REDUCTION OF UROPORPHYRINOGEN DECARBOXYLASE BY ANTISENSE RNA EXPRESSION AFFECTS ACTIVITIES OF OTHER ENZYMES INVOLVED IN TETRAPYRROLE BIOSYNTHESIS AND LEADS TO LIGHT-DEPENDENT NECROSIS

We introduced a full-length cDNA sequence encoding tobacco (Nicotiana tabacum) uroporphyrinogen III decarboxylase (UROD; EC 4.1.1.37) in rev erse orientation under the control of a cauliflower mosaic virus 35S p romoter derivative into the tobacco genome to study the effects of der egulated UROD expression on tetrapyrrole biosynthesis. Transformants w ith reduced UROD activity were characterized by stunted plant growth a nd necrotic leaf lesions. Antisense RNA expression caused reduced UROD protein levels and reduced activity to 45% of wild type, which was co rrelated with the accumulation of uroporphyrin(ogen) and with the inte nsity of necrotic damage. Chlorophyll levels were only slightly reduce d (up to 15%), indicating that the plants sustained cellular damage fr om accumulating photosensitive porphyrins rather than from chlorophyll deficiency. A 16-h light/8-h dark regime at highlight intensity stimu lates the formation of leaf necrosis compared with a low-light or a 6- h high-light treatment. Transgenic plants grown at high light also sho wed inactivation of 5-aminolevulinate dehydratase and porphobilinogen deaminase, whereas the activity of coproporphyrinogen oxidase and the 5-aminolevulinate synthesizing capacity were not altered. We conclude that photooxidation of accumulating uroporphyrin(ogen) leads to the ge neration of oxygen species, which destabilizes other enzymes in the po rphyrin metabolic pathway. This porphyrin-induced necrosis resembles t he induction of cell death observed during pathogenesis and air pollut ion.