A Mutation in the LPAT1 Gene Suppresses the Sensitivity of fab1 Plants to Low Temperature

Abstract

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant grows as well as wild type at 22°C, but after transfer to 2°C fab1 plants cannot maintain photosynthetic function and die after 5 to 7 weeks at 2°C. A fab1 suppressor line, S7, was isolated in a screen that identified mutants that remained alive after 16 weeks at 2°C and were able to flower and produce seed after return to 22°C. Relative to wild type, S7 plants had reduced levels of 16:3 fatty acid in leaf galactolipids, indicating reduced synthesis of chloroplast glycerolipids by the prokaryotic pathway of lipid metabolism. The suppressor mutation was identified, by map-based and candidate-gene approaches, as a hypomorphic allele of lysophosphatidic acid acyltransferase1 (lpat1), lpat1-3. LPAT1 encodes the enzyme that catalyzes the second reaction in the prokaryotic pathway. Several lines of evidence indicate that damage and death of fab1 plants at 2°C may be a result of the increased proportion of phosphatidylglycerol (PG) in fab1 that are high-melting-point molecular species (containing only 16:0, 18:0, and 16:1,D3-trans fatty acids). Consistent with this proposal, the lpat1-3 mutation strongly affects the fatty acid composition of PG. The proportion of highmelting-point molecular species in PG is reduced from 48.2% in fab1 to 10.7% in fab1 lpat1-3 (S7), a value close to the 7.6% found in wild type.There are two distinct pathways in higher plants for the synthesis of membrane glycerolipids. Both pathways are initiated by the synthesis of 16:0-acyl carrier protein (16:0-ACP) from acetyl-CoA by plastid acetylCoA carboxylase and enzymes of the fatty acid synthase. 16:0-ACP may be elongated by one additional cycle of the fatty acid synthase and then desaturated by the stromal 18:0-ACP desaturase (Lindqvist et al., 1996), so that 16:0-ACP and 18:1-ACP are the primary products of plastid fatty acid synthesis (Ohlrogge and Browse, 1995). The prokaryotic pathway located in the plastid envelope begins with the synthesis of 18:1-lysophosphatidic acid by acyl-ACP:glycerol-3-P acyltransferase. Then 16:0-ACP:lysophosphatidic acid acyltransferase (LPAT) completes the synthesis of phosphatidic acid (PA) that is a key intermediate in glycerolipid synthesis. The PA in the prokaryotic pathway is used for the synthesis of phosphatidylglycerol (PG) and, in some plants, as a precursor via diacylglycerol for the synthesis of monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD), and sulfoquinovosyldiacylglycerol (SQD), the four major glycerolipids of the photosynthetic thylakoid membranes. The alternative eukaryotic pathway begins with the hydrolysis of acyl-ACPs and the synthesis of 16:0-CoA and 18:1-CoA in the plastid envelope for export to the endoplasmic reticulum, where they are used for the synthesis of phosphatidylcholine (PC) and other phospholipids characteristic of the various extrachloroplast membranes of the cell. In addition, the diacylglycerol moiety of PC is returned to the chloroplast envelope-possibly in the form of PA (Be...

Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold

“…3B and 4B). This phenotype is unlike that of fab1 lpat1-3 and fab1 lpat1-4; in those double mutants, older leaves senesce prematurely but new leaves remain green in chilling conditions (Kim et al, 2010; Fig. 1A).…”

“…The S106 allele of lpat1 contains a C to T transition at nucleotide 689 of the open reading frame, creating a missense mutation at Pro-230, converting it to Leu (P230L; Table I). We have previously determined that the fab1 suppressor in the S7 line from our screen is a mutation in LPAT1, lpat1-3 (Kim et al, 2010). To determine whether the newly identified P230L mutation, designated lpat1-4, is responsible for suppression of the fab1 low-temperature phenotype, we crossed plants of the S106 and S7 lines.…”

“…To better understand the changes in lipid composition that contribute to the fab1 phenotype and the effects they have on the photosynthesis machinery, we conducted a screen for mutations that suppress the collapse of photosynthesis and death of fab1 plants that occur at low temperature (Barkan et al, 2006). Two of the suppressors have been described previously, and both are mutations in genes encoding enzymes in the chloroplast-localized prokaryotic pathway of glycerolipid synthesis, one in fad5 (Barkan et al, 2006) and one in lpat1 (Kim et al, 2010). Figure 6 shows the main reactions of the prokaryotic pathway together with the five mutations (in red) that act as suppressors of the fab1 low-temperature phenotype.…”

“…We have previously shown that mutations affecting the prokaryotic pathway in S31 (fab1 fad5-2) and S7 (fab1 lpat1-3) lines act to suppress the fab1 low-temperature phenotype (Barkan et al, 2006;Kim et al, 2010). For this reason, we hypothesized that the mutation affecting the prokaryotic pathway in S96 might be the fab1 suppressor and chose to map the locus based on segregation of the low-16:3 phenotype.…”

“…Previously, we have characterized suppressor mutants deficient in the second enzyme of the prokaryotic pathway, lpat1-3, and in the MGD 16:0 D7 desaturase (fad5; Barkan et al, 2006;Kim et al, 2010). Here, we use map-based cloning and full genome sequencing to identify the genetic loci of two additional suppressors from our screen in lines S106 and S96.…”