Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant.
A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) I. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P700 upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded HinfI fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT-->TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.
Schaffner
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C
Institute of Genetics, Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany.
Laasch
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Hagemann
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000235:genetics*; D018744:DNA, Plant; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045331:Photosystem I Protein Complex; D010940:Plant Proteins / Q000235:genetics*; D010944:Plants; D017354:Point Mutation
D018744:DNA, Plant; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; C089909:photosystem I, psaB subunit; C021261:thylakoid polypeptides