Detection of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylase transcripts in maize leaves by in situ hybridization with sulfonated cDNA probes.

This report outlines an efficient in situ hybridization method for locating specific mRNAs in tissue cryosections using sulfonated cDNA probes. The method involves chemical modification of DNA probes by insertion of a sulfone radical on cytosine residues, which generates a specific epitope. Sulfonated DNA is then detected by using indirect immunochemical procedure. Alternatively, antibodies conjugated to fluorescein or to alkaline phosphatase were used for mRNA detection. In situ hybridization was developed to study aspects of mesophyll and bundle sheath cell differentiation in maize leaves. Our results indicate that phosphoenolpyruvate carboxylase (PEP C) mRNA is restricted to mesophyll cells, and the nucleus-encoded mRNA of the small subunit (SSU) ribulose 1,5-bisphosphate carboxylase (RuBP C) is limited to the cytosol of bundle sheath cells. Thus, using in situ hybridization, we have demonstrated that the differential distribution of PEP C and RuBP C proteins in the two cell types also reflects the location of their mRNAs. These data imply either a tissue-specific transcriptional regulation or a selective mRNA degradation.