andl - plant physiology · proximal to the incident light andl those distal to it. similarly, the...

ACTION AND TRANSMISSION SPECTRA OF PHYCOMYCES 1,M. DELBRtCK AND W. SHROPSHIRE, JR.3CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA

In this paper measurements are presented of actionspectra for the photoresponses of sporangiophores ofPhycomyces an(d of the transmission spectra of thegrowing zone of single sporangiophores in stage IVb (7) and of the cell wall. Compared to other organ-isms, Phyconmyces offers several definite advantagesfor such measurements. There are two distinctphotoresponses, the growvth response and the tropicresponse, and there are many indications, particularlythe action spectra presented here, that these two re-sponses are mediated by the same pigment. Thegrowth response occurs under conditions of symmetricirradiation when the intensity varies with time. andthe tropic response occurs with an irradiation pro-gram which may be constant in time, but asymmetricwith respect to azimuth angle, i.e., the angle aroundthe axis of the sporangiophore. Crudely speaking.the growth response measures the azimuthal summa-tion of photoeffects, while the tropic response measuresthe azimuthal asymmetry. The combination of thesetwo measurements permits an estimate of the influ-ence of screening pigments lying betwzeen the receptorsproximal to the incident light andl those distal to it.Similarly, the measurement of transmission spectraof the intact sporangiophore and of the cell wall per-mits some conclusions with respect to the absorbingmaterials present in various locations.

Another advantage of Phycomyces is that the op-tics of the system are relatively simple. The sensitiveregion closely approximates a cylindrical lens withuniform refractive index. The importance of the di-optric properties of this lens for the tropic effect wasclearly demonstrated 40 years ago by Buder (2), whoshowed that immersion of the specimens in a mediumwith refractive index higher than that of the cellcontents reversed the sign of the tropic response.Thus, the dioptric properties, both refractive and re-flective, and not scattering or absorption have the de-cisive influence in producing the azimuthal asymmetryin the sporangiophore.

Two years ago, Curry and Gruen (10) discoveredthat the tropic sensitivity of the sporangiophores ex-tends deep into the ultraviolet region and that thetropic response reverses sign aroundl 300 mu. Thereversal has a simple explanation. There are screen-ing substances in the growing zone which, for wave-

Receive( June 22, 1959.2 Research supported by fundls provided by the National

Science Founidation Grant G-2847.Present address, Smithsonian Institution, Washing-

ton, D. C.

lengths shorter than 300 mA, effectively shield thedistal side of the sporangiophore from the incidentlight. For these short wavelengths, then, the spor-angiophore can be stimulated truly unilaterally, incontrast to the visible region, where both sidles arestimulated, the distal more than the proximal wlhenthe sporangiophore is irradiated unilaterally.

METHODS

CONCEPT OF "VISUAL" PIGMENT Any responseof an organism to light may be expected to involveat least one substance which undergoes a plioto-chemical reaction which ultimately, through a mloreor less complicated chain of events, leads to the ob-servable response. This substance will be referredto here as the "visual" pigment. The general purposeof making measurements of action spectra is to obtaininformation regarding the absorption spectrum of thisvisual pigmzent. Howvever, the action spectrum ofthe effect may differ from the action spectrum of thepigment because of the intervention of screening pig-ments or of self screening. These difficulties will bediscussed after presenting the data. The action spec-trum of the pigment is usually equated with its absorp-tion spectrum. This equation is not necessarily valid,since the quantum yield may vary with wxavelength.

EQTUAL RESPONSE VERSUS EQUAL FLUX ACTIONSPECTRA Visual pigment absorbs different wave-lengths witlh different absorption coefficients. Thisvariability may be exhibited either as a (lifference inthe response at various wavelengths under conditionsof equal energy or quantum flux, or as a difference influx needed to produce equal responses. Both meth-ods have been used by various authors and both arelikely to disclose at least the gross features of the ab-sorption spectrum. The equal response method, hoN-ever, seems preferable for quantitative work becauseit does not involve the uncertainty of translating (lif-ferences in response into differences in amount ofthe primary photochemical product. The measure-ments presented here are based entirely upon the prin-ciple of equal response.

NULL VERSUS FINITE RESPONSE The next ques-tion concerns the choice of size of the "equal response".If there were no perturbations from side effects, suchas screening, any size response should give the sameaction spectrum. The perturbations show up in adifferent dependence of the response on the flux atdifferent wavelengths. A clear example of this maybe found in the work of Shropshire and WithroT (16)

194

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

DELBRUCK AND SHROPSHIRE, JR.-ACTION AND TRANSMISSION SPECTRA

on the action spectrum of phototropic tip-curvature ofAvena. Such perturbations can be interpretedproperly only if a great deal of information is avail-able besides the action spectrum. Quite aside fromthese perturbations, the question of the amount ofresponse which should be chosen to obtain the mostaccurate measurements still remains. One conditionis that the response should vary as strongly as possiblewith the flux. Another condition, well known inphysical measurements, is that the response shouldvary symmetrically with the flux. This condition isbest realized if the response is a null response, varyingsymmetrically between negative and positive valuesfor small deviations from the null. Such a methodeliminates perturbations arising from variability insensitivity between specimens.

GROWTH RESPONSE NULL DETER-MINATIONS Forgrowth response the condition of null response wasrealized by irradiating the specimen during alternateequal intervals of 5 minutes from a standard lightsource and a monochromatic source. If these twosources appear equally bright to the specimen, therewill be no growth response. This is the null condi-tion. If the standard light appears brighter than thetest light, there will be a periodic growth responsewhose maximum occurs about 7.5 minutes after thestandlard light is turned on; i.e., in the middle of thetest light period. If the test light appears brighter,there also will be a periodic growth response, but witha phase shift of 1800.

The symmetry of the irra(liation was assured byrotating the specimen continuously at 2 rpm. Thearrangement for doing this while maintaining accuratepositioning has been described previously (9). Thespeed of 2 rpm is sufficiently fast to eliminate tropiceffects and sufficiently slow to avoid mechanical prob-lems. Figure 1 illustrates the irradiation programand the actual responses from minute to minute undervarious conditions of relative size of the test and stand-ard stimulus. The method by which the flux pro-ducing the null response was interpolated from a seriesof such measurements is illustrated in figure 2 andexplained in the figure legend.

TROPIC NULL DETERMINATIONS For the tropicresponse the null method is more obvious and hasbeen used by previous authors4 (5). The standardlight impinges from one side and is matched againstthe monochromatic test light. The monochromaticlight impinges from the other side, if test and standardproduce tropic effects of the same sign, and from thesame side, if they produce tropic effects of oppositesign. It is important that the two beams of light donot come in at an angle of 1800 from each other, butat a lesser angle. If the angle is 1800 and the sourcesare of equal effectiveness, the specimens are indiffer-ent phototropically; i.e., they are at equilibrium inwhichever direction they grow (11). Another condi-

4 See addendum

tion, only imperfectly realized in these experiments,is that the irradiation ought to be symmetric withrespect to the vertical in order to eliminate perturba-tions from geotropism (12). Arrangement of thelight sources is shown diagrammatically in figure 3.The equilibrium intensity of the test beam was dle-termined by changing the intensity until the equilibri-um position was obtained and maintained for 3 hoursunder continuous irradiation. Variations in the di-rection of growth of a single specimen during constantirradiation and variations between specimens amountto ± 100. This variation sets a limit to the accuracyin a single determination of the matching quantumflux of + 10 %. The matching quantum flux was(letermined for one to three sporangiophores at eachwavelength. The sensitive zone of each sporangio-phore was continuously centered to within ± 2 mmin all directions from the center of the field to eliminatethe effect of small intensity gradients in the fiel(l.

EQuIPMENT FOR STIAMULATION The standardstimulus was provided by a tungsten lamp with aCorning heat filter [1-69] and a Corning blue filter[5-61] arranged in tandem. Arrangement of thelight source, stage, microscope, etc., as well as theintensity scale, have been described in detail previously(11). For most of the measurements the intensityof this irradiation was I = -8. Unilateral I = -8corresponds in effectiveness to a monochromatic fluxat 440 my of 0.022 Mv/cm2.

The test stimulus was provided by a uniform field(1.5 X 2.0 cm) of monochromatic flux obtained 30cm from the exit slit of a Beckman DU spectrophoto-meter. Only the light sources and the dispersingelements of this instrument were used. The tungstenlamp was used for wavelengths longer than 330 mAand the hydrogen lamp for shorter wavelengths. Theintensity of the monochromatic flux is proportionalto the square of the slit width (since entrance and exitslit vary in parallel) and this intensity control, afterverifying its validity, was used in preference to neutralfilters. The rncaximumn half-intensity bandwidth, ascalculated from Beckman instrument data, was 5 mA.In most cases it was considerably smaller. The wave-length calibration was checked by isolating the brightlines from a medium pressure mercury arc source.The test light impinged horizontally from one sideonly, while the standard light impinged 60° from thevertical, from one or both sides.

CALIBRATION OF ENERGY FLUX The plate cur-rent, suitably amplified, of a calibrated RCA 935phototube was used to measure the monochromaticflux. The sensitivity of the phototube was determinedfrom 240 to 405 m/n by the Experimental Tube Divisionof RCA, using the strong mercury lines. For longerwavelengths tlhe tube was calibrated by comparisonwith a Kipp thermopile and Leeds and Northrup gal-vanometer system, which in turn had been calibratedagainst a secondary irradiation standard. From thesensitivity data the quantum fluxes for a 0.10 mm slitwere tabulated at 5 my intervals.

195


PLANT PHYSIOLOGY

TEST*

TIME (min)

CO.E

fIoz3: 8

co1

6 -z

O 2TES

'I~-

0

0:

TEST330 M."

TIME (min)

TIME (min)

(b)Log SLIT WIDTH (a)

OcO4 126

x 10'-I

0

08

I

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+10

0

-10

TESTX ' 330mMu

(

196

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DELBRUCK AND SHROPSHTRE, JR.-ACTION AND TRANSMISSION SPECTRA

TRANSMISSION SPECTRA Mounting specimensAfter determining the action spectra, which showedpronounced peaks, an attempt was made to detect thevisual pigment directly by the absorption of lightduring its passage across the sporangiophore. Thedioptric properties of the specimens in air make itdifficult to arrange a suitable geometry of the lightpaths in the optical system. These difficulties are-much reduced by immersing the specimens in a mediumof matching refractive index which is also transparentand non-toxic for the specimen. Pure fluorochemicalFC-43, perfluorotributylamine, (Minnesota Miningand MIanufacturing Co.) closely approximates theseconditions. Specimens completely submerged in itgrow at a normal rate for many hours and give normalgrowth responses. Its refractive index is 1.29 whichis close to that of the cell contents (1.34 from ourmeasurements).

It is convenient to work with sporangiophoreswxvhich have been severed from their mlycelium in orderto mount them on a photographic plate or a microscopestage. Such specimens retain their turgor for manyhours but do not grow when mounted in air or FC-43.Gruen (15) discovered that isolated sporangiophoresgrowN and respond normally for many hours whentheir base is immersed in water. We have foundthat when the growing zone is in contact with water,growth ceases quickly and turgor is lost. Thus, forthe present purposes. it was desirable to have speci-mens with their feet in water and their tops in FC-43.MIicrochambers (figure 6a) were constructed withtw-o partitions, separated by silicone grease. An iso-lated sporangioplhore is placed with the top half inone partition, where it is immersed in FC-43. and thelower half in the other partition, where it is immersedin water. Such specimens grow at a normal rate of3 nmm 'hr with little or no lag (lue to shock of immer-

sion, give normal growth responses, and can be ob-served continuously under a high power microscope.This arrangement permits a check whether or notthe transmission of non-growing isolated specimensdiffers from that of growing specimens. No differen-ces were found. Thus, the non-growing specimensgive a valid comparison picture to the action spectra.which naturally require growing specimens.

Numerous methods for the transmission measure-ments were tried and the two relatively most success-ful ones are presented. For both mlethods the speci-mens were immersed in FC-43.

SHADOWGRAPH METHOD The specimen is placeddirectly on the emulsion of a photographic plate andirradiated from above with nearly parallel monochro-matic light. The shadowgraph thus produced is eval-uated densitometrically. Since the specimens are only0.1 mm in diameter and the plates have to be scannedacross the specimen, this method requires fine grainemulsion and high resolution- densitometry. Themethod is laborious in requiring a new specimen foreach wavelength and densitometric calibrations foreach wavelength setting. It is particularly useful inthe ultraviolet since no optics are involved in takingthe shadow graphs; the method is informative withregard to the amount of light scattered by the cellcontents under various angles.

Turgid, isolated sporangiophores were placed onKodak medium lantern slide plates, imbedded in FC-43, covered with quartz coverslips supported by 0.5mm spacers, and exposed to monochromatic flux.FC-43 can be left in contact with a photographicemulsion for long periods without affecting it. Ex-posures were such that the half-intensity bandwi(dthswere 5 my or less and the background blackening wasat the top of the linear range for a plot of optical den-

FIG. 1 (upper 2 rowcs). Growth response, null method. A. Directions of irradiation. The specimen is ro-tated at 2 rpm to make the irradiation azimuthally symmetric. B, C, and D. Growth speeds during successive1 min intervals. Above each response curve the irradiation program is indicated. The points in the responsecurves represent averages over three successive cycles. Average values during the first half of the cycle are re-peated to facilitate the drawing of a smooth curve through the points. In B, with the slit of the monochromatorat 0.07 mm, the test light is weaker than the standard light. The response curve has a minimum near the middleof the standard irradiation. In C, with the slit of the monochromator at 0.14 mm, the test light is stronger thanthe standard light. The maximum of the response occurs near the middle of the standard irradiation. In D, witha slit of 0.10 mm, the two irradiations are nearly matched. To evaluate the response, the total elongations dur-ing each of three standard (S) and three test (T) periods were determined. The difference between these twoquantities, S-T, is negative in B, positive in C, and ideally zero at the growth response null. This condition is ap-proximated in D. The horizontal lines are the average growth speeds during standard and test periods.

FIG. 2 (louwer left). Interpolation method for determining the growth response null. The quantity S-T, obtainedas explained in figure 1, is plotted versus the logarithm of the slit width. From the total of all measurements atall wavelengths it was found that S-T near the null varies linearly with the logarithm of the slit width, with a slope3. Assuming this relation, every experimental point in this graph may be used to estimate a slit width giving nullresponse. These estimates were averaged for five to ten sporangiophores at each wavelength to estimate the match-ing quantum flux.

FIG. 3 (lowcer right). Arrangement of the light sources for determining the tropic null. The dotted line indicatesthe direction of equilibrium. Due to geotropism, this direction is slightly closer to the vertical than the bisector ofthe two directio'ns of growth produced by each of the sources separately.

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PLANT PHYSIOLOGY

FIG. 4. Shadowgraph method for determining trans-mission spectra. The specimen, immersed in FC-43, wasplaced directly on the emulsion of the photographic plateand irradiated with a parallel beam of monochromaticlight. The shadowgraph was obtained at 380 mA and isenlarged 45 x. Light bands at the margins of the spor-angiophore are due to the residual difference between therefractive indices of the medium (1.29) and the cellcontents (1.34). The residual lens effect produces anincreased intensity in the center of the shadowgraph cal-culated to be 14

sity (0. D.) versus the logarithm of exposure. Ex-posures were varied by changing exposure times atconstant intensity. The gamma (y) of the lanternslides and suitable exposure times wiere determinedfor the range from 240 to 540 m,, in 10 iniy steps overmost of the range ancl 5 mny steps near the maxima,minima, and inflection points of the action spectra.The plates were scanned 1 to 2 nim below the spor-angiumll with a micr-odensitolmieter capable of meas-uring fiells 1.2 X 25A (figs 4 an(d 5). The same in-strument was use(d to (letermine the y of the plates.

Below 300 mA so little light is transmitted (dueto absorption) that background and(l specimen cannotbe compared in a single photograph. For these pointslonger exposures were made until the transmittedlight gave appreciable blackening and the O.D. wasestimated by comparing the exposure times used withthose needed to produce similar background blacken-ing. At very high O.D.'s this procedure is subjectto errors from scattering, so that only lower limits ofthe O.D. can be estimatedl.

DIRECT METHOD In the second miietlhod the totaltransmitte(l lighlt is measured directly. A 2 mm sec-tion of the exit slit of the monochromator is imagedwith a 5 in focal length lens on the growing specimenand is carefully aligned parallel to the specimen axis.A slit width is chosen such that the image of the slitis narrow compared to the sporangiophore dliameter.The light paths are therefore collimated to nearlythe same degree as in the shadowgraph method. Thetotal transmitted light (the undeviated and a largeproportion of the scattered light) is collected by a mi-croscope objective andl nmeasured by a photometerwlhose sensitive element intercepts the image of theobserved section of the specimen. This methodl isconvenient since the entire wavelength range can bescanne(l within a few minutes. It is also mlore ac-curate than the shadlowgraph method since it does notrequire elaborate calibrations at each wavelength andavoids interspecimen variability. This gain is offset,however, by extreme requirements in alignment, fo-cusing, positioning andl by chromiiatic aberrations inthe optics. These factors introduce errors wlliclhseem to be particularly bad in the near-ultraviolet.The method could not be used at all below 400 miy.

The specimen, contained in a microclhamiiber onthe mechanical stage of a Reichert microscope, wasaligned using the regular niiicroscope optics (fig 6b).The eyepiece and top part of the tube were then re-moved and the inmage produced by a 45x objectiveprojected via a totally reflecting prism onto the sensi-tive surface of a 1P21 photomultiplier tube of aln E1-dorado photometer. The specimen was moved inand out of the beam by fine motions of the stage coln-trolled by reproducible front and back stops. At eaclhwavelength setting the background was adjusted tozero by varying the fine sensitivity control of theplhotometer and the O.D. of the specinmen in the grow-ing zone alone or belowv the growing zone was dleter-mined directly by mov-ing it into the beamii. Uncler-these conclitions anyV light tranismliitte(d (lirectl oirscattered within the specimeln into a forwar(d anigleless than about 450 was interceptedl by the objectivelens (N.A. - 0.65) and projecte(l onto the sensitivesurface.

RESULTSGROWTII RESPONSES AND ADAPTATION AT DIFFER-

ENT WAVELENGTHS The question of w,vhether or notthe specimens exhibited any qualitative differencesin their responses to different wavelengths, aside fronithe reversal of phototropism. was examiiined first.Figure 7 shows that the growth responses elicited bystimuli of different wavelengths are exactly conm-parable, even (leep in the ultraviolet where the stim-ulating light penetrates only a short (listance into thespecimen. Furthermore the ultraviolet is comparableto the visible in its adapting effects. As is wellknown (11), growth responses (lependl upon the ratioof the stimulus size to the level of adaptation, andthe latter, at equilibrium, is proportional to the inteni-sity of the adapting light. Light of any wavaelengtht

198


DELBRUCK AND SHROPSHIRE, JR.-ACTION AND TRANSMIISSION SPECTRA

was found to be interchangeable for either stimulusor adaptation, providing the intensity was adjustedby the calibration factor corresponding to the growthresponse action spectrum.

TROPIC RESPONSES AT DIFFERENT \VAVELENGTHSIn contrast to the growth response there are grossqualitative changes in the tropic response at differentwavelengths. The most obvious of these is the rever-sal of the sign of the tropic response below 300 my.

In the visible a short unilateral stimulus of moder-ate size produces a tropic response for which thetime of onset is very nearly the same as that of thegrowth response (3). A strong unilateral stimulusproduces at best a feeble and somewhat delayed tropicresponse. This phenomenon was discovered by Castle(4) and named phototropic indifference. Castle in-terpreted it by assuming that a strong stimulus givesa saturated growth response on both the proximaland the distal side and, therefore, no differential be-tween the two sides.

Below 300 mA, the distal side is shielded. Underthese conditions any stimulus capable of producing agrowth response should also give a tropic response.This is indeed the case. Below 300 my a strong, shortunilateral stimulus gives a strong, transient, negativetropic response. The response begins around 4minutes after the stimulus and results in a maximumtilt of 70° or more. The maximum rate of tilting isabout 80/min and occurs at 5 to 7 minutes after thestimulus. The- miaximnum tilt occurs at 11 to 15 min-utes and the tilt is back to zero at about 30 minutes.These are precisely the characteristics expected for astrictly unilateral growth response.

Similarly, continued unilateral stimulation at 280mA gives a continued negative tropic response, witha maximum rate of tilting greater than 20°/min.

For specimens immersedl in FC-43 the match ofrefractive indices is so close that no nhototropic re-snonse occurs even after prolonged unilateral irradia-tion with blue light. Irradiation with wavelengthsshorter than 300 mu, however, produces a strong neZa-tive tropic reaction. This is to be expected, since thespecimen is stimulated exclusively on the side proximalto the impinging light.

GROWTH RESPONSE AND TROPIC RESPONSE ACTIONSPECTRA Figure 8 shows the results of these meas-urements, obtained by the null method described above.The matching intensity was the same (or a smallmultiple) in all cases, I = -8; i.e., close to the lowerend of the "normal range" (11). For each wave-length measured, the ordinate gives the reciprocalquantum flux which matches the standard light. Forwavelengths below 300 my the matching reciprocalquantum fluxes for the tropic responses are enteredas negative values to indicate that the test light im-pinged from the same side as the standard light.

TRANSMISSION SPECTROSCOPY OF GROWING ZONEAs pointed out above, the shadowgraph method wasthe only one available below 400 mA. This method

was also used for longer wavelengths but was lessprecise than the direct method. The precision for theshadowgraph method was ± 0.1 O.D. unit, while forthe direct method the values were reproducible for agiven specimen to within 0.01 O.D. unit. In figure9 the measurements in the ultraviolet are given bothfor intact sporangiophores and for cell walls fromwhich the contents had been squeezed out.

The O.D. rises sharply slightly above 300 miyi(13). Dennison has identified the principal substancein the sap which absorbs in the ultraviolet as gallicacid. Since the onset of absorption of this substanceagrees with our O.D. findings, the absorption curveof gallic acid, adjusted to equal O.D. at 290 mA isgiven in figure 9. The match between the two curvesis remarkably good above 250 mA. It is poor below250 mn, where the expected O.D. of gallic acid de-creases wlhile the observed O.D. continues to remainhigh. Presumably, other substances contribute to theO.D. in this region.

The concentration of gallic acidl neededl to producethe indicated O.D. in a pathlength equal to the diamii-eter of the sporangiophore (100 A) is about 5 mg/mi.This is close to the value estimate(d by Dennison forthe concentration of gallic acid in the extracted sapof wchole sporangiophores (13). These facts stronglysuggest that the internal screen, at least near the neu-tral point, is gallic acid.

For later discussion of the inversion of photo-tropism the O.D. contributed by gallic acid in the crit-ical region between 300 and 330 my is given intable I.

The absorption of the cell wall (fig 9) shows theabsorption peak reduced by a factor of about ten.This is believed to be due to residual contents noteliminated during the squeezing process.

In the visible the results were quite variable, andare therefore not plotted. A few fairly regular fea-tures could be discerned, however. Generally, a back-ground O.D. of about 0.1 was found. This is pre-sumably due in large part to nonspecific absorptionand in small part to reflection losses and residualscattering not collected by the microscope objective.Superimposed on this background is a slight absorp-tion, amounting to a maximum O.D. of about 0.03,beginning at about 530 mA, and giving maxima around500, 460 and 430 mi. In sporangiophores grown ona lactate medium, this characteristic absorption ismuch stronger, contributing in some cases O.D.'s be-tween 0.1 and 0.2. It is likely that the substance con-

TABLE IOPTICAL DENSITY OF GALLIC ACID (5AIG/ML FOR

100 A PATHLENGTH)

300310320330

O.D.0.700.240.050.006

199


PLANT PHYSIOLOGY

0 SPOR~~~?UIANGjIOPHORE ~II_

oL

(A)

40

o-IIII

320

5O280 8 48513 xx~~~~~-3 0 0000~~~~~~~~~~~~0XX

0 --H + - -l-Hf --H250 XI 350 400 450 500

E 500

r-A I 1TlME(min)0 GROWTH RESPONSE

FIG. U.(upper lef).Mirodnstoetr tacng(1mm elw hebas o te porngumaTROPIC RESPONSE

0-X

.5

0 2 4 6 8 10 12 14 16 .6

TIME (min) WAVELENGTH (mkL)

FIG. 5 (upper left). Microdenisitometer tracing (1 mm below the base of the sporangium) across the sporangio-phore of the original shadowgraph of figure 4. Irregularities in the tracing are due to grain in the emulsion. Theprincipal point exhibited is the sharpness of the edge of the sporangiophore, indicating that scattering does Iiotaffect the optics of the sporangiophore appreciably.

FIG. 6 (upper right). Microchamber for direct transmission measurements. A. Top view of chamber. B. Thefield imaged by the microscope objective onto the sensitive element of the photometer, showing the image of theslit of the monochromator (hatched area) in relation to the specimen.

200

80

60

r


DELBRUJCK AND SHROPSHIRE, JR.-ACTION AND TRANSMIISSION SPECTRA

3.0

I,-

zw

a

-J

4C.)

a.0

2.0

1.01-

0.'J200 250 300

WAVELENGTH (rmFIG. 9. Transmission spectra of the

the sporangiophore and of the primaryregion. The dotted line is the opticalacid (5 mg/ml l00A pathlength). Opethe O.D. of intact growing zone andtransmission curve of the cell wall withtruded cell contents. The data are corre

effect mentioned in the legend to figur(

tributing these three peaks is 3-carotentene can be isolated from the extractsabsorption peaks, except for a slight w

The cell wall had no characteristic;than 1 %) in the visible and near-u

when the specimens had been carefullbefore extruding the cell contents.

No bleaching by blue light could bewhen the specimen was growing a

normally and had been thoroughly da

DISCUSSIONThe principal aim of this discussion

what can and cannot be concluded witaction spectrum of the visual pigmening are the main points in our data:

I. The growth and tropic action spectra areidentical from the upper limit at 500 mA to 360 mL,and show peaks at 385, 455, and 485 mi.

II. Below 300 my the tropic action spectrum isa negative image of the growth action spectrum, en-larged at the peak (280 my) by a factor 6.5.

III. The cell wall does not show a characteristicabsorption.

IV. Beyond 400 mA the O.D. of the growing zoneof a sporangiophore, whether non-growing, or grow-ing and reactive, whether dark adapted or not, showsno obvious peaks which could be correlated with those

00 o Q o o o of the action spectra.0 V. The O.D. begins to rise sharply near the in-

350 400 version point and reaches a maximum (about 2.5) atIFL) the 280 mA peak.growing zone of These findings are discussed in terms of the follow-cell wall in this ing postulates which are somewhat weaker than thosedensity of gallic originally proposed by Blaauw (1):n circles denote Growth responses are transient and occur as asolid circles the result of changes in the intensity of irradiation. Aimperfectly ex- positive intensity transient (a step-up or a pulse-up)

cted for the lens produces a positive growth response. Any photo-4. tropic effect in Phycomyces is conditioned by a bi-

lateral asymmletry of light energy absorbed. (A puz-zling feature of the tropic response to continuous uni-lateral irradiation is that it is not transient, like the

ae, since icaro- growth response to a step-up in the intensity, but per-and has simlfar sists beyond the time at which the growth response

absorption (less transients disappear. This has been discussed in dle-iltraviolet, (es tail by Cohen and Delbriick (8)).lyarkdaplt,even For evaluating the effectiveness of light absorbed[y dark adapted we have to take into account its precise azimuthal dis-

tribution (6). Thus, for unilateral irradiation in airobserved, even where the total light absorbed on the proximal sidekd desponding probably slightly exceeds the amount absorbed on the

distal side, the distal absorption is nevertheless moreeffective due to the advantage conferred upon the dis-tal side by the focusing effect. We have no a priori

l is to determine measure of the magnitude of this advantage, but will:h respect to the attempt to evaluate it from our data. Let us sayit. The follow- that the focusing effect in air gives the distal side the

stimulus 1+a when the proximal side receives unit

FIG. 7 (lozwer left). Growth responses at different wavelengths. The specimens were rotated at 2 rpm andcontinuously adapted by irradiation with standard light. They were stimulated every 10 min for 30 sec with mono-

chromatic light of suitable intensity. The points are averages for three cycles. The growth responses obtained atdifferent wavelengths are similar. Maximum response occurs at 5.5 min after the stimulus. The response time isindependent of the average growth rate and of the size of the response. The correlation between wavelength andaverage growth rate of the specimen is accidental.

FIG. 8 (lozwer right). Action spectra for growth response and tropic response. For the tropic response data,the matching quantum fluxes were determined with a precision of + 10 %. The precision of the growth response

data was somewhat better. The maxima at 280 and 385 mA and a broad region of high sensitivity between 440 anid490 myL are unambiguous. The dip between the two maxima at 445 and 485 mu could be due to a combination of ex-

perimental errors and screening effects, as explained in the text. Otherwise the action spectra, where they coincide,are believed to represent faithfully the action spectrum of the visual pigment. The distortions below 300 mA are

due to internal screening by gallic acid. The growth response peak at 280 mA is low by at least a factor two, as-

compared to the peak in the action spectrum of the visual pigment (see text).

I I>

0 N0 6 %oI ot

I %I \I %

I %

/

_ / \~~O)OO0

I *Ivoo

201

^ r% L


PLANT PHYSIOLOGY

stimulation. Superimposed on this adlvantage is a

factor. 1-b, accounting for any attenuation of the

light in its passage across that part of the sporangio-

phore between the proximal and the distal receptors.

Such an attenuation could be due to the visual pig-

ment itself and to other substances. The focusing ad-

vantage a is presumiiably independent of wavelength(neglecting (lispersion), while the attenuation b de-

penlIs strongly on the wavelength. In the visible the

attenuatioln is small compared to unity, according to

point 4 above. Since the reversal occurs between

320 andi 310 mn, w\here the total O.D. is still small

compared to unity and the absorption duie to gallicacid contributes anl O.D. between 0.05 and 0.24 (see

table I). we conclude that the focusing advantage is

also smlall compared to unity. Tn the visible, then,

both a and b are small an(d we canl neglect squared

terms. Thus, the ratio of the effects on the two sidesis

distal: proximal = (1+a -b): 1.

The effects of various types of screening on thetw o action spectra may now be considered syste-

matically.

I. INTERNAL SCREEN, ATTENUATION SLIGHT,h<<1 The growth action spectrum will not be af-fected, since it compares the sum of the effects on

proxinmal and distal sides, standard versus test.

Screening and focusinig contribute only second order

correction terms.In contrast, the tropic action spectruml mlay be

affected, since the null condition requires that the dif-ferentials are matched. Let the quantunm flux of thestan(lar(i light and also the absorption coefficient ofthe visual pigment for this light be unity. Let thequantum flux of the test light at equilibrium be x

and the absorption coefficient of the visual pigmentfor this light be P. Let b, and h,N be the attenuationsof standard and test light. Thenl the effects on thetwvo si(les will be:

standard side 1 + 8 x(l+a-bx)test sidle 3 x + 1+a-hb

For tropic equilibrium, these two exlpressions mutst

he equal. This giv-es for the action spectrum 1 /x theexpressionl

1/x , (<-bx)/(a-bS).This equation says that the actionl spectrum anid the

absol-ption spectrumll of the screeninig pigml-ent will be

in an inverse relation to each other. The action

spectrum (1/x) will have a dip wvhere the screeningpigment (bh) has a m11axim11um. The reason for this

is obvious: the screen increases the intensity differen-

tial whlere its spectruml has a mlaximum i: it therebyreduces the a(lvantage conferre(d upon the distal side

bh the focusing effect.This effect is appreciable only where bx is com-

pairable to a. In our (lata the effect is obvious as

the inrversion point is approaclhe(l. As the O.D. of

the screen rises and( becomies comp)arable to the focus-

ing advantage the tropic action spectrum drops off to

zero.

Equally significant is the fact that we find nosuch screening effect in the visible as evidenced bythe strict parallelism of the two action spectra (point1). This indicates that there is no internal screen-ing affecting appreciably the a(lvantage due to focus-ing.

Self-screening by the visual pigment, if it isslight, has qualitatively similar effects. It, too, willnot affect the growth action spectrum, but will affectthe tropic action spectrum inversely, when it is com-parable to the focusing advanltage. Again w,Ne canconclude from the parallelism of the twvo action spec-tra that self-screening in the visible is negligible col-pared to the focusing advantage (less than 10 % ofthe latter). In sum, we conclude that the internalattenuation across the sporangiophore in the visible,both due to self- and foreign-screening. is negligible.

II. INTERNAL SCREEN, ATTENUATION STRONG.1-b< <1 The growth action spectrum will be af-fected by a factor two, since the standlard light actson both sides while the test light reaches the proximalside only. To match the standard light, therefore, thetest light has to provide a quantum flux twice higlherthan corresponds to the absorption coefficient of thevisual pigment, and the action spectrum is low by afactor two compared to the visual pigment absorp-tion spectrum.

For the tropic action spectrum the situation isquite different. Consider a tropic equilibrium whenvisible light impinges from the same side as light of.say, 280 niy. The visible light acts at the rate oneon the proximal side and 1 +a on the (listal side.The ultraviolet acts on the proximal side only. Tobalance the asymmetry in the stimulation produced bythe standard light it must act at the rate a. Thus, insuclh a combination, the ultraviolet (loes not actuallymatclh the standardc light in its direct effect on thevisual pigmiient, but only compensates the dlifferentialof this effect across the sporangioplhore. Therefore.the quantum flux needced for tropic balance is low bythe factor a, and the action spectrumiii highi by thefactor 1/a.

Combining these two corrections we are nowr ina position to estimate a numerical value for the focus-ing advantage a. To do this we nmust raise the growthaction value by the factor two an(l comiipare it then to

the tropic action value, which is l/a timles larger.This gives for a the value 0.3.

III. EXTERNAL SCREENS Any screening sub-stance located entirely external to the photoreceptorswill affect the growth and the tropic action spectrumnequally and increase the quantum fluxes needed to

match the test stimulus. Thus the action spectrashould show dips where the screens show peaks. Un-fortunately, w\e do not know w\here the visual pig-ment is located and, therefore, also do not know whiclhregions are to be considlered external. The cell wallis certainly external andl it (loes not show any char-acteristic absorption (point 3). However, this (loesnot suffice as a justification for ignoring the possible

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DELBRUCK AND SHROPSHIRE, JR.-ACTION AND TRANSMISSION SPECTRA

effects of external screens. It may well be that ,B-carotene and gallic acid lie, at least in part, external

to the visual pigment. The distortions from this

source could be appreciable. In the ultraviolet. if

only 10 % of the gallic acid were located external to

the photoreceptor it would cut off half the flux at

280 mA and make both action spectra low by a factor

two. In the blue, if all the blue absorbing pigments

as measured by the total O.D. were entirely external

to the photoreceptor, half on each side, the action

spectra would be depressed by an O.D. of 0.015. This

depression would affect the action spectra by 3 to

4 % at its absorption peaks and might be responsiblefor some of the dip in the action spectra separatingthe peaks at 455 and 485 mA.

The principal conclusions regarding the action

spectrum of the visual pigment of Phvcomyces are

the following:In the visible and near-ultraviolet both action

spectra give a rather faithful image of the visualpigment action spectrum, not distorted by internal.external, or self-screening. Distortions from internalscreens are entirely negligible. External ones mightgive distortions of a few percent; hardly noticeablewithin the accuracy of our measurements. The only

significant region that might be affected by this cor-

rection is the dip separating the maxima at 455 and485 mA.

The total absorption of light in its passage betweenthe photoreceptors on the two sides of the sporangio-phore must be less than 10 % of the focusing ad-vantage. Since this is less than the directly measuredattenuation across the whole sporangiophore and sincethe cell wall has no measurable O.D., we must inferthat the measured attenuation is due to pigments lo-cated peripherally but inside the cell wall. Thismakes it seem likely that the photoreceptors are not

located at the cell wall but some distance inside. Per-haps they lie at the interface between protoplasm andvacuole or at some interface inside the protoplasm.

Regarding the chemical nature of the visual pig-ment we can rule out s-carotene with some confidence,because of the peaks at 280 and 385 my in the actionspectra. Various flavin compounds and other sub-stances mentioned in the literature (14) whose ab-sorption spectra show greater similarity with theaction spectra are possible candidates. With theaction spectrum given it would seem hopeful to isolatethe visual pigment and identify it by chemical means.

Our attenmpts in this direction have not vet been suc-

cessful. The O.D. produced by visual pigment must

be very small; it does not show up in the transmissionmeasurements, and self-screening does not affect thedifferential across the sporangiophore responsible forthe tropic reaction.

The discussion of the interrelationship betweenthe action spectrum of the growth response and thatof the tropic response illustrates how strongly bothspectra, and particularly the tropic spectrum, can be

modified by screening pigments. In Phycomycesthere happens to be no appreciable distortion in the

visible, though a very strong one below 350 mrI.In Avena the work of Shropshire and Withrow (16)has shown that the tropic action spectrum is stronglyaffected by screening pigments even in the visible.To correct for these screening effects. it would benecessary to have a clear notion of the cause andmagnitude of the bilateral asymmetry in the absenceof screening pigments, similar to the notion of thefocusing advantage in the case of Phycomyces. Wedo not have such a theoretical basis for Avena. Wesuspect that. here. too. dioptric effects are involved.since Ziegler (17) has shown that the phototropismfor Avena reverses sign when the specimens are im-mersed in paraffin oil. \Ve have confirmed this effectand have found that the extreme tip of the coleoptile,where most of the sensitivity resides, is sufficientlytransparent to permit dioptric effects to be operative.A detailed growth action spectrumii wvould lhelp clarifythis question.

SUM MIARY

Detailed action spectra of the growth response an 1of the tropic response of the sporangiophores of Phy-comyces in stage IVb are given for 10 mA intervalsfrom 250 to 300 my and for 5 my intervals from 300to 500 m/. Both spectra have been determined bynull response methods. The two spectra are identicalin the visible and near-ultraviolet and show maximaat 280, 385, 455, and 485 mfi. Below 300 my thetropic response reverses sign and shows a peak at 280myL. This peak is 6.5 times larger than a correspond-ing peak in the growth action spectrum.

Transmission spectroscopic data in the growingzone have been obtained for single sporangiophores.In the ultraviolet these were obtained from shadow-graphs and in the visible by direct photometric meas-

urements. In the ultraviolet a strong absorption isfound, beginning near the phototropic inversion pointand paralleling the absorption of gallic acid, a sub-stance which had previously been detected by Denni-son in the vacuole. In the visible region trans-

mission, including most of the scattered light, is never

more than about 75 %. Only a minute part of thesetransmission losses can be due to absorption by thevisual pigment. Most of the losses must occur peri-pheral to the visual pigment, but not in the cell wall.

The growth responses are qualitatively identicalthroughout the spectrum. The tropic responses, be-sides reversing sign below 300 mnyA are also strongerin this region than in the visible. These facts, as wellas the quantitative relationship between the two actionspectra, can be explainedl satisfactorily as mediatedlby the screening effect of an internal screen which is

principally due to gallic acidl.

ACKNTOWNLEDCF.MENTS

The authors are much in(lebted to Mrs. Lois Edgarfor technical assistance, to Dr. Burton L. Henke,Physics Department Pomona College, for permissionto use his sensitive microdlensitometer. and to Dr. John

203


PLANT PHYSIOLOGY

Tyler for the use of the facilities at the Scripps Insti-tute of Oceanography, La Jolla, Cal., in calibratingthe thermopile.

ADDENDUMA paper by G. M. Curry and H. E. Gruen has just

appeared (Proc. Natl. Acad. Sci. 45: 797. 1959) inwhichl action spectrum data for the phototropic re-sponse of Phycomyces are presentedl. The gross fea-tures of the results of these authors are similar toours. Differences in detail may be accounted for bythe nmethod used by Curry and Gruen, involving masscultures and the (letermination of balance points be-tween the standard and the test light source which areset up 2 meters apart. This method, which we hadtested ain(l rejected. involves two (lisadvantageousfeatures apparently not appreciated by the authors:

I. Mass cultures lead to mutual shading, an(dthis produces spurious balance points near the middleof any series of cultures set up together.

Ii. The intensity ratio received from the twosources varies with position, as (1 +x) 2/(1-X) 2,where x is the displacement from the center, takingthe distance from the center to one of the sourcesas unit. This is a very rapid variation, approximately1+4x for small displacements fronm the center. Thus,2.5 cm (lisplacenment changes the ratio by 10 %. Aglance at table I of the cited paper shows that balancepoints in repeat measurements differed in some casesby as nmuch as eight times this amount.

In view of these criticisms, the contention of Curryan(d Gruen of a (louble peak in the blue, characteristicof the absorption of :-carotene, cannot be consiHleredas substantiated by their measurements.

LITERATURE CITED1. BLAAU\\W, A. H. 1909. Die Perzeptioii des Lichtes.

Rec. trav. bot. neerl. 5: 209-372.2. BUDER, J. 1918. Die Inversion des Phototropismus

bei Phycomyces. Ber. deut. bot. Ges. 36: 104-105.3. CASTLE, E. S 1930. Phototropism and the light-

sensitive system of Phycomyces. Jour. Gen.Physiol. 13: 421-435

4. CASTLE, E. S. 1931. Phototropic "Indifference"and the light-sensitive system of Phycomyces. Bot.Gaz. 91: 206-212.

5. CASTLE, E. S. 1931. The phototropic sensitivity ofPhycomyces as related to wave-length. Jour. Gen.Physiol. 14: 701-711.

6. CASTIE, E. S. 1933. The physical basis of the posi-tive phototropism of Phycomyces. Jour. Gen.Physiol. 17: 49-62.

7. CASTLE, E. S. 1942. Spiral growth and the reversalof spiraling in Phycomyces and their bearing onprimary wall structure. Amer. Jour. Bot. 29:664-672.

8. COHEN, R. and M. DELBRi7CK 1959. Photoreactionsin Phycomyces: Growth and tropic responses tothe stimulation of narrow test areas. Jour. Gen.Physiol. 42: 677-695.

9. COHEN, R. and M. DELBR6iCK 1958. Distributioni ofstretch and twist along the growing zone of thesporangiophores of Phycomyces during normiialgrowth and during periodic illumination. jotur.Cellular Comp. Physiol. 52: 361-388.

10. CURRY, G. M. and H. E. GRUEN 1957. Negativephototropism of Phycomyces in the ultra-violet.Nature 179: 1028-1029.

11. DELBRUCK, M. and W. REICHARDT 1956. Systemanalysis for the light growth reactions of Phy-comyces. Cellular Mechanisms in Differentiationand Growth. Rudnick, Ed. Princetoni UniversityPress. Pp. 3-44.

12. DENNISON, D. S. 1958. Studies onl phototropicequilibrium and phototropic-geotropic equilibriumin Phycomyces. Thesis, California Institute ofTechnology.

13. DENNIsoN, D. S. 1959. Gallic acid in Phycomycessporangiophores. Nature 184: 2036.

14. GALST'ON, A. 1959. Phototropism of stems, rootsand coleoptiles. Handbuch der Pflanzenphysiologie17(1) : 492-529.

15. GRUEN, H. 1959. Growth anid development of iso-lated Phycomyces sporangiophores. Plant Phy siol.34: 158-168.

16. SHROPSHIRE, W., JR. and R. B. WIT1HROW 1958.Action spectrum of phototropic tip-curvature ofAvena. Plant Physiol. 33: 360-365.

17. ZIEGLER, H. 1950. Inversion phototropischer Reak-tionen. Planta 38: 474-498.

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