Plant Physiol. (1992) 98, 89-960032-0889/92/98/0089/08/$01 .00/0

Received for publication May 30, 1991Accepted August 8, 1991

Growth Stimulation by Catecholamines inPlant Tissue/Organ Cultures1

Calixto M. Protacio, Yao-ren Dai, Eldrin F. Lewis, and Hector E. Flores*

Department of Plant Pathology and Biotechnology Institute, The Pennsylvania State University,University Park, Pennsylvania 16802

ABSTRACT

Addition of catecholamines at micromolar concentrationscaused a dramatic stimulation of growth of tobacco (Nicotianatabacum) thin cell layers (TCLs) and Acmella oppostHffolia "hairy"root cultures. A threefold increase in the rate of ethylene evolutionwas observed in the catecholamine-treated explants. Aminooxy-acetic acid and silver thiosulfate, inhibitors of ethylene biosyn-thesis and action, respectively, reduced the growth-promotingeffect of dopamine. However, these compounds alone could alsoinhibit the growth of the TCL explants. When ethylene in theculture vessel was depleted by trapping with mercuric perchlo-rate, dopamine-stimulated growth was still obtained, suggestingthat ethylene does not mediate the dopamine effect. Dopaminepotentiated the growth of TCLs grown in Murashige and Skoogmedium supplemented with indoleacetic acid (IAA) and kinetin.When IAA was replaced by 2,4-dichlorophenoxyacetic acid, do-pamine addition showed no growth-promoting effect. Instead,2,4-dichlorophenoxyacetic acid stimulated the growth of TCLexplants to the same extent as that obtained with IAA plusdopamine. Because synthetic auxins do not appear to be sub-strates for IAA oxidizing enzymes, we hypothesized that cate-cholamines exert their effect by preventing IAA oxidation. Con-sistent with this explanation, dopamine (25 micromolar) inhibitedIAA oxidase activity by 60 to 100% in crude enzyme extracts fromtobacco roots and etiolated corn coleoptiles, but had no effecton peroxidase activity in the same extracts. Furthermore, additionof dopamine to TCL cultures resulted in a fourfold reduction inthe oxidative degradation of [1-'4C]IAA fed to the explants.Because the growth enhancement by catecholamines is observedin both IAA-requiring and IAA-independent cultures, we suggestthat these aromatic amines may have a role in the regulation ofIAA levels in vivo.

The catecholamines dopamine, norepinephrine, and epi-nephrine and their precursors phenylethylamine and tyramine(Fig. 1) are relatively widespread in the plant kingdom. Thesearomatic compounds and their derivatives have been detectedin 44 plant families, including at least 29 species grown forhuman consumption (banana, sugar beet, avocado, citrus,etc.) (28). High levels of norepinephrine have been reportedin the pulvini and tendrils of at least six plant species (3). In

'This work was supported by a research assistantship to C.M.P.from the Department ofPlant Pathology and Biotechnology Institute,The Pennsylvania State University, and a grant from the NationalScience Foundation to H.E.F. (EET-873078).

animal cells, aromatic monoamines serve very defined func-tions. For example, dopamine and norepinephrine are wellknown neurotransmitters, and epinephrine (adrenalin) is ahormone involved in rapid responses to stress (7). In contrast,the biochemistry and physiological significance of aromaticmonoamines in plants are poorly understood.The biosynthesis and catabolism of catecholamines has

been studied in several systems. In the peyote cactus Lopho-phora williamsii, phenylalanine is hydroxylated to tyrosine,which is further hydroxylated to DOPA2 or decarboxylated totyramine (25). Alternatively, p-hydroxylation of phenylethyl-amine can also give rise to tyramine (28). Dopamine may bederived either from hydroxylation of tyramine, as in Musasapientum, or from decarboxylation of DOPA, as in Cytisusscoparius (29). Dopamine is synthesized via DOPA in Portu-laca callus (9). The rate of catecholamine biosynthesis andaccumulation is much higher in dark- than in light-growncallus. Labeled tyramine and dopamine fed to plant cellcultures were metabolized mainly through oxidative polym-erization; 0.5 to 2% of the labeled dopamine was detected asCO2 (22).

Several reports suggest that catecholamines may interactwith plant hormones. For example, GA3 can induce hypocotylelongation in lettuce seedlings (17). This response is sup-pressed by removal of the cotyledons, and thus appears torequire the presence ofa factor contributed by this organ (17).The cotyledon factor was later identified as dopamine andshown to stimulate GA3 action in isolated lettuce hypocotyls(18). Exogenous dopamine (5-100 AM) stimulated ethylenebiosynthesis in illuminated chloroplast lamellae from sugarbeet leaves (8). In this study, dopamine was shown to functionas a cofactor for monovalent oxygen reduction necessary forethylene formation. Studies in Lemna suggest that aromaticmonoamines can affect flowering. Oota (24) reported thatflowering was promoted in L. gibba G3 by DL-epinephrine at10-8 M, and at 106 M by norepinephrine, counteracting theinhibitory effects of sugars. Khurana et al. ( 19) found that L-epinephrine and L-norepinephrine (10-4 M) induced morefloral primordia and promoted floral development whenadded to L. paucicostata prior to floral induction.

Recent studies in our laboratory have focused on polyaminemetabolism and function during flower formation in TCLsfrom tobacco inflorescences (1 1). In addition to polyamines

I Abbreviations: DOPA, dihydroxyphenylalanine; TCLs, thin celllayers; STS, silver thiosulfate; AOA, aminooxyacetic acid; FIM,flower-inducing medium; DA, dopamine; Kin, kinetin.

89

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021

Plant Physiol. Vol. 98, 1992

Figure 1. Structures of aromatic monoamines used in this study.

and their hydroxycinnamic acid amides, we observed thepresence of phenylethylamine and tyramine (C.M. Protacio,H.E. Flores, unpublished observations). Martin-Tanguy et al.(21) have previously reported that these compounds are thepredominant monoamines in the apical part of vegetativetobacco plants, and that phenylethylamine titer undergoes adramatic decline at the onset of flower initiation. Based onthese correlations and previous reports (19, 24), we studiedthe effect of aromatic monoamines on growth and flowerformation in tobacco TCLs. The results reported in this papershow that the addition of various monoamines can cause adramatic stimulation of growth in TCLs as well as in organcultures. We discuss these findings and experiments aimed atelucidating the mechanism for this growth stimulation.

MATERIALS AND METHODS

Experimental Systems

Epidermal TCLs excised from tobacco (Nicotiana tabacum)inflorescences were cultured in a medium conducive to directflower bud formation (30). Briefly, TCLs (2 x 10 mm) fromtobacco Samsun NN were cultured in Murashige and Skoogsalts (23), 100 mg/L myo-inositol, 400 ,ug/L thiamine, 3%sucrose, and 1 uM each ofIAA and Kin. The pH was adjustedto 5.8 and 0.2% Gelrite (Scott Labs) was added before auto-claving (15 min at 121°C); 25 mL of medium per dish wasdispensed into 100 x 15 mm plastic Petri dishes (VWR).Four TCLs were placed on each Petri dish, with 4 to 6 rep-licate plates per treatment. The cultures were kept undercontinuous white light (100 gmol m-2 s-') at 30°C. After14 d, the TCLs were harvested and fresh and dry weightsdetermined."Hairy root" cultures of Acmella oppositifolia obtained by

transformation with Agrobacterium rhizogenes were providedby Dr. Martin Hjortso (Louisiana State University) and main-tained in liquid Gamborg's B5 medium (13). Two-centimeterlong root tips from 2-week-old cultures were placed in solidGamborg's B5 medium containing 10 to 50 ,UM phenylethyl-amine, dopamine, or norepinephrine. Root weight was meas-ured after 18 d of culture.

Chemicals

Norepinephrine, epinephrine, AOA, IAA, Kin, and sodiumthiosulfate were purchased from Sigma; dopamine was pur-chased from Aldrich. Ten millimolar aqueous stock solutionswere made and sterilized through 0.2 ,um Nalgene filters. Allstocks were stored at -20°C.STS was prepared fresh according to the method of Reid et

al. (26). Briefly, 4.0 mL of 0.1 M Na2S203 5H20 was mixedwith 1 mL 0.1 M Ag NO3 and diluted to 10.0 mL with distilledwater to make a 10 mM STS stock.

Mercuric perchlorate was prepared according to Young etal. (33) by diluting 1.72 mL of 70% perchloric acid (Fisher)to 2.5 mL and adding 0.542 g of mercuric oxide (Baker). Thesolution was made up to 10 mL with deionized water andfilter-sterilized. For the experiments involving ethylene trap-ping, 2 mL aliquots were added to autoclaved clear glass vialsand placed inside the culture flasks before sealing with rubbersepta.

[1-'4C] IAA (specific activity 55 mCi/mmol) was obtainedfrom American Radiolabelled Chemicals Inc. at a concentra-tion of 0.1 mCi/mL.

Ethylene Measurement

Two TCL explants were cultured axenically in 125 mLEhrlenmeyer flasks containing 50 mL medium and sealedwith rubber septa. Air samples (1 mL) were drawn andmeasured every other day for 14 d; four flasks were used pertreatment. To measure the rate of ethylene evolution, eightexplants were cultured per flask. The vessels were sealed withcotton plugs. On the fifth day, the flasks were opened undera transfer hood, the air inside the flask was purged using asterile syringe, and each flask was sealed with a rubber septum.After 1 h, ethylene concentration in the flask was measuredusing a Hewlett-Packard model 5830A gas chromatographwith a flame ionization detector and activated alumina col-umn. Fresh and dry weights were subsequently determined.A parallel set of samples was treated in the same way for thesubsequent time point (ninth day). To determine the effect ofcatecholamines on ethylene evolution by Acmella roots, themedium of 2-week-old cultures was replaced with fresh Gam-borg's B5 medium to which different aromatic monoamineswere added. Ethylene content was assayed as above.

IAA Oxidase Assay

Crude extracts were prepared from tobacco roots and etio-lated corn coleoptiles. One-month-old tobacco roots fromgreenhouse-grown Samsun NN plants were thoroughlywashed in water and surface-sterilized by dipping them in70% ethanol for 2 min, followed by 15 min in 20% Clorox.The roots were rinsed three times in sterile water, then frozenin liquid nitrogen, and ground to a fine powder. An aliquotwas weighed out for extraction while the remaining samplewas stored in a -80°C freezer. Corn seeds were grown insterile vermiculite in the dark. Six-day-old etiolated corncoleoptiles were harvested and ground in liquid nitrogen.The enzyme was extracted by adding 0.5 g of the fine

powder to 1 mL of ice-cold homogenization buffer (50 mmHepes, pH 7.5, with 1 mM PMSF) in glass homogenizers. The

_I

Rl _ CH-CH2-N\R2

-I R2 R, RA R COMPOUND

H H H H H Phenylediylamine

OH H H H H Tyranmine

OH OH H H H Dopamine

OH OH OH H H Norepinephrine

OH OH OH H CH3 Epinephrine

90 PROTACIO ET AL.

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021

CATECHOLAMINES IN PLANT TISSUE/ORGAN CULTURES

extract was centrifuged at 16,000g for 20 min at 4°C. Thesupernatant was dialyzed through a 12 kD cut-off membrane(Sigma) against 200 volumes of 1 mM Hepes, pH 7.5, over-night at 4°C. This fraction was used to assay the soluble IAAoxidase activity. The pellet was washed three times with ice-cold homogenization buffer and vacuum infiltrated for 5 minduring the last wash. The washed pellet was extracted in 50mM Mes buffer, pH 6.0, containing 100 mm CaCl2 and 1 mM

PMSF, followed by vacuum infiltration for 5 min. Buffer (0.5mL) was added for every gram of ground tissue originallyhomogenized. This preparation was centrifuged as above, andthe supernatant fraction was likewise dialyzed overnightagainst 200 volumes of 1 mM Mes, pH 6.0, at 4°C. Thisfraction was used to assay the ionically-bound IAA oxidaseactivity. The dialyzed enzyme extracts were stored at -80°Cand assayed within a few days because a significant decreasein activity was observed upon prolonged storage. Stock solu-tions of peroxidase (type II, Sigma) were prepared in 10 mmphosphate buffer, pH 7.0, at 1 mg/mL.The IAA oxidase assay was modified from the procedure

of Beffa et al. (4). The assay mix contained 40 mM K2HPO4,pH 6.0, 50 ,M p-coumaric acid, 100 Mm IAA, 100 AM MnCl2,and 150 IuL enzyme extract; distilled water was added to a

final volume of 1 mL. The reaction was started by addingMnCl2 and incubating the mixture in a water bath shaker at25°C in the dark. After 10 min, the tubes were immediatelyplaced on ice, and 0.5 mL of 20% perchloric acid was added.The tubes were allowed to stand 10 min, then 0.5 mL of thissolution was added to 1 mL of Salkowski reagent (1 mL 0.5M FeCl3(,)/50 mL 35% perchloric acid). The mixture was

vortexed, and the color was allowed to develop for 30 min at25C. Absorbance at 540 nm was measured in a BeckmanDU-65 spectrophotometer. Experimental samples were pre-pared to fit within the linear range (20-120 ,mol) of the IAAstandard curve. Each assay was performed twice with dupli-cate samples per treatment. Protein was quantified by theprotein-dye binding method of Bradford (5) using bovine y

globulin as a standard.

Peroxidase Assay

Peroxidase activity was measured according to Shannon etal. (27) by monitoring the change in absorbance at 460 nmdue to the enzyme-catalyzed oxidation of o-dianisidine in thepresence ofH202. The assay mix contained 0.5% o-dianisidine(0.05 mL), 0.1 M H202 (0.1 mL), enzyme extract (0.1 mL),and 0.01 M phosphate buffer, pH 6.0 (2.0 mL). The reactionmixture was incubated at 30°C for 10 min, and the reactionstopped by adding 0.75 mL 40% (v/v) perchloric acid.

Decarboxylation of [1-14C]IAA in TCL Explants

Liquid FIM was prepared without IAA and autoclaved for15 min at 121°C. After cooling, [1_'4C]IAA was added at a

ratio of 0.275 MuCi/5 mL, for a final concentration of 1 ,MIAA. After filter sterilization, 25 ,uM dopamine was added tohalf of the medium and 5 mL aliquots were dispensed into20 x 100 mm test tubes. Plastic closure caps (Magenta Corp.)used to seal the tubes were pierced with a sterile 1.5-inch, 22-gauge needle. Labeled CO2 released from IAA was trapped on

filter paper discs (0.25 inch diameter, Schleicher & Schuell)impregnated with 2 N potassium hydroxide (50 ML/disc); twodiscs were impaled per needle. The needle hub was pluggedwith cork stoppers to prevent "'CO2 escape.

Three TCLs were cultured per tube and four replicates wereused for each time point. Controls that did not contain any

explants were also set up. The tubes were placed in a 30°Cwater bath under a chemical hood to simulate conditions inthe tissue culture room. The hood lights were supplementedwith white fluorescent light to give continuous 45 Mmol. m-2s-I illumination. At each sampling time, the filter paper discswere placed in Omnivials (Wheaton) containing 3 mL scin-tillation cocktail (Ecoscint, National Diagnostics) and countedfor 10 min in a Beckman LS 5000 TA model. All experimentswere repeated at least once; in most cases, three to fourrepetitions were done.

RESULTS

Effect of Catecholamines on Growth and FlowerInitiation of TCLs

Of all the compounds listed in Table I, only the structurescontaining two hydroxyl groups on the aromatic ring showedgrowth-promoting activity. Tobacco TCLs respond to lowconcentrations (25-50 ,M) ofdopamine by showing fast callusgrowth and radial expansion (Fig. 2). Dopamine additionincreased TCL fresh weight two- to sixfold over control values(Tables I and II). Endogenous norepinephrine levels rangedfrom 0.3 to 8.3 yg/g fresh weight in six plant species (3). InPortulaca, the endogenous dopamine content was reported tobe 39 Ag/g dry weight (9). By comparison, dopamine addedat 5 gM to the cultures is equivalent to 24 ,g/Petri dish or 25mL of medium.

In contrast with previous reports of promotion of floweringby catecholamines (19, 24), we did not observe any stimula-tion of flower initiation in our system. Dopamine and nor-

epinephrine addition inhibited both floral and vegetative budinitiation while promoting callus formation. In the TCL sys-tem, the tissues are apparently already competent to undergo

Table I. Effect of Aromatic Monoamine Addition on Growth ofTobacco Samsun NN TCLs and A. oppositifolia Hairy Root Cultures

TCL Weight8 Root WeightMonoamine FehFresh Dry (Fresh)b

mg/explant mg/root tip

Control 194 ± 13 22 ± 1 25 ± 2Phenylethylamine 112 ± 7 15 ± 1 60 ± 10Tyramine 112 ± 8 15 ± 1 NDCDopamine 1336 ± 75 124 ± 6 53 ± 13Norepinephnne 1187 ± 57 111 ± 5 47 ± 4Epinephrine 843 ± 68 91 ± 5 ND

All the aromatic monoamines were added at 25 Mm for the tobaccoTCLs. Values represent means ± SE (n = 16). b Monoamines at10 Mm were incorporated into solid B5 medium at the start of culture.Values represent fresh weight measurements from three separateexperiments; roots were harvested after 18 d culture. Values repre-sent means ± SE (n = 12). c ND, not determined.

91

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021

Plant Physiol. Vol. 98, 1992

150 5 10Days

Figure 2. Effect of dopamine concentration on the growth of tobaccoTCLs. Explants were grown in a flower-inducing medium for 14 dunder continuous light (100 Mmol _ m-2 s-1).

flower formation (30), whereas in the studies with Lemna, thecatecholamines were added at the time of flower induction.Thus, it is possible that if catecholamines affect flowering,they may be effective only at the induction stage.

Dopamine Addition Causes Increased EthyleneProduction

Based on our observation that the catecholamine-treatedexplants showed increased radial expansion, one of the tripleeffects attributed to ethylene (14), we followed ethylene evo-

Table II. Effect of AOA and STS on the Dopamine-Induced Growthof Samsun NN TCLs

All explants were grown on FIM, with additions as indicated. Valuesrepresent means ± SE (n = 16). Weight is given in mg/explant.

AOA STSTreatment

Fresh weight Dry weight (Fresh Weight)

Control (FIM) 455 ± 22 56 ± 2 266 ± 20FIM + 25 AM DA 1112 ± 28 100 ± 2 616 ± 35FIM + 25 AM DA + 10uM 731 ± 42 63 ± 3 334 ± 12

inhibitorFIM + inhibitor alone 222 ± 16 25 ± 2 61 ± 3

.6

3cm

3~-

e0

cam

-

10CO0o

w)

60 -

50-

40-

30-

20-

10'

0'

* 5th DayE 9th Day

Control

I B

25pM Dopamine

Figure 3. A, Total ethylene accumulated per flask from 2 TCLs oftobacco Samsun NN. Explants were grown under the same condi-tions as in Figure 2 except that the culture vessels used were 125mL Erhlenmeyer flasks sealed with rubber septa. B, Effect of dopa-mine on the rate of ethylene evolution by tobacco Samsun NN TCLs.See "Materials and Methods" for experimental details. Four replicateswere used for each treatment. Values represent means ± SE (n =16).

lution by dopamine-treated TCLs (Fig. 3A); over three timesas much ethylene was accumulated in the culture vessels bythe dopamine-treated explants compared with the controls.Addition of 10 uM AOA, an inhibitor of ethylene synthesis,reduced the ethylene levels and explant growth to valuessimilar to the control. Because the high levels of ethyleneevolved by dopamine-treated explants could reflect a simplecorrelation with the increased mass of these explants, wemonitored the actual rate of ethylene production, as shownin Figure 3B. The dopamine-treated explants evolved at leastthree times higher ethylene than the controls. A clear stimu-lation of ethylene release was also observed in catecholamine-

92 PROTACIO ET AL.

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021

CATECHOLAMINES IN PLANT TISSUE/ORGAN CULTURES

Table Ill. Effect of Ethylene Trapping on the Dopamine-InducedGrowth of Tobacco TCLs

Explants were cultured in 125 mL flasks sealed with rubber septa.Ethylene was measured after 14 d.

WeightTreatment Ethylene

Fresh Dry

mg/explant pL flask

Control 781 ± 68 78.5 ± 7.2 1.70 ± 0.34Control + 0.2 mL MP8 768 ± 93 78.0 ± 8.2 0.01 ± 0.01Control + 1.0 mL MP 867 ± 73 84.1 ± 6.7 0Control + 2.0 mL MP 1094 ± 117 108.4 ± 7.6 025 gM DA 1372 ± 120 104.1 ± 6.1 16.78 ± 0.7225Mm DA + 0.2 mL MP 1070 ± 234 117.7 ± 19.7 0.22 ± 0.0925Mm DA+ 1.0mLMP 1246±95 96.3±5.2 0.30±0.1425Mm DA + 2.0 mL MP 1316 ± 148 102.6 ± 10.9 0.02 ± 0.02a Mercuric perchlorate.

treated hairy root cultures. Addition of dopamine resulted inthe accumulation of0.78 uL ofethylene per flask as comparedwith 0.45 ML/flask in the control after 24 h culture. Norepi-nephrine and phenylethylamine also stimulated ethylene for-mation (0.75 and 0.70 ML/flask, respectively).The above observations led us to probe whether ethylene

may in fact mediate the growth promotion caused by cate-cholamines. We tested this possibility using inhibitors ofethylene biosynthesis and ethylene action, ethylene addition,and ethylene trapping. As shown in Table II, the addition ofAOA greatly reduced the growth-promotive effect of dopa-mine. Although this AOA concentration can indeed reducethe amount of ethylene produced by the explants (Fig. 3A), itis unlikely that the reduced growth of the dopamine-treatedexplants in the presence ofAOA is due to the lower levels ofethylene produced. When AOA alone was added to TCLexplants cultured on FIM, growth was also inhibited (TableII), suggesting that AOA may affect other processes besideethylene synthesis. Similar results were obtained with STS, an

inhibitor of ethylene action (26). Growth stimulation bydopamine could be reversed by the addition of 10 ,uM STS(Table II). However, STS inhibited growth when added aloneat 10 ,uM. Because of the inhibitory effects exhibited by AOAand STS on the control TCL explants, the results of the aboveexperiments are difficult to interpret, but do not argue stronglyfor the direct involvement of ethylene in the catecholamine-stimulated growth.

If the promotion of growth by catecholamines is mediatedby ethylene, it should be possible to obtain a growth responseby ethylene addition. Accordingly, we introduced ethylenegas into the tissue culture vessels. Addition of 0.5 ML/Lethylene at the start of culture, a concentration that is wellabove the concentration range commonly used to achieveepinasty (1), had no effect on the growth of the TCL explants(data not shown). Conversely, we tested the observed corre-

lation by trapping ethylene in mercuric perchlorate. As shownin Table III, growth stimulation by dopamine can be obtainedeven if298% ofthe ethylene produced is trapped by mercuricperchlorate. Thus, our results suggest that even though eth-ylene production is enhanced during the catecholamine-stim-

ulated growth response, this gaseous plant hormone does notappear to mediate the response.

Do Aromatic Monoamines Interact with Plant Hormones?

The results shown above were obtained with a tissue culturesystem that depends on exogenous hormones for growth and/or differentiation. If, as suggested below, catecholamines exerttheir growth-promoting effects through interactions with planthormones, it would be useful to establish interactions betweenexogenously added amines and endogenous hormones. Tothis end, we used transformed hairy root cultures that are

capable of growth in the absence of any exogenously addedhormones. As shown in Table I, the growth ofA. oppositifoliahairy root culture was stimulated by catecholamines. Rootfresh weight was twofold higher than the controls when phen-ylethylamine and its derivatives were added at a concentrationof 10 ,tM. This effect was also observed in cultures growing inliquid medium (C.M. Protacio, H.E. Flores, unpublishedresults), although in this case the cultures appear to have a

narrow window of competence for response to dopamine.The possible interactions of catecholamines with plant hor-

mones are further examined in the experiment shown in TableIV. Dopamine caused the expected growth stimulation inTCLs grown in FIM (Murashige and Skoog medium supple-mented with IAA and Kin), but had no growth-promotingeffect on explants grown in basal medium. Whereas IAA andKin showed the expected synergistic effect, Kin alone was

unable to promote growth, with or without dopamine addi-tion. However, IAA did cause a substantial growth response(Table IV); furthermore, the IAA effect was significantlyenhanced by dopamine. This result strongly suggests that thegrowth-promoting effect of catecholamines occurs throughtheir interaction with exogenous or (in the case of root cul-tures) endogenous auxin.

Possible Mechanism of Action

Catecholamines are dihydroxyphenols, substituted in posi-tions meta- and para- of the aromatic ring (Fig. 1). Previous

Table IV. Effect of Dopamine on Tobacco Samsun NN TCLs as aSubstitute for Auxin or Cytokinin

Basal medium is FIM without any hormones added (see "Materialsand Methods"). IAA and Kin were always added at 1 AM. Valuesrepresent means ± SE from four replicate Petri dishes containing fourexplants.

WeightTreatment

Fresh Dry

mg

Basal + 1 M IAAand Kin (FIM) 332 ± 42 38 ± 4Basal + 1M IMAA and Kin + 25AM DA 627 ± 49 70 ± 5Basal 26±2 4±0.4Basal+25gMDA 26±1 4±0.3Basal+Kin 30±3 4±0.2Basal + Kin + 25,M DA 32 ± 1 4 ± 0.3Basal + IAA 104 ± 10 13 ± 1Basal + IAA + 25AM DA 164 ± 22 13 ± 2

93

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021

Plant Physiol. Vol. 98, 1992

800

600

xca)

.9

400

200

0

FIM FIM+25pM DA 2,4-D 2,4-D+25pM DA

Figure 4. Effect of substituting 2,4-D for IAA on growth of tobaccoTCLs. FIM contains 1 AM each of IAA and Kin; 2,4-D was added at 1yM. Values represent means + SE (n = 16).

reports have shown that a wide variety of synthetic dihydrox-yphenols can inhibit IAA oxidase (15, 20). These observationssuggested a possible mode of action for the catecholamineeffects, namely that these compounds promote growth of invitro cultured explants by preventing the oxidation of exoge-

nous/endogenous IAA. If this is the case, then dopaminewould have no effect on explants cultured in the presence ofsynthetic auxins, which are not degraded by IAA oxidases. Asshown in Figure 4, dopamine can promote the growth ofTCLs in the presence of IAA, but is ineffective when IAA isreplaced with 2,4-D. This latter compound is sufficient to

induce a growth response comparable with that obtained byIAA plus dopamine.Our hypothesis was further tested by studying the effect of

dopamine on IAA oxidation in vitro and in vivo. IAA oxidase/peroxidase was extracted from plant sources reported to con-tain high levels of these enzymes, and assayed for IAA oxidaseactivity using the Salkowski reagent (4). In vitro IAA oxidaseactivity from corn coleoptiles and tobacco roots was inhibited100 and 62%, respectively, by 25 ,uM dopamine (Table V).The inhibition of IAA oxidase by dopamine appears to bespecific, as this compound had no effect on the peroxidaseactivity in the same extracts. Based on these results, extractsfrom 1- to 3-week-old tobacco TCL cultures were assayed forIAA oxidase, but we were unable to detect any activity.Because the IAA oxidase assay based on the Salkowski methodmay not be sensitive enough to detect activity in the tobaccoTCLs, we followed the metabolism of labeled IAA in thetissue culture explants. The TCLs were cultured in the pres-ence of 1 jtM [I-'4C]IAA, and the release of labeled CO, wasmonitored. The in vivo oxidative decarboxylation of IAA wasgreatly reduced from 36 ± 2 (control) to 9 ± 3 nmol/g freshweight when 25 uM dopamine was present after 12 h culture.The same trend was observed after 24 h; the amount ofIAA degraded in dopamine-treated cultures (10 ± 3) was

only one-fourth of the control (39 ± 1 nmol/g fresh weight).These data suggest that an IAA oxidase system in tobaccoTCLs is operative in vivo and can be inhibited by exogenous

dopamine.

DISCUSSION

We have shown that catecholamines can greatly stimulatethe growth of plant tissue and organ cultures at micromolarconcentrations. To our knowledge, this is the first report thatcatecholamines may have such an effect on plant systems. A

Table V. Effect of Dopamine on IAA Oxidase and Peroxidase Activities of Extracts from Three Sources

Enzyme Ex t SCrude Enzyme Crude Enzyme ActivityEnzyme Extract Specific Activity Activity (+ 25 uM DA)

jAg protein/mL units/mg protein

IAA oxidaseaTobacco roots

Soluble 430 681 293 ± 25 102 ± 9Ionic 243 370 90 ± 22 36 ± 10

Corn coleoptilesSoluble 534 0 0Ionic 649 538 349 ± 36 0

Commercial peroxidaseb 414 ± 1 0PeroxidasecCorn ionically bound 649 43 28 ± 1 29 ± 0

peroxidaseTobacco soluble 430 37 16 ± 1 15 ± 1

peroxidaseCommercial peroxidaseb 9 ± 0 10 ± 1

aOne unit of IAA oxidase activity is equivalent to 1 gmol IAA metabolized by 1 mL extract in 10min. b Used 100 MG from 1 mg/mL stock dissolved in 10 mm phosphate buffer, pH 7.0. c Oneunit of peroxidase activity is equivalent to a change in absorbance of 1.0 OD at 460 nm by 1 mL extractin 10 min.

94 PROTACIO ET AL.

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021

CATECHOLAMINES IN PLANT TISSUE/ORGAN CULTURES

recent study by Christou and Barton (6) reported that dopa-mine and epinephrine at 10 mm were toxic to callus culturesof tobacco and three other plant species. Because lower con-centrations were not tested in that study, our results are notdirectly comparable. However, we have observed similar toxiceffects on tobacco TCLs exposed to high concentrations (1mM) of dopamine (C.M. Protacio, H.E. Flores, unpublishedresults).

Because the catecholamine-treated tissues showed increasedrates ofethylene evolution that paralleled the growth response,we tested the hypothesis that ethylene in fact mediates thecatecholamine effect. Even though AOA and STS could par-tially reverse the growth-stimulating effect of dopamine intobacco TCLs, these compounds by themselves were inhibi-tory to growth, suggesting that these inhibitors may have othereffects beside interfering with ethylene synthesis or action,respectively. In fact, AOA is well known to affect pyridoxalphosphate-dependent enzymes (2). When mercuric perchlo-rate was used to trap the ethylene evolved by the TCLexplants, we were still able to obtain growth stimulation bydopamine (Table III). Thus, we concluded that ethylene doesnot mediate the response to catecholamines in our system.However, the high ethylene level produced by dopamine-treated tissues is most likely responsible for the morphologicalchanges observed, i.e. radial expansion of the explants. If, asdiscussed below, promotion of growth by catecholamines isdue to their inhibition of IAA oxidation, it is not surprisingthat catecholamine-treated explants produce higher levels ofethylene, for it is well known that IAA can promote ethylenesynthesis (2). Carrot tissues transformed with Agrobacteriumalso show high ethylene production correlated with the expres-sion of the transfer-DNA tms genes, which code for enzymesinvolved in IAA synthesis (16).As our results clearly show, dopamine can inhibit IAA

oxidation in vitro as well as in vivo (Table V). These datasupport our hypothesis that the growth-promoting effect ofcatecholamines is due to their inhibition ofIAA degradation,resulting in higher levels of auxin. Dihydroxyphenols havebeen known for over three decades to inhibit IAA oxidation(15, 20), and compounds that share these characteristics withcatecholamines are active in the TCL system. When caffeicand chlorogenic acid (25 gM) were added to tobacco TCLs,they stimulated ethylene production and growth at levelscomparable to dopamine (C.M. Protacio, H.E. Flores, unpub-lished results). The limited information available suggests thatcatecholamine metabolism in plants may be tightly regulated.Catecholamine biosynthesis has been shown to be under lightcontrol in Portulaca callus, and presumably a diurnal rhythmregulates the endogenous level of these amines (9). DOPAand dopamine accumulation is stimulated in the dark, andthe conversion of dopamine to noradrenalin also occurs onlyin the dark (9). Based on these metabolic features and theirwidespread distribution in higher plants (28, 29), we proposethat catecholamines may regulate auxin catabolism in vivo.Galston and Dalberg (12) suggested that the endogenousrhythm in IAA levels may result from the close coordinationof an IAA synthesizing system and IAA oxidase. It should beof interest to determine if IAA and catecholamine levels arecoordinately regulated in plant tissues. Our results also pro-vide evidence for the existence of an IAA oxidase system that

is distinct from peroxidases. As shown in Table V, catechol-amines inhibit IAA oxidase, but not peroxidase, activity. Arecent report has shown that IAA oxidase and peroxidase canbe partially resolved by size-exclusion chromatography (4).Our hypothesis may also provide a link between catechol-

amines and seemingly unrelated observations. For example,high levels of norepinephrine have been reported in pulviniand tendrils of several plants (3). Rhythmic plant movementsinvolving the pulvini of leguminous leaves and other nycti-nastic leaves have been thought to be controlled by auxin (32,10). The elongation of lettuce hypocotyls can be enhanced bydopamine (18), and this effect is competitively inhibited bythe antiauxin trans-cinnamic acid (31). Thus, it is possiblethat in both systems catecholamines are controlling the levelsof endogenous IAA by preventing oxidation.

ACKNOWLEDGMENTS

We wish to thank Dr. Fayek Negm, Dr. Richard Arteca, and Dr.Robert H. Hamilton for their suggestions and technical help, Dr.Robert Slocum for providing the protocol for the IAA oxidase assay,and Paula Sgrignoli for technical and editorial help.

LITERATURE CITED

1. Abeles FB (1973) Ethylene in Plant Biology. Academic Press,New York, p 121

2. Adams DO, Yang SF (1979) Ethylene biosynthesis: identificationof 1 -aminocyclopropane- 1 -carboxylic acid as intermediate inthe conversion of methionine to ethylene. Proc Natl Acad SciUSA 76: 170-174

3. Applewhite PB (1973). Serotonin and norepinephrine in planttissues. Phytochemistry 12: 191-192

4. Beffa R, Martin HV, Pilet PE (1990) In vitro oxidation ofindoleacetic acid by soluble auxin-oxidases and peroxidasesfrom maize roots. Plant Physiol 94: 485-491

5. Bradford MM (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal Biochem 72: 248-254

6. Christou P, Barton KA (1989) Cytokinin antagonist activity ofsubstituted phenylethylamines in plant cell cultures. PlantPhysiol 89: 564-568

7. Darnell J, Lodish H, Baltimore D (1990) Molecular Cell Biology,Ed 2. Scientific American Books, New York, pp 796-798

8. Elstner EF, Konze JR, Selman BR, Stoffer C (1976) Ethyleneformation in sugar beet leaves. Plant Physiol 58: 163-168

9. Endress RA, Jager A, Kreis W (1984) Catecholamine biosyn-thesis dependent on the dark in betacyanin-forming Portulacacallus. J Plant Physiol 115: 291-295

10. Ferni MG (1951) Fluorescence and photoinactivation of indole-acetic acid. Arch Biochem Biophys 31: 127-131

11. Flores HE, Protacio CM, Signs MW (1989) Primary and sec-ondary metabolism of polyamines in plants. Rec Adv Phyto-chem 23: 329-393

12. Galston AW, Dalberg LY (1954) The adaptive formation andphysiological significance of indole acetic acid oxidase. Am JBot 41: 373-380

13. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirementsof suspension cultures of soybean root cells. Exp Cell Res 50:148-151

14. Goeschl JD, Rappaport L, Pratt HK (1966) Ethylene as a factorregulating the growth of pea epicotyls subjected to physicalstress. Plant Physiol 41: 877-884

15. Goldacre PL, Galston AW, Weintraub RL (1953) The effect ofsubstituted phenols on the activity of the indoleacetic acidoxidase of peas. Arch Biochem Biophys 43: 358-373

16. Goodman TC, Montoya AL, Williams S, Chilton M-D (1986)Sustained ethylene production in Agrobacterium-transformed

95

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021

Plant Physiol. Vol. 98, 1992

carrot disks caused by expression of the T-DNA tms geneproducts. J Bacteriol 167: 387-388

17. Kamisaka S (1973) Requirement of cotyledons for gibberelicacid-induced hypocotyl elongation in lettuce seedlings. Isola-tion of the cotyledon factor active in enhancing the effect ofgibberellic acid. Plant Cell Physiol 14: 747-755

18. Kamisaka S (1979) Catecholamine stimulation of the gibberellinaction that induces lettuce hypocotyl elongation. Plant CellPhysiol 20: 1199-1207

19. Khurana JP, Tamot BK, Maheshwari N, Maheshwari SC (1987)Role of catecholamines in promotion of flowering in a shortday duckweed, Lemna paucicostata 6746. Plant Physiol 85:10-12

20. Lee TT, Starratt AN, Jevnikar JJ (1982) Regulation of enzymicoxidation of indole-3-acetic acid by phenols: structure-activityrelationships. Phytochemistry 21: 517-523

21. Martin-Tanguy J, Tepfer D, Paynot M, Burtin D, Heisler L,Martin C (1990) Inverse relationship between polyamine levelsand the degree of phenotypic alteration induced by the Ri TL-DNA from Agrobacterium rhizogenes Plant Physiol 92:912-918

22. Meyer E, Barz W (1978) Degradation of phenylethylamines inplant cell suspension cultures. Planta Med 33: 336-344

23. Murashige T, Skoog F (1962) A revised medium for rapid growthand bioassays with tissue cultures. Physiol Plant 15: 473-497

24. Oota Y (1974) Removal of the sugar inhibition of flowering in

Lemna gibba G3 by catecholamines. Plant Cell Physiol 75:63-68

25. Paul AG (1973) Biosynthesis of peyote alkaloids. Llyodia 36:36-45

26. Reid MS, Paul JL, Farhoomand MB, Kofranek AM, Staby GL(1980) Pulse treatments with silver thiosulfate complex extendthe vase life of cut carnations. J Am Soc Hortic Sci 105:25-27

27. Shannon LM, Kay E, Lew JY (1966) Peroxidase isozymes fromhorseradish roots. I. Isolation and physical properties. J BiolChem 241: 2166-2172

28. Smith TA (1977) Phenethylamine and related compounds inplants. Phytochemistry 16: 9-18

29. Smith TA (1980) Plant amines. In EA Bell, BV Charlwood, eds,Secondary Plant Products, Encyclopedia of Plant Physiol. NewSeries Vol 8. Springer Verlag, Berlin, pp 433-460

30. Tran Thanh Van M (1973) Direct flower neoformation fromsuperficial tissue of small explants of Nicotiana tabacum.Planta 115: 87-92

31. van Overbeek J, Blondeau R, Horne V (1951) Trans-cinnamicacid as an antiauxin. Am J Bot 38: 589-595

32. Yin HC (1941) Studies on the nyctinastic movement ofthe leavesof Carica papaya. Am J Bot 28: 250-261

33. Young RE, Pratt HK, Biale JB (1952) Manometric determina-tion of low concentrations of ethylene. Anal Chem 24:551-555

96 PROTACIO ET AL.

Dow

nloaded from https://academ

ic.oup.com/plphys/article/98/1/89/6087820 by guest on 25 August 2021