BIOSYNTHESIS ROOT - Plant Physiology · PLANT PHYSIOLOGY Total acid-hydrolyzable nicotinic acid content of the roots was measured by a bioassay procedure using Lactobacillus plantarum

BIOSYNTHESIS OF ANABASINE AND OF NICOTINE BY EXCISEDROOT CULTURES OF NICOTIANA GLAUCA 1, 2,3

MARIE L. SOLT, R. F. DAWSON, AND D. R. CHRISTMANDEPARTMENT OF BOTANY, COLUMBIA UNIVERSITY, NEW YORK, NEW YORK, AND DEPARTMENTS OF CHEMISTRY AND

BIOLOGY, BROOKHAVEN NATIONAL LABORATORY, UPTON, L. I., NEW YORK

Investigations of Nicotiana alkaloid biogenesishave tended thus far to center about nicotine as thearchetype of the group. Sufficient progress has beenmade to justify extending both the methods and theconcepts of these investigations to other members ofthe alkaloid complex. The case of anabasine isespecially attractive for the following reasons.

First, anabasine is a minor alkaloid accompanyingnicotine in Nicotiana tabacum L. However, it is themajor alkaloid in N. glauca Grah. where it is accom-panied by moderate amounts of nicotine. The latterspecies thus presents opportunity for investigating theformation of both substances in one and the samesystem using the known characteristics of nicotineproduction as convenient internal points of reference.

Second, it was shown earlier (6) that relativeanabasine and nicotine producing capacities are notshared equally by all portions of the plant body. EJx-tension of this aspect of the study may be expectedto reveal the degree of separation and perhaps also todelimit the compartment associated with the produc-tion of each alkaloid.

Third, the excised root system of N. glautca wouldappear to afford an ideal experimental tool for in-vestigating the rate stability of alkaloid production.Elsewhere, we report the fact (9) that supplying pre-cursors of nicotine to excised root cultures of N.tabacum does not increase nicotine yields. The pres-ent svstem might be used to learn whether nicotineproduction could be diverted to anabasine production,or vice versa, by supplying the corresponding pre-cursor in excess.

MATERIALS AND METHODSEXCISED ROOT CULTURES: A clonal line of Nico-

tiana glauca Grah. was established from one sterileseedling. White's medium (18) was employed withthe customary additions of copper and molybdenum(16). The behavior of this clone in sterile cultureis worth noting.

In the first passages, the clone exhibited pro-nounced dominance of the primary root such thatbranching of the second and higher orders was cur-tailed. The primary root grew rapidly and traversed

1 Received April 18, 1960.2 Work completed in part under the auspices of the

United States Atomic Energy Commission at ColumbiaUniversity under contract AT-30-1-1778 and at Brook-haven National Laboratory and of the Tobacco IndustryResearch Conmmittee.

3 The work on nicotinic acid recovery was performedby Miss Dorothy Hufnagel, Tobacco Industry ResearchCommittee Fellow in Botany at Columbia, 1957 to 1959.

the periphery of the culture flask (125 ml Erlenmyer)many times to form a coiled arrangement. Culturesremaining in this state were designated subclone A.After more than a year of culture, however, the domi-nance of the primary root diminished, and root branch-es of secondary and higher orders became common.In the latter condition, the cultures were similar inappearance to the excised root cultures of N. tabacumL. var. Turkish which have been grown for manyyears in this laboratory. Such cultures were isolatedand maintained through subsequent passages as sub-clone B. The relation between root morphology andalkaloid production rates is unknown. The data re-corded herein have been obtained with the use ofsubclone B except where otherwise indicated. Formore than a year following initial isolation of theclone, the cultures exhibited a strong tendency to re-generate shoot buds.

GROWTH AND ALKALOID PRODUCTION: For meas-uring growth and alkaloid production rates, suitablenumbers of cultures were prepared, incubated at 30+ 1° C, and harvested in lots of 50 each at stated in-tervals. Such measurements were made in two sepa-rate experiments.

ADDING INTERMEDIATES: In general, new culturesin lots of 50 to 100 were prepared and incubated for1 week prior to adding intermediates. The intermedi-ates were filter sterilized through Pyrex fritted glassdisks (UF) or autoclaved depending upon compoundlability. Incubation was continued for an additional4 weeks after adding the intermediates.

Nonlabeled intermediates were supplied to observethe effects upon relative yields of anabasine and nico-tine. In one series, DL-ornithine and L-lysine weresupplied separately at the level of 1.0 mg per cultureto duplicated lots; controls without added precursorwere provided in quadruplicate. In a second series,lysine and ornithine were again supplied as above butwith additions of 0.3 mg of nicotinic acid per culture.Figures for the effects of supplying nicotinic acidalone were secured in triplicate from other experi-ments in which the alkaloids were separated and as-sayed gravimetrically as the dipicrates. Ratios ofanabasine to nicotine yields were calculated, and theresulting figures, despite their heterogeneous origins,were subjected to an analysis of variance using a2 X 3 factorial design with the usual modificationsfor unequal lot numbers.

For measuring the available nicotinic acid in theroot cultures during the exponential period of growth,two experiments were completed in each of which fivelots of 20 cultures each were harvested on the 11th,14th, 17th, and 20th days, respectively, of the passage.

887

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

Total acid-hydrolyzable nicotinic acid content of theroots was measured by a bioassay procedure usingLactobacillus plantarum (Orla-Jensen) Holland,strain 17-5, as test organism (2). In another experi-ment, 337 ug of nicotinic acid were added to each of20 cultures and the amount remaining on the 20thday ascertained by assay.

LABELED INTERMEDIATES: L-Lysine-C14 (2.3 x

107 dpm/mg) was purchased from Schwarz Labora-tories. Inc. DL-Ornithine-2-C14 monohydrochloride(8.2 X 106 dpm/mg) was purchased from Tracerlab.Nicotinic acid labeled with tritium in position 2 was

prepared from 2-bromo-3-picoline by methods de-scribed elsewhere (9). Two preparations were em-

ployed with specific activities 2.25 X 105 and 2.57X 105 dpm/mg, respectively. Nicotinic acid ring-labeled with tritium was also prepared by the reactionLi" (n, a))H3 in the Brookhaven reactor (1, 21, 22).The specific activity of the latter after purificationand dilution (9) was 2.65 X 105 dpm/mg. 6-Tritiumnicotinic acid was prepared from 6-bromo-3-picoline(9) with specific activity 2.16 X 10' dpm/mg.

Circumstances surrounding the addition of thesecompounds to the culture medium were similar to

those already described in a preceding section except

for such details as are indicated in table I.

ISOLATION AND SEPARATION OF ALKALOIDS: Theroots were removed from the culture flasks andwashed with distilled water on a Buchner funnel. Thewashings were combined with the spent culture fluids.The roots were ground with a little trichloroacetic acidand then extracted twice with boiling water. Thefiltered aqueous extracts were combined with thespent culture fluids and washings and the volume re-

duced at lowered pressure. The alkaloids were ex-

tractecd from the alkalinized concentrate continuouslywith ether for 72 hours.

The ethereal extracts were concentrated to lowvolume and transferred to columns packed underbuffer-saturated ether with Johns-Manville Hyflo-SuperCel moistened (0.2 ml/g) with the same 0.5 mipotassium phosphate buffer (pH 6.6) (7). Ethersaturated with buffer was passed through this columnun(ler pressure and fractions collected automatically.When the nicotine fraction had left the column, thesolvent was changed to ether-chloroform (1: 1) andanabasine was eluted. In certain cases, when theanabasine was just off the column, the solvent was

changed to pure chloroform and nornicotine eluted inthe next two collections.

For assay, the different collections were combinedaccording to alkaloid composition and transferred toN HCl in a separatory funnel. The nicotine fractionwas steam-distilled onto an Amberlite IRC-50 resincartridge and eluted with HCl to remove nonvolatileimpurities (7). Anabasine and nornicotine fractionsdid not require distillation for purposes of assay.

The amounts of each alkaloid were estimated spectro-photometrically (19) using physical constants estab-lished in this laboratory.

When the alkaloids were to be isolated as picrates,the fractions were steam distilled into aqueous picricacid solution (nicotine from excess MgO and ana-basine from excess NaOH and NaCl). The distillateswere taken to low volume under reduced pressure,and the picrates dissolved by boiling with the leastamount of water and then allowed to crystallize atroom temperature. Solubility corrections were ap-plied when calculating picrate recoveries for esti-mating radiochemical yields. In still other cases, theinitial ethereal extracts were applied directly to filterpaper sheets (Whatman No. 1) for chromatography.

ISOTOPE ANALYSIS: Samples of the alkaloid pic-rates were assayed for C14 and for tritium by combus-tion and proportional gas counting (3, 4, 5, 20). Re-sults are expressed as radiochemical yield, that is, theproportion of radioactivity originally supplied to thecultures which was recovered in a given alkaloid frac-tion. Specific activities of tritium-labeled com-pounds were corrected for isotope decay.

PAPER CHROMATOGRAPHY OF ALKALOIDS: Ethe-real solutions of the alkaloids were concentrated tolow volume and applied to sheets of Whatman No. 1filter paper which had been impregnated earlier with0.2 M acetate buffer (pH 5.6). The chromatogramswere run with tert-amyl alcohol saturated with buffer(17). p-Aminobenzoic acid and cyanogen bromidewere the spot reagents. In some cases, the chromato-grams were cut into strips and scanned for radioactivespots with a windowless, flowing-gas proportionalcounter attached to an automatic strip chart recorder.

PREPARATION AND ADDITION OF NICOnNES: Pyr-rolidine-C14-nicotine was prepared by supplying 9.2mg of DL-ornithine-2-C14 monohydrochloride (2.84X 107 dpm/mg C) as obtained from Tracerlab to 25cultures of the excised roots of Nicotiana tabacumvar. Turkish. After 18 days, the nicotine was isolatedfrom these cultures as described above. This nicotinehad an activity of 1.36 X 106 dpm/mg C. The radio-chemical yield was 0.11. Dewey, Byerrum, and Ball(10) and Leete (13) have shown that the activitvcontributed by ornithine-2-C'4 to nicotine is locatedalmost entirely in the pyrrolidine ring.

Pyridine-C14-nicotine was obtained by supplyingto similar root cultures of N. tabacumn nicotinic acidring-labeled with C14 by nuclear recoil methods(1,9,21). A total of 3.75 mg (sp. act. 1.93 X 103dpm/mg C) was added to 25 cultures. The nicotineproduced in the presence of this acid possessed anactivity of 1.86 X 102 dpm/mg C. It has been shownelsewhere (9) that pyridine ring label of nicotinic acidis recoverable as pyridine ring label of nicotine.

Pyrrolidine-C14-nicotine (10.1 mg and 5.07 X104 dpm) was supplied as the hydrochloride to 79 cul-tures of excised roots of N. glauca for 38 days. Like-wise, pyridine-C14-nicotine (8.6 mg and 6.85 X 102dpm) was supplied, also as the hydrochloride, toanother 79 cultures for 38 days. Each group of cul-tures was harvested in two approximately equal sub-

888


SOLT ET AL-BIOSYNTHESIS OF ANABASINE AND NICOTINE

lots. The alkaloid picrates were isolated and assayedfor C14 as described above.

Nonlabeled nicotine was prepared by steam dis-tilling nicotine dipicrate (Distillation ProductsIndus.) into HCI. The alkaloid was supplied as thehydrochloride (pH 4.0) to two lots of 50 cultureseach. Two lots of cultures, 50 each, served as con-trols.

GROWTH MEASUREMENTS: Growth of the rootsin sterile culture was measured by weighing the ma-terial obtained at each harvest after drying in aventilated oven at 600 C.

RESULTS

ALKALOID PRODUCTION RATES: In the early partof the culture period, the roots made anabasine andnicotine in the proportion of 2 to 1 (fig 1). Towardthe end of each passage, however, the ratio hadchanged to 4 to 1. These figures fall in the rangealready established for relative nicotine and anabasineproduction capacities of N. glauca roots as estimatedby the use of reciprocal grafts (6).

Actual production of nicotine increased exponen-tially with elapsed time during the first 20 days asdid also root dry weight. For reasons outlined bySolt (16), however, it is convenient to eliminate the

la

16

14-

l-J

'a

0r

121-

10-

a

6

4-

2

0

DRY WEIGHT

0

1- A Io/-

5 10 15

time variable and to consider directly the linear rela-tion that exists between nicotine production and rootdry weight. In the present instance, once the cul-tures in two different passages began to grow at anexponential rate, namely, at 10 to 15 days from theinitial inoculation up to the 40th day, the yield of nico-tine was 2.72 + 0.214 /g per mg root dry weight(statistic is standard error of estimate). This figureis approximately 1/llth the constant relating nicotineyield to growth in root cultures of N. tabacum.

Solt has shown that the individual branch of cul-tured roots of N. tabacum grows and produces nico-tine at constant rates (16). Since growth occurs ator near the tip of the individual root branch, it seemsreasonable to conclude that nicotine production mayalso occur there and that the rate dependency of nico-tine production upon growth may rest upon restrictionof both processes to a common morphological com-partment. Solt has, in fact, shown experimentallythat this is the case in the root tip of N. tabacum,and there seems to be no doubt that the same conclu-sion may be drawn in the case of N. glauca. Con-ceptually, the Nicotiana root tip may thus be regardedas a moving point source for both new tissues andnicotine.

Actual production of anabasine did not follow asimple relationship with growth in terms of dry weight

w

Drn~D

a.

20 25 30 35 40

DAYSFIG. 1.

passage.

140

120 -

100 _ ANABASINE

so

so /

40

0

20NICOTINE

0 5 O 15 20 25 30 35 40DAYS

Mean dry weight and alkaloid yields of excised root cultures of Nicotiana glauca in the course of one

*~~~~~~ a I

889


PLANT PHYSIOLOGY

increase. Instead, the production of this alkaloidoccurred at a progressively faster rate than that ofroot dry weight and continued at a relatively high leveleven after growth rate had become virtually nil inthe last stages of each passage. The implication thatanabasine production may be a property of maturedroot tissues was examined in the following way. Itwas assumed A: that the compartment for anabasineproduction is the entire root organ possibly but notnecessarily exclusive of the growing tips; B: that pro-duction of the alkaloid proceeds at a uniform ratethroughout all parts of the compartment and for theduration of the culture period, and C: that growthproceeds at a relatively constant rate also during thisperiod. The amount of anabasine produced in anygiven period of time would, therefore, be proportionalboth to the amount of elapsed time and to the totalmass of matured root tissues. Obviously, if the thirdassumption is even approximately true, the specificrelationship between these three variables may bestated as follows. At any given moment the anabasinecontent of a root culture in micrograms is equal to aproportionality constant multiplied by the product ofthe root dry weight in milligrams and the half-time ofthe culture in days. When this calculation is madefor the period 10 to 40 days, there is obtained a pro-portionality constant of 0.40 + 0.008. The verysmall standard error of estimate indicates a smallamount of dispersion about the calculated line of re-gression and therefore validates the suggested rela-tionship. It is necessary only to comment that growthfollows a sigmoid relationship with respect to timeand is not strictly linear as assumed. The shape ofthe curve is such, however, that departure from linear-ity is not great. Thus, the line of least squares re-lating these two variables possesses a slope of 0.584mg per day with a standard error of estimate ± 0.037.The latter figure is equivalent to a coefficient of varia-tion 6.34 %. The fact that nicotine and anabasinesynthesis may occur in different compartments is ofinterest in connection with the use of different pre-cursors for these syntheses as described below.

INCORPORATING LABELED PRECURSORS: In anearlier paper (9) we have described the incorporationof labeled nicotinic acids into the pyridine ring ofnicotine by excised root cultures of N. tabacum.

In the present case, 2-tritium-nicotinic acid wasincorporated into both nicotine and anabasine, result-ing in radiochemical yields (table I) commensuratewith the relative levels of production of these twoalkaloids. Oxidation of one preparation of tritium-labeled anabasine to nicotinic acid gave a recovery of88 % of the tritium contained in the anabasine. It isclear that most of the nicotinic acid ring label hadentered the pyridine ring of anabasine. Evidently,the pyridine rings of nicotine and anabasine bothoriginate with nicotinic acid in N. glauca.

The pathway of conversion of nicotinic acid pvri-dine ring to nicotine pyridine ring was shown earlier(9) to involve loss not only of the carboxyl group butalso of the hydrogen atom in the 6 ring position.When nicotinic acid labeled with tritium in position6 was supplied to root cultures of N. glauca, only 1.1 %of the radioactivity was recovered as labeled nicotineand anabasine (table I).

Ornithine has been shown to provide the pyrrolid-ine ring of nicotine in N. tabacum (10, 13). Orni-thine-2-C14 supplied to the glauca cultures gave morethan 15 times as much label in the nicotine subse-quently produced as in the anabasine (table I). De-spite this expected outcome, actual radiochemicalyields were much lower than expected. Possiblv inN. glauca ornithine is more rapidly metabolized byother pathways than appears to be the case in N.tabacumn. Leete (14) has shown that lysine is theprecursor of the piperidine ring of anabasine. Testsconducted in our sterile root culture system confirmedthis result, for 100 times as much label was introducedinto anabasine from lysine than into nicotine (tableI). It may be concluded, therefore, that nicotine bio-synthesis in N. glauca very likely follows the samepathway as it does in N. tabacum. The anabasinebiosynthesis pathway shares with the nicotine path-

TABLE IINCORPORATION OF LABELED INTERMEDIATES INTO ALKALOIDS BY EXCISED ROOTS OF NICOTIANA GLAUCA

COMPOUND SUPPLIED

SPECIFIC CONCEN-ACTIVITY TRATION**

dpm/mg mg/ml

PERIOD ALKALOIIOF

CONTACT ANABASINE

DAYS mg

:D YIELDS* RADIOCHEMICAL YIELDS

NICOTINE ANABASINE NICOTINE

mg

Nicotinic acid-H3Nicotinic acid-2-H3Lysine-C' 4

Nicotinic acid-2-H3Nicotinic acid-2-H3Nicotinic acid-5-H3DL-Ornithine-2-C14

265,000257,000333,000

221,000221,000172,960

8,300,000

0.0050.0070.0017

0.0120.0200.00970.009

Subclone A213926

Subclone B42453427

* As the crystalline dipicrates.** In the culture medium (total vol/culture, 30 ml).

COMPOUND

145110

7157

783710851

0.220.420.29

0.080.050.0080.001

23173524

0.050.120.003

0.020.020.0030.015

890



way a similar use of nicotinic acid; but, as Leete hasshown, it departs from the latter in using a homologof ornithine, namely lysine, as the precursor of thepiperidine ring.

One interesting point may be noted in passing.Subclone A gave extraordinarily high radiochemicalyields and specific activities when supplied with labeledalkaloid precursors (table I). This unusual correla-tion between morphological and biochemical para-meters merits further consideration in terms of theidentification of the factors which are rate-limitingfor alkaloid synthesis in these excised root systems.In the latter connection, note that none of the alkaloidprecursors supported an increase in nicotine or ana-

basine yield. Apparently, the labeled intermediateswere incorporated into nicotine and anabasine simplyby pool dilution.

NICOTINIc ACID CONTENT OF ROOT TISSUES: To-tal acid-hydrolyzable nicotinic acid content of the rootcultures (table II) increased exponentially withelapsed time during the first 20 days of each passagewhen the cultures were transferred at sufficientlyshort intervals (21 days) to maintain both growthand alkaloid production under steady state conditions(cf. 16). The proportionality constants (logs) forgrowth in dry weight (0.078) and for nicotinic acidcontent (0.072) were essentially equal. Therefore,it is convenient once more to consider chemical com-

position as a simple linear function of root dry weight.The proportionality constant relating mean nicotinicacid content per culture to mean root dry weight per

culture was 0.127 0.009 Mg per mg.

Since nicotinic acid is a precursor of both ana-basine and nicotine, it seems reasonable to combinethe yields of both into a single figure for alkaloidyield by the root culture of N. glauca. At 40 daysof age, the culture had produced alkaloids (anabasineplus nicotine) at an average rate of 9.78 Mg per mg

root dry weight. The comparable figure for nicotineyield by the excised root culture of N. tabacum is29.05 Ag per mg. The ratio between these two fig-ures is 2.95. The nicotinic acid content of the N.glauca root culture was 0.127 Mg per mg root dryweight. Again, the comparable figure for tabacumis 0.298 ,g per mg. The ratio between these twofigures is 2.35. Obviously, the concentration of to-tal available nicotinic acid (by acid hydrolysis) in the

tissues of these two types of roots is rather closelyrelated to the actual amounts of pyridine alkaloidswhich they produce. Whether or not this correlationwould extend to other comparisons within the genusNicotiana is unknown.

When nicotinic acid (337 Mg/culture) was suppliedto N. glauca root cultures on the 8th day of thepassage, 210 lAg was recovered in the culture mediumon the 20th day. Nine ,ug were recovered from theroots. At 20 days, one single root culture not sup-plied with nicotinic acid would have contained aboutone Ag of nicotinic acid (cf. above). Consequently, anet recovery of 218 Ag was realized. The difference,120 Ag per culture or 36 % of the original amount sup-plied, was presumably metabolized by the roots.Radiochemical yields calculated on the basis ofamounts of labeled nicotinic acid which disappearedwill obviously be somewhat greater than those listedin table I.

Although labeled nicotinic acids supplied to theroot cultures were always incorporated into nicotine,the failure of such additions to affect nicotine or ana-basine yields in the quantitative sense is noteworthyas has been indicated above. The present observa-tions indicate quite clearly that, whether nicotinic acidis supplied to the cultures or not, there does not occura rate-limiting step for alkaloid production anywherein the reaction sequence up to and including nicotinicacid formation. In this sense, the observed correla-tion between total alkaloid production and nicotinicacid content of the root tissues of the species underdiscussion (see above) is entirely coincidental.

ATTEMPTED ALTERATION OF RELATIVE RATES OFANABASINE AND NICOTINE PRODUCTION: Lysine andornithine were supplied separately to the root cultureswith and without nicotinic acid, and the ratios of ana-basine yield to nicotine yield were calculated (tableIII).

The customary statistical analysis of these ratiosrevealed the occurrence of wholly negligible variancefor the comparison nicotinic and no nicotinic acid andfor interaction between pyridine and pyrrolidine-piperidine precursors. Variance for the comparisonpyrrolidine-piperidine with no pyrrolidine-piperidineprecursors yielded a ratio of 0.81 in the F-test withthe variance within treatments. Even this falls farshort of the value 19.37 required for significance at

BLE IINICOTINIC ACID CONTENT OF ROOT CULTURES WITH AND WITHOUT ADDED NICOTINIC ACID

TREATMENT DAYS MEAN ROOT MEAN NICOTINIC ACID CONTENT/CULTUREDRY WT ROOTS CULTURE MEDIUM

mg Mg MgNone 8 1.10 0.150 1

11 2.30 0.25014 4.70 0.575 ...

17 7.83 0.90 ...

20 8.87 1.18 ...Nicotinic acid added(337 Mg/culture) 20 9.4 9.15 210

891


PLANT PHYSIOLOGY

ALKALOID CONTENT OF ROOT

TABLE IIICULTURES SUPPLIED WITH NONLABELED PRECURSORS

MEAN DRY MEAN CONTENT/CULTURE CONC IN ROOTS RATIO OFTREATMENT WT ANABASINE

OF ROOTS NICOTINE ANABASINE NICOTINE ANABASINE TO NICOTINE

mg tggg/gmg tg/mg

No additions 19.6 43 105 2.2 5.4 2.419.0 42 121 2.2 6.4 2.9

Ornithine 23.6 53 165 2.2 7.0 3.1Lysine 23.3 58 181 2.5 7.8 3.1

22.4 60 160 2.7 7.1 2.7

No additions 26.3 83 189 3.2 7.2 2.224.8 63 166 2.5 6.7 2.7

Ornithine + niCOtiniC acid 26.2 77 198 2.9 7.6 2.625.7 57 184 2.2 7.2 3.2

Lysine + niCOtiniC acid 25.7 80 234 3.1 9.1 2.928.7 78 257 2.7 9.0 3.3

Nicotinic acid ... 71 241 ... ... 3.4... 56 126 ... ... 2.3

_... 91 281 ... ... 3.1

the 95 % level of probability and the prevailing de-grees of freedom (8 and 2).

It might have been supposed, since nicotinic acid(the common precursor) concentration is evidentlynot a limiting factor, that the production of one or

the other alkaloid could have been favored by supply-ing its specific precursor in excess. Such expectationwas not realized. Possibly, lack of interplay betweenthe two alkaloid-producing mechanisms may have re-

sulted from their separation into different compart-ments within the root (cf. above). Possibly, also,there are specificities in the pathways of synthesisof the two alkaloids which preclude such interplay.Leete (14) has shown, for example, that lysine isconverted to anabasine via an unsymmetrical inter-mediate, whereas he and Lamberts et al (12,15) haveshown that ornithine traverses a symmetrical inter-mediate on the pathway to nicotine. Whatever thereason, the stability of alkaloid production rates in thisexperimental system is extraordinary and deservingof further experimental attention.

METABOLISM OF ADDED NICOTINES: Pyridine-

C14-nicotine was recovered from the root cultures tothe extent of 60 % of the amount originally added.The corresponding figure for pyrrolidine-Cl4-nicotinewas only 43 % (table IV). The chemical basis fora differential turnover rate for the two rings is notknown but may hold considerable interest for futureinvestigation.

The above figures include some radioactivity thatappeared in the anabasine fraction from the SuperCelcolumns. However, examination of paper chromato-grams made from the alkaloid fractions as they came

off the columns indicated the presence of radioactivitynot in anabasine but in the small amounts of nornico-tine that accompanied anabasine. Experiments inwhich nonlabeled nicotine was added to similar cul-tures revealed that the quantities of nornicotine didnot increase as a consequence of nicotine feeding(table V). Therefore, the appearance of activity innornicotine indicates ready transfer of methyl groups

between pyridyl-pyrrolidyl residues without con-

comitant changes in the actual pool size of nornicotine.For this reason, and because of the known biosynthetic

LE IVRECOVERIES OF RADIOACTIVITY AFTER SUPPLYING NICOTINE LABELED WITH CARBON 14 IN PYRIDINE AND PYRROLIDINE

RINGS, REsPECTIVELY, TO EXCISED ROOT CULTURES OF NICOTIANA GLAUCA

COMPOUND SUPPLIED AMOUNT ALKALOID YIELDS**SUPPLIED ANABASINE NICOTINE

mg mg mg

Nicotine-pyridine-C14 3.96 2.8 3.4 0.69'^ " "9p3.47 2.9 2.7 0.51

Nicotine-pyrrolidine-C14 4.14 3.4 3.4 0.43"i "~ "p 3.92 4.6 3.8 0.44

* Includes all radioactivity recovered in nicotine and nornicotine (cf. text).** As free bases.

892



relationship between nicotine and nornicotine (7),the recoveries of radioactivity listed above may, forthe present purpose, be assigned to nicotine.

When nonlabeled nicotine was added to the cul-tures (table V), 74 % was recovered after 38 days.This quantity is higher than the recovery of pyridine-ring-labeled nicotine given above. However, the con-centration of added alkaloid in the tracer experimentwas half that employed in the present experiment.The mean amounts of nicotine to disappear per culturewere 55 Ag in the latter case and 42 /g in the former.Evidently, the fraction of added nicotine to disappearis related to the amount originally supplied. In theexperiments described here, the amounts of nicotineadded to the culture solutions were of the order ofmagnitude of the amounts normally contained in thecultures at the end of a passage. Consequently, thelosses described herein may be expected to exceedthose which would occur in the root culture to whichno nicotine is added.

DISCUSSIONThe unity of alkaloid biosynthesis in the Nicotiana

genus is indicated in the present instance by the utili-zation of nicotinic acid as precursor of the pyridinerings of nicotine and of anabasine. Variations inthe reduced ring were accounted for by using orni-thine and its homolog lysine as precursors. The latterresults confirm in a microbially sterile system the re-sults already reported in greater detail by Leete (13)and by Dewey, Byerrum, and Ball (10) using wholetobacco plants.

By employing a system in which both nicotine andanabasine are produced concurrently, it has beenpossible to show that the two alkaloids are probablysynthesized in separate portions of the root mass.Nicotine production, strictly correlated ratewise withgrowth, occurs in the root tips, while anabasine pro-duction seems to occur primarily in matured roottissues. Nicotine production in the excised root ofN. tabacum also occurs in the root tip where growthis a prominent physiological activity. The rate ofproduction of nicotine in both systems is so closelydependent upon growth rate as to suggest an obliga-tory dependency. In chemical terms, this wouldmean that the rate of a single chemical reaction is

limiting for both processes. Consequently, nicotineproduction becomes a facet of the biochemistry ofgrowth, a rather new notion in the field of plantphysiology. It is tempting to speculate that success-ful identification of the rate-limiting step for nicotineproduction might open a backdoor into the still ob-scure biochemistry of plant growth and development.

Evidently, the rate-limiting step for nicotine pro-duction must lie at a point on the sequential reactionpathway between nicotinic acid plus ornithine or ly-sine and nicotine or anabasine, respectively. Thereis no present indication of the identity of this rate-limiting step, although several obvious possibilitiessuggest themselves for experimental test. One isthe production rate of a postulated 1,6-dihydronico-tinic acid (8) and the other is the production rate ofthe piperidine or pyrrolidine ring precursor from ly-sine or ornithine, respectively.

The rate stability of anabasine production parallelsthat of nicotine production (8, 16). Failure to obtaina diversion of alkaloid synthesis from one to the otherby adding lysine and ornithine separately is a casein point. It may be that the same step is rate-limitingfor both alkaloids. If so, it is difficult to understandwhy nicotine production should be so intimately re-lated to growth rate while anabasine production isnot. By way of speculation, the unusual regenerativeproperties of N. glauca may be cited (11). It seems

entirely possible that matured root tissues of thisspecies could retain a limited portion of the sequen-tial reaction pathways necessary for growth or de-velopment sufficient to permit continued pyridinealkaloid production.

There is an appreciable turnover of nicotine inthese root cultures, but the exact magnitude cannotbe estimated from the data given. Owing to the ex-ponential nature of growth and of nicotine accumula-tion, it is evident that nicotine turnover would bestbe measured by adding small amounts of nicotine ofhigh specific activity at intervals throughout the cul-ture period. Such intervals and the amounts of ma-terial to be added should be so chosen as to coincideapproximately with the course of nicotine accumula-tion in the culture.

In conclusion, the excised root cultures of Nico-tiana glauca afford an excellent experimental systemfor investigating the kinetics, the compartmentaliza-

TABLE VEFFECTS OF ADDED NICOTINE: UPON ALKALOID COMPOSITION

OF EXCISED ROOT CULTURES OF NICOTIANA GLAUCA

TREATMENT MEAN DRY WT ALKALOID CONTENT/CULTUREOF ROOTS/CULTURE NICOTINE ANABASINE NORNICOTINE

mg /4 g

No additions 22.6 68 164 4Pt VP 21.5 67 165 5Nicotine added

(213 /Ag/culture) 23.2 200 172 4Nicotine added

(213 Ag/culture) 23.1 207 154 6

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PLANT PHYSIOLOGY

tion and the pathways of pyridine alkaloid biosyn-thesis. The kinetics and the sites of synthesis are

quite distinct in the two cases, anabasine and nicotine.So, also, are the pathways of their biosynthesis be-yond the step in which nicotinic acid is prepared forreaction with the piperidine or the pyrrolidine ringprecursor. The nature of the link between the process

by which these alkaloids originate and the fundamentalgrowth chemistry of the plant offers a new and stimu-lating subject for further study.

SUM MARY

The simultaneous production rates of anabasineand of nicotine by excised root cultures of Nicotianaglau?ca Grah. have been analyzed in relation to rootgrowth rate. It is shown that nicotine productionis simply proportional to root dry weight yields butthat anabasine production is proportional to the prod-uct of root dry weight and the length of the cultureperiod. These relationships are interpreted to indicatea restriction of anabasine and nicotine synthesizingfunctions to different compartments in the root system.

By using isotopically labeled nicotinic acids it hasbeen shown that the pyridine ring of anabasine andvery likely also that of nicotine are produced in themanner already demonstrated for the origin of nicotinein N. tabacum. This transformation involves lossof the hydrogen atom in position 6 of the pyridinering of nicotinic acid.

The preferential incorporation of labeled ornithineinto nicotine and of labeled lysine into anabasine was

interpreted to afford confirmation, in a microbiallysterile system, of the prior observations of R. U.Byerrum and of E. Leete and their coworkers.

The tissue concentrations of nicotinic acid duringgrowth of the root cultures was followed by a micro-biological method of assay. Nicotinic acid content ofthe roots was simply proportional to root dry weight.Of the nicotinic acid added to the cultures, 64 % was

recovered by microbiological assay. It was concluded,therefore, that the concentrations of nicotinic acid as

employed in our tracer experiments have not beenrate-limiting for alkaloid biosynthesis.

It was not possible to increase the chemical yieldsof anabasine or of nicotine or to influence the relativeproportions in which they are produced by supplyingnicotinic acid alone or in combination with ornithineor lysine. The rate stability of alkaloid biosynthesisin this experimental system was emphasized.

By adding labeled nicotine to the cultures, it wasshown that there is some turnover of this alkaloid.The losses lay between 26 and 40 % at the dosagelevels employed.

LITERATURE CITED1. ANDERSON, R. C., E. PENNA-FRANCA and A. P.

WOLF. 1954. Brookhaven National LaboratoryQuarterly Progress Report. October 1 to Decem-ber 31.

2. AsSOCIATION OF VITAMIN CHEMISTS. 1947. Meth-ods of Vitamin Assay. Interscience Publishers,Inc., New York.

3. CHRISTMAN, D. R. 1957. Tritium counting inglass proportional counting tubes. Chemist An-alyst. 46: 5-6.

4. CHRISTMAN, D. R., N. E. DAY, P. R. HANSELL, andR. C. ANDERSON. 1955. Improvements in isotopiccarbon assay and chemical analysis of organic com-

pounds by dry combustion. Anal. Chem. 27: 1935-1939.

5. CHRISTMAN, D. R. and A. P. WOLF. 1955. Inherenterrors and lower limit of activity detection in gas-phase proportional counting of carbon 14. Anal.Chem. 27: 1939-1941.

6. DAWSON, R. F. 1944. Accumulation of anabasinein reciprocal grafts of Nicotiana glauca and to-mato. Amer. Jour. Bot. 31: 351-355.

7. DAWSON, R. F. 1951. Alkaloid biogenesis. III.Specificity of the nicotine-nornicotine conversion.Jour. Amer. Chem. Soc. 73: 4218-4221.

8. DAWSON, R. F. 1960. Biosynthesis of the Nico-tiana alkaloids. Sigma Xi Lecture. AmericanScientist (in press).

9. DAWSON, R. F., D. R. CHRISTMAN, A. D'ADAMO,M. L. SOLT, and A. P. WOLF. 1960. The bio-synthesis of nicotine from isotopically labeled nico-tinic acids. Jour. Amer. Chem. Soc. 82: 2628-2633.

10. DEWEY, L. J., R. U. BYERRuM, and C. D. BALL.1955. The biosynthesis of the pyrrolidine ring ofniicotine. Biochim. Biophys. Acta. 18: 141-142.

11. KEHR, A. E. and H. H. SMITH. 1954. Genetictumors in Nicotiana hybrids. In: Abnormal andPathological Plant Growth. Brookhaven Symposiain Biology No. 6. Pp. 55-78.

12. LAMBERTS, B. L., L. J. DEWEY, and R. U. BYERRUM.1959. Ornithine as a precursor for the pyrrolidinering of nicotine. Biochim. Biophys. Acta. 33:22-26.

13. LEETE, E. 1955. The biogenesis of nicotine. Chem.and Ind. 537.

14. LEETE, E. 1956. Biogenesis of nicotine and ana-

basine. Jour. Amer. Chem. Soc. 78: 3520-3523.15. LEETE, E. and K. J. SIEGFRIED. 1957. The biosyn-

thesis of nicotine. III. Further observations on

the incorporation of ornithine into the pyrrolidinering. Jour. Amer. Chem. Soc. 79: 4529-4531.

16. SOLT, M. L. 1957. Nicotine production and growthof excised tobacco root cultures. Plant Physiol.32: 480-484.

17. Tso, T. C. and R. N. JEFFREY. 1953. Paperchromatography of alkaloids and their transforma-tion products in Maryland tobacco. Arch. Biochem.Biophys. 43: 269-285.

18. WHITE, P. R. 1938. Cultivation of excised rootsof dicotyledonous plants. Amer. Jour. Bot. 25:

348-356.19. WILLITS, C. O., M. L. SWAIN, J. A. CONNELLY, and

B. A. BRICE. 1950. Spectrophotometric deter-

mination of nicotine. Anal. Chem. 22: 430-434.20. WILZBACH, K. B., L. KAPLAN, and W. G. BROWN.

1953. The preparation of gas for assay of tritiumin organic compounds. Science 118: 522-523.

21. WOLF, A. P. and R. C. ANDERSON. 1955. Radio-

active anthracene-C14 and acridine-C14 from the

neutron irradiation of acridine. Jour. Amer.

Chem. Soc. 77: 1608-1612.22. WOLFGANG, R., F. S. ROWLAND, and C. N. TURTON.

1955. Production of radioactive organic com-

pounds with recoil tritons. Science 121: 715-717.

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BIOSYNTHESIS ROOT - Plant Physiology · PLANT PHYSIOLOGY Total acid-hydrolyzable nicotinic acid content of the roots was measured by a bioassay procedure using Lactobacillus plantarum