Phosphatidylglycerol and chilling sensitivity in plants

The hypothesis that molecular species of thylakoid phosphatidylglycerol containing two saturated fatty acids (disaturated phosphatidylglycerol) confer chilling sensitivity upon plants was tested by analyzing the fatty acid composition of phosphatidylglycerols isolated from leaves of a range of plants expected to have different sensitivities to chilling temperatures.

`Saturated' fatty acids (palmitate plus stearate plus hexadeca-trans-3-enoate) as a proportion of total phosphatidylglycerol fatty acids varied from 51 to 80 mole per cent in the plants analyzed but appeared to be rigidly fixed for a given plant species, being unaffected by leaf maturity or by environment.

Hexadeca-trans-3-enoate occurred only at the sn-2 position, whereas C-18 fatty acids occurred only at the sn-1 position of thylakoid phosphatidylglycerol. Therefore, the proportion of disaturated molecular species could be predicted accurately from the total fatty acids of phosphatidylglycerol.

Disaturated molecular species accounted for <25% of the total phosphatidylglycerol from leaves of chilling-resistant plants and for 50 to 60% of the phosphatidylglycerol in leaves from some of the most chilling-sensitive plants. However, not all chilling-sensitive plants contained high proportions of disaturated phosphatidylglycerol; solanaceous and other 16:3-plants and C₄ grasses may be important exceptions. Nonetheless, proportions of disaturated phosphatidylglycerol increased concomitantly with increasing chilling sensitivity of plants within a genus.

     

The effect of changing the composition of phosphatidylglycerol from thylakoid polar lipids of oleander and cucumber on the temperature of the transition related to chilling injury

The composition and phase behavior of some lipid classes and mixtures of thylakoid polar lipids were measured to investigate their role as determinants of the temperature of the transition associated with chilling injury. For Nerium oleander L., a plant which acclimates to growth temperature, a mixture of the phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG) showed transition temperatures of 22° and 10° C for plants grown at 45° and 20° C, respectively. This difference was similar to the 9 Celsius degrees differential in the transition of the polar lipids and indicated that the PG and-or the PG-SQDG mixture could be the major determinants of the transition temperature. Reconstitution of the PG-SQDG mixture from 20°-grown oleander with the galactolipids from 45°-grown plants, however, reduced the transition temperature by only 4 Celsius degrees. This indicates that some, low-melting-point lipids, which are structurally capable of forming a co-gel with the high-melting-point lipids, also play a role in determining the temperature of the transition and that the composition of these low-melting-point lipids also changes with growth temperature. More specific information on the role of PG was obtained using polar lipids from Cucumis sativus L., a chilling-sensitive plant. For this material the transition in the polar lipids was reduced from 9° to 5° and 4° C when the transition of the PG was reduced from 32° to 25° and 22° C. This was accomplished by reducing the proportion of disaturated molecular species in PG from 78 to 56 and 44 mol% by the addition of a fraction of the PG enriched in unsaturated molecular species. The data indicate that the transition temperature of the polar lipids of cucumber would be reduced to below 0° C, typical of a chillinginsensitive plant, when the transition temperature of PG was reduced to 15° C and this would occur at 21 mol% of disaturated molecular species. It is concluded that the transition in the thylakoid polar lipids, associated with chilling injury, involves both high- and low-meltingpoint lipids but can be reduced when the transition temperature of the high-melting-point component is reduced.Abbreviations DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulfoquinovosyldiacylglycerol     

Temperature-dependent phase behavior of phosphatidylglycerols from chilling-sensitive and chilling-resistant plants

Seven major lipid classes were isolated from leaves of chilling-sensitive and chilling-resistant plants, and the temperature-dependent phase behaviors of their aqueous dispersions were studied by a fluorescence polarization method using trans-parinaric acid and its methyl ester. Phosphatidylglycerols from the chilling-sensitive plants went from the liquid crystalline state into the phase separation state at about 30°C in 100 mm NaCl and at about 40°C in 5 mm MgCl₂. In contrast, phosphatidylglycerols from the chilling-resistant plants went into the phase separation state at a much lower temperature. The other classes of lipids remained in the liquid crystalline state at all temperatures between 5°C and 40°C regardless of the chilling sensitivity of the plants, except sulfoquinovosyl diacylglycerol from sponge cucumber in which phase separation seemed to begin at about 15°C. Compositions and positional distributions of fatty acids of the lipids suggest that the phosphatidylglycerols from the chilling-sensitive plants, but no other lipids, contained large proportions of molecular species which undergo phase transition at room temperature or above. The thermotropic phase behaviors and the fatty acid compositions suggest that, among the major lipid classes from leaves of the chilling-sensitive plants, only phosphatidylglycerol can induce a phase transition. Since a major part of this lipid in leaves originates from the chloroplasts, phase transition probably occurs in the chloroplast membranes.     

Chilling-Induced Heat Evolution in Plants

Increases in respiration, particularly via the alternative pathway, are observed in response to chilling. These increases result in increased heat evolution. We have measured increases in heat evolution in response to chilling in a number of plant species using a microcalorimeter. After 8 h of exposure to 8[deg]C, heat evolution in a variety of chilling-sensitive species increased 47 to 98%. No increase in heat evolution was seen with the extremely chilling-sensitive ornamental Episcia cupreata Hook. Heat evolution increased only 7 to 22% in the chilling-resistant species. Increases in heat evolution were observed when plants were chilled in constant light or in the dark, but not when plants were chilled at high humidity. Increased capacity to produce respiratory heat after exposure to chilling temperatures may contribute to the cold-acclimation process.     

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.     

Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves

The FAD7 gene, a gene for a chloroplast [omega]-3 fatty acid desaturase, is responsible for the trienoic fatty acid (TA) formation in leaf tissues. The TA content of the leaf tissue of the 25[deg]C-grown transgenic tobacco (Nicotiana tabacum cv SR1) plants, in which the FAD7 gene from Arabidopsis thaliana was overexpressed, increased uniformly by about 10%. Fatty acid unsaturation in all major leaf polar lipid species increased in the 25[deg]C-grown FAD7 transformants but was approximately the same between the control plants and the FAD7 transformants when grown at 15[deg]C. Therefore, the overexpression of the exogenous FAD7 gene leads to the same consequence in the tobacco plants as the low-temperature-induced TA production that may be catalyzed by an endogenous, temperature-regulated chloroplast [omega]-3 fatty acid desaturase. In the 25[deg]C-grown control plants, the chilling treatment caused symptoms of leaf chlorosis and suppression of leaf growth. The 25[deg]C-grown FAD7 transgenic plants conferred alleviation of these chilling-induced symptoms. A reductions of the chilling injury similar to that of the FAD7 transformants was also observed in the 15[deg]C-preincubated control plants. These results indicate that the increased TA production during chilling acclimation is one of the prerequisites for the normal leaf development at low, nonfreezing temperatures.     

Compositional and Thermal Properties of Thylakoid Polar Lipids of Nerium oleander L. in Relation to Chilling Sensitivity

The polar lipid classes from thylakoids of Nerium oleander L. were studied with the aim of relating changes in their composition and thermal behavior with reported changes in the transition temperature of their polar lipids and chilling sensitivity of their leaves. With an increase in growth temperature, the transition temperature of phosphatidylglycerol increased from 16°C to 26°C, and for sulfoquinovosyldiacylglycerol from 19°C to 24°C. Transitions in the other lipid classes were below −10°C for plants grown at both growth temperature. The major changes in the molecular species of phosphatidylglycerol, with increasing growth temperature, were an increase in 1-oleoyl-2-palmitoyl phosphatidylglycerol from 21 to 39% and a decrease in 1-oleoyl-2-trans-3-hexadecanoic phosphatidylglycerol from 51 to 25%. Although the disaturated species increased from 8 to 23%, the maximum was less than that reported for chilling-sensitive plants. There was no change in the sum of the palmitic, hexadeca-trans-3-enoic and stearic acids. Dipalmitoyl sulfoquinovosyldiacylglycerol increased from 12 to 20% and 1-linolenoyl-2-palmitoyl sulfoquinovosyldiacylglycerol decreased from 40 to 30%. It is concluded that the increase in the transition temperature of the polar lipids and the sensitivity of acclimated oleander plants to chilling could not be predicted by the absolute sum of the saturated fatty acids or disaturated molecular species in phosphatidylglycerol. The polar lipid transition appears to be a product of mixing of both high and low melting-point lipids.     

Molecular Species Composition of Phosphatidylglycerols from Chilling-Sensitive and Chilling-Resistant Plants

The molecular species of phosphatidylglycerols from leaves of9 species of chilling-sensitive plants and 12 species of chilling-resistantplants were analyzed by gas-liquid chromatography and gas chromatography-massspectrometry. The sum of the contents of the dipalmitoyl plusthe 1-palmitoyl-2-(trans-3-hexadecenoyl) species of phosphatidylglycerolranged from 3 to 19% of the total of this lipid in the chilling-resistantplants, and from 26 to 65% in the chilling-sensitive plants.These findings suggest that these two molecular species of phosphatidylglycerolsare closely associated with the chilling sensitivity of theplants. The biochemical difference between the chilling-sensitiveand the chilling-resistant plants is discussed in terms of theactivities of enzymes involved in phosphatidylglycerol biosynthesisin the chloroplasts. (Received August 19, 1982; Accepted November 19, 1982)     

A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18°C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury—leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of ¹⁴CO₂ into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury.     

Arrhenius plots and the involvement of thermotropic phase transitions of the thylakoid membrane in chilling impairment of photosynthesis in thermophilic higher plants

Abstract Breaks and discontinuities in Arrhenius plots of physiological and physical properties of thylakoids are not diagnostic of thermotropic lipid phase transitions of the membrane. Bulk lipid transitions, as first inferred by the membrane phase transition hypothesis, do not occur in any higher plant at chilling temperatures. Solidification of some varying, but always minor, fraction of the total membrane lipid does take place. However, the presence of minor domains of solid thylakoid membrane lipid at chilling temperatures is not unique to chilling sensitive plants but is also found in tolerant species. Minor solidification may in some plants, or groups of plants, be controlled by the specific molecular species of phosphatidylglycerol only recently investigated. In plants containing little, or no, phosphatidylglycerol with this positional distribution of fatty acids, other yet unknown constituents of the membrane must fill a similar function, since DSC thermograms indicate minor solidification also in isolated, unperturbed thylakoids from chilling tolerant species. However, chilling induced phase transitions, or other perturbations, of the thylakoid membrane are not the reason for the chilling lability of net photosynthesis in the intact plant. This conclusion follows from detailed comparison between photosynthetic membranes isolated from prechilled plants and the effects of chilling exposure on CO₂ fixation of the whole plant. Damage at the level of the thylakoid membrane does occur, although not to the extent where it can account for the proportionally much larger damage to CO₂ fixation.     

Compositions and Positional Distributions of Fatty Acids in Phospholipids from Leaves of Chilling-Sensitive and Chilling-Resistant Plants

The compositions and positional distributions of fatty acidsin the major leaf phospholipids of phosphatidylglycerol, phosphatidylcholineand phosphatidylethanolamine were analyzed by gas-liquid chromatographyand enzymic hydrolysis, and chilling-sensitive and chilling-resistantplants were comparcd with respect to the relative contents ofpalmitic and trans-³-hexadecenoic acids in the separated phospholipids.A distinct difference between these plants was found in thefatty acid compositions of phosphatidylglycerol, in which thesum of palmitic and trans-³-hexadecenoic acids ranged from 60to 78% of the total fatty acids in 8 species of chilling-sensitiveplants, and from 50 to 57% in 11 species of chilling-resistantplants. The only exception among the chilling sensitive plantsin this respect was the tomato, in which the sum of palmiticand trans-³-hexadecenic acids in phosphatidylglycerol amountedto 54%. The fatty acid compositions and the positional distributionsof fatty acids in phosphatidylglycerol suggest that the occurrenceof high proportions of dipalmitoyl and 1-palmitoyl-2-(trans-³-hexadecenoyl)species in this lipid is correlated with the susceptibilityto chilling of the leaves of higher plants. In the compositionsand positional distributions of fatty acids in phosphatidylcholineand phosphatidylethanolamine, no difference was found betweenthe chilling-sensitive and chilling-resistant plants. ¹ Present address: Department of Biology, Faculty of Science,Universityof Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received May 21, 1982; Accepted June 25, 1982)     

Chilling sensitivity in higher plants: the role of phosphatidylglycerol

Abstract A compilation of data on the level of high melting point fatly acids in the phosphalidylglycerol of leaves of higher plants suggests that the content of these acids is not directly related to the chilling sensitivity of the plant. Within a given plant family the level of high melting point fatty acids in phosphatidylglycerol appears to be relatively constant, although the individual species may differ widely in their susceptibility to low temperature. It seems possible that differences observed in the levels of high melting point fatty acids in the phosphatidylglycerols of chilling-sensitive and chilling-tolerant plants may be largely due to choice of experimental material.     

A suppressor of fab1 challenges hypotheses on the role of thylakoid unsaturation in photosynthetic function

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 degrees 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 degrees C, the suppressors remained viable after 16 weeks in the cold, as judged by their ability to resume growth following a return to 22 degrees 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 Delta7 desaturase. A double mutant produced by crossing fab1 to the original fad5-1 allele also remained alive at 2 degrees 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 KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

The fab1 mutant of Arabidopsis is partially deficient in activity of beta-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16:0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a cDNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size-exclusion chromatography and was severely impaired in condensation activity. These results suggest that the molecular defect in fab1 plants is a structural instability of the KAS2 gene product induced by insufficient space for the imidazole ring of the mutant phenylalanine residue.     

Lipid response to short-term chilling shock and long-term chill hardening in Jatropha curcas L. seedlings

Low non-freezing temperature is one of the major environmental factors that affect metabolism, growth, development and geographical distribution of chilling-sensitive plants, Jatropha curcas, a chilling-sensitive plant, which is considered as a sustainable energy plant with great potential for biodiesel production. Our previous studies showed that short-term chilling shock at 5 °C for 4 h and long-term chill hardening at 12 °C 1 or 2 days could improve chilling tolerance of J. curcas seedlings, but lipidomic response to chilling shock and chill hardening has not been elucidated. In this study, membrane lipid composition change in J. curcas seedlings during chilling shock and chill hardening was investigated by liquid chromatography-electrospray ionization-mass spectrometry (LC–ESI–MS) approach. The results indicated that the relative abundances of nine classes and 72 species of membrane lipids, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), lysophosphatidylcholine (lysoPC) and lysophosphatidylglycerol (lysoPG), two glycolipids digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG) and a sulfoquinovosyldiacylglycerol (SQDG), were significantly changed, and the degree of unsaturation of above-mentioned cellular membrane lipids with fatty acid differing in chain lengths and the number of double bonds also increased in varying degrees during chilling shock and chill hardening. These results suggested that remodeling and increase in the degree of unsaturation of membranes lipids may be a common physiological basis for short-term chilling shock- and long-term chill hardening-induced chilling tolerance of J. curcas seedlings.     

Low and High Temperature Limits to PSII : A Survey Using trans-Parinaric Acid, Delayed Light Emission, and F(o) Chlorophyll Fluorescence

Many studies have shown that membrane lipids of chilling-sensitive plants begin lateral phase separation (i.e. a minor component begins freezing) at chilling temperatures and that chilling-sensitive plants are often of tropical origin. We tested the hypothesis that membranes of tropical plants begin lateral phase separation at chilling temperatures, and that plants lower the temperature of lateral phase separation as they invade cooler habitats. To do so we studied plant species in one family confined to the tropics (Piperaceae) and in three families with both tropical and temperate representatives (Fabaceae [Leguminosae], Malvaceae, and Solanaceae). We determined lateral phase separation temperatures by measuring the temperature dependence of fluorescence from trans-parinaric acid inserted into liposomes prepared from isolated membrane phospholipids. In all families we detected lateral phase separations at significantly higher temperatures, on average, in species of tropical origin. To test for associated physiological effects we measured the temperature dependence of delayed light emission (DLE) by discs cut from the same leaves used for lipid analysis. We found that the temperature of maximum DLE upon chilling was strongly correlated with lateral phase separation temperatures, but was on average approximately 4°C lower. We also tested the hypothesis that photosystem II (PSII) (the most thermolabile component of photosynthesis) of tropical plants tolerates higher temperatures than PSII of temperate plants, using DLE and F_o chlorophyll fluorescence upon heating to measure the temperature at which PSII thermally denatured. We found little difference between the two groups in PSII denaturation temperature. We also found that the temperature of maximum DLA upon heating was not significantly different from the critical temperature for F_o fluorescence. Our results indicate that plants lowered their membrane freezing temperatures as they radiated from their tropical origins. One interpretation is that the tendency for membranes to begin freezing at chilling temperatures is the primitive condition, which plants corrected as they invaded colder habitats. An alternative is that membranes which freeze at temperatures only slightly lower than the minimum growth temperature confer an advantage.     

Chill-Induced Changes in the Activity and Abundance of the Vacuolar Proton-Pumping Pyrophosphatase from Mung Bean Hypocotyls

Changes in the properties of extractable vacuolar H+-pumping pyrophosphatase (V-PPase) and vacuolar ATPase activities in chilling-sensitive seedlings of mung bean (Vigna radiata) were investigated. Following chilling at 4[deg]C for 48 h, both hydrolytic and proton-pumping activities of the V-PPase increased 1.5- to 2-fold over controls and remained elevated even after 72 h at low temperatures. Vacuolar ATPase levels did not change significantly throughout the chilling regime. However a large increase in alcohol dehydrogenase activity during chilling suggests a shift toward fermentative metabolism, which can be expected to decrease ATPase activity in situ. Western blotting of vacuolar membrane-enriched fractions from control and treated plants has confirmed that the changes in V-PPase activity are mirrored by increases in the amount of pump protein. Results suggest a specific role for the V-PPase in protecting chill-sensitive plants from the injurious effects of low temperatures via the maintenance of the proton gradient across the vacuolar membrane.     

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.     

Correlation between the Circadian Rhythm of Resistance to Extreme Temperatures and Changes in Fatty Acid Composition in Cotton Seedlings

Fluctuations in fatty acid composition were examined in cotton (Gossypium hirsutum L. cv Deltapine 50) leaves during light-dark cycles of 12:12 h and under continuous light and were correlated to the rhythmic changes in chilling (5[deg]C) resistance (CR) and heat (53[deg]C) resistance (HR). The chilling-resistant and chilling-sensitive phases developed in the dark or the light period, respectively, and this rhythm persisted under continuous light for three cycles. The heat-resistant phase developed in the light period and an additional peak of HR occurred in the middle of the dark period. Under continuous light, only one peak of HR developed, lasting from the middle of the subjective night to the middle of the subjective day. The amounts of palmitic and oleic acids were constant during the light-dark cycle and under continuous light, but those of linoleic and linolenic acids fluctuated, attaining a high level in the middle of the dark period or the subjective night, and a low level in the middle of the light period or the subjective day. A low temperature of 20[deg]C induced CR and affected changes in fatty acid composition similar to those that occurred during the daily CR phase. A high temperature of 40[deg]C induced HR but did not affect changes in fatty acid composition. The results in their entirety show that the CR that develops rhythmically as well as the low-temperature-induced CR coincide with increased levels of polyunsaturated fatty acids. No correlation is found between changes in fatty acid composition and the HR that develops rhythmically or the high-temperature-induced HR.