The subcellular and intra‐organelle recognition of nuclear and chloroplast transcripts in developing leaf cells

Abstract

We have developed and optimized methods for the in situ hybridization to mRNA and rRNA to enable the specific localization of transcripts within the leaf cell to cytoplasmic or to organelle compartments. Polyethylene glycol embedded leaf tissue was probed using digoxigenin labelled RNA probes and the hybridization products visualized using anti-digoxigenin alkaline phosphatase, nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. The specificity and discrimination of the technique is illustrated by the discrete localization of three transcripts: (a) the chloroplast encoded Rubisco large subunit mRNA; (b) the nuclear encoded 25S ribosomal RNA; and (c) a nuclear encoded transcript bearing strong homology to the cs gene putatively responsible for the chelation of magnesium by protoporphyrin. Each transcript is shown to have a precise intracellular distribution as follows: a is confined to the chloroplast, b is confined to the cytoplasmic compartment and c is within the cytoplasmic compartment, with enhanced localization round the chloroplast. We also describe the expansion of the method to visualize a specific mRNA and its related protein within the same cell or same organelle in the same section. This is achieved using sequential probing and employing two indicator fluorochromes with different emissions to facilitate simultaneous specific recognition of endogenous transcripts and proteins. The application of these techniques is discussed