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.

     

Fatty acids of lipids from cultured soybean and rape cells

Lipids from cultured cells, leaves and seeds of two varieties each of soybean (Glycine max) and oil seed rape (Brassica napus) were separated into neutral lipids, glycolipids and phospholipids and their fatty acids were analysed. Usually, the fatty acid composition differed between corresponding fractions from cultured cells, leaves and seeds. Differences were least marked in (i) the phospholipids from cultured cells and leaves of soybean and (ii) the neutral lipids from cultured cells and seeds of rape. In the cultured cells, the fatty acid composition of the phospholipids differed from that of the glycolipids and neutral lipids, and fatty acids of chain length greater than C₁₈ comprised a large proportion of the fatty acids of the glycolipids.     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Further evidence for an elongation-decarboxylation mechanism in the biosynthesis of paraffins in leaves

In isolated tobacco leaves l-valine-U-¹⁴C gave rise to labeled even-numbered isobranched fatty acids containing 16 to 26 carbon atoms and iso C₂₉, iso C₃₁, and iso C₃₃ paraffins. l-Isoleucine-U-¹⁴C on the other hand produced labeled odd-numbered anteiso C₁₇ to C₂₇ fatty acids and anteiso C₃₀ and C₃₂ paraffins. Trichloroacetic acid inhibited the incorporation of isobutyrate into C₂₀ and higher fatty acids and paraffins without affecting the synthesis of the C₁₆ and C₁₈ fatty acids. Thus the very long branched fatty acids are biosynthetically related to the paraffins. In Senecio odoris leaves acetate-1-¹⁴C was incorporated into the paraffins (mainly n-C₃₁) only in the epidermis although acetate was readily incorporated into fatty acids in the mesophyll tissue. Similarly only the epidermal tissue incorporated acetate into fatty acids longer than C₁₈ suggesting that the epidermis is the site of synthesis of both paraffins and the very long fatty acids. In broccoli leaves n-C₁₂ acid labeled with ¹⁴C in the carboxyl carbon and ³H in the methylene carbons was incorporated into C₂₉ paraffin without the loss of ¹⁴C relative to ³H. Since n-C₁₈ acid is known to be incorporated into the paraffin without loss of carboxyl carbon these results suggest that the condensation of C₁₂ acid with C₁₈ acid is not responsible for n-C₂₉ paraffin synthesis in this tissue. Thus all the experimental evidence thus far obtained strongly suggests that elongation of fatty acids followed by decarboxylation is the most likely pathway for paraffin biosynthesis in leaves.     

Lipid and Fatty Acid composition of chloroplast envelope membranes from species with differing net photosynthesis

Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was similar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C_16:0, 41 and 36%) and linolenic (C_18:3, 29 and 40%) acids for spinach and sunflower; palmitic (77%) and stearic (C_18:0, 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative.     

Galactolipid Synthesis in Vicia faba Leaves: IV. Site(s) of Fatty Acid Incorporation into the Major Glycerolipids

The fatty acids of the major glycerolipids of Vicia faba leaves were analyzed immediately following ¹⁴CO₂ feeding. The leaves were fractionated into chloroplast and cytoplasmic fractions and the location of radioactivity in the fatty acids determined. The results indicate that the major site of incorporation of fatty acids is in the phospholipids. Phosphatidylcholine contained the highest level of radioactivity in the cytoplasmic fraction, whereas phosphatidylglycerol contained radioactivity in both the chloroplast and cytoplasmic fractions. The galactolipids contained very little radioactivity in comparison, this radioactivity being confined to high speed centrifugal fractions believed to contain the envelopes of the chloroplast. Our results suggest that phosphatidylcholine is a major site of incorporation of fatty acids (mainly in oleic acid) in the cytoplasm, whereas phosphatidylglycerol is also a site of incorporation involving both oleic and palmitic acids, inside and outside the chloroplast.     

Biosynthesis and desaturation of prokaryotic galactolipids in leaves and isolated chloroplasts from spinach

Mono- and digalactosyldiacylglycerol (MGDG and DGDG) were isolated from the leaves of sixteen 16:3 plants. In all of these plant species, the sn-2 position of MGDG was more enriched in C₁₆ fatty acids than sn-2 of DGDG. The molar ratios of prokaryotic MGDG to prokaryotic DGDG ranged from 4 to 10. This suggests that 16:3 plants synthesize more prokaryotic MGDG than prokaryotic DGDG. In the 16:3 plant Spinacia oleracea L. (spinach), the formation of prokaryotic galactolipids was studied both in vivo and in vitro. In intact spinach leaves as well as in chloroplasts isolated from these leaves, radioactivity from [1-¹⁴C]acetate accumulated 10 times faster in MGDG than in DGDG. After 2 hours of incorporation, most labeled galactolipids from leaves and all labeled galactolipids from isolated chloroplasts were in the prokaryotic configuration. Both in vivo and in vitro, the desaturation of labeled palmitate and oleate to trienoic fatty acids was higher in MGDG than in DGDG. In leaves, palmitate at the sn-2 position was desaturated in MGDG but not in DGDG. In isolated chloroplasts, palmitate at sn-2 similarly was desaturated only in MGDG, but palmitate and oleate at the sn-1 position were desaturated in MGDG as well as in DGDG. Apparently, palmitate desaturase reacts with sn-1 palmitate in either galactolipid, but does not react with the sn-2 fatty acid of DGDG. These results demonstrate that isolated spinach chloroplasts can synthesize and desaturate prokaryotic MGDG and DGDG. The finally accumulating molecular species, MGDG(18:3/16:3) and DGDG(18:3/16:0), are made by the chloroplasts in proportions similar to those found in leaves.     

Tests Whether a Head to Head Condensation Mechanism Occurs in the Biosynthesis of n-Hentriacontane, the Paraffin of Spinach and Pea Leaves

Isobutyrate-1-¹⁴C and l-isoleucine-U-¹⁴C fed through the petiole labeled the surface lipids of broccoli leaves, but the incorporation was much less than from straight chain precursors. Not more than one-third of the ¹⁴C incorporated into the surface lipids was found in the C₂₉ paraffin and derivatives, whereas more than two-thirds of the ¹⁴C from straight chain precursors are usually found in these compounds. The small amount of ¹⁴C incorporated into the paraffin fraction was found in the n-C₂₉ paraffin rather than branched paraffins showing that the ¹⁴C in the paraffin must have come from degradation products. Radio gas-liquid chromatography of the saturated fatty acids showed that, in addition to the n-C₁₆ acid which was formed from both branched precursors, isoleucine-U-¹⁴C gave rise to branched C₁₅, C₁₇, and C₁₉ fatty acids, and isobutyrate-1-¹⁴C gave rise to branched C₁₆ and C₁₈ acids. Thus the reason for the failure of broccoli leaf to incorporate branched precursors into branched paraffins is not the unavailability of branched fatty acids, but the absolute specificity of the system that synthesizes paraffins, probably the elongation-decar-boxylation enzyme complex. Consistent with this view, no labeled branched fatty acids longer than C₁₉ could be found in the broccoli leaf. The branched fatty acids were also found in the surface lipids indicating that the epidermal layer of cells did have access to branched chains. Thus the paraffin synthesizing enzyme system is specific for straight chains in broccoli, but the fatty acid synthetase is not.     

Manipulating membrane Fatty Acid compositions of whole plants with tween-Fatty Acid esters

This paper describes a method for manipulating plant membrane fatty acid compositions without altering growth temperature or other conditions. Tween-fatty acid esters carrying specific fatty acids were synthesized and applied to various organs of plants growing axenically in glass jars. Treated plants incorporated large amounts of exogenous fatty acids into all acylated membrane lipids detected. Fatty acids were taken up by both roots and leaves. Fatty acids applied to roots were found in leaves, while fatty acids applied to leaves appeared in both leaves higher on the plant and in roots, indicating translocation (probably in the phloem). Foliar application was most effective; up to 20% of membrane fatty acids of leaves above the treated leaf and up to 40% of root membrane fatty acids were exogenously derived. Plants which took up exogenous fatty acids changed their patterns of fatty acid synthesis such that ratios of saturated to unsaturated fatty acids remained essentially unaltered. Fatty acid uptake was most extensively studied in soybean (Glycine max [L.] Merr.), but was also observed in other species, including maize (Zea mays L.), mung beans (Vigna radiata L.), peas (Pisum sativum L.), petunia (Petunia hybrida L.) and tomato (Lycopersicon esculentum Mill.). Potential applications of this system include studying internal transport of fatty acids, regulation of fatty acid and membrane synthesis, and influences of membrane fatty acid composition on plant physiology.     

Epicuticular wax composition in relation to aphid infestation and resistance in red raspberry (Rubus idaeus L.)

Epicuticular waxes from the aphid-resistant red raspberry (Rubus idaeus) cultivar Autumn Bliss and the aphid-susceptible cultivar Malling Jewel were collected from the newly emerging crown leaves, and also from the group of four more mature leaves immediately below the crown. Resistance and susceptibility status of the leaves to infestation by the large raspberry aphid, Amphorophora idaei, were determined by bioassay with the insect just prior to collection of the wax. Analysis showed the waxes to consist of a complex mixture of free fatty acids; free primary alcohols and their acetates; secondary alcohols; ketones; terpenoids including squalene, phytosterols, tocopherol and amyrins; alkanes and long chain alkyl and terpenyl esters. Compositional differences which may relate to A. idaei-resistance status were noticeably higher levels of sterols, particularly cycloartenol, together with the presence of branched alkanes, and an absence of C₂₉ ketones and the symmetrical C₂₉ secondary alcohol in wax from the resistant cultivar Bliss. There were also differences between the cultivars in the distribution of individual amyrins and tocopherols and in the chain length distribution for homologues of fatty acids, primary alcohols and alkanes, and these may also be related to resistance to A. idaei. Emerging leaves had lower levels of primary alcohols and terpenes, but higher levels of long-chain alkyl esters, and in general, more compounds of shorter chain-length than the more mature leaves. During bioassay A. idaei displayed a preference to settle on the more mature leaves. This may be due to greater wax coverage and higher levels of the compounds of shorter chain length found in the newly emerged younger leaves at the crown of the plant.     

Signature Lipids and Stable Carbon Isotope Analyses of Octopus Spring Hyperthermophilic Communities Compared with Those of Aquificales Representatives

The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12, Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus, and Aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. The n-C_20:1 and cy-C₂₁ fatty acids dominated all of the Aquificales, while the alkyl glycerol ethers were mainly C_18:0. These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C₁₈ and C₂₀ alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C_20:1 and cy-C₂₁, plus a series of iso-branched fatty acids (i-C_15:0 to i-C_21:0), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in ¹³C relative to source water CO₂ by 10.9 and 17.2‰, respectively. The C_20–21 fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6‰, respectively. The biomass of T. ruber grown on CO₂ was depleted in ¹³C by only 3.3‰ relative to C source. In contrast, biomass was depleted by 19.7‰ when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3‰). The depletion in the C_20–21 fatty acids from the PSC indicates that Thermocrinis biomass must be similarly depleted and too light to be explained by growth on CO₂. Accordingly, Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Lipids of the tropical seagrass Thallassia hemprichii

The component sterols, alcohols, hydrocarbons, monocarboxylic, α,ω-dicarboxylic and α- and ω-hydroxy acids from the leaves and roots of the tropical seagrass Thallassia hemprichii are reported. The leaves contained significant concentrations of cholest-5-en-3β-ol, a sterol not normally detected in either higher plants or seagrasses. The lower abundance of polyunsaturated fatty acids found in both the leaves and roots compared to other seagrass species may be a result of the warmer waters from which this species was collected. Solvent-extractable, long-chain (> C₂₂)α,ω-diacids, α- and ω-hydroxy and monocarboxylic acids were also isolated from the leaves. The distribution pattern of these lipids should enable these components along with other distinctive components to be used as chemical markers for this seagrass.     

Turnover of Fatty Acids during Natural Senescence of Arabidopsis, Brachypodium, and Switchgrass and in Arabidopsis β-Oxidation Mutants

During leaf senescence, macromolecule breakdown occurs and nutrients are translocated to support growth of new vegetative tissues, seeds, or other storage organs. In this study, we determined the fatty acid levels and profiles in Arabidopsis (Arabidopsis thaliana), Brachypodium distachyon, and switchgrass (Panicum virgatum) leaves during natural senescence. In young leaves, fatty acids represent 4% to 5% of dry weight and approximately 10% of the chemical energy content of the leaf tissues. In all three species, fatty acid levels in leaves began to decline at the onset of leaf senescence and progressively decreased as senescence advanced, resulting in a greater than 80% decline in fatty acids on a dry weight basis. During senescence, Arabidopsis leaves lost 1.6% of fatty acids per day at a rate of 2.1 μg per leaf (0.6 μg mg⁻¹ dry weight). Triacylglycerol levels remained less than 1% of total lipids at all stages. In contrast to glycerolipids, aliphatic surface waxes of Arabidopsis leaves were much more stable, showing only minor reduction during senescence. We also examined three Arabidopsis mutants, acx1acx2, lacs6lacs7, and kat2, which are blocked in enzyme activities of β-oxidation and are defective in lipid mobilization during seed germination. In each case, no major differences in the fatty acid contents of leaves were observed between these mutants and the wild type, indicating that several mutations in β-oxidation that cause reduced breakdown of reserve oil in seeds do not substantially reduce the degradation of fatty acids during leaf senescence.     

Effects of two sodium salts on fatty acid and essential oil composition of basil (Ocimum basilicum L.) leaves

The effects of two sodium salts on growth, fatty acids, and essential oil compositions were investigated in a medicinal and aromatic plant, Ocimum basilicum cultivated in hydroponic medium. Plants were subjected to an equimolar concentration of Na₂SO₄ (25 mM) and NaCl (50 mM) for 15 days. Our results showed that leaf growth rate was more depressed by 25 mM Na₂SO₄ than by 50 mM NaCl. The total fatty acid contents did not show any change in plants. α-Linolenic, palmitic, and linoleic acids were the major fatty acids. The identification of basil leaf fatty acids has not been previously studied and this work revealed the predominance of polyunsaturated fatty acids. Under both salts, leaf fatty acid composition remained unchanged. Regarding the essential oil yield, it decreased significantly by 28 % under 25 mM Na₂SO₄ and showed an increase by 27 % under 50 mM NaCl. The major volatile compound in leaves was linalool with 34.3 % of total essential oil constituents, followed by eugenol (19.8 %), 1.8-cineole (14.4 %) and methyl eugenol (5.2 %). Further, levels of eugenol and methyl eugenol were most modulated by salt, and the negative correlation between these two compounds reflects the stimulation of O-methyltransferase activity under both salts.     

Hydrocarbons and wax esters from seven species of mangrove leaves

Seven species of fresh mangrove leaves were found to contain saturated normal and branched chain hydrocarbons, mostly between C₁₆ and C₃₆ with both odd and even carbon numbers. Significant quantitative variations were found between species. Wax esters were found to contain fatty acids with chain lengths between C₁₂ and C₂₂. Palmitic (16:0) and stearic (18:0) acids were the major component saturated fatty acids, whereas, oleic (18:1) and linolenic (18:3) acids were the major unsaturate α-acids. Chain lengths of the alcohols of wax esters were between C₁₄ and C₃₆. Significant quantitative and minor qualitative differences were noted in the alcohol composition of wax esters. Hydrocarbon and wax ester compositions were characterised by the presence of low M, components in high proportions.     

Free Fatty acids regulate two galactosyltransferases in chloroplast envelope membranes isolated from spinach leaves

Effects of MgCl₂ and free fatty acids (FFA) on galactolipid:galactolipid galactosyltransferase (GGGT) and UDP-galactose: 1,2-diacylglycerol galactosyltransferase (UDGT) in chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.) leaves were examined. GGGT activity was sigmoidally stimulated by MgCl₂ with a saturated concentration of more than 5 millimolar. Free α-linolenic acid (18:3) caused a drastic increase in GGGT activity under limiting concentrations of MgCl₂, without affecting its maximum activity at higher MgCl₂ concentrations. Free 18:3 alone did not affect the GGGT activity. The effective species of FFA for the stimulation of GGGT activity in the presence of MgCl₂ were unsaturated 16- and 18-carbon fatty acids. GGGT activity was also stimulated by 18:3 in the presence of MnCl₂, CaCl₂ and a high concentration of KCl in place of MgCl₂. UDGT activity was hyperbolically enhanced by MgCl₂ with a saturated concentration of 1 to 2 millimolar. In contrast to GGGT, UDGT was severely inhibited by 18:3, and MgCl₂-induced stimulation was completely abolished by 18:3. Unsaturated 16- and 18-carbon fatty acids were more inhibitory to UDGT than the saturated acids. The dependence of GGGT activity on monogalactosyldiacylglycerol (MGDG) and MgCl₂ concentrations was identical in the envelope membranes isolated from non- and ozone (0.5 microliter/liter)-fumigated spinach leaves, indicating that GGGT remained active in the leaves during ozone fumigation. The results are discussed in relation to the regulation of galactolipid biosynthesis by the endogenous FFA in the envelopes and to the involvement of GGGT in the triacylglycerol synthesis from MGDG in ozone-fumigated leaves.     

Neutral lipids of leaves and stems of Trifolium repens

The neutral lipids of white clover leaves and stems have been separated into wax esters, free fatty acids, free fatty alcohols, free sterols, triglycerides and hydrocarbons. The wax esters were mainly of C₁₈ di- and tri-unsaturated fatty acids and C₃₀ fatty alcohol. Linolenic acid was the predominant free fatty acid and triacontanol was the principal free fatty alcohol. Of the hydrocarbons, C₂₉ and C₃₁ were present in the largest amounts.     

Content of fatty acids ofChlorella kessleri in a deep tank fermentation

Chlorella kessleri cultivated in a deep tank contained 4.8% of non-polar lipid; 51% of this fraction represents saturated fatty acids, 7% unsaturated fatty acids. Our investigation of the fatty acids profile demonstrated even- and odd-numbered saturated and unsaturated fatty acids ranging from C₁₂ to C₂₀. Unlike in otherChlorella species, stearic acid was the dominant fatty acid found. Also shown was an elevated C_16:0 fatty acid content and a reduced level of unsaturated fatty acids.     

Effects of Substituted Pyridazinones (San 6706, San 9774, San 9785) on Glycerolipids and Their Associated Fatty Acids in the Leaves of Vicia faba and Hordeum vulgare

The fatty acids of the major glycerolipids from the leaves of Vicia faba and Hordeum vulgare plants treated with three different concentrations of pyridazinone derivatives were analyzed. These compounds showed multiple effects on the levels of lipids and pigments. At low concentrations, the primary effect of San 9785 was on the level of linolenic acid (18:3) in the galactolipids of V. faba, whereas the effect of San 6706 was primarily on the trans-Δ³-hexadecenoic acid (16:1) content in phosphatidylglycerol. At higher concentrations, the two compounds reduced the content of both fatty acids in the leaves. The results appear to indicate a differential effect of these herbicides on fatty acid accumulation and a difference in susceptibility of two fatty acids in the species examined. Electron microscopic studies revealed that two herbicides caused different abnormalities in V. faba chloroplast ultrastructure.