Mitochondrial Glycolysis in a Major Lineage of Eukaryotes.

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

Physiology, phylogeny, early evolution, and GAPDH.

The chloroplast and cytosol of plant cells harbor a number of parallel biochemical reactions germane to the Calvin cycle and glycolysis, respectively. These reactions are catalyzed by nuclear encoded, compartment-specific isoenzymes that differ in their physiochemical properties. The chloroplast cytosol isoenzymes of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) harbor evidence of major events in the history of life: the origin of the first genes, the bacterial-archaeal split, the origin of eukaryotes, the evolution of protein compartmentation during eukaryote evolution, the origin of plastids, and the secondary endosymbiosis among the algae with complex plastids. The reaction mechanism of GAPDH entails phosphorolysis of a thioester to yield an energy-rich acyl phosphate bond, a chemistry that points to primitive pathways of energy conservation that existed even before the origin of the first free-living cells. Here, we recount the main insights that chloroplast and cytosolic GAPDH provided into endosymbiosis and physiological evolution.

A "green" phosphoribulokinase in complex algae with red plastids: evidence for a single secondary endosymbiosis leading to haptophytes, cryptophytes, heterokonts, and dinoflagellates.

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.

The GapA/B gene duplication marks the origin of Streptophyta (charophytes and land plants).

Independent evidence from morphological, ultrastructural, biochemical, and molecular data have shown that land plants originated from charophycean green algae. However, the branching order within charophytes is still unresolved, and contradictory phylogenies about, for example,the position of the unicellular green alga Mesostigma viride are difficult to reconcile. A comparison of nuclear-encoded Calvin cycle glyceraldehyde-3-phosphate dehydrogenases (GAPDH) indicates that a crucial duplication of the GapA gene occurred early in land plant evolution. The duplicate called GapB acquired a characteristic carboxy-terminal extension (CTE) from the general regulator of the Calvin cycle CP12. This CTE is responsible for thioredoxin-dependent light/dark regulation. In this work, we established GapA, GapB, and CP12 sequences from bryophytes, all orders of charophyte as well as chlorophyte green algae, and the glaucophyte Cyanophora paradoxa. Comprehensive phylogenetic analyses of all available plastid GAPDH sequences suggest that glaucophytes and green plants are sister lineages and support a positioning of Mesostigma basal to all charophycean algae. The exclusive presence of GapB in terrestrial plants, charophytes, and Mesostigma dates the GapA/B gene duplication to the common ancestor of Streptophyta. The conspicuously high degree of GapB sequence conservation suggests an important metabolic role of the newly gained regulatory function. Because the GapB-mediated protein aggregation most likely ensures the complete blockage of the Calvin cycle at night, we propose that this mechanism is also crucial for efficient starch mobilization. This innovation may be one prerequisite for the development of storage tissues in land plants.

Origin, evolution, and metabolic role of a novel glycolytic GAPDH enzyme recruited by land plant plastids.

NAD-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a cytosolic marker enzyme of eukaryotes (GapC; EC 1.2.1.12). Land plants possess an additional NADP+-dependent enzyme (EC 1.2.1.13) within their chloroplasts which is composed of two subunits, GapA and GapB. Another plastid GAPDH enzyme (GapCp) was recently discovered in gymnosperms and ferns. This novel GapCp is closely related to cytosolic GapC and displays glycolytic NAD+ cosubstrate specificity. Here we show that this new gene GapCp is also present and actively expressed in angiosperms, mosses, and liverworts. Phylogenetic analyses of the available GapC and GapCp sequences suggest that the gene duplication giving rise to GapCp occurred in ancestral charophyte algae. The data are also consistent with a monophyletic origin of charophytes and land plants and further support the view that land plants arose from a Coleochaete-like green alga. Northern hybridizations were employed to study the expression of the genes GapCp, GapC, GapA, and GapB in green and nongreen tissues from pepper (Capsicum annuum). The results demonstrate that GapCp mRNAs are mainly expressed in red pepper fruit and roots, in which the transcript levels of photosynthetic GapA and GapB are downregulated. This suggests that in flowering plants GapCp plays a specific role in glycolytic energy production of nongreen plastids such as chromoplasts and leukoplasts and that angiosperms may be the only land plants where glycolysis is absent in green chloroplasts.

Light-dependent anaerobic induction of the maize glyceraldehyde-3-phosphate dehydrogenase 4 (GapC4) promoter in Arabidopsis thaliana and Nicotiana tabacum.

The maize glyceraldehyde-3-phosphate dehydrogenase 4 (GapC4) promoter confers strong and specific anaerobic gene expression in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum). Here we show that the promoter is also anaerobically induced in Arabidopsis thaliana. Histochemical analysis demonstrates that the promoter is anaerobically induced in roots, leaves, stems and flower organs. Surprisingly, the strong anaerobic induction of the promoter is dependent on light and on the substitution of oxygen with carbon dioxide. High carbon dioxide concentration alone does not induce the promoter in the presence of oxygen and light. If anaerobic conditions are generated under complete darkness or if plants are submerged, no induction above background is observed. When transgenic tobacco harbouring a GapC4 promoter-reporter gene construct is analysed for light dependent anaerobic induction, the results are indistinguishable from those with arabidopsis. The implications for using the GapC4 promoter as an anaerobic reporter for monitoring alterations in the anaerobic signal transduction pathway are discussed.

Anaerobic induction of the maize GapC4 promoter in poplar leaves requires light and high CO2.

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 ( GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula x P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO2 atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO2 and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO2 concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.