Defective synthesis of porphyrins in barley plastids caused by mutation in nuclear genes

Abstract

Seedlings of certain lethal chlorophyll mutants in barley accumulate precursors of protochlorophyllide upon feeding with δ-aminolevulinate in the dark. The porphyrins accumulated in these mutant shoots and their wild types have been identified by thin-layer chromatography and spectroscopy. Mutants at three xantha loci and one albina locus accumulate mainly protoporphyrin. Mutants at two other xantha loci accumulate, in addition to protoporphyrin, uroporphyrin or magnetism protoporphyrin and its monomethyl ester, respectively. A new method for the quantitative determination of porphyrins in mixtures, utilising regression analysis of the spectra, is described. The total amount of porphyrins accumulated by the shoots of any one of the xantha mutants fed δ-aminolevulinate is the same as the wild type; the albina mutant accumulates less. About 90% of the porphyrin produced in the wild type is protochlorophyllide and the rest protoporphyrin. In mutants at the xan-f, xan-g, xan-h and alb-e loci, 80–97% of the total accumulated porphyrins is protoporphyrin and 3–20% protochlorophyllide. The mutant xan-u 21 produces 35% of the total prophyrins as uroporphyrin, whereas xan-l 35 accumulates 48% as magnesium protoporphyrins. None of the mutants is absolutely blocked in protochlorophyllide synthesis when fed δ-aminolevulinate. It is concluded that mutations in the nuclear genes xan-f, -g and -h of barley block the early steps between protoporphyrin and protochlorophyllide. The mutant xan-l 35 has a metabolic block after magnesium protoporphyrin monomethyl ester, whereas the more pleiotropic mutant xan-u 21 contains a major lesion in the pathway between uroporphyrin and protoporphyrin. The latter mutant is also deficient in the conversion of protoporphyrin to protochlorophyllide as well as in carotenoid synthesis. Besides a general reduction in porphyrin and carotenoid synthesis, alb-e 16 has a specific block in the conversion of protoporphyrin to protochlorophyllide. On the basis of a comparative survey of mutants accumulating chlorophyll precursors in photosynthetic organisms, it is suggested that the chlorophyll-synthesising enzymes of algae and seed-plants occupy at least two compartments in the chloroplast: one containing the enzymes converting δ-aminolevulinate to protoporphyrin and the other forming protochlorophyllide from protoporphyrin. The mutants provide evidence that in seed-plants every intermediate in chlorophyll synthesis can regulate the formation of δ-aminolevulinate, whereas in algae only protochlorophyllide and chlorophyll can be ascribed such a function.