Fat metabolism in higher plants XLIX fatty acid biosynthesis by subcellular fractions of higher plants

A method is described for the rapid and comprehensive subcellular fraction-ation of plant tissue using a combination of differential and discontinuous Ficoll gradient centrifugation. The procedure has been used to study the synthesis of fatty acids from acetate-1-¹⁴C or malonyl CoA-1,3-¹⁴C, by fractions of germinating pea and lupin seeds and developing avocado fruit, castor bean and safflower seeds. Particle free supernatants of seeds synthesize fatty acids from¹⁴C-malonyl CoA in the presence of added cofactors. Since acetyl CoA carboxylase activity is absent the utilization of¹⁴C-acetate by these fractions is minimal. Other particulate fractions show different activities depending on seed types. Active fractions include the low speed particulate of pea and lupin, the pea microsomes, the avocado mesocarp chloroplasts, and the fat fractions of castor bean and safflower. Individual fractions produce characteristic patterns of acids; especially noteworthy is oleic acid biosynthesis by soluble enzymes of castor bean and safflower from¹⁴C-malonyl CoA. Some characteristics of the avocado supernatant, pea supernatant, and castor bean fat fraction synthesizing systems are compared. As a result of these studies, generalizations derived from work with mammalian or bacterial systems cannot be applied to higher plants. Presented in part at the AOCS Meeting, Houston, May 1971.     

General resistance to sterol biosynthesis inhibitors inSaccharomyces cerevisiae

Screening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessivefen1-1 mutation was characterized by a 1.5-fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. Thefen1-1 mutation was linked toMAT locus on chromosome III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5-fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. Thefen1-1 mutation permits viability in aerobiosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild typeFEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that thefen1-1 mutation eliminates the specific ergosterol requirement in yeast.     

Regulation of the sequencing in sterol biosynthesis

The evolutionary development and sequencing of events in the biosynthetic pathway leading to sterols is reviewed and illustrated by recent experiments from the author's laboratory. One of 12 papers being published from the “Sterol Symposium” presented at the AOCS Meeting, New Orleans, April 1970.     

Absence of sterol biosynthesis in oyster tissue culture

An oyster heart tissue culture was used to investigate the ability of the American oyster (Crassostrea virginica) to synthesize sterols from [¹⁴C]acetate. Lipids were extracted from cultured oyster heart cells and no label was found in the purified fatty acid fraction, 90% of which was located in the saturated and monounsaturated fractions. Although these results cannot rule out the possibility of sterol synthesis in other oyster tissues, this and previous evidence are in support of the hypothesis that the American oyster is unable to synthesize sterols.     

Aspects related to mevalonate biosynthesis in plants

We purified and characterized a membrane-associated enzyme system from radish (Raphanus sativus L.) that is capable of converting acetyl-CoA into 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). The enzyme system apparently comprises acetoacetyl-CoA thiolase (EC 2.3.1.9) and HMG-CoA synthase (EC 4.1.3.5). Its activityin vitro can be strongly stimulated by Fe^II. When ferrous ions are applied chelated with ethylenediaminetetraacetate, citrate or adenosine 5′-triphosphate (ATP), the stimulation is further increased. Stimulation is due to a higher catalytic efficiency as indicated by an increase in V_max, whereas the affinity of the enzyme towards acetyl-CoA remains constant (K_m=6 μM). A considerable portion of HMG-CoA lyase activity is associated with the same membranes. HMG-CoA lyase (EC 4.1.3.4) is also solubilized and partially co-purified. Its activity requires comparatively high concentrations of Mg²⁺. The conversion of HMG-CoA to mevalonic acid is catalyzed by HMG-CoA reductase (EC 1.1.1.34) that is associated with the same membranes. By cDNA encoding theArabidopsis HMG-CoA reductase, we isolated a corresponding gene from a cDNA library newly established from etiolated radish seedlings. This full-length cDNA, referred to as λcRS3, encodes a polypeptide of 583 amino acids with a molecular mass of about 63 kDa. The hydropathy profile suggests the presence of two hydrophobic membrane-spanning domains within the N-terminal 165 amino acids. The carboxy-terminal part, where the catalytic site resides, is highly conserved in all eukaryotic HMG-CoA reductase genes sequenced so far. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

The biosynthesis of cyclopropane and cyclopropene fatty acids in higher plants (Malvaceae)

The biosynthesis of cyclopropane and cyclopropene fatty acids has been investigated in immature seeds, leaves and callus tissue cultures of several species of Malvaceae. Chemical characterization of labeled cyclopropane and cyclopropene fatty acids obtained from incubations withl-[¹⁴CH₃] methionine confirmed that the ring methylene group was derived from the methyl group of methionine. The variation with time in the distribution of radioactivity in the products of incubations with [¹⁴CH₃] methionine and [2-¹⁴C] acetate suggested that the pathway involved initial formation of dihydrosterculic acid from oleic acid with subsequent desaturation to sterculic acid and α-oxidation to malvalic and dihydromalvalic acids. Direct evidence in favor of this pathway was provided by the conversion of [1-¹⁴C] oleic acid to dihydrosterculic and sterculic acids and by the desaturation of [1-¹⁴C] dihydrosterculic acid to sterculic acid, the first time that these processes have been demonstrated in higher plants. No conversion of [1-¹⁴C] stearolic acid to sterculic acid could be obtained under the same conditions. The presence of an active fatty acid α-oxidation system was demonstrated in the callus cultures.     

Developmental regulation of sterol biosynthesis inZea mays

Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods fromZea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-¹⁴C]mevalonic acid, [2-¹⁴C]acetate, and [2-³H]acetate), and stable isotopes (D₂O), we discovered that hydroxymethylglutaryl CoA reductase is not “the” rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24α-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an activede novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3β-ol. Examination of the mass spectrum and¹H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed. Dedicated to the distinguished insect steroid biochemist Dr. James A. Svoboda on occasion of his sixtieth birthday. Based on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids”, Atlanta, Georgia, May 1994.     

AY-9944 inhibition of sterol biosynthesis inChlorella emersonii

WhenChlorella emersonii, a green alga, was cultured in the presence of 20 ppm AY-9944, a number of sterols accumulated which appear to be intermediates of sterol biosynthesis in this organism. The sterols isolated include 14α-methyl-ergost-8-en-3β-ol, 14α-methyl 24S-stigmast-8-en-3β-ol, 14α-methyl ergosta-8,24(28)-dien-3β-ol and 4α, 14α-dimethyl 24S-stigmast-8-en-3β-ol. Smaller quantities of several other sterols were found in addition to the normally occurring Δ⁷, chondrillasterol and Δ⁷. Control cultures were found to contain, in addition to the normally occurring sterols, smaller quantities of most of the sterols isolated from AY-9944 inhibited cultures. AY-9944 is a specific inhibitor of Δ⁷ in cholesterol biosynthesis in animals. However, sinceC. emersonii terminates sterol biosynthesis one step prior to the Δ⁷ step, AY-9944 apparently inhibits sterol biosynthesis prior to this step in this organism. The accumulation of 14α-methyl sterols in treated cultures suggests that AY-9944 is an effective inhibitor of the 14α-methyl removal inC. emersonii. Scientific Article No. A1865, Contribution No. 4775 of the Maryland Agricultural Experiment Station.     

Inhibition of sterol biosynthesis in chlorella ellipsoidea by AY-99441

WhenChlorella ellipsoidea was grown in the presence of 4 ppm AY-9944, complete inhibition of Δ⁵-sterol biosynthesis was achieved. However total sterol production remained unaltered. As a result a number of sterols accumulated that appear to be intermediates in sterol biosynthesis. These sterols were described and identified as (24S)-5α-ergost-8(9)-en3β-ol, (24S)-5α-stigmast-8(9)-en-3β-ol, 4α-methyl-(24S)-5α-ergosta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-stigmasta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-ergost-8(9)-en-3β-ol and (24S)-4α-methyl-5α-stigmast-8(9)-en-3β-ol. The occurrence of these sterols inChlorella ellipsoidea is the first time they have been noted in biological material. The accumulation of these sterols in treated cultures indicates that AY-9944 is an extremely effective inhibitor of the Δ⁸→Δ⁷ isomerase and the Δ¹⁴ reductase of these plants. The occurrence of small amounts of other sterols in treated cultures has led to a proposed pathway for thebiosynthesis of sterols inChlorella ellipsoidea. Scientific Article No. A1775, Contribution No. 4565 of the Maryland Agricultural Experiment Station.     

Developmental regulation of sterol biosynthesis inCucurbita maxima L.

Twenty-two sterols were identified by capillary gas chromatography and capillary gas chromatography/mass spectroscopy inCucurbita maxima grown under green-house conditions. Both whole plants and individual tissues (leaves, stems, roots, cotyledons, flowers) were analyzed at weekly intervals during the 12-week development of the plant. In whole plants, sterol accumulation parallels plant growth except for a period in the mid-life cycle where there is a reduction in the amount of sterol accumulated on a total sterol/plant and mg sterol/g dry wt basis. This reduction in the amount of sterol is coincident with the visual onset of flowering. During development, the percent contribution of each class of sterol (Δ^5_, Δ^7_, Δ^0_-sterols) remains relatively constant. However, the percent contribution of an individual sterol species varies depending on the tissue examined and the developmental period selected for analysis. While the young plant (<2 weeks) possesses elevated levels of sterols with the Δ²⁵⁽²⁷⁾-double bond, the trend was toward a reduction in the amounts of these sterols with development. Leaves and stems accumulate large quantities of 24ζ-ethyl-5α-cholesta-7,22-dien-3β-ol (7,22-stigmastadienol) and 24ζ-ethyl-5α-cholest-7-en-3β-ol (7-stigmastenol), while roots accumulate only 7,22-stigmastadienol as their principal sterol. Male flowers and roots were found to contain elevated levels of Δ^5_-sterols.     

The steric requirements for sterol inhibition of tetrahymanol biosynthesis

Many naturally occurring sterols are accumulated and metabolized byTetrahymena pyriformis. In most cases, the sterols are desaturated to give^Δ5,7,22-derivatives. Compounds with an ethyl, but not with a methyl, substituent at C-24 are dealkylated. Exposure of the ciliates to the appropriate sterol sharply curtails the synthesis of the native pentacyclic triterpenoid alcohols, tetrahymanol and diplopterol. An analysis with modified sterols has revealed several additional features that are required for desaturation at C-7,8 and C-22,23 and for inhibition of tetrahymanol biosynthesis. The presence of atrans-17(20)-double bond, which eliminates free rotation at C-20 and fixes C-22 to the right of the nucleus, does not interfere with desaturation, while thecis- or left-handed isomer is not metabolized. Thecis-Δ¹⁷⁽²⁰⁾-isomer is, however, an effective inhibitor of tetrahymanol biosynthesis, although less so that thetrans-counterpart. When a methyl or hydroxyl group at C-20 protrudes to the front of the molecule in the right-handed conformation, metabolism is reduced or abolished. Shortening (by one C-atom) or lengthening of the sterol side chain has little effect on the ability of the compounds to inhibit tetrahymanol biosynthesis or to support growth, as long as the overall length of the side chain does not exceed seven carbons from C-20. The presence of a 7α-, 7β-, 20α-, 20β-, or a 25-hydroxy group in the cholesterol molecule sharply inhibits desaturation and curtails the effectiveness of the compound as an inhibitor of tetrahymanol biosynthesis. The 7- or 22-keto derivatives seem to act in a fashion similar to the hydroxy derivatives, but these compounds show greater inhibition of growth. 20-Methylcholesterol, however, is a potent inhibitor of synthesis, which suggests that the polarity of the substituent of C-20 is more important than bulk. Many sterols can effectively replace tetrahymanol in the membranes of these ciliates. However, several of the compounds, which inhibit synthesis, appear to be physiologically inappropriate, and poor growth results. An example of the latter class is 20-methylcholesterol. Finally, a class of sterols, represented by 20α-hydroxycholesterol and 7-ketocholesterol, does not severly inhibit tetrahymanol synthesis but leads to growth inhibition and surface abnormalities. These sterols apparently lead to a disordered membrane, even in the presence of tetrahymanol.     

The effect of cerulenin on sterol biosynthesis inSaccharomyces cerevisiae

Cerulenin specifically inhibited fatty acid biosynthesis inSaccharomyces cerevisiae without having an effect on sterol formation. Ergosterol was not required for cell growth in the presence of cerulenin (1 μg/ml). The addition of fatty acids to the growth medium reduced the amount of ergosterol formed by 45%; further addition of cerulenin to the media had no effect on the amount of ergosterol synthesized by the cells. The incorporation of³H from³H₂O into ergosterol was not affected by cerulenin whereas incorporration into fatty acids was inhibited by 90%.     

The effct of cerulenin on sterol biosynthesis in Saccharomyces cerevisiae.

Cerulenin specifically inhibited fatty acid biosynthesis in Saccharomyces cerevisiae without having an effect on sterol formation. Ergosterol was not required for cell growth in the presence of cerulenin (1 microgram/ml). The addition of fatty acids to the growth medium reduced the amount of ergosterol formed by 45%; further addition of cerulenin to the media had no effect on the amount of ergosterol synthesized by the cells. The incorporation of 3H from 3H2O into ergosterol was not affected by cerulenin whereas incorporation into fatty acids was inhibited by 90%.     

Effects of triarimol and tridemorph on sterol biosynthesis inSaprolegnia ferax

The effects of two fungicides, triarimol and tridemorph, on sterol biosynthesis inSaprolegnia ferax were examined. Cultures grown in the presence of triarimol accumulated lanosterol. Tridemorph-treated cultures accumulated Δ⁸-sterols including zymosterol, fecosterol and stigmasta-8-24(28)-dienol. The latter is a new sterol. A proposed scheme is given for sterol biosynthesis inS. ferax showing points of inhibition of triarimol and tridemorph. Results point to the intermediacy of lanosterol but not cycloartenol in sterol synthesis inSaprolegnia.     

Effects of triparanol and AY-9944 upon sterol biosynthesis inChlorella

The growth rates of three species ofChlorella were inhibited by triparanol and AY-9944.Chlorella emersonii was noticeably more resistant to both inhibitors than the other species. A large number of sterols were isolated and identified in inhibited cultures. Ca. 18 of these were identified from nature for the first time. Triparanol resulted in inhibition of the removal of the 14α methyl group. It also inhibited the second alkylation of the side chain and, in one species, strongly inhibited the Δ⁸ → Δ⁷ isomerase reaction. InC. ellipsoidea, triparanol also inhibited Δ¹⁴-reductase and Δ⁷-reductase. The introduction of the Δ²² double bond was inhibited by both drugs. The effect of AY-9944 was similar to that of triparanol inC. emersonii, but it was an extremely effective Δ¹⁴-reductase inhibitor inC. ellipsoidea. These various types of inhibition of sterol synthesis indicate a lack of specificity of both drugs inChlorella and suggest that primitive plants such as these may be valuable as test organisms in an evaluation of the activity of potential inhibitors of sterol biosynthesis.     

The biosynthesis of polyunsaturated fatty acids in plants

This paper is a review of some of the work being done at the author's laboratory. The phospholipids and glycolipids of the alga,Chlorella vulgaris, have been implicated in fatty acid transformations such as chain elongation and desaturation. Labeling studies with [¹⁴C] acetate have shown that newly synthesized galactosyl glycerides have mainly saturated fatty acids. Subsequent to de novo synthesis, a series of alterations of fatty acid structure takes place within the same glycolipid molecules. The specific incorporation of [¹⁴C] oleic acid intoChlorella phosphatidyl choline provides a convenient model system for studying the lipid dependent desaturation of oleic to linoleic acid. The inhibitor of fatty acid desaturation, sterculic acid, only inhibits the conversion of oleate into linoleate if added before the precursor fatty acid has been incorporated into a complex lipid. Studies with isomeric monoenoic fatty acids have suggested that there are two enzymes which catalyze the formation of linoleic from oleic acid. One measures the position of the second double bond from the carboxyl group, the other, from the methyl end of the chain. The latter enzyme probably requires the complex lipid substrate.     

Design of resilient processing plants—II Design and control of energy management systems

The “standard heat exchanger network problem” has been surprisingly difficult to solve. It is only now that simple and reliable methods have evolved to synthesize the most efficient network of heat exchangers to heat and cool known process streams between stated temperature bounds. This has taken over a decade of research and scores of publications.The “resilient heat exchanger network problem” requires a solution that can cope with the uncertainties of industrial design. Fixed flow rates and temperature bounds rarely occur industrially. Rather, an industrial heat exchanger network problem necessarily involves ranges of flow rates and ranges of temperature bounds, and must include ease of operation and control.In this paper we make several fundamental advances in the design of resilient heat exchanger networks. (1) An efficient design procedure is developed to yield resilient designs which handle fluctuations within the condition of maximum energy efficiency. (2) A control structure and operating policy are developed to adjust flow distributions in the network to meet temperature constraints with minimum utility usage.These developments are based on several new theorems concerning resiliency in network design.     

Metabolic costs of terpenoid accumulation in higher plants

The net value of any plant trait can be assessed by measuring the costs and benefits associated with that trait. While the other contributors to this issue examine the possible benefits of terpenoids to plants, this article explores the metabolic costs of terpenoid accumulation in plants in the light of recent advances in terpenoid biochemistry. Terpenoids are more expensive to manufacture per gram than most other primary and secondary metabolites due to their extensive chemical reduction. The enzyme costs of making terpenoids are also high since terpenoid biosynthetic enzymes are apparently not shared with other metabolic pathways. In fact, plant cells may even possess more than one set of enzymes for catalyzing the basic steps of terpenoid formation. Terpenoids are usually sequestered in complex, multicellular secretory structures, and so storage costs for these substances are also likely to be substantial. However, not all of the processes involved in terpenoid accumulation require large investments of resources. For instance, the maintenance of terpenoid pools is probably inexpensive because there is no evidence that substantial quantities of terpenes are lost as a result of metabolic turnover, volatilization, or leaching. Moreover, plants may reduce their net terpenoid costs by employing individual compounds in more than one role or by catabolizing substances that are no longer needed, although it is still unclear if such practices are widespread. These findings (and other facets of terpenoid biochemistry and physiology) are discussed in relation to the assumptions and predictions of several current theories of plant defense, including the carbonnutrient balance hypothesis, the growth-differentiation balance hypothesis, and the resource availability hypothesis.     

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-¹⁴C and mevalonate-5-³H into squalene, C₃₀ sterols, and C₂₇ sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of³H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas¹⁴C-acetate conversion was reduced to ca. 35% of control values during the same period.³H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as¹⁴C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from¹⁴C-acetate and³H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.