Diatoms make a considerable contribution to the production of oxygen and biomass in the world’s oceans and aquatic ecosystems. However, up until now little is known about the molecular biology and biochemistry of these eukaryotic algae. Prof. Peter Kroth and his team at the University of Constance are hoping to shed more light on these algae. The team has recently been involved in the deciphering of the Phaeodactylum tricornutum genome, research that revealed quite a few surprising results.
Diatoms are one of the most common types of phytoplankton found in the upper layers of rivers, lakes and oceans, where they carry out photosynthesis and are used by zooplankton and fish as food. Botanists have been well aware of their existence ever since microscopes were invented. Ernst Haeckel, a 19th century evolutionary biologist and illustrator, sketched the diatoms in their full glory, referring to them as "Schachtellinge" (see: Artforms of Nature, 1904) due to their silica shells (frustules) which overlap one another like the two parts of a hat box. "However, research initially concentrated to a greater extent on the classical microorganisms, for example the bacterium E. coli, the fruit fly Drosophila melanogaster that was used as an insect model, the thale cress Arabidopsis thaliana that was used as a model for higher plants and mice and rats, that were models for vertebrates," said Prof. Peter Kroth explaining why very little knowledge existed about the molecular biology and biochemistry of diatoms.
Over the last few years, technological progress and the enormous DNA sequencing capacities associated with this progress have given rise to the possibility of expanding the existing knowledge on diatoms and the physiology and biochemistry of other organisms using molecular analyses. "The development of a genetic transformation system for diatoms about ten years ago has considerably increased researchers' interest in this group of organisms," explains the biochemist. Diatoms are related to brown algae, and obtained their chloroplasts from red algae.
Kroth and his team of researchers are investigating several species of diatoms, including Phaeodactylum tricornutum, Skeletonema costatum and Thalassiosira pseudonana. Their investigations involve both laboratory experiments where algae culture conditions can be clearly defined, and student field trips to Lake Constance to gather samples. Although all diatoms are unicellular organisms, they have completely different lifestyles,” said Prof. Peter Kroth, explaining that some diatoms live close to the shore on sand or stone surfaces where they can move around whilst excreting carbohydrates. Others live as plankton in the upper layers of lakes and oceans. The diatoms’ physiology and biochemistry can also vary considerably from one diatom to another. “It is known that the photosynthetic behaviour of some diatoms is similar to that of so-called C3 plants (e.g. Arabidopsis), while the photosynthetic behaviour of others is more similar to that of C4 plants (e.g., maize),” said the Constance researcher, adding that the major difference is how the diatoms bind carbon dioxide in order to carry out photosynthesis.
The genomic investigations of Prof. Peter Kroth and his team have recently shown that Phaeodactylum has an unusually large number of CO2-fixing enzymes in unexpected compartments. “Our findings have also given us information about another unusual feature of diatoms, namely how they deal with high light intensities,” said Kroth. According to the researchers’ findings, planktic diatoms are transported rapidly with the ascending current from areas lacking light into the more sunlit water areas close to the surface. “Diatoms are efficient in converting light energy into thermal energy, thereby rendering light energy harmless,” said the researcher.
Recently, Kroth’s team of researchers were decisively involved in the annotation of the Phaeodactylum tricornutum genome at the Joint Genome Institute (JGI) in California, which threw up some rather unexpected findings. “Many Calvin cycle enzymes are present in multiple copies and it is still unclear whether the function of these copies is to compensate the missing regulation through thioredoxins by way of differential gene expression, or whether the duplication has something to do with gene transfer processes during secondary endocytobiosis activities,” said Prof. Peter Kroth.
The researchers are now looking into why these organisms have retained all these genes. Expression analyses (EST databases) have shown that the genes are actively expressed in the cells. “In multicellular organisms, isogenes and isozymes are often expressed in specific tissues according to certain states of development; however, in diatoms, the expression of such genes might depend on external conditions,” said the researcher explaining that one of the five known fructose-bisphosphate aldolases in Phaeodactylum is highly expressed during iron deficiency. Kroth further explained that the higher number of isozymes might also suggest a better adaptation to environmental changes, which would partially explain the evolutionary success of diatoms.
The most important photosynthetic pathway, the Calvin cycle (reductive pentose phosphate pathway) in plant plastids is light-regulated, so that it does not occur simultaneously with the oxidative pentose phosphate pathway, which would lead to the direct release of the newly bound CO2. “Therefore, the enzymes of the reductive pentose phosphate pathways in plants are switched on by the redox enzyme thioredoxin during light conditions, while the enzymes of the oxidative pentose phophate pathway are only active during darkness,” said Prof. Peter Kroth. He also explained that their findings show that many of the enzymes that are activated in plants by way of the protein thioredoxin through light-dependent reduction, do not underlie redox regulation in diatoms. In diatoms, however, only one enzyme of the Calvin cycle, fructose-1,6-bisphosphatase, seems to be switched on in this way. “This could be enough to regulate the Calvin cycle in diatoms since the oxidative pentose phosphate pathway in diatoms was translocated from the plant plastids into the cytosol during evolution,” said Prof. Peter Kroth.
Diatoms have some metabolic particularities that are related to their unusual evolution. While cyanobacterial symbiosis gave rise to the plastids of all algae, diatoms and related algae developed as a result of the incorporation of a complete algal cell into a host cell and its subsequent degradation to a plastid. “This involved comprehensive genomic rearrangements, including rearrangements of the metabolic pathways,” said Prof. Peter Kroth. For example, diatoms have a urea cycle that is found in animals but not in plants. Elementary pathways, such as nucleotide biosynthesis for example, were translocated from the plastids into the cytosol. This of course also means that new transport pathways had to be established for the metabolites.
“We have been able to show that in the case of a nucleotide translocator for example, the diatoms have taken over the gene of an intracellular parasitic bacterium,” explained the Constance biochemist. "The redundancy resulting from the secondary uptake of cells as well as lateral gene transfer has in some cases led to a higher number of isogenes whose importance for the metabolism is still not understood. We were also able to identify several genes in which enzymes for subsequent reactions were fused, leading to double enzymes in the cell, which in turn potentially increased the catalytic conversion rates."
Prof. Peter Kroth and his team have found that some metabolic pathways in the cells of diatoms are distributed differently from those in the cells of green land plants. “In contrast to green plants in which the addressing signals for important compartments such as chloroplasts and mitochondria differ only marginally (transit sequences) and cannot always be predicted with the same certainty, the corresponding addressing signals for the same organelles differ considerably in diatoms,” said Kroth. Kroth and his team found a highly conserved amino acid motif (AFAP motif) for plastids. The computer-assisted predictions that the researchers carry out are thus quite good and can subsequently be tested through the microscopic localisation of GFP fusion proteins.
The Constance researchers were also surprised to find that in Phaeodactylum tricornutum many genes resembled animal genes and not plant genes, and additionally that there were a large number of bacterial genes in the genome. According to Kroth, about 7.5 per cent of the genes in Phaeodactylum tricornutum are more similar to bacteria than to plants or animals. “This indicates that horizontal gene transfer between completely different organisms plays a much greater role than inheritance in the evolution of diatoms and other organisms than has previously been assumed,” said Kroth further assuming that the high proportion of bacterial genes in diatoms might be the result of the nutrition of the non-photosynthetic host cells. “Many unicellular organisms live on the uptake of bacteria that are subsequently digested. This might of course mean that the DNA of the prey can easily enter the cells,” said Kroth. “Genetic transformation experiments with different organsims have shown that DNA can quickly migrate and integrate into the cell nucleus as soon as the DNA has reached the cytosol. If this happens very frequently, these genes will eventually also receive nuclear promoters (gene switches) during evolution and be able to be expressed.
Another important aspect is the rapid evolution of diatoms, whose major lines separated about 90 million years ago. The genomes of the two representatives of these groups, Thalassiosira and Phaeodactylum, differ more from each other than the genomes of fish and humans who separated into different lineages about 550 million years ago.
Prof. Peter Kroth does not believe that diatoms will be able to make a great contribution to stopping global climate change despite their important role in the earth's carbon dioxide budget. “Iron fertilisation experiments carried out at the Alfred Wegener Institute in 2009 have shown that the iron sulphates lead to a temporary bloom of diatoms, which then bind a great deal of CO2 through photosynthesis. However, the algae are eaten up very quickly, which leads to the quick release of CO2. The researchers agree that while such experiments are suited to finding answers to basic questions, they are not suited to large-scale experiments for ecological reasons,” said Kroth.