SUBCELLULAR VISUALIZATION OF GENE TRANSCRIPTS ENCODING KEY PROTEINS OF THE CHLOROPHYLL ACCUMULATION PROCESS IN DEVELOPING CHLOROPLASTS

The coordination of the synthesis of chlorophyll (Chl) and light-harve sting Chl proteins was determined by observing the sequence of appeara nce of the specific mRNAs for the nuclear genes CHLH, Por, and Lhcb12 (AB180). CHLH encodes a magnesium protoporphyrin chelatase subunit th at is involved in the first committed step in Chl biosynthesis; For en codes protochlorophyllide oxidoreductase, which catalyzes the penultim ate and only light-dependent step in Chl biosynthesis; and Lhcb12 enc odes light-harvesting Chl a/b binding protein of the type-1 light-harv esting complex of photosystem II. Using digoxigenin-labeled antisense and sense RNA probes and a highly sensitive in situ hybridization tech nique, we have visualized the first appearance of the specific mRNAs i n postmitotic mesophyll cells of developing 7-d-old wheat leaves (Trit icum aestivum cv Maris dove). The transcripts for CHLH and POR are det ectable in the youngest (18 h postmitotic) leaf tissue containing divi ding cells; light-harvesting complex of photosystem II transcripts app ear 12 h later. This is consistent with a requirement for;accumulation of Chl before synthesis of Chl a/b binding protein can proceed at a h igh rate. All of the transcripts are most abundant in mesophyll cells. In the first leaf the POR message is initially restricted to the pali sade, but 12 h later it is also present in the spongy mesophyll cells. All three transcripts aggregated around the surface of the chloroplas ts, suggesting that translation may occur preferentially in the vicini ty of the target organelle for the primary translation products.