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. 2018 May 29;9:691.
doi: 10.3389/fpls.2018.00691. eCollection 2018.

Flagella-Associated WDR-Containing Protein CrFAP89 Regulates Growth and Lipid Accumulation in Chlamydomonas reinhardtii

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Free PMC article

Flagella-Associated WDR-Containing Protein CrFAP89 Regulates Growth and Lipid Accumulation in Chlamydomonas reinhardtii

Qiulan Luo et al. Front Plant Sci. .
Free PMC article

Abstract

WD40-repeat (WDR) domain-containing proteins are subunits of multi-protein E3 ligase complexes regulating various cellular and developmental activities in eukaryotes. Chlamydomonas reinhardtii serves as a model organism to study lipid metabolism in microalgae. Under nutrition deficient conditions, C. reinhardtii accumulates lipids for survival. The proteins in C. reinhardtii flagella have diverse functions, such as controlling the motility and cell cycle, and environment sensing. Here, we characterized the function of CrFAP89, a flagella-associated WDR-containing protein, which was identified from C. reinhardtii nitrogen deficiency transcriptome analysis. Quantitative real time-PCR showed that the transcription levels of CrFAP89 were significantly enhanced upon nutrient deprivation, including nitrogen, sulfur, or iron starvation, which is considered an effective condition to promote triacylglycerol (TAG) accumulation in microalgae. Under sulfur starvation, the expression of CrFAP89 was 32.2-fold higher than the control. Furthermore, two lines of RNAi mutants of CrFAP89 were generated by transformation, with gene silencing of 24.9 and 16.4%, respectively. Inhibiting the expression of the CrFAP89 gene drastically increased cell density by 112-125% and resulted in larger cells, that more tolerant to nutrition starvation. However, the content of neutral lipids declined by 12.8-19.6%. The fatty acid content in the transgenic algae decreased by 12.4 and 13.3%, mostly decreasing the content of C16:0, C16:4, C18, and C20:1 fatty acids, while the C16:1 fatty acid in the CrFAP89 RNAi lines increased by 238.5 to 318.5%. Suppressed expression of TAG biosynthesis-related genes, such as CrDGAT1 and CrDGTTs, were detected in CrFAP89 gene silencing cells, with a reduction of 16-78%. Overall our results suggest that down-regulating of the expression of CrFAP89 in C. reinhardtii, resulting in an increase of cell growth and a decrease of fatty acid synthesis with the most significant decrease occurring in C16:0, C16:4, C18, and C20:1 fatty acid. CrFAP89 might be a regulator for lipid accumulation in C. reinhardtii.

Keywords: E3 ubiquitin ligase; RNA interference silencing; WDR proteins; lipid metabolism; triacylglycerol.

Figures

FIGURE 1
FIGURE 1
CrFAP89 was a novel WD40 repeat-containing protein. (A) Gene structure of the CrFAP89. The blue rectangle indicates the 5′- or 3′-UTR regions, the green boxes show exons, and the lines show the introns. (B) The WDR domain is a conserved sequence at the C-terminus of CrFAP89 shown as triangles, and magenta boxes show unnamed domains with low complexity. (C) Neighbor joining-phylogenetic unrooted tree of CrFAP89 constructed by MEGA7. (D) Predicted 3D protein structure of CrFAP89 using Phyre2 software. The magenta and blue lines show the α-helix and β-sheet, respectively, β1 to β7 denote the interaction hubs composed of the seven β-sheets. The yellow arrow points to one view of the second interaction hub.
FIGURE 2
FIGURE 2
The expression patterns of CrFAP89 under deprivation of nitrogen, sulfur, and iron. The different letters of each column indicates that they were significantly different at P ≤ 0.05, ∗∗P ≤ 0.01 according to one-way ANOVA.
FIGURE 3
FIGURE 3
The growth of CrFAP89-RNAi algal lines. (A) RNA-mediated interference of CrFAP89 in CrFAP89i-6, -61 compared with the control transformed with an empty vector (Maa7-34, 66) and wild-type (cc425), confirmed using real-time PCR. The different letters of each column indicate that they were significantly different at P ≤ 0.05, ∗∗P ≤ 0.01. (B) The growth curves and (C) liquid cultures of the CrFAP89-RNAi cell lines and the control transformed with an empty vector grown in HSM medium. The different letters of each lines indicate they were significantly different at P ≤ 0.05. (D) Growth of CrFAP89-RNAi and control lines under nutrition starvation conditions. Growth in HSM served as a control, while HSM with nutrition element deficiency, including low sulfur (–S), nitrogen (–N), and iron (–Fe), were used to induce stress. The numbers listed along the left indicate the initial inoculum cell density.
FIGURE 4
FIGURE 4
Lipid accumulation in CrFAP89-RNAi lines and control. (A) Lipid content in CrFAP89-RNAi lines and the control transformed with an empty vector grown in HSM medium. The different letters of each line indicate that they were significantly different at P ≤ 0.05. (B) Fluorescence microscopy images of CrFAP89-RNAi cells and control cells grown for 9 days in HSM and (C) after N-deprivation for 4 days to visualize TAG by Nile Red staining. Red indicates chlorophyll autofluorescence, and yellow indicates the lipid bodies. (D) Expression analysis of genes encoding key enzymes in lipid biosynthesis in CrFAP89-RNAi lines and control. The different letters of each column indicates they were significantly different at P ≤ 0.05, ∗∗P ≤ 0.01 according to one-way ANOVA.

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