Planta
Planta 143, 323-328 (1978)
9 by Springer-VerIag 1978
The Circadian Rhythm in Bryophyllum Leaves: Phase Control by Radiant Energy Philip J.C. Harris* and Malcolm B. Wilkins Botany Department, Glasgow University, Glasgow G12 8QQ, U.K.
Abstract. A 4-h exposure to white light from fluorescent lamps can shift the phase of the rhythm of CO2 output in leaves of Bryophyllum fedtschenkoi Hamet & Perr. otherwise kept in continuous darkness. The position in the cycle at which irradiation occurs determines the magnitude and direction of the phase shift. Red and white light induce similar advances or delays in the phase, but blue and far-red irradiation have no effect. Far-red irradiation given simultaneously with, or immediately after, exposure to red light, modifies the phase-shift induced by red light alone. Radiation in the red and far-red regions of the spectrum interacted in several experimental r6gimes, but complete red/far-red reversibility was not observed. The evidence suggests that phytochrome is the receptor molecule involved in the induction of phase-shifts by light. Key words: Bryophyllum - Circadian rhythm - Photocontrol - Phytochrome.
Introduction
In continuous darkness, detached leaves of Bryophyllure fedtschenkoi exhibit a circadian rhythm in their rate of carbon dioxide output. The phase of this rhythm is shifted by exposure to white light between the peaks but not by exposure near the apex of a peak (Wilkins, 1960). Investigation of the phase-shift induced by a 4-h exposure to monochromatic radiation at a particular time in the cycle between the peaks showed activity to be confined to wavelengths between 560 and 700 nm, with a maximum at 660 nm. This wavelength dependence suggests that phytochrome is the photoreceptor involved in the phasePresent address: Department of Biological Studies, Lanchester Polytechnic, Priory Street, Coventry, CV1 5FB, U.K. *
shifting response (Wilkins, 1973). Red and white light are similar in their effects on the period of the rhythm (Harris and Wilkins, 1976), and in entraining the rhythm to cycles of light and darkness (Harris and Wilkins, 1978). In the latter case, complete red/far-red reversibility, indicative of phytochrome involvement, was observed. Entrainment of the rhythm, and modification of its period, have also been achieved with far-red radiation (730 nm), although wavelengths longer than 700 nm are less effective than those in the red region of the spectrum. The photoreceptors associated with the control of circadian rhythms have been investigated in a number of species. Although the rhythms in different species display generally similar responses to light, the photoreceptors appear to be diverse (Hastings and Sweeney, 1960; Sargent and Briggs, 1967; Wilkins, 1973). In some organisms different pigments even appear to be involved in controlling different aspects of the rhythm. For example, chlorophyll may be the photoreceptor involved in the initiation of the petal movement rhythm of KalanchoO blossfeldiana by transfer from light to darkness or vice versa (Karv6 et al., 1961), while another pigment appears to be responsible for shifting the phase of this rhythm (Schrempf, 1975). Different regions of the spectrum are active in shifting the phase of the leaf movement rhythms of Coleus blumeii x C.frederici (Halaban, 1969) and Phaseolus multiflorus (Bfinning and Moser, 1966), and in both species different spectral zones appear to be active at different times in the cycle. These findings suggest the occurrence in some organisms of complex control mechanisms apparently involving more than one photoreceptor. This paper reports a study of light-induced phaseshifts in the Bryophyllum rhythm, and of the photoreceptor pigment associated with this response.
0032-0935/78/0143/0323/$01.20
324
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in
80 6C 40 20 0 60 40 20
Material and Methods Plants of Bryophyllum (Kalancho~)fedtschenkoi Hamet et Perrier were grown in a heated glass-house at a minimum temperature of 18 ~ C. Experimental plants were transferred from the glass-house to a controlled environment room and maintained for at least 7 days with an 8-h photoperiod provided by white and warm-white fluorescent lamps at an incident radiant flux density of 47.3 J m - 2 s - ~ . The temperature was 25 ~ C during the photoperiod and 15 ~ C in darkness. The rate of carbon dioxide output of leaves into initially carbon-dioxide-free air was measured with Grubb-Parsons SB1 or SB2 infra-red gas analysers as described by Wilkins (1973). Monochromatic radiation in 25-nm wide spectral bands was obtained with Bausch and Lomb, High Intensity grating monochromators. Two layers of Cinemoid Orange (No. 5) filter were inserted in the beam when the monochromators were set at wavelengths longer than 560 nm to eliminate overlapping blue light of other order spectra. The far-red radiation used in red-far-red reversal experiments was obtained by passing the columnated beam from a 45-W tungsten halogen lamp through a Corning 7-69 far-red filter. Leaves were detached from the plants, weighed, placed in chambers and transferred to constant darkness and a temperature of 15 _+0.1 ~ at 1600 h, the end of the photoperiod. In each experiment two samples of leaves were irradiated while a third was kept in darkness. The traces given to illustrate the results are representative of data obtained in at least two, but usually more, independent experiments. The rate of carbon dioxide output of the leaves was calculated in pg CO2 h l g (fresh weight)-t and plotted hourly against the time of day.
Results
Bryophyllum
40 20 v,
0
z 80 9~ 60 4O 20 0 o
qJ
40 20
Phase Control by Single Light Treatments The relative effectiveness of a standard exposure to radiant energy in shifting the phase of the rhythm was assessed as a function of the time in the cycle at which it was applied. Leaves were exposed for 4 h to white fluorescent light at a flux density of 0.6 Jm-2s -1. One and a half cycles of the rhythm were scanned at intervals of 1 or 2 h beginning at 0400 h, 12 h after the leaves had been transferred from the growth room to continuous darkness. The results of several experiments are shown in Figure 1. The phase of the rhythm was advanced by light treatments which began in the trough before the first peak of the rhythm just before the rate of CO2 output began to rise from its minimum value (Fig. 1 A and B). No phase shift was induced when the mid-point of the light treatment occurred 22-24 h after the leaves were placed in darkness (Fig. 1 C), whereas the phase was delayed by treatments applied after the first peak of carbon dioxide output (Fig. 1 D). In Figure 1 D a small peak of CO2 output occurred soon after the end of the light period. As the light interruptions were given later in this part of the cycle, the minor peak became larger until it clearly constituted the advanced second peak (Fig. 1 E and F). Irradia-
i
80 60 40 20 OI
F
,
,~ "~
,
o
6
."~
t e'~" t ' "
6'6'6' Time of day
Fig. 1A-F. Effects of a 4-h exposure to white fluorescent light (0.6 J m - 2 s - I) on the phase of the circadian rhythm of carbon dioxide output in Bryophyllurn leaves otherwise kept in continuous darkness. The times of the light treatments are shown by the shaded bars. The rhythms in control, unirradiated leaves are shown by the broken lines. 0 = midnight
tion during the second cycle had essentially the same effect as in the first cycle. The combined results of this series of experiments are shown in Figure 2. When the phase of the rhythm was reset, a peak of carbon dioxide emission occurred between 21.5 and 29 h after the end of the light interruption. The magnitude of the phase-shift induced by the light
Bryophyllum
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in I
46
I
4o
I" 9 9
42 v
9
38
I~
~- 34
I~
.~" ,~'
9
9
20
:
/'
0"
I
I
iil}
I
I
I "" I 9
30
~ ~i:;! ;' i~ /
"
6o '
,.,,::!::! ,~-, .
40[
i ~i !
20
! ;?:!
.,.........
.
.
.
9 I
=~ 26 "6
~
I
9
~I
9
22
r 9
18
.@~ ~
"1
:.
14
',
. . . . . . . .
o
9 I I I
I
610
410
20
0
~I0
100
120
Time (h)
Fig. 2. The time of occurrence of peaks of carbon dioxide output from Bryophyllum leaves exposed to white light for 4 h at the times indicated by the shaded bar. The times of the mid-points of the light treatments are shown on the ordinate. The times at which peaks occur in control, unirradiated leaves are shown by the broken lines. Each point is the mean of at least two, and in most cases more, individual measurements. The times of occurrence of peaks of the control rhythms are the means of 24 experiments
_'..
325
..--, !i::i
40
40
20
20 ~
0
0 :i
40
40
20
2C
D
' iii
s i
i
I ~:: i
.... I
,
I i I i
0:22
i
0
0
i
i
i
0
0
0
Time of day
Fig. 4 A - D . Effects of exposure to 4 h red light A, 4 h far-red radiation B, 4 h of red light followed by 4 h of far-red radiation C, and red and far-red radiation simultaneously for 4 h D, on the phase of the circadian rhythm in Bryophyllumleaves. The times of the irradiations are shown by the shaded bars. Control rhythms in leaves in continuous darkness are shown by the broken lines. 0 = midnight
~: 0 c~ u
40
4(
20
20
0
0
0
0
0
_. ~ ,,, , !iii
0
0
F
0
0
0
Time of day
between 0400 and 0800 h in the same cycle (Fig. 3 E). The 450-nm and 730-nm spectral bands had no measurable effect on the phase of the rhythm at either time in the cycle (Fig. 3 A, C, D and F).
Fig. 3 A-F. Effectiveness in inducing phase-shifts, of a 4-h exposure to monochromatic radiation in spectral bands centred at 450 nm A and D, 660nm B and E, and 7 3 0 n m C and F. The times of irradiation are shown by the shaded bars. Rhythms in leaves in continuous darkness are shown by the broken lines. 0 = m i d n i g h t
Interaction of Red and Far-red Radiation in Phase Control
treatments was virtually the same in three successive cycles. A new steady-state was thus attained, at the latest 21.5-29 h after the end of the light treatment. Leaves kept in prolonged darkness were exposed to monochromatic radiation for 4 h at a quantum flux density of 47 pE cm-28 1. Spectral bands were selected to include wavelengths (660 nm) previously found to be active in shifting the phase, and those (450 nm and 730 nm) which were not (Wilkins, 1973). The effectiveness of each band in shifting the phase was tested at several times in the cycle; the results are shown in Figure 3. The 660-nm spectral band induced a phase delay of 3 4 h when applied between 2000 and 0000 h during the second day in darkness (Fig. 3B) and a phase advance of 6 h when applied
Exposing leaves for 4 h to red light (660 nm) at a radiant flux density of 85 m J m - 2 s - 1 resulted in a phase-shift of about 12 h (Fig. 4A) whereas exposure to far-red radiation (7.8 j m - 2 s -1) had no effect (Fig. 4B). Exposure to red light for 4 h, followed immediately by an exposure to far-red radiation for 4 h, resulted in a marked phase-shift (Fig. 4C), although its magnitude was less than that obtained with red light alone (Fig. 4A). Simultaneous exposure to red and far-red radiation for 4 h (Fig. 4 D ) virtually abolished the circadian rhythm of carbon dioxide output and it was not possible to assess whether a phase-shift was induced. The lack of clear red/far-red reversibility in these experiments might be attributed to the length of exposure being sufficient for the phase-shifting process to proceed beyond the reversible photochemical
P.J.C. Harris and M.B. Wilkins: Circadian R h y t h m in BryophylIum
326
,"-.
A
...
[3
10 C 3O
40
,. t
iiii
A
E
,..
20
;ii'~: ,;- .... 0
,'",
20
"
,
ir
i
...---. .......... i
,
i
,
i
J= ,
i
lC
~n
'x-
i
I
i
i
2c lC 0
i
,
I:;:;
,
i
I
i
,
i
eo
,
i
c
G 3C
i
i
D
~_ 40 20
I:?
0 0
::~: 0
'
:
,
'
, 0
~ O
0
L 0
L
,
0
i
0
0
0
0
T i m e of d a y
Time of day
Fig. 5 A - C . Effects of exposing leaves of Bryophyllum for 4 h to:5 rain of red light alternating with 5 rain of darkness A, 5 rain of red light alternating with 5 rain of far-red radiation B, the r~gime in A superimposed on a 4-h continuous exposure to far-red radiation C. Times of irradiation are shown by the shaded bars. The broken lines show the r h y t h m in unirradiated, control leaves. 0 = midnight
Fig. 6 A - D . Effect of exposing leaves of Bryophyllum for 4 h to :-10 s of red light alternating with 30 s of darkness A, 10 s of red light aiternating with 30 s of far-red radiation B, the r6gime used in A superimposed upon a continuous exposure to far-red radiation for 4 h C and D. Times of irradiation are shown by the shaded bars. The broken lines show the r h y t h m in unirradiated leaves. 0 = midnight
stages. In an attempt to obviate this difficulty leaves were exposed for 4 h to rapid alternation of red light and darkness. Alternation of 5 min of red light with 5 rain of darkness for 4 h, induced a distinct phaseshift (Fig. 5 A). When 5 min of red light alternated with 5 rain of far-red radiation for 4 h (Fig. 5 B) there was not a clear abolition of the phase-shift effect induced by red light alone, but, rather, a considerable reduction of the amplitude of the rhythm to the extent that it was scarcely discernible in subsequent darkness. Continuous exposure of leaves to far-red radiation for 4 h superimposed on the 5 rain red/5 min darkness r6gime caused a loss of the circadian nature of the rhythm; small peaks appeared at 12-h intervals (Fig. 5 C). Again, a clear reversal of the effectiveness of red light by an exposure to far-red radiation could not be found in so far as the induction of phase-shifts was concerned. There is no doubt, however, that the far-red radiation interacts in some way with the red light and leads to a loss of the circadian nature of the rhythm. Several different irradiation r~gimes were used over a period of 4 h to test for red/far-red reversibility. For example, 10 s of red light followed by 30 s of darkness led to the induction of a substantial phaseshift (Fig. 6A), but 10 s of red light followed by 30 s of far-red radiation led, once again, to the virtual
abolition of the circadian rhythm (Fig. 6B). When this irradiation r6gime was applied to leaves with a simultaneous and continuous exposure to far-red radiation, the result was variable and difficult to interpret. Either, a very slight phase-shift was induced in the rhythm (Fig. 6C), or the rhythm again appeared to lose its circadian nature altogether (Fig. 6D).
Discussion
A 4-h exposure to light can reset the phase of the rhythm of carbon dioxide metabolism in leaves of Bryophyllum but the magnitude and direction of the phase-shift depends on the time in the cycle at which the exposure occurs. The phase-response of Bryophyllum to a light stimulus is thus basically similar to that reported for a number of other organisms (Aschoff, 1965; Pittendrigh, 1965). In an earlier study in which light was applied at only two times in the cycle (Wilkins, 1960), it was found that the phase was reset so that the next peak of carbon dioxide emission occurred a specific time after the end of a light treatment. It is now apparent from this more detailed investigation, that the time between the end of the light treatment and the occurrence of a peak
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in Bryophyllurn of carbon dioxide output is not constant but varies between 21.5 and 29 h, depending on the time in the cycle at which the leaves are irradiated. Furthermore, this peak may not necessarily be the first after the end of the light treatment; in some instances a peak occurs a few hours after the end of the light treatment but still within the same circadian cycle (e.g. Fig. 1 E and F). The occurrence of a peak 21.5 to 29 h after the end of the light interruption permits the prediction that at some point in the cycle a light treatment should be without effect on the phase of the rhythm. This has been observed in the present and previous (Wilkins, 1960) studies. While the net effect of the light treatment is to advance or delay the phase of the rhythm it is difficult, in practice, to distinguish unequivocally between phase advances and phase delays when the light perturbations are applied during some parts of the cycle. This is because in a number of experiments a temporary increase in the rate of carbon dioxide release occurred during, or soon after, the leaves were irradiated. Similar peaks of carbon dioxide emission appear during entrainment of the rhythm to light-dark cycles (Harris and Wilkins, 1976). It is not clear whether the increased rate of carbon dioxide output represents a direct effect of radiant energy on the biochemical processes monitored as the overt rhythm, or an effect on the basic circadian oscillator. When the phase of rhythms in organisms such as Drosophila (Pittendrigh and Bruce, 1957) is shifted by a single exposure to light, there may occur several transient cycles before the phase attains a new steadystate. Advancing phase-shifts often pass through several transient cycles while delaying phase-shifts tend to reach their final values more rapidly (Aschoff, 1965). In other organisms such as Gonyaulax, a new stable phase is reached immediately after either delaying or advancing shifts (Hastings and Sweeney, 1958). In Bryophyllum a new stable phase is attained rapidly; the phase difference, whether an advance or delay, between the rhythm reset by a light perturbation and the control rhythm persisting in darkness is virtually the same in each of the three cycles following the stimulus. At no point in the cycle was blue or far-red radiation active in shifting the phase of the rhythm, whereas red light was active at all points of the cycle where a phase-shift could be induced by white light. It thus seems probable that the same photoreceptor is responsible for mediating both phase advances and phase delays. This is apparently not so in all organisms, since in both Phaseolus (B/inning and Moser, 1966) and Coleus (Halaban, 1969) different wavelengths of radiant energy are effective in inducing phase-shifts at different times in the circadian cycle.
327 Several kinds of experimental approach, using sequential or simultaneous exposure to red light and far-red radiation, showed that these spectral regions interact in their effects on the rhythm. This finding suggests the involvement of phytochrome in the response. The red/far-red interaction quite frequently did not take the form of a clear reversal of the effectiveness of red by far-red radiation, but rather the virtual abolition of the rhythm when far-red radiation was applied. The abolition of the rhythm may arise in a number of ways. For example, far-red radiation may interact with red to abolish oscillation of the basic circadian system in each cell of the leaf. On the other hand, total reversal of the phase-shift induced by red light may occur in some cells and not others. This would result in the loss of the overt rhythm because the rhythms in some cells would be approximately 12 h out of phase with those in other cells. The occurrence of small peaks at 12-h intervals in some phase-shift experiments could result from such cellular desynchronization. The leaves are 3 5 mm thick and it is not possible to ensure that each constituent cell is equally irradiated. The photocontrol of a number of aspects of the Bryophyllum rhythm has now been investigated (Wilkins, 1960, 1973; Harris and Wilkins, 1976, 1978). Blue light is totally without effect on the phase and period of the rhythm and does not induce entrainment. Modification of the period (Harris and Wilkins, 1976) and entrainment of the rhythm (Harris and Wilkins, 1978) can be achieved with both monochromatic red (660 nm) and far-red (730 nm) radiation, but the latter does not modify the transient (Harris and Wilkins, 1976) or induce phase-shifts when applied as a single 4-h perturbation. The observation of red/far-red reversibility in the entrainment of the rhythm (Harris and Wilkins, 1978), and an interaction of these spectral bands in its phase-shift response, leave little doubt that phytochrome is the photoreceptor involved in each aspect of the control of the Bryophyllum rhythm by light. Explanation of the different sensitivities of the phase-shift and entrainment responses to the 730-nm spectral band should perhaps be sought in terms of phytochrome action rather than in the possible involvement of other pigments. Phytochrome also appears to be the pigment involved in the photocontrol of the rhythms of carbon dioxide output in Lernna (Hillman, 1971) and of movement in excised pulvini of Samanea (Simon et al., 1976). There is also evidence that phytochrome controls the rhythms in sensitivity to flower induction in Xanthium (Salisbury, 1965; Denney and Salisbury, 1970) and in germination promotion in Sphaerocarpos donelli (Steiner, 1969). It may also be involved in the phaseshift response of the KalanchoO petal movement
328
rhythm (Schrempf, 1975). While phytochrome appears to be involved in photocontrol of circadian rhythms in many higher plants, it is by no means the only photoreceptor pigment associated with rhythmic systems in plants. Phytochrome is not the photoreceptor in the alga Oedogonium cardiacum (Btihnemann, 1955), the photosynthetic marine dinoflagellate Gonyaulax polyedra (Hastings and Sweeney, 1960), or the fungus Neurospora crassa (Sargent and Briggs, 1967). Pigments other than phytochrome also appear to be involved in the photocontrol of rhythms in some higher plants such as Portulaca (Karv6 and Jigajinni, 1966) and Coleus (Halaban, 1969).
References Aschoff, J. : Response curves in circadian periodicity. In : Circadian Clocks, pp. 95 111, Aschoff, J., ed., Amsterdam: North Holland 1965 Btihnemann, F.: Das endodiurnale System der Oedogoniumzelle IV. Die Wirkung verschiedener Spektralbereiche auf die Sporulation and Mitoserhythmik. Planta 46, 227-255 (1955) Bfinning, E., Moser, I.: Response-Kurven bei der circadianen Rhythmik yon Phaseolus. Planta 69, 101 110 (1966) Denney, A., Salisbury, F.B.: Separate clocks for leaf movement and photoperiodic flowering in Xanthium strumarium L. Plant Physiol. 46, Suppl. 26 (1970) Halaban, R.: Effects of light quality on the circadian rhythm of leaf movement of a short-day-plant. Plant Physiol. 44, 973 977 (1969) Harris, P.J.C., Wilkins, M.B. : Light-induced changes in the period of the circadian rhythm of carbon dioxide output in Bryophyllurn leaves. Planta 129, 253-258 (1976) Harris, P,J.C., Wilkins, M.B.: Evidence of phytochrome involvement in the entrainment of the circadian rhythms of CO2 metabolism in Bryophyllum. Planta 138, 271 278 (1978) Hastings, J.W., Sweeney, B.M.: A persistent diurnal rhythm of luminescence in Gonyaulax polyedra. Biol. Bull. 115, 440-458 (1958)
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in Bryophyllum Hastings, J.W., Sweeney, B.M.: The action spectrum for shifting the phase of the rhythm of luminescence in Gonyaulaxpolyedra. J. Gen. Physiol. 43, 697-706 (1960) Hillman, W.S.: Entrainment of Lemna CO2 output through phytochrome. Plant Physioi, 48, 770-774 (1971) Karv6, A., Engelmann, W., Schoser, G.: Initiation of rhythmical petal movements in KalanchoO blossfeldiana by transfer from continuous darkness to continuous light or vice versa. Planta 56, 700-711 (1961) Karv6, A.D., Jigajinni, S.G. : Pigment system involved in the photoperiodic entrainment of circadian leaf movements. Naturwissenschaften 53, 181 (1966) Pittendrigh, C.S. : On the mechanism of the entrainment of a circadian rhythm by light cycles. In : Circadian Clocks, pp. 277-297, Aschoff, J., ed., Amsterdam: North Holland 1965 Pittendrigh, C.S., Bruce, V.G.: An oscillator model for biological clocks. In: Rhythmic and Synthetic Processes in Growth, pp. 75-109, Rudnik, D., ed., Princeton: Princeton University Press 1957 Salisbury, F.B. : Time measurement and the light period in flowering. Planta 66, 1 26 (1965) Sargent, M.L., Briggs, W.R.: The effects of light on a circadian rhythm of conidiation in Neurospora. Plant Physiol. 42, 1504-1510 (1967) Schrempf, M.A. : Eigenschaften und Lokalisation des Photoreceptors ffir phasenverschiebendes St6rlicht bei der B1/itenblattbewegung yon Kalancho~ blossfeldiana (v. Poelln). Doctoral Thesis, Universitfit Tfibingen, W-Germany 1975 Simon, E., Satter, R.L., Galston, A.W.: Circadian rhythmicity in excised Samanea pulvini. II. Resetting the clock by phytochrome conversion. Plant Physiol. 58, 421-425 (1976) Steiner, A.M. : Changes of the endogenous rhythm in phytochrome mediated spore germination of the liverwort Sphaerocarpos during spore after ripening. Z. Pflanzenphysiol. 61, 184-191 (1969) Wilkins, M.B. : An endogenous rhythm in the rate of CO2 output of Bryophyllum. II. The effects of light and darkness on the phase and period of the rhythm. J. exp. Bot. 11,269 288 (1960) Wilkins, M.B. : An endogenous rhythm in the rate of carbon dioxide output of Bryophyllum. VI. Action spectrum for the induction of phase shifts by visible radiation. J. exp. Bot. 24, 488 496 (1973)
Received 4 August; accepted 10 August 1978
Planta 143, 323-328 (1978)
9 by Springer-VerIag 1978
The Circadian Rhythm in Bryophyllum Leaves: Phase Control by Radiant Energy Philip J.C. Harris* and Malcolm B. Wilkins Botany Department, Glasgow University, Glasgow G12 8QQ, U.K.
Abstract. A 4-h exposure to white light from fluorescent lamps can shift the phase of the rhythm of CO2 output in leaves of Bryophyllum fedtschenkoi Hamet & Perr. otherwise kept in continuous darkness. The position in the cycle at which irradiation occurs determines the magnitude and direction of the phase shift. Red and white light induce similar advances or delays in the phase, but blue and far-red irradiation have no effect. Far-red irradiation given simultaneously with, or immediately after, exposure to red light, modifies the phase-shift induced by red light alone. Radiation in the red and far-red regions of the spectrum interacted in several experimental r6gimes, but complete red/far-red reversibility was not observed. The evidence suggests that phytochrome is the receptor molecule involved in the induction of phase-shifts by light. Key words: Bryophyllum - Circadian rhythm - Photocontrol - Phytochrome.
Introduction
In continuous darkness, detached leaves of Bryophyllure fedtschenkoi exhibit a circadian rhythm in their rate of carbon dioxide output. The phase of this rhythm is shifted by exposure to white light between the peaks but not by exposure near the apex of a peak (Wilkins, 1960). Investigation of the phase-shift induced by a 4-h exposure to monochromatic radiation at a particular time in the cycle between the peaks showed activity to be confined to wavelengths between 560 and 700 nm, with a maximum at 660 nm. This wavelength dependence suggests that phytochrome is the photoreceptor involved in the phasePresent address: Department of Biological Studies, Lanchester Polytechnic, Priory Street, Coventry, CV1 5FB, U.K. *
shifting response (Wilkins, 1973). Red and white light are similar in their effects on the period of the rhythm (Harris and Wilkins, 1976), and in entraining the rhythm to cycles of light and darkness (Harris and Wilkins, 1978). In the latter case, complete red/far-red reversibility, indicative of phytochrome involvement, was observed. Entrainment of the rhythm, and modification of its period, have also been achieved with far-red radiation (730 nm), although wavelengths longer than 700 nm are less effective than those in the red region of the spectrum. The photoreceptors associated with the control of circadian rhythms have been investigated in a number of species. Although the rhythms in different species display generally similar responses to light, the photoreceptors appear to be diverse (Hastings and Sweeney, 1960; Sargent and Briggs, 1967; Wilkins, 1973). In some organisms different pigments even appear to be involved in controlling different aspects of the rhythm. For example, chlorophyll may be the photoreceptor involved in the initiation of the petal movement rhythm of KalanchoO blossfeldiana by transfer from light to darkness or vice versa (Karv6 et al., 1961), while another pigment appears to be responsible for shifting the phase of this rhythm (Schrempf, 1975). Different regions of the spectrum are active in shifting the phase of the leaf movement rhythms of Coleus blumeii x C.frederici (Halaban, 1969) and Phaseolus multiflorus (Bfinning and Moser, 1966), and in both species different spectral zones appear to be active at different times in the cycle. These findings suggest the occurrence in some organisms of complex control mechanisms apparently involving more than one photoreceptor. This paper reports a study of light-induced phaseshifts in the Bryophyllum rhythm, and of the photoreceptor pigment associated with this response.
0032-0935/78/0143/0323/$01.20
324
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in
80 6C 40 20 0 60 40 20
Material and Methods Plants of Bryophyllum (Kalancho~)fedtschenkoi Hamet et Perrier were grown in a heated glass-house at a minimum temperature of 18 ~ C. Experimental plants were transferred from the glass-house to a controlled environment room and maintained for at least 7 days with an 8-h photoperiod provided by white and warm-white fluorescent lamps at an incident radiant flux density of 47.3 J m - 2 s - ~ . The temperature was 25 ~ C during the photoperiod and 15 ~ C in darkness. The rate of carbon dioxide output of leaves into initially carbon-dioxide-free air was measured with Grubb-Parsons SB1 or SB2 infra-red gas analysers as described by Wilkins (1973). Monochromatic radiation in 25-nm wide spectral bands was obtained with Bausch and Lomb, High Intensity grating monochromators. Two layers of Cinemoid Orange (No. 5) filter were inserted in the beam when the monochromators were set at wavelengths longer than 560 nm to eliminate overlapping blue light of other order spectra. The far-red radiation used in red-far-red reversal experiments was obtained by passing the columnated beam from a 45-W tungsten halogen lamp through a Corning 7-69 far-red filter. Leaves were detached from the plants, weighed, placed in chambers and transferred to constant darkness and a temperature of 15 _+0.1 ~ at 1600 h, the end of the photoperiod. In each experiment two samples of leaves were irradiated while a third was kept in darkness. The traces given to illustrate the results are representative of data obtained in at least two, but usually more, independent experiments. The rate of carbon dioxide output of the leaves was calculated in pg CO2 h l g (fresh weight)-t and plotted hourly against the time of day.
Results
Bryophyllum
40 20 v,
0
z 80 9~ 60 4O 20 0 o
qJ
40 20
Phase Control by Single Light Treatments The relative effectiveness of a standard exposure to radiant energy in shifting the phase of the rhythm was assessed as a function of the time in the cycle at which it was applied. Leaves were exposed for 4 h to white fluorescent light at a flux density of 0.6 Jm-2s -1. One and a half cycles of the rhythm were scanned at intervals of 1 or 2 h beginning at 0400 h, 12 h after the leaves had been transferred from the growth room to continuous darkness. The results of several experiments are shown in Figure 1. The phase of the rhythm was advanced by light treatments which began in the trough before the first peak of the rhythm just before the rate of CO2 output began to rise from its minimum value (Fig. 1 A and B). No phase shift was induced when the mid-point of the light treatment occurred 22-24 h after the leaves were placed in darkness (Fig. 1 C), whereas the phase was delayed by treatments applied after the first peak of carbon dioxide output (Fig. 1 D). In Figure 1 D a small peak of CO2 output occurred soon after the end of the light period. As the light interruptions were given later in this part of the cycle, the minor peak became larger until it clearly constituted the advanced second peak (Fig. 1 E and F). Irradia-
i
80 60 40 20 OI
F
,
,~ "~
,
o
6
."~
t e'~" t ' "
6'6'6' Time of day
Fig. 1A-F. Effects of a 4-h exposure to white fluorescent light (0.6 J m - 2 s - I) on the phase of the circadian rhythm of carbon dioxide output in Bryophyllurn leaves otherwise kept in continuous darkness. The times of the light treatments are shown by the shaded bars. The rhythms in control, unirradiated leaves are shown by the broken lines. 0 = midnight
tion during the second cycle had essentially the same effect as in the first cycle. The combined results of this series of experiments are shown in Figure 2. When the phase of the rhythm was reset, a peak of carbon dioxide emission occurred between 21.5 and 29 h after the end of the light interruption. The magnitude of the phase-shift induced by the light
Bryophyllum
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in I
46
I
4o
I" 9 9
42 v
9
38
I~
~- 34
I~
.~" ,~'
9
9
20
:
/'
0"
I
I
iil}
I
I
I "" I 9
30
~ ~i:;! ;' i~ /
"
6o '
,.,,::!::! ,~-, .
40[
i ~i !
20
! ;?:!
.,.........
.
.
.
9 I
=~ 26 "6
~
I
9
~I
9
22
r 9
18
.@~ ~
"1
:.
14
',
. . . . . . . .
o
9 I I I
I
610
410
20
0
~I0
100
120
Time (h)
Fig. 2. The time of occurrence of peaks of carbon dioxide output from Bryophyllum leaves exposed to white light for 4 h at the times indicated by the shaded bar. The times of the mid-points of the light treatments are shown on the ordinate. The times at which peaks occur in control, unirradiated leaves are shown by the broken lines. Each point is the mean of at least two, and in most cases more, individual measurements. The times of occurrence of peaks of the control rhythms are the means of 24 experiments
_'..
325
..--, !i::i
40
40
20
20 ~
0
0 :i
40
40
20
2C
D
' iii
s i
i
I ~:: i
.... I
,
I i I i
0:22
i
0
0
i
i
i
0
0
0
Time of day
Fig. 4 A - D . Effects of exposure to 4 h red light A, 4 h far-red radiation B, 4 h of red light followed by 4 h of far-red radiation C, and red and far-red radiation simultaneously for 4 h D, on the phase of the circadian rhythm in Bryophyllumleaves. The times of the irradiations are shown by the shaded bars. Control rhythms in leaves in continuous darkness are shown by the broken lines. 0 = midnight
~: 0 c~ u
40
4(
20
20
0
0
0
0
0
_. ~ ,,, , !iii
0
0
F
0
0
0
Time of day
between 0400 and 0800 h in the same cycle (Fig. 3 E). The 450-nm and 730-nm spectral bands had no measurable effect on the phase of the rhythm at either time in the cycle (Fig. 3 A, C, D and F).
Fig. 3 A-F. Effectiveness in inducing phase-shifts, of a 4-h exposure to monochromatic radiation in spectral bands centred at 450 nm A and D, 660nm B and E, and 7 3 0 n m C and F. The times of irradiation are shown by the shaded bars. Rhythms in leaves in continuous darkness are shown by the broken lines. 0 = m i d n i g h t
Interaction of Red and Far-red Radiation in Phase Control
treatments was virtually the same in three successive cycles. A new steady-state was thus attained, at the latest 21.5-29 h after the end of the light treatment. Leaves kept in prolonged darkness were exposed to monochromatic radiation for 4 h at a quantum flux density of 47 pE cm-28 1. Spectral bands were selected to include wavelengths (660 nm) previously found to be active in shifting the phase, and those (450 nm and 730 nm) which were not (Wilkins, 1973). The effectiveness of each band in shifting the phase was tested at several times in the cycle; the results are shown in Figure 3. The 660-nm spectral band induced a phase delay of 3 4 h when applied between 2000 and 0000 h during the second day in darkness (Fig. 3B) and a phase advance of 6 h when applied
Exposing leaves for 4 h to red light (660 nm) at a radiant flux density of 85 m J m - 2 s - 1 resulted in a phase-shift of about 12 h (Fig. 4A) whereas exposure to far-red radiation (7.8 j m - 2 s -1) had no effect (Fig. 4B). Exposure to red light for 4 h, followed immediately by an exposure to far-red radiation for 4 h, resulted in a marked phase-shift (Fig. 4C), although its magnitude was less than that obtained with red light alone (Fig. 4A). Simultaneous exposure to red and far-red radiation for 4 h (Fig. 4 D ) virtually abolished the circadian rhythm of carbon dioxide output and it was not possible to assess whether a phase-shift was induced. The lack of clear red/far-red reversibility in these experiments might be attributed to the length of exposure being sufficient for the phase-shifting process to proceed beyond the reversible photochemical
P.J.C. Harris and M.B. Wilkins: Circadian R h y t h m in BryophylIum
326
,"-.
A
...
[3
10 C 3O
40
,. t
iiii
A
E
,..
20
;ii'~: ,;- .... 0
,'",
20
"
,
ir
i
...---. .......... i
,
i
,
i
J= ,
i
lC
~n
'x-
i
I
i
i
2c lC 0
i
,
I:;:;
,
i
I
i
,
i
eo
,
i
c
G 3C
i
i
D
~_ 40 20
I:?
0 0
::~: 0
'
:
,
'
, 0
~ O
0
L 0
L
,
0
i
0
0
0
0
T i m e of d a y
Time of day
Fig. 5 A - C . Effects of exposing leaves of Bryophyllum for 4 h to:5 rain of red light alternating with 5 rain of darkness A, 5 rain of red light alternating with 5 rain of far-red radiation B, the r~gime in A superimposed on a 4-h continuous exposure to far-red radiation C. Times of irradiation are shown by the shaded bars. The broken lines show the r h y t h m in unirradiated, control leaves. 0 = midnight
Fig. 6 A - D . Effect of exposing leaves of Bryophyllum for 4 h to :-10 s of red light alternating with 30 s of darkness A, 10 s of red light aiternating with 30 s of far-red radiation B, the r6gime used in A superimposed upon a continuous exposure to far-red radiation for 4 h C and D. Times of irradiation are shown by the shaded bars. The broken lines show the r h y t h m in unirradiated leaves. 0 = midnight
stages. In an attempt to obviate this difficulty leaves were exposed for 4 h to rapid alternation of red light and darkness. Alternation of 5 min of red light with 5 rain of darkness for 4 h, induced a distinct phaseshift (Fig. 5 A). When 5 min of red light alternated with 5 rain of far-red radiation for 4 h (Fig. 5 B) there was not a clear abolition of the phase-shift effect induced by red light alone, but, rather, a considerable reduction of the amplitude of the rhythm to the extent that it was scarcely discernible in subsequent darkness. Continuous exposure of leaves to far-red radiation for 4 h superimposed on the 5 rain red/5 min darkness r6gime caused a loss of the circadian nature of the rhythm; small peaks appeared at 12-h intervals (Fig. 5 C). Again, a clear reversal of the effectiveness of red light by an exposure to far-red radiation could not be found in so far as the induction of phase-shifts was concerned. There is no doubt, however, that the far-red radiation interacts in some way with the red light and leads to a loss of the circadian nature of the rhythm. Several different irradiation r~gimes were used over a period of 4 h to test for red/far-red reversibility. For example, 10 s of red light followed by 30 s of darkness led to the induction of a substantial phaseshift (Fig. 6A), but 10 s of red light followed by 30 s of far-red radiation led, once again, to the virtual
abolition of the circadian rhythm (Fig. 6B). When this irradiation r6gime was applied to leaves with a simultaneous and continuous exposure to far-red radiation, the result was variable and difficult to interpret. Either, a very slight phase-shift was induced in the rhythm (Fig. 6C), or the rhythm again appeared to lose its circadian nature altogether (Fig. 6D).
Discussion
A 4-h exposure to light can reset the phase of the rhythm of carbon dioxide metabolism in leaves of Bryophyllum but the magnitude and direction of the phase-shift depends on the time in the cycle at which the exposure occurs. The phase-response of Bryophyllum to a light stimulus is thus basically similar to that reported for a number of other organisms (Aschoff, 1965; Pittendrigh, 1965). In an earlier study in which light was applied at only two times in the cycle (Wilkins, 1960), it was found that the phase was reset so that the next peak of carbon dioxide emission occurred a specific time after the end of a light treatment. It is now apparent from this more detailed investigation, that the time between the end of the light treatment and the occurrence of a peak
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in Bryophyllurn of carbon dioxide output is not constant but varies between 21.5 and 29 h, depending on the time in the cycle at which the leaves are irradiated. Furthermore, this peak may not necessarily be the first after the end of the light treatment; in some instances a peak occurs a few hours after the end of the light treatment but still within the same circadian cycle (e.g. Fig. 1 E and F). The occurrence of a peak 21.5 to 29 h after the end of the light interruption permits the prediction that at some point in the cycle a light treatment should be without effect on the phase of the rhythm. This has been observed in the present and previous (Wilkins, 1960) studies. While the net effect of the light treatment is to advance or delay the phase of the rhythm it is difficult, in practice, to distinguish unequivocally between phase advances and phase delays when the light perturbations are applied during some parts of the cycle. This is because in a number of experiments a temporary increase in the rate of carbon dioxide release occurred during, or soon after, the leaves were irradiated. Similar peaks of carbon dioxide emission appear during entrainment of the rhythm to light-dark cycles (Harris and Wilkins, 1976). It is not clear whether the increased rate of carbon dioxide output represents a direct effect of radiant energy on the biochemical processes monitored as the overt rhythm, or an effect on the basic circadian oscillator. When the phase of rhythms in organisms such as Drosophila (Pittendrigh and Bruce, 1957) is shifted by a single exposure to light, there may occur several transient cycles before the phase attains a new steadystate. Advancing phase-shifts often pass through several transient cycles while delaying phase-shifts tend to reach their final values more rapidly (Aschoff, 1965). In other organisms such as Gonyaulax, a new stable phase is reached immediately after either delaying or advancing shifts (Hastings and Sweeney, 1958). In Bryophyllum a new stable phase is attained rapidly; the phase difference, whether an advance or delay, between the rhythm reset by a light perturbation and the control rhythm persisting in darkness is virtually the same in each of the three cycles following the stimulus. At no point in the cycle was blue or far-red radiation active in shifting the phase of the rhythm, whereas red light was active at all points of the cycle where a phase-shift could be induced by white light. It thus seems probable that the same photoreceptor is responsible for mediating both phase advances and phase delays. This is apparently not so in all organisms, since in both Phaseolus (B/inning and Moser, 1966) and Coleus (Halaban, 1969) different wavelengths of radiant energy are effective in inducing phase-shifts at different times in the circadian cycle.
327 Several kinds of experimental approach, using sequential or simultaneous exposure to red light and far-red radiation, showed that these spectral regions interact in their effects on the rhythm. This finding suggests the involvement of phytochrome in the response. The red/far-red interaction quite frequently did not take the form of a clear reversal of the effectiveness of red by far-red radiation, but rather the virtual abolition of the rhythm when far-red radiation was applied. The abolition of the rhythm may arise in a number of ways. For example, far-red radiation may interact with red to abolish oscillation of the basic circadian system in each cell of the leaf. On the other hand, total reversal of the phase-shift induced by red light may occur in some cells and not others. This would result in the loss of the overt rhythm because the rhythms in some cells would be approximately 12 h out of phase with those in other cells. The occurrence of small peaks at 12-h intervals in some phase-shift experiments could result from such cellular desynchronization. The leaves are 3 5 mm thick and it is not possible to ensure that each constituent cell is equally irradiated. The photocontrol of a number of aspects of the Bryophyllum rhythm has now been investigated (Wilkins, 1960, 1973; Harris and Wilkins, 1976, 1978). Blue light is totally without effect on the phase and period of the rhythm and does not induce entrainment. Modification of the period (Harris and Wilkins, 1976) and entrainment of the rhythm (Harris and Wilkins, 1978) can be achieved with both monochromatic red (660 nm) and far-red (730 nm) radiation, but the latter does not modify the transient (Harris and Wilkins, 1976) or induce phase-shifts when applied as a single 4-h perturbation. The observation of red/far-red reversibility in the entrainment of the rhythm (Harris and Wilkins, 1978), and an interaction of these spectral bands in its phase-shift response, leave little doubt that phytochrome is the photoreceptor involved in each aspect of the control of the Bryophyllum rhythm by light. Explanation of the different sensitivities of the phase-shift and entrainment responses to the 730-nm spectral band should perhaps be sought in terms of phytochrome action rather than in the possible involvement of other pigments. Phytochrome also appears to be the pigment involved in the photocontrol of the rhythms of carbon dioxide output in Lernna (Hillman, 1971) and of movement in excised pulvini of Samanea (Simon et al., 1976). There is also evidence that phytochrome controls the rhythms in sensitivity to flower induction in Xanthium (Salisbury, 1965; Denney and Salisbury, 1970) and in germination promotion in Sphaerocarpos donelli (Steiner, 1969). It may also be involved in the phaseshift response of the KalanchoO petal movement
328
rhythm (Schrempf, 1975). While phytochrome appears to be involved in photocontrol of circadian rhythms in many higher plants, it is by no means the only photoreceptor pigment associated with rhythmic systems in plants. Phytochrome is not the photoreceptor in the alga Oedogonium cardiacum (Btihnemann, 1955), the photosynthetic marine dinoflagellate Gonyaulax polyedra (Hastings and Sweeney, 1960), or the fungus Neurospora crassa (Sargent and Briggs, 1967). Pigments other than phytochrome also appear to be involved in the photocontrol of rhythms in some higher plants such as Portulaca (Karv6 and Jigajinni, 1966) and Coleus (Halaban, 1969).
References Aschoff, J. : Response curves in circadian periodicity. In : Circadian Clocks, pp. 95 111, Aschoff, J., ed., Amsterdam: North Holland 1965 Btihnemann, F.: Das endodiurnale System der Oedogoniumzelle IV. Die Wirkung verschiedener Spektralbereiche auf die Sporulation and Mitoserhythmik. Planta 46, 227-255 (1955) Bfinning, E., Moser, I.: Response-Kurven bei der circadianen Rhythmik yon Phaseolus. Planta 69, 101 110 (1966) Denney, A., Salisbury, F.B.: Separate clocks for leaf movement and photoperiodic flowering in Xanthium strumarium L. Plant Physiol. 46, Suppl. 26 (1970) Halaban, R.: Effects of light quality on the circadian rhythm of leaf movement of a short-day-plant. Plant Physiol. 44, 973 977 (1969) Harris, P.J.C., Wilkins, M.B. : Light-induced changes in the period of the circadian rhythm of carbon dioxide output in Bryophyllurn leaves. Planta 129, 253-258 (1976) Harris, P,J.C., Wilkins, M.B.: Evidence of phytochrome involvement in the entrainment of the circadian rhythms of CO2 metabolism in Bryophyllum. Planta 138, 271 278 (1978) Hastings, J.W., Sweeney, B.M.: A persistent diurnal rhythm of luminescence in Gonyaulax polyedra. Biol. Bull. 115, 440-458 (1958)
P.J.C. Harris and M.B. Wilkins: Circadian Rhythm in Bryophyllum Hastings, J.W., Sweeney, B.M.: The action spectrum for shifting the phase of the rhythm of luminescence in Gonyaulaxpolyedra. J. Gen. Physiol. 43, 697-706 (1960) Hillman, W.S.: Entrainment of Lemna CO2 output through phytochrome. Plant Physioi, 48, 770-774 (1971) Karv6, A., Engelmann, W., Schoser, G.: Initiation of rhythmical petal movements in KalanchoO blossfeldiana by transfer from continuous darkness to continuous light or vice versa. Planta 56, 700-711 (1961) Karv6, A.D., Jigajinni, S.G. : Pigment system involved in the photoperiodic entrainment of circadian leaf movements. Naturwissenschaften 53, 181 (1966) Pittendrigh, C.S. : On the mechanism of the entrainment of a circadian rhythm by light cycles. In : Circadian Clocks, pp. 277-297, Aschoff, J., ed., Amsterdam: North Holland 1965 Pittendrigh, C.S., Bruce, V.G.: An oscillator model for biological clocks. In: Rhythmic and Synthetic Processes in Growth, pp. 75-109, Rudnik, D., ed., Princeton: Princeton University Press 1957 Salisbury, F.B. : Time measurement and the light period in flowering. Planta 66, 1 26 (1965) Sargent, M.L., Briggs, W.R.: The effects of light on a circadian rhythm of conidiation in Neurospora. Plant Physiol. 42, 1504-1510 (1967) Schrempf, M.A. : Eigenschaften und Lokalisation des Photoreceptors ffir phasenverschiebendes St6rlicht bei der B1/itenblattbewegung yon Kalancho~ blossfeldiana (v. Poelln). Doctoral Thesis, Universitfit Tfibingen, W-Germany 1975 Simon, E., Satter, R.L., Galston, A.W.: Circadian rhythmicity in excised Samanea pulvini. II. Resetting the clock by phytochrome conversion. Plant Physiol. 58, 421-425 (1976) Steiner, A.M. : Changes of the endogenous rhythm in phytochrome mediated spore germination of the liverwort Sphaerocarpos during spore after ripening. Z. Pflanzenphysiol. 61, 184-191 (1969) Wilkins, M.B. : An endogenous rhythm in the rate of CO2 output of Bryophyllum. II. The effects of light and darkness on the phase and period of the rhythm. J. exp. Bot. 11,269 288 (1960) Wilkins, M.B. : An endogenous rhythm in the rate of carbon dioxide output of Bryophyllum. VI. Action spectrum for the induction of phase shifts by visible radiation. J. exp. Bot. 24, 488 496 (1973)
Received 4 August; accepted 10 August 1978
