One-hundred ten cycles of divergent selection for grain protein concentration in maize has produced populations with the known phenotypic extremes for this trait. Compared to an average of 8-12% grain protein, Illinois High Protein (IHP) kernels contain over 32% protein and Illinois Low Protein (ILP) kernels contain only 4% protein. Recurrent selection in this experiment has most dramatically affected both the expression and accumulation of the 19- and 22-kD α-zein proteins, but the molecular mechanisms underlying these phenotypic changes are still unknown. Analysis of individual α-zein gene expression is complicated by the high copy number and sequence homology of the α-zein genes. Here we employ red fluorescent protein (RFP) reporter lines (see http://maiaze.tigr.org/cellgenomics/index.shtml), where the RFP is fused to an intact 22-kD α-zein gene, floury2. This allows for tracking expression and protein accumulation of an individual zein gene. When the floury2-mRFP construct is introgressed into IHP and ILP the kernels appear pink in color, with the intensity of pink coloration corresponding to known levels of zein accumulation in these strains. In order to further characterize floury2-mRFP protein accumulation within the developing kernel, images were taken of both IHP and ILP kernels 24 days after pollination using the LSM710 Zeiss confocal laser scanning microscope. No floury2-mRFP expression was evident in the pericarp, aleurone or embryo, consistent with known zein accumulation patterns, but rather exclusively in endosperm tissue. Both IHP and ILP exhibit the strongest expression in the subaleurone cells with a progressive reduction in expression into the starchy endosperm. However, expression extends much further into the starchy endosperm in IHP (>20 cellular layers) compared to ILP (<5 layers). The fL2-mRFP construct appears to follow normal spatial and developmental patterns of zein gene expression and protein accumulation, and will be a useful tool for studying α-zein regulation in the future.
