The pgp1 mutant locus of Arabidopsis encodes a phosphatidylglycerolphosphate synthase with impaired activity

Abstract

Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants. Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation. In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol. Details on the plant-specific pathways in different organelles are scarce. Here, we describe a phosphatidylglycerol biosynthesis-deficient mutant of Arabidopsis, pgp1. The overall content of phosphatidylglycerol is reduced by 30%. This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (EC 2.7.8.5) isoform encoded by a gene on chromosome 2. The mutant shows an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoform. Mutant plants are pale green, and their photosynthesis is impaired. This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants.

Figure 1

Growth and morphology of Arabidopsis wild type and pgp1 mutant. A, Six-week-old plants grown for 10 d on agar-solidified Murashige and Skoog medium with 1% (w/v) Suc followed by photoautotrophic growth on soil. B, Three-week-old plants grown on agar-solidified Murashige and Skoog medium with 1% (w/v) Suc. C, Three-week-old plants grown on soil. Note the yellow tissues in the center of the mutant rosette.

Figure 2

Lipid phenotype of pgp1 and molecular defect. Sections of a lipid chromatogram (top panel) and a DNA gel of PCR products (bottom panel) of wild type, the pgp1 mutant, and two independent transgenic pgp1 lines transformed with the PGP1 wild-type cDNA (pgp1/cPGP1) are shown. Lipids were analyzed by TLC and stained by exposure to iodine. Monogalactosyldiacylglycerol (MGDG), serving as loading control, and PG are shown. B, Restriction length polymorphism in the pgp1 mutant locus. Sequence comparison of the wild-type (PGP1) and mutant (pgp1) locus showing the mutated BamHI site (top). The nucleotide number refers to the GenBank accession number of the BAC clone T16B24 (accession no. AC004697). Genomic Southern blot of the wild type and the pgp1 mutant probed with the PGP1 gene (bottom). Restriction digests are indicated. Numbers indicate the size (in kilobases) of the fragments marked by arrows.

Figure 3

The PGP1 locus on chromosome 2 of Arabidopsis. A, Genetic map showing SSLP markers (positions on map), experimentally determined map distances, and recombinant chromosomes/total chromosomes analyzed for each marker. B, BACs from the genome sequencing project and markers. C, Structure of the PGP1 gene (top) and cDNA (bottom). The gray box represents the exon carrying the mutation in pgp1. A, Poly(A) tail; ATG, start codon; TAG, stop codon; TP, transit peptide.

Figure 4

Active site mutation of PGP synthase in the pgp1 mutant. A, Alignment of different Arabidopsis PGP1 paralogs (PGP2 and PGP3) and bacterial PGP synthase orthologs. GenBank accession numbers (Arabidopsis gene numbers) for the respective protein sequences are: Arab-PGP1, AAC28995 (At2g3920); Arab-PGP2, CAB82698 (At3g55030); Arab-PGP3, CAB80852 (At4g04870); E. coli, P06978; Rhodobacter sphaeroides, AAC44003; Bacillus subtilis, I39950; and Synechocystis sp., S76208. The arrow indicates the Pro (Pro-170) to Ser change in the pgp1 mutant. B, Reduced specific activity of the PGP1-Ser-170 mutant protein. Total activity in extracts of the E. coli YA5512 PGP synthase-deficient mutant expressing wild-type PGP1 cDNA (squares) or mutant pgp1 cDNA (circles) was determined. The two graphs represent single representative experiments. Equal amounts of protein were used. In addition, the mean specific activity (±se) for three independent PCR constructs is provided. The PGP synthase activities in YA5512 and YA5512 containing pQE32 were not detectable.

Figure 5

Chloroplastic PGP synthase and galactolipid biosynthetic activities in wild type and pgp1 mutant. Isolated and ruptured chloroplasts were either incubated with labeled glycerol-3-phosphate (Gro-3-P) and CDP-diacylglycerol (PGP synthase assay, left two lanes) or with labeled UDP-Gal (galactolipid biosynthesis, right two lanes). An autoradiograph of a thin layer chromatogram of labeled lipid extracts is shown. Identified lipids were digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglycerol (MGDG), phosphatidylglycerol (PG), and phosphatidylglycerolphosphate (PGP).