Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii

Abstract

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%-17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%-21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.

Fig. 1

Phylogenetic analysis of the lipid phosphate phosphatase protein family C. reinhardtii CrPAP2, Arabidopsis AtLpp1p, AtLpp2p, and AtLpp3p proteins, S. cerevisiae Dpp1p and Lpp1p proteins, all available mammalian lipid phosphate phosphatase proteins, Drosophila Wunen protein, as well as uncharacterized putative lipid phosphate phosphatase-like proteins identified in A. thaliana, D. melanogaster, C. elegans, and Schizosaccharomyces pombe genomes, were used for comparison. The tree was built using the neighbor joining method, wherein point accepted mutation (PAM) distances were computed based on reliably aligned sites. The SwissProt accession numbers (in brackets) designate all protein sequences. The length of horizontal branches is such that the evolutionary distance between two proteins is proportional to the total length of the horizontal branches that connect them. Bootstrap values are shown at the nodes. H.s. Lpp1p, Lpp2p, and Lpp3p: Homo sapiens Lpp1p, Lpp2p, and Lpp3p; S.c.Dpp1p: Saccharomyce scerevisiae Dpp1p; S.p.Dpp1p: Schizosaccharomyces pombe Dpp1p; R.n. Lpp1p and Lpp3p: Rattus norvegicus Lpp1p and Lpp3p; M.m. Lpp1p and Lpp2p: Mus musculus Lpp1p and Lpp2p; D.m.: D. melanogaster lipid phosphate phosphatase; C.e.: C. elegans lipid phosphate phosphatase; C.p.Lpp1p: Cavia porcellus Lpp1p

Fig. 2

Biomass (a), lipid content (b), and mRNA levels (c) of CrPAP2 in HSM and HSM-N medium The mRNA levels of C. reinhardtii CC425 samples grown in the indicated medium for 1, 2, 3, or 4 d, were analyzed using RT-PCR. +N: cells cultivated in N-sufficient HSM medium; −N: cells cultivated in N-free HSM medium. Data are expressed as mean±SD (n=3)

Fig. 3

Biomass (a), lipid content (b), and mRNA levels (c) of CrPAP2 RNAi transgenic algae strains Maa7-4 (10, 19), pMaa7IR/XIR transgenic algae strains; PAP2-RNAi-3 (10, 56), pMaa7IR/CrPAP2 IR transgenic algae strains. Data are expressed as mean±SD (n=3)

Fig. 4

Microscopic observation of CrPAP2 transgenic algae strains (250× Nikio 80i) Above: bright field, for cell morphology; Below: dark field, for oil droplets. After 4 d of cultivation in HSM medium, fewer oil droplets of CrPAP2 RNAi transgenic algae were found. Maa7-19, pMaa7IR/XIR transgenic algae strain number 19; PAP2-RNAi-56, pMaa7IR/CrPAP2 IR transgenic algae strain number 56

Fig. 5

Biomass (a), lipid content (b), mRNA level (c), and PAP activity (d) assays of CrPAP2 over-expression transgenic algae in HSM medium pCAMBIA-2 (8, 16), pCAMBIA1302 transgenic algae strains; pCAMBIA-PAP2-4 (26, 60), pCAMPAP2 transgenic algae strains. Data are expressed as mean±SD (n=3)

Fig. 6

Lipid content in a transgenic algae line detected by Nile Red staining (250× Nikio 80i) Above: bright field, for cell morphology; Below: dark field, for oil droplets. After 4 d of cultivation in HSM medium, more oil droplets of CrPAP2 transgenic algae were found. pCAMBIA-16, pMCAMBIA1302 transgenic algae strain number 16; pCAMBIA-PAP2-60, pCAMPAP2 transgenic algae strain number 60

Fig. 7

Expression of CrPAP2 in E. coli BL21 and in vitro enzyme activity assay After induction by IPTG and cultivation for 0, 2, or 4 h, total protein was harvested and run on SDS-PAGE. (a) Expression of CrPAP2 in E. coli BL21; (b) The purified GST-CrPAP2; (c) In vitro enzyme activity assay of GST-CrPAP2. GST, E. coli BL21 transformed with pGEX-6p-1; GST-CrPAP2, E. coli BL21 transformed with plasmid pGEX-6p-1-PAP2 to express the fusion protein GST-CrPAP2. The induction time of the cultured cells is indicated by 0, 2, 4, and 6 h. The GST and GST-CrPAP2 fusion proteins are indicated by the arrow