Pathway for lipid A biosynthesis in Arabidopsis thaliana resembling that of Escherichia coli

Abstract

The lipid A moiety of Escherichia coli lipopolysaccharide is a hexa-acylated disaccharide of glucosamine that makes up the outer monolayer of the outer membrane. Arabidopsis thaliana contains nuclear genes encoding orthologs of key enzymes of bacterial lipid A biosynthesis, including LpxA, LpxC, LpxD, LpxB, LpxK and KdtA. Although structurally related lipid A molecules are found in most other gram-negative bacteria, lipid A and its precursors have not been directly detected in plants previously. However, homozygous insertional knockout mutations or RNAi knock-down constructs of Arabidopsis lpx and kdtA mutants revealed accumulation (or disappearance) of the expected monosaccharide or disaccharide lipid A precursors by mass spectrometry of total lipids extracted from 10-day old seedlings of these mutants. In addition, fluorescence microscopy of lpx-gfp fusions in transgenic Arabidopsis plants suggests that the Lpx and KdtA proteins are expressed and targeted to mitochondria. Although the structure of the lipid A end product generated by plants is still unknown, our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli.

Fig. 1.

Enzymatic synthesis of lipid A in E. coli and evidence for a similar pathway in A. thaliana. The green asterisks indicate that significant orthologs of the E. coli enzymes are present in A. thaliana. These proteins show 27–41% sequence identity over their full-lengths (Table 1).

Fig. 2.

Chromosomal locations of the putative Arabidopsis lipid A genes. The gene locations were obtained from The Arabidopsis Information Resource database. Multiple AtLpxC genes may have arisen by duplication.

Fig. 3.

Detection of the lipid A precursor 2,3-diacylglucosamine 1-phosphate in A. thaliana. (A) Negative-ion LC–ESI/MS analysis of lipids, eluting from a normal phase LC column between minutes 22.7 and 23.3, from 10-day old Col-0 wild-type seedlings. (B) Corresponding analysis of the lipids from 10-day old seedlings of the atlpxb-1 mutant. The m/z 710.43 ion peak that accumulates in this mutant corresponds to the [M-H]^- ion of 2,3-diacylglucosamine 1-phosphate (lipid X) with the same acyl chain composition as E. coli lipid X (Fig. 1). (C) ESI/MS/MS analysis of the lipid X [M-H]^- ion at m/z 710.43, which accumulates in the atlpxb-1 mutant. The fragment ions are the same as those seen for E. coli lipid X. The mass spectra were acquired using a high resolution QSTAR XL quadrupole time-of-flight tandem mass spectrometer (Applied Biosystems).

Fig. 4.

Quantification of 2,3-diacylglucosamine 1-phosphate and other lipid A precursors in Arabidopsis mutants. MRM analysis and quantification of 2,3-diacylglucosamine 1-phosphate (lipid X) (A), UDP-2,3-diacyl-GlcN (B), disaccharide 1-phosphate (C), and lipid IV_A (D) was carried out using lipids extracted from the indicated 10-day old seedlings of three parental wild types, six Arabidopsis insertional mutants, and one RNAi transgenic line. These Arabidopsis lipid A precursors, which have the same acyl chain compositions and molecular weights as their E. coli counterparts (Fig. 1), were detected using the precursor/product ion pairs shown in Fig. S3. Peak areas were normalized to the major phosphatidylethanolamine molecular species (C16∶0/C18∶2) present in the same sample (Fig. S3). Error bars represent standard deviations of three biological replicates. The full LC–MRM tracings for each precursor/product ion pair are shown in Figs. S4, S6, S7, and S8. All LC–MRM experiments were performed using a 4,000 Q-Trap hybrid triple quadrupole linear ion trap mass spectrometer, equipped with a Turbo V ion source (Applied Biosystems).

Fig. 5.

Subcellular localization of GFP-fusion proteins of AtLpxA, AtLpxC1.1, AtLpxD1, AtLpxD2, AtLpxB, and AtLpxK. Roots from seven-day old Arabidopsis plants expressing the indicated GFP fusion proteins (green) were stained simultaneously with MitoTracker (red). The GFP and MitoTracker images were merged to show the colocalization of the GFP signal and mitochondria. Col WT, the Col-0 WT without the GFP transgene; 35S∷GFP, overexpression of GFP cDNA under the control of 35S CaMV promoter in the Col-0 background. Scale bar: 20 μm.