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On the track of fascinating diatoms
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.
Algae researcher Prof. Peter Kroth (right) and his colleague Dr. Ansgar Gruber holding a petri dish containing
Phaeodactylum transformants. © University of Constance
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.
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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.
Genomic investigations using different unicellular organisms
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.
Phaeodactylum tricornutum analysis leads to unexpected results
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
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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.
Plastids, which are the photosynthetic organelles of all algae, are believed to have developed through primary
endosymbiosis, where a host cell incorporates a cyanobacterium and converts it into an organelle. An important
aspect is the transfer of many genes (black arrows) into the nucleus of the host cell. The proteins required in the
plastids, whose genes are now in the cell nucleus (approx. 2000 – 3000 proteins), must now be imported into the
organelles (red arrows). Such primary plastids are found in green algae (e.g., Chlamydomonas reinhardtii), red algae
and land plants. It is assumed that secondary endosymbiosis occurred in diatoms and related algae, in which a
complete eukaryotic red alga was taken up by a host cell and transformed into an organelle. Except for the plastids,
all cellular components of the endosymbiont were degraded, leaving the membrane behind, which in turn requires
all proteins and metabolites to be transported across four membranes. Some of the genes of the endosymbiont as
well as bacterial genes are also found in the nuclear genome of Phaeodactylum. (N: nucleus; P: plastids; ER:
endoplasmic reticulum) © Peter Kroth
Exciting conclusions about diatom evolution
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,
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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.
Genes with bacterial characteristics
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.
Diatoms to rescue us from a global climate change?
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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.
Further information:
University of Constance
Prof. Dr. Peter Kroth
Tel.: +49 7531 88-4816
Fax: +49 7531 88-3042
E-mail: peter.kroth(at)uni-konstanz.de
Article
22-Oct-2009
mst
BioLAGO
© BIOPRO Baden-Württemberg GmbH
The article is part of the following dossiers
A green view - plant genome research
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