Palmelloid Formation and Cell Aggregation Are Essential Mechanisms for High Light Tolerance in a Natural Strain of Chlamydomonas reinhardtii

Abstract

Photosynthetic organisms, such as higher plants and algae, require light to survive. However, an excessive amount of light can be harmful due to the production of reactive oxygen species (ROS), which cause cell damage and, if it is not effectively regulated, cell death. The study of plants' responses to light can aid in the development of methods to improve plants' growth and productivity. Due to the multicellular nature of plants, there may be variations in the results based on plant age and tissue type. Chlamydomonas reinhardtii, a unicellular green alga, has also been used as a model organism to study photosynthesis and photoprotection. Nonetheless, the majority of the research has been conducted with strains that have been consistently utilized in laboratories and originated from the same source. Despite the availability of many field isolates of this species, very few studies have compared the light responses of field isolates. This study examined the responses of two field isolates of Chlamydomonas to high light stress. The light-tolerant strain, CC-4414, managed reactive oxygen species (ROS) slightly better than the sensitive strain, CC-2344, did. The proteomic data of cells subjected to high light revealed cellular modifications of the light-tolerant strain toward membrane proteins. The morphology of cells under light stress revealed that this strain utilized the formation of palmelloid structures and cell aggregation to shield cells from excessive light. As indicated by proteome data, morphological modifications occur simultaneously with the increase in protein degradation and autophagy. By protecting cells from stress, cells are able to continue to upregulate ROS management mechanisms and prevent cell death. This is the first report of palmelloid formation in Chlamydomonas under high light stress.

Keywords: Chlamydomonas reinhardtii; aggregation; high light; natural variation; palmelloid; protein.

Figure 1

Growth of two Chlamydomonas strains exposed to high light stress. (A) Cells were diluted to a density of 2 × 10⁶ cells mL⁻¹ and subsequently subjected to 5-fold serial dilutions. Cells were then spotted on TAP medium plates and cultivated under 50 and 1500 µmol photons m⁻² s⁻¹. Photos were taken after one week. (B) Cells were diluted to a density of 2 × 10⁶ cells mL⁻¹ in liquid TAP medium and exposed to high light of 1500 µmol photons m⁻² s⁻¹ over a period of 5 days.

Figure 2

Growth and maximum quantum efficiency of PSII (F_v/F_m) of Chlamydomonas. Cells were grown under (A) low light at 50 µmol photons m⁻² s⁻¹ and (B) high light at 1500 µmol photons m⁻² s⁻¹. The cell density of each strain was assessed to compare the growth of the two strains over a period of 5 days. The F_v/F_m values of the high-light-tolerant strain (CC-4414) and the high-light-sensitive stain (CC-2344) were measured under low light (C) and high light (D). All data are means ± SD (n = 3). Significant differences between the two strains on the same day are indicated with asterisks (*) (p < 0.05).

Figure 3

Pigment contents and composition of Chlamydomonas under low light. Photosynthetic pigments were measured from cells grown under high light at 1500 µmol photons m⁻² s⁻¹ and reported as (A) chlorophyll a, (B) chlorophyll b, (C) carotenoids, (D) total chlorophyll, (E) chlorophyll a/b ratio, and (F) carotenoid/chlorophyll ratio. All data are means ± SD (n = 3). Significant differences between the two strains on the same day are indicated with asterisks (*) (p < 0.05).

Figure 4

Pigment contents and composition of Chlamydomonas under high light treatment. Photosynthetic pigments were measured from cells grown under high light at 1500 µmol photons m⁻² s⁻¹ and reported as (A) chlorophyll a, (B) chlorophyll b, (C) carotenoids, (D) total chlorophyll, (E) chlorophyll a/b ratio, and (F) carotenoid/chlorophyll ratio. All data are means ± SD (n = 3). Significant differences between the two strains on the same day are indicated with asterisks (*) (p < 0.05).

Figure 5

Growth of Chlamydomonas under oxidative stress. Cells were diluted to a density of 2 × 10⁶ cells mL⁻¹ and subjected to 5-fold serial dilutions. Cells were spotted on TAP, TAP with Rose Bengal (RB), and TAP with hydrogen peroxide (H₂O₂). Plates were incubated under 50 µmol photons m⁻² s⁻¹, and photos were taken after 5 days.

Figure 6

Chlamydomonas responses under oxidative stress. Cells were grown under high light at 1500 µmol photons m⁻² s⁻¹. (A) Lipid peroxidation was measured by the thiobarbituric acid reacting substance (TBARS) method. Oxidative stress was evaluated by detecting malondialdehyde (MDA) levels in cells. (B) The activity of superoxide dismutase (SOD) was measured to evaluate ROS scavenging activities. (C) Non-photochemical quenching (NPQ) of Chlamydomonas. All data are means ± SD (n = 3). Significant differences between the two strains are indicated with asterisks (*), while significant differences between each day and day 0 within the same strain are indicated with plus signs (+) (p < 0.05).

Figure 7

Venn diagrams showing proteins differentially expressed under low light of 50 µmol photons m⁻² s⁻¹ and two days after exposure to high light of 1500 µmol photons m⁻² s⁻¹. Comparison between low light and high light for CC-4414 (A). Comparison between low light and high light for CC-2344 (B). Comparison of proteins exclusively expressed in high light of CC-4414 (237 proteins) and CC-2344 (20 proteins) (C).

Figure 8

Gene Ontology (GO) enrichment analysis of proteins exclusively expressed in CC-4414 exposed to high light of 1500 µmol photons m⁻² s⁻¹ for 2 days. The GO terms are categorized as a biological process (top), cellular component (middle), or molecular function (bottom). The number of proteins for each GO term is indicated at the end of each bar graph.

Figure 9

Predicted interaction network of significant proteins exclusively found in Chlamydomonas CC-4414 under high light treatment. STRING software was used to obtain a network of protein interactions.

Figure 10

Morphological changes in Chlamydomonas were monitored via microscopy. (A) Chlamydomonas cells were cultured in TAP medium and placed under low light of 50 µmol photons m⁻² s⁻¹ and high light of 1000 and 1500 µmol photons m⁻² s⁻¹ for 2 days. Black arrows indicate examples of palmelloid structures in CC-4414. (B) A close-up image of palmelloid cells. (C) Percentage of palmelloid cells. Significant differences between the two strains are indicated with asterisks (*), while significant differences between each high light intensity and low light within the same strain are indicated with plus signs (+) (p < 0.05).