Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus

To play an essential role in C₄ photosynthesis, the maize C₄ phosphoenolpyruvate carboxylase gene (PPCZm1) acquired many new expression features, such as leaf specificity, mesophyll specificity, light inducibility and high activity, that distinguish the unique C₄ PPC from numerous non-C₄ PPC genes in maize. We present here the first investigation of the developmental, cell-specific, light and metabolic regulation of the homologous C₄ PPCZm1 promoter in stable transgenic maize plants. We demonstrate that the 1.7 kb of the 5-flanking region of the PPCZm1 gene is sufficient to direct the C₄-specific expression patterns of -glucuronidase (GUS) activity, as a reporter, in stable transformed maize plants. In light-grown shoots, GUS expression was strongest in all developing and mature mesophyll cells in the leaf, collar and sheath. GUS activity was also detected in mesophyll cells in the outer husks of ear shoots and in the outer glumes of staminate spikelets. We did not observe histological localization of GUS activity in light- or dark-grown callus, roots, silk, developing or mature kernels, the shoot apex, prop roots, or pollen. In addition, we used the stable expressing transformants to conduct and quantify physiological induction studies. Our results indicate that the expression of the C₄ PPCZm1-GUS fusion gene is mesophyll-specific and influenced by development, light, glucose, acetate and chloroplast biogenesis in transgenic maize plants. These studies suggest that the adoption of DNA regulatory elements for C₄-specific gene expression is a crucial step in C₄ gene evolution.