A SPLICE-SITE MUTATION GIVES RISE TO A MUTANT OF THE C-4 PLANT AMARANTHUS-EDULIS DEFICIENT IN PHOSPHOENOLPYRUVATE CARBOXYLASE ACTIVITY

The molecular nature of a mutant of the C-4 plant Amaranthus edulis th at has been shown to contain only 5% of the normal activity and protei n of phosphoenolpyruvate carboxylase (PEPC) (Dever et al., 1995) has b een investigated. Using Northern blot analysis, it has been shown here that the PEPC transcripts are produced in the mutant. In-vitro transl ation of these transcripts generated two products immunoprecipitable b y a PEPC N-terminus-specific antibody. One of these products has the s ize of the complete PEPC polypeptide, the other is 9 kDa smaller and w as not revealed when using a PEPC C-terminus-specific antibody. In the mutant plant, using the same N- and C-terminus-specific antibodies, o nly the larger polypeptide was immunodetected, whilst at a very low le vel. A sequence analysis of the suspected faulty region of the mRNA re vealed 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 trun cated protein due to a stop codon generated by a frame shift in the tr anslation. Finally, the sequencing of the boundary between the last in tron and exon showed that these inaccurate splicings result from a mut ation in the genuine canonical 3'AG splicing site. (C) 1998 Elsevier S cience B.V. All rights reserved.