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Phosphoribulokinase (PRK) is an essential enzyme of photosynthetic eukaryotes which is active in the plastid-located Calvin cycle and regenerates the substrate for ribulose-bisphosphate carboxylase/oxygenase (Rubisco). Rhodophytes and chlorophytes (red and green algae) recruited their nuclear-encoded PRK from the cyanobacterial ancestor of plastids. The plastids of these organisms can be traced back to a single primary endosymbiosis, whereas, for example, haptophytes, dinoflagellates, and euglenophytes obtained their "complex" plastids through secondary endosymbioses, comprising the engulfment of a unicellular red or green alga by a eukaryotic host cell. We have cloned eight new PRK sequences from complex algae as well as a rhodophyte in order to investigate their evolutionary origin. All available PRK sequences were used for phylogenetic analyses and the significance of alternative topologies was estimated by the approximately unbiased test. Our analyses led to several astonishing findings. First, the close relationship of PRK genes of haptophytes, heterokontophytes, cryptophytes, and dinophytes (complex red lineage) supports a monophyletic origin of their sequences and hence their plastids. Second, based on PRK genes the complex red lineage forms a highly supported assemblage together with chlorophytes and land plants, to the exclusion of the rhodophytes. This green affinity is in striking contrast to the expected red algal origin and our analyses suggest that the PRK gene was acquired once via lateral transfer from a green alga. Third, surprisingly the complex green lineages leading to Bigelowiella and Euglena probably also obtained their PRK genes via lateral gene transfers from a red alga and a complex alga with red plastids, respectively.

Cryptomonads are complex microalgae which share characteristics of chromophytes (chlorophyll c, extra pair of membranes surrounding the plastids) and rhodophytes (phycobiliproteins). Unlike chromophytes, however, they contain a small nucleus-like organelle, the nucleomorph, in the periplastidial space between the inner and outer plastid membrane pairs. These cellular characteristics led to the suggestion that cryptomonads may have originated via a eukaryote-eukaryote endosymbiosis between a phagotrophic host cell and a unicellular red alga, a hypothesis supported by rRNA phylogenies. Here we characterized cDNAs of the nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from the two cryptomonads Pyrenomonas salina and Guillardia theta. Our results suggest that in cryptomonads the classic Calvin cycle GAPDH enzyme of cyanobacterial origin, GapAB, is absent and functionally replaced by a photosynthetic GapC enzyme of proteobacterial descent, GapC1. The derived GapC1 precursor contains a typical signal/transit peptide of complex structure and sequence signatures diagnostic for dual cosubstrate specificity with NADP and NAD. In addition to this novel GapC1 gene a cytosol-specific GapC2 gene of glycolytic function has been found in both cryptomonads showing conspicuous sequence similarities to animal GAPDH. The present findings support the hypothesis that the host cell component of cryptomonads may be derived from a phototrophic rather than a organotrophic cell which lost its primary plastid after receiving a secondary one. Hence, cellular compartments of endosymbiotic origin may have been lost or replaced several times in eukaryote cell evolution, while the corresponding endosymbiotic genes (e.g., GapC1) were retained, thereby increasing the chimeric potential of the nuclear genome.

Genes for glycolytic and Calvin-cycle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher eukaryotes derive from ancient gene duplications which occurred in eubacterial genomes; both were transferred to the nucleus during the course of endosymbiosis. We have cloned cDNAs encoding chloroplast and cytosolic GAPDH from the early-branching photosynthetic protist Euglena gracilis and have determined the structure of its nuclear gene for cytosolic GAPDH. The gene contains four introns which possess unusual secondary structures, do not obey the GT-AG rule, and are flanked by 2- to 3-bp direct repeats. A gene phylogeny for these sequences in the context of eubacterial homologues indicates that euglenozoa, like higher eukaryotes, have obtained their GAPDH genes from eubacteria via endosymbiotic (organelle-to-nucleus) gene transfer. The data further suggest that the early-branching protists Giardia lamblia and Entamoeba histolytica--which lack mitochondria--and portions of the trypanosome lineage have acquired GAPDH genes from eubacterial donors which did not ultimately give rise to contemporary membrane-bound organelles. Evidence that "cryptic" (possibly ephemeral) endosymbioses during evolution may have entailed successful gene transfer is preserved in protist nuclear gene sequences.

Nuclear protein coding sequences from gymnosperms are currently scarce. We have determined 4 kb of nuclear protein coding sequences from gynosperms and have collected and analyzed > 60 kb of nuclear sequences from gymnosperms and nonspermatophytes in order to better understand processes influencing genome evolution in plants. We show that conifers possess both biased and nonbiased genes with respect to GC content, as found in monocots, suggesting that the common ancestor of conifers and monocots may have possessed both biased and nonbiased genes. The lack of biased genes in dicots is suggested to be a derived character for this lineage. We present a simple but speculative model of land-plant genome evolution which considers changes in GC bias and CpG frequency, respectively, as independent processes and which can account for several puzzling aspects of observed nucleotide frequencies in plant genes.

Algae are a heterogeneous group of photosynthetic eukaryotes traditionally separated into three major subdivisions: rhodophytes, chlorophytes, and chromophytes. The evolutionary origin of rhodophytes or red algae and their links to other photosynthetic and nonphotosynthetic eukaryotes have been a matter of much controversy and speculation. Here we present the first cDNAs of nuclear protein genes from red algae: Those encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Chondrus crispus. A phylogenetic analysis including GAPDH gene sequences from a number of eukaryotic taxa, cyanobacteria, and purple bacteria suggests that chloroplasts and rhodoplasts together form a monophyletic group of cyanobacterial descent and that rhodophytes separated from chlorophytes at about the same time as animals and fungi. The composite GAPDH tree further demonstrates that chloroplast and cytosolic GAPDH genes are closely related to their homologs in cyanobacteria and purple bacteria, respectively, the presumptive ancestors of chloroplasts and mitochondria, thereby firmly establishing the endosymbiotic origin of these nuclear genes and their fixation in eukaryotic cells before the rhodophyte/chlorophyte separation. The present data are in conflict with phylogenetic inferences based on plastid-encoded rbcL sequences supporting a polyphyletic origin of rhodoplasts and chloroplasts. Comparison of rbcL to GAPDH phylogenies suggests that rbcL trees may be misleading because they are composed of branches representing ancient duplicated (paralogous) genes.

Higher plants process two distinct, nuclear gene-encoded glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins, a Calvin-cycle enzyme active within chloroplasts and a glycolytic enzyme active within the cytosol. The gene for the chloroplast enzyme was previously suggested to be of endosymbiotic origin. Since the ancestors of plastids were related to cyanobacteria, we have studied GAPDH genes in the cyanobacterium Anabaena variabilis. Our results confirm that the nuclear gene for higher plant chloroplast GAPDH indeed derives from the genome of a cyanobacterium-like endosymbiont. But two additional GAPDH genes were found in the Anabaena genome and, surprisingly, one of these sequences is very similar to nuclear genes encoding the GAPDH enzyme of glycolysis in plants, animals, and fungi. Evidence that the eukaryotic nuclear genes for glycolytic GAPDH, as well as the Calvin-cycle genes, are of eubacterial origin suggests that eukaryotic genomes are more highly chimeric than previously assumed.

One gene and two cDNAs encoding three different beta-tubulins (TUB1, TUB2, TUB3) of pea have been cloned and sequenced. The derived amino acid sequences show between 92% and 96% identity relative to one another and to most other beta-tubulins of higher plants and green algae. Two notable extremes are the high similarity of 98% between pea TUB3 and maize beta-tubulin 2 and the relatively low similarity (90%) of the hypocotyl-specific beta-tubulin 1 of soybean to the pea sequences. These similarities do not reflect the molecular phylogeny but rather differences in evolutionary rate of beta-tubulins which are differentially regulated during plant development. Genomic Southern blots reveal a beta-tubulin gene family in pea with at least four separate members including two TUB1 genes, one TUB2 gene and one TUB3 gene. This contradicts an earlier report by Raha et al. (Plant Mol Biol 9: 565-571, 1987) suggesting a tandem repeat organization of tubulin genes in pea. The pea TUB1 gene has two introns in identical positions compared to the beta-tubulin genes from Arabidopsis and soybean. In an attempt to reconstruct the universal ancestor of all present-day tubulin genes the intron positions in 38 different alpha- and beta-tubulin genes from plants, animals, fungi and protozoa were compared. This comparison shows that the primordial gene probably had many introns (more than 20) separating 'protoexons' of 15 to 20 codons in agreement with the 'exon theory of genes'. It also supports the view that, during the course of evolution, introns have shifted and were deleted preferentially in the 3' part of the genes. Similar observations have been made previously for other genes. They can be interpreted in terms of a homologous recombination of genes with their modified (incorrectly spliced) and reverse-transcribed pre-mRNAs.

Extracts from 13 different angiosperm species (spinach, mustard, pea, bean, tomato, cucumber, pumpkin, maize, sorghum, rye, wheat, oats, barley) were submitted to electrophoresis under nondenaturing conditions and stained for enzyme activities of cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases by a modified tetrazolium test of high sensitivity. Zymograms of the cytosolic enzyme revealed a single band of similar electrophoretic mobility for all but one species, the tomato, which displayed an ordered set of five different bands. In contrast, zymograms of the chloroplast dehydrogenase are highly different, containing between two and five distinct bands of variable electrophoretic mobilities according to the plant species examined. This variability of the native chloroplast enzyme is paralleled by a remarkable interspecific heterogeneity of the enzyme with respect to subunit size and number, as shown by dodecylsulfate electrophoresis of the purified chloroplast enzyme from 11 different angiosperm species. The present data suggest that cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases belong to two separate protein families of different evolutionary rate. While the cytosolic enzyme is probably an extremely conservative protein like the corresponding enzymes from animals, yeast and bacteria, the chloroplast enzyme seems to change rather rapidly during evolution.

The light-regulated nuclear gene encoding subunit A of chloroplast glyceraldehyde-3-phosphate dehydrogenase (subunit GAPA, gene Gpa1) from maize is extremely G + C rich (67% in codons). The genomic surroundings of this gene have been characterized together with the sequences of two strongly conserved Gpa pseudogenes isolated from a genomic maize library by differential cDNA hybridization. The comparisons show that the high G + C content of the maize gene is maintained independently of the surrounding noncoding sequences, which are G + C poor (42%), and only as long as the gene encodes a functional protein. After nonfunctionalization, Gpa pseudogenes rapidly loose G + C mainly due to enhanced turnover of CpG and CpXpG methylation sites. These results suggest that the maize Gpa1 gene is under strong functional GC pressure, due to constraints (CpG island) probably exerted at the transcriptional level. They also indicate that Gpa pseudogenes are methylated and that methylation was either the cause or the immediate consequence of their nonfunctionalization. It can be concluded further that the progenitor of pseudogenes 1 and 2 was a second functional Gpa gene (Gpa'), which, after duplication, accelerated in evolutionary rate due to relaxation of selective constraints. This is in agreement with the neutral theory of evolution. Comparison of Gpa intron sequences reveals a gradient of divergence: the more 3' the position of an intron the more its sequence has diverged between the three Gpa genes. A speculative model is presented explaining these observations in terms of a homologous recombination of genes with their reverse-transcribed pre-mRNAs.