Evolution rbcS genes in Solanum tuberosum : Conservation of transit peptide and exon shuffling during evolution ( plant gene family / ribulose bisphosphate carboxylase / potato / intron )

Five genes of the rbcS gene family ofSolanum huberosum (potato) were studied. One of these is a cDNA clone; the other four are located on two genomic clones representing two different chromosomal loci containing one (locus 1) and three genes (locus 2), respectively. The intron/exon structure of the three genes in locus 2 is highly conserved with respect to size and position. These genes contain two introns, whereas the gene from locus 1 contains three introns. Although in most cases the amino acid sequences in the transit peptide part of different rbcS genes from the same species varied considerably more than the corresponding mature amino acid sequences, one exception found in tomato and potato indicates that the transit peptide of rbeS could have a special function. A comparison of the rbeS genes of higher plants with those of prokaryotes offers suggestive evidence that introns first served as spacer material in the process of exon shuffig and then were removed stepwise during the evolution of higher plants. Ribulose bisphosphate carboxylase (RbuP2 carboxylase; E.C. 4.1.1.39), the key enzyme in the dark reaction of photosynthesis (for a review, see ref. 1), is composed of eight large (LSU) and eight small (SSU) subunits in higher plants, which are both essential for the enzymatic activity (2). The corresponding genes are located in different compartments. The rbcL gene that codes for LSU is part of the chloroplast genome (3), leading to a copy number of several thousand genes per mesophyll cell (4). The rbcL-encoded mRNA is translated inside the organelle on plastid ribosomes. In contrast, the SSU is encoded in the nucleus and the rbcS-encoded mRNA is translated in the cytoplasm into a precursor protein containing an amino-terminal extension, named transit peptide, that mediates the transport of the polypeptide into the chloroplast and is cleaved off during or shortly after transport (for a review, see ref. 5). The mature SSU assembles in the chloroplast stroma with the LSU to form the holoenzyme. In all plants studied, several copies of rbcS genes have been found (6-8). We would like to understand the reason for the existence of gene families in plants. Due to the fundamental importance of RbuP2, the rbcS gene family provides a significant example with which to probe the relevance of gene families in plants. Comparative studies of gene structures and sequences give information relevant to this question. Conservation of amino acid sequence in different species can provide indications for a functional role of conserved regions in SSU proteins. We chose to study the rbcS genes from Solanum tuberosum (potato) since several members of the rbcS gene family from other solanaceous species are available for interspecies comparison. In addition, we have extended our comparison to rbcS genes of other higher plants and prokaryotes. We find different types ofgenes with respect to intron number, the positions of which are, however, highly conserved within higher plants. The distribution of homology between prokaryotic and eukaryotic genes leads to a scheme for the evolution of the rbcS genes that supports the idea of exon shuffling and stepwise removal of introns and confirms the endosymbiotic theory. MATERIALS AND METHODS Plant Material. The following lines from the potato collection of the Max-Planck-Institut fMr Zuchtungsforschung were used: the diploid line HH 1201/7 (cDNA) and the haploid line AM 5793 (genomic library) (9). DNAs and Vectors. Nuclear DNA was prepared according to Bedbrook (10). The synthesis and isolation of cDNA rbcS c was described as L900 by Eckes et al. (11). The construction of the genomic library was described elsewhere (9). Genomic clones were subcloned in pUC8 or in the M13 phage vectors mp18 and mpl9 (12). Phage A propagation, screening, and DNA isolation were performed as described by Rosahl et al. (9). Sequencing. The DNA sequence was determined by using the chain-termination method described by Sanger et al. (13) on M13-derived templates (14). Data Handling. For sequence comparisons, the Wisconsin computer group program package (15) and the GENEXPERT package (16) were used on a VAX computer (Digital Corporation).