The green microalga Chlamydomonas reinhardtii has a single ω-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids

Abstract

The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids.

Figure 1.

Total fatty acid composition of the mutant 1C11 (crfad7) and the wild type (WT). Values are shown as mol % of total fatty acids and are means of three biological replicates. Error bars denote 95% confidence intervals. Fatty acids are represented by total carbon numbers:total numbers of unsaturations (position of unsaturations counted from the carboxyl end).

Figure 2.

Quantification of major membrane lipid classes of the crfad7 mutant. A, Content of major polar membrane lipids. B, Fatty acid composition of MGDG in the wild type (WT) and the mutant crfad7. C, Fatty acid composition of PtdEtn in the wild type and the mutant crfad7. Strains were grown under standard conditions. Values represent averages of three biological replicates. Error bars denote 95% confidence intervals.

Figure 3.

Structural features of CrFAD7. The presence of three conserved His boxes, a typical feature of membrane-bound desaturases, is underlined. The predicted chloroplast transit peptide (cTP) is boxed in the N terminus.

Figure 4.

Molecular genetic analyses of the mutant crfad7. A, PCR amplification of the Cre01.g038600 sequence from genomic DNA prepared either from the wild type (WT) or the crfad7 mutant. B, Insertion of the transposon TOC1 113 bp upstream of the ATG start codon of Cre01.g038600 in the mutant crfad7 revealed after DNA sequencing of the PCR product. C, Quantitative RT-PCR analyses of the transcript level of CrFAD7 in the wild type and the mutant crfad7. Expression levels of CrFAD7 were normalized to the housekeeping gene RACK1 and compared with the level in the wild type (set to 1). Error bars represent sd based on three biological replicates, each biological replicate consisting of three technical replicates.

Figure 5.

Complementation of crfad7 by nuclear expression of CrFAD7. Fatty acid composition analyses for three independent nuclear complementation lines are shown. Values are means of three biological replicates, and error bars represent 95% confidence intervals. WT, Wild type.

Figure 6.

Subcellular localization of CrFAD7 by immunofluorescence analysis. A, CrFAD7 protein is fused to 3xHA in its C terminus. B, Western-blot analysis of total cellular protein, soluble fraction, and membrane fraction using the anti-HA antibodies. Proteins extracted from one representative complemented line are shown. C, Confocal images of the crfad7 mutant when complemented by CrFAD7-HA. Cells were excited at 488 nm, and emission was collected at 498 to 515 nm for FITC and at 656 to 714 nm for chlorophyll. [See online article for color version of this figure.]

Figure 7.

Analysis of lipid molecular species of the wild type, crfad7, and the nuclear and plastidial complemented lines. Relative abundance for each lipid molecular species is shown. (The complete data set is also presented in Supplemental Table S1.) It is calculated as an area percentage of all molecular species present in that particular lipid class. Values are means of three biological replicates and two technical replicates, and error bars represent 95% confidence intervals. crfad7:cpCrFAD7, plastidial complemented lines; crfad7:nuCrFAD7, nuclear complemented lines. Note that the stereospecific position of each acyl group on the glycerol backbone could not be assigned.

Figure 8.

Regulation of ω-3 fatty acid synthesis by light and temperature in wild-type C. reinhardtii. A, Quantitative RT-PCR analyses of CrFAD7 transcript for wild-type cells when cultivated in the light or under dark. B, Fatty acid compositional analysis of the cells harvested from light- or dark-grown cultures. C, Quantitative RT-PCR analyses of CrFAD7 transcript for wild-type cells when cultivated at 15°C, 25°C, and 35°C. D, Fatty acid compositional analysis of the cells after being subjected to 15°C, 25°C, and 35°C for 48 h. Wild-type cells were cultivated in TAP medium at 25°C until midlog phase, then they were either subjected to changes in luminosity (in the dark or under a light intensity of 100 µmol photons m⁻² s⁻¹) or transferred to two other temperatures (15°C or 35°C) for 48 h before being sampled for analyses. Error bars represent sd based on three biological replicates. Transcription level was calculated the same way as described in Figure 4C.

Figure 9.

Low ω-3 fatty acid content causes PSII to be more tolerant to high temperature. The effect of short exposure to high temperature (45°C) on the photosynthetic efficiencies of the wild type (WT) and the crfad7 mutant as measured by the chlorophyll fluorescence parameter F_v/F_m after 0, 30, and 60 min of growth at 45°C is shown. Error bars represent sd based on three biological replicates.

Figure 10.

Possible models involving the plastidial CrFAD7 for the desaturation of ω-3 fatty acids present in extraplastidic membrane lipids. A, CrFAD7 acts on the plastid envelope at plastid-ER contact sites (red dashed area). B, The ω-3 fatty acids are synthesized in the plastid, clipped off the plastidic lipids through a specific lipase(s), and exported out into the ER as acyl-CoAs via the classical route for 16:0/18:0/18:1. ACP, Acyl carrier protein; IM, inner plastid envelope; OM, outer plastid envelope. [See online article for color version of this figure.]