Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthe sis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous io n by ferrochelatase directs protoporphyrin IX towards the formation of chlo rophyll and heme, respectively. A full length cDNA clone encoding a ferroch elatase was identified from a Nicotiana tabacum cDNA library. The encoded p rotein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in s troma and thylakoids confirm its close relationship to the previously descr ibed Arabidopsis thaliana plastid-located ferrochelatase (FeChll). A 1700-b p tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allo wing investigation into the consequences of selective reduction of the plas tidic ferrochelatase activity for protoporphyrin IX channeling in chloropla sts and for interactions between plastidic and mitochondrial heme synthesis . Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity-dependent formation of necrotic leaf lesions. In comparison with wild-type plants the total ferrochelatase acti vity was decreased in transgenic lines leading to an accumulation of photos ensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in p lastids but not in mitochondria of transgenic plants. By means of the speci fically diminished ferrochelatase activity consequences of the selective in hibition of protoheme formation for the intracellular supply of heme can be investigated in the future.