Plant Physiol. (1980) 66, 215-2190032-0889/80/66/02 15/05/$00.50/0

Production of Hexanal and Ethane by Phaeodactylum triconutumand Its Correlation to Fatty Acid Oxidation and Bleaching ofPhotosynthetic Pigments'

Received for publication September 14, 1979 and in revised form February 14, 1980

BRIGITTE SCHOBERT AND ERICH F. ELSTNERInstitutffur Botanik und Mikrobiologie, Lehrstuhlffur Botanik, Technische Universitat, D-8000 Munchen 2,West Germany

ABSTRACT MATERIALS AND METHODS

In a Ught-dependent reaction (3.5 kilolux) at pH 5, the evolution ofhexanal, ethane, and ethylene has been established with cell suspensionsof the diatom, Phaeodactylwn ticornutum During this process, chlorophylland carotenoids are partially bleached. Addition of25 milUmolar a-Unolenicacid or 12 millimolar docosahexaenoic acid yield total pigment destructionand enhancement of ethylene and ethane formation (by about 150 and7,600%, respectively), whereas hexanal production decreases by 70%.Eicosapentaenoic acid, the major polyunsaturated fatty acid in diatoms,stimulates both ethane and hexanal formation (by about 1,400 and 130%,respectively), but reduces ethylene production (by about 60%). This com-petition suggests that the production of the volatile compounds is closelyconnected, although hexanal and ethylene obviously possess differentunsaturated fatty acids as precursors. Both the kind of the fatty acids andtheir relative amounts seem to determine the pattern of the evolvedhydrocarbons. The presence of 10 millimolar propylgallate inhibits theevolution of the volatile compounds by about 80%, indicating that radicalformation might play a key role in this light-dependent cascade of reactions.

The production of several volatile compounds as a consequenceof fatty acid peroxidation has been reported by many authors (5,21). Furthermore, in vivo experiments with unicellular algaeshowed that lipid peroxidation is correlated with bleaching ofphotosynthetic pigments and destruction of the electron transportsystem (1, 4). Riely et al. (17) introduced a new and simple methodfor following fat oxidation in intact animals after CC14 treatmentor in tissue homogenates by measuring ethane formation. Thismethod was also used for higher plants (6). Ethane and.ethyleneproduction by leaf homogenates is stimulated in the presence ofa-linolenic acid (11, 14), which is assumed to be a precursor ofboth hydrocarbons. In contrast to higher plants and most otheralgal species, a-linolenic acid is only a minor constituent ofdiatomlipids, but they contain a considerable amount of several otherpolyunsaturated fatty acids (12, 15). Therefore, this algal speciesseemed to be an interesting object to study and correlate hydro-carbon liberation and fatty acid composition. This paper describesthe conditions for fatty acid peroxidation in the diatom Phaeo-dactylum tricornutum, the subsequent breakdown of cellular ultra-structure, the bleaching of photosynthetic pigments and the evo-lution of three volatile hydrocarbons from their possible precur-sors.

'This work was supported by the Deutsche Forschungsgemeinschaft.

Strain and Culture Conditions. The diatom Phaeodactylumtricornutum was purchased and cultivated under continuous light(3.5 kilolux, warm-white fluorescent lamp) in a medium (pH 8),its composition described previously (18). The algae for bothcultivation and experiment were kept in a temperature-controlledroom at 18 C.

Determination of n-Hexanal, Ethane, and Ethylene. Algal sus-pensions were centrifuged for 10 min at 2,000g and resuspendedin nutrient solution with 50 mm potassium biphthalate-NaOHbuffer (pH 5) (unless otherwise indicated). Four ml algal suspen-sion, corresponding to 0.1 ml wet-packed cells, were placed intoFembach flasks of about 15 ml volume. The glass vessels weresealed with serum rubber stoppers and the suspension continu-ously stirred during the incubation time of 24 h (unless otherwiseindicated) under constant illumination (3.5 kilolux) or, whenindicated, dark conditions were maintained by screening the flaskswith a black box. Five parallel samples were used for eachmeasuring point. The volatile hydrocarbons n-hexanal, ethane,and ethylene from the headspace of the flasks were determined bygas chromatography with a Varian 1400 GC as described earlier(6). The integrals of the peak areas are expressed in units; 103 unitscorrespond to I pmol of ethane or ethylene. Since no conversionfactor was available for hexanal, the relative amounts of all threehydrocarbons are given in units. Under the experimental condi-tions (column: Poropak 80, temperature: 70 C) the retention timesof the individual gases were (min): ethylene, 1.07; ethane, 1.23;and n-hexanal, 4.45.

Identification of the Gases. Ethane and ethylene were identifiedby their retention times and by comparison with authentic gases(Linde, Munich, FRG). n-Hexanal was identified with a Finnigan3200 mass spectrometer, directly coupled to a gas chromatograph.Column: 75 m Reoplex; temperature program: 50-160 C; t = 2 C/min; gas flux: 2.6 ml He/min. The molecular ion of n-hexanal wasidentified by comparison with a pure reference substance. Theretention time was established with vaporized authentic n-hexanal.

Determination of Pigments. Chl a and carotenoids were ex-tracted, separated and quantitatively determined as describedelsewhere (9).

Electron Microscopy. Electron microscopic studies were per-formed as outlined elsewhere (19).

Materials. Eicosapentaenoic acid methylester was obtainedfrom Serva, Heidelberg, FRG. All the other fatty acids wereproducts of Sigma. Only fresh material was used and the sealedglass tubes were opened immediately before assay, since the blanksof ethane production from a-linolenic acid increase significantlywith aging. Only the pure fatty acids have been added to the flasksand they were found to be satisfactorily emulsified by stirring the

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SCHOBERT AND ELSTNER

G"hexanal

lU X -10

etylene -

7 6 5 4 pH

FIG. 1. pH-dependence of hexanal, ethane, and ethylene productionby cell suspensions of P. tricornutum. (The algal medium contained thebuffer systems: pH 7, 50 mm Hepes/NaOH; pH 6 and pH 5.5, 50 mMMes/NaOH; pH 5 to pH 4, 50 mi K-biphthalate/NaOH).

3.

.0 2

I,oX 1

x

1-

2 4 8 16 24l hoursl 32

FIG. 2. Time course of hexanal, ethane, and ethylene formation by cellsuspensions of P. tricornutum.

algal suspensions. The presence of detergents or emulsifiers wasavoided, because they accelerate the degradation of the cells.DABCO2 was purchased from EGA-Chemie, Steinheim, FRG,and propylgallate from Aldrich-Europe, Nettetal, FRG. DOPAwas obtained from Sigma.

RESULTS

Effect of pH. The production of hexanal, ethane and ethyleneat different pH values in the algal medium is demonstrated in

2Abbreviations: DABCO: 1,4-diazabicyclo-(2,2,2)-octane; DOPA: 3,4-dihydroxyphenylalanine.

0 lightU dark

06

-g1.0 10IN0

x

0.2

ethylene ethane hexanatFIG. 3. Influence of light (3.5 kilolux) on hexanal, ethane, and ethylene

production by cell suspensions of P. tricornutum.

Table I. Influence of Increasing Concentrations of a-Linolenic Acid onEthane, Ethylene, and Hexanal Formation by Cell Suspensions of P.

tricornutum

Gas ProductionSample

Ethylene Ethane Hexanal

units x 10 6/ml packed cellsControl 0.59 ± 0.09 1.84 ± 0.37 22.35 ± 3.90+ 5 mm a-Linolenic acid 0.82 ± 0.07 6.69 ± 1.60 7.55 ± 0.80+1O mM a-Linolenic acid 0.90 ± 0.14 11.11 ± 2.01 4.50 ± 0.77+25 mM a-Linolenic acid 1.51 ± 0.15 187.25 ± 40.23 5.45 ± 1.41

FIG. 4. Gas chromatogram, showing the retention times of ethylene,ethane, and hexanal and the relative amounts of the volatile hydrocarbonsin the absence and presence of 25 mM a-linolenic acid.

Figure 1. A significant liberation of the volatile hydrocarbons isrestricted to acidic pH values, starting at pH 6 and exhibiting amaximum at pH 5. Several overlapping buffer systems have beenused with essentially identical results (data not shown), indicatingthat the observed hydrocarbon evolution is merely a pH effect.Although the production of the gases is further increased at pH 4,this condition seemed too unphysiological and pH 5 was used forthe subsequent investigations. Hexanal is the major volatile hy-drocarbon found under these conditions.Time Course. The production of hexanal, ethane, and ethylene

continues for approximately 30 h until a maximum value isreached (Fig. 2).

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HEXANAL AND ETHANE PRODUCTION

Table II. Effect of Dfferent Unsaturated Fatty Acids on Ethylene, Ethane, and Hexanal Production by CellSuspensions of P. tricornutum

Gas ProductionSample

Ethylene Ethane Hexanal

units x 106/mlpacked cellsControl 0.64 ± 0.10 2.43 ± 0.26 22.29 ± 1.80+ 12 mm Palmitoleic acid (16:1 A9) 0.44 ± 0.08 0.42 ± 0.04 5.21 ± 0.62+ 12 mm Elaidic acid (18:1 A9 tr.) 1.49 ± 0.29 3.22 ± 0.35 19.87 ± 3.72+ 12 mM Oleic acid (18:1 A9, w9) 0.57 ± 0.09 1.15 ± 0.19 10.68 ± 1.56+ 12 mm Erucic acid (22:1 w9) 1.20 ± 0.20 3.20 ± 0.65 15.49 ± 3.10+ 12 mm Nervonic acid (24:1 w9) 1.43 ± 0.13 3.88 ± 0.45 18.87 ± 3.20+ 12 mm Linoleic acid (18:2 w6) 0.62 ± 0.08 1.12 ± 0.10 4.98 ± 1.78+ 12 mM Arachidonic acid (20:4 w6) 0.93 ± 0.17 1.20 ± 0.24 4.96 ± 0.93+ 12 mm Eicosapentaenoic acid (20:5 w3) 0.25 ± 0.06 36.91 ± 4.78 52.88 ± 0.82+ 12 mM Docosahexaenoic acid (22:6 w3) 1.67 ± 0.41 188.82 ± 55.18 6.31 ± 1.56+ 25 mm a-Linolenic acid (18:3 w3) 1.51 ± 0.15 187.25 ± 40.23 5.45 ± 1.41

Table III. Effects ofpH, Light, and a-Linolenic Acid on the Destructionof Photosynthetic Pigments in Cells of P. tricornutum

Pigment

Sample Fuco- 8i-Caro-Chl a xan-

thine tene

mg/ml packed cellspH 8, light 24 h 1.19 0.59 0.056pH 5, light 24 h 0.68 0.38 0.038pH 5, light 3 h + 10 mM a-Linolenic acid 0.13 0.23 0pH 5, light 24 h + 10 mM a-Linolenic acid 0 0 0

Table IV. Influence ofDABCO, DOPA, Propylgallate, and EDTA on theProduction of Ethylene, Ethane, and Hexanal by Cell Suspensions of P.

tricornutum

Addition. 10 mm fmal con-Inhibition of Gas ProductionAddition (10 mtM final con -______________

centration) Ethylene Ethane Hexanal

% of control

DABCO 0 0 27DOPA 0 58 32Propylgallate 76 91 89EDTA 40 43 37

Light Dependence. In addition to acidic pH light is necessaryfor the liberation of the volatile hydrocarbons. In the dark, theproduction of all three gases is strongly reduced (Fig. 3).

Effect of Different Unsaturated Fatty Acids. Since a-linolenicacid has been established as a precursor ofethane in higher plants,its influence on the formation of hexanal, ethane and ethylene byP. tricornutum was investigated. The presence of a-linolenic acidin the incubation mixture strongly stimulates the production ofethane, moderately enhances the formation of ethylene whereashexanal formation is inhibited to a considerable extent (Table I,Fig. 4). The stimulation is not proportional to the inhibition,however, because both the 6- and 100-fold enhancement ofethaneproduction is correlated with an approximate 75% reduction ofhexanal formation.

Because a-linolenic acid is only a minor constituent of diatomlipids (12, 15) and therefore probably not the actual precursor ofethane in P. tricornutum, the influence of nine different unsatu-rated fatty acids on hydrocarbon formation has been investigated.Table II shows that 12 mm docosahexaenoic acid strongly stimu-lates ethane production, reduces hexanal formation and is wellcomparable to the effect of 25 mm a-linolenic acid. Eicosapenta-

enoic acid (12 mM) stimulates both ethane and hexanal formation,but reduces ethylene production. All the other unsaturated fattyacids tested, either do not drastically enhance or even reduce theformation of the hydrocarbons.

L-Methionine has been established as a precursor of ethylene inhigher plants and several microorganisms (3, 13, 20). In contrast,an incubation of P. tricornutum with L-methionine (100 mm or 10mm final concentration) for 24 h has no influence on the produc-tion of ethylene or the other hydrocarbons. The uptake of thisamino acid by the diatoms was confirmed with [14C]methionine(data not shown).

Influence on Photosynthetic Pigments. During the incubationat pH 5 the dark-brown algal suspension becomes greenish indi-cating a change in pigment composition. Chl a, fucoxanthin, and,8-carotene have been determined as representative photosyntheticpigments (9). Table III shows that the formation of the volatilehydrocarbons is correlated with the destruction of these pigments.The presence of a-linolenic acid accelerates this process.

Effect of Radical Scavengers. To get insight whether free radicalintermediates are involved in the observed effects, radical andsinglet oxygen scavengers have been added to the incubationmixture. The singlet oxygen quencher DABCO (8) slightly pre-vents the production of hexanal (Table IV). Both ethane andhexanal production is inhibited by about 60 and 30%o, respectively,in the presence ofDOPA, an o-diphenol which reacts with certainoxygen radicals (7). The radical scavenger propylgallate effectivelyinhibits ethylene, ethane, and hexanal formation. The productionof the gases is likewise reduced in the presence of EDTA (byabout 40%), indicating that metal catalysis might be involved.

Ultrastructural Changes. Fatty acid oxidation, gas formation,and pigment bleaching are accompanied by ultrastructuralchanges of the algal cells (Fig. 5 B 1), which are most evident inthe chloroplast (Fig. 5 B2). The thylakoids are totally destroyedand osmiophilic granules are accumulated, indicating pathologicallipid accumulation.

DISCUSSIONWith initially intact cells of P. tricornutum the oxidation of

unsaturated fatty acids can be induced in the light and at pHvalues lower than 5.5. Ethylene, ethane, and hexanal have beendetermined as their volatile breakdown products. Both a-linolenicacid (18:3 3) and docosahexaenoic acid (22:6 w3) stimulateethane and ethylene production. However, the 22:6 w3 acid is theprobable precursor of these breakdown products, since P. tricor-nutum is described to contain approximately I1% docosahexaenoicacid (of the total fatty acid content) and only 0.2% a-linolenic acid(12, 15). Eicosapentaenoic acid (20:5 03), the major fatty acid inP. tricornutum (12, 15) has been established as the probable

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SCHOBERT AND ELSTNER

Al B1

A. _w B2FIG. 5. Section through an oval cell-type of P. tricornutum (Al, B 1) and detail of the chloroplast (A2, B2). Electron micrograph, OsO4 fixation,

poststained with lead. Al (magnification 1.4 x I04: 1), A2 (magnification 4.4 x lO4: 1): pH 8, 3.5 kilolux, 24 h (control). B I (magnification 1.7 x 104: 1),B2 (magnification 4.3 x 106:1): pH 5, 3.5 kilolux, 24 h.

precursor of the breakdown product n-hexanal. All the fatty acids,involved in the production of the volatile hydrocarbons, belong tothe w3 family. Several other unsaturated fatty acids, of the w6,w9 and A9 family show either inhibitory or very small stimulatoryeffects. It is not known, whether they are not oxidized under theexperimental conditions or they yield breakdown products whichare not perceived by the applied measurements. Not only thestructure of the unsaturated fatty acids, but also their relativeamounts seem to determine their breakdown pattern, since com-petition reactions have been demonstrated. It is obvious from theelectron microscopic investigations, showing totally destroyedchloroplasts, that fatty acids from membranes are oxidized. Thisresult is also supported by earlier observations (16). In addition,fatty acids of reserve lipids may contribute to the described effect.The cellular destruction originates in the production of fatty

acids hydroperoxides. Their existence has been described forseveral in vivo and in vitro systems and the involvement of alipoxygenase in producing the hydroperoxides is a widely distrib-

uted phenomenon (5, 21). The oxidation of polyunsaturated fattyacids and the activity of the lipoxygenase in aqueous solution iswell measurable by the coupled oxidation of fl-carotene (2).However, the light dependence of hexanal and ethane productionby P. tricornutum points to a nonenzymic formation of radicalintermediates which may induce a series of oxidative reactions.Acid pH-conditions are used for several purposes in plant

physiology, e.g. the incubation of cells or tissues with hydrolyticenzymes for plant cell wall digestion. The investigated systemprovides useful insight that under these conditions membraneartifacts can easily be obtained.

Acknowledgments-We would like to thank Prof. Ziegler and Mrs. Blase,Munchen. for performing electron microscopy, and Prof. Rapp and Dr. Knipser,Siebeldingen, for the mass spectroscopic determination of n-hexanal.

LITERATURE CITED

1. ASAMI S, T AKAZAWA 1978 Photooxidative damage in photosynthetic activitiesof Chromatium vinosum. Plant Physiol 62: 981-986

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HEXANAL AND ETHANE PRODUCTION

2. BEN Aziz A, S GROSSMAN, J ASCARELLI, P BUDOWSKI 1971 Carotene-bleachingactivities of lipoxygenase and heme proteins as studied by a direct spectropho-tometric method. Phytochemistry 10: 1445-1452

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8. FOOTE CS 1976 Photosensitized oxidation and singlet oxygen: Consequences inbiological systems. In WA Pryor, ed, Free Radicals in Biology, Vol. 2. Aca-demic Press, New York, pp 85-133

9. HAGER A, H STRANSKY 1970 Die Carotinoidmuster und die Verbreitung deslichtinduzierten Xanthophyllcyclus in verschiedenen Algenklassen. Arch Mik-robiol 73: 77-89

10. HITCHCOCK C, BW NICHOLS 1971 Plant Lipid Biochemistry. Academic Press,New York

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Acta 116: 264-27813. KONZE JR, EF ELSTNER 1976 Pyridoxalphosphate-dependent ethylene produc-

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15. MORENO VJ, JEA DE MORENO, RR BRENNER 1979 Biosynthesis of unsaturatedfatty acids in the diatom Phaeodactylum tricornutum. Lipids 14: 15-19

16. MEAD JF 1976 Free radical mechanisms of lipid damage and consequences forcellular membranes. In WA Pryor, ed, Free Radicals in Biology, Vol 1.Academic Press, New York, pp 51-68

17. RIELY CA, G COHEN. M LIEBERMAN 1974 Ethane evolution: a new index of lipidperoxidation. Science 183: 208-210

18. SCHOBERT B 1977 The influence of water stress on the metabolism of diatoms II.Z Pfanzenphysiol 85: 451-461

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