From molecular manipulation of domesticated Chlamydomonas reinhardtii to survival in nature

1Chlamydomonas reinhardtii is a single-celled green alga found in temperate soil habitats (Figure 1). It has proven to be such a powerful model for dissecting fundamental processes in biology that investigators have dubbed it the 'green yeast'. Ehrenberg described the genus Chlamydomonas in 1833, and Dangeard the species C. reinhardtii in 1888. Chlamydomonas was found suitable for genetic studies in the early 20th century, while the development of C. reinhardtii as a model organism dates to the 1950s when the first mutants were generated.

2Various features make C. reinhardtii an excellent laboratory species. It grows vegetatively as a haploid, which allows mutant phenotypes to be expressed immediately. Under optimal conditions, C. reinhardtii grows so rapidly that its numbers can double approximately every 8 hours. The fact that it can grow in the dark on acetate-containing medium while retaining a functional photosynthetic apparatus, has allowed even light-sensitive photosynthesis mutants to be isolated. The motile cilia of this photosynthetic eukaryote share the same structure and many of the same constituent proteins as those of mammals, and so research into its motility prompted studies that greatly advanced our understanding of cilium dysfunctions in humans. Furthermore, C. reinhardtii can be induced to sexually reproduce in the laboratory, making it easy to introduce multiple traits into a single haploid strain (e.g. to generate double or triple mutants). The power of C. reinhardtii as a model organism was further elevated by the advent of genetic transformation, the establishment of a full nuclear genome sequence, the construction of a genome-wide library of mapped, indexed insertional mutants and CRISPR-mediated targeted gene disruptions ( and references therein).

3Studies of C. reinhardtii have enabled numerous landmark discoveries and advances. One remarkable example is the discovery of intraflagellar transport of granule-like particles and the roles of motor proteins in the process ( and references therein). Furthermore, structural analyses of wild type and mutants with defective cilia have massively contributed to our knowledge of the building blocks of these structures, their organization and their function. These analyses also led to classic studies that demonstrated that abnormal cilia could cause human genetic diseases such as polycystic kidney disease. Additionally, acetate-requiring mutants (often unable to perform photosynthesis) have immensely advanced our understanding of photosynthesis, especially the ordering of electron carriers in the photosynthetic electron transport chain (e.g. ). Two core proteins of photosystem II (D1 and D2) were first identified in C. reinhardtii and later proposed to be key components of this photosystem’s reaction centers. More recently, a central role of the STT7 kinase in photosynthetic state transitions, and a key function of the xanthophyll cycle in nonphotochemical quenching were first established in C. reinhardtii.

4The field of optogenetics experienced a recent quantum leap with the discovery of channelrhodopsins in C. reinhardtii. When expressed in other cells, these gated ion channels can be stimulated with light to activate various processes, including neuronal activity. Sophisticated genetic, biochemical and cell biological analyses of C. reinhardtii are currently being performed to understand the cell cycle, basal bodies/centrioles function, pyrenoid structure, mechanisms associated with photoreceptor function and light acclimation and organismal interactions in ecosystems. Finally, C. reinhardtii is being exploited to study the evolution of multicellularity, especially with respect to multicellular algal species of the order Volvocales.

5C. reinhardtii can unambiguously be identified by sequencing internal transcribed spacers (ITS) or various phylogenetically informative genes. Yet many ecological studies have relied on light microscopy to identify Chlamydomonas species (sensu lato – see Box 1). Typically, two anterior cilia and a cup-shaped chloroplast harboring a pyrenoid have been sufficient criteria for a cell to be considered a Chlamydomonas sp. This morphology-based identification may be reliable to the level of genus, but rarely to the species level since many species look very similar. For these reasons, at times we omit species designations and simply note the organism as Chlamydomonas sp. A routine use of genetic taxonomic markers in the future would improve our knowledge of the geographical distribution of C. reinhardtii and related species and allow for more precise classifications.

6Based on traditional taxonomic criteria, the genus Chlamydomonas (sensu lato) contains more than 500 species. In the course of taxonomic revisions, which are still in progress, Chlamydomonas (sensu stricto) is comprised of three species. Accordingly, the taxonomy of some species mentioned in this article, such as C. nivalis or C. euryale, may be revised in the future. Furthermore, our use of the designations 'Chlamydomonas sp.' and 'Chlamydomonas spp.' refers to one or more Chlamydomonas species, respectively, which were typically not classified to the level of species and may not be C. reinhardtii. The majority of the contemporary C. reinhardtii laboratory strains were derived from a single zygote isolated from a potato field in Massachusetts in 1945. The sequencing of 39 common laboratory strains shows that they fall into five genetically distinct lineages from two parents or haplotypes. Under laboratory conditions, mutations accumulate at a rate of ~0.03 division-1 genome-1, corresponding to one mutation every 30 generations. In addition, removal of C. reinhardtii from its natural environment, including cultivation in the laboratory or cryopreservation, may unintentionally select for specific traits. For example, C. reinhardtii is often grown on medium containing ammonium as a nitrogen source, which allowed for the evolution of mutants (nit1, nit2) unable to utilize nitrate. For these reasons, the isolates domesticated for decades in the laboratory may only loosely correspond to wild C. reinhardtii strains. Furthermore, we do not know if the laboratory strains are still capable of surviving in the wild. To examine the ecological significance of laboratory findings, it will be important to isolate additional wild C. reinhardtii strains and characterize their behavior both in the field and in culture.

7While Chlamydomonas spp. (not identified at the species level) occur widely in temperate, subtropical and tropical soils, confirmed C. reinhardtii has only been found in temperate soils in Northern America and Japan. It occurs in cultivated fields but appears absent from many other habitats, suggesting it prefers nutrient-rich, disturbed soils. Most contemporary laboratory strains have emanated from a single soil isolate collected in 1945 (Box 1). Light typically penetrates only millimeters into the soil, depending on factors such as the soil structure and moisture content. Therefore, photosynthetic microbes are generally most abundant in the upper few millimeters where they can harvest light energy, although in some instances they can be present in soil layers where there is essentially no light. Chlamydomonas spp. are even present in biological soil crusts where they help stabilize the surface of drylands, contribute to primary production and potentially act as pioneer species.

8All unambiguously identified C. reinhardtii isolates were collected from soil habitats (T. Pröschold, personal communication), yet Chlamydomonas spp. are also commonly found in the pelagic zone of lakes, where they sometimes form spring blooms. The term 'pelagic zone' refers to the water column of lakes and oceans not on or near the lake or ocean bottom. Chlamydomonas spp. are usually motile, and although this has an energetic cost, it gives them a competitive advantage in lakes that have stratified into distinct layers as a consequence of seasonal changes in temperature. Under conditions of stratification, motile algae often ascend toward the lake surface during the day to optimize their exposure to sunlight. During the night, they tend to descend to access the nutrient-rich environment below the surface. Indeed, this pattern of vertical movement has been observed for the population of Chlamydomonas sp. in a small Finnish lake.

9Environmental conditions and the availability and distribution of natural resources differ substantially in soils and lakes. Phosphorous, for example, is likely to be limiting to the growth of organisms in lakes and geologically old soils, while nitrogen limitation is more common in young soils. Light availability and grazing pressure by predators represent additional key environmental differences between soil and lake habitats. Consequently, these two habitats require distinct adaptations and life history strategies to optimize fitness, and it is still an open question as to whether specific pelagic strains of C. reinhardtii exist in lakes (Box 2).

10Chlamydomonas spp. other than C. reinhardtii are adapted to a wide range of habitats. For example, Chlamydomonas eustigma is an acidophilic species isolated from acid mine drainage, Chlamydomonas euryale is found in temperate marine environments, Chlamydomonas spp. have been isolated from Antarctic ice, and some members of the genus Chlamydomonas are carotenoid-rich organisms present on the surface of snow, giving it a red appearance. A Chlamydomonas sp. has even been identified in the air at 1,100 meters above the ground: this and other algae can be dispersed by wind over extended distances. Taken together, several reports provide information on the biogeographical distribution of C. reinhardtii and other Chlamydomonas spp. However, there is little knowledge of the abundance and variations of Chlamydomonas spp. in different soil types, the dynamics of these natural populations over daily or seasonal cycles, and their physiological capabilities.

11The chloroplast and mitochondrial genomes of C. reinhardtii have been sequenced and are 206 and 15.8 kb, respectively. Since the sequence of the nuclear genome was first published, the scientific community has focused some effort on elevating the quality of the genome sequence and improving its assembly and annotation. The current version 5.5 nuclear genome is 111 Mb, which is similar in size to the genome of the model land plant Arabidopsis thaliana. Recently, whole-genome sequences for more than 50 additional laboratory strains and field isolates were generated. The sequences of 12 field isolates confirmed earlier reports that with a nucleotide diversity (π) of ~3%, the C. reinhardtii genome is among the most polymorphic of all eukaryotes. The field strains, isolated from various locations in the United States and Canada, genetically group into three distinct populations, with gene flow between populations sufficiently low to allow the populations to adapt to their local environments. The low ratio of genome-wide non-synonymous to synonymous substitutions (0.58) further indicates that natural selection efficiently eliminates C. reinhardtii alleles of low fitness. Whole-genome and epigenome sequencing has also been used to examine adaptation in the laboratory under changing environmental conditions.

12Forming zygotes likely allows C. reinhardtii to survive when conditions become harsh. In the laboratory, gametogenesis can be induced by nitrogen starvation in conjunction with specific light conditions; both signals may inform the cell of deteriorating environmental conditions (see below). The fusion of haploid gametes results in diploid zygotes that can develop over several days into highly resistant, dormant zygospores (Figure 2). When nitrogen is added back to the medium, the zygotes germinate in the light, undergo meiosis and typically release four haploid cells that resume vegetative growth. Dormant zygospores can remain viable in soil for many years and survive freezing, desiccation and probably other forms of harsh environmental conditions. This extraordinary resistance is associated with the multilayered cell wall of the zygospores, which contains a durable lipid polymer structurally similar to those found in million-year-old microfossils (described for Chlamydomonas monoica; ). Furthermore, sexual reproduction can increase the rate of adaptation of C. reinhardtii to new or changing environmental conditions, particularly if the population and genetic diversity within the population are large.

13What are the geographic origins of C. reinhardtii? What are its current geographic and vertical distributions? How do populations of C. reinhardtii quantitatively change over time and what factors impact these changes? What are the major mechanisms of C. reinhardtii dispersal? For example, are aquifers common routes for the transport of C. reinhardtii over long distances?

14What is the genetic variability within and between C. reinhardtii populations? What are the relationships among populations of the various Chlamydomonas species?

15Do specific pelagic strains of C. reinhardtii exist in lakes? If so, do they have major differences in their life histories, physiologies and genome sequences compared to soil-dwelling strains?

16What are the typical division rates of vegetative C. reinhardtii cells in the wild? How frequently does sexual reproduction occur in natural populations? How common are dormant zygospores in the environment, and where do they occur? Are zygospores typical overwintering forms, and do they also have an increased resistance to challenging biotic interactions?

17What are the most common biotic interactions of C. reinhardtii in the environment (competing photosynthetic microbes, grazers, bacteria, fungi)? In what ways does C. reinhardtii communicate with its neighbors (e.g. infochemical signals)? What is the metabolic significance of these interactions?

18How often and under what situations do cells shed their cilia in nature? Is there a selective advantage of deciliation in response to stress?

19Does C. reinhardtii associate with biofilms on soil particles and, if so, how are the algal cells organized within the biofilm community?

20Following gamete fusion, a pair of homeodomain transcription factors initiates the genetic program for zygote development. The first zygote-specific genes are induced within minutes of gamete fusion, with hundreds of additional genes activated over the next few hours. A gene encoding a polyketide synthase is induced two days after zygote formation and is critical for the zygote-to-zygospore transition, probably because it participates in the biosynthesis of the cell wall lipid polymer. Several stages of the sexual cycle, including gamete formation and maintenance and zygote germination, depend on light and involve regulation by three different photoreceptors.

21Similar to Saccharomyces cerevisiae, we know little about the life cycle of C. reinhardtii in its natural environment. For example, there is no quantitative data on the frequency of sexual reproduction relative to vegetative growth. Yet, nitrogen is thought to become limited more commonly in soils than lakes. This notion is congruent with nitrogen limitation being a major cue for zygospore formation in nature, but we are not aware of any data on zygospore induction in natural soil environments. The occurrence of clonal cultures of opposite mating types that are derived from a single zygospore isolated from dry soil provides additional evidence for a critical role of zygospores during desiccation. Freezing resistance of zygospores and their more efficient germination under long-day conditions compared to short-day conditions suggests that zygospore formation is an overwintering strategy. If true, the question arises as to whether or not nitrogen limitation and day length are adequate cues to herald the approach of winter, or whether, for example, a decrease in soil moisture content or temperature can also induce zygospore formation in C. reinhardtii.

22C. reinhardtii not only orients itself with respect to light, but can also swim upward in complete darkness. This negative gravitaxis may facilitate orientation and movement of the cells at night or in the soil environment, potentially helping the alga locate areas with more favorable conditions of illumination following daybreak. Furthermore, vegetative cells are attracted to ammonium, nitrite and nitrate ( and references therein). Chemotaxis towards ammonium is strongest during the night, whereas phototaxis towards the light is strongest during the day, with both processes regulated by the circadian clock. Finally, the hypothesis that the circadian clock depends on gravity or a magnetic field was refuted by experiments performed with C. reinhardtii on a space shuttle under microgravity conditions.

23Cilia enable C. reinhardtii to swim in an aqueous medium, and also glide on solid surfaces. Gliding motility may be important when C. reinhardtii resides within a thin water film that coats soil particles. The gliding speed of C. reinhardtii is ~1 µm s-1 whereas the average forward swimming speed is 100-200 µm s-1. Under various stress conditions, such as acidification of the medium, C. reinhardtii loses or sheds its cilia when a specific break point near the base of the cilium is activated. When conditions improve, the cilia regenerate. The biological reason for deciliation is still a mystery, but various hypotheses have been put forth. Deciliation is observed in a wide range of different cell types; for example, inhalation of irritant chemicals can lead to deciliation of respiratory epithelial cells in mammals. Therefore, it seems likely that a predetermined break point is an ancient and inherent property of every cilium. Consequently, deciliation may not confer a selective advantage, but might be a consequence of pathological conditions that cause over-stimulation of the ciliary disassembly process. On the other hand, the ciliary membrane of C. reinhardtii is in direct contact with the environment (not protected by cell wall) and therefore, deciliation may reduce the entrance of noxious compounds into cells. Deciliation may also allow cells to escape when their cilia are stuck to the surface of a predator. Studying deciliation in the natural environment holds the promise of new insights into selection pressures that led to its evolution.

24The ability of C. reinhardtii to grow under heterotrophic and fermentative conditions might be an adaptation to soil environments where there can be both low light and low oxygen. Anoxic/hypoxic conditions are likely to mostly occur at night when there is no photosynthesis to release oxygen and the soil microbes are respiring. Under anoxic conditions, C. reinhardtii can use glycolysis to yield energy, which is sustained by fermentation metabolism and the release of reduced organic compounds. C. reinhardtii has recently been shown to activate a variety of different pathways that result in the formation of many fermentation products including formate, lactate, acetate, acetyl-CoA, succinate, hydrogen and glycerol. While some regulatory elements involved in anoxic metabolism are known, little is understood about what controls the various pathways associated with fermentation and the ways in which these pathways are integrated.

25In nature, C. reinhardtii is continuously in contact with other organisms, including competitors, predators, pathogens, parasites, commensals or mutualists. Most molecular details concerning these interactions, which likely involve chemical signaling, nutrient exchange and receptor-mediated processes, have not been examined. In lakes, the various Chlamydomonas spp. successfully compete with many other pelagic algal species for light and nutrients; rapid growth of Chlamydomonas spp. likely compensates for severe grazing losses, such as during periods of rapid proliferation of filter feeders, like water fleas (cladocerans). High rates of algal growth demand high nutrient levels. The concentrations of dissolved nutrients during the growing season are usually highest after spring mixing, and therefore, the abundance of Chlamydomonas spp. in temperate lakes often shows a strong peak in spring or summer. In addition, the absence of filter feeders and the presence of more selective feeders in the soil may result in lower grazing losses and less seasonal differences in abundance patterns.

26Predation of C. reinhardtii by zooplankton such as Daphnia, a highly efficient filter feeder, rotifers, and protists such as Tetrahymena or Peranema (Figure 3), has been shown to occur in the laboratory. These predators either live exclusively in the pelagic zone of lakes, or at least more commonly in this habitat compared to soils. In the soil, animals such as earthworms or springtails, and protists are typical predators of microscopic algae, but there is currently little specific information on predators of C. reinhardtii. The formation of large aggregates of C. reinhardtii cells is a general and probably non-specific defense strategy by which the alga may avoid ingestion. For example, the rotifer Brachionus calyciflorus triggers the formation of so-called palmelloid colonies. These colonies are aggregates of C. reinhardtii that may form as a consequence of the failure of the mitotically dividing mother cell to release the daughter cells from its encapsulating cell wall. This phenomenon may be triggered by stress under conditions in which zygospore formation is not possible. On the other hand, C. reinhardtii can actively aggregate in the presence of the predatory protist Peranema trichophorum. A P. trichophorum culture filtrate was able to induce algal aggregation, suggesting that C. reinhardtii senses an unidentified substance (a kairomone) that is released by the predator.

27C. reinhardtii is also a prey for soil bacteria. The bacterium Pseudomonas protegens can surround and immobilize algal cells (Video 1); it secretes a cyclic lipopeptide that triggers an increase in calcium levels inside C. reinhardtii cells with subsequent deciliation. This antagonistic interaction inhibits algal growth and probably leads to the death of most of the algal cells; the bacteria may acquire trace metals from the dying cells. Furthermore, small molecules from C. reinhardtii activate quorum sensing in Pseudomonas aeruginosa. It will be important to determine if algal cells also produce quorum-sensing mimics that influence P. protegens. Finally, while no viral pathogens of C. reinhardtii have been reported, it seems likely that they exist. The areas of algal-bacterial and algal-viral interactions are fertile for more probing basic research.

28Several beneficial interactions of C. reinhardtii have been described, including interactions with growth-promoting bacteria and even mutualism (e. g.;; ). These findings provide the basis for future studies that address regulatory mechanisms and identify specific compounds that impact the biology of C. reinhardtii in nature. One compound synthesized by prokaryotes and used by many algae is vitamin B12. Although C. reinhardtii does not depend on vitamin B12 to grow, it can obtain the compound from bacteria and use it as a cofactor in a pathway for methionine biosynthesis that is thermal tolerant. Indeed, under elevated temperatures, B12-providing bacteria increase the fitness of the alga. A mutualism was also observed between C. reinhardtii and S. cerevisiae in sealed microtiter plates, with the algae trading reduced nitrogen for CO2. While the significance of these interactions may be uncertain, they, and many yet to be discovered, likely shape the ways in which C. reinhardtii navigates in a complex biosphere.

29Although C. reinhardtii has been studied in the laboratory for many decades, we do not know the extent to which results from the laboratory reflect growth, life cycle and behavior of this alga in nature (Box 2). As a model system, C. reinhardtii is almost exclusively grown as a pure culture, a situation almost never encountered in the 'wild'. Returning a laboratory strain of C. reinhardtii to its native habitat would reveal whether domestication caused it to lose its ability to survive within the dynamic fabric of nature. Molecular analyses of the reintroduced strain could also reveal changes in the cells' physiology that underlie the loss of fitness in natural habitats, as well as other changes potentially associated with its adaptation to laboratory conditions.

30Field surveys are often hampered by difficulties in assessing the metabolic state of the cells and in establishing key inter-organismal interactions. However, harnessing the full potential of meta-omics and single-cell technologies could provide a fuller appreciation of the physiological status of cells as they experience environmental fluctuations and the dominant interactions that shape the life of C. reinhardtii. Expanding this understanding will require time-resolved data on the geographical occurrence of C. reinhardtii in different habitats, its genetic potential and population genetics, and dissection of biotic and abiotic interactions. Such studies could then be extended to include analyses performed under controlled laboratory conditions that closely align with conditions encountered in the field, using innovative methods such as microfluidics to mimic conditions of the soil and other complex environments.