Plant Physiol. (1992) 99, 1090-10980032-0889/92/99/1 090/09/$01 .00/0

Received for publication October 4, 1991Accepted January 27, 1992

A New Bioassay for Auxins and Cytokinins'Wout Boerjan, Chris Genetello, Marc Van Montagu*, and Dirk Inze

Laboratorium voor Genetica (W.B., C.G., M.V.M.) and Laboratoire Associe de l'lnstitut National de la RechercheAgronomique (D.I.), Universiteit Gent, B-9000 Gent, Belgium

ABSTRACT

The authors have developed a sensitive bioassay that can beused to detect auxins as well as cytokinins. The bioassay is basedon the expression in transformed tobacco (Nicotiana tabacum)mesophyll protoplasts of a chimeric gene, consisting of the up-stream sequences of the Agrobacterium tumefaciens gene 5, cou-pled to the coding sequence of the i6-glucuronidase. The expressionof this gene is induced by the presence of both auxin and cytokininin the culture medium. Using this assay, indole-3-acetic acid wasdetected at 5 x 10-' molar, whereas trans-zeatin could be detectedat 5 x 101 molar. The assay can be performed in microtiter plates,allowing numerous samples to be analyzed simultaneously. Only2.5 x 105 protoplasts are required for one individual assay in 250microliters of culture medium and for qualitative results, the re-action is readily visualized by ultraviolet light.

Auxins and cytokinins are phytohormones that play a

major role in plant growth and development (6). The primarydetection of a novel auxin or cytokinin in plant extractsdepends on a suitable bioassay. Bioassays are also used tomonitor the purification process and to evaluate the activityof purified or synthetic hormones. The most sensitive bioas-say for cytokinins is based on the greening of cucumbercotyledons. The detection limit for a range of cytokinins isapproximately 5 x 10`0 M (8). The tobacco callus (25) andthe soybean callus bioassay (20) are also relatively sensitive,with a detection limit for kinetin of approximately 5 x 10'M, but both assays require 3 to 5 weeks for growing the callustissues prior to evaluation. The more rapid but less specificAmaranthus betacyanin bioassay (3) (detection limit for ki-netin between 10' to 10' M) is also frequently used. In thecase of auxins, the most commonly used bioassays are theAvena coleoptile curvature test with a detection limit of 1.5X 10' M IAA (29), or the less sensitive Avena section (4) or

pea split-intemode (30) tests.The expression of several plant genes has been shown to

be dependent on the presence of phytohormones (14, 19,27). Coupling a promoter of such a gene to the codingsequence of an easily detectable marker gene such as guswould allow the establishment of a sensitive bioassay. Maurel

'This work was supported by grants from the 'A.S.L.K.-Kanker-fonds, the Services of the Prime Minister (I.U.A.P. 120C0187), andthe 'Vlaams Actieprogramma Biotechnologie' (174KP490). D.I. is a

Research Director of the Institut National de la Recherche Agrono-mique (Paris, France).

et al. (19) showed a 20- to 100-fold increase of GUS2 activitywhen mesophyll protoplasts of rolB-gus-transformed tobaccoplants were incubated in auxin-containing culture medium.We have analyzed the hormonal response of a chimeric geneconsisting of the Agrobacterium tumefaciens T-DNA gene 5promoter (Pg5) fused to the gus coding sequence in mesophyllprotoplasts of transgenic tobacco plants. The gene 5 is derivedfrom the TL region of the A. tumefaciens plasmid pTiAch5 (9),and synthesis of the gene 5-encoded protein in transgenictobacco results in overproduction of indole-3-lactate, anauxin antagonist (17). Using a chimeric construct with the ocscoding region as a marker, it was shown that Pg5 is highlyactive in callus tissue grown on a medium with a high auxin,low cytokinin ratio (16). When callus was grown on a mediumwith auxin alone, no promoter activity could be detected.Upon investigating different plants in the absence of exoge-nous growth substances, the chimeric gene was shown to beexpressed at a low level in young leaves and was not ex-pressed in fully developed leaves (16). These phenomenasuggested that the gene 5 might be under hormonal control.

In this paper, we show that the expression governed bythe gene 5 promoter in transgenic protoplasts is easily con-trolled by altering the levels of auxin and cytokinin supple-ments. The results of these experiments led to the devel-opment of a sensitive bioassay for auxins as well as forcytokinins.

MATERIALS AND METHODS

Plasmid Construction and Transformation of Tobacco

A chimeric gene was constructed consisting of the promoterof the gene 5 of the Agrobacterium tumefaciens octopineplasmid pTiAch5 (9), coupled to the coding region of the gusgene (12). For this construct, pGUS1 (21) was digested withHindIII-XbaI and the 2559-base pair fragment consisting ofthe coding sequence of the gus gene and the 3'ocs sequencewas cloned into the HindIII-XbaI-linearized binary vectorpGSC1706 (21), yielding the plasmid pGUSlB1. This con-struct was mobilized by the helper plasmid pRK2013 to A.

2Abbreviations: GUS, #l-glucuronidase; T-DNA, transferred DNA;ocs, octopine synthase; BAP, 6-benzylaminopurine; 1-NAA, 1-naph-thaleneacetic acid; PAA, phenylacetic acid; oClPAA, o-chloro-phen-ylacetic acid; mCIPAA, m-chloro-phenylacetic acid; pClPAA, p-chloro-phenylacetic acid; pFPAA, p-fluoro-phenylacetic acid; pBrPAA,p-bromo-phenylacetic acid; pIPAA, p-iodo-phenylacetic acid; pi-cloram, 4-amino-3,5,6-trichloropicolinic acid; 2-NAA, 2-naphthal-eneacetic acid; NAM, naphthalene acetamide; MU, 4-methyl-umbel-liferone; RIA, radioimmunoassay.

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NEW BIOASSAY FOR AUXINS AND CYTOKININS

tumefaciens strain C58ClRif containing the vir plasmidpGV2260 by triparental mating, and was used for transfor-mation of tobacco (Nicotiana tabacum cv Petit Havana SR1)by leaf disc transformation.

Protoplast Preparation

Leaf protoplasts of in vitro grown Pg5-gus-transformed N.tabacum cv Petit Havana (SR1) were prepared overnight indigest medium (Gamborg's B5 medium [Flow Laboratories],0.4 M Suc, pH 5.6, 0.5% cellulase R10, 0.2% macerozymeR10, no phytohormones). After three washings in culturemedium (Gamborg's B5 medium, 0.4 M Suc, pH 5.6, 750 mg/L CaCl2.2H20, 250 mg/L NH4NO3, 250 mg/L Xyl, no phy-tohormones), all protoplasts were collected at a density of2.5 x 107/mL.

Bioassay in Petri Dishes

Protoplasts (106) were incubated in 5 mL of culture mediumcontaining the compound to be tested using 5-cm diameterPetri dishes. To test for auxin or cytokinin activity, 0.3 mg/LBAP or 0.3 mg/L 1-NAA, respectively, were added to theculture medium. After 72 h of culture in the growth chamber(22-250C, 5000 lux, 16-h/8-h light/dark cycle), the proto-plasts were collected by centrifugation (50g; 5 min) andtransferred to an Eppendorf tube. This tube was then placedin a horizontal position and after 10 min, the rest of theculture medium was removed using a 200-.uL pipette, leavinga dense protoplast suspension in the tube. The protoplastswere resuspended and sonicated in 1 volume of GUS extrac-tion buffer (12). The samples were centrifuged twice for 5min in an Eppendorf centrifuge and the protein concentrationof the supematant was measured using the Bio-Rad proteinassay (Bio-Rad Laboratories GmbH, Munchen, Germany).Fluorimetric GUS assays were performed as described (12)using 4-methyl-umbelliferyl glucuronide (Sigma) as sub-strate.

Bioassay in Microtiter Plates

Protoplasts (2.5 x 105; this is 10 ML of a dense protoplastsuspension) were incubated in 250 gL of culture mediumcontaining the compound to be tested. To test for auxin orcytokinin activity, 0.3 mg/L BAP or 0.3 mg/L 1-NAA had tobe present in the culture medium, respectively. After 72 h ofculture, 220,uL of culture medium were slowly removed and40,uL of GUS extraction buffer was added. The solution waspipetted up and down to lyse the protoplasts. The microtiterplates were centrifuged for 6 min at 1000 rpm (130g) (ALC4230; Apparecchi per Laboratori Chimici, Milano, Italy). Thesupernatant was placed in a fresh well and the proteinconcentration was determined (Bio-Rad). Determination ofprotein concentration is not absolutely required when per-forming qualitative assays, e.g. to determine auxin or cytoki-nin activity in a series of HPLC fractions derived from acomplex mixture of compounds. Equal amounts (30-40 tsg)of proteins (or equal volumes of the protein extract, whenthe protein concentration was not determined) were subjectedto 0.5 mm 4-methyl-umbelliferyl glucuronide final concentra-

tion (Sigma). The reaction was done at room temperature,followed on a UV tray, and photographed using a green filter.Alternatively, quantitative data could be obtained spectro-photometrically using the method described by Jefferson (12).For this, the supernatants were supplemented with 1 mM p-nitrophenyl-glucuronide. The reaction was followed bya microtiter plate reader (340 ATTC; SLT Lab Instru-ments, Abcoude, The Netherlands) and the values obtainedin the linear part of the curves were corrected for proteinconcentration.

Bioassay of Extracts of Plant Tissues

An extract of crown gall tissue (tms2-; SR1 3132; 28) wasenriched for cytokinins by a modification of the methoddescribed by Akiyoshi et al. (1). Fresh crown gall tissue wasground in liquid nitrogen and extracted with 10 mL/g meth-anol containing 10 mm 2-mercaptoethanol. The extract wasfiltered (0.45 g; Sartorius, Gottingen, Germany), ninefolddiluted with sodium acetate, pH 5.2, and passed throughDEAE-Sephacel (Pharmacia, Uppsala, Sweden). The cytoki-nins were enriched by adsorption onto an octadecyl silicacartridge (SEP-PAK C18; Waters Associates-Millipore, Mil-ford, MA). The cartridge was washed with 10% acetonitrileand the cytokinins eluted with 50% acetonitrile. The solutionwas dried, redissolved in the bioassay culture medium, filtersterilized, and used in dilutions in the bioassay.

Phenol Extraction of Plant Tissue

Crown gall tissue (tms2-; SR1 3132; 28) was ground inliquid nitrogen and resuspended in 10 mL/g TE buffer (TE =10 mm Tris, 1 mm EDTA) (pH 8). This solution was extractedwith one volume of phenol:chloroform (1:1 v/v). The waterphase was filtered (0.45 ,u; Sartorius), ether extracted, anddried. The powder was redissolved in the bioassay culturemedium, filter sterilized, and used in dilutions in the bioassay.

RESULTS

Construction of a PgS-gus Gene Fusion and Transfer intoTobacco

A binary vector was constructed containing a chimeric geneconsisting of the complete 1050-base pair upstream se-quences of the gene 5 of the A. tumefaciens octopine plasmidpTiAch5 TL region, coupled to the coding sequence of the gusgene (see 'Materials and Methods'). A schematic represen-tation of the T-DNA construct is given in Figure 1. Thischimeric gene, which will be further referred to as Pg5-gus,was transformed to N. tabacum cv Petit Havana SR1 using A.

LB Pg5 gus 3'ocs

1II35S nptil 3'g7 RB

I_

Figure 1. Schematic representation of the Pg5-gus T-DNA con-struct. LB, RB, left and right border of T-DNA; Pg5, 5'-upstreamsequence of gene 5; gus, coding sequence of fl-glucuronidase;3'ocs, 3' end of octopine synthase gene; 35S, cauliflower mosaicvirus 35S promoter; nptil, coding sequence of neomycin phospho-transferase 11; 3'g7, 3' end of the T-DNA of gene 7.

IW.S.

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Plant Physiol. Vol. 99, 1992

1000 -

750

GUS activity

_0 h 72 h

500

250

v-NAA -NAA +NAA -NAA +NAA-BAP -BAP -BAP +BAP +BAP

Figure 2. Auxin and cytokinin dependence of the expression of thePg5-gus construct. Pg5-gus protoplasts were incubated in culturemedium without hormones, with the addition of 10-6 M 1-NAA,10-6 M BAP, or 10-6 M 1-NAA and 10-6 M BAP. GUS activity was

measured just after protoplast preparation (time 0) and after 72 hof incubation. GUS activity is in pmol of MU/100,g of protein/min.

medium, a typical increase in GUS activity of about 10-foldis observed (Fig. 2).

Optimal 1-NAA and BAP Concentration for MaximalInduction of the Pg5-gus Construct

To establish conditions with the best response to additionof a particular compound, we determined the optimal 1-NAAand BAP concentration for maximal induction of Pg5-gusexpression. For this, the concentration of 1-NAA and BAPhad to be ranged from 0.0 mg/L to 0.7 mg/L. Because thisexperiment requires a large number of individual incubationsand, hence, a large amount of protoplasts and time, we

established a procedure using microtiter plates (see 'Materialsand Methods'). With this method, a large number of individ-ual assays could be performed simultaneously. Maximal GUSactivity was detected when 0.2 to 0.5 mg/L 1-NAA and 0.1to 0.3 mg/L BAP were included in the culture medium. Thedata of a typical experiment are shown in Table I. In thefollowing experiments, 0.3 mg/L (1.5 x 10-6 M) 1-NAA and0.3 mg/L (1.3 X 10-6 M) BAP were used as optimal auxin andcytokinin concentrations.

tumefaciens (see 'Materials and Methods'). Several independ-ent transformants were obtained. The results described belowwere obtained with one transformant and have been con-

firmed independently with five other ones (data not shown).

Auxins as well as Cytokinins Are Required to InduceExpression of the Pg5-gus Fusion in Transgenic TobaccoProtoplasts

Protoplasts were prepared from leaves of the in vitro grownPg5-gus plants. A low GUS activity was detected in proteinextracts of freshly prepared protoplasts as measured with thefluorimetric assay (12) (Fig. 2). In most experiments, a 72-hincubation of protoplasts in the absence of an auxin and a

cytokinin led to a slight decrease in GUS activity. Cultivationof the protoplasts in a medium containing either auxin (106M 1-NAA) or cytokinin (106 M BAP) for 72 h resulted in a

slight increase in GUS activity. In contrast, when both 106

M 1-NAA and 106 M BAP were included in the culture

Dose-Response Experiments

Because the chimeric Pg5-gus gene is induced by a combi-nation of auxins and cytokinins in mesophyll protoplasts,one can determine the activity range of cytokinins using a

fixed concentration of auxin (1.5 x 106 M 1-NAA) and byvarying the concentration of cytokinin. Alternatively, bychanging the concentration of a particular auxin and using a

fixed concentration of cytokinin (1.3 X 106 M BAP), one can

follow the activity pattern of this particular auxin. In thisway, a range of auxins, cytokinins, and related compoundsas well as other hormones were analyzed.A fluorimetric assay in microtiter plates allows a rapid

screening for biologically active compounds. A typical ex-

ample is shown in Figure 3. In the upper row (row B of theplate), the optimal concentration of 1-NAA together with a

concentration of trans-zeatin, ranging from 5 x 10-11 M up to5 x 10' M, was included in the culture medium. As can beseen, a visual inspection of the fluorescence provides a quickestimation of the active concentration. A significant increase

Table I. Optimal Concentrations of 1-NAA and BAP for Maximal Pg5-gus Induction1 -NAA and BAP concentrations ranged between 0.0 and 0.7 mg/L. The experiment was performed

as described in 'Materials and Methods" using the microtiter plate reader. Maximal GUS activity wasset as 100.

1-NAABAP

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.0 5 5 6 6 8 6 4 70.1 10 44 71 100 100 77 41 430.2 11 45 89 70 91 83 46 630.3 8 44 91 77 81 69 51 470.4 8 24 51 45 37 48 38 470.5 9 43 52 42 52 48 47 440.6 5 46 50 43 41 43 47 500.7 3 36 42 57 48 57 41 40

I I

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NEW BIOASSAY FOR AUXINS AND CYTOKININS

O-.)()cb -\ (o 4-)

42~4- 12 4-,<'~~f-~r4

t - ze at in -

'

3-

I 3)

.4,~

J] N AA D <6

gI BA PI-=1.3 10-6

- c o n t r o I s

1 2 3 4 5 7 8 Y 10 11 12

Figure 3. Evaluation of trans-zeatin, GA3, 2,4-D, and PAA in themicrotiter plate bioassay. The assay was performed as described in"Materials and Methods." In rows B and C, wells contain 1.5 x 10-6M 1 -NAA and an increasing concentration of trans-zeatin (B3-B9) or

GA3 (C5-C9). In rows D and E, the wells contain 1.3 x 10-6 M BAPand an increasing concentration of 2,4-D (D4-D9) or PAA (E6-E9).Well F2 is devoid of phytohormones. Well F7 contains both 1.5 x

106 M 1-NAA and 1.3 X 10-6 M BAP. Black wells are empty. Allconcentrations are in molar units.

in GUS activity is detected when 5 X 10-9 M trans-zeatin isincluded in the medium. An optimum is reached at 5 x 106

M. Auxin in combination with GA3 does not induce theexpression of Pg5-gus (row C). In row D, the optimal concen-

tration of BAP together with a concentration of 2,4-D ranging

between 5 X 10-10 M and 5 X 10' M is included in the culturemedium to evaluate the active concentration of 2,4-D. Anincrease of GUS activity is observed when 5 X 108 M 2,4-Dis included in the medium, whereas an optimum is reachedat 5 X 106 M 2,4-D. However, PAA in combination withBAP is less active in inducing Pg5-gus gene expression. Thefirst response to PAA is seen only at 5 X 10-6 M, whereas a

high expression is observed at a concentration of 5 X 10' M.

It is also clear from Figure 3 that auxin alone or cytokininalone added to the culture medium slightly induces theexpression of the Pg5-gus fusion (compare F2 with B2 andD2).To further evaluate the specificity of the bioassay, we

analyzed a series of substituted phenylacetic acids. As seen

in Figure 4, biological activity depends on the nature andposition of the substituents. pIPAA and pBrPAA have no or

little activity. Picloram, a synthetic herbicide, has a highactivity in a broad concentration range. 1-NAA, mClPAA,pClPAA, pFPAA, pBrPAA, and pIPAA lead to partial or

complete cell death at a concentration of 5 X 10-4 M. Theresults correlate very well with the growth-promoting activi-ties of these compounds as assayed by Caboche et al. (5).

Precise quantitative dose-response data can be obtainedusing a larger amount of protoplasts (106) in a larger volume(5 mL) (see 'Materials and Methods') of culture mediumusing 5-cm diameter Petri dishes. Table II summarizes thedetection limit and optimal concentration of a large numberof compounds tested.The naturally occurring auxins IAA and the synthetic aux-

ins 1-NAA and p-chloro-phenoxyacetic acid show approxi-mately the same dose-response curves. The dose-responsecurve for IAA is shown in Figure 5. These auxins can be

detected in concentrations of 5 X 1o-8 M and the optimalconcentration to induce Pg5-gus expression is about 10-6 M.Indole-3-butyric acid and 2-NAA can also be detected atconcentrations of 5 X 108 M, but the optimal concentrationis about one order of magnitude higher than that of IAA (seealso Fig. 6). Indole 3-pyruvic acid, a precursor in the biosyn-thesis of IAA, can be detected at concentrations of 5 X 10-7M. However, another precursor of IAA, tryptamine, did notinduce expression of Pg5-gus (Table III).Indole-3-acetamide,a compound that is only synthesized in plants transformedwith the T-DNA of A. tumefaciens or in microorganisms suchas Pseudomonas savastanoi, can also be detected at concentra-tions of 5 X 1iO' M, but its optimal concentration is morethan 5 X 10-5 M. Moreover, the level of induction measuredat the optimal concentration is much lower than the levelconferred, for instance, by 1-NAA. A comparable observationwas made with NAM. This compound could only be detectedat a concentration of 5 X 10-6 M, but the GUS activity at theoptimal concentration was low as compared to 1-NAA. Avery low level of induction by the natural IAA conjugate IAAL-aspartic acid is measured at concentrations of more than 5X 10-6 M. This low level of activity is probably due to slowhydrolysis to free IAA (10). PAA, another naturally occurringauxin, can only be detected at concentrations about 1 X 10-6M. However, strong induction is measured at 7 X 10' M. Atconcentrations of 5 x 10' M, PAA becomes partially cyto-toxic. Thus, the activity range of phenylacetic acid is relativelynarrow as compared with 1-NAA or 2,4-D. The detectionlimit of 2,4-D is about 2.5 X 10-8 M and the optimal concen-tration is more than 4.5 X 10-5 M. L-Tryptophan, benzoicacid, tryptamine, and tryptophol were not able to induceexpression of the Pg5-gus fusion; neither were GA3, ABA,1-aminocyclopropane-1-carboxylate, and the major alfalfa-specific Nod signal of Rhizobium meliloti, NodRm-1 (18;Table III).

A A

r: p A -

MCI P'l A -

PC 'Ae-

93O\Q<%-)V'4-2. .c

<e 4-) ) <D) )

6~~~~~~~~~~ ~~~ ~~~ ~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Figure 4. Evaluation of 1-NAA, substituted phenylacetic acids, andpicloram in the microtiter plate bioassay. The assay was performedas described in "Materials and Methods." All assays contain 1.3 x

106 M BAP and the concentration of auxins as indicated. Due tocytotoxicity, the wells with 5 x 10-4 M 1-NAA, mCIPAA, pCIPAA,pFPAA, pBrPAA, and pIPAA contained only half of the amount ofproteins present in all other wells. Control in well a contains no

hormones and control in well b contains only 1.3 x 10'6 M BAP. Allconcentrations are in molar units.

A,.B

nF

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pFPAApBrPAA

-pIPAAp c I o r a m

controls

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Plant Physiol. Vol. 99, 1992

Table II. Evaluation of Different Hormones and Hormone Analogs Using the Pg5-gus ProtoplastBioassay

Detection limit and optimal concentration were determined using the bioassay in Petri dishes asdescribed in "Materials and Methods." For each compound, a series of dilutions that differed by oneorder of magnitude was tested. Detection limits were estimated as the minimal concentration leadingto a significant increase in GUS activity. The GUS activity (in pmol of MU/100 Ag of protein/min) atthe optimal concentration was determined by comparing all the tested compounds at their optimalconcentration in one assay.

Detection Limit Optimal Concentration Relative GUS ActivityHormones (estimated) for Gene 5-gus at Optimal(Estimated) Induction Concentration

Auxins1-NAA 5.0 x 10-8 M 1.5 X 10-6 M a2-NAA 5.0 x 10-8 M 5.4 X 10-5M bIAA 5.0 x 10 8M 5.5 x 10-6 M b

p-Chloro-phenoxyacetic acid 5.0 x 10-8 M 5.0 x 10-6 M c

lndole-3-butyric acid 5.0 X 10-8 M 25.0 x 10-5 M c

lndole-3-pyruvic acid 5.0 x 10-7 M >5.0 x 10-5 M blndole-3-acetamide 5.0 x 10-7 M >5.0 x 10-5 M e

IAA-L-aspartic acid 3.0 x 10-6 M >3.4 x 10-' M e

NAM :5.0 x 10-6 M >5.0 x 10 5 M dPAA 1.0 x 10-6 M 7.0 x 10-5 M b2,4-D 2.5 x 10-8 M >4.5 x 10-5 M b

CytokininsKinetin c5.0 x 10-10 M 2.5 X 10-6 M atrans-Zeatin c5.0 x 10-11 M 3.5 x 10-8 M aIsopentenyl adenine c5.0 x 10-9 M 5.0 x 10-6 M aBAP c4.5 x 10i9 M 1.5 x 10-6 M aa 880 to 1099, b 660 to 879, 440 to 659, d 220 to 439, 0 to 219.

The cytokinins kinetin, isopentenyladenine, BAP, andtrans-zeatin all display similar dose-response curves (withrespect to their shape). Generally, the cytokinins can bedetected at lower concentrations than the auxins; trans-zeatinis the most active and could be detected at 5 x 10-11 M. Thedose-response curve for trans-zeatin is given in Figure 7.

The Pg5-gus Gene Is Expressed at a Higher Level in YoungLeaves as Compared to Fully Developed Ones

During the course of our experiments, we observed thatthe absolute levels of GUS activity in response to exogenouslyapplied hormones as well as the basal GUS activity differedsubstantially between different protoplast batches. To deter-mine whether this is due to the developmental state of theleaves from which protoplasts were derived, protoplasts wereprepared from the small upper leaves and lower, fully devel-oped ones of the same in vitro grown plant. GUS activitywas measured (a) just after protoplast preparation, (b) after72 h of incubation in culture medium without hormones, (c)with the addition of auxin only, and (d) with the addition ofcytokinin only.From Table IV it is clear that younger leaves have a higher

basal level of GUS activity than mature ones. Moreover, thelevel ofGUS activity increased substantially when protoplastsof these young leaves were incubated with only auxin or onlycytokinin, whereas there was only a slight induction of GUSactivity when the same treatment was done on protoplastsderived from mature leaves. A significant increase in GUSactivity was even detected when protoplasts of young leaves

were incubated for 72 h in the absence of exogenouslyapplied hormones.

Additionally, fluorimetric GUS analyses were done onextracts of leaves of greenhouse-grown plants. Figure 8 showsa schematic representation of the obtained fluorimetric data.Immature, nonflowering plants contain a higher GUS activityin the younger leaves than in the fully developed ones,whereas mature flowering plants show approximately equalGUS activity in all leaves. However, GUS activity is high inthe small leaves at the base of both nonflowering and flow-ering plants.

GUS ACTIVITY600

500

400

300

200]

I100A

10-4 I10-7 10-1 I 0 10

[IAA](M)

Figure 5. Dose-response curve for IAA. The experiment was per-formed as described in "Materials and Methods" using the Petridish method. GUS activity is in pmol of MU/100 Ag of protein/min.

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NEW BIOASSAY FOR AUXINS AND CYTOKININS

°oN1\ (O4 I

-I . . .

_!~t%~r%r!% r

_:)/~4i , 1

800

600

2 - N A A1 - N A A [B A PJ=1.3*10-6

400

200Figure 6. Comparison of the biological activity of 1-NAA and 2-NAA. The experiment was performed as described in "Materialsand Methods." All wells contain 1.3 x 10-6 M BAP and the concen-tration of auxins is as indicated. All concentrations are in molarunits.

GUS ACTIVITY

0 10-il lo-lo10 10- I010 10 10 10 10 10-3[t ZEATIN] (M)

Bioassay of Extracts of Plant Tissue

Because we were able to detect very small quantities ofpure hormones using the Pg5-gus protoplast assay, we were

interested in evaluating this assay on plant extracts. For thispurpose, an extract of a crown gall tumor (tms2-; SR13132;28) was enriched for cytokinins by a modification of themethod described by Akiyoshi et al. (1) (see 'Materials andMethods'). This tissue has been shown to contain 500 to1500 pmol.g'1 fresh weight zeatin riboside equivalents (22).Using the fixed concentration of auxin (0.3 mg/L 1-NAA),we were able to detect a significant increase in Pg5-gusexpression with an aliquot of the extract corresponding to 25mg of crown gall tissue (Table V). Even by performing a

simple phenol/chloroform extraction on this crown gall tissue(see 'Materials and Methods'), which is a very crude extrac-tion without a specific enrichment for cytokinins, we were

able to detect a significant increase in GUS activity with an

Table l1l. Compounds That Do Not Induce Pg5-gus GeneExpression at the Indicated Concentration RangeThe GUS activity was measured at the concentration shown, or

as the concentration of each compound was increased by succes-sive orders of magnitude for the indicated range. ACC, 1-amino-cyclopropane-1-carboxylate; NodRm-1, alfalfa-specific Nod signal(18).

Compound

L-TryptophanBenzoic acidTryptamineTryptopholGA3GA3GA3ABAABAABAACCACCACCNodRm-1NodRm-1NodRm-1

Concentration

5 x 10-68 x 10-76 x 10-96 x 10-93 x 10-73 x 10-93 x 10-94 x 10-84 X 10-94 x 10-9x 10-9

1 X 10-6

M + 1.3 x 10O6 M BAP-*8 x 10-5 M + 1.3 x 10-6 M BAP-.6 X 10 5M + 1.3 x 10 6 M BAP

6 x 10-5 M + 1.3 x 10-6 M BAP3 x 105 M

-.3 x 10 5 M + 1.3 x 10-6 M BAP-3 x 1O-'M + 1.5 x 10-6 M 1-NAA4 X 10-5 M4 X 105 M + 1.3 x 10-6 M BAP4 x 10-5M + 1.5 x 10-6 M 1-NAA

--1 X 10o- M

M + 1.3 X 10O6 M BAP1 X 10-6 M + 1.5X 10-6 M 1-NAA1 X 10 10 _.1 X 10-7MX 0-10 1 X 10-7 M +1.3 X 10-6M BAPX 100 1 X 10-7 M + 1.5 x 10-6 M 1-NAA

Figure 7. Dose-response curve for trans-zeatin. The experimentwas performed as described in "Materials and Methods" using thePetri dish method. GUS activity is in pmol of MU/100 Ag of protein/min.

aliquot of the extract (water phase) corresponding to 100 mgof crown gall tissue (data not shown).

DISCUSSION

We developed a bioassay that can be used to detect auxinas well as cytokinin activity. The system is based on theinduction by auxin and cytokinin of a chimeric Pg5-gus fusionin tobacco mesophyll protoplasts. The method has severaladvantages over other bioassays. First, unlike most bioassaysthat are specific for one type of hormone, the Pg5-gus pro-toplast bioassay can detect auxin as well as cytokinin activityusing identical procedures. A second advantage is that theprotoplasts are essentially derived from one cell type, themesophyll cell. It is thus expected that the response will bemuch more uniform than alternative bioassays based onintact, multicellular tissues such as cotyledons, callus, coleop-tiles, or stem segments. Moreover, by using protoplasts andliquid culture medium, one avoids potential problems oftransport of different hormones or conjugates in the tissue.This is a particular problem of the Avena curvature test (29).Furthermore, the protoplast batch can be divided equally toperform individual incubations with different hormone con-centrations. Each batch will thus have exactly the same levelof background activity, thereby allowing detection of verylow hormone concentrations. Third, because the bioassay,when performed in microtiter plates, uses only a smallamount of protoplasts (2.5 X 105), numerous samples can beanalyzed simultaneously. A final advantage is that the assayis performed in a small volume (250 ,L), reducing the amountof growth factor to be tested to a minimum.We defined dose-response curves for several auxins, cyto-

kinins, and related compounds such as analogs and precur-sors, and tested the activity of other hormones as ABA, GA3,and 1-aminocyclopropane-1-carboxylate. In general, the clas-sic auxins (1-NAA, IAA, 2,4-D, indole-3-butyric acid) can bereproducibly detected in the range of 5 x 10-8 M. The activitiesof indole-3-acetamide and NAM might be due to the hydro-

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Plant Physiol. Vol. 99, 1992

Table IV. The Expression of Pg5-gus Is Developmentally RegulatedThe experiment was performed as described in 'Materials and Methods" using the bioassay in

Petri dishes. Protoplasts were prepared from the top and mature leaves of an axenically grown plantand cultured in medium without hormones, with addition of 0.3 mg/L 1-NAA or 0.3 mg/L BAP.

1 -NAA/BAP Hours of Incubation GUS ActivityConcentration in Culture Medium

mg/L pmol MUIOO1,g protein/min

Young leaves 0/0 0 1480/0 72 198

0.3/0 72 3350/0.3 72 479

Mature leaves 0/0 0 620/0 72 62

0.3/0 72 700/0.3 72 73

lytic conversion of these compounds to IAA and 1 -NAA,respectively, by aspecific endogenous hydrolases or, alter-natively, they might act as auxins, as has been proposedpreviously (7, 15). In several studies of auxin action, 2-NAAis considered as a biologically inactive auxin analog. It hasbeen shown that the capacity of tobacco pedicel explants toform auxin aspartate conjugates depends on the presence ofa biologically active auxin (26). In those experiments, theconjugate 2-NAA-aspartate was not formed when the ex-plants were treated with 2-NAA unless 1-NAA was addedprior to or simultaneously with 2-NAA. In corn coleoptiletissue, 2-NAA is able to enter the tissue as efficiently as 1-NAA, but is unable to move in the polar transport system,and is at the same time very ineffective as a growth promoterof coleoptile segments (11). 2-NAA is not capable of inducingtransmembrane potential differences in tobacco protoplasts.However, it can reverse the hyperpolarizing effect of 1 -NAA,suggesting it can compete with 1-NAA at the plasma mem-brane sites involved in the membrane response (2). Cabocheet al. (5) showed that a minimum concentration of 2.5 x 10-7M 2-NAA can induce proliferation of tobacco mesophyllprotoplasts. It has been shown by Karlin-Neumann (13) thatthe height of the shoot of Arabidopsis seedlings was severelyreduced when these seedlings, transformed with a chimerictms2 gene under control of the cabl40 promoter and suppliedwith 2-NAM, were grown under intermittent red light ascompared to dark-grown ones; again, this suggests a biolog-ical activity of 2-NAA. In our bioassay, 2-NAA clearly showsbiological auxin activity (Fig. 6). The differences in dose-response curves for 1-NAA in Figures 4 and 6 probablyreflect differences in the physiological state of the differentprotoplast batches (see below). In comparison with 1-NAA,the optimum for 2-NAA is shifted toward higher concentra-tions, whereas the GUS activity at the optimal concentrationis lower (Fig. 6; Table II). However, L-tryptophan, benzoicacid, tryptamine, and tryptophol could not induce Pg5-gusexpression. The most active cytokinin tested was trans-zeatin;this compound could be detected at 5 x 10-11 M. The detectionlimit for pure t-zeatin is thus of the same order of magnitudeas detected by RIA and HPLC-MS/single-ion monitoring (H.Van Onckelen, personal communication). For pure IAA, ourbioassay is approximately one order of magnitude less sen-

sitive as compared to RIA and HPLC-MS/single-ion moni-toring (H. Van Onckelen, personal communication).The dose-response curves are obtained with mesophyll

protoplasts and might substantially differ with other tissuesources. It has been shown that in the case of IAA, differentdose-response curves have been obtained when differenttissues were analyzed. In general, maximal root elongation isachieved with approximately 5 x 10-10 M IAA, whereas shootelongation occurs maximally at about 10'6 M. Moreover,many tissues do not respond to exogenously applied hor-mones, whereas other tissues show a drastic response (15).Furthermore, when analyzing one particular tissue, differentgenes might respond differently to a given auxin/cytokininratio. The Pg5-gus fusion is induced by the presence of bothauxin and cytokinin in the culture medium, whereas a roiB-gus fusion is induced in transgenic tobacco mesophyll pro-toplasts by auxin alone, but not by cytokinin alone (19).

Identical experiments performed with the same protoplast

GUS activity1200

1000_ IMMATURE

80i MATURE800 L

TOP BASE TOP BASE

Figure 8. Dependence of the expression of Pg5-gus on the devel-opmental state of the plant. GUS activity was determined in allindividual leaves of an immature, nonflowering plant and a fullygrown, flowering one. The main vascular bundles were removedbefore preparing the extract. Both plants were greenhouse-grownand derived from the same in vitro propagated transgenic line. GUSactivity is in pmol of MU/100 ,ug of protein/min.

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NEW BIOASSAY FOR AUXINS AND CYTOKININS

Table V. Bioassay of Extracts of Crown Gall tms2- Tissue

1-NAA Concentration Extract of Crown Hours of Incubation GUS ActivityGall Tissue tms2-a in Culture Medium

mg/l mg pmol MU/IOO ug protein/min

o o 0 2010 0 72 1560.3 0 72 3330.3 2.5 72 3600.3 25 72 4820.3 250 72 1083

a This value corresponds to the amount of crown gall tissue.

batch differ by no more than 10% in the absolute levels ofGUS (examined for the bioassay in Petri dishes; data notshown). However, variation is observed between identicalexperiments performed with protoplasts of different prepa-rations (in the absolute amount of GUS produced as well asin background GUS activities and dose-response curves). Thisis most probably due to the physiological conditions anddevelopmental state of the plants from which protoplastshave been derived. It is possible that the higher basal GUSactivity in immature Pg5-gus tobacco leaves and the higherlevel of induction of Pg5-gus expression in protoplasts ofimmature leaves upon addition of either auxin or cytokininalone correlate with the endogenous auxin and cytokininlevels in these tissues; higher auxin and cytokinin levels havebeen reported to occur in young leaves (24). An alternativepossibility is that these young tissues might be more sensitiveto auxin and cytokinin than older tissues. Santoni et al. (23)have shown that the sensitivity of the H+-ATPase-mediatedproton translocation by plasma membrane vesicles in re-sponse to exogenously applied auxin is highly dependent onthe plant age from which the membranes were prepared.Our results correlated well with those of Koncz and Schell(16). They also showed that the expression of the gene 5 iscorrelated with the state of development of the tissues har-boring the gene: no activity was detected in fully developedleaves, whereas young leaves showed a low level of activity.When protoplasts derived from mature leaves were used toperform the bioassay, the Pg5-gus fusion was only inducedwhen both auxins and cytokinins were included in the culturemedium and the background GUS activity was low. Hence,fully developed, mature leaves should be used to performthe bioassay and the appropriate negative controls; namely,assays of either auxin alone or cytokinin alone should beperformed to determine the background GUS activity. Ingeneral, experiments with protoplasts derived from matureleaves give very good results.Our data clearly show that the expression of a chimeric

gene 5-gus construct is dependent on the presence of an auxinand a cytokinin in the culture medium. Recently, Korber etal. (17) have demonstrated that the gene 5 product convertstryptophan into indole-3-lactate, which is an auxin antago-nist. These authors suggested that the gene 5 product playsa role in fine tuning the auxin/cytokinin ratios in crown galltissue. It is thus not inconceivable to propose that gene 5expression would respond directly to auxins and cytokinins.However, we cannot exclude that some other natural com-

pounds, which have no structural homologies with the clas-sical auxins and cytokinins, also have activity in our bioassay.To further evidence the specificity of the bioassay, we ana-lyzed a series of substituted PAA derivatives (Fig. 4). Theresults obtained with the Pg5-gus bioassay correlate very wellwith the cell growth-promoting activities of these compounds(5). In the Pg5-gus bioassay, we observed the following orderof detection limits: picloram > 1-NAA> mClPAA > oClPAA> pClPAA > pFPAA > PAA > pBrPAA > pIPAA, withpicloram being the most active; whereas in the cell growth-promoting assay, the following order was obtained: picloram> 1-NAA > mClPAA > oClPAA > pFPAA > PAA >pClPAA, pBrPAA, pIPAA. Biological activity of pClPAA wasonly detected in the Pg5-gus bioassay and not in the cellgrowth assay. This is possibly a consequence of the narrowlyactive concentration range before cytotoxicity occurs.

For the determination and isolation of active compoundsfrom plant extracts, one has to bear in mind that compoundsin the plant extract can reduce the response of the Pg5-gusbioassay. Therefore, it is advisable to use the bioassay as aqualitative tool during purification processes. The Pg5-gusbioassay can be of great use in combination with HPLC. Forquantitative analysis of biologically active fractions, one willhave to make a standard curve for each compound and foreach experiment, because different protoplast batches canrespond differently, as discussed above.Due to the possibility of analyzing numerous samples

simultaneously, the bioassay can be utilized to screen fornew growth factors with auxin or cytokinin activity, e.g., incombination with HPLC. Alternatively, the bioassay couldbe used to identify enzymes that convert an inactive com-pound into an active auxin or cytokinin.

ACKNOWLEDGMENTS

We are grateful to Martin Crespi for preparing the plant extracts,Michel Caboche for providing the PAA derivatives, and A. Kondorosifor providing the Rhizobium meliloti Nod factor, NodRm-1. We thankHarry Van Onckelen, Martin Crespi, and Allan Caplan for criticalreading of the manuscript, Martine De Cock for typing it, and KarelSpruyt, Vera Vermaercke, and Stefaan Van Gijsegem for figures andphotographs.

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