In this study, we have analyzed about 3000 expressed genes from the scaly green flagellate Mesostigma viride
. We compared the expressed genes with the complete genomes from the angiosperms Arabidopsis thaliana
and Oryza sativa
, the chlorophyte Chlamydomonas reinhardtii
, the red alga Cyanidioschyzon merolae
and the diatom Thalassiosira pseudonana
, as well as the ESTs from the moss Physcomitrella patens
, and the red alga Porphyra yezoensis
. Altogether, the Mesostigma
proteome is more similar to the embryophytes than to Chlamydomonas
, although Mesostigma
are both flagellate unicells. Mesostigma
shares more genes with the embryophytes than with Chlamydomonas
, including several enzymes confined to the streptophytes (e.g. GAPDH B, [Cu-Zn] superoxide dismutase), and the average identity of shared proteins is higher between Mesostigma
and the embryophytes than between Mesostigma
. Therefore, we consider Mesostigma
to be a member of the streptophytes, although Mesostigma
clearly shares some ancestral characters with chlorophytes. Plastidic (with the exception of the Calvin cycle) and mitochondrial functions e.g. seem to be more conserved between Mesostigma
and chlorophytes than between Mesostigma
and embryophytes, i.e. these functions are more derived in embryophytes, probably due to adaptation of embryophytes to the terrestrial habitat. In contrast, other cellular functions except for the cytoskeleton are more conserved between Mesostigma
and embryophytes than between Mesostigma
. Interestingly, in previous phylogenetic analyses plastidic and mitochondrial genes failed to show a clear relationship between Mesostigma
and the streptophytes [14
], whereas actin and nuclear-encoded SSU rDNA phylogenies support the notion that Mesostigma
is a member of the streptophytes [10
]. The different evolutionary rates for different cellular functions observed in this study might explain this discrepancy.
We calculated the average identity (AI) values from automatically generated BLAST output alignments. Automatically derived alignments are prone to errors. However, we believe that our approach is justified for the following reasons: (1) the BLAST alignments cover only the conserved parts of proteins and our calculated AI values indicate that in most alignments more than half of the amino acids are identical enhancing the quality of the automatically produced alignments; (2) although small mistakes may occur, they are insignificant given the high number of amino acids used to calculate the AI. On average the BLAST alignments contained about 150 amino acids and therefore about 45,000 amino acid positions were used in the constrained data set. In large data sets small unbiased errors become irrelevant [27
]. Our results indicate that at least 100 (better are 150–200) expressed genes have to be used to obtain statistically significant results. It could be argued that our analysis uses only similarity values and no real evolutionary distances. AI values can be easily converted into evolutionary distances using an approximation given by Kimura [28
], with the effect that the differences between the various organisms become larger but no changes occur in the order of relatedness (included in Table ). We conclude that the AI of proteins shared between different organisms represents a reasonable measure of evolutionary relatedness, if sufficiently large data sets are used.
In the following, we briefly discuss some major differences in coding potential observed between the different photosynthetic eukaryotic organisms.
11 of 18 proteins included in supplemental Table 1 [see Additional file 1
] which are shared only by Mesostigma
are associated with flagellar functions such as axonemal dyneins or components of the IFT (intra-flagellar transport) machinery. Most likely, the angiosperms lost these proteins during evolution together with the ability to produce flagellate cells. The absence of these proteins in the ESTs from the moss Physcomitrella
, is presumably due to the fact that ESTs from developing spermatozoids are not available.
Proteins shared by Mesostigma
and the embryophytes but not present in chlorophytes perform diverse functions. There are some well known biochemical differences between chlorophytes and streptophytes such as the presence of (Cu-Zn) superoxide dismutase [29
] and glycolate oxidase in streptophytes [31
] but not in chlorophytes. In addition, streptophytes use the DXP and mevalonate pathways for isoprene biosynthesis whereas chlorophytes posses only the DXP pathway [33
]. For all these functions, we find molecular support in our expressed gene data set except for the mevalonate pathway of isoprene biosynthesis. Two genes matched two different enzymes of the DXP pathway; however, no matches for the MVA pathway were obtained, although the presence of this pathway has been demonstrated biochemically [33
]. This could be due to the selective expression of one or the other pathway under different environmental conditions.
Remarkably, our list of proteins uniquely shared by Mesostigma and the embryophytes includes several proteins involved in steroid biosynthesis (e.g. a 3-oxo-5-beta-steroid dehydrogenase and a C-4 sterol oxidase), a homeobox protein of the knox family and proteins of the F-box family. The latter protein family underwent a dramatic expansion in the embryophytes (Arabidopsis has more than 700 members of this family).
Our expressed protein data set contains sequences similar to a protein involved in vitamin B-12 metabolism (present in rhodophytes and chlorophytes), an arginine kinase and a ARL6 protein, the latter two are absent in chlorophytes, embryophytes and red algae. It has been shown that arginine kinase is part of the ATP regeneration system in cilia of Paramecium
lacks arginine kinase and recently Pazour et al. [35
] showed that enzymes of the late glycolytic pathway are present in the flagella of Chlamydomonas
, suggesting that the ATP required for flagellar function is produced by the glycolytic pathway in Chlamydomonas
. The ARL6 protein has been implicated in protein translocation at the rER [36
], although its exact function is still not known.
There are some typical embryophyte pathways that we failed to detect in Mesostigma
, e.g. sucrose metabolism, hexokinase, and enzymes of cellulose biosynthesis. There are no reports about the presence of sucrose metabolism and hexokinase in green algae in the literature, whereas embryophyte-like Ces genes (catalytical subunit of cellulose synthase) have been reported in the streptophyte alga Mesotaenium
]. Although we cannot exclude that Mesostigma
lost these genes, we do expect to find theses genes in the genome of Mesostigma
Evolution of photosynthesis and photorespiration
It is well known that embryophytes and chlorophytes differ in important aspects of photosynthesis and its regulation, and in photorespiration (e.g., presence of GAPDHB, number of enzymes regulated by thioredoxin, glycolate oxidase vs. glycolate dehydrogenase, and presence or absence of (Cu-Zn) superoxide dismutase).
Table summarizes the available information on the regulation of plastidic proteins by the thioredoxin system. The number of thioredoxin-regulated proteins has apparently increased during evolution and Mesostigma
in this respect most closely resembles the embryophytes. Similarly, the peroxisomes of Mesostigma
have been biochemically characterized as "leaf-type peroxisomes" [38
] in full agreement with our EST-data. In contrast, chlorophytes lack glycolate oxidase and photorespiration involves only chloroplast and mitochondrial enzymes [38
]. Interestingly, red algae possess a peroxisomal glycolate oxidase whereas the other enzymes of the photorespiratory cycle are located in the mitochondrion [32
]. Thus, it seems likely that at the onset of streptophyte evolution major changes occurred in the regulation of the Calvin cycle and the subcellular organization of photorespiration. What might have been the driving force for these changes? We note that rhodophytes and chlorophytes both presumably evolved in a marine environment [red algae in a coastal benthic habitat, whereas chlorophytes proliferated as marine phytoplankton [39
]]. Streptophyte algae most likely originated in a freshwater/brackish environment. In contrast to their marine counterparts, they had to deal with much higher light intensities and fluctuating environmental conditions such as salinity and temperature. With higher temperature, the rate of photorespiration increases. The observed changes in regulation of the Calvin cycle and photorespiration might be adaptations to this stress. It is possible that these adaptations to a shallow freshwater/brackish environment prepared streptophytes to colonize the terrestrial habitat later during evolution. In this respect we note that in extant chlorophytes activation of carbon concentrating mechanisms (CCM) is the dominant reaction to compensate for increased photorespiratory losses [38
]. In contrast, streptophytes are able to channel large amounts of glycolate through the photorespiratory cycle [38
]. According to Badger and Price [40
] CCMs did not evolve until 400 million years ago, long after streptophytes had evolved and the colonization of the terrestrial habitat by streptophyte algae took place. Therefore during the palaeozoic era with reduced CO2
- and increased O2
] streptophyte algae might have had an advantage over chlorophyte algae allowing them to colonize the terrestrial habitat during that time.
Table 8 Regulation of plastidic enzymes by the thioredoxin system. Proteins similar to embryophyte plastidic thioredoxin-regulated proteins were identified in the genomes of Cyanidioschyzon, Chlamydomonas, and the ESTs of Mesostigma using the BLASTP or BLASTX (more ...)