Lipid involvement in oleoyl CoA desaturase activity of Fusarium oxysporum microsomes

Desaturation of oleoyl CoA by the microsomal fraction of Fusarium oxysporum hyphal cells required O2, NADPH, MgCl2, and the addition of either bovine serum albumin or the 105 000g supernatant fraction. In the absence of reduced nucleotide, [14C]oleoyl CoA was rapidly incorporated into phospholipid and triacylglycerol and hydrolyzed to free fatty acids. After addition of NADPH, oleate was desaturated at the normal rate. Analysis of the distribution of [14C]oleate and [14C]linoleate between different lipid classes revealed that phosphatidylcholine and phosphatidylethanolamine were labeled with [14C]linoleate before any other lipid class. These results are consistent with oleoyl phospholipid being a direct intermediate in the desaturation of oleoyl CoA. The preference of the oleoyl-desaturase for NADPH, the relatively high pH optimum of 8.2, and the sensitivity to thenoyltrifluoroacetone inhibition suggest that some components of the microsomal electron-transport chain are common to both the oleoyl desaturase and stearoyl CoA desaturase systems in this fungus.     

Oleate metabolism in microsomes from developing leaves ofPisum sativum L.

Microsomes from young leaves of pea,Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA:lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties.Abbreviations FA unesterified fatty acid (s) - PC phosphatidylcholines - 18:1 oleoyl moieties - 18:2 lmoleoyl moieties Dedicated to Professor Helmut K. Mangold, Bundesanstalt für Fettforschung, Münster, on his 60th birthday     

Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed.

1. [14C]Oleoyl-CoA was metabolized rapidly and essentially completely by microsomal preparations from developing safflower (Carthamus tinctorius) cotyledons, and most of the [14C]oleate was incorporated into 3-sn-phosphatidylcholine. 2. In aerobic reaction mixtures containing NADH2 the [14C]oleate in 3-sn-phosphatidylcholine was converted into [14C]linoleate without any change in the specific radioactivity of the lipid. Over a 60 min incubation period the extent of conversion of [14C]oleoyl phosphatidylcholine into [14C]linoleoyl phosphatidylcholine was generally greater than 60%. The rate of desaturation of endogenous [14C]oleoyl phosphatidylcholine labelled from [14C]oleoyl-CoA was much greater that of exogenous [14C]dioleoyl phosphatidylcholine the specific radioactivity of the oleoyl moiety of the lipid remained constant, indicating that labelled and unlabelled oleate were desaturated at the same rate. On this assumption an initial rate of desaturation of about 15 nmol of oleate desaturated/min per mumol of 3-sn-phosphatidylcholine was estimated. 4. [14C]Oleate esterified at positions 1 and 2 of both endogenous and exogenous 3-sn-phosphatidylcholine was desaturated. 5. Attempts to demonstrate the presence of an oleoyl-CoA desaturase in safflower microsomal fractions by the appearance of linoleoyl-CoA in reaction mixtures were inconclusive.     

Lipid metabolism in microsomal fraction from photosynthetic tissue. Effects of catalase and hydrogen peroxide on oleate desaturation.

On incubation of microsomal fraction from pea (Pisum sativum L.) leaves with ammonium [1-14C]oleate or [1-14C]oleoyl-CoA in the presence of ATP, CoA, Mg2+ and NADH, the major reactions observed were those catalysed by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. The reaction catalysed by oleoyl phosphatidylcholine desaturase was specifically inhibited by H2O2, and this inhibitory effect was overcome by catalase (EC 1.11.1.6).     

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography     

Oleate from triacylglycerols is desaturated in cold-induced developing sunflower (Helianthus annuus L.) seeds

For the first time, an active fatty-acid metabolism is indicated for triacylglycerols (TAG) of developing sunflower (Helianthus annuus L.) seeds. When the developing seeds were transferred to low temperature, the total amount of oleate found in TAG decreased as that of linoleate increased, while the contents of total lipids and TAG remained unchanged. These results suggest that oleate from TAG was used for desaturation. This occurred first in microsomal TAG, but after a long cold period it was observed mainly in the oil-body fraction. Thesn-2 position of TAG was preferentially enriched in linoleate. Apparently, more linoleate than necesary for the maintenance of membrane fluidity was synthesized at the expense of TAG oleate.     

Solubilisation of oleoyl-CoA thioesterase,oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase: The oleate desaturase system of pea leaf microsomes

Membrane-bound enzymes involved in oleate metabolism in microsomes from pea (Pisum sativum L.) leaves were solubilised using detergents, such as n-octyl glucoside, Triton X-100, digitonin or cholate. The detergents were found to be inhibitory to oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Detergent removal by dialysis resulted in the restoration of activity of both the solubilised oleoyl-CoA thioesterase and oleoyl-CoA:phosphatidylcholine acyltransferase. The putative components of the oleoyl phosphatidylcholine desaturase system were also partially solubilised.     

Interaction of free fatty acids with the erythrocyte membrane as affected by hyperthermia and ionizing radiation

The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (100), lauric (120), myristic (140), oleic (cis-181) and elaidic (trans-181) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37°, but also at 42° and 47°C lauric acid (120) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis.     

Oleate desaturation and acyl turnover in sunflower (Helianthus annuus L.) seed lipids during rapid temperature adaptation

In-vivo experiments with developing sunflower (Helianthus annuus L.) seeds demonstrated that oleate desaturase activity was stimulated by low temperature (10 °C), repressed by high temperature (30 °C) and rapidly restored by returning the seeds to low temperature. Within time periods of 2–4 h, in which the de-novo fatty acid synthesis was negligible, the percentages of oleate (18:1) and linoleate (18:2) were modified in the seed lipids as a consequence of temperature adaptation. When the seeds were transferred to low temperature, the 18:2 content increased in all lipids from both microsomal membranes and oil bodies. After shifting to high temperature, the overall 18:2 content remained constant, but the 18:2 content decreased in diacylglycerols, phosphatidylcholine (PC) and other polar lipids of the two fractions and also in triacylglycerols (TAGs) of the microsomes but increased in TAGs of the oil bodies. The results indicate that the mechanism for the rapid adaptation of sunflower seeds to temperature changes involves (i) the synthesis or activation of oleate desaturase at low temperature and the reversible inhibition of this enzyme at high temperature and (ii) the exchange of 18:1 and 18:2 between TAGs and PC. Under both low and high temperature, 18:1 is transferred from reserve TAGs to PC and 18:2 is transferred from PC to reserve TAGs. At low temperature, 18:1 is desaturated to 18:2 thus allowing the enrichment of membrane lipids with 18:2, the excess being stored in reserve TAGs. At high temperature, however, and provided that oleate desaturase is repressed, the membrane lipids become enriched in 18:1 and the oil-body TAGs become enriched in 18:2. Received: 11 August 1997 / Accepted: 10 November 1997     

Radiolabelling studies of fatty acids in pisum sativum and Vicia faba leaves at different temperatures

Leaves of pea and broad bean plants were incubated with acetate-[¹⁴C] at temperatures varying from 7 to 34°. No significant difference was observed in the distribution of radioactivity between phosphatidylcholine and the galactosylglycerides in pea with different temperatures. However, increasing temperatures increased the labelling of phosphatidylcholine in broad bean leaves, at the expense of polar lipids other than the galactosylglycerides. The incubation temperature had no significant effect on the pattern of labelling of the fatty acids of the major leaf lipids. A correlation was seen in the specific radioactivity of oleate and linoleate in phosphatidylcholine and, especially, in the galactosylglycerides. The data emphasise the rapid equilibration of oleate and linoleate (which probably occurs by transacylation) between the two galactosylglycerides and phosphatidylcholine in leaf tissues.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower

The utilisation of [¹⁴C]glycerol 3-phosphate and [¹⁴C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C₁₈-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.Abbreviation BSA bovine serum albumin     

Influence of photoperiod,temperature and host plant on the production of diapause eggs inPetrobia (Tetranychina) harti (Acari: Tetranychidae)

Petrobia harti (Ewing) diapauses in the egg stage. Adult females lay either diapause or nondiapause eggs. On the University of Thessaloniki campus (41°N), the mite was found to develop on leaves ofOxalis corniculata L. throughout the year, while no mites were found on leaves ofOxalis articulata Savigny growing in the same area. In the laboratory the mite could be maintained equally well on detached leaves of both plant species, kept on wet cotton-wool.Forty to 90% females laying diapause eggs (dlf) were produced when the mites developed under LD 1212 and 19±1 °C, or LD 168 and 19±1 °C or 25±1 °C on leaves ofO. articulata detached from plants grown in the open in various seasons. Under the same conditions, a very low to zero percentage ofdlf was produced onO. corniculata. By rearing certain feeding stages on one of these twoOxalis hosts, and the other feeding stages on the other host, various percentages ofdlf were obtained. These percentages were the net effect of the antagonistic action of the twoOxalis species.By rearing the mites at LD 8.515.5, LD 1212 or LD 168 and a temperature of 19±1 °C onO. articulata leaves renewed every 3 days, or every 16–18 days, or not at all, it could be shown that diapause induction or aversion is caused by the direct effect of photoperiod on the mites, and not by an effect through the host leaves.When wholeO. articulata plants were grown under LD 168 and 19±1 °C in the laboratory, or developed in the open during April and May, flowers were produced, while under LD 1212 no flowering occurred. In the laboratory under diapause-inducing conditions, higher percentages ofdlf were produced on leaves detached from flowering plants than on leaves detached from plants not flowering.OnO. articulata leaves at 20 °C, photoperiods with photophases equal to or longer than 12 h induced from 70 to 80%dlf, while photoperiods with photophases equal to or shorter than 10.9 h induced very low to zero percentages. By transferring different chrysalis stages from a diapause-inducing (LD 1212) to a diapause-averting (LD 8.515.5) photoperiod, and vice versa, it was found that the nymphochrysalis through deutonymph stages were sensitive to photoperiod, the deutochrysalis and deutonymph being the most sensitive.Under an LD 1212 photoperiod, a temperature of 20 °C induced diapause, whereas 25 °C, 30 °C, or a daynight thermoperiod of 25 °C18 °C suppressed it.     

The utilization and desaturation of oleate and linoleate during glycerolipid biosynthesis in olive (Olea europaea L.) callus cultures

Callus cultures from olive (Olea europaea L.) were used to study characteristics of desaturation in this oil-rich tissue. The incorporation of [1-(14)C]oleate and [1-(14)C]linoleate into complex lipids and their further desaturation was followed in incubations of up to 48 h. Both radiolabelled fatty acids were rapidly incorporated into lipids, especially phosphatidylcholine and triacylglycerol. Radiolabelling of these two lipids peaked after 1-4 h, after which it fell. In contrast, other phosphoglycerides and the galactosylglycerides were labelled in a more sustained manner. [1-(14)C]Linoleate was almost exclusively found in the galactolipids. With [1-(14)C]linoleate as a precursor, the only significant desaturation to linolenate was in the galactolipids. Monogalactosyldiacylglycerol was the first lipid in which [1-(14)C]linoleate and [1-(14)C]linolenate appeared after incubation of the calli with [1-(14)C]oleate and [1-(14)C]linoleate, respectively. The presence of radioactivity in the plastidial lipids shows that both [1-(14)C]oleate and [1-(14)C]linoleate can freely enter the chloroplast. Two important environmental effects were also examined. Raised incubation temperatures (30-35 degrees C) reduced oleate desaturation and this was also reflected in the endogenous fatty acid composition. Low light also caused less oleate desaturation. The data indicate that lysophosphatidylcholine acyltransferase is important for the entry of oleate and linoleate into olive callus lipid metabolism and phospholipid:diacylglycerol acyltransferase may be involved in triacylglycerol biosynthesis. In addition, it is shown that plastid desaturases are mainly responsible for the production of polyunsaturated fatty acids. Individual fatty acid desaturases were differently susceptible to environmental stresses with FAD2 being reduced by both high temperature and low light, whereas FAD7 was only affected by high temperature.     

The incorporation of oleic and linoleic acids and their desaturation products into the glycerolipids of maize leaves

[1-¹⁴C]Oleic and [1-¹⁴C]linoleic acids were rapidly desaturated when incubated with maize leaves from 8-day-old plants and the labeled fatty acids, and their desaturation products, were rapidly incorporated into glycerolipids. Oleic acid was desaturated to linoleate at the rate of 0.7 nmol/100 mg tissue/h and further desaturated to linolenate at about one-third this rate. The rates of linolenate formation were similar when either oleic acid or linoleic acid was the substrate although there was a 2-h lag period when oleic acid was substrate. When radioactive oleic, linoleic, and linolenic acids were substrates, phosphatidylcholine was the most extensively labeled glycerolipid followed by monogalactosyldiacylglycerol. The relative rates of incorporation of label into individual glycerolipids are consistent with a movement of labeled fatty acids from phosphatidylcholine to monogalactosyldiacylglycerol and then to diagalactosyldiacylglycerol. The rates of labeling of phosphatidylcholine oleate and of phosphatidylcholine linoleate are consistent with a precursor-product relationship in that there was a delayed accumulation of phosphatidylcholine linoleate relative to that of phosphatidylcholine oleate and phosphatidylcholine linoleate continued to accumulate while phosphatidylcholine oleate declined. Linoleate formed from oleate was widely distributed in glycerolipids but neither phosphatidylcholine linolenate nor linolenate-containing diacylglycerol was detected at short and intermediate incubation times when either oleic or linoleic acid was substrate. The kinetics of incorporation of linoleate and linolenate into monogalactosyldiacylglycerol suggest a transfer of linoleate from phosphatidylcholine. The initial rate of accumulation of labeled linolenate in monogalactosyldiacylglycerol was very similar to the rate of desaturation of linoleate and it is suggested that desaturation of linoleate occurs while associated with monogalactosyl-diacylglycerol.     

High-Oleate Oilseeds Fail to Develop at Low Temperature

The fad2 mutants of Arabidopsis thaliana are deficient in activity of the endoplasmic reticulum oleate desaturase that is the main enzyme responsible for polyunsaturated lipid synthesis in developing seeds of oil crops. A comparison of wild-type and fad2 seeds developing on heterozygous (FAD2/-) plants was used as a model for genetically engineered high-oleate oilseeds of species such as soybean and canola. When fad2 seeds developed at normal temperatures (22[deg]C), they showed high viability compared to wild-type seeds. When a portion of seed development took place at 6[deg]C, germination of the wild-type siblings remained high but germination of fad2 segregants declined considerably. This was true even when exposure to low temperature was limited to the final stages of seed filling and maturation. Compared to wild-type seeds, fully viable fad2 seeds produced at 22[deg]C had reduced lipid contents and were slower to germinate at 10 and 6[deg]C. Taken together, these results indicate that for some oilseed species at least, molecular genetic manipulation of oleate levels in the oil may result in plant lines with unacceptable performance in the field.     

Molecular Cloning and Stress-Dependent Expression of a Gene Encoding ω3-Fatty Acid Desaturase in the Microalga Dunaliella salina

A fragment of the gene des3-1 encoding 3 fatty acid desaturase was cloned from a cDNA library of the unicellular green galophilic alga Dunaliella salina. The comparative phylogenetic analysis of 3-desaturase amino acid sequences from diverse organisms placed the desaturase of D. salina between cyanobacteria and higher plants in the evolutionary range of desaturases. The expression of des3-1 was studied in D. salina cells exposed to low temperatures, high irradiance, and high CO₂ concentrations. Lowering the external temperature from 32 to 22°C produced a transient increase in the level of specific mRNA. Considerable accumulation of mRNA for 3-desaturase was also observed when CO₂ concentration in gas–air mixture was raised from 2 to 10%. An irradiation increase from 70 to 500 mol/(m² s) did not affect the level of specific mRNA. The latter evidence presumes that in Dunaliella cells, this desaturase is probably located in the endoplasmic reticulum, rather than in the chloroplast.     

Involvement of polyamines in cytoplasmic male sterility of stem mustard (Brassica juncea var. tsatsai)

Changes in carbon isotope composition(¹³C) and leaf morphology associated withvegetative phase change were monitored in Metrosiderosexcelsa Sol. ex Gaertn. (family Myrtaceae). Plants of threeontogenetic states were used: juvenile seedlings, micropropagated plants in arejuvenated state, and reproductively mature plants bearing leaves with adultcharacteristics. The effects of temperature regime (32/24 °C,24/16 °C, and 16/8 °C day/night) and plantarchitecture (branched and single-stemmed plants) were studied in two separateexperiments. Although both juvenile and rejuvenated plants exhibited juvenileleaf morphology at the start of the experiments, there was no differencebetweenleaf ¹³C in these plants and that in adultplantsat this time (mean ca. –27%). Vegetative phase change occurred injuvenileand rejuvenated plants grown at 24/16 °C, and there was acorresponding increase in leaf ¹³C (from ca.–27% to –23%) in these two groups of plants. Leaf¹³C in adult plants remained relatively constant(ca. –26%) at 24/16 °C. There was little change in leaf¹³C in all plant states maintained at 32/24°C or 16/8 °C, and vegetative phase change didnot occur in juvenile and rejuvenated plants grown under these two temperatureregimes. Rejuvenated plants grown in a greenhouse also exhibited a progressivedevelopment of adult leaf morphology, accompanied by an increase in leaf¹³C, an effect that was more pronounced insingle-stemmed (from –26.4% to ca. –24%) than in branched plants. Itis suggested that increasing ¹³C in juvenile andrejuvenated plants undergoing phase change is a result of reduced sink strengthin single-stemmed plants, and to a lesser extent within each branch of branchedplants, causing reduced stomatal conductance and photosynthesis.     

Biosynthesis of gamma-linolenic acid in cotyledons and microsomal preparations of the developing seeds of common borage (Borago officinalis).

The developing seeds of Borago officinalis (common borage) accumulate a triacylglycerol oil that is relatively rich in the uncommon fatty acid gamma-linolenate (octadec-6,9,12-trienoic acid). Incubation of developing, whole, cotyledons with [14C]oleate and [14C]linoleate showed that the gamma-linolenate was synthesized by the sequential desaturation of oleate----linoleate----gamma-linolenate. Microsomal membrane preparations from the developing cotyledons contained an active delta 6-desaturase enzyme that catalysed the conversion of linoleate into gamma-linolenate. Experiments were designed to manipulate the [14C]linoleate content of the microsomal phosphatidylcholine. The [14C]linoleoyl phosphatidylcholine labelled in situ was converted into gamma-linolenoyl phosphatidylcholine in the presence of NADH. The substrate for the delta 6-desaturase in borage was, therefore, the linoleate in the complex microsomal lipid phosphatidylcholine, rather than, as in animals, the acyl-CoA. This was further confirmed in experiments that compared the specific radioactivity of the gamma-linolenate, in acyl-CoA and phosphatidylcholine, that was synthesized when [14C]linoleoyl-CoA was incubated with microsomal membranes, NADH and non-radioactive gamma-linolenoyl-CoA. The delta 6-desaturase was positionally specific and only utilized the linoleate in position 2 of sn-phosphatidylcholine. Analysis of the positional distribution of fatty acids in the endogenous microsomal sn-phosphatidylcholine showed that, whereas position 1 contained substantial linoleate, only small amounts of gamma-linolenate were present. The results shed further light on the synthesis of C18 polyunsaturated fatty acids in plants and in particular its relationship to the regulation of the acyl quality of the triacylglycerols in oilseeds.