A splice site mutation gives rise to a mutant of the C4 plant Amaranthus edulis deficient in phosphoenolpyruvate carboxylase activity

The molecular nature of a mutant of the C-4 plant Amaranthus edulis that has been shown to contain only 5% of the normal activity and protein of phosphoenolpyruvate carboxylase (PEPC) (Dever et al., 1995) has been investigated. Using Northern blot analysis, it has been shown here that the PEPC transcripts are produced in the mutant. In-vitro translation of these transcripts generated two products immunoprecipitable by a PEPC N-terminus-specific antibody. One of these products has the size of the complete PEPC polypeptide, the other is 9 kDa smaller and was not revealed when using a PEPC C-terminus-specific antibody. In the mutant plant, using the same N- and C-terminus-specific antibodies, only the larger polypeptide was immunodetected, whilst at a very low level. A sequence analysis of the suspected faulty region of the mRNA revealed incorrect splicing of the last intron of the PEPC pre-mRNA. Two mis-splicings have been identified, both occurring after an AG site, one leading to a protein lacking five amino acids, the other to a truncated protein due to a stop codon generated by a frame shift in the translation. Finally, the sequencing of the boundary between the last intron and exon showed that these inaccurate splicings result from a mutation in the genuine canonical 3'AG splicing site. (C) 1998 Elsevier Science B.V. All rights reserved.