Plant Physiol. (1986) 82, 479-4840032-0889/86/82/0479/06/$0l1.00/0

The Biosynthesis of a Novel Nicotine Alkaloid in the Trichomesof Nicotiana stocktonii1

Received for publication March 21, 1986

ERNO ZADOR2 AND DAVY JONES*Department ofEntomology, University ofKentucky, Lexington, Kentucky 40546

ABSTRACT

N-Hydroxyacylnornicotine, newly discovered from fresh plant tissue,was found entirely in the trichome exudate produced at the epidermis ofthe aerial part of Nicotiana stocktonii. Nicotine and nornicotine, but notN-hydroxyacylnornicotine, were present inside of the trichomes as wellas other internal parts of the plant. Only nicotine was found in bleedingsap squeezed from cut roots or stems. Feeding of leaves with 2'-'4C-labeled nicotine primarily yielded labeled nicotine, nornicotine, and N-hydroxyacylnoraicotine. When similarly labeled nornicotine was fed toleaves as a precursor, a labeled N-hydroxyacylnornicotine was obtained,with a higher specific activity than with the 12'-4Cinicotine feeding.Based on these results, a synthesis route is suggested where nicotine isconverted in the leaf to nornicotine, followed by trichome conversion ofnornicotine to N-hydroxyacylnornicotine, and rapid secretion of thisproduct.

,.V. 1

I1

.:

!4kThe presence of a new type of tobacco alkaloid from freshplant tissue has been recently reported from the genus Nicotiana,section Repandae. This group of alkaloids contains an acylatednitrogen on the pyrrolidine ring. The major component amongthem is the [iso-N'(3-hydroxy- 12-methyltetradecanoyl)]-nomi-cotine (20). The N-acylnornicotine type of pyridine alkaloids hasbeen found earlier only in air-cured or harvested and aged leavesof N. tabacum (13, 14). In the plant kingdom the N-acylatedpyrrolidine ring is very rare (12). Also, this type of nicotinealkaloid is selectively toxic to the larvae of Manduca sexta, incontrast to other tobacco herbivores (7, 10, 18).We regarded the above mentioned nicotine alkaloid of Repan-

dae as an interesting subject of studies because (a) of its novelty,(b) the biosynthesis of this chemical reveals a previously rarelyconsidered aspect of plant physiology and (c) its potential use asa model for a new approach to genetically engineering of plantsfor insect resistance. We initiated a study on the distribution andsynthesis of the alkaloid in the plant, and in the present work wereport the localization of N-hydroxyacylnornicotine to the exu-date and its probable synthesis from nicotine via nornicotine.

MATERIALS AND METHODS

Plants. The Nicotiana stocktonii plants were grown in thegreenhouse in pots using standard soil mixture and were fertilized

' Supported, in part, by National Institutes of Health grant GM 33995and United States Department of Agriculture Cooperative Agreement.This paper is published in connection with a project of the KentuckyAgricultural Experiment Station (58-43YK-5-0034) and is published withthe approval of the Director.

2 Permanent address: Institute of Genetics, Biological Research CenterHungarian Academy of Sciences, Szeged pf. 521 H-6701.

Am.

'sF

a

a b i

2

3.3

S c d s

FIG. 1. The major nicotine alkaloids obtained (a) by washing leaveswith acetonitrile for 5 min, (b) by extracting internal tissue after aceto-nitrile washing, (c) by washing leaves with methylene chloride for 1 min,and (d) by extracting internal leaf alkaloids after methylene chloridewashing. S = standards: 1, N-Hydroxyacylnornicotine; 2, nicotine; 3,nornicotine.

(20-20-20, NPK) once per 2 weeks. All of the plants were 40 to50 cm high and had 12 to 20 leaves at the time ofthe experiments.

Extraction of Nicotine Alkaloids. Eight grams of plant leaves,stems, petioles, or roots were cut into small pieces and ground

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Plant Physiol. Vol. 82, 1986

ll,*_M

a 1 00.um

.._I

I. o FIG. 2. Scanning EM picture of (a) intactleaf surface, (b) individual glandular tri-chome, (c) leaf surface washed with aceto-nitrile for 5 min, (d) individual trichomefrom the acetonitrile washed surface, (e)leaf surface washed with methylene chlo-ride for 1 min, and (0 leaves on the plantafter acetonitrile (small arrow) and meth-ylene chloride (large arrow) wash for theabove described durations.

Table I. N-Hydroxyacylnornicotine Production by Leaves ofthe SameSurface Area after Washing with Acetonitrilefor 20 Seconds

The data are the average ± SE of three experiments.Content after Washing

Oh 24h 96h 144h

nmol ± SE27±4.2 101.3± 17.2 396±29.9 353±26.3

in a mortar with sand in 50 ml chloroform containing 1 mlNH40H. The homogenized material was kept overnight at roomtemperature and then filtered through cloth and partitionedagainst 3x30 ml water acidified with HCO to pH 2. The chloro-form fraction was evaporated under vacuum and when the leaveshad not been previously washed with acetonitrile this fractionwas the source of N-hydroxyacylnornicotine. The acidic water

was collected, the pH adjusted to 12 with NaOH and partitionedagainst chloroform. The latter was then evaporated (base frac-tion). This fraction was the source of the nicotine and thenornicotine.

Collection of Bleeding Sap. Well watered plants were eithercut at the border of the stem and the root, or near the top of thestem. Two gl of sap was squeezed out from the cut surfaces andcollected with a micropipette containing or not containing ace-tonitrile.

Plant Surface Extraction. Leaves, petioles, stems, and rootswere dipped into 170 ml acetonitrile for 20 s without breakingthe surface cuticle. The dipping time was longer if it was necessaryfor the total surface extraction (see text). The surface wash withmethylene chloride was carried out as for the acetonitrile wash.TLC Chromatography. TLC plates (Kieselgel 60) were devel-

oped in chloroform:methanol (100:40) and chloroform:methanol:NH40H (90:10:1) solvents, sprayed with p-aminoben-

f

480 ZADOR AND JONES

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ALKALOID BIOSYNTHESIS IN THE TRICHOMES

I

1

9.

1

2

2

3

a b

a bc d ef s

FIG. 3. The nicotine alkaloids obtained in a surface wash of roots (a),stems (c), and petioles (e) with acetonitrile for 5 min. The internal extractof roots (b), stems (d), and petioles (f) after surface washing with aceto-nitrile. Standards (s) as in Figure 1.

zoic acid or aniline or benzidine and put into cyanogen bromidevapor. The alkaloids were identified by RF value and color withthe help of standards (nicotine [Sigma]; nornicotine, providedby L. P. Bush; N-hydroxyacylnornicotine, provided by R. F.Severson). For radiolabeled compounds, [2'-'4C]nicotine wasprovided by L. P. Bush and labeled nornicotine obtained byfeeding this nicotine to leaves for extraction (see below).

Feeding Experiments. Leaves of the same expanded surfacearea were cut from the plant at the base of the petiole, washedfor 20 s with acetonitrile, and provided with sterile distilled H20.For feeding, either [2'-'4C]nicotine (10,000 cpm, 52 mCi/nmol)or [2'-'4C]nornicotine (5,000 cpm, 3.3 ,Ci/nmol) was added in100 Al sterilized distilled H20. After the leaves took up thisvolume they were transferred into distilled H20 until the time ofextraction.

s I

*2

33

c s

2

2

d e s

. I

FIG. 4. The major nicotine alkaloid content of (a) an acetonitrilewash of 8 mm2 abaxial leaf surface from which the trichomes wereremoved, (b) all trichomes collected from the same 8 mm2 leaf surfacebefore acetonitrile wash, (c) the exudate collected from 100 trichomes,(d) 500 trichomes collected from the leaf surface washed first for 5 minwith acetonitrile, (e) 500 trichomes collected from the intact leaf surface.Standards (s) as in Figure 1.

Quantitation of Cold and Labeled Alkaloids. For estimation ofN-hydroxyacylnornicotine production the leaves were washedfor 5 min with acetonitrile. The surface wash was evaporatedand then dissolved in 3 ml chloroform. The chloroform waspartitioned against 3x 1 ml acidified water with HCO (pH 2), thendried with Na2SO4 and evaporated. This fraction was the prepar-ative source of N-hydroxyacylnornicotine. The acidic water wasmade basic with NaOH (pH 12) and partitioned against 3x 1 mlchloroform, which was collected and evaporated. This fractioncontained only a trace amount of nicotine and did not containN-hydroxyacylnornicotine. The internal nicotine and nornico-tine content of homogenized leaves was chloroform extracted,the chloroform evaporated, the alkaloid residue TLC purified,eluted, dried and dissolved in 100 gl ethanol. The quality ofalkaloids in each of the above mentioned fractions was deter-mined by TLC chromatography; the quantity was estimated byvisual comparison of the thin layer chromatograms to dilutionsof standards. Aliquot parts of the obtained alkaloid concentrateswere either diluted up to 1 ml with 0.1 N HCO and quantitated

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48ADRANONSPlantPhysiol. Vol. 82, 1986

2

3

a b s

FIG. 5. The nicotine alkaloid content of bleeding sap of the root (a)and the stem (b). Standards (s) as in Figure 1.

by A at 260 nm, or measured for radioactivity. The leaf surfacealkaloid fractions were similarly treated.

Obtaining Labeled Nornicotine. Radioactive nornicotine was

obtained from tobacco leaves of N. tabacum L. Tobacco Intro-duction 11 12 (T.I.I1 12) fed with (2'-'4C]nicotine. The norni-cotine was recovered and TLC purified as described above andhad 3.3 MCi/nmol specific activity. This material was fed toleaves of N. stocktonii like the labeled nicotine.

RESULTS

Typically, alkaloids from species of Nicotiana have beenwashed from the leaf surface with methylene chloride (19). Weobserved that CH2Cl2 extracts nicotine and N-hydroxyacylnor-nicotine (Fig. 1, lane c), destroys the leaf surface (Fig. 2e) andcauses the leaf to collapse (Fig. 2f). This result suggests that the

methylene chloride extracts not only the surface but also theinternal tissue and it is consistent with another recent reportabout the effect of this solvent (11).The exudate containing N-hydroxyacylnornicotine can be se-

lectively washed from the leafwith acetonitrile (7). This chemicalextracts only a trace amount of nicotine (Fig. 1, lane a) and itsonly visible effect on the leaf surface is the elution of exudatedrops from the glandular trichomes (Fig. 2, c and d). The leafsurvives the 5 min acetonitrile treatment (Fig. 2f), and it is ableto secrete the N-hydroxyacylnornicotine again (Table I). Thisresult shows that the acetonitrile is a more specific agent to elutethe N-hydroxyacylnornicotine from the leaf surface than themethylene chloride. Therefore, the acetonitrile was used to collectsurface alkaloids in the following experiments.The surface wash of the root did not contain N-hydroxyacyl-

nornicotine (Fig. 3, lane a). The surface of petioles and stemscontained this alkaloid (Fig. 3, lanes c and e), as did the leafsurface extract (Fig. 1, lane a); the amount of this substance wasabout half of that of the internal nicotine. There was moreacylated nornicotine at the surface of leaf than petiole and stem.The internal tissue of surface washed leaves, petioles, stems, androots contains nicotine, nornicotine, and some unidentified al-kaloids, one of which is apparently anabasine (byRF and color).The order of internal alkaloid content per fresh weight was higherin the root than the leaf, the stem, and the petiole, respectively.About 90% of the internal total alkaloid content was nicotineand nornicotine. The nornicotine concentration was about 15%of nicotine in theleaf, higher in the root, but lower in the stem,and petiole, which is consistent with other reports (2, 4, 17).None of these internal parts of plant organs contain N-hydroxy-acylnornicotine (Fig. 3, lanes b, d, f). This result shows that theN-hydroxyacylnornicotine is localized mainly, if not entirely, atthe surface of the aerial part of the plant.To identify more exactly the localization of the N-hydroxy-

acylnornicotine, the exudate drops were removed from the glan-dular trichomes, with a drawn out microcapillary pipette con-taining acetonitrile, and analyzed for nicotine alkaloids. Theyonly contained N-hydroxyacylnornicotine in significant amounts(Fig. 4, lane c). This result confirms that this substance is local-ized in the exudate drops.From a known area of the abaxial leaf surface all trichomes

were collected with fine tweezers and the remaining surface areawas washed. The washing solution and the collected exudatedrops were analyzed for nicotine alkaloids. Both samples hadN-hydroxyacylnornicotine (Fig. 4, lanes a and b). These resultsare consistent with the report that exudate drops fall as a depositon the leaf cuticle(1).

Since the N-hydroxyacylnornicotine was found entirely in thetrichome exudate it is probably synthesized in the trichomesimmediately before being secreted in the exudate. If this is true,the alkaloid should not be found inside the trichomes in asignificant quantity. As a test of this hypothesis 500 trichomeswere collected from the washed and unwashed leaf surface andanalyzed for nicotine alkaloid content. The trichomes of thewashed surface contained nicotine and nornicotine, but no de-tectable amount of N-hydroxyacylnornicotine, while those of theunwashed leaf possessed this alkaloid as well as nicotine andnornicotine (Fig. 4, lanes d and e). This result again supports thehypothesis that the N-hydroxyacylnornicotine is secreted intothe exudate, which can be washed from the trichomes (Fig. 2,lanes c and d), and the synthesis of this material takes placeshortly before secretion. The presence of nornicotine and nic-otine inside the trichomes suggested that they might be involvedin the synthesis of this new substance and this hypothesis wasthen tested.To identify the alkaloid precursor(s) transported to the leaves

in vivo the bleeding sap from the root and the stem was squeezedout, collected with a microcapillary tube, with or without aceto-

482 ZADOR AND JONES

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ALKALOID BIOSYNTHESIS IN THE TRICHOMES

Table II. Radioactivity and Specific Radioactivity RecoveredA. Radioactivity recovered from major nicotine alkaloids fractions after feeding of leaves with

[2'-'4C]nicotine. The data are the average ± SE of three experiments.Time Radioactivity of OtherAfter N-Hydroxyacyl- Com-

Feeding Nicotine Nornicotine nornicotine ponents

h cpm ± SE6

244022.6 ± 477.5

2307.33 ± 265.672 ± 21.1

209 ± 42.2130.5 ± 43.1

57 ± 15.896.3 ± 9.645.3 ± 14.08

B. Specific radioactivity recovered from major nicotine alkaloids after feeding of leaves with [2'-'4C]nicotine.The data are the average ± SE of three experiments.

Time Specific Radioactivity ofAfter N-Hydroxyacyl-

Feeding Nicotine Nomicotine nornicotine

h cpm/lmol6 799.6 ± 101.4 33.1 ± 12.9 109.6 ± 49.9

24 719.1 ± 122.7 109.4 ± 23.4 23.4 ± 6.96

Table III. Radioactivity and Specific RadioactivA. Recovery of radiolabel from the major nici

fractions within 24 h after feeding [2'-'4C]norniccThe data are the average ± SE of three expe

Radioactivity of

N-Hydroxyacyl-Nicotine Nornicotine nomicotine

26.3 ± 9.4 221 ± 26.5cpm ± SE

21.6 ± 3.8

B. Specific radioactivity of major nicotine alkaloiafter [2'-'4C]nornicotine feeding to leaves. The

average ± SE of three experiments.Specific Radioactivity of

Nicotine Nornicotine N-Hydroxyac'cpm/,qmol

58.1 ±4.1 692.0± 141 162.6

nitrile, and analyzed. Both ofthe sources containin a significant amount (Fig. 5). Based on thileaves were fed with [2'-'4C]nicotine. About:radioactivity was recovered, and 95% of this maform of nicotine, nornicotine, or N-hydroxyacylible II). This result suggests either the indirecnicotine via nornicotine into the hydroxyacylatesimultaneous direct conversion of nicotine intoyacylnornicotine and nornicotine. To test betsnative hypotheses the leaves were fed with [2'-'A significant portion of the radioactivity waN-hydroxyacylnornicotine (Table III). Furthermactivity of N-hydroxyacylnornicotine was highebeled nornicotine than labeled nicotine feeding,the nornicotine, not nicotine, is the directN-hydroxyacylnornicotine production.

DISCUSSION

The localization and the synthesis of N-hydr4tine is described here in one of the species of secThis substance, only recently described from fr

iity Recovered (7), has been completely missed in many surveys (8, 17, 21, 22).otine alkaloid The failure ofthe alkaloid to be detected in fresh tissue previously)tine to leaves. may be because this chemical is hardly soluble in water andriments. cannot be recovered from the so-alled base fraction, as can the

other nicotine alkaloids (24).Other N-Hydroxyacylnornicotine is found in the exudate secreted by

Components the glandular trichomes at the epidermis and its synthesis hasdirect connection with the pool of nicotine alkaloids. Nicotine,which is the main alkaloid in the internal tissue, is produced bythe root in N. glauca, N. tabacum, and N. glutinosa, and trans-

283.3 ± 90.3 ported to the aerial part of the plant where it is demethylated tonornicotine (4). This synthesis route was also found in N. stock-

ds within 24 h tonii in the present study. Nomicotine appears to be the substratedata are the of an acyltransferase ( 15) and after acylation is secreted imme-

diately into the exudate by the glandular cells of the trichomes.The plausibility of nornicotine as the direct precursor toN-hydroxyacylnornicotine is strengthened when it is noted that

ylnornicotine the specific activity ofadministered nornicotine was 16,000 timesless than that ofthe administered nicotine, yet the specific activity

t 34.7 of the N-hydroxyacylnornicotine obtained from nornicotine washigher than for nicotine. The amounts of endogenous nicotineand nornicotine in these leaves prior to feeding was in the same

led only nicotine order of magnitude. Exudate on the leaf surface may not contains result isolated enzymes for alkaloid degradation, since no alkaloidal breakdown35% of the fed products soluble in acetonitrile were detected in the exudate.tterial was in the Thus, the N-hydroxyacylnornicotine can build up in the exudatenornicotine (Ta- to a very high concentration for presentation to herbivorousconversion of insects, without degradation or toxicity to the plant.

d alkaloid or the The other substrate in this conversion, the long chain fattyboth N-hydrox- acid, was reported earlier from the chloroplast (3, 6, 23). Theteen these alter- latter investigators also suggested that the electrondense globules'4C]nornicotine. observed by EM might be a deposit ofinsoluble lipids. Therefore,is recovered as Akers et al. (1) who also found these globules proposed that thekore, the specific chloroplast ofthe glandular cells may have a metabolism directed-r in case of la- toward lipid rather than starch. These results suggest a situationsuggesting that in which the enzyme activity producing N-hydroxyacylnornico-precursor for tine in Repandae is based on the fatty acid and the nicotine

alkaloid pathway being present.It is also of interest that the N-hydroxyacylnornicotine is

heterogeneous with respect to length of the acylchain (C-12 toC-16, Ref. 20). Thus, the enzyme activity is not very specific to

oxyacylnornico- the C- 14 chain length. Presently, it is not known whether or not-tion Repandae. the hydroxylation of the acyl chain is essential for the acylationesh plant tissue or toxicity. The isolation of the enzyme(s) of this pathway in

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484 ZADOR AND JONES

trichomes is in progress (9). If the acylation is due to a singleenzyme it will make this system highly attractive for new ap-proaches to plant genetic engineering. That is, to achieve novelproduction of an insecticidal nonprotein factor (7, 8) one needsto transfer only the last enzyme of a biosynthetic route (insteadof a whole pathway) into a plant which contains the appropriateprecursor (e.g. cultivated tobacco produces nornicotine). A num-ber of other solanaceous plants related to Nicotiana possesstrichome secretions toxic to some insects (5). Thus, conceptsdeveloped using the N. stocktonii system may in the future beapplied to solanaceous food plants as well.

Acknowledgments-The authors would like to thank Professors E. Leete and L.P. Bush for fruitful discussions and suggestions on the manuscript, and to ProfessorL. P. Bush for providing the labeled nicotine.

LITERATURE CITED

1. AKERS CP, JA WEYBREW, RC LONG 1978 Ultrastructure of glandular trichomesof leaves of Nicotiana tabacum L. cv Xanthi. Am J Bot 65: 282-292

2. ALWORTH WL, H RAPOPORT 1965 Biosynthesis of the Nicotiana alkaloids inNicotiana gluttinosa. Arch Biochem Biophys 112: 45-53

3. BAILEY JL. AG WHYBORN 1963 The osmiophilic globules of chloroplast. II.

Globules of the spinach-beet chloroplast. Biochim Biophys Acta 78: 163-169

4. DAWSON RF 1945 An experimental analysis of alkaloid production in Nico-tiana: the origin of nornicotine. Am J Bot 32: 416-423

5. DIMOCK MB, GA KENNEDY 1983 The role of glandular trichomes in theresistance ofLvcopersicum hirsltum F. glabradum to Heliothis zea. EntomolExp Appl 33: 263-268

6. GREENWOOD AD, RM LEECH, JP WILLIAMS 1963 The osmiophilic globules ofchloroplasts. 1. Osmiophilic chloroplasts and their isolation and compositionin Viciafaba L. Biochim Biophys Acta 78: 148-162

7. HUESING JE, D JONES 1986 A new form ofantibiosis in Nicotiana. Phytochem-istry. In press

8. JEFFREY RN 1959 Alkaloid composition of species of Nicotiana. Tobacco Sci3: 89-93

9. JONES D, HUESING J, ZADOR E, HEIM C 1986 The tobacco-insect model systemfor genetically engineering plants for non-protein insect resistance factors. InLaw J, ed, Molecular Entomology, UCLA Symposia on Molecular and

Plant Physiol. Vol. 82, 1986

Cellular Biology, New Series, Vol 49. Alan R Liss, New York. In press10. JONES D, GA JONES, T HAGEN, E CREECH 1985 Wild species of Nicotiana as a

new source of tobacco resistance to the tobacco hornworm, Manduca sexta.Entomol Exp Appl 38: 157-169

1 1. KEENE CK, GJ WAGNER 1985 Direct demonstration of duvatrienediol biosyn-thesis in glandular heads of tobacco trichomes. Plant Physiol 79: 1026-1032

12. LOTTER HL 1984 Untersuchung der Struktur-Wirkungsbeziehung antihepato-toxischer Naturstoffe (Silybin-Antamanid) durch Rontgenstrukturanalyse. ZNaturforsch 39c: 535-592

13. MIYANO M, H MATSUSHITA, N YASUMATSU, K NISHIDA 1979 N-isopropylnor-nicotine in burley tobacco (Nicotiana tabacum). Agric Biol Chem 43: 2205-2206

14. MIYANO M, N YASUMATSU, H MATSUSHITA, K. NIsHIDA 1981 1'-(6-hydroxy-octanoyl)nornicotine and 1'-(7-hydroxyoctanoyl)nornicotine, two new al-kaloids from Japanese domestic tobacco. Agric Biol Chem 45: 1029-1032

15. MURAKOSHI F, A SANDA, J HAGIMIWA, H TOMASU, SH OMIYA 1978 An acetyl-Co A cytisine N-acyltransferase in Sophora seedlings. Chem Pharm Bull 26:809-8 12

16. PARR JC, R THURSTON 1968 Toxicity of Nicotiana and Petunia species tolarvae of the tobacco homworm. J Econ Entomol 61: 1525-1531

17. SAITOH F, M NOMA, N KAWASHIMA 1985 The alkaloid contents of sixtyNicotiana species. Phytochemistry 24: 477-480

18. SCHMELTZ J 1971 Nicotiana and other tobacco alkaloids. In M Jacobson, DGCrosby, eds, Naturally Occurring Insecticides. Marcel Dekker, Inc, NewYork, pp 99-135

19. SEVERSON RF, RF ARRENDALE, OT CHORTY, AW JOHNSON, DM JACKSON,GR GWYNN, JF CHAPLIN, MG STEPHENSON 1984 Quantitation ofthe majorcuticular components from green leaf of different tobacco types. J AgricFood Chem 32: 566-570

20. SEVERSON RF, JE HUESING, D JONES, RF ARRENDALE, VA SISSON 1987Identification of a tobacco hornworm antibiosis factor from the cuticulae ofNicotiana section Repandae. J Chem Ecol. In press

21. SMITH HH 1942 Alkaloids in certain species and interspecific hybrids ofNicotiana. J Agric Res 65: 347-359

22. SMITH HH, DV ABASHIAN 1963 Chromatographic investigations on the alka-loid content of Nicotiana species and interspecific combinations. Am J Bot50: 435-447

23. STUMPF PK, AT JONES 1963 The biosynthesis of long chain fatty acids bylettuce chloroplasts. Biochem Biophys Acta 70: 20-32

24. Tso TC, RN JEFFREY 1959 Biochemical studies on tobacco alkaloids. The fateof labelled tobacco alkaloids supplied to Nicotiana plants. Arch BiochemBiophys 80: 46-56

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