Two closely related genes of Arabidopsis encode plastidial cytidinediphosphate diacylglycerol synthases essential for photoautotrophic growth

Abstract

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth.

Figure 1.

Overview of the predicted CDS proteins encoded by the five genes of Arabidopsis. The transmembrane domains are shown as boxes and based on consensus prediction (Arai et al., 2004).The bend in the structure of CDS4.2 indicates a deletion of nine amino acids due to alternative splicing.

Figure 2.

Functional complementation of Saccharomyces cerevisiae mutant YBR029c lacking CDP-DAG synthase activity by expression of Arabidopsis CDS cDNAs under control of the PGAL promoter. Yeast suspensions of wild-type (WT) and mutant strain containing one of the given Arabidopsis CDS cDNAs were stepwise diluted 10 times and spotted on 2% Gal (A) and 2% Glc (B) and incubated at 28°C for 48 h. cDNAs are termed according to the proteins they encode (see Fig. 1). Unlike wild-type cells, mutant cells were only able to grow when expression of the Arabidopsis gene constructs was induced by Gal.

Figure 3.

Activities and subcellular localization of Arabidopsis CDS isoforms expressed in the yeast mutant strain YBR029c. Incorporation rates of [³H]CTP into CDP-DAG by mitochondrial and microsomal fractions of yeast cells harboring the given CDS isoform are depicted as mean values and sds of three independent preparations.

Figure 4.

Subcellular localization of Arabidopsis CDS isozymes expressed as fusion proteins with RFP or GFP in tobacco BY2 cells. A, BY2 cell that expresses CDS4.1-RFP fusion protein was counterstained with Mitotracker green. The RFP signal is shown. B, Same cell as in A, but the Mitotracker green signal is shown. C, Merge of images A and B. D, RFP signal of a BY2 cell expressing CDS5-GFP fusion protein as a positive control because CDS5 has been shown to be located in the chloroplast envelope. E, Enlargement of part of A. F, RFP signal of a BY2 cell expressing CDS4.2-RFP fusion protein. G, GFP signal of a BY2 cell expressing CDS5-GFP fusion protein as positive control. H, GFP signal of plastids within a BY2 cell expressing CDS5-GFP fusion protein. [See online article for color version of this figure.]

Figure 5.

Certain properties of CDS4.1 and CDS5 heterologously expressed in yeast. A, Enzymic activity of CDS4.1 (white circles) and CDS5 (black circles) in dependence on CTP concentrations. B and C, Enzymic activities of CDS4.1 (B) and CDS5 (C) as a function of the concentrations of three different PA species (triangles, dioleoyl; squares, dipalmitoyl species; diamonds, species from egg lecithin).

Figure 6.

Analysis of the cds4 and cds5 T-DNA insertion alleles. A, Positions of the T-DNA insertions are indicated relative to the exon-intron structure of CDS genes. Exons are indicated with black bars and introns with thin lines. Primers used for the analysis of the locus (LP and RP) and the respective transcripts (fw and rev) are indicated by arrows. The T-DNAs are shown as white boxes with LB designating the left border. B, Expression of CDS genes in cds4 and cds5 mutants in comparison to the wild type (WT). CDS transcripts were determined by RT-PCR. The lower of the two bands amplified with the CDS4-specific primers is due to the CDS4.2 transcript having a short deletion of 27 nucleotides at the 5′ region of exon 2.

Figure 7.

Phenotype of Arabidopsis cds4 cds5 mutants. A, Two-week-old seedlings segregating in a progeny of heterozygous mutant plants were cultivated on agar-solidified MSG medium with 3% (w/v) Suc. Homozygous double mutant seedlings are easily recognized by their pale greenish-yellow color and their retarded growth. B, Close-up of a wild-type (left) and mutant seedling (right) shown in A. C to F, Plants grown for 2 weeks on agar with Suc followed by 2 weeks of growth on earth. C, The cds4 cds5 mutant. D, The cds4 cds5 mutant expressing a chimeric CDS4 construct under the control of the 35S promoter that encodes CDS4.1 as a GFP fusion protein. E, The cds4 cds5 mutant expressing the open reading frame of CDS5 under the control of the 35S promoter. F, A wild-type plant. [See online article for color version of this figure.]

Figure 8.

Ultrastructure of chloroplasts of cds4 cds5 mutant (A, C, and E) and wild-type plants (B, D, and F). A and B, Chloroplast of mesophyll cells. C and D, Thylakoid membranes of the chloroplast. E and F, Mitochondrium and part of the chloroplast.

Figure 9.

Membrane lipid composition of wild-type (white bars) and cds4 cds5 (gray bars) mutant plants. After a 5-week cultivation on agar-solidified MSG medium with Suc, lipids were extracted from the plants, separated by thin-layer chromatography, and quantified by gas-liquid chromatography of fatty acid methyl esters derived from the individual glycerolipods. Three replicates were analyzed and sds are indicated. PC, Phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; DGD, digalactosyldiacylglycerol; MGD, monogalactosyldiacylglycerol.

Figure 10.

In vivo [³³P]phosphate labeling pattern of phosphoglycerolipids of the cds4 cds5 mutant and the wild type. After an 18-h incubation with labeled phosphate, lipids were extracted from the mutant (lanes A and C) and wild-type plants (B and D) and separated by thin-layer chromatography, and radioactively labeled lipids were visualized with a Bioimager and identified by cochromatography with authentic lipids (PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine).