A mutant of Arabidopsis deficient in desaturation of palmitic Acid in leaf lipids

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by elevated amounts of palmitic acid and a decreased amount of unsaturated 16-carbon fatty acids as a consequence of a single nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids suggested that the mutant is deficient in the activity of a chloroplast ω9 fatty acid desaturase which normally introduces a double bond in 16-carbon acyl chains esterified to monogalactosyldiacylglycerol (MGD). The mutant exhibited an increased ratio of 18- to 16-carbon fatty acids in MGD due to a change in the relative contribution of the prokaryotic and eukaryotic pathways of lipid biosynthesis. This appears to be a regulated response to the loss of chloroplast ω9 desaturase and presumably reflects a requirement for polyunsaturated fatty acids for the normal assembly of chloroplast membranes. The reduction in mass of prokaryotic MGD species involved both a reduction in synthesis of MGD by the prokaryotic pathway and increased turnover of MGD molecular species which contain 16:0.     

Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase.

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by reduced levels of polyunsaturated 18-carbon fatty acids and an increased proportion of oleate as a consequence of a single recessive nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids demonstrated that all the major phospholipids of the extrachloroplast membranes are affected by the mutation, whereas the chlorplast lipids show fatty acid compositions only slightly different from those of wild type plants. These results are consistent with the parallel operation of two pathways of lipid synthesis in plant leaf cells (the prokaryotic pathway in the chloroplast and the eukaryotic pathway in the endoplasmic reticulum) and with genetic evidence (Browse, J., Kunst, L., Anderson, S., Hugly, S., and Somerville, C.R. (1989) Plant Physiol 90, 522-529) that an independent 18:1/16:1 desaturase operates on chloroplast membrane lipids. Direct enzyme assays confirmed that the mutant plants are deficient in the activity of a microsomal oleoyl-phosphatidycholine desaturase and demonstrated that this desaturase is the major enzyme responsible for the synthesis of polyunsaturated phospholipids. Despite this deficiency in 18:1-desaturase activity, mutant plants contained relatively high levels of 18:3 in their leaf phospholipids. This finding is interpreted as additional evidence that considerable two-way exchange of lipid occurs between the chloroplast and endoplasmic reticulum and that this exchange allows the chloroplast desaturases to provide lipids containing 18:3 to the extrachloroplast compartment, thus partially alleviating the deficiency in 18:1 desaturase activity.     

A Mutant of Arabidopsis Deficient in the Elongation of Palmitic Acid

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by an increased level of 16:0 and a concomitant decrease of 18-carbon fatty acids as a consequence of a single recessive nuclear mutation at the fab1 locus. Quantitative analysis of the fatty acid composition of individual lipids established that lipids synthesized by both the prokaryotic and eukaryotic pathways were affected by the mutation. Direct enzyme assays demonstrated that the mutant plants were deficient in the activity of 3-ketoacyl-acyl carrier protein synthase II; therefore, it is inferred that fab1 may encode this enzyme. Labeling experiments with [14C]acetate and lipase positional analysis indicated that the mutation results in a small shift in the partitioning of lipid synthesis between the prokaryotic and eukaryotic pathways. Synthesis of chloroplast lipids by the prokaryotic pathway was increased with a corresponding reduction in the eukaryotic pathway.     

A mutant of Arabidopsis deficient in c(18:3) and c(16:3) leaf lipids

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 16:3 and 18:3 fatty acids and has correspondingly increased levels of the 16:2 and 18:2 precursors due to a single recessive nuclear mutation. The kinetics of in vivo labeling of lipids with [¹⁴C]acetate and quantitative analysis of the fatty acid compositions of individual lipids suggests that reduced activity of a glycerolipid n-3 desaturase is responsible for the altered lipid composition of the mutant. The effects of the mutation are most pronounced when plants are grown at temperatures above 26°C but are relatively minor below 18°C, suggesting a temperature-sensitive enzyme. Since the desaturation of both 16- and 18-carbon fatty acids is altered, it appears that the affected enzyme lacks specificity with respect to acyl group chain length and that it is located in the chloroplast where 16:3-monogalactosyldiglyceride is synthesized. Because the degree of unsaturation of all the major glycerolipids was similarly affected by the mutation, it is inferred that either the affected desaturase does not exhibit head group specificity or there is substantial transfer of trienoic acyl groups between different lipid classes. Both chloroplast and extrachloroplast lipids are equally affected by the mutation. Thus, either the desaturase is located both outside and inside the chloroplast, or 18:3 formed inside the chloroplast is reexported to other cellular sites.     

Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C16 and C18 acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [14C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C18-sn-2-C16 (C18/C16) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C18-sn-2-C18 (C18/C18) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C18/C18 species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C18/C18 species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Elucidation of the Biosynthesis of Eicosapentaenoic Acid in the Microalga Porphyridium cruentum (II. Studies with Radiolabeled Precursors)

In the course of the study of the biosynthesis of the fatty acid eicosapentaenoic acid (EPA) in the microalga Porphyridium cruentum, cells were pulse-labeled with various radiolabeled fatty acid precursors. Our data show that the major end products of the biosynthesis are EPA-containing galactolipids of a eukaryotic and prokaryotic nature. The prokaryotic molecular species contain EPA and arachidonic acid at the sn-1 position and C16 fatty acids, mainly 16:0, at the sn-2 positions, whereas in the eukaryotic species both positions are occupied by EPA or arachidonic acid. However, we suggest that both the eukaryotic and prokaryotic molecular species are formed in two pathways, [omega]6 and [omega]3, which involve cytoplasmic and chloroplastic lipids. In the [omega]6 pathway, cytoplasmic 18:2-phosphatidylcholine (PC) is converted to 20:4[omega]6-PC by a sequence that includes a [delta]6 desaturase, an elongation step, and a [delta]5 desaturase. In the minor [omega]3 pathway, 18:2-PC is presumably desaturated to 18:3[omega]3, which is sequentially converted by the enzymatic sequence of the [omega]6 pathway to 20:5[omega]3-PC. The products of both pathways are exported, as their diacylglycerol moieties, to the chloroplast to be galactosylated into their respective monogalactosyldiacylglycerol molecular species. The 20:4[omega]6 in both eukaryotic and prokaryotic monogalactosyldiacylglycerol can be further desaturated to EPA by a chloroplastic [delta]17 ([omega]3) desaturase.     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

Metabolism of Unsaturated Monogalactosyldiacylglycerol Molecular Species in Arabidopsis thaliana Reveals Different Sites and Substrates for Linolenic Acid Synthesis

Synthesis of unsaturated monogalactosyldiacylglycerol (MGDG) was examined in a mutant of Arabidopsis thaliana (L.) Heynh. containing reduced levels of hexadecatrienoic (16:3) and linolenic (18:3) acids in leaf lipids. Molecular species composition and labeling kinetics following the incorporation of exogenous [¹⁴C]fatty acids suggest that at least two pathways and multiple substrates are involved in desaturation of linoleic acid (18:2) to 18:3 for production of unsaturated galactolipids. A reduction in 18:3/16:3 MGDG and an increase in 18:2/16:2 MGDG, together with labeling kinetics of these molecular species following the incorporation of exogenous [¹⁴C]12:0 fatty acids, suggests that a chloroplastic pathway for production of 18:3 at the sn-1 position of MGDG utilizes 18:2/16:2 MGDG as a substrate. This chloroplastic (prokaryotic) pathway is deficient in the mutant. When exogenous [¹⁴C]18:1 was supplied, a eukaryotic (cytoplasmic) pathway involving the desaturation of 18:2 to 18:3 on phosphatidylcholine serves as the source of 18:3 for the sn-2 position of MGDG. This eucaryotic pathway predominates in the mutant.     

A Study of Phospholipids and Galactolipids in Pollen of Two Lines of Brassica napus L. (Rapeseed) with Different Ratios of Linoleic to Linolenic Acid

The phospholipids and galactolipids of the pollen-coat and internal domains of two lines of Brassica napus, Wesroona and IXLIN, with different linoleic/linolenic acid ratios (18:2/18:3) have been characterized by normal phase silica high performance liquid chromatography and gas liquid chromatography. The polar lipids of the pollen-coat are similar to leaf lipids in the high proportion of galactolipids (almost 50%) and the fatty acids; 18:3, palmitic (16:0) and hexadecatrienoic (16:3). In contrast, the pollen internal domain, although rich in 18:3, 18:2 and 16:0, is composed primarily of phosphatidyl-choline, -ethanolamine, and -inositol whose 18:2/18:3 ratio is correlated with that of the seed generation. The difference between the two divergent 18:2/18:3 ratio lines is most evident in the internal domain phospholipids. The 18:2/18:3 ratio of the galactolipids of both pollen domains is not significantly effected by the line genotype. The results are interpreted in terms of the previously described `prokaryotic' and `eukaryotic' plant desaturation pathways (PG Roughan, CR Slack [1982] Annu Rev Plant Physiol 33: 97-132). We propose that the eukaryotic pathway is the major desaturation pathway providing polyunsaturated fatty acids to the haploid-specified internal domain in which the IXLIN genotype modifies the activity of the sn-2 linoleoyl phosphatidylcholine desaturase/s of the endoplasmic reticulum. In the diploid-specified pollen-coat, our evidence suggests that a combination of the prokaryotic and eukaryotic pathways contribute polyunsaturated fatty acids.     

The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis

A mutant of Arabidopsis thaliana with reduced content of C_18:3 and C_16:3 fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO₂-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.     

Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic Acid unsaturation

A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast ω9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO₂ fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28°C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.     

A Mutant of Arabidopsis with Increased Levels of Stearic Acid

A mutation at the fab2 locus of Arabidopsis caused increased levels of stearate in leaves. The increase in leaf stearate in fab2 varied developmentally, and the largest increase occurred in young leaves, where stearate accounted for almost 20% of total leaf fatty acids. The fatty acid composition of leaf lipids isolated from the fab2 mutant showed increased stearate in all the major glycerolipids of both the chloroplast and extrachloroplast membranes. Although the stearate content was increased, the fab2 mutant still contained abundant amounts of 18:1, 18:2, and 18:3 fatty acids. These results are consistent with the expectations for a mutation partially affecting the action of the stromal stearoyl-acyl carrier protein desaturase. Positional analysis indicated that the extra 18:0 is excluded with high specificity from the sn-2 position of both chloroplast and extrachloroplast glycerolipids. Although stearate content was increased in all the major leaf membrane lipids, the amount of increase varied considerably among the different lipids, from a high of 25% of fatty acids in phosphatidylcholine to a low of 2.9% of fatty acids in monogalactosyldiacylglycerol.     

Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants

Photosynthetically active chloroplasts retaining high rates of fatty acid synthesis from [1-¹⁴C]acetate were purified from leaves of both 16:3 (Solanum nodiflorum, Chenopodium album) and 18:3 plants (Amaranthus lividus, Pisum sativum). A comparison of lipids into which newly synthesized fatty acids were incorporated revealed that, in 18:3 chloroplasts, enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD) were significantly less active than in 16:3 chloroplasts. In contrast, labeling rates of MGD from UDP-[¹⁴C]gal were similar for both types of chloroplasts.

The composition and positional distribution of labeled fatty acids within the glycerides synthesized by isolated 16:3 and 18:3 chloroplasts were similar and in each case only a C18/C16 diacylglycerol backbone was synthesized. In nodiflorum chloroplasts, C18:1/C16:0 MGD assembled de novo was completely desaturated to the C18:3/C16:3 stage.

Whereas newly synthesized C18/C18 MGD could not be detected in any of these chloroplasts if incubated with [¹⁴C]acetate after isolation, chloroplasts isolated from acetate-labeled leaves contained MGD with labeled C18 fatty acids at both sn-1 and sn-2 positions. Taken together, these results provide further evidence on an organellar level for the operation of pro- and eucaryotic pathways in the biosynthesis of MGD in different groups of plants.

     

Phosphatidylglycerol Composition Is Central to Chilling Damage in the Arabidopsis fab1 Mutant

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0, resulting from decreased activity of 3-ketoacyl-ACP synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains >40% high-melting-point molecular species (HMP-PG; molecules that contain only 16:0, 16:1-trans, and 18:0 fatty acids)—a trait associated with chilling-sensitive plants—compared with <10% in wild-type Arabidopsis. Although they do not exhibit short-term chilling sensitivity when exposed to low temperatures (2°C to 6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. To test the relevance of HMP-PG to the fab1 phenotype, we used transgenic 16:0 desaturases targeted to the endoplasmic reticulum and the chloroplast to lower 16:0 in leaf lipids of fab1 plants. We produced two lines that had very similar lipid compositions except that one, ER-FAT5, contained high HMP-PG, similar to the fab1 parent, while the second, TP-DES9*, contained <10% HMP-PG, similar to the wild type. TP-DES9* plants, but not ER-FAT5 plants, showed strong recovery and growth following 75 d at 2°C, demonstrating the role of HMP-PG in low-temperature damage and death in fab1, and in chilling-sensitive plants more broadly.

In higher plants, the chloroplast membranes that host the light harvesting and electron transport processes of photosynthesis have a characteristically high number of double bonds in the glycerolipid acyl chains. Only ∼10% of the fatty acids that compose the hydrophobic core of the thylakoid bilayer lack double bonds altogether, whereas >80% are polyunsaturated, having two or three double bonds (Ohlrogge et al., 2015). The photosynthetic light reactions produce reactive oxygen species as by-products, and these can degrade polyunsaturated fatty acids, so it is assumed that highly unsaturated membranes are required to support photosynthesis (McConn and Browse, 1998).The glycerolipids in chloroplast membranes are synthesized by two separate pathways. (Browse et al., 1986; Ohlrogge and Browse, 1995). Synthesis de novo of fatty acids takes place in the stroma of chloroplasts, producing 16:0 esterified to acyl carrier protein (ACP). A large proportion of this 16:0-ACP is elongated by 3-keto-acyl-ACP synthase II (KASII) to 18:0-ACP, which is in turn desaturated by stearoyl ACP desaturase to produce 18:1-ACP (Lindqvist et al., 1996; Carlsson et al., 2002). The fatty acids from 16:0-ACP and 18:1-ACP may be used within the chloroplast in the prokaryotic pathway (Kunst et al., 1988; Kim and Huang, 2004) to produce phosphatidic acid (PA). Some of this PA intermediate is used for synthesis of phosphatidylglycerol (PG; Ohlrogge and Browse, 1995; Wada and Murata, 2007), which is the only chloroplast glycerolipid that is produced solely by the prokaryotic pathway. In some plants, including Arabidopsis (Arabidopsis thaliana), PA is also converted to diacylglycerol (DAG), which is the precursor for the synthesis of the other chloroplast glycerolipids, monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD), and sulfoquinovosyldiacylglycerol (SQD; Browse et al., 1986; Ohlrogge and Browse, 1995; Ohlrogge et al., 2015).The second route for chloroplast glycerolipid synthesis, the eukaryotic pathway, begins with export of 16:0 and 18:1 from the chloroplast as CoA thioesters. (Li et al., 2015). In the endoplasmic reticulum (ER), these fatty acids are rapidly incorporated into phosphatidylcholine (PC) by acyl exchange (Bates et al., 2007), and are also used (via PA and DAG intermediates) for the synthesis of all the phospholipids of the extrachloroplast membranes of the cell (Ohlrogge et al., 2015). In addition however, the DAG moiety of PC can be returned to the chloroplast and contribute to the production of MGD, DGD, and SQD required for thylakoid synthesis (Benning, 2009; Roston et al., 2012). The ER-to-chloroplast flux of lipid is reversible to some extent (Browse et al., 1989, 1993).With the exception of the first Δ9 double bond in 18:1-ACP, all the double bonds in the acyl chains are introduced after the initial synthesis of glycerolipid molecules. In Arabidopsis, this involves the action of seven fatty acid desaturases that are integral membrane proteins in the chloroplast and ER (Ohlrogge and Browse, 1995; Wallis and Browse, 2010). Characterization of Arabidopsis fatty acid desaturation (fad) mutants deficient in one or more of these desaturases has shown that the high level of thylakoid unsaturation is essential to photosynthetic function (Murakami et al., 2000; Routaboul et al., 2000). For example, fad2 fad6 double-mutant plants are unable to synthesize polyunsaturated fatty acids and cannot grow autotrophically; however, when grown on Suc as a carbon source, the double mutants are robust plants showing strong leaf and root development (McConn and Browse, 1998). These results indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids; photosynthesis is the one process that requires high levels of polyunsaturation. Mutants with smaller changes in unsaturation are often similar to the wild type under typical growth-chamber conditions and reveal their phenotypes only under more extreme conditions (Wallis and Browse, 2002, 2010). Several mutants grow more slowly and become chlorotic at temperatures in the range 2°C to 10°C (Hugly and Somerville, 1992; Routaboul et al., 2000), indicating a role for fatty acid composition in maintaining photosynthesis at these low temperatures.Like other species native to temperate regions, Arabidopsis is chilling resistant and able to grow at temperatures close to 0°C. By contrast, many tropical and subtropical plant species are chilling sensitive and suffer sharp reductions of photosynthesis and extensive tissue damage after even short exposure to low temperatures. Many of the world’s most important crops, including rice (Oryza sativa), maize (Zea mays), and soybean (Glycine max) are chilling sensitive, so a better understanding of the biochemical and genetic factors contributing to this sensitivity has the potential to enhance sustainable food production (Nishida and Murata, 1996; Iba, 2002; Thakur et al., 2010). One hypothesis proposes that chilling sensitivity is a result of the fatty acid composition of chloroplast PG. It is based on the observation that many chilling-sensitive plants contain >30% of PG molecules with only saturated or trans unsaturated fatty acids—16:0, 18:0, and 16:1-Δ3trans (16:1t)—at both the sn-1 and sn-2 positions of the glycerol backbone, referred to as high-melting-point molecular species (HMP-PG; Murata, 1983; Barkan et al., 2006). This name alludes to the fact that HMP-PG species can induce a phase change from liquid crystalline (typical of biological membranes) to gel phase at temperatures well above 0°C and thereby disrupt membrane and cellular function (Murata and Yamaya, 1984). Chilling-resistant plants have <10% HMP species in chloroplast PG (Murata et al., 1982; Murata, 1983; Roughan, 1985).One perspective on the role of HMP-PG in plant temperature responses has come from our investigations of the fatty acid biosynthesis1 (fab1) mutant of Arabidopsis. In this mutant, a hypomorphic mutation in the gene encoding KASII reduces elongation of 16:0-ACP to 18:0-ACP (Carlsson et al., 2002), producing plants that have increased levels of 16:0 in all membrane glycerolipids (Wu et al., 1994). In particular, fab1 plants contain HMP-PG at levels (∼40% to 50% of total PG) similar to those of many chilling-sensitive plant species (Wu and Browse, 1995). Nevertheless, the fab1 mutant does not show typical symptoms of chilling sensitivity and is unaffected, in comparison to wild-type controls, by a range of chilling treatments that kill chilling-sensitive plants; instead, fab1 plants only show a collapse of photosynthesis after >10 d of exposure to 2°C, with the plants dying after several weeks at low temperature (Wu and Browse, 1995; Wu et al., 1997).We have previously screened for genetic suppressors of the fab1 low-temperature phenotype. Most, though not all, of the suppressor mutations substantially reduce the proportion of saturated fatty acids in PG, consistent with the notion that HMP-PG causes eventual death of fab1 plants in the cold (Barkan et al., 2006; Kim et al.,2010; Gao et al., 2015). However, all the suppressors have additional changes, relative to fab1, in the fatty acid compositions of membrane lipids that prevent a clear linkage between reductions in HMP-PG and improved low-temperature survival.Here, we have taken a new approach to investigating the role of HMP-PG in damage and death of fab1 plants at chilling temperatures by using a 16:0-CoA desaturase from Caenorhabditis elegans, FAT-5 (Watts and Browse, 2000), and a glycerolipid desaturase, DES9*15, derived from a cyanobacterial enzyme by directed evolution (Bai et al., 2016). When expressed in the fab1 mutant background, both the FAT-5 enzyme targeted to the ER and the DES9*15 enzyme targeted to the chloroplast reduced leaf 16:0 to near-wild type levels. The fatty acid compositions of individual leaf lipids in plants of both transgenic lines were very similar, with the sole exception of PG. Plants expressing the FAT-5 desaturase retained high levels of HMP-PG, similar to fab1, while plants expressing the DES9*15 enzyme had HMP-PG lowered to levels close to those of the wild type. Like the fab1 mutant, fab1 plants expressing a 16:0 desaturase in the ER lost photosynthetic function over 28 d of exposure to 2°C and showed little capacity for recovery and growth after longer periods at low temperature. By contrast, plants expressing a 16:0 desaturase targeted to the chloroplast retained substantial photosynthetic function, even after 75 d at 2°C, and were subsequently able to resume growth, flower, and set seed upon return to 22°C. These results provide the most direct evidence yet that high levels of HMP-PG cause gradual loss of photosynthesis and eventual death of plants at chilling temperatures.     

Expression of a gene for plastid ω-3 fatty acid desaturase and changes in lipid and fatty acid compositions in light- and dark- grown wheat leaves

The leaves of monocotyledonous plants create a developmental sequence of cells and plastids from the base to the apical portion. We investigated fatty-acid and lipid compositions in successive leaf sections of light- and dark-grown wheat (Triticum aestivum L. cv. Chihoku) seedlings. The most notable change in the fatty acid composition was the increase of linolenic acid (18:3) with maturation of leaf cells, which occurred both in light- and dark-grown leaf tissues. In light-grown leaves, the increase of 18:3 with maturation was mainly attributed to the increase of monogalactosyldiacylglycerol (MGD) and also to the increase of the 18:3 level of MGD. In dark-grown leaves, the increase of 18:3 in the leaf apex was caused by the increase of the levels of MGD and digalactosyldiacylglycerol (DGD) and also by the increase of the 18:3 levels of within these two lipids. Since MGD and DGD are mainly found in plastid membranes, these findings indicate that both the synthesis of galactolipids and the formation of 18:3 these lipids take place during plastid development. The plastid ω-3 fatty acid desaturase is responsible for the formation of 18:3 in plastid membrane lipids. To investigate the regulation of desaturation, we isolated a gene for wheat plastid ω-3 fatty acid desaturase (TaFAD7). The mRNA level of TaFAD7 in light-grown leaves was much higher than that in dark-grown leaves. During the greening of etiolated leaves the level of TaFAD7 mRNA increased significantly, accompanied by an increase of the 18:3 level of total fatty acids. On the other hand, the levels of TaFAD7 mRNA were almost the same in all the leaf sections of both light- and dark-grown leaf tissues. These results suggest that the effect of the expression of the TaFAD7 gene on the increase of the 18:3 level is different between the leaf development under continuous light- or dark-conditions and the light-induced greening process of etiolated leaves. The increase of 18:3 content of MGD (or MGD and DGD) with maturation is apparently regulated not solely by the level of TaFAD7 mRNA.     

Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity

The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for Δ12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the Δ12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the Δ12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.     

The sulfolipids 2'-O-acyl-sulfoquinovosyldiacylglycerol and sulfoquinovosyldiacylglycerol are absent from a Chlamydomonas reinhardtii mutant deleted in SQD1

The biosynthesis of thylakoid lipids in eukaryotic photosynthetic organisms often involves enzymes in the endoplasmic reticulum (ER) and the chloroplast envelopes. Two pathways of thylakoid lipid biosynthesis, the ER and the plastid pathways, are present in parallel in many species, including Arabidopsis, but in other plants, e.g. grasses, only the ER pathway is active. The unicellular alga Chlamydomonas reinhardtii diverges from plants like Arabidopsis in a different way because its membranes do not contain phosphatidylcholine, and most thylakoid lipids are derived from the plastid pathway. Here, we describe an acylated derivative of sulfolipid, 2'-O-acyl-sulfoquinovosyldiacylglycerol (ASQD), which is present in C. reinhardtii. Although the fatty acids of sulfoquinovosyldiacylglycerol (SQDG) were mostly saturated, ASQD molecular species carried predominantly unsaturated fatty acids. Moreover, directly attached to the head group of ASQD was preferentially an 18-carbon fatty acid with four double bonds. High-throughput robotic screening led to the isolation of a plasmid disruption mutant of C. reinhardtii, designated Deltasqd1, which lacks ASQD as well as SQDG. In this mutant, the SQD1 ortholog was completely deleted and replaced by plasmid sequences. It is proposed that ASQD arises from the sugar nucleotide pathway of sulfolipid biosynthesis by acylation of the 2'-hydroxyl of the sulfoquinovosyl head group. At the physiological level, the mutant showed increased sensitivity to a diuron herbicide and reduced growth under phosphate limitation, suggesting a role for SQDG and/or ASQD in photosynthesis as conducted by C. reinhardtii, particularly under phosphate-limited conditions.