Plant Physiol. (1977) 59, 948-952

Blue Light-induced Absorbance Changes in Membrane Fractions

from Corn and Neurospora 2

Received for publication September 30. 1976 and in revised form February 11, 1977

ROBERT D. BRAIN, JOHN A. FREEBERG,3 CHARLES V. WEISS,4 AND WINSLOW R. BRIGGS

Department of Plant Biology, Carnegie Institution of Washington, Stanford, California 94305

ABSTRACT

Blue light-induced absorbance changes were measured from differen-tially centrifuged membrane fractions from dark-grown coleoptiles ofZea mays L., and mycelia from an albino mutant of Neurospora crassa

Actinic irradiation caused changes in absorbance consistent with a flavin-mediated reduction of a b-type cytochrome. Both corn and Neurosporashowed similar light-minus-dark difference spectra, dose response

curves, and kinetics of dark recovery after irradiation. The photoreduci-ble cytochrome system from Neurospora showed the same distribution as

the activity of a sodium-stimulated adenosine triphosphatase, thought tobe a plasma membrane marker, in differential centrifugation experi-ments. The fraction showing the absorbance change did not co-sedimentwith the mitochondria, nor with the endoplasmic reticulum. Comparisonof absorption spectra of fully oxidized, partially reduced, and fullyreduced preparations showed that approximately a 30% reduction of thecytochromes involved with the process was needed to obtain the light-induced absorbance changes.

There is currently a substantial body of literature concerningthe effects of blue light on biological processes in higher plantsand fungi. Only recently has any real progress been made towardthe identification of the blue light photoreceptor associated withthese effects (see ref. 6). Poff and Butler (16) studied blue light-induced A changes indicative of a Cyt reduction in Phycomycesand Dictyostelium, and Munioz and Butler (15) have recentlyextended these findings to intact mycelia of Neurospora. Both ofthese reports implicate a flavin-mediated reduction of a b-typeCyt in the blue light photoreception process, with a flavoproteinmoiety as the actual photoreceptor. The implication comes fromsimilarity of the action spectrum for the cytochrome reductionwith that for many other well characterized blue light responses(15).

Briggs (5) reported briefly on light-induced A changes in Zeamembrane fractions separated by differential centrifugation.However, he reported only on recovery oxidation followingactinic irradiation. More recently, Schmidt and Butler (21) havereported on such changes in a pelletable fraction in Neurosporathat partially resemble the responses found by Mufnoz and Butler(15) in intact Neurospora mycelia. The only attempt at fraction-

' This work has been supported by National Science FoundationUndergraduate Research Award No. EPP75-04559 to R. D. B. andCarnegie Institution of Washington, Dept. of Plant Biology, Stanford,Calif. 94305.

2 CIW-DPB Publication No. 578.: Present address: Department of Biology, University of Massachu-

setts, Boston, Mass. 02215.4 Present address: Dept. of Biology, Menlo School, Atherton, Calif.

94025.

ation in that report was the separation of soluble from pelletablecomponents. Mufioz and Butler (15) did report light-inducibleCyt reduction in soluble extracts of Neurospora, but they studiedsupernatant rather than pelletable activity, observed reductionboth of Cyt b and c, and had to add FMN or FAD in order toobserve the reaction. It seems likely that they were studying thesame phenomenon as Schmidt and Butler (20) who found photo-reduction of Cyt c in the presence of added flavins in a modelsystem. Widell and Bjorn (24) have reported light-induced Achanges in another higher plant system, intact wheat coleoptiles,but the responses measured were not the same as those moni-tored by Briggs (5) or Munioz and Butler (15).The purposes of the study reported here were: (a) to isolate

and characterize partially purified membrane fractions of Neuro-spora with a photoreduction system similar to that found inintact mycelia by Munoz and Butler; (b) to study further theblue light-induced A changes found in corn membrane fractionsand compare them to similar changes found in Neurospora; and(c) to determine qualitatively the redox state necessary forobservation of these changes. A preliminary account of thesestudies has appeared elsewhere (3, 7).

MATERIALS AND METHODS

Preparations. The carotenoidless strain of Neurospora crassaused (albino-timex) was isolated by M. L. Sargent from a crossbetween al-2 (FGSC No. 99) and an invertaseless bd (FGSCNo. 1959) obtained from the Fungal Genetics Stock Center,California State University, Humboldt, Arcadia, Calif. Cultureswere maintained by transferring small amounts of conidia every7 days to a 250-ml Erlenmeyer flask filled with 75 ml of solidmedium: 2% (v/v) Vogel's solution (23), 2% (w/v) Difco Bacto-agar, 1.5% (w/v) sucrose, and 1.5% (w/v) Sigma casein hydroly-sate. The cultures used in this study had undergone at least threedark transfers, and were grown at 30 C in light-tight incubators.To prepare the mycelia, approximately 70-ml liquid suspen-

sions were made of the 7-day-old agar cultures, and added to a2,000-ml Erlenmeyer flask, filled with 1,000 ml of liquid me-dium (the same as solid medium except dextrose instead ofsucrose, and no agar). The cultures were grown aerobically inthe dark at 30 C for 24 hr at 250 rpm in an incubating rotaryshaker (New Brunswick Scientific, model G-25).

Approximately 40 g of mycelia were harvested by suctionfiltration, torn into small strips, and placed into a 125-ml Nal-gene canister (Nagel Co.) adapted for a B. Braun M.S.K. me-chanical cell homogenizer (Bronwill Co.), with a 100-mm layerof 0.5-mm glass beads on the bottom. The canister was filled towithin 20 mm of the top with extraction buffer: 250 mm sucrose,100 mM N-morpholinopropane sulfonic acid (MOPS),5 14 mM

Abbreviations used: MOPS: N-morpholinopropane sulfonic acid;ATPase: adenosine triphosphatase; NPA: 1-N-naphthylphthalamic acid;DTT: dithiothreitol; nKP: n thousand x g pellet; nKS: n thousand x gsupernatant.

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CYTOCHROME REDUCTION IN CORN AND FUNGI

2-mercaptoethanol, 3 mm EDTA, and 0.1 mM MgCI2, titratedto pH 7.4 with KOH. The mycelia were then homogenized byvigorous shaking for 90 sec, with the canister surrounded by highpressure liquid CO2 to provide cooling. All procedures to thispoint were performed under dim red safelights (Kodak I-A). Allsubsequent operations were done under dim green safelights(maximum emission at 530 nm, no measurable emission above560 nm or below 500 nm).The homogenate was centrifuged at 2,000, 9,000, 20,000,

and 50,000g for 10, 15, 30, and 75 min, respectively. Thepellets were designated 2KP, 9KP, 2OKP, and 5OKP respec-tively. The 9, 20, and 5OKPs were respectively resuspended in0.2, 0.1, and 0.05 ml resuspension buffer/g fresh weight ofmycelia. Resuspension buffer was the same as extraction buffer,except with 25 mm instead of 100 mm MOPS, with no 2-mercapetoethanol, and adjusted to pH 7. The 2KP, consistingmainly of cell walls and larger fragments, was discarded. Thepellets were ground exhaustively in a glass-glass tissue homoge-nizer with their resuspension buffer at 4 C, and considered readyfor testing. Five-ml aliquots of the supernatants from the 2,000and 50,000g spins were also kept (designated 2KS and 5OKS,respectively). All assays were performed on these fractions aswell. Samples were either kept on ice or handled in a cold roomat 4 C until used.Corn seedlings, Zea mays L. (WF9 x Bear 38), obtained from

Bear Hybrid, Decatur, Ill., were grown for 4 days in the dark at27 C in nearly 100% humidity. During the first 3 nights, theseedlings were exposed to 2 hr of red light. The corn washarvested by removing the coleoptiles (average length 10 mm,without primary leaves) and immersing them in 4 ml extractionbuffer/g fresh weight (same as extraction buffer for Neurosporaexcept with 25 mm instead of 100 mm MOPS).

Harvesting and all following operations were done undergreen safelights and from 0 to 4 C. The coleoptiles werechopped by razor blades for 5 min, and the resulting brei groundin a mortar and pestle for an additional 5 min. The suspensionwas then filtered through fine nylon cloth, and the filtrate centri-fuged at 500, 9,000, and 21,000g, all for 15 min. The pelletfrom the 21,000g spin (21KP) was resuspended in 0.17 mlresuspension buffer (same as before)/gram fresh weight of co-leoptiles, and was the only Zea fraction used in the experimentsreported here. Finally, the resuspended pellet was ground ex-haustively in a glass-glass tissue homogenizer with resuspensionbuffer.

Spectrophotometric Measurements. Light-induced A changeswere measured on a Perkin-Elmer model 356 dual wavelengthspectrophotometer in the dual beam mode. Except for differ-ence spectrum measurements, the beams were set at 410 and423 nm, respectively. Thus, the readout wasA at 423 nm minusthat at 410 nm, in most cases. ForA change measurements withwavelengths fixed, the half-bandwidth for Neurospora was 5 nm,and for corn, 3 nm. Absorption spectra were measured on thesame instrument in the split beam scanning mode, with a halfbandwidth of 5 nm. White tissue paper provided a light-scatter-ing reference.

Actinic irradiation of the samples was from a tungsten-iodidesource, filtered through a Corning 7-96 broad band blue filter, atan effective intensity of 1.9 x 104 ergs x cm2 x sec-1 (correctedfor IR by measuring energy with and without an IR-transmitting,visible absorbing filter, Corning 7-56.

Assays. Proteins were measured by the technique of Lowry etal. (13). Cyt c oxidase activity, used as a mitochondrial markerenzyme, was measured by the method of Applemans et al. (1).NADH-dependent Cyt c reductase activity, used as an ERmarker, was determined by the assay of Lord et al. (12). Thesodium-stimulated ATPase activity was measured by the methodof Hedman (8), corrected for background phosphate concentra-tion in both the sodium-stimulated and unstimulated samples.

RESULTS

Common Effects. Characteristic blue light-induced A changesafter 60 sec actinic irradiation for the 21 and 20 KPs of corn andNeurospora, respectively, are shown in Figures 1 and 2. Becauseof the overlap between actinic and measuring beam wavelengths,we did not measure A during irradiation, and thus could onlyobserve changes following the end of actinic exposure. Thechange monitored in both cases isA at 423 nm minus that at 410nm. The loss of A observed after illumination is similar to thatfor the Cyt oxidation shown by Munioz and Butler (15) followingirradiation of intact Neurospora mycelium. The half-lives atroom temperature are approximately 35 sec. Since the instru-ment response time plus pen response upon reactivating thephotometer was only a fraction of a second, it was possible toobtain quantitative information about response magnitude fol-lowing different light doses and at different measuring beamwavelengths.Dose response curves plotting magnitude of A change at the

end of actinic irradiation versus length of illumination were madefor the 21 and 2OKPs of Zea and Neurospora, respectively, andare shown in Figure 3. A measurable change occurs with as littleas 1 sec irradiation, with between 5 and 10 sec yielding half-saturation, and nearly full saturation of the response occurringby the end of a 60-sec blue light exposure. Thus, a 60-secirradiation period was used in all further assays measuring pho-toreduction.

E

c

0

E

cC

(1

-0

0

A0

O 1 2 3 4 5 6Time, min

7 8 9

FIG. 1. Light-induced A changes in the resuspended 21,000g pelletfrom a homogenate of corn coleoptiles (about 3 mg protein/ml). Upwardarrows: actinic light on; downward arrows: actinic light off.

Ec0

c

(1)

N

Qa-0

-0

2 3 4 5 6 7 8

Time,min

FIG. 2. Light-induced A changes in the resuspended 20,000g pelletfrom a homogenate ofNeurospora mycelium (about 200 mg protein/ml).Upward arrows: actinic light on; downward arrows: actinic light off.

0.002A

t 4 t4

1i--

0.02 A

t 4 f 4

I

949Plant Physiol. Vol. 59, 1977

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BRAIN ET AL. Plant Physiol. Vol. 59, 1977

E

c

0

E

c

LC\

v

01a;

Irradiation time. sec

FIG. 3. Dose response curves, signal height at end of actinic irradia-tion versus duration of irradiation, for resuspended 21,000g pellet fromcorn and 20,000g pellet from Neurospora mycelium, normalized to showsimilarity. x: Neurospora; 0: corn.

Light-minus-dark difference spectra were measured for the 21and 2OKPs from the two organisms, respectively. The spectrawere obtained by setting one measuring beam at a standardreference wavelength, and varying the other from one actinicexposure to the next. For Neurospora, this standard wavelengthwas 500 nm, chosen because preliminary experiments showed a

minimal light-induced A change at that wavelength (when mea-

sured against 600 nm). Although a reference wavelength of 600nm or longer might have been preferable (light at 500 nm showsslight action in Neurospora, see ref. 18), we were unable tobalance the two beams of the Perkin-Elmer spectrophotometerwhen the reference and variable wavelengths were too far apart.The reference wavelength for corn was 410 nm. The reason forthis latter choice was that the light-induced A changes for corn

were very small (compare Figs. 1 and 2), and preliminary experi-ments showed a difference minimum near 410. Since our read-out was the difference between two wavelengths, the wave-lengths were deliberately set to maximize the light-induced dif-ference to gain the highest resolution for determining the posi-tion of the 423 band. After every four measurements, a standard410 nm versus 423 nm measurement was made to determine ifthe blue light effect was changing with time. When a significantchange was found, the results were not used and the samplediscarded.The 21KP preparation of corn was not nearly as stable as the

2OKP of the fungus. Hence, it was not possible to obtain exten-sive data from any one preparation. Partial light-minus-darkdifference spectra were obtained on three separate occasions,and one is shown in Figure 4. Figure 5 shows a more completespectrum obtained from a 2OKP of Neurospora. In both Figures4 and 5, there is a maximum near 423 nm and a minimum near

448 nm. From one preparation to the next, a consistent maxi-mum at 423 nm was found, but a minimum at 410 nm did notalways occur, and the relative size of the 448 nm minimum was

variable. The Neurospora preparation illustrated was completelylacking the 410 minimum. Although the corn difference spec-trum shown in Figure 4 shows no minimum, this is only because410 was a reference wavelength, as mentioned above. Separatemeasurements against stable reference wavelengths revealed aconsistent minimum near 410 nm, and a stronger minimum at450 nm. The Neurospora sample also shows a small increase in Anear 556 nm, indicative of a Cyt a-band. This is not present inFigure 4 for corn because instability of the Zea preparation andthe small size of the spectral change made measurements at theselonger wavelengths unfeasible. Both of these spectra are similarto those shown by Munioz and Butler (15), and are suggestive ofa flavin-mediated reduction of a b-type Cyt. Variation from one

preparation to the next could simply involve variation in theratio of flavin to Cyt.Cytochrome Measurements. When freshly resuspended, the

Cyt both in the 21KP from corn and the 20KP from Neurosporawere almost completely oxidized. In this redox condition, no

light-induced A signals like those in Figures 1 and 2 could bemeasured. A short time after the onset of measurement, a

reduction of the Cyt was observed. When this reduction becamestabilized, signals were obtainable. However, the system was not

completely reduced, for the addition of sodium dithionite pro-

duced a further reduction. After such dithionite reduction, sig-nals were again not obtainable.

Figure 6 shows room temperature absorption spectra for thethree redox conditions mentioned above. Figure 6A shows the20KP for Neurospora nearly fully oxidized. After the sponta-neous reduction, the absorption spectrum appeared as in Figure6B. This spectrum shows the emergence of an a-band near 556nm, and a shift in the Soret maximum from 411 nm to about 418nm. Figure 6C shows the pellet in its fully reduced form, afterthe addition of dithionite. Further absorption between 500 and560 nm has appeared, along with a further Soret shift to 425 nm.

The onset of the spontaneous reduction is variable, but usuallywithin 30 min after the beginning of measurement.The importance of this intermediate redox state in obtaining

light-induced A changes was particularly well demonstrated byone experiment with Zea. In that study, the 2OKP was initiallyoxidized, and failed to yield light-induced signals. It sponta-neously became partially reduced, and became stabilized at an

intermediate redox state, at which characteristic light-induced Achanges were obtainable. After approximately 75 min, the sam-

q)

c

a

-0

0

.C

5)0)

-0

0)E

0

0.004

0.003

0.002

0.001 F

0

-0.001410 430 450 470

Wavelength, nm

FIG. 4. Light-minus-dark difference spectrum for the resuspended21,000g pellet from corn coleoptiles. Reference wavelength, 410 nm.

-C-c

c

5)

5)

a

5)

0

a

E

C

0

0

5l)-0

A

40

0.08

0.06

0.04

-0.02

420 460 500 540

Wavelength, nm

580

FIG. 5. Light-minus-dark difference spectrum for the resuspended20,000g pellet from Neurospora mycelium. Reference wavelength 500nm.

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CYTOCHROME REDUCTION IN CORN AND FUNGI

ple spontaneously became further reduced, and after that timeno signals could be observed. Then, about 10 min later, itbecame slightly oxidized, and restabilized at a state yieldinglight-induced Cyt reduction. After 30 min, it became furtheroxidized, and again would not yield light-induced A changes.Addition of DTT (to 1 mM) brought about a slight reduction,and once again, signals were obtainable.

Localization of the Response. Table I lists the distribution ofvarious marker enzymes on a per mg protein basis among thecentrifuged fractions mentioned for Neurospora. Table II liststhese same markers on a total activity basis, with the last column

0)u

na-QL.0U)-Q

350 400 450 500

Wavelength, nmFIG. 6. Absorption spectra from a single resusj

from Neurospora. A: spectrum from a fresh prefollowing its pipetting into the cuvette; B: sarroughly 30 min, partially reduced and yielding stchanges; C: same preparation after addition of a inite. Increased UV A caused by dithionite. Theplaced vertically for clarity.

TABLE I. Distribution of marker enzymes and light-induc

various cell fractions from Neurospora crassa - ass

Fraction Protein Absorbanie Na Dependent CytoChange Adenosine Tri- c ox

phosphatase

mg/ml relative mm PO4 x min mm x

x 10 x

2KS 224 15.1 2 5.0 11.39KP 185 49.0 (38) 11.3 (39) 65.8

20KP 169 130.1 (100) 29.0 (100) 31.850KP 228 64.0 (49) 14.4 (50) 21.550KS 188 2.7 0.72 0.8

1Arbitrarily measured as mm signal height on the A = 0.01 seiscale pen deflection.

2Figures in brackets represent amounts relative to the 201K,

951

TABLE II. Distribution of marker enzymes and light-inducible cytochrome in the

various cell fractions from Neurospora crassa - total activity

Fraction Protein Absorbanie Na+ Dependent Cytochrome NADH DependentChange Adenosine Tri- c Oxidase Cytochrome c

phosphatase

mg relative mm PO4 mx in mm x sec m xsec-3x 10 x 10

2KS 15,900 24.0 7.97 18.0 3.929KP 1,850 7.4 2.08 12.1 0

20KP 1,020 10.3 2.93 3.2 0.000155OKP 910 4.7 1.31 1.9 0.58SOKS 2 12,030 1.4 0.27 1.0 3.57

% Recovery 99 99 82 100 100

1Arbitrarily measured as - signal height on the A = 0.01 sensitivity scale for fullscale pen deflection.

2Percent of amount present in the 2KS.

indicating how much activity was lost from the amount initiallypresent in the 2KS. Neither the mitochondria (Cyt c oxidase) northe ER (NADH-dependent Cyt c reductase) has a distributionlike that for the light-induced A change. However, the corre-spondence is excellent between the distribution of the signal, andthe sodium-stimulated ATPase. This correspondence could indi-cate association of the photoreception system with the plasmamembrane (see under "Discussion").Table II shows that most of the initial activity for each enzyme

can be accounted for at the end of the experiment, as can the Achanges, with the exception of the ATPase, which showed a lossof 17.4%.

DISCUSSION

One of the aims of this study was to compare the resultsobtained from membrane fractions of Neurospora to those foundin intact Neurospora mycelia by Mufioz and Butler (15). Com-

B parisons of kinetics of oxidation after actinic exposure and light-minus-dark difference spectra indicate that the same photore-duction process is operating in both systems. In addition, thelight-induced responses for the 21KP of corn also closely resem-ble the responses for the 2OKP of Neurospora, suggesting thatwe are measuring the same photoreception system in both orga-nisms. Widell and Bjorn (24) found a light-induced A change in

c wheat coleoptiles that has a light-minus-dark difference mini-mum where our systems show a maximum (near 423 nm), and a

550 600 maximum where our responses show a minimum (near 450 nm).Dark recovery after actinic irradiation was much slower, andWidell and Bjom's action spectrum was dissimilar to that mea-

pended 20,000g pellet sured by Munioz and Butler (15) for their A change. On theparation immediately latter grounds, it seems unlikely that it is related to phototropicme preparation after photoreception.trong light-induced A The 2OKP of Neurospora had both the highest specific activity

few crystals of dithio- for a Na+-stimulated ATPase, and the greatest magnitude of Athree curves are dis-change activity on a per mg protein basis. The A change is clearlynot correlated with the distribution of markers for mitochondria,

ible cytochrome in the ER, nor, for that matter, the soluble fraction. It seems likely thatays per mg protein at least in Neurospora, the photoactive fraction is in the plasma-ays membrane. Monovalent cation-stimulated ATPases are knownochrome NADH Dependent

cidase cytochrome c to be specific markers for plasma membrane in erythrocytes (I 7,reductase 22) for example, and Scarborough (19) and more recently Bow-

secj1 mM(x sec 1 man and Slayman (2) have located such ATPase activity in10-3 x 10-3 plasma membranes purified from the slime mutant of Neuro-

2.42 spora. As yet, little can be said about the specific localization of(207) 0 (0) the photoactivity in corn, since there is still considerable uncer-(100) 0.015 (100) 40

(67) 6.38 (4.3 x ) tainty about the validity of plasma membrane markers in higher2.97 plants (10).

nsitivity scale for full There is an important difference between the studies reportedhere and those of Mufioz and Butler (15) and Schmidt and

normalized to 100. Butler (21) with in vitro systems. In the latter two cases, no

Plant Physiol. Vol. 59, 1977

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952 BRAIN ET AL.

activity could be obtained without the addition of exogenousflavin. In the present study, no added flavin was required (al-though preliminary experiments showed that with repeatedwashing, bound flavin could be removed from the pellets andlight-inducible Cyt reduction was concomitantly lost). There ispresumably sufficient bound flavin in the particulate fraction tosuffice without further addition. That the flavin is the photore-ceptor in the present work is strongly suggested by the observa-tion (Brain, unpublished) that phenylacetic acid, known to forma fairly stable covalent adduct with the triplet state of flavins(11), completely blocks the Cyt photoreduction in the resus-pended 2OKP from Neurospora.One of the striking features of the Neurospora system is its

ability to become poised and maintain itself at an intermediateredox state, in some cases for as long as 8 hr. The redox behaviorof the preparation provides another similarity between our stud-ies and those of Munioz and Butler (15). They harvested theirmycelia from conditions of ample oxidizable carbon; hence uponinitiation of measurement, their preparations were nearly fullyreduced. No signals were obtainable at that time. They inserteda 4-hr waiting time into their protocol, allowing the initiallyreduced Cyt to become partially oxidized. At the end of 4 hr,light-induced changes were observable.

In our preparation, the Cyt were nearly fully oxidized at thebeginning of measurements. We could not obtain any signalsinitially either. By allowing the sample to stand for a time, weobtained reducing conditions, and the initially oxidized Cyt be-came partially reduced. Thus, between the two studies, the netresult is the same, i.e. an intermediate redox state must bereached before signals can be obtained.

Figure 6 shows this process well. In Figure 6A, no a-band isdiscernible, while in Figure 6B, where signals are obtainable, theemergence of an a-band can be seen. This correlation was foundto be invariant. In five separate studies with Neurospora, ab-sorption spectra were taken before and after attainment of acondition yielding light-induced Cyt reduction, and in all fivecases signal appearance was concomitant with partial a-bandemergence. Figure 6C shows a fully reduced preparation, andthe absorption spectrum is different. The a- and Soret bandsfrom Figure 6B are shifted to longer wavelengths. This resultsuggests that both a partial and a selective Cyt reduction occursto make the sample susceptible to blue light-induced photore-duction. Further spectral and chemical studies are under way tocharacterize the Cyt which becomes reduced in the light.There seem to be at least two equally viable explanations for

the necessity of an intermediate redox state. It is possible thatthe components of the photoreception system we are measuringmust both accept and pass along electrons. To accomplish this,neither a fully reduced nor fully oxidized state would be favored,but an intermediate potential would allow maximal efficiency.

Another possibility is that we are measuring the end productof the first reaction of a two-step dismutase reaction, i.e. we are

monitoring AH in:

H+ + e1- + A AHH+ + AH + e2-* AH2

Thus, AH would be at an intermediate reduction/oxidation po-tential.

Neither the present paper nor the current literature providesconvincing evidence that this flavin-Cyt complex is the photore-ceptor system for the various blue light effects in higher plantsand fungi beyond the similarity of action spectra. Indeed,Schmidt and Butler (20) report on an artificial system whichmimics most, but not all of the characteristics reported here.Evidence is clearly required that links the behavior of the frac-

Plant Physiol. Vol. 59, 1977

tions studied in the present work to the behavior of some bluelight responses. Brain and Briggs (4) report preliminary observa-tions with a mutant of Neurospora, poky, known to be deficientin b-type Cyt (14). The double mutant poky-timex shows sub-stantially less photosensitivity than timex alone. Recently Brain(unpublished) has shown that poky-timex 2OKP yields 1.2% asmuch signal as wild type, has less than 1% as much light sensitiv-ity, and has about 16% normal b-type Cyt. These experimentswill be published in detail elsewhere.

Since the light-induced Cyt reduction described by Munioz andButler (15) and in the present paper requires a restricted inter-mediate redox state, it seems unlikely that the reduction ob-served could represent precisely the primary act of photorecep-tion. The primary action could, for example, involve either alight-induced change in the redox state of the system or in therate of electron flow through it. In any case, the observedreduction provides a convenient assay for this blue light-sensitivesystem both in vivo and in vitro.

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