Galactolipid deficiency and abnormal chloroplast development in the Arabidopsis MGD synthase 1 mutant

Abstract

The lipid monogalactosyl diacylglycerol (MGD) is a major structural component of photosynthetic membranes in chloroplasts. Its formation is catalyzed by the enzyme MGD synthase. In many plants, MGD derives from two different biosynthetic pathways: the prokaryotic pathway, which operates entirely within the plastid, and the eukaryotic pathway, which involves steps in the endoplasmic reticulum. Here, we describe the identification and characterization of an Arabidopsis mutant with a defective MGD synthase gene (MGD1). The mutant was identified in a screen of T-DNA lines for individuals with defects in chloroplast biogenesis. It has a yellow-green phenotype that correlates with a approximately 50% deficiency in total chlorophyll per plant. A single T-DNA insertion is located adjacent to the transcription initiation site of the MGD1 gene, and the abundance of MGD1 mRNA is reduced by 75% compared with wild type. Correlation between steady-state MGD1 transcript levels and MGD synthase activity (also reduced by 75% in mgd1) suggests that MGD1 is the most important MGD synthase in green tissues. The amount of MGD in mutant leaves is reduced by 42% compared with wild type. MGD from the mutant contains 23% less 16:3 fatty acid and 10% more 18:3 fatty acid. Because 16:3 is a characteristic feature of MGD from the prokaryotic pathway, it is possible that MGD1 operates with some preference in the prokaryotic pathway. Finally, the MGD-deficiency of mgd1 is correlated with striking defects in chloroplast ultrastructure, strongly suggesting a unique role for MGD in the structural organization of plastidic membranes.

Figure 1

Visible phenotype of the mgd1 mutant. (A) Photograph showing 23-day-old seedlings. Plants were grown in vitro in continuous white light. The wild type is shown on the left and the mgd1 mutant is shown on the right. (B) Chlorophyll measurements. Plants were grown in vitro in continuous white light for either 7 or 14 days. Chlorophyll was extracted by using N,N′-dimethylformamide and determined photometrically according to described procedures (26). Presented values are means from six measurements of 10 seedlings (7 days) or 13 measurements of 2 seedlings (14 days) ± SE.

Figure 2

Structure of the MGD1 gene. (A) Schematic representation of the gene disrupted in the mgd1 mutant. The depicted gene encodes the MGD synthase enzyme, MGD1. Filled boxes correspond to translated regions of the MGD1 transcript; open boxes correspond to untranslated regions of the MGD1 transcript. Exons are numbered from 1 to 8. The T-DNA insertion is represented by a triangle. Indicated features include the T-DNA left border (LB), the translation initiation codon (ATG), the translation termination codon (Stop), and the polyadenylation site [p(A)]. (B) Alignment of the MGD1 and csMGD1 amino acid sequences. Dashes indicate gaps introduced to maximize alignment. Residues conserved between both sequences are shaded.

Figure 3

Biochemical phenotype of the mgd1 mutant. (A) Lipid composition of wild-type and mutant leaves. Total leaf lipids were extracted and the different lipid classes were separated by TLC and quantified. Indicated lipids are monogalactosyl diacylglycerol (MGD), phosphatidylglycerol (PG), digalactosyl diacylglycerol (DGD), sulfolipid (SL), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). (B) MGD synthase and DGD synthase activities of wild-type and mutant chloroplasts. The rate of incorporation of [¹⁴C]galactose into galactolipids in isolated chloroplasts was determined. Units are pmol UDP-[¹⁴C]galactose/mg chlorophyll/min. (C) Fatty acid composition of MGD from wild-type and mutant leaves. MGD isolated in A above was subjected to fatty acid methyl ester quantification. (A–C) Presented values are means from three (wild type) or six (mgd1) independent measurements ± SE.

Figure 4

Ultrastructure of mgd1 plastids. Electron micrographs of representative plastids from (A) 5-day-old etiolated wild-type cotyledons, (B) 5-day-old etiolated mgd1 cotyledons, (C) 5-day-old light-grown wild-type cotyledons, (D) 5-day-old light-grown mgd1 cotyledons, (E) 23-day-old light-grown wild-type leaves, and (F) 23-day-old light-grown mgd1 leaves. A and B are at higher magnification than C–F. (Bars = 1 μm.)