We have isolated five members of the multigene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase in petunia and examined their expression in petunia leaves. Of the five rbcS genes, two (ssu11A and ssu8) are expressed at high levels in petunia leaves. Northern analysis using gene specific oligonucleotide probes revealed that ssu11A accounts for 40% of the total rbcS transcripts in petunia leaves while ssu8 accounts for 4 to 5% of the total rbcS transcripts. Structural comparisons of ssu8 and ssu11A revealed that the coding sequence of ssu8 is interrupted by three introns, while the coding sequence of ssu11A is interrupted by two introns. The positions of the first two introns are identical, the third intron in ssu8 is located in a highly conserved region of the protein. The 5' and 3' flanking sequences of ssu11A are highly homologous to the 5' and 3' flanking sequences of ssu8. S1 nuclease mapping was used to locate the start of transcription of ssu8 and ssu11A and showed that ssu8 mRNA leader differs in sequence from the ssu11A mRNA leader.

We have mapped the transcription start sites of three petunia rbcS genes and reassayed the relative expression levels of the petunia rbcS genes using the technique of primer extension. This analysis was performed specifically to address the confusion in the literature concerning the relative expression levels of the two petunia rbcS genes, SSU301 and SSU11A. The primer extension analysis reported here confirms our previous results, specifically that the rbcS gene, which we term SSU301, gives significantly higher levels of steady state RNA than any of the other rbcS genes in leaf tissue from 10 week old petunia plants.

The sequences in the petunia genome which encode the small subunit polypeptides of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase have been characterized. Sequence analysis of four cDNA clones indicate that there are several distinct genes transcribed in leaf tissue. There is 8-9% nucleotide divergence between the transcripts however these changes do not alter the encoded amino acid sequence. Examination of nuclear DNA by Southern hybridization and analysis of cloned small subunit genes confirm that there are a number of different genes which encode this single protein.

Of the eight nuclear genes in the plant multi-gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500-fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady-state mRNA in each of the organs examined. We discuss the differences in the steady-state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage.