Gene expression in NCS2 mutant plants

The maternally inherited Nonchromosomal stripe (NCS) mutants in maize are characterized by variable leaf striping,

poor growth, and decreased yields (Shumway and Bauman, Genetics 55:33; Coe, Maydica 28:151). It has been shown that the phenotypically distinguishable NCS2 and NCS3 mutants have distinct alterations in their mitochondrial DNA (mtDNA) restriction enzyme profiles, relative to their common progenitor cytoplasm, cms-T (Newton and Coe, PNAS 83:7363). To determine whether the observed DNA alterations in the NCS2 mutant are functionally significant, we used several approaches. Proteins synthesized by mitochondria isolated from mutants and from related plants with normal growth have been compared. NCS2 mitochondria synthesize very reduced amounts of a single polypeptide (approximately 24 kD).

Clones containing previously characterized plant mitochondrial genes were kindly provided by Sam Levings, Chris Leaver, Dave Stern and Axel Brennicke. They were used to test whether any of these genes had altered structure or expression in NCS2. Southern blot analysis with labeled probes for cytochrome oxidase subunits I, II and III; ATPase subunits 6, 9 and alpha; cytochrome b; URF1 and ribosomal RNA genes demonstrated that the DNA regions with these genes (or homologous sequences) appear to be the same in NCS2 mutant and related normal plants. Northern blot analysis of mitochondrial RNAs, using these same probes, showed similar transcripts were produced in NCS2 and related non-mutants.

An 11kb NCS2-specific HindIII fragment was cloned and tested by Southern and Northern blot analysis. It detects the NCS2 mtDNA alterations observed with several different restriction enzymes (it is not homologous to the NCS3 alterations.) This probe hybridizes to an aberrant set of transcripts in NCS2 mitochondria, relative to normal mitochondria.

Thus, in NCS2 mutants, the synthesis of one mitochondrial protein is reduced and one set of transcripts, homologous to the NCS2-altered region of mtDNA, is also aberrant.

Heidi Feiler and Kathleen Newton
 
 

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