actin binding activities could not be determined. Recombinant inbred lines varying in eEF1A content were developed from a cross between a high (Oh51Ao2) and a low (Oh545 o2) eEF1A inbred. The parental inbreds and RILs with the highest and lowest eEF1A content were used to investigate patterns of gene expression and protein synthesis. Transcript profiling with an endosperm EST microarray identified about 110 genes coordinately regulated with eEF1A. These genes encode proteins involved in several biological structures and processes, including the cytoskeleton, the endoplasmic reticulum and the protein synthesis apparatus. The content of alpha-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. Thus, higher levels of eEF1A may be related with a more extensive cytoskeletal network surrounding the rough ER and increased translation of mRNAs encoding cytoskeleton-associated proteins, all of which contribute significantly to the lysine content of the endosperm