Photoperiodism and Circadian Rhythms

KARL C. HAMNER University of California, Los Angeles, California

Many of you were present at the conference on aspect... In retrospect, the necessity of accepting the photoperiodism held in Gatlinburg, Tennessee, in theory of an endogenous rhythm to explain photo- October 1957, the title of which was "Photoperiodism periodic responses with regard to flowering does not and Related Phenomena in and " [1] seem to be very great yet!" I believe my own work and At the conference I presented a paper based upon work the work of my students in addition to Btinning's which led me to believe that photoperiodism in plants work provides fairly conclusive evidence that endoge- was affected by an endogenous rhythm. Prior to the nous rhythms participate in the photoperiodic reaction, conference I had read some of the literature on endoge- and that something very close to Bfinning's hypothesis nous rhythms, but at the conference I was astounded may be the case. by the papers presented which indicated that scientists working with a wide variety of responses had obtained RHYTHMIC RESPONSES TO CYCLE LENGTH a tremendous fund of information on rhythms unrelated Twenty years ago Snyder [4], working with me, to photoperiodism. Because of the fact that the data obtained results which indicated that the flowering which I presented was partly that of my students, and response of Biloxi (Glycine max) was rhythmic, had been previously unpublished, and because I depending upon the amount of interrupted darkness recognized that I could not discuss the results critically interspersed between the high intensity light period in relation to all of the information available, I re- and the continuous dark period. He obtained maximum quested the withdrawal of publication of my paper. flowering on a 24-hour cycle and on a 48-hour cycle of After three additional years of work I feel that exten- treatment. At the time the results seemed rather sive new information is necessary to understand the confusing to us, and we agreed that they should be complex mechanism of biological clocks in relation to published without comment. Needless to say, Biinning photoperiodism in plants. However, I feel that I can noticed these data in Snyder's paper, and they were present conclusive data indicating that the biological taken by him to indicate strong support for his theory clock is participating in the photoperiodic response. of endogenous rhythms. We did not return to this Btinning [2] first proposed that endogenous rhythms problem until about 1953. At that time two of my participated in the photoperiodic reaction. He de- students, first Blaney [5] and later Nanda [6, 7, 8, 9], veloped his hypothesis as a result of his experiments on carried out rather extensive investigations of the leaf movement, and postulated that the photoperiodic photoperiodic responses of Biloxi soybean in relation responses of plants involved the same endogenous to the length of the light period, the length of the dark rhythm (or biological clock) that caused leaf move- period, and the length of the cycle of treatment. ments. In both long-day plants and short-day plants he Biloxi soybean is a short-day . It may be grown assumed that the flowering response depended upon the in the vegetative condition under long days in the green- time at which the plants were exposed to light in re- house. If transferred to short day, initiation of lation to the oscillation of the rhythm. He spoke of a takes place after three short days of treatment, and the photophil phase arid a scotophil phase of the rhythm, number of nodes which produce flowers is directly and postulated that short-day plants and long-day proportional to the number of short days received up plants flowered only if exposed to light during the to about 10. It has been our practice, therefore, to give proper phase of the rhythm. Btinning's hypothesis was the plants 7 cycles of treatment, using as a control a not accepted and, in fact, was actively opposed by many standard treatment of 7 cycles of short day, each short scientists for many years. At the Gatlinburg conference day consisting of S hours of high intensity light, fol- in 1957, and later at the International Botanical lowed by 16 hours of continuous darkness. The controls Congress at Montreal in 1959, several people disputed usually produce between 40 and 50 flowering nodes per very vigorously with Bihming and myself. As recently ten plants, and any variation from the standard short as 1959 in a review, Doorenbos and Wellenseik [3] day usually produces a reduction in the number of state: "Although one of the merits of Biinning's flowering nodes, thus providing a basis for comparison theory is that it suggests an explanation of the difference of the effectiveness of the different treatments. A between SDP (short-day plants) and LDP (tong-day typical cxperiment is shown in Fig. 1. We have per- plants), there is little experimental evidence on this formed many such experiments, and the results of a 269 270 HAMNER

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Zo 80 r~ o ~ ~ "' 2O LIJ r.~ 3:60 o 2: B . I0 4O u- I-- 0 0 I.-- I I I I I t,l 0 6 12 18 24 30 36 4~ w 20 48 54 60 66 7'2 I1:

i 0 12 24 36 48 60 72 CYCLE DURATION - HOURS o (~ NO. OF POINTS ON ONE POSITION 124 6 FIGURE 3. Summary of responses shown in Fig. 2. Each LIGHT DARK curve of Fig. 2 was adjusted so that all curves coincided CYCLE DURATION - HOURS at the 24-hour cycle. This figure is used as standard FIGURE 1. Typical experiment on flowering response of control curve in the discussion. Biloxi soybean in relation to cycle length. Each treat- ment consisted of seven cycles, each cycle containing an o---o JAPANESE MORNING GLORY 8 hr. high-intensity light period. The length of the dark a.---A SOYBEAN vor. PEKING periods in the various treatments varied to give cycles (9 of differing length. Ten plants were used in each treat- _z 20 I1: ment. w

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r o 50 ~w z Q 0 z J ~ o _J I,- / " 30 i 0 21 24 30 36 42 48 54 60 66 72 if) IiJ CYCLE DURATION - HOURS ~ 20 o z //..\ %..~~ FIGURE 4. Flowering responses of Japanese morning ._1 I0 ~/~,,~'- -- glory (Pharbitus nil) and Peking soybean (Glycine max. p. 0 var. Peking) with cycles of various length. Treatments I,- are similar to those of Fig. 1 except that Pharbitus O 16 24 32 40 48 56 64 72 received only 2 cycles of treatment. CYCLE DURATION- HOURS FIGURE 2. Six response curves of Biloxi soybean (control curves in various experiments cited in text). Treatments plants, Japanese morning glory (Pharbitus nil) and similar to those shown in Fig. 1. Peking soybean (Glycine max var. Peking). It seems clear that the pattern of response in both plants is number of these are shown in Fig. 2. It is obvious that very similar to that of Biloxi soybean. We have done a the flowering response follows a rhythmic pattern, great deal of work with Xanthium, but our results with depending upon the length of the cycle used. These this plant have not been quite so satisfactory. This experiments have been performed over a period of 4 or particular plant flowers as a result of exposure to a 5 years, and of course the curves are not exactly single short day. In fact, if the plant is taken from superimposed. However, Fig. 3 shows the summary of long-day conditions in the greenhouse and exposed to a all of the results with the data adjusted so that the single long dark period and returned to long day, it magnitude of the flowering peak on the 24-hour cycle flowers. The curves in Fig. 5 represent results of Raven (i.e., with an 8-hour light period and a 16-hour dark [11], and we have repeatedly obtained similar results. period) is the same for all of the experiments. It may be We have additional evidence, Finn [12], too volumi' seen from this curve that the results from all of our nous and indirect for review here, which also convinces experiments are indeed remarkably uniform with us that an endogenous rhythm is participating in the respect to the rhythmic pattern in relation to cycle photoperiodic responses of Xanthium. lengths. We also have evidence that the photoperiodic While these data indicate the participation of an responses of long-day plants involve an endogenous endogenous rhythm in the photoperiodic response of rhythm [12, 13]. If we expose long-day plants to the Biloxi soybean, the question arose concerning the same sort of treatment that we used for the short-day responses of other photoperiodically sensitive plants. plants, namely, expose them to 8 hours of light in each We do have evidence that a similar situation cycle and vary the length of the cycle by a varying prevails in other short-day plants. In Fig. 4 are pre- amount of darkness in each cycle, then we obtain the sented the unpublished data [10] for two other short-day results shown in Fig. 6. In this case, in contrast to the PHOTOPERIODISM AND CIRCADIAN RHYTHMS 271

O .o a o HYOSDYAMUS NISER ~.Zlo_ 5 ~o'" "" .... "" w r A----/I SILENE ARMERIA

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0 a ~ i 1 == ==so II 24 36 48 12 24 38 48 60 72 DARK PERIOD " HOURS CYCLE DURATION HOURS FIGURE 5. Flowering response of Xanthium pen(n)~ F1GURE 6. Flowering responses of and sylvanicum in two experiments when exposed to a single Silene armeria when given eight hours of high-intensity dark period of various durations at two different temper- light and varying durations of darkness in each cycle atures. The degree of response was determined by similar to Fig. 1. Treatments continued for 50 days. measuring influorescence development two weeks after Data from Finn [12]. treatment by the method described by Lincoln et al. [34]. The dotted line gives our best estimate of the re- sponses at long dark periods with normal temperature...... STANDARD CONTROL CURVE Empty circles and triangles at 25~ and solid circles r DARK PERIOD " 23"C and triangles at 4~ Data from the work of Raven [11]. A~te~DARK PERIOD- IO'C z ,,=, 4o short-day plants, flowering is inhibited by cycle lengths ~ 30 of 24 hours. With cycles shorter than 24 hours or ..'; . longer than 24 hours, the plants . In fact, with ci 7 ..y 0.. ..'p 0

Hyoscyamus, flowering on cycle lengths of 36 to 42 -1 Ir hours is almost as rapid as on continuous light. To o emphasize the point, these long-day plants flower I- o~ rapidly in spite of the fact that they are receiving CYCLE DURATION - ROURS approximately 30 hours of darkness for each 8 hours of FIGURE 7. Effect of dark-period temperature and cycle light. In Silene a rhythmic response is obtained which is length on flowering response of Biloxi soybean. The 12 hours out of phase with the soybean rhythm. It is standard control curve is obtained from Fig. 3. Treat- ments are similar to those of Fig. 1. Data after Nanda obvious that the length of the cycle to which the plants and Hamner [8]. arc exposed is more important than the amount of light which the plants receive. Unfortunately, data of this kind are difficult to obtain, not only because of the of treatment at normal temperatures, it required from difficulties involved in manipulation of the experiment, 8 to 10 short days at low temperature to bring about but also because of the fact that on the longer cycles, induction. However, at the low temperature the where the plants receive relatively little light, many of critical day length (or critical night length) was affected the plants die before a response can be measured. only slightly. In other words, it took more short days to induce flowering at a low temperature, but the quality TEMPERATURE EFFECTS of the effective short day was not greatly changed. It One of the characteristic features of endogenous appeared, therefore, that the reactions which took rhythms that have been studied in various organisms place in the dark period and determined the critical has been the remarkable temperature independence. length of the dark period had a very low temperature Temperature coefficients of from 0.8 to 1.2 have been coefficient. Recently we have reinvestigated this reported. With respect to the photoperiodic response, phenomenon in relation to endogenous rhythms and the temperature independence of the "timing mecha- find effects very similar to those produced by tempera- nism" has been a puzzling feature. Perhaps a better ture on other endogenous rhythms that have been dis- way to state the problem is to quote a statement from cussed in this symposium. For example, some of the Pittendrigh and Bruce [14], "One of the most chal- effects of temperature on the rhythmic response of lenging features of the Drosophila data has been the Biloxi soybean [8] are presented in Fig. 7. It may be strange mixture of temperature dependence and inde- noted that a change of temperature has relatively little pendence they have revealed." The same statement effect upon the periodicity of the rhythm, but does have could be made with respect to the photoperiodic a marked effect upon the magnitude of the oscillation, response. We discovered as early as 1940 [15] that, and also that a change in temperature brings about a although Xanthium flowered as a result of one short day phase shift. That is, maximum flowering at low tern- 272 HAMNER

such short cycles just before the main light period of each cycle produced little effect upon the response. On the other hand, the insertion of such short cycles be- (.~ - , '" I tween the main light period and the 16-hour dark ." .-'" ,,, - / period of each cycle resulted in a rhythmic response. o,,ol ^ :..... Nanda and Hamner [6, 9] have conducted rather I--'l:-vla~. , extensive experiments on the effects of light perturba- ,,m 50fC ~ ::""" :'" tions on the photopcriodic response of Biloxi soybean. In Fig. 8, result~ arc plotted so as to show the relation- ~),o ! ! z --.~. ~6 i ship between the effects of light perturbations given at 4oL___~L_ various times during the dark period in cycles of differing lengths. The curve of response without light perturbations, Fig. 3, is superimposed for comparison. The interrelationship between the effects of a light 0 24 48 0 24 48 72 CYCLE DURATION - HOURS perturbation and the time at which it is given in the cycle is clearly shown. It may be noted that, in every FIGURE 8 A-H. Flowering response to light perturba- tions given at different times in cycles of various lengths. case where a light perturbation is given 16 hours after Length of cycle is given on the right hand side of each the beginning of the cycle (that is, 16 hours after the figure. Flowering of controls (no light perturbation) beginning of the main light period and 8 hours after is represented by horizontal dash line. Each point on the end of the light period), no flowering was obtained. each curve represents flowering on that cycle length Furthermore a light perturbation given eight hours with a perturbation given at that point in the dark period. The perturbation consisted of 30 rain. of 1000 ft. before the end of the dark period resulted in marked c. illumination. The standard response curve from inhibition, particularly at the shorter cycles. It is Fig. 3 is superimposed as the dotted line. Data from equally clear that the effect of the light perturbation is Nanda and Hanmer [9]. rhythmic. In certain cycles the light perturbation may stimulate flowering. The stimulation is especially marked in those cycles whose length is such as to fall perature was obtained at cycle lengths of 36 and 66 within the inhibitory phase of the rhythmic response. hours instead of 24 and 48. Thus, cycle lengths of 30-36 hours (Fig. 8 B, C) and 60 hours (Fig. 8 F) produce very little flowering because LIGHT PERTURBATIONS these cycle lengths fall within the dips in the rhythm Another characteristic feature of endogenous rhythms of the control curve. Perturbations given at particular in many organisms is the striking effect of a light points in these cycles cause marked stimulation, perturbation given during an otherwise long dark presumably by removing the adverse effect of cycle period. In 1938 [16] we demonstrated the very great length. It may be noted that certain of these curves effect of a small light perturbation on the photo- represent duplication of work of others, but taken periodic response. When plants were exposed to short together, they represent a considerable extension of day, a small interruption of the dark period with light the previous reports on the effects of light perturbations inhibited flowering in short-day plants and stimulated given at different times during a long dark period. flowering in long-day plants. Since these initial reports, While all of the light perturbation experiments of there have been numerous light perturbation experi- this type indicate that the endogenous rhythm is ments in connection with photoperiodism [17, 18, 19, disturbed by the light signal, they provide relatively 20, 21, 22, 23, 24]. Bfinning has reviewed some of this little information as to the exact nature of the disturb- work in this symposium and has added several recent ance. For this reason another type of experiment was experiments of his own. In spite of all of these reports, designed which enables one to obtain a clearer picture plant physiologists in general have not been willing to of the effects of the perturbation on the rhythm. The accept the results as conclusive evidence that the light method used and the results obtained are shown in perturbation was affecting an endogenous rhythm. I Fig. 9. believe the work discussed below, a large part of which In Fig. 10 the results are plotted to show the rela- is as yet unpublished, provides an opportunity for the tionship between the effects of a single light perturba- critical evaluation of the effect of light perturbations tion given at a specific time in each cycle in relation to in relation to endogenous rhythms and the biological the length of the cycle. The standard control curve of clock. Fig. 3, adjusted to conform in magnitude to the controls Snyder [4] interrupted the light-dark alternations of a of this experiment, is superimposed on each of the short-day treatment with varying amounts of short other curves. It is clear from these curves that there is a cycles consisting of a brief exposure to light followed by striking interaction between the main eight-hour light three hours of darkness. He found that the insertion of period and the light perturbation. It appears that the PHOTOPERIODISM AND CIRCADIAN RHYTHMS 273

CYCLE NO. OF NODES DURATION FLOWERINO A ..". . HOUR: 4 l(/'30min" ligh! brlok ..~-: ...... I I0 -I':. "

a / "... :-"... LF --- 17 o 10 14. i i / ":...... "" m30 c -'"i "".. .-" 0 8 12 z IO -, ~,,~: V ~30 D :"" ]~.. ] " IO 8 16 '-I . .7 08 24 48. 72 O8 24 48 72 CYCLE DURATION - HOURS FIGURE 10 A-H. Data from Fig. 9. Standard control curve from Fig. 3, adjusted in magnitude to fit control curve of this experiment, is superimposed as dotted line. Each point on experimental curve represents flow- 19 ering response at that cycle length. Dashed lines repre- sent data from published work [5, 6].

14 84u ~3 showing that endogenous rhythms participate in a 421 tremendous variety of biological responses, it seems logical to assume that photoperiodism also involves the participation of the biological clock. Based upon this I0 22 assumption, it seems worthwhile to examine the known 8 4O 3 facts concerning photoperiodism and attempt to relate them to our knowledge of circadian rhythms. 6 BI)NNING'S HYPOTHESIS It may be recalled in this connection that Biinning, who has studied endogenous rhythms for some thirty years, has postulated [25] for the photoperiodic response FIGURE 9. Experimental design and results of experi- ment to show the effect on flowering of a light perturba- that "The regulation of the endogenous cycles by the tion given at specific times after the beginning of a light-dark periods, as is well known, makes the short- cycle in cycles of varying duration Light perturbation: day plants scotophil in the second half of the day, the 30 min. of 1000 ft. c. illumination. Each cycle repeated long-day plants photophil. But now we understand that 7 times. Data from Nanda and Hamner [9]. this is not due to a 12-hour phase difference of the basic cycles in the two types." basic rhythmic response of flowering is determined to a Btinning thus postulates that during the second half large extent by the main light period since all of the of the day the short-day plants are in the scotophil curves tend to resemble the control curve However, the phase of an endogenous rhythm and that exposure to light perturbation affects the oscillation of this curve light during this phase inhibits flowering. On the other and when given toward the end of a long dark period hand, the long-day plants during this same phase of the may cause a displacement of the curve to the extent endogenous rhythm are in the photophil phase, and that, in Fig. l0 G, a phase shift of approximately 12 exposure to light during this phase stimulates flowering. hours has occurred. Light perturbation given during As far as the author is aware, the effects of low in- the early part of the dark period has a marked de- tensities of light in shortening the dark period of a pressing effect upon the oscillations of the curve. In light-dark cycle or in interrupting such a dark period fact, in Fig. l0 B, where the light perturbation is given produce exactly the opposite effects in the two groups eight hours after the end of the main light (i.e., 16 of plants. Furthermore, the action spectrum of this hours after the beginning of the cycle), flowering is light effect in both groups seems to be identical [26, 27]. almost completely inhibited regardless of the length of We may therefore assume that the differences in the associated dark period or cycle. response between the long-day plants and the short-day In view of the preceding evidence, together with the plants involve a different reaction on the part of the abundant evidence presented at this symposimn plant to the same internal rhythmic oscillations. An 274 HAMNER

~o 4. HR. PHOTOPERIOD perturbation given about eight hours before the next 0---0 8 HR. ,, main light period would inhibit flowering since the z I0 H R. " light perturbation itself would be expected to initiate ~: 40 /~ X'--"X 12 HR. w 3= the scotophil phase of the rhythm. However, such an ] 3o interpretation does not fit with the results of Blaney tt. and Hamner [5] shown in Fig. 11, in which maxinmm ,.o, ao flowering occurs on a 24-hour cycle regardless of the O z IO length of the photoperiod or main light period in each cycle. It is obvious, therefore, that certain difficulties V- 0 o are involved in the application of Btinning's hypothesis 16 20 24 28 32 CYCLE DURATION - HOURS to fit all of the data. FIGURE 11. Flowering response of Biloxi soybean in COUPLED OSCILLATOR SYSTEM relation to cycle length with photoperiods (main light period) of differing durations. Data from Blaney and Pittendrigh and Bruce [14] on the basis of many Hamner [5]. years' work on endogenous rhythms in Drosophila and other organisms have postulated a coupled oscilla- tor system. They state: "The A oscillator is self- examination of the data obtained with Biloxi soybean sustaining; it can be coupled with and entrained by lends considerable support to the hypothesis that the light regime of the environment; when free-running exposure to light induces a rhythmic oscillation of some in aperiodic condition, its phase can be shifted by sort within the plant which has a period of about 24 single light signals; it is temperature independent; and hours. Furthermore, it would appear that exposure to it is coupled with and drives a second B oscillation. a small amount of light about 16 hours after the The B oscillator is the system whose rhythmic behavior beginning of this oscillation causes a very marked is more immediately reflected in the fly's overt rhythm; damping effect. Thus a light perturbation given 16 in some sense the fly reads phase (hence time) from the hours after the beginning of the main light period, in motion of B." This coupled oscillator system offers Fig. 10 B, caused a nearly complete inhibition of several attractive features. Oscillator A, since it is flowering regardless of the length of this cycle. Light reset by light, would be expected to be free-running perturbations given in phase with the main light during a long dark period and is supposed to be tem- period (i.e., 24 hours after the beginning of the main perature insensitive. The results of Nan da and Hamner light period) have no effect on the flowering response shown in Fig. 7 indicate the period of the oscillation is unless these perturbations consist of 30 minutes or temperature independent since the two peaks are 24 more of fairly intense light. If the light intensity and hours apart. However, a temperature dependent duration is high enough, they begin to add to the over- component is shown by the fact that tile rhythm is all response [10]. If the perturbations, coming in phase. delayed and that the peaks come about 12 hours out are of low intensity and of short duration, they neither of phase with the peaks at normal temperature. How- add nor detract from the over-all response. This must ever, it is the B oscillator (which is supposed to be mean that the general magnitnde of flowering or the temperature sensitive) in which we are interested here magnitude of the oscillations are determined by the at the moment. main light period. If the light perturbations come during In 1940 I demonstrated [15] that, in order to stimu- the latter part of a long dark period, the perturbation late flowering in either long-day or short-day plants, the may cause a phase shift in the oscillation but not in the main light period of the inductive day must contain high magnitude or degree of oscillation (see Fig. 10 F, G, HI. light intensities. The light energies required for the If we accept the Btinning hypothesis for the moment, stimulation of flowering in either group of plants is we could say that at the beginning of the main light many thousand times the energy requirements for the period a rhythm is initiated which, about 16 hours light perturbation effect. It has been generally assumed later, would be at the maximum amplitude of the that this high intensity light effect (main light period) scotophil phase, thus explaining the results in Fig. 10 B. was simply photosynthesis. However, in 1942 I raised However, Fig. 10 E, F, G, H, shows that maximum some questions concerning this interpretation [28], and flowering is obtained when a dark period of approxi- I believe the questions I raised then still hold today. mately 16 hours follows a light perturbation. One Let us consider the high intensity light reaction might assume that the beginning of the dark period purely in terms of circadian rhythms. In the first place, initiates the scotophil phase of the rhythm, and such high intensity light seems to participate in rhythmic an interpretation would indeed fit many of the facts. responses other than the photoperiodic response. For One would then expect that a light perturbation given example, Hastings and Sweeney [29] working with about eight hours after the end of the main light Gongaulax have found a loss in overt rhythmicity when period would inhibit flowering, and also that a light the organism is subjected to constant bright light, but PHOTOPERIODISM AND CIRCADIAN RHYTHMS 275

a retention of rhythmicity in constant dim light. It appears that the rhythmic leaf movement in plants [30] ~50 t /t," PHOTOPERIOD30-32 C" which gradually fades away when the plant is main- ~40t PHOTOPERIOD tained in constant darkness or in low intensity light may be restored only by exposures to high intensity light. There is abundant evidence that the effectiveness of , ,-., o 0 8 16 24 32 40 high intensity light in the photoperiodic response PHOTOPERTOD-HOURS increases with time during the first few hours of expo- FIGURE 12. Effect of duration and temperature of the sure [5, 10, 15, 31]. Sirohi and Hamner [10] obtained photoperiod (main light period) on flowering of Biioxi evidence with Biloxi soybean that the maximum soybean. Each photoperiod was associated with a dark period of sufficient length to give maximum values. Data effectiveness of the high light intensity was reached from Blaney and Hamner [5]. with light periods of about seven hours. Their work would not preclude the possibility that, at sufficiently high light intensities, the maximum effectiveness is temperature as at normal temperatures if sufficient time reached in a six-hour light period. With Biloxi soybean is allowed. In terms of a rhythm or oscillation we would at normal temperature Hamner [15] and Blaney and say that the low temperature has phase shifted the Hamner [5] have found that lengthening the high rhythm to a marked degree. We have also applied short intensity light period beyond a certain point will periods of low temperature at various places during a result in no flowering regardless of the length of the light-dark cycle [5]. It was found that such an applica- associated dark period. In contrast to this, Snyder tion of low temperature regardless of where it comes [32] found that the long-day plant Plantago flowered during the light-dark cycle seems to phase shift the regardless of the length of the cycle if the high intensity rhythmic flowering response. The inclusion of a few light period of each cycle was beyond a certain length. hours of low temperature either during the light period Thus, both long-day and short-day plants require or during the dark period causes the peaks of flowering periodic stimulation with high intensity light in order to appear in longer cycles than would be expected at to flower. If, however, the high intensity light period is normal temperatures. too long, the long-day plants flower and the short-day It appears, therefore, that there is a temperature plants fail to flower regardless of the associated dark sensitive as well as a temperature independent compo- period lengths. nent in the rhythmic response. If we assume two This high intensity light effect is quite temperature separate oscillator systems A and B as postulated sensitive. Mann [31] has shown the high temperature above, it is tempting to speculate that oscillator B, the coefficient when high intensity light is used to cause temperature dependent system, is stimulated when the stimulation of flowering in Xanthium. With Biloxi plant is exposed to high intensity light. One of the soybean, we have shown [6] that low temperature difficulties of interpretation when considering the high during the high intensity main light period may greatly intensity light effect is the fact that conditions which decrease the amplitude of the rhythmic flowering bring about the high intensity light effect also produce response. the low intensity light effect. At the moment I can only In addition we have found another effect of tempera- say that the coupled oscillator system may in some ture on the high intensity main light period [5]. Light way be involved in the response. periods of 16 hours or more at normal temperatures, as CONCLUSIONS mentioned, inhibit flowering regardless of the length of the associated dark period. At low temperatures, high It seems obvious that in the photoperiodic response of intensity light periods of 16 hours appear to produce plants we are dealing with an endogenous rhythm or the optimum effect on flowering and light periods as rhythms by means of which the plant tells time; that is, long as 32 hours produce some flowering. While our the perception of day length depends upon the rhythms. data are quite incomplete, the best interpretation of our Furthermore, it would appear that the identical data at present is summarized in Fig. 12. The effect of rhythm or clock is read by the short-day plant in temperature on the main light period in stimulating exactly the opposite fashion as by the long-day plant. flowering and the effect of the length of the main light One is tempted to speculate that conditions which period in inhibiting flowering indicate a very high damp out some oscillator system inhibit flowering in temperature coefficient of the high intensity light short-day plants and that the same damping out of the effect. It may be noted that low temperature seems to oscillator causes flowering in long-day plants. at least double the length of time necessary for the As far as the author is aware, experiments of the types main light period to reach its maximum effectiveness, described above for Biloxi soybean and other plants but the degree of effectiveness of the main light period have not been carried out to any extent with animals. in stimulating flowering is at least as high at low There is no reason to believe that the photoperiodic 276 HAMNER responses in animals are not the same as the responses 14. PITTENDRIGH,C. S., and V. G. BRUCE. 1959. Daily in plants, but a decision cannot be made until appropri- rhythms as coupled oscillator systems and their relation to thermotropism and photoperiodism. ate experiments are performed. It is hoped that the pp. 475-505. Photoperiodism and Related Phenomena apparatus we have recently developed [33] for exposing in Plants and Animals, ed. Withrow. Washington: organisms to different regimens of light-dark cycles A.A.A.S. may stimulate the accumulation of data with many 15. HAMNER, K. C. 1940. Interrelation of light and dark- organisms similar to that described above. It is felt ness in photoperiodic induction. Bot. Gaz., 101: 658-687. that such further studies in photoperiodism may 16. HAMNER, K. C., and J. BONNER. 1938. Pholoperi- elucidate the complex mechanisms of the biological odism in relation to hormones as factors in floral clock. initiation and development. Bot. Gaz., 100: 388- 431. ACKNOWLEDGEMENTS 17. CARR, D. J. 1952. The photoperiodic behavior of short-day plants. Physiol. Plantarum, 5: 70-84. Finally, I wish to express my sincere appreciation to 18. CARR, D. J. 1952. A critical experiment on Biinning's G. S. Sirohi, T. Hoshizaki, and Bruce Carpenter for theory of photoperiodism. Zeit. f. Naturf., 7b: their critical help in preparing this manuscript and 570-571. 19. CLAES, H., and A. LANG. 1947. Die bliitenbildung making the figures. yon Hyoscyamus niger in 48-sttindiger lichV dunkel-rhythmen und in zyklen mit aufgeteilter REFERENCES lichtphasen. Zeit. f. Naturf., 2b: 56. 1. International Symposium on: Photoperiodism and 20. CLAUSe, H., and W. RAU. 1956. Uber die bliiten- Related Phenomena in Plants and Animals, held at bildung yon Hyoscyamus niger und Arabidopsis Gatlinburg, Tennessee, and sponsored by the U. S. thaliana in 72-stunden zyklen. Zeit. f. Botan. National Science Foundation. Publication No. 55 ~4: 437-454. for the American Association for the Advance- 21. SCHWABE, W. W. 1955. Photoperiodic cycles of ment of Science. 1959. lengths differing from 24-hours in relation to the 2. BUSSING, E. 1936. Die endonome tagesrhythmik ale endogenous rhythm. Physiol. Plantarum, 8: grundlage der photoperiodischen reaktion. Ber. 273-278. deutsch, bot. Gee., 5~: 590. 22. MELCHERS, G. 1956. Die beteiligung der endonomen 3. DOORENBOS, J., and S. J. WELLENSIEK. 1959. 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periods in cycles of any desired duration. Plant realize the limitation of Btinning's hypothesis. I have Phys., 85: 276-278. already pointed out specific difficulties involved in 34. LINCOLN, R. G., K. A. RAVEN, and K. C. HAMNER. 1956. Certain factors influencing the expression of explaining some results on the basis of Btinning's the flowering stimulus in Xanthium. Part I. Trans- hypothesis (see my comment on Bfinsow's paper). location and inhibition of the flowering stimulus. Hamner has also pointed out such difficulties. In view Bot. Gaz., 117: 193-206. of the above fact, Pittendrigh's suggestion for light DISCUSSION break e• based on a di-diurnal or tri-diurnal system is contradictory. SmoHi: Pittendrigh in his comment has brought up the Use of the odd cycles to further elucidate the entrain- point that Hamner's approach with odd cycles has less merit phenomenon to study endogenous rhythms is one bearing in analyzing Bfinning's hypothesis than of the basic tools to elucidate the entrainability of Biinsow's approach of light break experiments. rhythms as indicated by many experiments reported in First I want to point out that Pittendrigh and Bruce this symposium. Its special significance in flowering in their publication (Gatlinburg Symposium 1957) said response is evident from Hamner's paper. that photoperiodic and thermoperiodic effects on I may further point out that Hamner has also used growth efficiency which are related to endogenous light break technique. He has not only indicated" the rhythms can not be properly elucidated as long as the results dealing with di-diurnal, tri-diurnal systems but interpretation is restricted to special terms of Bfinning's has additional information on intermediate diurnal views that the endogenous cycles are comprised of systems. distinct photophil and scotophil fractions. They further stated that most fruitful insights and suggestions for PITTENDRIGH: It is clear that Mr. Sirohi completely new experiments arise from a broader picture given by a misunderstood the comparison I made between Biin- generalized oscillation model. They specifically empha- sow's approach and Hamner's. The merits of Biinsow's sized the entrainment phenomenon, on the basis of are noted in his own reply (pg. 260) to an earlier which they attempted the interpretation of the above- question of Sirohi, and so need not be itemized here. mentioned relationship. Although they did not take The crucial point is the simultaneous analysis of a) a into consideration the flowering response in their photoperiodic response and b) a known circadian discussion, it is apparent from their views that they rhythm in one and the same organism.