Algae Volume 17(1): 59-68, 2002 The Chloroplast rpl23 Gene Cluster of Spirogyra maxima (Charophyceae) Shares Many Similarities with the Angiosperm rpl23 Operon Jungho Lee* and James R. Manhart Department of Biology, Texas A&M University, College Station, TX, 77843-3258, U.S.A. A phylogenetic affinity between charophytes and embryophytes (land plants) has been explained by a few chloro- plast genomic characters including gene and intron (Manhart and Palmer 1990; Baldauf et al. 1990; Lew and Manhart 1993). Here we show that a charophyte, Spirogyra maxima, has the largest operon of angiosperm chloroplast genomes, rpl23 operon (trnI-rpl23-rpl2-rps19-rpl22-rps3-rpl16-rpl14-rps8-infA-rpl36-rps11-rpoA) containing both embryophyte introns, rpl16.i and rpl2.i. The rpl23 gene cluster of Spirogyra contains a distinct eubacterial promoter sequence upstream of rpl23, which is the first gene of the green algal rpl23 gene cluster. This sequence is completely absent in angiosperms but is present in non-flowering plants. The results imply that, in the rpl23 gene cluster, early charophytes had at least two promoters, one upstream of trnI and another upstream of rpl23, which partially or completely lost its function in land plants. A comparison of gene clusters of prokaryotes, algal chloroplast DNAs and land plant cpDNAs indicated a loss of numerous genes in chlorophyll a+b eukaryotes. A phylogenetic analysis using presence/absence of genes and introns as characters produced trees with a strongly supported clade contain- ing chlorophyll a+b eukaryotes. Spirogyra and embryophytes formed a clade characterized by the loss of rpl5 and rps9 and the gain of trnI (CAU) and introns in rpl2 and rpl16. The analyses support the hypothesis that the rpl23 gene cluster and the rpl2 and rpl16 introns of land plants originated from a common ancestor of Spirogyra and land plants. Key Words: Charophytes, Chloroplast, embryophytes, Introns, operon, promoter, rpl2, rpl16, rpl23, Spirogyra Coleochaete orbicularis of the Coleochaetales, and Chara INTRODUCTION and Nitella of the Charales. Among these taxa, Spirogyra and Sirogonium of the Zygnematales do not contain More than 450 million years ago, land plants evolved chloroplast tufA like land plants while the others have from a green algal ancestor (Graham 1993; Kenrick and the gene in their chloroplast genomes (Baldauf et al. Crane 1997). A group of green algae called charophytes 1990). Spirogyra chloroplast DNA (cpDNA) also has an (the Charophyceae) are generally accepted as the closest unusual structure of rps12 and rps7 (Lew and Manhart algae to land plants based on analysis of phenetic and 1993), that contains two introns including a trans-splic- molecular data (Mishler et al. 1994). There are nuclear ing intron. This unusual structure of rps12 and rps7 has and chloroplast genome characters that support a close been documented only in Spirogyra and land plants. relationship of charophytes and land plants. The question remains whether or not charophyte Charophytes and embryophytes have tufA (Baldauf et al. chloroplasts have additional chloroplast genomic charac- 1990) in their nuclear genome while non-charophyte ters that support the hypothesized close relationship green algae have tufA in their chloroplast genome only. between charophytes and embryophytes. The gross Some charophytes have chloroplast trnA and trnI group structure of Spirogyra chloroplast DNA, the only charac- II introns (Manhart and Palmer 1990), which are absent terized charophyte cpDNA, differs from those of land in non-charophyte green algae. The charophyte taxa plants (Palmer 1991) by the absence of the inverted included in these studies were Spirogyra and Sirogonium repeats (IRs) and the order of the limited number of of the Zygnematales (Mattox and Stewart sensu 1983), mapped genes (Manhart et al. 1990). Among land plants, Pinus does not contain large IRs (Wakasugi et al. 1994). *Corresponding author ([email protected] or Therefore, Spirogyra is worth investigating in detail in [email protected]) order to reveal possible similarities or transitional states 60 Algae Vol. 17(1), 2002 Fig. 1. Alignment of the ribosomal gene cluster (S10, spc, α operons) in E. coli, Mycoplasma, Synechocystis, a cyanelle, algal and land plant chloroplasts. [E. coli (Blattner et al. 1997), Mycoplasma genitalium (Fraser et al. 1995), Synechocystis (Kaneko et al. 1996), the cyanelle of Cyanophora (Stirewalt et al. 1995) and cpDNAs of Odontella (Kowallik et al. 1995), Guillardia (Douglas and Penny 1999), Porphyra (Reith et al. 1995), Cyanidium (Glockner et al. 2000), Euglena (Christopher and Hallick 1989; Hallick et al. 1993), Chlorella (Wakasugi et al. 1997), Nephroselmis (Turmel et al. 1999), Mesostigma (Lemieux et al. 2000) and land plants (see Table 1)] S10, spc, and α are three E. coli operons. | : gene absence =: separate between land plants and non-charophyte green algae. The land plant rpl23 gene cluster shows similarities to Land plants have a unique gene cluster, trnI-rpl23- three consecutive ribosomal operons (S10, spc, and alpha rpl2-rps19-rpl22-rps3-rpl16-rpl14-rps8-infA-rpl36-rps11- operons) of E. coli (Tanaka et al. 1986) with differences in rpoA, that is found in all completely sequenced land gene and intron content. The genes of the E. coli operons plant cpDNAs. In angiosperms, the gene cluster is a are also clustered together in other eubacteria with a few transcriptional unit (Kanno and Hirai 1993), the rpl23 variations in gene contents (Fig. 1). The same gene operon (Sugiura 1992). In the bryophyte, Marchantia, a groupings are found in Synechocystis, a cyanobacterium, distinct eubacterial promoter region is present upstream and its gene contents are more similar than those of E. of trnI and there is no other distinct eubacterial promoter coli to the ribosomal gene cluster of chloroplasts and the region in the rpl23 gene cluster (Ohyama et al. 1988). So it cyanelle of Cyanophora. The cyanobacterial gene order is likely that the rpl23 gene cluster in Marchantia is also a likely represents the ancestral condition from which the single operon. gene clusters found in cyanelle and chloroplast DNAs Lee & Manhart: Chloroplast rpl23 Gene Cluster of Spirogyra maxima 61 Fig. 2. rpl23 gene cluster and its physical map in Spirogyra cpDNA. Closed boxes indicate genes/exons and open boxes indicate introns. were derived. Similar gene clusters are found in the cluster in any chloroplast or cyanelle genome. The cyanelle of the glaucophyte (Cyanophra paradoxa) and introns in land plants and euglenoids were probably chloroplast genomes of rhodophytes (Porphyra purpurea introduced independently and relatively recently. and Cyanidium caldarium), a chromophyte (Odontella None of genes in the rpl23 operon has been investigat- sinensis), a cryptophyte (Guillardia theca). Porphyra and ed in charophyte cpDNAs. Therefore, we have mapped, Odontella cpDNAs contain most of the cyanobacterial cloned, and sequenced a region corresponding to the genes in the same order (Fig. 1). The cyanelle of rpl23 gene cluster in Spirogyra cpDNA in order to deter- Cyanophra paradoxa has two separate gene clusters but mine whether it resembles that of land plants or other their combined content is similar to single clusters found known algal cpDNAs regarding gene content, structure in Porphyra and Odontella cpDNAs. In addition, a chloro- and intron presence/absence. This gene cluster was plast-like plasmid of Plasmodium (McFadden et al. 1997) characterized in Spirogyra to improve our understanding also contains a gene cluster similar to those of Porphyra of chloroplast genome evolution and phylogenetic rela- and Odontella cpDNAs. tionships in green plants. Euglena, Chlorella, Nephroselmis and Mesostigma cpDNAs have rpl23 gene clusters (Fig. 1) similar to those MATERIALS AND METHODS in land plants. These algal rpl23 gene clusters are distin- guished from land plants by the absence of trnI (CAU) at Genomic DNA and cloned cpDNA fragments of the 5’ end of the gene clusters and the presence of rpl5 Spirogyra maxima (UTEX LB2495) in pBluescript II SK+ and rps9. The Euglena rpl23 operon differs from all other (Stratagene), as previously described (Manhart et al. taxa in the complete absence of E. coli alpha operon 1990) were used for this study. The rpl23 gene cluster genes, the presence of roaA (ribosomal operon-associated was found in 5.0 kb ClaI, 5.3 kb ClaI, and 4.8 kb PstI frag- gene), and the addition of trnI-rps14 at the 3’ end of the ments by sequencing both ends of the clones (Fig. 2). The operon (Christopher and Hallick 1990; Jenkins et al. three cpDNA fragments of Spirogyra maxima containing 1995). Chlorella, Nephroselmis and Mesostigma in the the members of rpl23 gene cluster were cut with the Chlorophyceae (Mattox and Stewart 1983) contain all restriction endonucleases EcoRI, PstI, ClaI, and XbaI genes of the angiosperm rpl23 operon except rpl22 and singly and in combination. The cut DNA fragments were trnI (CAU). Chlorella, Nephroselmis and Mesostigma are cloned into pBluescript II SK+. Ligations were done the only chlorophyll a + b taxa that have rps9 but it is using ligase and buffer supplied by Boehringer found in Synechocystis and non-green plastids (Fig. 1). Mannheim, Germany. The plasmids were transformed Introns have been documented in several of the genes into E. coli strain DH5α and plasmid DNAs were puri- in Fig. 1 but only in land plants and euglenoids. In most fied using Quiagen Plasmid Midi Kit (Quiagen, land plants, group II introns with the same insertion Germany). The cloned DNAs were first manually sites have been documented in rpl2 and rpl16. The rpl23 sequenced using the Sequenase kit (United States operon genes of Euglena gracilis contain numerous Biochemical, Cleveland, Ohio) with 7 pM of T3 and T7 introns that are unique for euglenoids (Hallick et al. primers, 100 ng of template, and S35 labeled dATP at 1993). Except for euglenoids and land plant cpDNAs, 37°C. For further manual sequencing, octomers of 50% introns have not been found in the genes of this gene GC were designed for sequencing as described by 62 Algae Vol.