The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm

When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction.     

A Semidian Rhythm in the Flowering Response of Pharbitis nil to Far-Red Light: II. The Involvement of Phytochrome

The semidian (~12 h) periodicity in the effect of far-red (FR) interruptions of the light period preceding inductive darkness on flowering in Pharbitis nil appears to be mediated by phytochrome: (a) promotion by interruptions 2 hours before inductive darkness (−2 hours) and inhibition at −8 hours are greater the higher the proportion of FR/R+FR during the interruption; (b) brief FR exposures followed by darkness are even more effective than FR throughout; (c) the effect of brief FR is reversed by subsequent R; (d) R interruptions of an FR background are most promotive at −8 hours, when FR is most inhibitory. Promotive FR interruptions at −2 or −14 hours shorten the critical dark period whereas inhibitory FR interruptions at −8 hours lengthen it. We conclude that the semidian rhythm is controlled by a `timing pool' of phytochrome FR absorbing form (Pfr) which disappears rapidly in darkness: four different estimates from our experiments indicate that Pfr was reduced to the level set by FR within 20 to 45 minutes in darkness. However, flowering may also be influenced by a `metabolic pool' of Pfr with a delayed loss in darkness, the time of which can be advanced or retarded by shifting the semidian rhythm.     

Flowering of Pharbitis nil is controlled by an endogenous semidian rhythm

Abstract Flowering of Pharbitis nil after an inductive dark period is greatly influenced by far-red (FR) irradiation during the preceding light period. The response to FR is rhythmic in otherwise constant conditions, and the period of the oscillation is approximately 12 h (i.e. semidian). The rhythm also appears to operate under daily light-dark cycles. The expression of this novel rhythm depends on the time from the beginning of FR pretreatment to the onset of the inductive dark period. The cotyledons are the site of response to both the pretreatment and inductive darkness, and both these conditions must be perceived by the same cotyledon.     

Shifts in the semidian rhythm of flowering do determine night-break timing and critical night length in Pharbitis nil

There is a semidian (≈12 h) rhythm in the flowering response of the short-day plant Pharbitis nil Choisy following 90 min exposure to either far-red light/darkness or a temperature drop (27 °C to 12 °C) given at various times in constant conditions before an inductive dark period. This semidian rhythmic response to the temperature-drop pretreatments in the light is also evident through the inductive dark period without change of phase. Furthermore, those pretreatments which increase flowering also advance the time of maximum sensitivity to red light (R) interruptions of the dark period by up to 1.5 h and shorten the critical night length. Conversely, pretreatments which reduce flowering delay the time of maximum R inhibition by up to 1.5 h and increase the critical night length by the same amount. However the phase of a circadian rhythm of flowering response had no effect on either the time of maximum R inhibition or the critical night length. Thus, the semidian rhythm determines both the time of maximum R inhibition and the critical night length in Pharbitis. Received: 8 November 1997 / Accepted: 7 January 1998     

Photoperiodic Entrainment Patterns in the CO(2) Output of Lemna perpusilla 6746 and of Several Other Lemnaceae

The CO₂ output of Lemna perpusilla 6746 in “skeleton photoperiods” consisting of alternating 10½-hour and 13-hour dark periods separated by ¼-hour illuminations was recorded under stable high and low nitrate conditions. The phase relationship finally attained between light schedule and output is the same regardless of which dark period is given first, but entrainment is more rapid (as is flowering) with an initial 13-hour dark period. In all respects other than bistability—the assumption of two different stable phase relationships depending on the initial dark period—both flowering and the course of CO₂ output conform to Pittendrigh's model derived from Drosophila eclosion rhythms, confirming the view that an endogenous circadian rhythm, or biological clock, underlies the photoperiodic control of flowering in this plant. Experiments with rigorous temperature control show that earlier results with long light exposures were in part due to temperature changes; in consequence, it is clear that entrainment patterns with high nitrate differ even more from those in low nitrate than was previously evident, and not simply by the addition of a “nitrate peak.” Other Lemnaceae tested with a few simple light-dark schedules in both types of media show a variety of responses, with no obvious correlation to photoperiodic response type.     

Action spectra for the light inhibition of flowering and its reversal inLemna paucicostata T-101

Lemna paucicostata Hegelm. T-101, a short-day plant, flowers when plants preirradiated with red light (R) for 24 h are subjected to inductive darkness for 72 h followed by two short-day cycles (6 h R+ 18 h dark). However, flowering is inhibited by blue-or far-red-light pulses applied at the beginning of the inductive dark period. These inhibitory light effects are fully reversible by a R pulse. The action spectra for the inhibitory light effect and for its reversal show that the light pulses act exclusively through phytochrome. It is concluded that a low level of Pfr at the beginning of the inductive dark period prevents flowering.Abbreviations R red (light) - B blue (light) - FR far-red (light)     

Investigation of the endogenous rhythm of flowering inChenopodium rubrum L.

Under the conditions applied in our laboratory 4 1/2 days old plants ofChenopodium rubrum require 2–3 photoperiodic cycles for maximal flowering response, whereas 2 1/2 days old plants are able to flower after having obtained a single inductive cycle. The period length of the free-running rhythm of flowering observed in 2 1/2 days old plants after a single transfer from light to darkness is 30h and the first peak of flowering occurs at about hour 12 in darkness. When a cycle consisting of 16h darkness and 8h light or of 8h darkness and 8h light precedes the long dark period the rhythm is rephased. Rephasing is greater when the light commenced to act on the positive slope of the first peak of the free running rhythm than when it impinged on the negative slope. With an 8h interruption of darkness by light rhythm phase is controlled by the light-on, as well as by the light-off signal. Feeding 0.4 M glucose during the long period of darkness enhanced the amplitude of the flowering response and, moreover, substituted for one photoperiodic cycle.     

Evidence that photoperiodic, dark time measurement in Pharbitis nil involves a circadian rather than a semidian rhythm

Experiments were carried out to determine whether a semidian (12 h) rhythm in flowering response operates in Pharbitis nil as the basis for photoperiodic time measurement. The effect of 5 min far-red light followed by 85 min dark (FRD) given 4, 8,14 and 22 h before the end of a 48 h photoperiod on night-break timing and critical night length was determined. When given 4 h before the end of a 48 h photoperiod, an interruption with FRD advanced the phase of the circadian rhythm in the night-break inhibition of flowering. In contrast, earlier interruptions of the photoperiod had no effect on the phase of the rhythm. The critical night length was modified by FRD given 4 h (shortened) or 8 h (lengthened) before the end of the photo-period; when given at other times FRD did not alter the critical night length. The results are discussed in relation to the basis for photoperiodic timekeeping, with particular reference to suggestions for the involvement of a semidian rhythm. A circadian model based on the concept of limit cycles is described.     

Effects of light quality on the circadian rhythm of leaf movement of a short-day-plant

Studies were made of the effects of blue, green, red and far-red (FR) light on the circadian rhythm of leaf movement of Coleus blumei × C. frederici, a short day plant. Under continuous illumination with blue light, there was a significant lengthening of the period of the rhythm to about 24.0 hr, as compared to 22.5 hr in continuous darkness. Under continuous red light, the period length was significantly shortened to 20.5 hr. Under continuous green or FR, the period length was not significantly different from the dark control. It was observed that under continuous FR illumination, the leaves tended to oscillate in a more downward position. Eight-hr red light signals were effective in advancing the phase of the rhythm as compared to a control under continuous green light. Blue light signals were effective in delaying the phase of the rhythm. FR light signals were ineffective in producing either delay or advance phase shifts. Far-red light did not reverse the effects of either red or blue light signals. On the basis of these results it is suggested, that pigments which absorb blue or red light, rather than phytochrome, mediate the effect of light on the circadian rhythm of leaf movement.     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbitis nil Chois

For dark-grown seedlings of Pharbitis nil capacity to flower in response to a single inductive dark period was established by 24 h white, far-red (FR) or ruby-red (BCJ) light and by a skeleton photoperiod of 10 min red (R)-24 h dark-10 min R. FR alone was ineffective without a brief terminal (R) irradiation, confirming that the form of phytochrome immediately prior to darkness is a crucial factor for flowering in Pharbitis. The magnitude of the flowering response was significantly greater after 24 h FR or white light (WL) (at 18° C and 27° C) than after two brief skeleton R irradiations, but the increased flowering response was not attributable to photosynthetic CO₂ uptake because this could not be detected in seedlings exposed to 24 h WL at 18° C. Photophosphorylation could have contributed to the increased flowering response as photosystem I fluorescence was detectable in plants exposed to FR, BCJ, or WL, but there were large differences between flowering response and photosystem I capacity as indicated by fluorescence. We conclude that phytochrome plays a major role in photoresponses regulating flowering. There was no simple correlation between developmental changes, such as cotyledon expansion and chlorophyll formation during the 24-h irradiation period, and the capacity to flower in response to a following inductive dark period. Changes in plastid ultrastructure were considerable in light from fluorescent lamps and there was complete breakdown of the prolamellar body with or without lamellar stacking at 27 or 18° C, respectively, but plastid reorganization was minimal in FR-irradiated seedlings.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing from of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

Time-Measuring Processes Involved in the Photoperiodic Response of Lemna paucicostata 441

Lemna paucicostata 441 exposed to a single dark period of variouslengths showed a rhythmic flowering response with a 22- to 24-hperiod, even when the dark period was preceded by continuouslight. The critical night length (about 12 h) was scarcely influencedby pretreatment with 8D–4L (8 h of darkness followed by4 h of light), 8D–8L or 8D–12L. However, the rhythmof the response in the second cycle was markedly damped by thepretreatment with 8D–4L or 8D–12L, and was slightlyamplified by 8D–8L. The flowering response to a red-light interruption given atdifferent times in the inductive dark period also showed circadianrhythmicity even when the dark period was preceded by continuouslight, and this rhythmicity was scarcely influenced by a dark-lighttreatment given prior to the inductive dark period. A red-lightinterruption given at the 6th or 14th hour of the dark periodmarkedly shifted the phase of the rhythm of the response tothe length of the following dark period (the former delayedand the latter advanced), but that given at the same phase markedlyweakened and disturbed the rhythmicity of the response to ared-light interruption given in the following dark period. (Received March 21, 1992; Accepted June 12, 1992)     

The effect of red light on the level of free IAA

Red light (660 nm) break which cancels the floral inductive effect of the dark period brought about a transitional rise in the level of free IAA in the shoot ofChenopodium rubrum. The higher content of IAA was then found in treated plants at the beginning of the following photoperiod, too. The red light treatment did not change the phase of endogenous rhythm fluctuations of free IAA.     

Floral initiation in Lolium temulentum L.: the role of phytochrome in the responses to red and far-red light

Summary The possibility that phytochrome is involved in the promotion of flowering by far-red light was investigated. The addition of far-red (FR) to a day extension with red (R) light promotes inflorescence initiation in Lolium. A 2-hour interruption with darkness also promoted flowering compared with the uninterrupted red light control; apex length was further increased by a 10-minute FR irradiation given before the 2-hour dark interruption and was decreased by 10-minutes of R light given in the middle: both FR promotion and R inhibition were reversed by R and FR respectively. Apex length increased approximately linearly with increasing duration of dark interruption up to at least 2 1/2 hours. When varying ratios of R:FR light were substituted for a 2-hour dark period, apex length was increasingly depressed as the % R was increased above 25%; no difference between 25% R/75% FR and 100% FR could be detected. Apex length was inversely linearly related to the calculated [Pfr]/[P] ratios above about 40% Pfr.FR promoted flowering when given during a 5-hour interruption of a day extension with R light but, between 0.25 and 0.90 J m² s^-1, there was no effect of intensity of FR; at 0.11 J m^-2 s^-1 apex length was shorter than at 0.25 J m^-2 s^-1 but longer than in darkness. When the duration of FR (from the beginning of a dark interruption of a day extension with R) was varied, apex length increased with increasing duration of FR up to 1 1/4 to 2 hours but further increasing the duration of FR did not promote flowering more.The results implicate phytochrome in the promotion of flowering by FR light. It has been demonstrated that a low [Pfr]/[P] ratio (less than present in 25% R/75% FR) is needed over a relatively long period of time: this explains why a relatively high proportion of FR light must be added to R for several hours in order to give maximum promotion of flowering. It is concluded that, in Lolium, the increased flowering response to FR light is brought about by a reduction of [Pfr]/[P] ratio at the appropriate time, although the possibility that another effect of far-red is also involved has not been rigorously excluded.     

Twilight effect: initiating dark measurement in photoperiodism of xanthium

Six experiments studied the effects of low levels of red and far-red light upon the initiation of measurement of the dark period in the photoperiodic induction of flowering in Xanthium strumarium L. (cocklebur), a short-day plant, and compared effects with those of comparable light treatments applied for 2 hours during the middle of a 16-hour inductive dark period. Red light, or red plus far-red, at levels that inhibit flowering when applied during the middle of the inductive dark period, either had no effect on the initiation of dark measurement (i.e., were perceived as darkness), or they delayed the initiation of dark measurement by various times up to the full interval of exposure (2 hours). Far-red light alone had virtually no effect either at the beginning or in the middle of the dark period. These results confirm that time measurement in the photoperiodic response of short-day Xanthium plants is not simply the time required for metabolic dark conversion of phytochrome. Results also suggest that the pigment system (phytochrome?) and/or responses to it may be significantly different as they function during twilight (initiation of dark measurement), and as they function during a light break several hours later. Possible mechanisms by which cocklebur plants detect the change from light to darkness are discussed.Comparing experimental results with spectral light measurements during twilight and with measurements of light from the full moon led to two conclusions: First, light levels pass from values perceived by the plant as full light to values perceived as complete darkness in only about 5.5 to 11.5 minutes, although twilight as perceived by the human eye lasts well over 30 minutes. Second, cocklebur plants probably do not respond to light from the full moon, even when most sensitive, 7 to 9 hours after the beginning of darkness.     

Rhythmic changes in ribonuclease activity in relation to nucleic acid synthesis in cotyledons ofChenopodium rubrum L. and to floral induction of the plants

Ribonuclease (RNAse) activity was investigated in cotyledons ofChenopodium rubrum plants subjected to various conditions of illumination (photoperiodic induction, continuous light, induction cancelled by interrupting the dark period by a light-break). At the end of the dark period of the single inductive cycles RNAse activity of induced plants was inferior to that of plants grown in continuous light. At the end of the first two cycles the activity was lowest after the interruption of the dark period by light. The investigation of the enzyme in 6h intervals showed rhythmic changes in activity to occur in induced plants. Enzyme activity followed a pattern opposed to this of nucleic acid (NA) synthesis in the cotyledons. In plants from continuous light the enzyme activity did not show any rhythm and in plants having obtained a light-break during the inductive period the rhythm was less distinct than in the induced ones. The period length of the endogenous rhythm of NA synthesis in the cotyledons is about half as long as this of flowering and the peaks of flowering coincide with the throughs of NA synthesis.     

Rhythms as photoperiodic timers in the control of flowring in Chenopodium rubrum L.

Summary In C. rubrum, the amount of flowering that is induced by a single dark period interrupting continuous light depends upon the duration of darkness. A rhythmic oscillation in sensitivity to the time that light terminates darkness regulates the level of flowering. The period length of this oscillation is close to 30 hours, peaks of the rhythm occurring at about 13, 43 and 73 h of darkness.Phasing of the rhythm by 6-, 12- and 18-h photoperiods was studied by exposing plants to a given photoperiod at different phases of the free-running oscillation in darkness. The shift in phase of the rhythm was then determined by varying the length of the dark period following the photoperiod; this dark period was terminated by continuous light.With a 6-h photoperiod the timing of both the light-on and light-off signals is shown to control rhythm phasing. However, when the photoperiod is increased to 12 or 18 h, only the light-off signal determines phasing of the rhythm. In prolonged periods of irradiation-12 to 62 h light—a durational response to light overrides any interaction between the timing of the light period and the position of the oscillation at which light is administered. Such prolonged periods of irradiation apparently suspend or otherwise interact with the rhythm so that, in a following dark period, it is reinitiated at a fixed phase relative to the time of the light-off signal to give a peak of the rhythm 13 h after the dusk signal.In daily photoperiodic cycles rhythm phasing by a 6-h photocycle was also estimated by progressively increasing the number of cycles given prior to a single dark period of varied duration.In confirmation of Bünning's (1936) hypothesis, calculated and observed phasing of the rhythm controlling flowering in c. rubrum accounts for the photoperiodic response of this species. Evidence is also discussed which indicates that the timing of disappearance of phytochrome P_fr may limit flowering over the early hours of darkness.     

Studies of the Involvement of an Endogenous Rhythm in the Photoperiodic Response of Hyoscyamus niger

An attempt was made to determine the involvement of an endogenous circadian rhythm in the flowering response of the long-day plant Hyoscyamus niger L. grown in a modified White's medium. Both variable-cycle-length and light interruption experiments were employed in this attempt. In the variable-cycle experiments, plants were subjected to light periods of 6, 12, or 18 hours followed by varying lengths of darkness. The total lengths of the cycles varied from 12 to 72 hours. In experiments utilizing a 6-hour photoperiod, a high level of flowering occurred in cycle lengths of 12, 36, and 60 hours. Flowering was suppressed in the 24-, 48-, and 72-hour cycles. When a 12-hour photoperiod was used the flowering response was low between 24 and 36 hours and flowering did not indicate a rhythmic response. When an 18-hour photoperiod was used, the flowering response was suppressed in the 36- and 60-hour cycles.

Light-break experiments were conducted to study further the flowering response in Hyoscyamus. These experiments consisted of a 6-hour main photoperiod followed by varying lengths of darkness to make cycles of 24, 48, and 72 hours. At given intervals the dark period was interrupted by 2-hour light breaks. In a 24-hour cycle, flowering was promoted when a light break was given at either the twelfth or eighteenth hour of the cycle. In a 48-hour cycle, flowering was strongly promoted by light breaks given near the beginning or at the end of the dark period. In a 72-hour cycle, light breaks given at the eighteenth, forty-second, and sixty-sixth hour of the cycle stimulated flowering as compared with light breaks given at the thirtieth and fifty-fourth hour. These results are indicative of the involvement of an endogenous rhythm in the flowering response of Hyoscyamus niger.

     

Phytochrome-mediated effects of near-ultraviolet radiation in the induction of flowering in etiolated Lemna paucicostata T-101, a short-day plant

Induction of flowering of etiolated Lemna paucicostata Hegelm. T-101, a short-day plant, was inhibited by far-red (FR) or blue light (BL) applied at the beginning of a 72-h inductive dark period which was followed by two short days. In either case the inhibition was reversed by a subsequent exposure of the plants to near-ultraviolet radiation (NUV), with a peak of effectiveness near 380 nm. Inhibition by BL or FR and its reversion by NUV are repeatable, i.e., NUV is acting in these photoresponses like red light although with much lower effectiveness. Thus, it is considered that NUV acts through phytochrome and no specific BL and NUV photoreceptor is involved in photocontrol of floral induction on this plant.Abbreviations BL blue light - FR far-red light - NUV near ultraviolet radiation - P red-absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - R red light     

Rhythmicity of Flowering in Pharbitis nil

When young seedlings of Pharbitis nil are grown under continuous light, except for a single inductive dark period, they flower to a varying degree, depending on when this dark period is given. Plants become sensitive to this induction approximately three days after the seedlings emerge from the soil. The expression of flowering varies in a rhythmic fashion for three or more cycles, when an inductive dark period is given at progressively later times. The time between maximum expression of flowering is 24 hours or somewhat longer. It appears necessary that the inductive dark period be of sufficient duration, to only partially induce the plants to flower for this rhythm to be expressed. Under the conditions employed in this study, this duration is 12 hours. If this rhythm is endogenous, it exists at least from when the plants emerged from the soil since no environmental cues are given after that time, and it raises questions of the interpretations of data from previous studies with this organism.     

Photoperiodic Control of Flowering in Dark-Grown Seedlings of Pharbitis nil Choisy : The Effect of Skeleton and Continuous Light Photoperiods

The control of night-break timing was studied in dark-grown seedlings of Pharbitis nil (Choisy cv. Violet) following a single continuous or skeleton photoperiod. There was a rhythmic response to a red (R) interruption of an inductive dark period, and the phasing of the rhythm was influenced by the preceding light treatment.

Following a continuous white light photoperiod of 6 hours or less, the points of maximum inhibition of flowering were constant in real time. Following a continuous photoperiod of more than 6 hours, maximum inhibition occurred at 9 and 32.5 hours after the end of the light period. The amplitude of the rhythm during the second circadian cycle was much reduced following prolonged photoperiods.

Following a skeleton photoperiod, the time of maximum sensitivity to a R interruption was always related to the second pulse of the skeleton, R₂, with the first point of maximum inhibition of flowering occurring after 12 to 18 hours and the second after 39 hours. Without a second R pulse, the time of maximum sensitivity to a R interruption was related to the initial R₁ pulse. A `light-off' or dusk signal was not mimicked by a R pulse ending a skeleton photoperiod; such a pulse only generated a `light-on' signal and initiated a new rhythm.

It is concluded that the timing of sensitivity to a R interruption of an inductive dark period in Pharbitis nil is controlled by a single circadian rhythm initiated by a light-on signal. After 6 hours in continuous white light, the phase of this rhythm is determined by the transition to darkness. Following an extended photoperiod, the timing characteristics were those of an hourglass; this seemed to be due to an effect on the coupling or expression of a single circadian timer during the second and subsequent cycles, rather than to the operation of a different timing mechanism.

In addition to the effects on timing, the photoperiod affected the magnitude of the flowering response.