Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth

Abstract

The sulfolipid sulfoquinovosyldiacylglycerol is one of the three nonphosphorous glycolipids that provide the bulk of the structural lipids in photosynthetic membranes of seed plants. Unlike the galactolipids, sulfolipid is anionic at physiological pH because of its 6-deoxy-6-sulfonate-glucose (sulfoquinovose) head group. The biosynthesis of this lipid proceeds in two steps: first, the assembly of UDP-sulfoquinovose from UDP-glucose and sulfite, and second, the transfer of the sulfoquinovose moiety from UDP-sulfoquinovose to diacylglycerol. The first reaction is catalyzed by the SQD1 protein in Arabidopsis. Here we describe the identification of the SQD2 gene of Arabidopsis. We propose that this gene encodes the sulfoquinovosyltransferase catalyzing the second step of sulfolipid biosynthesis. Expression of SQD1 and SQD2 in Escherichia coli reconstituted plant sulfolipid biosynthesis in this bacterium. Insertion of a transfer DNA into this gene in Arabidopsis led to complete lack of sulfolipid in the respective sqd2 mutant. This mutant showed reduced growth under phosphate-limited growth conditions. The results support the hypothesis that sulfolipid can function as a substitute of anionic phospholipids under phosphate-limited growth conditions. Along with phosphatidylglycerol, sulfolipid contributes to maintaining a negatively charged lipid-water interface, which presumably is required for proper function of photosynthetic membranes.

Figure 1

The pathway for sulfolipid biosynthesis in Arabidopsis. Two enzymes, SQD1 and SQD2, are specific to this process and catalyze the reactions as indicated. DAG, diacylglycerol; R, fatty acyl groups; SQDG, sulfoquinovosyldiacylglycerol; UDP-Glc, UDP-glucose; UDP-SQ, UDP-sulfoquinovose.

Figure 2

Structure of the SQD2 gene carrying the T-DNA insertion (A), induction of SQD2 expression after phosphate deprivation (B), and lack of expression of SQD2 in the sqd2 mutant (C). (A) Exons are numbered as referred to in the text. A small portion of the bacterial artificial chromosome (BAC) F7J8 sequence is shown with nucleotide numbers referring to GenBank accession no. AL137189.2. The arrow indicates the T-DNA insertion point. (B) Plants were grown on agar-solidified medium for ≈20 days. As loading control, one of the rRNA bands stained with ethidium bromide is shown. (C) Plants were grown on soil for ≈25 days. An actin cDNA (GenBank accession no. M20016.1) was used as a probe for control purposes. WT, wild type.

Figure 3

Two-dimensional separation of lipids extracted from wild-type (A) and sqd2 mutant (B) leaves. The lipids were visualized by iodine. (Inserts) Autoradiographs in the area of the sulfolipid spots. Leaves were incubated with [³⁵S]sulfate to label sulfolipid. Lipids were separated as described for A and B. The small circles in the lower-right corners indicate the origins, and the arrows point toward the position of the sulfolipid. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; SQDG, sulfoquinovosyldiacylglycerol.

Figure 4

Plasmid constructs used for the expression of the SQD1 and SQD2 genes in E. coli (A) and separation of lipids from different E. coli strains (B). (A) The used vector plasmid is indicated in brackets for each construct. The open boxes indicate the His tag, and the black boxes indicate plant sequences. P, promoter; T, terminator. (B) The four left lanes represent extracts from E. coli harboring no plasmid or plasmids as indicated. An Arabidopsis leaf lipid extract was included for lipid-identification purposes. The lipids were visualized by α-naphthol, which primarily stains glycolipids. Other lipids are visualized because of light charring. The abbreviations are as described in the Fig. 3 legend.

Figure 5

Growth curves for wild type (filled diamonds, solid lines) and sqd2 mutant (filled circles, broken lines) on agar-solidified medium with 1 mM (A) or no (B) phosphate added. Eight plants were weighed for each time point. To rule out effects of secondary mutations, one time point (12 days) was repeated with an sqd2 mutant line transformed with the SQD2 cDNA (open diamonds) containing sulfolipid and the original sulfolipid-deficient sqd2 mutant (open circles). At least 15 plants are represented in each point in this experiment. Standard errors are indicated where they exceed the size of the symbols.