Chlamydomonas reinhardtii Alternates Peroxisomal Contents in Response to Trophic Conditions

Abstract

Chlamydomonas reinhardtii is a model green microalga capable of heterotrophic growth on acetic acid but not fatty acids, despite containing a full complement of genes for β-oxidation. Recent reports indicate that the alga preferentially sequesters, rather than breaks down, lipid acyl chains as a means to rebuild its membranes rapidly. Here, we assemble a list of potential Chlamydomonas peroxins (PEXs) required for peroxisomal biogenesis to suggest that C. reinhardtii has a complete set of peroxisome biogenesis factors. To determine involvements of the peroxisomes in the metabolism of exogenously added fatty acids, we examined transgenic C. reinhardtii expressing fluorescent proteins fused to N- or C-terminal peptide of peroxisomal proteins, concomitantly with fluorescently labeled palmitic acid under different trophic conditions. We used confocal microscopy to track the populations of the peroxisomes in illuminated and dark conditions, with and without acetic acid as a carbon source. In the cells, four major populations of compartments were identified, containing: (1) a glyoxylate cycle enzyme marker and a protein containing peroxisomal targeting signal 1 (PTS1) tripeptide but lacking the fatty acid marker, (2) the fatty acid marker alone, (3) the glyoxylate cycle enzyme marker alone, and (4) the PTS1 marker alone. Less than 5% of the compartments contained both fatty acid and peroxisomal markers. Statistical analysis on optically sectioned images found that C. reinhardtii simultaneously carries diverse populations of the peroxisomes in the cell and modulates peroxisomal contents based on light conditions. On the other hand, the ratio of the compartment containing both fatty acid and peroxisomal markers did not change significantly regardless of the culture conditions. The result indicates that β-oxidation may be only a minor occurrence in the peroxisomal population in C. reinhardtii, which supports the idea that lipid biosynthesis and not β-oxidation is the primary metabolic preference of fatty acids in the alga.

Keywords: Chlamydomonas; PEX; PTS1; fatty acids; fluorescence microscopy; glyoxylate cycle enzymes; microalgae; peroxisomes; β-oxidation.

Figure 1

C. reinhardtii is not able to grow on exogenous fatty acids. (A) C. reinhardtii culture cell density after 3 d following transfer from TAP medium to minimal medium (MM), TAP, or MM with the addition of butyric acid (C4, BTA) in Tween 80 (FA dispersing agent, Tween), valeric acid (C5, Val A) in Tween 80, or Tween 80 alone. About 5 × 10⁶ cells of strain CC124 or CC4425 were transferred to each new medium for each experiment. The graph shows averages ± standard error mean of three independent experiments. (B) Growth of C. reinhardtii and C. vulgaris cultured on agar plates without light. C. reinhardtii CC124, CC5082, and C. vulgaris (UTEX#395) were cultured for 4 d in TAP, then streaked on agar plates containing either acetic acid (C2 FA, left) or palmitic acid (C16 FA, right) and placed in the dark for 20 d. Tween is a dispersant for palmitic acid but did not affect growth with the acetic acid present. Growth was not observed when C16 FA was used as a sole carbon source in the dark (right Petri plate). (C) Growth of Chlamydomonas CC5082 cultured with different carbon sources. CC5082 was cultured for 14 days in liquid MM that contained Tween and other carbon sources, such as acetic acid, palmitic acid, and glucose. The cells were cultured with or without light. Notice that the cells proliferate in the dark only when acetic acid is in the medium.

Figure 2

Co-localization of FL-C16 and CFP-SLI (PTS1 containing peroxisomal targeting protein) was identified in C. reinhardtii. Accumulations of FL-C16 and peroxisomal marker proteins after the cells were cultured in TAP for 24 h in illuminated conditions. Thirty optical sections of each 20 µm z-stack are projected into a plane image with maximum intensity. CFP-SLI: CFP fused to the PTS1 signal peptide serin-leucine-isoleucine. CIS2-RFP: Twenty-five N-terminal amino acid sequences of glyoxylate cycle enzyme citrate synthase 2 (CIS2) fused to RFP. CIS2-CFP: CIS2 fused to CFP. A compartment that contains CIS2-RFP and CFP-SLI but not FL-C16 is shown with a white circle. A pair of compartments that contain FL-C16 and CFP-SLI but not CIS2-RFP are shown with an arrowhead (upper panel). Notice that CIS2-CFP and CIS2-RFP are all colocalized (lower panel), suggesting that non-colocalization between CFP-SLI and CIS2-RFP (marked with the arrowhead) is not an artifact by protein expression or imaging acquisition.

Figure 3

Statistical analysis revealed alteration of peroxisomal content by light conditions. (A) Schematic of fluorescently labeled palmitic acid (FL-C16) pulse experiment. C. reinhardtii expressing both CFP-SLI and CIS2-RFP was cultured in TAP with FL-C16 for 8 h, then transferred to a fresh medium under four different conditions for 24 h before imaging: TAP with continuous light, minimal medium (without acetate, MM) with continuous light, TAP in the dark, and MM in the dark. (B) Localization of FL-C16 and peroxisomal marker proteins after 24 h in each condition. Thirty optical sections of each 20 µm z-stack are projected into one image with their maximum intensity. Co-localizations of all three markers are rarely observed, indicated by white arrows. (C,D) Thirty optical sections within the 20 µm z-axis were obtained by confocal microscopy. Colocalization in the optical sections was analyzed with ComDet (ImageJ plugin for analyzing colocalization of bright intensity spots). Mean of the spot number, standard error of the mean, numbers of cells, and optical sections for the analysis are, TAP with continuous light (TAP Light: mean 9.7, S.E. 0.47, 8 cells, 20 sections), MM with continuous light (MM Light: mean 5.75, S.E. 0.46, 7 cells, 32 sections), TAP in the dark (TAP Dark: mean 12.1, S.E. 2.78, 3 cells, 6 sections), and MM in the dark (MM Dark: mean 6.94, S.E. 0.76, 5 cells, 19 sections). (C) Compartments detected in an optical section are reduced when the cells are cultured without acetic acid. A total number of compartments in an optical section detected by ComDet is presented as a box plot with individual data points. Notice that the number of compartments is reduced when the cells are cultured without acetic acid (MM) regardless of light conditions. A letter on the top of each bar indicates mean comparisons for each pair using Student’s t-test. Levels not connected by the same letters are significantly different (p < 0.05). (D) Compartments change contents depending on culture conditions. Colocalization rates in the compartments are calculated, based on the culture conditions (TAP Light: n = 194, TAP Dark: n = 105, MM Light: n = 73, MM Dark, n = 132).