Mutants defective in conversion of Mg protoporphyrin monomethyl ester to protochlorophyllide

The orobanche locus (oro), phenotypically brownish and very necrotic, is defective in conversion of Mg-protoporphyrin monomethyl ester (Mg-proto me) to protochlorophyllide (pchlide). Last spring a number of additional oro mutants, apparently uncharacterized genetically, were obtained from Maize Genetics Cooperation. In order to clarify this situation allele tests of these mutants (excluding oro-1-6577) were carried out. On the basis of their superficial phenotypic resemblance, sienna and l*-4923 were also included in this test. The following results were obtained:

oro = oro-4889 = oro-8081 = oro-6474 = oro-64-4589-4 oro2 sienna l*-4923

Beyond these preliminary observations, more extensive work has been carried out only on oro and oro2. A dominant modifier of oro, Orom, has also been investigated.

Light grown, both oro and oro2 are very necrotic. Tissue that does develop appears to bleach in a manner corresponding to the light, dark cycle. No porphyrin pigments are detected in such plants:

Table.

It is likely that pigments produced by oro and oro2 are immediately photo-oxidized, resulting in cellular disruption and necrosis.

Dark grown, such plants develop perfectly normal leaf tissue and accumulate a small amount of Mg-proto me corresponding approximately to the amount of protochlorophyllide accumulated by normal plants:

Table.

*nM extinction coefficients of Kahn et al., (1976) in: Genetics and Biogenesis of Chloroplasts and Mitochondria, Amsterdam: Elsevier/North Holland Biomedical Press, pp. 119-131, were used throughout this report to quantitate precursors.

This is in contrast with the observation that completely blocked mutants defective in the conversion of protoporphyrin-IX (proto) to Mg-proto accumulate no detectable proto. It would appear that Mg-proto me and pchlide are not as important in regulating the chlorophyll biosynthetic pathway as proto.

When these mutants, dark-grown, are fed d-aminolevulinic acid (ALA) for 24 hr, a mixture of pigments, including proto, Mg-proto and their monomethyl esters, is accumulated. The protoporphyrins are spectrally indistinguishable from their monomethyl ester so for purposes of calculation they are treated as units. The total amount of pigments accumulated by these plants is extremely variable due mainly to variation in uptake of ALA:

Table.

However, the relative proportions of proto's and Mg-proto's are approximately equal (Figure 1A & B).

Figure 1.

Two possible explanations for the fact that more than one locus is involved in the same step are envisioned. Firstly, since there are actually several reactions involved in the conversion of Mg-proto me to pchlide these mutants may represent defects in subunits or components of a complex that normally carries out this conversion. Secondly, reactions after the formation of proto are carried out in the chloroplast membranes. Specific disruption of the membrane where enzymes or pigments normally bind might result in the same phenotypic expression.

All mutant alleles at the oro locus, involved in the allele tests reported here, are influenced in a similar manner by the modifier gene, Orom. This modifier acts in a dominant fashion and is genetically independent of oro:

Table.

*The unmodified oro sources represent a total of seedlings counted from crosses of the various oro alleles to oro/+ : Orom/orom plants used in allele tests.

In the presence of Orom, oro plants bypass the lesion to some extent and produce some chlorophyll in light (first table). Nearly normal leaf development is observed in these plants and a luteus phenotype is typical in greenhouse material. Unfed dark grown material accumulates at least a normal level of pchlide (second table); however, when fed ALA modified oro seedlings accumulate a mixture of pigments including pchlide as well as the pigments accumulated by oro (third table, Figure 1C). The concentration of porphyrins in oro:Orom plants is extremely high compared to controls. The significance of this is unclear. These data suggest that Orom is not integrated with the primary chlorophyll biosynthetic system but may be involved in an alternative pathway. It does not appear that normal plants are affected by Orom. 75% of the normal plants in this experiment should have contained the modifier, however, these control values are similar to those of other experiments. The effect of the modifier on normal plants and the extent of its presence in normal stocks has not been systematically studied. It will be interesting to determine the effect of Orom on oro2 and normal plants. (Thanks to Dr. Lambert for providing adequate seed for the allele tests reported).

Peter Mascia


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