Metamorphosis of a unicellular green alga in the presence of acetate and a spatially structured three-dimensional environment

Abstract

Introduction

Microalgae are microscopically small eukaryotic photosynthetic organisms that live in oceans, freshwater, soil, or even ice. They represent a polyphyletic group with diverse evolutionary origin and are involved in a multitude of processes of vast ecological importance. Together with the prokaryotic blue-green algae (cyanobacteria), microalgae contribute to about half of the global carbon fixation (Field et al., 1998; Brodie et al., 2017).

In the past decades, several algal genomes have been sequenced, leading to the development of genetically tractable microalgal models (Grossman, 2007; Brodie et al., 2017; Falciatore et al., 2020). One of the first well-developed algal models is the green microalga Chlamydomonas reinhardtii that was first isolated from a potato field in the USA (Harris, 1989). In its natural habitat, it is typically found in wet soil, coexisting with other microorganisms (Sasso et al., 2018). However, in laboratory settings, it is commonly cultivated axenically in liquid culture. The biciliate C. reinhardtii cell has a cup-shaped chloroplast including a primitive visual system at its edge near the cell equator, called eyespot. Within the chloroplast, there is also a pyrenoid that contains RuBisCO and is surrounded by a starch sheet (Harris, 1989; Sasso et al., 2018). Chlamydomonas reinhardtii reproduces asexually by mitosis in the form of haploid vegetative cells that have mating type plus or minus. Under unfavorable environmental conditions (lack of a nitrogen source), C. reinhardtii initiates a sexual life cycle, allowing sexual crosses (reviewed in Sasso et al., 2018).

Chlamydomonas reinhardtii serves as a model for studying different biological processes, such as photosynthesis and its acclimation, the function, biogenesis and length of cilia or light-driven processes (Grossman, 2000; Petersen et al., 2022; Sakato-Antoku & King, 2022; Ishikawa et al., 2023). The genome of C. reinhardtii has been sequenced (Merchant et al., 2007) and recently a new version from a wild-type strain was released (Craig et al., 2023). Additionally, many molecular tools are available for this alga as well as large-scale mutant libraries (Li et al., 2019; Schroda, 2019; Fauser et al., 2022).

Although there is a pressing demand for easily and rapidly cultivatable model algae in laboratory settings, we must be aware that the growth conditions in the laboratory do not reflect their predominant natural habitat. Some algae, as for example C. reinhardtii, live in soil, a highly complex habitat. A broad biodiversity of soil organisms including microorganisms has recently been highlighted, with estimates that even 59% of species on Earth live in soil (Anthony et al., 2023). Chlamydomonas reinhardtii typically coexists with other microorganisms, which influence its fitness, for example by secreting vitamin B₁₂ or toxins such as orfamide A, a cyclic lipopeptide, or protegencin, a polyyne (Hotter et al., 2021; Bunbury et al., 2022; Hou et al., 2023). Predation, nutrient availability, and water turbulence were even shown to shape transitions from unicellular to multicellular green algae (Cornwallis et al., 2023).

Here, we get a step closer to nature-like conditions for a soil microalga using C. reinhardtii as a model. The genus Chlamydomonas, including the species C. reinhardtii, are repeatedly found in rice paddy field soil (Barrett & Koch, 1982; Ghasemi et al., 2008; Lin et al., 2013) as well as in rice wash water (Szydlowski et al., 2019; Carrasco Flores et al., 2024). Thus, we took rice paddy soil, a natural habitat of C. reinhardtii as an example. We grew the algae in a medium containing acetate, the most abundant organic carbon intermediate in rice soil, within a light–dark cycle (Hori et al., 2007; Shen et al., 2014). Chlamydomonas reinhardtii can use acetate as a carbon source and grows mixotrophically in the presence of light (Harris, 1989). Moreover, we introduced an environment with spatially structured three-dimensional components (S3-D; Fig. 1a) simulating sandy rice soil (Chinachanta et al., 2020). RNA-Seq data reveal strong transcriptional reprogramming in S3-D that coincides with changes at the morphological and molecular level. Also, polymicrobial interactions are altered.

Fig. 1

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The growth of the green microalga Chlamydomonas reinhardtii is enhanced in a spatially structured three-dimensional environment (S3-D) and its transcriptome undergoes major changes in S3-D in the presence of acetate. (a) Setup used for the growth of the microalgal cells in S3-D (see the Materials and Methods section for details). (b–e) The algae were grown in syringe tubes bedded with glass beads of different sizes (+GB) or in pure liquid culture (−GB, see green arrow in (b)). The different colors indicate pure liquid cultures or S3-D cultures with different ratios of small and big beads as indicated. Photographs of C. reinhardtii cultures in S3-D were taken on days 0, 5 and 10 for mixotrophic growth in Tris-Acetate-Phosphate (TAP) medium (b) and on days 0, 8 and 18 for photoautotrophic growth in minimal medium without acetate (d). Brown arrows indicate the accumulation of algal cells at the top of the syringe tubes in (d). (c, e) Mixotrophic (c) and photoautotrophic (e) growth of C. reinhardtii is significantly enhanced in S3-D compared to pure liquid culture (indicated by the green lines). Error bars indicate SDs based on three independent biological replicates per condition. The statistical significance analysis is depicted in Supporting Information Table S1. (f, g) Principal component analysis (PCA) (f) and Volcano plot (g) of samples taken from liquid and S3-D (mixotrophic growth) using only small beads. (f) PCA; dots represent three independent biological replicates R1, R2 and R3; control samples are indicated in green color and samples in S3-D in magenta color. (g) Volcano plot showing the relative amounts of nearly 18 000 transcripts identified by RNA-Seq from three independent biological replicates after algal growth in liquid culture compared to S3-D. Genes that have a |log₂ (fold change)| ≥ 1 and an adjusted P-value of ≤ 0.01, are considered significant. The orange spots represent significantly upregulated, and the blue spots significantly downregulated transcripts in S3-D. (h, i) Changes in transcript abundances isolated from cells grown mixotrophically in pure liquid (−GB) vs cells grown in S3-D (only small or only big beads or 2 : 2 small : big beads). Error bars indicate SDs. Asterisks indicate significant differences as calculated by Student's t-test: ***, P < 0.001; ****, P < 0.0001. (h) Transcript encoding the ciliary ammonium transporter AMT1D. (i) Transcript encoding the ciliary ion channel KCN6.