An Endogenous Rhythm in The Rate of Carbon Dioxide Output ofBryophyllum. IV. Effect of Intensity of Illumination on

Entrainment of The Rhythm byCycles of Light & Darkness 1

Malcolm B. Wilkins 2

Department of Botany, King's College, London, W.C.2., Great Britain

IntroductionExcised leaves of the succulent plant Bryophyllum

fedtschenkoi show an endogenous circadian rhythm intheir rate of carbon dioxide emission (17,18). Therhythm occurs when leaves kept at a constant tem-perature and in an air stream initially free fromcarbon dioxide are transferred from light to pro-

longed darkness. The entrainment of the basicoscillating system in Bryophyllum leaf cells by variouscycles of light and darkness and the extent to whichentrainment depends upon the light intensity in thecycles are described in this paper.

Exposure of an organism to cycles other than 24hours in length can modify the period of its endoge-nous circadian rhythm. When this occurs the periodusually becomes either equal to, or a multiple of, thatof the stimulating cycles. The former phenomenonis termed "entrainment" and the latter is a specialtype of entrainment known as "frequency demulti-plication".

Both plant and animal rhythms can be entrainedby cycles of light and darkness having a period otherthan 24 hours. In most organisms such entrainmentoccurs only when the amount by which the perioddiffers from 24 hours lies within certain limits, andthese appear to be different in different organisms.When the period of the light-dark cycles is less than24 hours, clearly defined limits of entrainment havebeen observed in all organisms investigated. For ex-

ample, the rhythm of leaf movement in Canavalia ensi-formis entrains to 8: 8 but not to 6: 6 hour cycles oflight and darkness (8), whereas that of petal move-

ment in Kalanchoe blossfeldiana entrains to 6: 6 butnot to 4: 4 hour cycles (5). On the other hand,when the period of the stimulating cycles exceeds 24hours limits of entrainment have been observed in a

few organisms (2,4, 12) but not in others (8,9, 10,14, 15). Entrainment data for plant and animalrhythms have recently been tabulated and discussedby Bruce (1).

1 Received March 23, 1962.2 Present address: Biological Laboratories, Harvard

University, Cambridge 38, Mass.

Although the limits of entrainment have been de-termined for a number of biological oscillating systemsand given considerable emphasis in the literature'(1, 3), the extent to which they are a function of thelight intensity in the stimulating cycles has not previ-ously been critically investigated. Furthermore, littleattention has been given to the possibility that the ob-served entrainment is the result only of a direct effectof light on the biochemical or physiological processbeing used to monitor the behavior of the unidentifiedbasic oscillating systems.

Materials & MethodsThe plants of Bryophyllum fedtschenkoi, R.

Hamet et H. Perr. de la Bath. were grown in a green-house in short days. Natural illumination, supple-mented throughout the year with a Philips high pres-sure mercury vapor lamp and four 100-w tungstenlamps, was given from 0800 hours until 1600 hours.The leaves were excised between 1500 hours and 1530hours and placed in glass tubes through which streamsof air, initially free from carbon dioxide, passed ata rate of 1.55 liters per hour. Three sample tubes,each containing three or four leaves, and a blank tubewere immersed in a water bath controlled to 26 +0.05 C. Beginning at 1600 hours two of the leafsamples were treated with the various cycles of lightand darkness while the third was kept in darknessas a control. The rate of carbon dioxide output ofthe leaves was automatically measured and recordedby an apparatus incorporating an infra-red gas ana-lyser (17, 20). The unfiltered white light in thestimulating cycles was provided by tungsten lamps,its intensity being measured in meter-candles (lux)with an Everett-Edgecombe autophotometer. Therate of carbon dioxide output of the leaves has beenplotted at hourly intervals against time of day. Insome figures both sample curves are shown togetherwith the control to indicate the reproducibility of theresults; in others only one sample curve is shownand the positions of the peaks of the control rhythmare indicated by vertical broken lines for clarity.The method for estimating the times of occurrenceof the peaks has been described in detail in previouspapers (17, 20). Throughout this paper the usual

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120 _ _ _____-%dioxide metabolism of the tissue, a lower intensity of

g'IV illumination (25 lux) was employed in later experi-

r00 Ait &-6t f nments. At this intensity the rhythm was entrained

\lt'A by 16: 16 hour cycles (fig 1B) as efficiently as in theprevious experiment (fig 1A). The phases of the

stimulating cycles in figures 1A and 1B differed

C 60CR; by 180 degrees but in each experinment the peaks ofcarbon dioxide emission occurred during the later

part of the dark portion of the cycle. The rhytlhn;wassimilarly entrained on exposing leaves to 20: 20

hour (fig 2A) andl 24:24 hour (fig 2B) cycles oflight (25 lux) and darkness. The peaks of carbon

20 (lioxide output occurred at intervals of 38 to 39 hourso and 46 to 48 hours, respectively.s 100 In each experiment an approximately 23-hour

period reappearedl when the leaves were transferre(d) 80 B from the entraining cycles to prolonged darkness.

6L 151 l | |, 1\ tX l | The tinme which elapsed between the onset of prolong-ed darkness and the next I)eak of the rhy,thmi was 16

40 -| l i, 1'' |_*8\( \tX hours in figure 1B, 15.5 hours in figure 2A and 15hours in figure 2B. These times are closely similar

to those previously reported (17, 18, 20).The rhythm of carbon dioxide emission in Bryo-

0 _ _ phyllum leaves is rapidly entrained by cycles of lightMn Mn Mn Mn Mn Mn Mn and darkness having a period greater than 24 hours.

TIME OF DAY No experiments were conducted in which the periodFigs. 1 A & B. Entrainment of the rhythm of carbon of the light-dark cycles exceeded 48 hours. It seems

dioxide output in Bryophyllum leaves on exposure to reasonable to assume from data already published16: 16 hour cycles of light and darkness (broken lines). (17, 18) that if the perio(d of the stimulating cyclesRhythms in leaves kept in continuous darkness are shown were further increased, the rhythm would be inhibitedby solid lines. The light intensity in the cycles was during the light phase and begin again during the3,000 lux (A) and 25 lux (B). The illumination pro- (lark phase. If the dark phase were sufficiently longgram is shown by the bar above each figure. Ordinate: (e.g. 48 hr), a second peak of the normal 22 to 23

Ag CO, given off/hour/g fr wt of tissue. Abscissa: hour rhythm would undoubtedly before the be-

time of day, Mn = midnight. ginning of the next light phase.- II. Effect of Stimulating Cycles Having a

Period Less Than 24 Hours. Regulation of the

convention of showing the length of the (lark phase ofthe cycle in italics (e.g. 20 :20) has been adopted.Each experiment was carried out at least twice.

Results

> I. Effect of Stimulating Cycles Having a PeriodGreater Than 24 Hours. The rhythnm was entrainedwhen leaves were exposed to 16: 16 hour cycles oflight and darkness in which the intensity was 3,000lux, the peaks of carbon dioxide output occurred atintervals of approximately 33 hours (fig 1A). Whenthe leaves vere later transferred to continuous dark-ness at 2400 hours the rhythm regained a circadianperio(l. The next peak occurred 17.5 hours after theonset of prolonged darkness and subsequent peaks atintervals of approximately 25 hours. In this experi-ment both sets of leaves showed a free-running periodapproximately two hours longer than the normalperiod at this temperature. This was the only ex-

periment in which a slightly longer period was ob-served.

To reduce complications introduced by the possi-bility of a direct effect of light on the rate of carbon

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TIME OF DAY.

Figs. 2 A & B. Entrainment of the rhythm of carbondioxide output on exposing Bryophyllum leaves to 20: 20hour (A) and 24: 24 hour (B) cycles of light and dark-ness (Broken lines). The light intensity was 25 lux.Rhythms in leaves in prolonged darkness are shown bysolid lines.

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WILKINS-ENDOGENOUS RHYTHM IN CO., OUTPUT

80 leaf cells without entraining the basic oscillating.tA ^ ~ ~~~~~~~~system.

60 " tA A ) , 17 ^ It is possible to determine whether or not theregulation of the rhythm shown in figures 3 A to D

40 * 1 z R E C \\ t -. , \ ,X ~ is due to entrainment of the unidentified basic oscil-20 M - lating system in the leaf cells by examining the phase

oi" .:\ . vl,<of the rhythms after the stimulating cycles are re-0 __ placed by continuous dlarkness. Previous experi-o60 s ments have shown the rhythm to be inhibited by light

and to begin again when darkness is restored. At39 40 _ ; l l l l ¢26 C the timle which elapses between the onset of pro-

20 T longed darkness and the first peak of the rhythm (thetransient) is 17 to 19 hours following ex'posure to

o * . ,_. _ _ - light for 3 to 8 hours (18, 19). If the basic oscil-0 lators are being entrainedl by the light-dark cycles a

'116 40 > A c similar interval would be expected between the be-0 ginning of continuous (larkness andl the next peako 20ci U, \; t\,/ \ 1t\ / St oof the rhythmii. On the other hand, if the cycles are

0o n not entraining the basic oscillating systenm, but affect-ing only the rate of carbon dioxide metabolislml of thetissue, the final phase of the rhythm will not he related

20 to the tinme at wlhiclh prolonge(d darkness begins, andthe Deaks of carbon (lioxidle output will occur at aboutthe usual time of dav. In other words, the oscillatorwoul function normally while the leaves were ex-

T M E OF DAY posed to the cycles but its activity, usually revealedFigs. 3 A-D. Entrainment of the rhythm of carbon by the rate of carbon (lioxide emission of the tissue,

dioxide output on exposing leaves to 8: 8 hour (A and Nwould be masked.B). 6: 6 hour (C) and 3: 3 hour (D) cycles of light and In the experiments shown in figures 3 A to D,darkness. In each experiment the light intensity was 17 to 19 hours elapsed between the onset of prolonged1,000 lux. For further explanation see text. Positions darkness and the next peak of the rhythml. Theseof peaks in the control rhythms are shown by vertical transients indicate that the basic oscillating systembroken linles. was entrainied by the various cycles of light alndt (lark-

ness. The evidence is, however, not conclusive forthe experiments shown in figures 3C anid 3D because

rhythm by cycles of light and darkness having a period the peaks of carbon dioxide output after the onsetless than 24 hours is shown in figures 3 A to D. In of continuous dlarkness occurred at approximiiatelyeach experiment the light intensity was 1,000 lux. the times of day (1100 hr & 0800 hr) expected hadThe phases of the 8: 8 hour cycles in figures 3A and the oscillators been functioning normally while the3B differ by 180 degrees and the two curves shown leaves were exposed to the stimulating cycles. Inin figures 3A are the results of experiments carried two further experiments, therefore, leaves were ex-

out on different occasions. posed to 3: 3 hour cycles of light, (1,000 lux) andThe rhythm was apparently entrained by 8: 8 darkness but transferred to prolonged darkness at

hour (figs 3 A & B), 6: 6 hour (fig 3C), and 3:3 dlifferent times of dlay (1000 hr in fig 4A, 0400 hr inhour (fig 3D) cycles, the peaks occurred at intervals fig 4B). In each experiment the rhytlhnm wvas en-of approximately 16, 12, and 6 hours, respectively. trained and 17 hours elapsed between the onset ofIn every case the rate of carbon dioxide emission of darkness andl the next peak of the rhythm.the tissue increased during the time that the leaves These results show that at an intensity of 1,000were illuminated and decreased while they were in lux the basic oscillating system in Bryophyllunm leavesdarkness. is entrained by 8: 8, 6:6 and 3:3 hour cycles of light

The modification of the period of the rhythm in and darkness.figures 3 A to D could be the result of the stimulating Although a circa(lian period reappeared after thecycles having entrained the basic oscillating system of onset of prolonged darkness, close inspection of thethe leaf cells. On the other hand, because the nature curves in figures 3A to D and 4A and B revealsof the basic oscillating system is not known, the ob- that the period dues not return to its normlial value ofserved results could be due to the stimulating cycles 22.4 + 0.4 hours at 26 C (20) for 2 to 3 days. Theaffecting only the biochemical process which is used interval betweein the first and second peaks after theto monitor the behavior of the unidentified basic oscil- onset of continuous dlarkness is slightly longer thanlating system. In these experiments, for example, those between subsequent ones. The means of thethe light-dark cycles might have had a direct effect intervals between successive peaks following the on-

upon the rate of carbon dioxide metabolism of the set of darkness in the curves shown in figures 3A to

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PLANT PHYSIOLOGY

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Mn Mn Mn Mn Mn MnTIME OF DAY

Figs. 4 A & B. Entrainment of the rhythm by 3: 3hour cycles in which the light intensity was 1,000 lux.Broken and solid lines show duplicate experiments. Forfurther explanation see text. Positions of the peaks inthe control rhythms are shown by vertical broken lines.

D and 4A an(l B are shown with their standarderrors in table I.

Table I

Changes in the Interval Between Successive PeaksAfter Onset of Prolonged Darkness*

No. of peak after onset 1 2 3 4of prolonged darkness

Interval between peaks 24.3 23.4 22.9in hr + 0.15 + 0.51 0.04

n 9 7 5

* For further explanation see text.

Leaves were also exposed to 6: 6 and 3: 3 hourcycles in which the light intensity was lower thanthat used in the experiments shown in figures 3A toD and 4A and B. The rhythm was entrained by6: 6 hour cycles with light at an intensity of 500 lux,the peaks occurred at approximately 12-hour intervals(fig 5A). On replacing the stimulating cycles byprolonged darkness the rhythm regained a circadianperiod and the next peak occurred 18 hours later at2200 hours. This indicates entrainment of the basicoscillating system in the leaf cells.

In contrast, entrainment of the rhythm did notoccur when leaves were exposed to either 3: 3 hourcycles in which the light intensity was 500 lux (fig5B) or 6: 6 hour cycles in which the intensity was

100 lux (fig 5C). The first peak of the rhythm oc-

curred at 1300 hours in figure 5B and at 0600 hoursin figure SC, but in each experiment the second peakoccurred 22 to 23 hours after the first one. In figure5B prolonged darkness began at 0400 hours, betweenthe second and third peaks of the rhythm. The in-terval between these peaks was approximately 25

hours but the time between the onset of darkness andthe next (3rd) peak was only 9 hours. The thirdpeak was slightly delayed by the onset of continuousdarkness but later peaks occurred at intervals of 23to 24 hours. In figure 5C prolonged darkness alsobegan at 0400 hours, but this was at the crest of thethird peak of the rhythm. The next peak occurred23 to 24 hours later and a subsequent one after an

interval of approximately 23 hours. The absence ofobvious entrainment of the rhythm of carbon dioxideemission, and the fact that the next peaks did not

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TIME OF DAY

Fig. 5. A, Entrainment of the rhythm by 6: 6 hourcycles in which the light intensity was 500 lux. B and C,show absence of entrainment in leaves exposed to 3: 3hour cycles in which the intensity was 500 lux (B) and6: 6 hour cycles in which the intensity was 100 lux (C).Positions of peaks in the control rhythms are shown byvertical broken lines.

occur 17 to 19 hours after the onset of continuousdarkness indicate that entrainment of the basic oscil-lating system in the leaf cells did not take place inthese two experiments. The occurrence of frequencydemultiplication while the leaves were exposed to thelight-dark cycles is unlikely because in neither ex-

periment was the period of the rhythm a multiple ofthat of the stimulating cycles.

Although the rhythms were not entrained in theexperiments shown in figures 5B and 5C the peaksof carbon dioxide output did not occur at their usual

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WILKINS ENDOGENOUS RHYTHMar IN CO., OUTPUT

timle of clav. When leaves kept at 26 C are transfer-red to prolonged darkness at 1600 hours after a nor-mal 8-hour day, the first peak of the rhythm occursat 1000 hours to 1100 hours the following morning.The first peak was 2 to 3 hours late in figure SB and4 to 5 hours early in figure 5C. It could be sug-gested that as the periods of the rhythms in these twofigures are close to 24 hours, this variation in thetime of occurrence of the first peaks is evidence offrequency demultiplication. However, other experi-ments have shown the periods of rhythms in leavesexposed to 3: 3 hour cycles to be approximately 19and 21 hours (figs 6A & B). The light intensitiesin these cycles were 300 lux (fig 6A) and 500 lux(fig 6B). A period of 21.5 hours was recorded ina similar experinment during treatment of the leaveswith 3: 3 hour cycles in which the light intensity was100 lux. The periods of the rhythms in these experi-ments are sufficiently different from 24 hours toeliminate the possibility that frequency demultiplica-tion is taking place.

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TIME OF DAYFig. 6A & B. Effects of exposing leaves to 3: 3

hour cycles in which the intensity of light was 300 lux(A) and 500 lux (B). Note the period is approximately19 hours in (A) and 21 hours in (B). Positions of peaksin control rhythms are shown by vertical broken lines.

The variation in the times of occurrence of thefirst peaks in the curves shown in figures 5B and Cand 6A and B, and the shorter periods found infigures 6A and B, are most probably due to some ofthe light exposures exerting a slight phase shiftingeffect on the basic oscillating system in the leaf cells.A light treatment of 3 or 6 hours duration shifts thephase of the rhythm when it is given between, butnot at the crests of, the peaks (18, 19). While re-petitive exposure to 3 or 6 hours' low intensity illu-

minationi dces not entrain the rhythm, those exposureswhich occur in that part of the cycle where the phaseis most sensitive to light could have a slight effecton the position of the next peak of the rhythm.

DiscussionThe rhythm of carbon dioxide output in Bryo-

phllum leaves is (lue principally to the periodic ac-tivity of the process which brings about the fixationof carbon (lioxide in darkness (16). It is not knownwhether this process is the basic oscillating system ofthe leaf cells or whether it is rhythllmic only becauseit is coupled to the basic oscillator. The observedentrainment of the rhythm of carbon dioxide emissioncould, therefore, be clue to the light-dark cycles affect-ing only the rate of carbon dioxide metabolism of theleaves and not the basic oscillating system. Thispossibility has been considered in section II of theResults and it has been concluded that the data pre-sented in this paper show that the basic oscillatingsystem in the leaf cells can be entrained by cycles oflight and darkness having periods ranging from 6to 48 hours.

Entrainment of the basic oscillators to cycleslonger than 24 hours occurs at high and low light in-tensities. Even at 25 lux, no upper limit of entrain-ment is observed as the period of the stimulatingcycles is increased to 48 hours. In contrast, whenthe period of the cycles of light and darkness is re-duced from 24 to 6 hours the limit of entrainment ofthe basic oscillating system in the leaf cells is a func-tion of light intensity. The existence of a sinmilarrelationship in other organismiis has not been estab-lished, although one may holcl for the basic oscillatorin Gon vaulax Pol vedra. The rhythm of luminescencein this dinoflagellate is entrained by 6: 6 hour cycles-with light at an intensity of 800 ft-c, but at 200 ft-centrainment is less distinct. However, the finalphase of the rhythm appears always to be determinedby the time the cultures are transferred to continuousdim light (6, fig 6). This suggests entrainment ofthe basic oscillator to 6: 6 hour cycles even at 200ft-c. Before entrainment could be established withcertainty at this light intensity it would be necessaryto replace the light-dark cycles by prolonged dim lightat different times of day to determine whether thenext peak of luminescence always occurs approxi-mately 18 hours after the change.

Hitherto, the limits of entrainment have been re-garded as a fixed characteristic of the basic oscillat-ing system in an organism rather than a function ofthe intensity of illumination in the stimulating cycles.In Bryophyllum, however, the lower limit of entrain-ment of the basic oscillator is determined by the levelof radiation energy in the entraining cycles. Untilit is established whether or not this is the case inother organisms, use of the term "limit of entrain-ment" without specifying the energy level (light in-tensity) employed should be discontinued.

In those experiments in which the rhythm in Bryo-phyllum leaves was not entrained by the light-dark

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PLANT PHYSIOLOGY

cycles of period less than 24 hours, it is possible thateither the basic oscillators were essentially unaffectedby the periodic stimulation or that frequency demulti-plication took place. The latter possibility can beeliminated because the periods of the rhythms of car-bon dioxide output in these cases were never multiplesof those of the stimulating cycles. Bruce (1) hasdrawn attention to the generalization that if a rhythmis readily shifted and quickly entrained by light, fre-quency demultiplication is less likely to occur. Theoscillator in Bryophyllum conforms to this generaliza-tion because of the absence of frequency demultiplica-tion, the rapidity vith which it is entrained by light-dark cycles and the ease with which its phase is resetby a single exposure to light (18, 19).

The reappearance of a circa(lian period in theBryophyllum rhythm after the entraining cycles arereplaced by prolonged darkness is typical of mostother rhythms. There are only two organisms inwhich this is apparently not the case. Under somecircumstances the rhythm of spore discharge in Pilo-boluis sphaerosporus appears to retain the period ofthe entraining cycles for a limited time after the onsetof a uniform environment (15). It is unlikely, how-ever, that this phenomenon is due to nmodification ofthe free-running period of the basic rhythmic process(19, 7). The alga Hydrodictyon reticulatimi is theonly organism in which the persistence of a rhythmequal in period to the entraining cycles has beenclearly established (11, 13).

The induction of phase shifts in the Bryophyllumrhythm by light perturbations has been discussed inprevious papers in terms of a model system oscillatingbetween limits A and B (17, 18, 20). This modelrepresents the unidentified basic oscillating system inthe cells. Oscillation of the system is apparentlystopped when leaves are transferredl from darkness tolight by the system being driven to, and held at, oneof its limits (A). When darkness is restored, oscil-lation begins again by the system nmoving towardslimit B. This results in the first peak of the rhythmoccurring a specific time after renmoval of the inhibi-tory condition. At 26 C this time decreases from17 to 19 hours to 14 to 15 hours as the previous ex-posure to light is extended from 8 to 50 hours (17,18, 20).

Entrainment of the rhythm of carbon dioxideemission can be explained in terms of the basic oscil-lating system being driven to limit A during theearly part of each light treatment and being held ator near this limit until the onset of the next darkphase. During each (lark phase the system will movetoward limit B. For short exposures to darknessthe system will move only a part of the way towardlimit B before being driven back to limit A with theonset of the next light phase. During the longestexposures to darkness, however, the system willactually reach limit B and return again to limit Ain the normal course of oscillation before the begin-ning of the next light phase. This type of mechan-ism can account for both the immediate reappearance

of a circadian period when the stimulating cycles arewithdrawn, and the occurrence of the next peaks atspecific times (transients) after the onset of a uni-form environment. The transients found in the pres-ent experiments are closely similar to those reportedearlier (17, 20) after previous exposure of leaves tolight for similar times.

In a previous paper it was shown that illuminatingleaves for 6 hours between the peaks changes thephase of the rhythm. At intensities above 8 lux themagnitude of the phase shift is determined not by thelength or intensity of the treatment but by the timeit ends. At 2 lux, on the other hand, the phase delayis proportional to the length of the treatnment. Thissuggests that the level of energy input required todrive the basic system from linmit B to limit A in6 hours is between 2 and 8 lux (18,19). In this pa-per it is found that entrainment of the rhythm bylight-clark cycles of period less than 24 hours is alsodependent upon light intensity. It will be noticed,however, that on reducing the intensity, entrainmentof the rhythm to 6: 6 hour cycles ceases between 500and 100 lux. Since both entrainment of the rhvthmto 6: 6 hour cycles and the induction of a phase shiftby a single 6-hour light stimulus between the peaksseem to depend upon light driving the basic oscillatorto limit A, it might have been expected that both ef-fects would be equally sensitive to light intensity.The leaves used in the present experiments weregrown, however, under a high pressure mercuryvapor lamp. The most likely explanation of the dif-ferent light sensitivity of the leaves used in this, andthe previous, investigation is that this procedure de-creased the sensitivity of the leaves to subsequentillumination.

SummaryI. Excised leaves of the succulent plant Brrvo-

phnlltu fedtschenkoi exhibit an endogenous circalianrhythmii in their rate of carbon dioxide emission whenthey are kept in darkness at 26 C. The entrainmentof this rhythm by various cycles of light and darkness,and the extent to which entrainment depends uponthe light intensity in the cycles have been investigated.

II. The rhythm is rapidly entrained by 16: 16hour cycles when the intensity is 3,000 lux and by16: 16, 20: 20, and 24: 24 hour cycles when the in-tensity is 25 lux. No limit of entrainment was ob-served on increasing the period of the stimlulatingcycles from 24 to 48 hours, even though a low lightintensity was used.

III. In contrast, the intensity of illumiiinationdetermines whether or not the rhythm is entrainedwhen the period of the light-dark cycles is less than24 hours. At 1,000 lux entrainment occurs to 8: 8,6: 6 and 3: 3 hour cycles. At 500 lux entrainmentoccurs to 6: 6 hour but not to 3: 3 hour cycles whileat 100 lux entrainment no longer occurs to 6: 6 hourcycles.

IV. In all experiments where the rhythm was

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WILKINS-ENDOGENOUS RHYTHM IN CO2 OUTPUT

entrained a circadian period reappeared when thelight-dark cycles were withdrawn and replaced byprolonged darkness. Furthermore, the final phaseof the rhythm was determined by the time at whichcontinuous darkness began; the next peak occurreda specific time after the onset of darkness.

V. The observed entrainment of the rhythm ofcarbon dioxide emission of the leaves in these experi-ments is due to entrainment of the unidentified basicoscillating system in the leaf cells and not merely to adirect effect of the light-dark cycles on the rate ofcarbon dioxide metabolism of the tissue.

VI. The possibility that frequency demultiplica-tion occurred when the system was not entrained hasbeen rejected on the grounds that the period of therhythm was never a multiple of that of the light-darkcycles.

VII. For light-dark cycles of period less than 24hours the limit of entrainment of the basic oscillatingsystem in Bryophyllum leaves is not a fixed char-acteristic but depends upon the light intensity in thecycles. The different limits of entrainment observedin various species of plants and animals might there-fore be attributed to the light intensities employed.Use of the term "limit of entrainment" should be dis-continued unless the level of radiation energy em-ployed is stated.

VIII. The mechanism of entrainment of theBryophyllum oscillators by light-dark cycles is dis-cussed and the present results are compared withthose of other authors.

AcknowledgmentsI wish to thank Professor T. A. Bennet-Clark, F.R.S.

for providing laboratory facilities for this work, and Mr.A. J. D. Cooke and Mr. J. P. Weight for their technicalassistance throughout the investigation. I am indebtedto the Royal Society for providing funds for the purchaseof the infra-red gas analyser.

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13. SCH6N, W. J. 1955. Periodische Schwankungender Photosynthese und Atmung bei Hydrodictyon.Flora (Jena) 142: 347-380.

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