Regulation of C4 Gene Expression in Developing Amaranth Leaves

Immunofluorescence microscopy and in situ hybridization were used to examine the expression of genes encoding C4 photosynthetic enzymes during early leaf development in the C4 dicotyledonous grain plant amaranth. During early developmental stages, the chloroplast-encoded large subunit and nuclear-encoded small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) were expressed in both bundle sheath and mesophyll cells in a C3-type pattern. The RuBPCase proteins and mRNAs became specifically localized to bundle sheath cells in the characteristic C4-type pattern as the leaves continued to expand and develop. Changes in the localization of the RuBPCase proteins corresponded closely with changes in the localization of their mRNAs, indicating that the cell-specific expression of genes encoding RuBPCase is controlled, at least in part, at the level of transcript accumulation. Genes encoding pyruvate orthophosphate dikinase were expressed specifically in mesophyll cells at all developmental stages examined. Immunolocalization with antibodies raised against phosphoenolpyruvate carboxylase (PEPCase) showed that this enzyme is present only in leaf mesophyll cells, even though RNA sequences with homology to PEPCase gene sequences were present in both bundle sheath and mesophyll cells. These results suggest that the regulation of genes encoding PEPCase in amaranth is complex and could involve the differential expression of divergent PEPCase genes or possibly regulation at the post-transcriptional level.