A Suppressor of fab1 Challenges Hypotheses on the Role of Thylakoid Unsaturation in Photosynthetic Function

Abstract

Leaf membrane lipids of the Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis 1 (fab1) mutant contain a 35% to 40% increase in the predominant saturated fatty acid 16:0, relative to wild type. This increase in membrane saturation is associated with loss of photosynthetic function and death of mutant plants at low temperatures. We have initiated a suppressor screen for mutations that allow survival of fab1 plants at 2°C. Five suppressor mutants identified in this screen all rescued the collapse of photosynthetic function observed in fab1 plants. While fab1 plants died after 5 to 7 weeks at 2°C, the suppressors remained viable after 16 weeks in the cold, as judged by their ability to resume growth following a return to 22°C and to subsequently produce viable seed. Three of the suppressors had changes in leaf fatty acid composition when compared to fab1, indicating that one mechanism of suppression may involve compensating changes in thylakoid lipid composition. Surprisingly, the suppressor phenotype in one line, S31, was associated with a further substantial increase in lipid saturation. The overall leaf fatty acid composition of S31 plants contained 31% 16:0 compared with 23% in fab1 and 17% in wild type. Biochemical and genetic analysis showed that S31 plants contain a new allele of fatty acid desaturation 5 (fad5), fad5-2, and are therefore partially deficient in activity of the chloroplast 16:0 D7 desaturase. A double mutant produced by crossing fab1 to the original fad5-1 allele also remained alive at 2°C, indicating that the fad5-2 mutation is the suppressor in the S31 (fab1 fad5-2) line. Based on the biophysical characteristics of saturated and unsaturated fatty acids, the increased 16:0 in fab1 fad5-2 plants would be expected to exacerbate, rather than ameliorate, low-temperature damage. We propose instead that a change in shape of the major thylakoid lipid, monogalactosyldiacylglycerol, mediated by the fad5-2 mutation, may compensate for changes in lipid structure resulting from the original fab1 mutation. Our identification of mutants that suppress the low-temperature phenotype of fab1 provides new tools to understand the relationship between thylakoid lipid structure and photosynthetic function.

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

“…Consistent with previous results (Wu et al, 1994;Barkan et al, 2006), all of the lipids in fab1 leaves had increased 16:0 compared with wild type. Three of the major membrane lipids, MGD, DGD, and PC, from S7 plants all showed a further increase in 16:0 relative to fab1.…”

“…Reductions in lipid synthesis by the prokaryotic pathway have previously been observed in the gly1 mutant, which is deficient in glycerol-3-P dehydrogenase activity (Miquel et al, 1998), and the act1 mutant, which is deficient in activity of the chloroplast acyl-ACP:glycerol-3-P acyltransferase (Kunst et al, 1988). However, test crosses of S7 with gly1 and act1 produced F 1 progeny with levels of leaf 16:3 similar to wild type, indicating that the lipid mutation in S7 is not allelic to either of these previously characterized mutants (Barkan et al, 2006). Here we describe identification of the S7 suppressor, by map-based cloning, as a new, hypomorphic allele of LPAT1 that encodes acyl-ACP:LPAT, the second enzyme in the prokaryotic pathway.…”

“…Interestingly, the leaf fatty acid composition of this new line contained only 1.8% 16:3 (Table III), considerably less than the 5.6% 16:3 measured in fab1 lpat1-3 (S7) leaves (Barkan et al, 2006). We interpret this difference as indicating that the reduced KASII activity caused by the fab1 mutation (Wu et al, 1994) results in an increase in 16:0-ACP, the substrate for LPAT1, which, in turn, increases synthesis of PA and prokaryotic lipids by the mutant LPAT1 enzyme encoded by lpat1-3.…”

“…This change in fatty acid composition of PG is significant because it likely reflects a decrease in highmelting-point species of PG (Roughan, 1985). We have previously proposed that high-melting-point PG contributes to the photosynthetic decline and death of fab1 at low temperature (Wu et al, 1997;Barkan et al, 2006). To test whether S7 plants contain substantially lower levels of high-melting-point PG than fab1, we submitted wild-type, fab1, and S7 leaf lipids for lipidomics analysis by mass spectrometry (Esch et al, 2007).…”

“…We initiated a suppressor screen for mutations that rescue fab1 from death at low temperatures (Barkan et al, 2006). One of the mutants isolated in this screen, S7, showed a reduced level of 16:3 in total leaf lipids, and this altered fatty acid composition cosegregated with the suppressive phenotype as a single, recessive mutation.…”