Planit Physiol. (1967) 42, 247-257

Transient Effects of Light on Auxin Transportin the Avena Coleoptile'

Robert M. Thornton2 and Kenneth V. Thimann2Biological Laboratories, Harvard University, Cambridge, Massachusetts

andThe University of California, Santa Cruz, California 95060

Received September 16, 1966.

Sumimary. The transport of indole-3-acetic acid-1-14C (IAA) through 4 mmsegments of etiolated Avena coleoptiles was studied as a function of time by applyingIAA in apical agar blocks and measuring the basal IAA export rate at 5-minuiteintervals. The transport velocity found in this way is at least 15 mm/hour at 260.Following a 30-minute equilibration period, the export rate is nearly constant for atleast 50 minutes at physiological donor concentrations. Exposure to about 3 X 105ergs/cm2 blue light for 15 minutes leads to a transient reduction in the export. Theexport rate reaches a minimum about 25 mintutes after the onset of illumination, thenrises to reach a maximum by 35 minutes, and subsequently declines again. The resultis a net export depression during the first 80 minutes, amounting to some 12 to 17 %.Its timing closely matches the timing of the light growth response elicited by thesame light dosage.

At higher IAA concentrations (0.5 and 1.8 mg/l), the export rate reaches a peakabout 60 minutes after the initial application of auxin, and thereafter declines rapidly.Light increases this decline in export rate, without causing peaks and troughs, andeven at 0.25 mg/l IAA the transient changes in export appear to be superimposed ona gradtual decline in export rate after illumination. Blue light is effective in allthese phenomena; the red far-red system appears to exert no effect. The resultsare discussed in relation to the mechanism of action of light both in the light growthresponse and in phototropism.

WVhen the stubapical part of the etiolated oatcoleoptile is exposed to a tunilateral pulse of strongblue light, its response includes a transient depres-sion in growth rate as well as eventual cturvaturetoward the light souirce. The light-growth response(LGR) does not need to occur for the plant tocturve (4), and it is well established that photo-tropic curvatuire involves lateral movement of auxin(3,16) rather than the changes in tissue sensitivity,atuxin produiction, or auxin movement that mightbe expected to produce a growth response. Thusthere is little basis for an assumption that theLGR produices phototropism.

OIn the other hanid, the close similarity betweenthe LGR and phototropism in terms of dose-re-sponse relations (51) stuggests that both responsesmight spring from the same basic mechanism. If

1 This ws-ork was supported in part by a NationalScience Foundation Predoctoral Fellowship to R. M.Thornton and in part by a grant to Dr. K. V. Thimannfrom National Scienice Foundation No. GB4337.

2Present address: Division of Natural Sciences, Uni-versity of Californiia, Santa Cruz, California 95050.

it could be shown that a light-aulxin responseparallels the LGR, just as lateral transport paral-lels phototropism, then it would almost surelyfollow that the LGR and phototropism have acommon origin. In that case the LGR could beviewed as the transient outptut of a system whoselong-term output is phototropism. The lack ofwork on the LGR dturing the past 25 years, com-pared to the nutmerouis stuidies of phototropism,appears to have resulted from a number of investi-gations (7, 20, 26, 27) in which the limited pre-cision of the Avena bioassay created a confusingand contradictory picture of the light-auxin re-sponse. The clarification of this response there-fore seemed of ftundamental importance. Withthe aid of labelled auxin at high specific activity,it has been possible in the present investigation tostudy auxin transport on a brief enough time scaleto provide an adequate parallel to the LGR.

Material and Methods

For auxin transport studies, seeds of Avenasativa var. Segeshavre (Svalov, Sweden), or in a

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PLANT Pl{YSIOLOGY

few experiments the American variety Victory,were hulled, soaked under room lights for 2 hours,sown on moist filter paper in closed bowls, andallowed to grow in darkness, at 260 and about 90 %relative humidity, for 69 to 75 hours. Red lightwas applied for the first 17 to 24 hours, afterwhich the plants received only intermittent ex-postures of brief duration to dim red light. Forstudies on the LGR, the same conditions wereemployed except that the seeds were planted invials of 1.5 % agar.

Duiring experiments, the coleoptiles were lightedby a 25w incandescent bulb covered with a sheetof Corning signal red glass, and placed 40 to 50cm away. Test illumination was given by a smallfluorescent lamp containing two 15w cool whiteblulbs. For blue, red and far-red light respectivelythis lamp was equipped with 2 sheets of DuPontblue cellophane, or 2 sheets of 0.3 cm red darkroomsafe-light plastic, or both. In some cases a 5 cmwater filter was added, with no visible effect onthe results. The absolute emission of the test lampvith the blue cellophane was determined with anEppley thermopile, calibrated against a Bureau ofStandards lamp and coupled to a Kintel electronicgalvanometer. The intensity at the transport as-semblies was 1000 ergs/cm2sec, of which 300 to400 ergs/cm2sec was estimated as blue light fromthe spectrum shown in figure 1. The emissionspectrum of the test lamp was determined with acalibrated selenium photocell coupled to the Kintelgalvanometer and placed at the 2 mm exit slit ofa Bausch and Lomb grating monochrometer throughwvhich the lamp radiation was passed.

For transport studies, coleoptiles were selectedwithin a size range of 5 mm about the mean ineach experiment, this mean in nearly all experi-ments lying between 2.5 and 3 cm, though in a fewcases it was 1.3 cm. No correlation was foundbetween mean coleoptile size and experimentalresults. After removal of the primary leaf, a4 ± 0.1 mm section was cut 1.5 to 2 mm below theapex. Each section was picked up on a lanolin-coated spatula and inserted tip-down in a luciteholder (8). Sets of 12 or 16 sections were pro-vided with apical (lower) and basal (upper) blocksof washed 1.5 % agar (16). In all experiments,2 sets were prepared in sequence and run at thesame time. The cutting time for each set was 10minutes, so that about 20 minutes elapsed betweeninitiation of cutting and application of auxin.

When both sets were complete, the lower blockswere replaced with agar donor blocks containingpurified (8) carboxyl-labelled 14C indoleacetic acid(16.9 C/mole, synthesized bv the method developedby Dr. B. B. Stowe) (21). At intervals of 5minutes + 5 seconds thereafter, the receiver(tipper) blocks were removed to etched aluminumplanchets and immediately replaced with freshblocks of agar.

500 600WAVELENGTH (MJI)

FIG. 1. Spectral emission of the test lamp. Ordinate:emitted light energy (arbitrary scale). Abscissa: lightwavelength in millimicrons. Solid line: emission throughthe blue filter. Dashed line: emission through the signalred filter. Dotted line: emission through the combinedblue and red filters.

Starting 30 to 35 minutes after auxin applica-tion, test illumination was provided with the lampset about 450 above the horizontal. A second lightdose, starting 60 to 65 minutes after auxini appli-cation, was generally included as a control andwas found on comparison with single-dose experi-ments to give no measurable effect during the85-minute experimental period. This lack of effectwas expected from the partial refractory period ofthe LGR (11) and indicates that the response tothe primary dose is not a thermal effect.

Since the light dose (10-15 min) was longerthan the 5 minute interval between changes ofreceivers, handling requirements made it necessaryto give both members of each pair of sets the samelight treatment, and to perform control and testexperiments at different times. These times werechosen at random over a 9-month period.

At the end of each experiment the planchetswere stored in a deep-freeze for 1 to 5 days, afterwhich the frozen agar was heated to 90 to 100°on a hotplate and spread evenly over the flat sur-face of the planchet with a curved spatula. Driedspecimens were heated for 5 to 10 minutes andeither restored to the freezer or immediatelycounted for 30 to 80 minutes on a Nuclear-Chicagoautomatic gas-flow counter with a background of3 to 5 CPM.

To prevent cross-contamination, the spatulasused in handling agar blocks were washed between

248

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TIIORNTON AND THIIINANN LIGHT AND AUXIN TRANSPORT

use. Rinses in 3 successive flasks of distilled waterwere found sufficient to reduice the transfer ofisotope to backgrouind levels (24). In general,restults have been converted to DPM assumingnegligible self-absorption; however, a correctionwas made (8) before cotunts from agar blocks of(liffering size were compared.

The absence of auxin destruiction during storageof the stock soluition, storage of dried planchets,and heating of planchets was verified by repeatcounts of samples. In no case was loss of radio-activity detectable.

For sttudy of the LGR, vials containing seed-lings were taped together in grouips of 6 or 7 andfastened to a glass plate. The set of vials wasplaced on a heavy brass stage andl viewed with a

horizontal travelling microscope calibrated in unitsof 10 ,u. Coleoptiles were marked with carborun-(tlm duist to provide apical height reference pointsan(d either left intact, or decapitated and suppliedwith 0.25 mg/l IAA in apical 1.5 % washed agarblocks. The illumination was as in the transportexperiments, except that in taking measuirementsthe plants were systematically moved relative to there(l working light, which in this case was a smallfluiorescent safe-light. Each height measurementwas dulplicated and the average height of eaclhcoleoptile determined at precisely known intervalsaveraging 5 minutes in length. At 90 minutes afterthe onset of the blue light exposure, phototropiccurvatuires were determined with a protractor heldbehind the plants. The mean curvatuire of a set

of 7 intact plants was about 100, uniformly dis-tributed along the coleoptile, verifying that thelight dosage lay in the second positive range ofphototropism (23). Suich cturvattires began abotit15 minutes after the onset of illumination and were

easily seen in the microscope. Thuis the measuredILGR's were somewhat exaggerated in amplitude.However, this is llnimportant in the present work,since it was (lesired only to reprodtuce the testconditions used in transport work and to determinewhether the illuiminatioin had anly effect on growth.That the observed responses agree in form and

timing with those resulting from symmetrical illu-mination (5) indicates that the responses had theirorigin in the LGR. The only ill effect resultingfrom uniiilateral illuimination is a false appearance

of slow growth rate recovery, (Ilue to the curvinig.

Results

System Characteristics and Dose Timing. Therelevance of these experiments depends on theextent to which the transport of exogenous IAAin cut sections resembles auxin transport in intactplants. Though exogenous IAA appears to move

in the normal channels (9), there is some uncer-

tainty regarding the effect of cuitting the tissue.While the accepted transport velocity (i.e., the

distance moved by an average auixin molectule inuinit time) is near 10 mm/hour in coleoptiles (25)and also in stem sectionls (17), the phototropicresponse appears to migrate 2 or 3 times as fastin intact plants (see fig 20 of 6; also 27, 24). Thuisit appears that injuiry may decrease the transportvelocity.

Forttunately, the present experiments clearlvshow that the transport velocity, at least for shorttimes, is greater than the accepted valuie. Figture2 shows the cuimulative aulxin export as a ftunctionof time. For most of the time the plot is linear,yielding an extrapolatedl intercept at 22 minutes,from which the velocity, compuited for 4-mm sec-tions by the method of van (ler Weij (25), is closeto the accepted 10 mm/houir. But this interceptis in error. Traces of ra(lioactivity first appearedin the third receiver, i.e. d(lring the tenth to fif-teenth minute after the application of auixiu, aindlmarked activity was present in the fifteenth totwentieth minuite.

The following metho(d was used to verify thereality of the rapid atuxin transport. If no radio-activity were exported uintil the fouirth .5-mintuteinterval, then on the average the couints dule tobackground shouild he identical in the second andthirdl receivers. Thus the cotunt ratio third: secondshotild average tunity. This ratio was determinedin each of 23 experinments. The (leviations fromunity were stubjected to a t-test (10), with theresuilts shown in table 1. With a 2-tailed test themeani deviation (liffers from tunity at 99.9 % con-fidence. Thtis the extra radioactivity in the thirdreceiver is not a statistical artifact.

Sinice atuxin could have begun to enter the thir(dr-eceiver at anv time between 10 and 15 minu1tesafter the start of the experiment, the initial trans-port velocity is betweeln 15 an(d 26 mm/houir. Avelocity even above 26 mm/houir cannot be ex-clu1ded, since it was impossible to determine inthese experiments whether the second receiver alsocontained auixin.

Table I. Comtparison Between Radioactiitv in th1Second and Third Receivers

Periods: 5 to 10 and 10 to 15 minutes after auixinlal)plicatiou, respectively. Four auxin doonor concenitra-tioIns were used. The data were pooled and suibjected toa t-test of the deviation of the ratio (CPM in receiver#3: CPM in receiver #2) from uinity, by the metlhodouitlined in Goldstein (10). t = 4.17, 19 d.f. The de-viationl of the ratio from unit is significanit at 99.9+ %confidence.

Donor concn N~To. of CPM in receiver #3(mg/l IAA) experiments CPM in receiver #2

0.13 6 1.170.25 4 1.050.40 2 1.330.50 8 1.39

249)

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PLANT PHYSIOLOGY

6

z

- 5<-0

4IF-L..uJ

IX-J

zo-.x 0-3w 0

z _i

;z0

O 10 20 30 40MINUTES AFTER AUXIN APPLICATION

. 8 _

0

u-I. 6

z

E .4

0

.2

0

w

4

0a.

w

w

4-Jw

1.2[

0.81

0.41

0.c0 20 40 60 80MINUTES AFTER AUXIN APPLICATION

50

20 40 60 80 20 40 60 80MINUTES

FIG. 2. (upper left). Net auxin export (CPM/coleoptile section) versus time (minutes) after the application of0.5 mg/l IAA to the apices of 4-mm coleoptile sections. Average of 4 experiments.

FIG. 3. (upper right). The effect of auxin donor concentration on the time course of auxini export rate. Thedonor concentrations (mg/l IAA) and the number of contributing experiments are shown at the right of each curve.Individual experiments were averaged, the results smoothed as in figure 4, and each export rate w-as divided by theamount exported during the first half hour to make all the curves concurrent for the first 25 minutes.

FIG. 4. (bottom). The time course of auxin export rates in individual experiments, as a function of light treat-ment. Each curve represents a single experiment. In the right-hand figure, blue light was appliied starting 35minutes after auxin application (see bar on abscissa). Left hand figure, no blue light. The data in each experi-ment were smoothed b averaging adjacent trios of points. Auxin donor concentration: 0.13 mg/l IAA.

It may be concluded, therefore, that exogenousauxin moves in excised tissue at essentially thesame velocity as the endogenous auxin in intactplants during the phototropic response.

The export of auxin from short sections isknown to decline almost to zero in a few hours,while the uptake and binding proceed linearly forseveral more hours (9). This suggests that an

auxin-degrading system becomes active at the basalcut surface of the sections. The auxin outputfrom old sections cannot therefore be taken as a

direct measure of the operation of the transportingsystem. As a practical matter, the stimulus inphotoresponse studies must be given early enoughso that the response takes place before the exporthas begun to decline.

The sharpness of the decline in export dependson the concentration of auxin in the donor as wellas on the length of the sections (9). For thisreason several auxin concentrations were tested inorder to find the optimal level for photoresponseexperiments.

250

1.8 mg/I (2)

0.25 mg/1(4)

0 0.13 mg/I (6)

I

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THORNTON AND THIMANN-LIGHT AND AUXIN TRANSPORT

Figure 3 shows the time courses of the exportof auixin when the donor concentrations were 0.13,0.25 and 1.8 mg/I. These experiments were per-formed without exposure to blue light. In a fewcases far-red light was applied, but was found tobe without effect. At 0.13 and 0.25 mg/l IAA theexport rate (van der Weij's transport capacity)was nearly constant between 35 and 65 minutesafter auxin application. In contrast, at 1.8 mg/lIAA there was no period of constant export rate.Preliminary experiments at 0.5 mg/l IAA (notshown in fig 3) always showed a similar sharpdecline.

The data in figure 3 were normalized so thatall curves became concurrent at times up to 30minutes after auxin application. During the sub-sequent period a 100 % change in donor concen-tration (from 0.13 to 0.25 mg/l IAA) yielded onlya 15 % change in the export rate. Thus relativelylarge errors in donor preparation can be toleratedwithout seriously affecting the shape or height ofthe normalized export curve.

It is apparenit that low donor concentrations arepreferable to high. In practice, 0.13 mg/l IAA(7 X 10-7 M) yielded the minimum convenientlymeasurable outptut in 5 minute collections. Theperiod of stable export is long enough to accom-modate a light-auxin response corresponding to theLGR if the plants are illuminated 30 to 35 minuitesafter atlxin application.

The first experiments with different irradianceswere directed towards the selection of an optimaldose. It was known that 15 minutes of illumina-tion brings about the maximal LGR (5). Bluelight was employed partly to limit temperaturechanges (7) and partly to provide a gross spectralcharacterization of the response. Doses lastingonly 10 minutes were found to elicit the sameresponse as those of 15 minutes, but reduction to2 to 3 minutes led to disappearance of the response.Variation of the test light intensity by a factor of2 with the use of a concave mirror or by movingthe lamp had no discernible effect on the results.

Effect of Light on Auxin Export. In compar-ing individual experiments (fig 4), it is apparentthat blue light induces an oscillation in the rate ofatuxin export.

For quantitative study of the oscillation, it isuiseful that tests and control experiments proceededunder identical conditions for the first half houtr.However, the total export during the first halfhour did vary from experiment to experiment, andit was therefore essential to relate the export rateswhich developed in later time periods with thosefound at the beginning. For this purpose the ex-port rate for each interval of time after supplyingauxin has been plotted against the total amountexported in the first half houlr. Figure 5 showsthat the plot is linear. However, after the tenth

Table II. The Effect of Light on Auxin Export RatesLeft half: experiments receiving a 10 or 15 minute dose of blue light starting 35 minutes after auxin application.

Right half: control experiments receiving no blue light stimulus. All auxin export rates are given as CPM/coleop-tile section/ 5 minutes. Each datum was gained from a single experiment (12 or 16 sections) bv averaging themeasured export rates at the desired time with the rates in neighboring 5-minute intervals.

Blue-treatedExport Rates:

35 Minafter start

Expt (beforeno stimulation)

A. Donor 0.13 mg/l IAA19 2.2720 2.0221 2.2022 2.44

Mean 2.23

B. Donor 0.25 mg/l IAA7 5.248 4.919 2.8110 3.12Li 1.80L2 1.88

Mean 3.29

C. Donor 1.8 mg,/I IAA01 5.8302 3.69

Mean 4.76

55 M inafter start(lhei-ht ofresponse)

2.542.402.202.132.32

5.044.383.363.191.891.913.29

8.005.026.51

Dif fer-ence inexportrates

+0.27+0.38

0.00-0.31

0.09

-0.20-0.53+0.55+0.07+0.09+0.03

0.00

+2.17+1.33+1.75

Exptno

ConrtrolExp,ort Rates:

Dif fer-ence in

35 Min 55 Mmin exportafter start after start rates

25262930

Mean

33363738MlM2

Mean

KlKZ'

Mean

2.152.453.013.292.72

4.812.692.612.391.491.372.56

5.254.064.65

2.712.873.653.483.17

5.663.133.383.421.801.753.19

7.485.506.49

+0.56+0.42+0.64+0.19+0.45

+0.85+0.44+0.77+1.03+0.31+0.38+0.65

+2.23+1.44+ 1.83

2il1

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PLANT PHYSIOLOGY

interval (i.e., the fiftieth mim) the points forilltuminated plants fall on a different line from thatfor the dark controls.

Separate test and conltrol regression linies havebeen (Irawn in figure 5, and(I in each plot thevertical separation of these lines is a quianititativemeasuire of the effect of light on the export rate.The vertical displacements in figuire 5 may also beuised to express the test export rate as percelnt ofcontrol. In this way the light-auxin response hasbeen comptuted for 3 auxin donor concentrations.The resuilts are shown in figuire 6.

The light-autxin response as shown in figure 6has the following featuires: A) a brief dip inaulxinl export occuirs with 10 to 15 minutte latency,a minimum abotut 20 minuttes after the onset ofillumination, arid a following recovery period lead-ing to a peak in the rate at abouit 30 minuites afterthe onset of illutmination; B) the prece(ling effect(leclines in amplituide with increasinig (lonor coni-centrationi an(l is absent at 1.8 mg/l JAA; C) the(lip aind recovery are superimpose(d onl a gradutial

decline in the rate of auxin export relative tocontrols (this is most pronounced at 1.8 mg/l IAA)as a result of this gradual decline, the transient(lip in atuxin export at 0.25 mg/I IAA lies entirelybelow the control export rate.

The reprodutcibility of the effects shown infiguire 6 has been examined in terms of the (if-ference between the export rates 35 an(d 55 miniutesafter the application of auxin, i.e. the pre-stimuluisrate and the rate at the peak of the photoeffect.These resuLlts are shown in table II. Both theindividuial differences an(d the means are consis-tenitly smaller at all donor concentrations in illui-minate(l than in control experiments.

Table II does not permit a valid statistical test,since the data have not been normalized. How-ever, 2 independent tests are possiible, as follows:A) If the average export rate after 35 mintutes iscompuited for each experiment, then the time re-qlire(l to achieve half the average rate can beletermine(l. This half-time falls near 20 minuttes,i.e. duiring the pre-stimuluis period. Sinice test and(l

2S

Intefrval 24

- t

Iuterv.a 2

.0

LIGHT

/ tervol I

I . .

20

12

4

0

el

latM vii 6

00

beorwsl II

nervoI13 I2 te 1al14

0 2 4 6 a ., 0 2 4 * * 10

0/ 2 t4r 615

0 2 4 6 8 10

nt*rvo 16

0 2 4 6 S 10

FIG. 5. Export rates at successive 5-minute time intervals after the application of auxin. Each plot showsthe export rate during the interval studied (Ordinate: CPM/coleoptile section/minute) versus the total export duringthe first half hour of the experiment (abscissa: CPM/section). Solid circles: individual control experiments (noblue light). Open circles: individual experiments receiving 15 minutes of blue light starting 35 minutes after aluxinapplication. Donor concentration; 0.25 mg/I IAA. The regression lines were drawn by eye.

4

25

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TIIORNTON AND THIMANN-LIGHT AND AUXIN TRANSPORT 253Table III. The Effect of Light on the Time (T) Required to Reach Half the Average Post-Stimulus Auxin

Export RateThe T values are in minutes after auxin application, alnd fall in the pre-stimuilus portioni of eaclh experimenit.

DoncControl

Expt Tno (mil) j

25 21.026 23.027 20.028 21.529 22.530 22.031 21.532 24.5

Mean 22.0

ir conlcIn. 0.13 mg/l IAABlue-treated

Deviation Expt Tfrom meani no (min)

1.0-1.0

2.00.5

-0.50.00.5

-2.5

192021222324

21.521.021.519.019.519.5

Deviatioiifrom mean

-1.2-0.7-1.2

1.30.80.8

20.3

Donor concn. 0.25 mg/l IAAControl Blue-treated

Expt T Deviation Expt T Deviationno (min) from mean no (min) from mean

33363738MlM2

20.523.021.021.023.523.5

1.6-0.9

1.11.11.41.4

78910LiL2

22.1

20.019.019.519.022.022.0

-0.31.30.81.31.71.7

20.3

control experiments are influenced by the samevariaables for the first 35 minutes, any consistent(lifference in half-time must 'be ascribed to changesin auxin export after the stimulus. The measuredhalf-times (T) at various auxin donor concentra-tions and light treatments are shown in table III.If the mean T is called D, the individual deviationsfrom the mean calle( d, and the control and bluelight experimental data distinguished by subscriptsc and b respectively, then the t-test for the effectof blue light on T employs the formtulae (10):

Dc - Db d2 ± d2bdFc ± fl 1Sd L nc + fb 2JL2 ncnb j

Where n represents the number of experimentalpoints. For donor concentrations of 0.13 and 0.25mg/I IAA respectively, table III yields t-values of2.40 and 2.25. Both of these signify that the effectof light is significant with a confidence valuebetween 97.5 and 99 % (one-tailed test). The dataat 1.8 mg/i IAA were too few to warrant a test.B) An idea of the magnitude of the photoeffectcan be obtained bv plotting the net post-stimultusexport of auxin (35-80 min after auxin applica-tion) against the net prestimulus export (0-30min). The resuilts are shown in figure 7. As infigure 5, the test and control points fall on separateregression lines. The vertical depression of theline is a measulre of the export depression. The(lepression was 14.9 % at 0.13 mg/l IAA and 17.2 %at 0.25 mg/l IAA. A straightforward statisticaitest of the net (lepression has been made availableby Dr. Carl Morris, and has been presented in(letail in referenice (24). An abbreviated form isincluded here as an appendix. From the formulae,the t-value for the light effect at 0.13 mg/l IAAis -4.12 (one-tailed test, 14 experiments), and at0.25 mg/l IAA is -4.58 (one-tailed test, 11 ex-periments). In both cases values of t from stand-ard tables (10) show that the hypothesis that thelines for the control and illuiminated plants arecoincident mav be rejected with better than 99.5 %

confidence; the depression in the test curve ishighly significant.

Light Grozeth Response. The transient effectof light on auxin export shown in figure 6 hasobvious parallels with the short LGR described by\Vent (26) and van Dillewijn (5). To determinewhether similar growth responses were eliciteduinder the present conditions, both intact plants andplants whose apices had been replaced with 0.25mg/l IAA in agar were stimulated for 10 or 15minutes with the same blue light as that usedl inthe transport experiments.

Figure 8 shows that illumination causes thegrowth rate to utndergo a brief dip, having a mini-mum about 20 minutes after the onset of illumina-tion. Thereafter the rate rapidly recovers andovershoots that of the controls. In intact plants,the overshoot gives way to a second and longerperiocl of subnormal growth, which was not seenin decapitated plants.

The results, shown in figure 8, agree with thoseof previous investigators (5, 11, 12, 26). Sincethe second period of depressed growth in the intactplant is due to a separate LGR system that ispresent only in the apex of the coleoptile, it is notpertinent to the auxin export experiments. In thedata shown here the length of the latent periodlies between 10 and 20 minuites, while Van Dillewijn(5), working at 200, gave the average latent perio(dfor the LGR as 12 minuites. Whether the perio(dof increased growth is stufficient to balance thatof decreased growth is an open quiestion, since theestimation of the control growth rate is not precise.

Tn form and timing, the short LGR is remark-ably similar to the transient dip in aulxin export inexperiments ilsing 0.13 mg/l IAA (fig 6). Thisis also the case at 0.25 mg/l if the graduial declineis taken into consideration. It may be concltudedthat, at low donor concentrations, the short LGRis paralleled by a closely similar light-atuxin re-sponse in ctut sections.

Spectral Origin of the Light-Autxrin Response.

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PLANT PHYSIOLOGY

0.13 MG /LIAA

A.

20 40 60 80

100 L --___ _

90. 0.25 MG/L

a', IAA

o00I. 90-zO ,le

40

8. 220 40 60 80

_------- 20

49

1.8 MG/L if

IAA I-10a

LIGHTC.20 40 60 80

MINUTES AFTER AUXIN APPLICATION0 20 40 g0 so

MINUTES AFTER FIRST READING

80 _

* CONTROLo BLUE-TREATED

14.9 %

I I

84

0.25 MG/L IAA

17.2 %

14 8

1.8 MG/L IAA

-7

12.6 %

I I

4

PRE-STIMULUS TRANSPORT, CPM/SECTIONFIG. 6. (upper left). The effect of blue light (bar on abscissa) on the auxin export rate at three auxin donor

concentrations. The export rates are expressed as percent of the rate in non-stimulated control experiments.FIG. 7. (bottom) The effect of blue light on the total auxin exported between the 30th and 80th minutes after

auxin application (post-stimulus transport). The data are plotted against net pre-stimulus transport (the total amountexported up to the 30th minute after auxin application). Each point is a single experiment. Solid circles, control; opencircles, light-treated exper,iments. Light dosage and timing as in figure 6. On each plot is shown the auxin donorconrcentration and the percent reduction in post-stimulus export due to light treatment.

100

90

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THORNTON AND THIMANN-LIGHT AND AUXIN TRANSPORT

In these experiments the red working light shouldhave saturated the red-light response system of thecoleoptile (2). The blue test lamp was chosenprimarily to limit possible heating effects fromlong-wavelength radiation during the test exposure.No difference in either time course or total auxinexport of the controls was detected whether or notthey were given weak far-red light in addition tothe red working light. Furthermore, the light-auxin response was not duplicated by a transientincrease in the red light intensity. Thus while thelight sources were not chosen to provide an actionspectrum, it is evident that the light-auxin responseis elicited by the blue end of the spectrum.

Discussion

Short-Termiii Auxin Export: Genertal. Thepresent experiments show a higher initial auxintransport velocity than has been derived from linearextrapolations. They also show that an equilibra-tion process is involved in establishing the plateauexport rate (fig 5). Here the export rates fromthe control experiments, when plotted against thenet export during the first half hour, fall on a linepassing through the origin. In contrast, the re-gression lines do not pass through the origin attimes later than about 35 minutes after the appli-cation of auxin. A downward shift of each exportplot would cause it to pass through the origin andwould leave it with the same slope as in the plotat 30 minutes, which already passes through theorigin. Thus the sections export auxin better after35 minutes than their performance during the firsthalf hour would have led one to expect. It maybe proposed that auxin was being shared betweenthe transport stream and another system during thefirst half hour, and that this sharing was completeby 35 minutes so that more auxin became availablefor export. The intercepts of control plots infiguire 5 do not change appreciably between 35 and80 minutes after aulxin application, suggesting thatthe auxin relations in the sections do not deterioratefor the first 80 minutes.

Poole and Thimann (18) also fouind that theuptake and release of auxin by coleoptile sectionsinvolve a fast and a slow component: the fastprocess is over in 30 minutes, agreeing with thededuction above that an equilibration process occursin the early stages of auxin transport. It is pos-sible that this process is related to the loading ofthe transport stream with labelled atuxin.

Effect of Light oni, Auxin Export. The presentdata show that strong doses of blue light tem-porarily reduce both the auxin export from coleop-tile sections and the growth rate of the coleoptile.The timing of the cardinal points is the same inthe growth and auxin responses.

However, a remark on the technique of meas-urement is in order. The growth response takesplace everywhere at once and is measured in sumat the apex of the coleoptile. On the other hand,the auxin response is measured only through theevents taking place at the basal cut surface, ex-cluding the contributions from upper regions whichrequire a finite transit time t,o reach the base.Unfortunately, kinetic analyses are difficult sincethe auxin-flow system is not in a steady state. Thesimplest interpretation is that the auxin relationsin the regions far from the basal cut surface donot contribute to the measured light-auxin response.This is not to say that no response takes placeabove the cuit surface, since the LGR is propor-tional to the length of coleoptile stimulate(d (26).But if all the zones respond with the same timerelations and the same percentage change in auxinoutput, then no wave of abnormal auxin transportshould occur, to be propagated down the coleoptile.Thus pending quantitative treatment, the similaritybetween the light-auxin and light-growth responsessuggests that the relative sensitivity to light is thesame all along the part of the coleoptile understudy.

Earlier attempts to find light-auxini responseshave met with varying results. Oppenoorth (13)made a number of studies on extractable auxin,but the role of extractable auxin in growth isdoubtfu1l (9, 28, 29).

Examining by bioassay the auxini exported inthe first half hour after exposure to strong light,Stewart and Went (20) failed to detect any effectof light on export. During this half hour only thenegative phase of the short LGR should have beenregistered had the plants been growing. Theseexperiments differ from ours in lacking an auxindonor during the collection period. Their negativefindings, therefore, suggest that an apical auixinsupply is essential to the appearance of a light-auxin response.

DuBuy (7) provided his coleoptile sections withauxin and divided the collection periodl into inter-vals so that comparisons like ours in table II couldbe made. In this way a relative depression ofsome 13 % was noted in the interval 30 to 75 min-utes after the onset of a 45-minute exposure to

FIG. 8. (upper right). The light growth response following exposure to blue light in growing coleoptiles. Thegrowth rate (microns/minute) is plotted against time (minutes). The dashed lines indicate the presumed course ofthe grow-th rate in the absence of light stimulation. The timing of the light dose is shown on each plot. A) averageresponse of 7 intact plants. B) average responses of 6 intact plants and 6 plants decapitated and supplied w-ith 0.25mg/l IAA in agar blocks.

?j5

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PLANT PHYSIOLOGY

strong blue light. DuBuy noted that the effectwould have been obscured by the experimentalvariation if he had employed only 1 long collectioninterval. The same would have been true in ourexperiments, as may be seen by averaging the datain figure 7.

Using labelled auxin, Pickard and Thimann (16)found that continuous illumination of corn coleop-tiles led to no significant change in net auxinexport over a 3-hour period as compared to darkcontrols. While this result excluded auxin decar-boxylation, the experiments were not designed toshow brief transient changes, and particularly notchanges involving a compensation, in analogy tothe LGR. The amplitude of the LGR in continuouslight (5) is such that the net reduction in auxinexport over a 3-hour period should be no morethan 2 % (24), far too smnall a change to be de-tectable.

The gradual export decline after illuminationis more difficult to interpret than is the transientmodification. It was mentioned above that ex-cision seems to induce the formation of an auxindestruction system at the basal cut surface incoleoptile sections, and that the speed at which thissystem develops depends on the auxin concentra-tion (fig 3). There was some early evidence,based on bioassay, that light promotes the develop-ment of the non-photolytic auxin degradationsystem in cut seetions (14), allowing auxin-freecoleoptile sections to increase the destruction ofauxin in agar blocks. The gradtual decline in ex-port after illumination appears to support thisearly work.

The transient light-auxin response is similarenough to the LGR to suggest that both have thesame cause. Two major possibilities are open;either light may directly inhibit the polar flow ofauxin (19), or light may stimulate the active up-take of auxin into lateral channels. In both casesa growth response should be recorded. The agentdirectly responsible for these changes might be,for instance, a transport enzyme. The continuedinpuit of auixin from the donor, or the apex, willcause the auxin to pile tip in the affected area,uintil its concentration becomes high enough to re-store the original flow rate. On retutrning todarkness the transport enzyme wouild recover andthe stored auxin be released. This mechanismaccouints for the growth overshoot in the LGR,sometimes known as the dark growth response.

A choice between these 2 proposals is not pos-sible on present information. If the polar flowwere directly inhibited, then unilateral illtuminationshould lead to transient phototropic movementsduring the LGR without implicating lateral trans-port of auxin. The extended lateral transport thatwould be required for long-term phototropism (16)would then presumably be provided either by anentirely separate mechanism or (less probably) bylateral auxin diffusion.

On the other hand, the action of light in photo-tropism favors the proposal that light would pro-mote the movement of auxin into lateral pools.It is well established that lateral auxin transportoccurs only with difficulty in coleoptiles that havenot been tropistically stimulated (28), and is pro-moted by phototropic and geotropic stimuli (16).It is evident that lateral transport proceeds by anactive mechanism analogous to the polar transportsystem, so that the establishment of long-termlateral transport would involve a transient periodof equilibration similar to that which is encounteredlin the early stages of movement of labelled auxinin the polar stream. Thus the LGR would be dueto a temporary imibalance between uptake of auxininto lateral channels and leakage back into thepolar stream. Cholodny's observation (4) that avery gradual increase in unilateral light intensityleads to phototropism without an LGR wouild thenbe expected.

To summarize, the short LGR is shown to beaccompanied by a light-auxin response of verysimilar form, together with a super-imposed declinein auxin output for which there is no knowncounterpart in growth. The variability in the re-sponse makes necessary a system of data normal-ization before statistical tests can be meaningfullyapplied, and the failure to provide such normal-ization accounts in large part for the previouscontradictory results in the literature.

There is at present no indication that the light-auxin response precedes the LGR or that it causesthe phototropic response in the case of unilateralillulmination. The most attractive possirbility so faris that light promotes an active transfer of auxinfrom the polar stream into lateral pools comprisingthe pathway for lateral transport in phototropism.With this expl-anation the LGR is viewed as aside-effect accountable as an equilibration phenom-enon, and both the LGR and phototropism may beinterpreted compatibly as component responses ofa single photosensitive system.

Statistical Test of the Effect of Light on NetPost-Stimulus Auxin Export. The following tech-nique was devised by Dr. Carl Morris, Universityof California, Santa Cruz, California.

Each control data point may be described hy 2measuires: the net post-stimuluis export z and thenet pre-stimtluis export x". The correspondingmeasuires for each test data point are y and x'.The mean values for x, y and z are denoted bysuiperscript bars. For a given auxin donor con-centration, the pooled variance s and the weightedregression parameter b are defined by the followingformulae:

Ab =

Sr"2 (n"c- 1)bz

sS"2 (nc-1) + S.t'2 (nb-1)(nb-2) S.,2 + (nc-2) S,..2

s =

nb + nc -4

256-

S.V2 (nb-1 ) by,

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THORNTON AND THIMANN-LI(

in which Ulb anid are, respectively, the numberof test and control data points; subscripts b anid crefer to test and control data respectively, and theremaininig variables are defined as in Goldstein(10).

The initial hypothesis in the present work was

that light, which temporarily decreases the growthrate, should also temporarily decrease the rate ofauixin export and therefore should lead to a netexport redluction. It is appropriate with this rea-

soning to apply a 1-tailed t-test in determining thesignificance of the observed export reduiction. Thetest hypothesis is that the regression lines for thetest and the control are concurrent, and a t-valutemore negative than 3.499 (for 7 degrees of free-(loni) or 3.169 (for 10 degrees of freedom) wouldsigniify that the test hypothesis must be rejecte(daniid that the export in blule-treated experiments hasileen redluced below the valute in conitrol experi-ments. At these t-values this con1cltusion1 mav be

drawnvwitlh 99.5 % conifidence.In the preselnt work, the formuila for t is:

t = (l/s) [vtA

(,1r' ")I 1tb + II

Acknowledgment

WVe are indebted to Dr. Carl Morris for providingthe statistical test.

Literature Cited

1. i\Rusz, WV. H. 1915. Untersuchunig jiher (len Plho-totrol)ismlltis. Rec. Trav. Bot. Neerl. 12: 44-216.

2. BRdIGGS, WV. R. 193. Red light, auxin relation-slhips, anid the phototropic responses of corn andoat coleoptiles. Am. J. Botany 50: 196-207.

3. BRIGGS, W. R. 1964. Plhototropism in higherplants. In: Photobiology. A. C. Giese, ed. Aca-demic Press, New York. p 223-71.

4. CHOLODNY, N. 1933. Beitrage zur Kritik derBlaauwschen Theorie des Phototropismus. Planta20: 549-76.

5. DILLEWIJN, C. VAN. 1927. Die Lichtwachstums-reaktionlen von Avena. Rec. Trav. Bot. Neerl.24: 307-582.

6. DOLK, H. E. 1936. Geotropism and the GrowthSubstance. Rec. Trav. Bot. Neerl. 33: 509-85.

7. DuBuy, H. G. 1933. Vber Wachstum und Photo-tropismus von Avena sativa. Rec. Trav. Bot. Neerl.XXX: 801-921.

8. GOLDSMITH, M. H. 1959. Clharacteristics of thetranslocation of indoleacetic acid in the coleoptileof Avena. Ph.D. Thesis, Harvard University.

9. GOLDSMITH, M. H. AND K. V. THIMANN. 1962.Some characteristics of movement of indoleaceticacid in coleoptiles of Avena. I. Uptake, destruc-tion, immobilization, and distribution of IAA dur-ing basipetal translocation. Plant Physiol. 37:492-505.

GHT AND AUXIN TRANSPORTr 257

10. GOLDSTEIN, A. 1964. "Biostatistics". Mac-MillanCo., New York.

11. KONINGSBERGER, V. J. 1922. Tropismus und Wachl-stum. Rec. Trav. Bot. Neerl. 19: 1-137.

12. KONINGSBERGER, V. J. 1923. Lichtintenisitiit unidLichtempfiindlichkeit. Rec. Trav. Bot. Neerl. 20:257-312.

13. OPPENOORTH, W. 1941. On tlle role of aulxin inphototropism and light growth reactions of Avenacoleoptiles. Rec. Trav. Bot. Neerl. 38: 287-372.

14. OVERBEEK, J. VAN. 1936. Light growth responiseand auxin curvatures of Avena. Proc. Nat]. Acad.Sci. 22: 421-25.

15. PICKARD, B. G. 1963. The role of inidole aceticacid in the tropisms of coleoptiles. Ph.D. Tlhcsis,Harvard University.

16. PICKARD, B. G. AND K. V. THIMANN. 1964. Traiis-port and distribution of auxin dulrinig tropisticresponses II. The lateral migratioln of autxini illphlototropism of coleoptiles. Plant Physiol. 39:341-50.

17. PILET, P. E. 1965. Actioni of gibberellic acid oliauxini transport. Nature 208: 1344- 45.

18. POOLE, R. AND K. V. THIMANN. 1964. Uptaklzeof indole-3-acetic acid and indole-3-acetonitrile bvAvena coleoptile sections. Plant Physiol. 39: 98-103.

19. SHEN-MIILLER, J. AND S. A. GORDON. 1966. Hor-monal relations in the phototropic response: III.The movement of 14C-labelled and endogenouisindoleacetic acid in phototropically stimulated Zcacoleoptiles. Plant Phvsiol. 41: 59-65.

20. STEWART, W. AND F. W. WENT. 1940. Liglhtstability of auxin in Avcna coleoptiles. Botani.Gaz. 101: 706-13.

21. STOWFE, B. B. 1963. Syn11thiesis of Highi SpecificActivity 14C_Carbox\ 1 Indoleacetic Acid anid of14C-Nitrile Indoleacetoniitrile. AAnal. Bioclhem. 5:107-15.

22. THIMANN, K. VT. 1960. Plan1t Growthi. In: FUn1-damental Aspects of Normal aniid MalignanltGrows th. WV. Nowiniski, ed. Elsevier, Amsterdamii.p 7484822.

23. THIMANN, K. V. AND G. M. CURRY. 1960. Pho-totropism and Phototaxis. Comp. Bioclhem. 1:243-309.

24. THORNTON, R. M. 1966. A Comparative stu1dy ofphototropism in Phycomyces and Avena. Ph.D.Thesis, Harvard University.

25. WEIJ, H. G. VAN DER. 1932. Der Mechanismu-ilsdes Wuchsstoff transportes. Rec. Trav. Bot. Neerl.29: 379-496.

26. WENT, F. WV. 1925. Concerning the difference insensibility of the tip and base of Avena to light.Koninkl. Ned. Akad. Wetenschap. Proc. 29: 1-7.

27. WENT, F. W. 1925. Die Erkliirung des photo-tropischen Kriummungsverlauf. Rec. Trav. Bot.N6erl. 25a: 484-89.

28. WENT, F. W. AND K. V. THIMANN. 1937. Phy5-tohormones. Macmillan and Co., New York.

29. WINTER, A. AND K. V. THIMANN. 1966. Boundindoleacetic acid in Avena coleoptiles. PlantPhysiol. 41: 335-42.

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