Non-Photosynthetic Alga Enhances the Reductive Evolution of Plastid Electron Transport System

A novel strain of non-photosynthetic Volvocales green algae, chlamydomonad sp. NrCl902. a Light microscopic observation of a cell. Bar = 10 μm. b Light microscopic observation of the orange eyespot, indicated by a closed double arrowhead, at the anterior part of a cell. Bar = 10 μm. c Transmission electron microscopy of non-photosynthetic plastids containing starch granules. Bar = 1 μm. Arrowheads show the membranes enclosing starch granules. S: starch granule, N: nucleus, M: mitochondrion, P: plastid. d DIC, e SYBR Green, f Mitotracker, g Merge. n: nuclear DNA. SYBR Green fluorescence signals other than the nuclear DNA are highly likely organellar DNAs. Bar = 5 μm. Double arrowheads indicate SYBR Green signal not overlapped with Mitotracker signal, suggesting presence of plastid DNA (pl nucleoid). Image Source:

By: Benison Zerrudo

Reductive evolution is the process by which genes are lost due to intracellular habitat (Wixon, 2001). This is evident in the organelle called plastid of non-photosynthetic algae and plants. Non-photosynthetic plastid retains various metabolic pathways involving redox reactions. However, there is a lack of knowledge about the underlying mechanism for balancing redox in functionally versatile non-photosynthetic plastids and the reductive evolution of plastid electron transport system. In this research, non-photosynthetic Chlamydomonad sp. green alga was isolated, cultured, and analyzed to reveal certain structural characteristics, phylogenetic similarities, and metabolic processes, expanding the knowledge of reductive evolution in plastids. This is important because symbiotic relationship is one of the major driving forces of evolution, and this cooperation among species allows them to survive differently than they would as an individual. The knowledge obtained from this study includes understanding evolution at the molecular level.

Plastids are the organelles that usually carry out photosynthesis. It also facilitates the synthesis of many classes of molecules which are needed for the plant to function (Bergthaller and Hollmann, 2007). According to endosymbiotic theory, heterotrophic eukaryote engulfed a cyanobacterium to form the first plastid. The formation of the first plastid occurred in the last common ancestor of land plants, green algae, red algae, and freshwater unicellular algae. Some algal lineages have complex plastids obtained through multiple endosymbiotic events. Researchers have shown interest in resolving the evolutionary events around the variety of modern plastids.

Photosynthesis converts light energy into ATP and NADPH that algae and plants use as their biochemical energy. The light to biochemical energy conversion occurs through the photosynthetic electron transport and the plastid ATP synthase complex. The ATP and NADPH are then used in the Calvin cycle and other various metabolic processes that are important in the photosynthetic plastid. Calvin cycle involves the process of converting carbon dioxide into glucose. Photosynthesis is beneficial because it allows plants and algae to produce their own energy, but some species of algae and plants have lost their ability to convert light energy into food source. Almost all photosynthetic lineages appear to include these non-photosynthetic species showing independent multiple losses of photoautotroph in the evolution of eukaryotes.

Among the species with non-photosynthetic plastid, the parasite Plasmodium falciparum is the most well-observed organism. P. falciparum is a unicellular protozoon and the deadliest Plasmodium that causes malaria in humans (Rich et. al., 2009). It has a plastid called apicoplast which do not facilitate photosynthesis but has few metabolic processes such as the production of heme, fatty acids, and other compounds. The apicoplast also retains the most reduced photosynthetic electron transport system. However, recent studies inflated the knowledge in the known functions of these non-photosynthetic plastids. For example, some species of diatoms have non-photosynthetic plastid that still maintain multiple redox reactions for glycolysis, amino acid synthesis, and pentose phosphate pathway, in addition to some functions in the apicoplast. Some genera of green algae such as Polytomella and Helicosporosium also have similar complex metabolic processes in non-photosynthetic plastid. Inversely, some golden algae seem to have lost certain amino acid and fatty acid synthesis in non-photosynthetic plastids carrying only the process of glycolysis and the synthesis of heme and iron-sulfur cluster. The novel Rhodelphidia of the red algal lineage also has non-photosynthetic plastid that only functions to produce heme and iron-sulfur cluster. Proteins that were encoded by the nucleus and imported through the plastid membranes are behind for all the functions mentioned above.

Apart from metabolic processes, few research studies have concentrated on the evolution of the electron transport system in non-photosynthetic plastids. Besides from photosynthetic linear electron transport, multiple branched pathways of electron transport are equipped in plastids that have photosynthetic capability to avoid deadly light damages caused by ineffective linear electron transport due to disparity ratio of reducing power and ATP. Chlororespiration may also contribute to balancing the redox reaction by regulating excess NADPH produced by biochemical reactions in plastid. Chlororespiration is the process by which oxygen is converted by a putative respiratory electron transfer chain in the thylakoid membrane to produced ATP (Bioblast).

Chlamydomonad sp. green alga was isolated from a freshwater sediment sample obtained from a paddy field in Japan. The cell was grown in a dark condition, and later deposited to Microbial Culture Collection of National Institute for Environmental Studies.

The experiment established and maintained a pure sterile clonal culture of the colorless Chlamydomonad sp. green alga. The medium used contains sodium acetate which is the only source of carbon/energy for the cells. Researchers observed the cell shape and structures using fluorescence/differential microscope following the staining of different structures and organelles. Ultrastructure was observed using transmission electron microscope. Membrane-bound structure surrounding starch granules, but lacking accumulation of thylakoid membranes was observed. This suggests that the Chlamydomonad sp. green alga with non-photosynthetic plastid can produce starch.

Gene sequence of the Chlamydomonad sp. green alga was retrieved and aligned with those of Volvocales species. Total DNA was extracted from the cell for library construction and DNA was sequenced. The complete plastid genome sequence was obtained, and the protein-coding genes were identified. Mitochondrial DNA segment was detected by comparing with the mitochondrial DNA sequence of Chlamydomonas reinhardtii and Chlamydomonas leiostraca. Using a database, C. reinhardtii sequences involved in several plastid functions were compared for similar sequences in the transcriptome data of the green alga. Plastid metabolic pathways of the Chlamydomonad sp. green alga were constructed by transcriptome analyses with C. reinhardtii chloroplast functions as references. Researchers detected DNA fragments encoding plastid-targeted proteins that are involved in most of the C. reinhardtii chloroplast functions but did not detect any functions related to photosynthesis such as photosynthetic thylakoid membrane complexes, chlorophyll synthesis, and carbon fixation. These complex metabolic pathways in the non-photosynthetic plastid may be fueled by metabolisms from the mitochondria/cytosol through plastid transporters.

Plastid-targeted proteins for electron transport system from other non-photosynthetic algae and plants were observed for transcriptome data. Such non-photosynthetic algae and plants include Polytomella, Monotropa, Nitzschia, and Spumella. The genome data of Helicosporidium, Rhodelphis, and Plasmodium was also observed. Plastid-targeting sequences of detected homologs were investigated, and phylogenetic analyses of specific proteins were performed. In gene phylogeny, the Chlamydomonad sp. green alga is closely related to Chlamydomonas pseudoplanoconvexa which is a photosynthetic species. The green alga was determined to be distantly related to non-photosynthetic Volvovales green algae such as Polytoma and Polytomella. Hence, Chlamydomonad sp. green alga is the third independent lineage of non-photosynthetic Volvocales species.

The research succeeded the assembly of complete 176-kb-long plastid genome of the non-photosynthetic Chlamydomonad sp. green alga. The genome does not include genes for photosynthetic thylakoid membrane complexes, the carbon fixation pathway, and chlorophyll synthesis. The proteins found in the plastid of the Chlamydomonad sp. green alga are related to proteolysis, transcription, translation, protein transport, and plastid division. The gene collection for encoding protein of the Chlamydomonad sp. green alga is completely similar with that of Polytoma uvella. Chlamydomonad sp. green alga and P. uvella have lost photosynthetic capability independently and the shared gene collection represents the convergent reductive plastid genome evolution after the loss of photosynthesis.

Carotenoids (yellow, orange, and red pigments) and other pigments were extracted and separated individually. Carotenoids were identified by their retention time and characteristic absorption spectra using certain detection equipment. Quinones were extracted from the cells of the Chlamydomonad sp. green algae for analysis. Quinone functions as electron and proton carriers in photosynthetic and respiratory electron transport chain (Nowicka and Kruk, 2010).

Homogentisate solanesyltransferase is a protein involved in the synthesis of plastoquinone, a type of quinone (Uniprot). DNA fragments for expressing homogentisate solanesyltransferase was obtained through reverse transcription of RNA taken from the Chlamydomonad sp. green algal cells. The DNA fragment was used as a template for double-strand RNA of homogentisate solanesyltransferase. Electroporation was performed with and without the homogentisate solanesyltransfer double-strand RNA for several Chlamydomonad sp. green algal cell suspension. Electroporation is defined as a technique to increase cell membrane permeability to hydrophilic molecules using electrical pulse (ScienceDirect). After electroporation, the cells were subjected to cell counting, reverse transcription polymerase chain reaction assays, plastoquinone detection, and quinone extraction.

The study showed that the central component for plastid electron transport systems is still retained in the novel strain of obligate heterotrophic Chlamydomonad sp. green algae. Genome and microscopic analyses showed that Chlamydomonad sp. green alga has non-photosynthetic plastids and a plastid DNA that does not carry gene for photosynthetic electron transport system. The non-photosynthetic green alga can synthesize carotenoid and plastoquinol, but no trace of chlorophyll pigments. Short-term RNA interference knockdown leads to decrease in plastoquinone. Chlamydomonad sp. green alga seems to have genes for an electron sink system. Other non-photosynthetic algae and land plants also possess key genes for this system which suggests a broad distribution of an electron sink system in their plastids. Plastoquinone pool and the involved electron transport systems reported in this study might be conserved for balancing the redox. It might represent an in-between step towards a more reduced set of the electron transport system in several non-photosynthetic plastids.

These findings that non-photosynthetic plastid in some algae and plants still retain several biochemical processes associated in redox reaction and carotenoid biosynthesis pathway should be significant. Successively, this proposes that the electron transport systems for balancing the redox that decrease excess reducing power and sink excess electrons might also be exclusive for certain processes of non-photosynthetic plastids. However, it is still unknown as to why metabolically versatile non-photosynthetic plastids still manage several redox reactions.

The new strain of non-photosynthetic Chlamydomonad sp. green alga retains synthesis of plastoquinone. A short-term RNA interference knockdown experiment discovered a gene linked to the synthesis of plastoquinone. The experiment also detected gene sequences for ferredoxin, plastidal, and plastoquinone-mediated electron sink systems, which may be required for carotenoid synthesis and balancing the redox in the non-photosynthetic plastid. Extensive observation suggests a broad distribution of the gene set for plastid electron transport systems that are simpler than that of plastids that are photosynthetic, but more complex than that of apicopasts. Researchers revealed a step for reductive evolution of photosynthetic electron transport systems that was previously uncovered, together with the evolution of photosynthetic algae to heterotrophic protists.

Understanding the mechanisms and processes in non-photosynthetic plastids fabricates a bridge of knowledge between photoautotrophic and heterotrophic pathways given that most people think that only plants perform photosynthesis. The study suggests that an organism may have cellular structure that had certain protein complexes at some point but disappeared through reductive evolution. Protein complexes we currently know may be associated with protein complexes that existed before and that a higher or more complex metabolic processes was occurring depending on the environment and the composition of the atmosphere. With the changing environment, we can extrapolate that there will be changes in protein complexes and metabolic pathways.


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