Plant Physiol. (1973) 52, 162-165 Mechanism of Plant Growth Stimulation by Naphthenic Acid EFFECTS ON NITROGEN METABOLISM OF PHASEOLUS VULGARIS L.' Received for publication February 7, 1973 D. J. WORT, J. G. SEVERSON, JR.,2 AND DAVID R. PEIRSON3 Department of Botany, University of British Columbia, Vancouver, British Columbia Canada ABSTRACT Fourteen-day-old Phaseolus vulgaris L. cv. Top Crop (bush bean) plants were sprayed with the plant growth stimulant, potassium naphthenate (20 mM). Seven days after treatment the contents of glutamic acid dehydrogenase, glutamic-ox- aloacetic transaminase, nitrate reductase, glutamine synthetase, and cytochrome oxidase in the trifoliate leaf blades of treated plants were significantly larger, and the specific activity of the last four was significantly greater. Potassium nephthenate (1 AM) in the assay solutions did not significantly alter the activity of these enzymes in the cell-free extracts of untreated plants. Leaf discs from treated plants did not incorporate "4C- leucine into protein more actively. The protein content of leaves of treated plants was 15.3% greater, and the percentages of 16 individual amino acids in the hydrolysates of the proteins of control and treated plants showed numerous differences. The major changes were greater percentages of glutamic acid, glycine, and proline, and smaller values of arginine, lysine, tyrosine, and leucine in protein of treated plants. The con- tent of ethanol-soluble (free) amino acids was greater by 7.5 %. The principal changes in content of these acids were larger percentages of arginine and lysine, and smaller values for glutamic acid, serine, and proline in the leaves of potassium naphthenate-treated plants. The content of DNA, measured 1, 2, and 3 weeks after a foliar application of potassium naph- thenate, was not significantly different from that of untreated plants, but the amount of RNA was significantly greater at all three times of measurement. The number and weight of green pods per plant 30 days after potassium naphthenate applica- tion were significantly larger, suggesting that the stimulative action of potassium naphthenate was in progress at the times of the assays. A mechanism, involving a genetic and a meta- bolic phase, is suggested for the stimulation of plant growth by naphthenate. Naphthenic acid is the name applied to the complex mixture of acids extracted from petroleum. The diesel oil fraction (180- 400 C) may contain 0.03% naphthenic acid. The structure of the individual higher molecular-weight acids in the mixture has yet to be determined, but it is known that others are car- boxylic derivatives of cyclopentane, cyclohexane, and cyclo- IThe research was supported by National Research Council of Canada grants to the senior author. 'Present address: Department of Biology, Saint Louis University, St. Louis, Mo. 63103. 'Present address: Department of Biology, Simon Fraser Univer- sity, Burnaby, B. C., Canada. heptane. The carboxyl group of most of the acids is separated from the saturated ring by an aliphatic side chain containing one or more methylene groups. In our laboratory, gas-liquid chromatography of naphthenates derived from the diesel fraction of a Venezuelan crude oil yielded a tracing in which most of the components appeared to be in the C. to C17 range. Within this range there were 25 major peaks. The acid mixture and some of its individual members have been shown to stimulate the vegetative and reproductive growth of a large variety of plants. Severson (16) has tabulated their effects on growth, yield, and composition of over 50 species. The stimulated growth of the green bush bean, Phaseolus vulgaris L. cv. Top Crop, following naphthenate application, has been shown to be accompanied by increased rates of photo- synthesis and respiration, and greater specific activity of phos- phorylase, phosphoglycerate kinase, nitrate reductase, and glutamic-pyruvic transaminase (5). The activity of catalase in grape (9) and of peroxidase and ascorbic acid oxidase in cotton (1) has also been found to be stimulated as a result of naph- thenate application. It has been suggested (5) that the stimulation of plant growth by naphthenates rests to a large extent on an increased flow of metabolic intermediates and a greater supply of energy as ATP, coupled with an enhanced activity of anabolic enzymes. The investigation described below uses Phaseolus vulgaris cv. Top Crop in a study of the effect of KNap' on the amount and activity of four enzymes of nitrogen metabolism and of cytochrome oxidase; the contents of DNA and RNA, amino acids, and protein; and the incorporation of '4C-leucine into protein. MATERIALS AND METHODS Growth of Plants. Five seeds of the bush bean Phaseolus vulgaris L. cv. Top Crop were sown in 15-cm pots of com- posted soil. Seven days later the seedlings were thinned to two per pot, and 5 days after this one plant was allowed to remain. As a result, the population was very uniform. The plants were grown in a growth room fitted with cool white fluorescent and 60-w incandescent lamps giving an in- tensity of 16.1 klux (1500 ft-c) at the plant tops. The photope- riod was 16 hr. Day/night temperatures were 26 + 1/21 ± 1 C, and the relative humidities were 60 to 70/70 to 80%. Treatment. Fourteen days after seed sowing, at which time the primary leaves were expanded and the trifoliate leaves were in tight bud, the aerial parts of half of the plants were sprayed to drip with 20 mm (4600 jug/ml) KNap' in 0.3% (v/v) Tween 20. Control plants were not sprayed, since previous work had shown that the application of water or of 0.3% Tween 20 re- sulted in no significant (P = 0.05) changes in vegetative or 'Abbreviation: KNap: potassium naphthenate. 162 Downloaded from https://academic.oup.com/plphys/article/52/2/162/6073829 by guest on 25 August 2021
Transcript
Plant Physiol. (1973) 52, 162-165
Mechanism of Plant Growth Stimulation by Naphthenic Acid
EFFECTS ON NITROGEN METABOLISM OF PHASEOLUS VULGARIS L.'
Received for publication February 7, 1973
D. J. WORT, J. G. SEVERSON, JR.,2 AND DAVID R. PEIRSON3Department of Botany, University of British Columbia, Vancouver, British Columbia Canada
ABSTRACT
Fourteen-day-old Phaseolus vulgaris L. cv. Top Crop (bushbean) plants were sprayed with the plant growth stimulant,potassium naphthenate (20 mM). Seven days after treatmentthe contents of glutamic acid dehydrogenase, glutamic-ox-aloacetic transaminase, nitrate reductase, glutamine synthetase,and cytochrome oxidase in the trifoliate leaf blades of treatedplants were significantly larger, and the specific activity of thelast four was significantly greater. Potassium nephthenate(1 AM) in the assay solutions did not significantly alter theactivity of these enzymes in the cell-free extracts of untreatedplants. Leaf discs from treated plants did not incorporate "4C-leucine into protein more actively. The protein content ofleaves of treated plants was 15.3% greater, and the percentagesof 16 individual amino acids in the hydrolysates of the proteinsof control and treated plants showed numerous differences.The major changes were greater percentages of glutamic acid,glycine, and proline, and smaller values of arginine, lysine,tyrosine, and leucine in protein of treated plants. The con-tent of ethanol-soluble (free) amino acids was greater by 7.5 %.The principal changes in content of these acids were largerpercentages of arginine and lysine, and smaller values forglutamic acid, serine, and proline in the leaves of potassiumnaphthenate-treated plants. The content of DNA, measured 1,2, and 3 weeks after a foliar application of potassium naph-thenate, was not significantly different from that of untreatedplants, but the amount of RNA was significantly greater at allthree times of measurement. The number and weight of greenpods per plant 30 days after potassium naphthenate applica-tion were significantly larger, suggesting that the stimulativeaction of potassium naphthenate was in progress at the timesof the assays. A mechanism, involving a genetic and a meta-bolic phase, is suggested for the stimulation of plant growthby naphthenate.
Naphthenic acid is the name applied to the complex mixtureof acids extracted from petroleum. The diesel oil fraction (180-400 C) may contain 0.03% naphthenic acid. The structure ofthe individual higher molecular-weight acids in the mixturehas yet to be determined, but it is known that others are car-boxylic derivatives of cyclopentane, cyclohexane, and cyclo-
IThe research was supported by National Research Council ofCanada grants to the senior author.
'Present address: Department of Biology, Saint Louis University,St. Louis, Mo. 63103.
'Present address: Department of Biology, Simon Fraser Univer-sity, Burnaby, B. C., Canada.
heptane. The carboxyl group of most of the acids is separatedfrom the saturated ring by an aliphatic side chain containingone or more methylene groups. In our laboratory, gas-liquidchromatography of naphthenates derived from the dieselfraction of a Venezuelan crude oil yielded a tracing in whichmost of the components appeared to be in the C. to C17 range.Within this range there were 25 major peaks.The acid mixture and some of its individual members have
been shown to stimulate the vegetative and reproductive growthof a large variety of plants. Severson (16) has tabulated theireffects on growth, yield, and composition of over 50 species.The stimulated growth of the green bush bean, Phaseolus
vulgaris L. cv. Top Crop, following naphthenate application,has been shown to be accompanied by increased rates of photo-synthesis and respiration, and greater specific activity of phos-phorylase, phosphoglycerate kinase, nitrate reductase, andglutamic-pyruvic transaminase (5). The activity of catalase ingrape (9) and of peroxidase and ascorbic acid oxidase in cotton(1) has also been found to be stimulated as a result of naph-thenate application.
It has been suggested (5) that the stimulation of plant growthby naphthenates rests to a large extent on an increased flow ofmetabolic intermediates and a greater supply of energy as ATP,coupled with an enhanced activity of anabolic enzymes.The investigation described below uses Phaseolus vulgaris
cv. Top Crop in a study of the effect of KNap' on the amountand activity of four enzymes of nitrogen metabolism and ofcytochrome oxidase; the contents of DNA and RNA, aminoacids, and protein; and the incorporation of '4C-leucine intoprotein.
MATERIALS AND METHODS
Growth of Plants. Five seeds of the bush bean Phaseolusvulgaris L. cv. Top Crop were sown in 15-cm pots of com-posted soil. Seven days later the seedlings were thinned to twoper pot, and 5 days after this one plant was allowed to remain.As a result, the population was very uniform.The plants were grown in a growth room fitted with cool
white fluorescent and 60-w incandescent lamps giving an in-tensity of 16.1 klux (1500 ft-c) at the plant tops. The photope-riod was 16 hr. Day/night temperatures were 26 + 1/21 ±1 C, and the relative humidities were 60 to 70/70 to 80%.
Treatment. Fourteen days after seed sowing, at which timethe primary leaves were expanded and the trifoliate leaves werein tight bud, the aerial parts of half of the plants were sprayedto drip with 20 mm (4600 jug/ml) KNap' in 0.3% (v/v) Tween20. Control plants were not sprayed, since previous work hadshown that the application of water or of 0.3% Tween 20 re-sulted in no significant (P = 0.05) changes in vegetative or
'Abbreviation: KNap: potassium naphthenate.162
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NAPHTHENATE EFFECTS ON BEAN N METABOLISM
reproductive growth of P. vulgaris, compared with unsprayedplants. The salt was prepared by neutralization of naphthenicacid (Eastman Organic Chemicals, Rochester, N. Y.) by KOH.The average mol wt of the acid was given as 230 and the acidvalue was 243.
Extraction and Assay of Enzymes. From the treated andfrom the untreated plants, three groups of six plants each weredrawn. The finely chopped and thoroughly mixed trifoliateleaves of each group of six plants were used, in the assay ofprotein content, by the method of Lowry et al. (10) and in thedetermination of the activities of the five enzymes. Duplicatemeasurements were made in each case.
Extraction. The enzyme extract for the measurement of ni-trate reductase activity was prepared by grinding 10-g samplesof the chopped and thoroughly mixed leaf blade material in aVirTis 45 homogenizer at medium high speed for 3 min at 0 to4 C. The grinding medium consisted of 45 ml cold 0.1 M phos-phate buffer, pH 7.8, containing 1 mM reduced glutathione. Thehomogenate was filtered through four layers of cheesecloth,and its volume was brought to 50 ml with grinding medium.The filtrate was centrifuged at 20,000g for 15 min at 0 to 4 C.The resulting cell-free supernatant was used for the assay.
For the assay of glutamic-oxaloacetic transaminase, glutamicacid dehydrogenase, and glutamine synthetase, the enzyme ex-tracts were prepared as above, except the grinding mediumconsisted of cold 33 mm tris buffer, pH 7.2, 3.3 mM L-cysteine-HCI, and 0.1 mM Na,EDTA. This extract was also used in themeasurement of cytochrome oxidase activity.
Assay. The activity of nitrate reductase was measured by theprocedure of Evans and Nason (4) as modified by Yang (21).The nitrite formed was measured spectrophotometrically at540 nm. The specific activity of nitrate reductase is nmolesnitrite formed/mg protein-hr. Glutamic-oxaloacetic transami-nase was assayed by the method outlined by Reitman andFrankel (15). The amount of oxaloacetic acid formed wasdetermined colorimetrically with a Klett-Summerson color-imeter equipped with a green (No. 54) filter. Specific activity ofglutamic-oxaloacetic transaminase is expressed as umolesoxaloacetic acid formed/mg protein-hr. The procedure ofLowry et al. (11) as modified by Harper and Paulsen (7), wasemployed in the determination of the activity of glutamic aciddehydrogenase. The NAD formed was indicated by the fluores-cence of samples measured in a Turner, Model 111, fluo-rometer equipped with a primary filter, spectral transmissionabout 370 nm, and a secondary filter whose spectral transmis-sion was about 480 nm. The specific activity of glutamic aciddehydrogenase is tumoles NAD formed/mg protein-hr. Gluta-mine synthetase activity was determined by the method of El-liot (3). From the absorbance at 500 nm, the amount of glu-tamylhydroxamic acid was ascertained. The specific activity ofglutamine synthetase is expressed as ,umoles glutamylhydrox-amic acid formed/mg protein-hr. The activity of cytochromeoxidase, expressed as absorbance change at 550 nm/min-mgprotein, was measured by the method of Fritz and Beevers (6).The in vitro effect of KNap on enzyme activity was de-
termined by the addition of sufficient KNap to the various as-say solutions to give a concentration of 1 ,uM (230 /-g/l) KNap.The cell-free supernatant obtained from plants whose foliagehad not been sprayed with KNap was used in the assays.DNA and RNA. The DNA and RNA contents of trifoliate
leaf blade material, from which the midribs had been removed,were measured by the method described by Bonner andZeevaart (2). A 5-g leaf sample was drawn from each of twogroups of five treated and from two groups of five untreatedplants. Duplicate measurements were made in each case. Theassay was done 7, 14, and 21 days after the foliar applicationof KNap.
Soluble Amino Acids, Protein, and Amino Acids of Protein.Fourteen days after treatment, the trifoliate leaves of eighttreated and eight control bush bean plants were used to de-termine the content of 80% ethanol-soluble amino acids, theprotein, and the amino acids of the hydrolyzed protein. Thesoluble amino acids and the protein were obtained from theleaves by the method described by Ranjan and Laloraya (14).A Beckman Spinco Model 120 amino acid analyzer was usedto measure the free amino acids and the amino acids resultingfrom the hydrolysis of the protein fraction. Protein was de-termined by the method of Lowry et al. (10).
Incorporation of 14C-L-Leucine. Seven days after the appli-cation of KNap, the ability of 1-cm discs, cut from the leafletsof trifoliate leaves of treated and control plants, to incorporate14C-L-leucine into protein was measured by the method de-scribed by Harper and Paulsen (7). Minor variations includedthe use of 20 jug of streptomycin sulfate/ml of incubationmedium which contained 0.55 iM 4"C-L-leucine with an activityof 1 Ac/7.5 ml of the medium; no 'C-L-leucine was includedin the grinding medium; and 0.3-ml aliquots of solubilized pro-tein were applied to pleated 4 cm X 7.5 cm Whatman No. 1filter papers. The samples were counted in a Nuclear Chicagoscintillation counter, Model 720. The scintillation fluid wastoluene-PPO, 1 liter-4 g. The results were expressed as nc/gfresh weight and as nc/mg protein. Six assays were made.
Yield of Pods. In order to determine if treatment was effec-tive in stimulating reproductive growth, eight treated and eightuntreated plants were allowed to grow for 30 days after appli-cation of the KNap. The number and fresh weight of the podsper plant were obtained.
Statistical Analysis. With the exception of those of aminoacids, all data were analyzed for statistical significance.
RESULTSPod Yield. The average pod numbers were 7.5 and 8.6, and
the pod weights per plant were 28.9 and 33.4 g for control andtreated plants, respectively. This represented an increase of14.7% in number of pods, and of 15.6% in weight of pods,both significant at P = 0.05. The increase in pod productionwas similar to that reported previously (19). The larger podyield suggests that the stimulative action of KNap was inprogress at the times of the various assays.Amino Acids and Protein. The protein content of the tri-
foliate leaves of treated bean plants was significantly (P =0.05) greater than that of control plants (Table I). The averagevalues in control and treated plants, respectively, were 19.1 and21.8 mg/g fresh weight or 146.9 and 169.4 mg/g dry weight.On the dry weight basis the increase in protein, resulting fromtreatment, was 15.3%.The weights of the individual amino acids in 100 mg of the
amino acid complex obtained by the hydrolysis of the proteinare given in Table I. The values are the averages of assay re-sults of two separate experiments. The relative amounts of the16 amino acids in the protein of the control and treated plantleaves are somewhat different. The major quantitative changesare the greater weights of glutamic acid, glycine, and proline,and the smaller amounts of arginine, lysine, tyrosine, and leu-cine.The content of free amino acids (soluble in 80% ethanol) in
the leaf cells of the plants to which KNap had been applied wasgreater by 7.5% (Table I). The values were 87.1 ,ug and 93.6ug/ 100 g fresh weight, and 670 ug and 720 ug/ 100 g dryweight in untreated and sprayed plants, respectively.
The principal changes in weight of individual amino acids in100 yg of 80% ethanol-soluble amino acids, resulting from
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WORT, SEVERSON, AND PEIRSON
Table I. Soluble Amino Acids, Proteini, anzd AminoAcids in the Proteint Hydrolysate of Trifoliate
Leaves of Bush Beanz PlantsPotassium naphthenate, 20 mm (4600 /g/ml), was applied to
the foliage 14 days after seed planting.
Ethanol-soluble Amino Amino Acids in ProteinAcids' Hydrolysate2
'Content of ethanol-soluble amino acids: as pg/100 g freshweight, control, 87.1; treated, 93.6; as ug/100 g dry weight, control,670.0; treated, 720.0.
2 Content of protein: as mg/g fresh weight, control, 19.1; treated21.8*; as mg/g dry weight, control, 149.6; treated, 169.4*; *Signifi-cantly different from control value at P = 0.05.
Table II. DNA and RNA int Trifoliate Leaf Blades of BlushBean Plants 7, 14, anzd 21 Days after Bein1g
Sprayed with Potassium NaphthenzatePotassium naphthenate, 20 mM (4600 4g/ml), was sprayed on
the foliage 14 days after seed planting.
Time after Treatment (days)Treatment Average V-alue
Not significantly different from control value at P = 0.05.* Significantly different from control value at P =0.05.** Significantly different from control value at P = 0.01.
treatment, were larger amounts of arginine and lysine, andsmaller values for glutamic acid, serine, and proline.DNA and RNA. The content of DNA and RNA in cells of
trifoliate leaves, determined weekly for 3 weeks, is given inTable II. Treatment of the plants with KNap resulted in anumerically greater content of DNA at each of the three timesof assay, but the values were not significantly different from
fresh weight, at each of the assay times, and the average ofthese values were significantly greater in treated plants. Overthe 3-week period the average content of DNA was 0.24 and0.28 mg/g fresh weight, and of RNA 1.33 and 1.68 mg/g freshweight, in control and treated plant leaves, respectively. Maxi-mum contents of both DNA and RNA were measured 1 weekafter treatment.
Incorporation of 14C-L-Leucine into Protein. Treatment ofplants with KNap did not result in a significant change (P =0.05) in rate of incorporation of radioactive leucine by leafdiscs, measured 7 days after application of the growth stimu-lant to the plant. The average activity values for control andtreated plants, respectively, were 627.1 and 720.5 nc/g freshweight, or 32.8 and 33.0 nc/mg protein, based on a proteincontent of 19.1 and 21.8 mg protein/g fresh weight of controland treated plant leaves, respectively.Enzyme Content and Specific Activity. The content of pro-
tein in the cell-free enzyme extracts was found to be signifi-cantly greater in the leaves of bush bean plants to which KNaphad been applied. As mentioned above, the means for controland treated plants, respectively, were 19.1 and 21.8 mg pro-
tein/g fresh weight, or 146.9 and 169.4 mg protein/g dryweight.The specific activities of three of the enzymes of N metabo-
lism, namely nitrate reductase, glutamic-oxaloacetic acid, andglutamine synthetase, were significantly greater in the leavesof KNap-treated plants (Table III). Only in the case of glu-tamic acid dehydrogenase was there no significant change inspecific activity, an increase in activity on a crude enzyme ex-
tract basis being offset by the increase in protein content. Thespecific activity of cytochrome oxidase in treated plant leaveswas greater by 69%, significant at P = 0.01. In no case was
the in vitro enzyme activity changed significantly by the addi-tion of KNap to the cell-free extract of untreated plant leaves.
DISCUSSION
The significantly larger content of protein in the leaves ofKNap-treated P. vulgaris plants is similar to the greater protein
Table III. Activity of Enzzymies ofN Metabolism anidCylochrome Oxidase in Trifoliate Leaves of BushBean Plants 7 Days after Being Sprayed with
Potassium Naphthenzate
Potassium naphthenate, 20 mm (4600 ,g/ml), was applied to thefoliage 14 days after seed planting. The specific activity units are
given in the text. For the in vitro measurements, potassium naph-thenate was added to the assay solutions to give 1 AM (230 Ag/l) atthe time of assay. Extracts of leaves of untreated plants were used.
Not significantly different from control value at P = 0.05.* Significantly different from control value at P = 0.05.
those of control plants. However, the weights of RNA per g ** Significantly different from control value at P = 0.01.
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NAPHTHENATE EFFECTS (
content of cobs and leaves of naphthenate-treated maize (22)and of the leaves of treated sugar beet plants (13).
Treatment resulted in an altered percentage composition ofleaf protein in terms of its amino acids, and also in the relativeamounts of individual ethanol-soluble amino acids. In Table Iit may be seen that arginine and lysine were present in largeramounts among the soluble amino acids of the leaves of KNap-treated plants, but in smaller quantities in the protein of theseplants. On the other hand treatment resulted in decreasedamounts of glutamic acid, serine, and proline in the pool ofsoluble acids, but a larger content of these acids in the proteinsof leaf cells. The weight of ethanol-soluble amino acids in theleaves of treated bush bean plants was greater by 7.5%.
Severson (17) reported that root tips removed from 7-day-oldP. vulgaris plants and placed for 6 hr in a medium containing10 /M (2300 tg/l) KNap were subsequently able to absorb andmetabolize larger amounts of the "C of a "C-glucose solution.Greater radioactivity was detected in eight of the nine ethanol-soluble amino acids he investigated, suggesting the biosynthesisof larger amounts of the eight acids. Peterburgsky and Kara-mete (13) also found that the content of each of 10 free aminoacids, on a dry weight basis, was greater in maize to whichnaphthenate solution had been applied. The over-all increasewas 15.3%. The relative amounts of the individual acids werenot given in the latter paper.
It would appear, then, that in stimulated plants the forma-tion of keto acids, their amination to form amino acids, andsubsequent transamination occurred more vigorously. Thissuggestion is borne out by the increased content and activity ofnitrate reductase, and glutamic-oxaloacetic transaminase (Ta-ble III), and glutamic-pyruvic transaminase (5), coupled withthe finding (5) that respiration in treated Top Crop bush beanplants is significantly enhanced. One consequence of the latterwould be a greater availability of keto acids. The increased ac-tivity of phosphoglyceric kinase (5) and cytochrome oxidase(Table III) suggests a greater ATP production in the glycolyticand electron transfer system phases of respiration. An increasedsupply of amino acids in the soluble pool and of reduced nu-cleotides and ATP from both respiration and photosynthesiswould make possible a stimulated synthesis of protein.The larger contents of RNA and DNA, in leaf cells of
treated plants, suggest that in one phase of its activity KNapmay influence the transcription/translation sequence at the ge-netic level. The specific RNAs in the larger amounts of totalRNA were not determined, but mRNA and sRNA may be in-volved. Thus the transfer of amino acids to ribosomes and theircondensation into protein at these organelles would be signifi-cantly influenced.The greater quantities and specific activities of numerous
enzymes and enhanced processes of metabolism in treated bushbean plants suggest that, in a second phase, the stimulative ac-tion of KNap functions at the metabolic level.
It was shown that leaf discs cut from KNap-treated plantsdid not incorporate significantly higher amounts of "C-leucineinto protein. Based on the findings of Holleman and Key (8)that higher concentrations of "C-leucine in the amino acid pooldiminished the incorporation of "C-leucine into protein bysoybean hypocotyls, it might well be that the greater contentof soluble leucine in the leaves of treated plants (Table I)would offer resistance to "C-leucine entrance and incorpora-tion.
Following the discovery that cyclohexanecarboxylic acid, anindividual member of the naphthenic acid mixture, was an
effective plant growth stimulant (20), Severson et al. (18)showed that cyclohexanecarboxylic acid, administered to leafdiscs of P. vulgaris, is converted to conjugates of glucose and
ON BEAN N METABOLISM 165
of aspartic acid. Padmanabhan (12) further found that whenradioactive cyclohexanecarboxylic acid is applied to the surfaceof a primary leaf of the bush bean plant it is quickly absorbedand is rapidly converted to the conjugate forms. These conju-gates, rather than the acid itself, move out of the leaf andthrough the plant, suggesting that the conjugated naphthenateis the biologically active form. If so, it affords an explanationfor the lack of in vitro stimulation of any of the five enzymesby the addition of KNap to the cell-free extract of leaves ofbush bean plants. Apparently, the acid per se was not stimu-lative and, under the conditions of the assays, its conjugationwith glucose or aspartate did not take place.
Acknouwledgments-The measurement of DNA and RNA by Dr. UshaPadmanabhan, and of amino acids by Dr. Iain Taylor (Department of Botany),and the statistical analyses by Dr. Anton Kozak (Faculty of Forestry) aregratefully acknowledged.
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