Nucleic acid synthesis inChenopodium rubrum L. during photoperiodic induction and its relation to endogenous rhythmicity

Beginning with the second inductive cycle the rate of nucleic acid (NA) synthesis in cotyledons and apical buds ofChenopodium rubrum is higher at the end of the dark period or 4h following transfer of the plants to light in induced plants than in non-induced ones. This is due to an increase in all NA fractions. The greatest difference between NA synthesis in induced and non-induced plants was observed at the end of the second (or sometimes third) inductivecycle. In the subsequent cycles the difference decreased or disappeared eventually. During photoperiodic induction NA synthesis shows a diurnal rhythm with a peak at the end of the dark and at the beginning of the light period. Rhythmicity of NA synthesis is endogenous. The period length of the endogenous oscillation is about 18 h. Interruption of the dark period by light causea amplitude of the first oscillation to be reduced and delays the appearance of the second peak. NA synthesis did not show rhythmicity in plants grown in continuous light. The significance of the observed phenomena for photoperiodic induction is being discussed.     

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.     

Fluctuations of Uridine Incorporation in the Shoot Apex ofChenopodium rubrum L. during Photoperiodic Induction

Uridine incorporation into the shoot apex of the short-day plantChenopodium rubrum was investigated during a 16 h period of darkness and the following transfer to light. Uridine incorporation during this single inductive cycle was compared to incorporation under non-inductive conditions of continuous light. After transfer of the plants from light to darkness RNA synthesis was reduced to about half after the first two hours. This occurred not only when the plants were precultivated in continuous light but also after an interruption of the dark period by light for 31/2 h. The low level of uridine incorporation was maintained for the whole duration of the dark period. Incorporation regained its initial level after exposure of the plants to light irrespective of the duration of the preceding dark period. After this immediate rise of uridine incorporation in plants transferred from darkness to light a slight temporary decrease was observed in light. In darkness the decrease of incorporation into the nucleoli was still more marked than the reduction of overall incorporation. After the termination of the dark period incorporation into the nucleolus rose slowly and extranucleolar incorporation was relatively enhanced during the first 10 h of light in induced plants. The fluctuations of RNA synthesis observed in the shoot apex during photoperiodic treatment may be regarded as a necessary condition for the transition from the vegetative to the reproductive state.     

Nucleic acid synthesis and effect of glucose on its kinetics in cotyledons ofChenopodium rubrum l. during photoperiodic induction

In cotyledons ofChenopodium rubrum L. polydisperse RNA is synthesized in the region of the low molecular weight RNAs during photoperiodic induction. After short-time labelling the rate of 4s RNA synthesis was always higher in induced plants than in plants having obtained a light-break in the middle of the dark period. When glucose was added to the nutrient medium during the dark period of a single photoperiodic cycle the rate of nucleic acid (NA) synthesis was higher in non-induced plants than in induced ones at the termination of the dark period. In plants induced by two cycles in the absence of glucose the rate of NA synthesis at the termination of the second dark period was higher in induced than in non-induced plants. This difference is due to the differential kinetics of NA synthesis during darkness. In plants induced in the presence of glucose the peak of the rhythm in NA synthesis was advanced by 4 h relative to that found in plants induced in the absence of sugar. Thus, the termination of the dark period coincided with the negative slope of the oscillation in plants induced in the presence of glucose, while in plants having obtained a light-break NA synthesis decreased only slightly after having attained its peak. In plants induced in the absence of glucose the termination of the dark period coincided with the peak in the rhythm in NA synthesis. The rhythm in NA synthesis of the cotyledons during the dark period of an inductive cycle is out of phase with the rhythm in flower initiation.     

Changes in the content of endogenous auxins in apical buds ofchenopodium rubrum L. Induced with respect to the endogenous rhythm in capacity to flower

The content of endogenous auxins was examined in apical buds ofChenopodium rubrum plants induced by a photoperiodic cycle of 16h darkness and 8h light followed by a dark period of various duration so as to correspond with either maximal or minimal flowering response in the endogenous rhythm in capacity to flower initiated by the photoperiodic treatment. Apical buds of potentially generative plants contained less auxins than apical buds of plants which remained in the vegetative state. Apical buds from plants treated with kinetin (1. 10^-3 M) and therefore remaining in the vegetative state showed an auxin level comparable to that of untreated plants exhibiting minimal flowering response irrespective of the duration of the second dark period. Plants cultivated on a sucrose solution (0.6 M) during the second dark period became generative even at the normal minimum of flowering. The auxin content of the apical buds was low, similarly as in untreated plants induced for a period leading to maximal flowering response. On the other hand, apical buds from plants grown on sucrose solution during a dark period leading to the manifestation of maximal flowering response showed a relatively high auxin content comparable to that found in untreated plants which had obtained a more extended induction by three photoperiodic cycles. The results are discussed with respect to the possible role of endogenous auxins in the regulation of the changes in growth correlations occurring in the shoot apex during photoperiodic induction and in the expression of the competence to flower.     

Retarding effect of inhibitors of protein and RNA synthesis on chlorophyll and protein breakdown

Cycloheximide, ethionine,p-fluorophenylalanine, 6-azauracil, 5-diazouracil and vanillin, applied at relatively high concentrations, retarded the yellowing of kale (Brassica oleracea L. var.acephala) leaf discs in darkness, and stimulated it in light. All the compounds inhibited protein synthesis and retarded protein breakdown. Cycloheximide,p-fluorophenylalanine, diazouracil and vanillin also inhibited the incorporation of uracil-¹⁴C into RNA of senescing discs. Abscisic acid and 2-chloroethylphosphonic acid accelerated yellowing both in darkness and in light and stimulated the protein breakdown in senescing discs. Abscisic acid inhibited the chlorophyll, protein and RNA synthesis in detached, greening cucumber cotyledons. There was no direct correlation between the activity of a given compound as an inhibitor of yellowing in darkness, and the degree of inhibition of RNA synthesis. The arrest of yellowing in darkness is possibly a consequence of the retarded rate of protein breakdown. Yellowing in light, on the contrary, is dependent on the actual rate of protein synthesis.     

Photoperiodic control of tetrasporangium formation in the red alga Rhodochorton purpureum

Six geographical isolates of Rhodochorton purpureum (Lightfoot) Rosenvinge (Rhodophyta, Nemalionales) formed tetrasporangia only in short days at 10°C. For most isolates, the critical day-length increased with latitude of origin from 9.5 h for an isolate from California to 14.5 h for one from Antarctica. Tetrasporangium production could be induced by 9–15 short-day cycles followed by a further 22–28 cycles in long days. A night-break consisting of 1 h of white light in the middle of a 16-h dark period inhibited the short-day response of isolates from low latitudes, but not those from higher latitudes. When a similar night-break was given in the middle of a 14-h dark period, however, the response of all isolates was at least partially inhibited. Night-breaks given at any time in the central 7 h of a 14-h dark period were equally inhibitory. Broad-band red light (0.3–0.4 mmol m^-2), given as a night-break, caused 50% inhibition of the short-day response. At a slightly higher photon exposure (0.6 mmol m^-2, given as 1 μmol m^-2 s^-1 for 10 min), narrow-band red (662 nm) and blue (448 nm) light caused similar inhibition, but green (547 nm) and far-red (731 nm) were ineffective as night-breaks. The inhibitory effect of a 10-min night-break with red light could not be reversed by subsequent exposure to an equal photon exposure of far-red light. These results add to the existing evidence that the pigments mediating photoperiodic responses among algae are more varied than those among flowering plants.     

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.     

Light Rquirements for Photoperiodic Sensitivity in Cotyledons of Dark-grown Pharbitis nil

The light requirements for induction of flowering by a long dark period were investigated in dark-grown seedlings of Pharbitis nil Chois, cv. Violet. The cotyledons bcame photoperiodically sensitive to a 24 h dark period by two 1 min red irradiations (6.3 μmol m⁻² S⁻¹) separated by a 24 h dark period. The reversibility of the effect of brief red irradiations, and the effectiveness of low energies of red irradiation suggest the involvement of phytochrome in the induction of photoperiodic sensitivity. Partial de-etiolation occurred after these brief periods of red irradiation but the seedlings were not capable of net CO₂ uptakeeven 7 h after the start of the main light period that followed the critical dark period. A changing response to the duration of the priod of darkness given between the two short red irradiations showed the the correct phasing of an endogenous photoperiodic rhythm is needed for the attainment of photoperiodic snsitivity.     

Circadian Timekeeping for the Photoperiodic Control of Budset in Picea abies (Norway Spruce) Seedlings

Romanian populations of Norway spruce are induced to set terminal buds by four inductive cycles of 8 h light/16 h darkness. To distinguish between circadian and hourglass timekeeping for the photoperiodic control of budset, seedlings were raised in continuous light at 300 µmol m^-2s^-1 at 20°C for 10 weeks. They were then exposed to an extended night regime consisting of three cycles of 8 h light/40 h dark with 4-h or 1-h nightbreaks (120 µmol m^-2s^-1) applied to groups of plants at intervals during the extended night. Following a final cycle of 8 h light/16 h dark to maximize budset, the plants were transferred to continuous light. Budset was delayed when the night-break was applied close to the critical nightlength (CNL) of 6–7 h or about 22–23 h later in the extended night, consistent with circadian rather than hourglass timekeeping. Confidence intervals were calculated for the times to maximum effect of the night-breaks.     

Changes in cotyledon mRNA during floral induction of Pharbitis nil CV. Violet

Floral induction in seedlings of Pharbitis nil Choisy cv. Violet, with one cotyledon removed, was manipulated by applying various photoperiodic treatments to the remaining cotyledon. Populations of polyadenylated RNA from treated cotyledons were examined to identify messages specifically involved in floral induction. The RNA was translated in vitro using a wheat-germ system, and the resulting translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Substantial qualitative and quantitative differences were found between mRNA from cotyledons of seedlings kept in continuous light (non-induced) and of seedlings given a 16-h dark period (induced). In contrast, inhibition of flowering with a night-break resulted only in one detectable, quantitative difference in mRNA.Abbreviations CL continuous light - kDa kilodalton - NB 16 h darkness+10 min red-light break, 8 h into the dark period - poly(A)⁺ RNA polyadenylated RNA (isolated by binding to a cellulose oligodeoxythymidine affinity column) - SD short day (16 h dark) - SDP short-day plant - SDS sodium dodecyl sulfate     

Different circadian rhythms regulate photoperiodic flowering response and leaf movement in Pharbitis nil (L.) Choisy

The phase shifting effect of red light on both the leaf movement rhythm, and on the rhythm of responsiveness of photoperiodic flower induction towards short light breaks (10 min red light), has been studied in Pharbitis nil, strain Violet, and comparisons between the two rhythms have been made. The phase angle differences between the rhythms after a phase shift with 2 or 6 h of red light given at different times during a long dark period were not constant. The results indicate the involvement of two different clocks controlling leaf movement and photoperiodic flower induction.Abbreviations DD continuous darkness - l:D x:y light/dark cycles with x hours of light and y hours of darkness - PPR rhythm of photoperiodic responsiveness towards light break     

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.     

Chloroplast Biogenesis: XXII. Contribution of Short Wavelength and Long Wavelength Protochlorophyll Species to the Greening of Higher Plants

The contribution of short and long wavelength membrane-bound fluorescing protochlorophyll species to the over-all process of chlorophyll formation was assessed during photoperiodic growth. Protochlorophyll forms were monitored spectrofluorometrically at 77 K during the first six light and dark cycles in homogenates of cucumber (Cucumis sativus L.) cotyledons grown under a 14-hour light/10-hour dark photoperiodic regime, and in cotyledons developing in complete darkness. In the etiolated tissue, short wavelength protochlorophyll having a broad emission maximum between 630 and 640 nm appeared within 24 hours after sowing. Subsequently, the long wavelength species fluorescing at 657 nm appeared, and accumulated rapidly. This resulted in the preponderance of the long wavelength species which characterizes the protochlorophyll profile of etiolated tissues. The forms of protochlorophyll present in etiolated cucumber cotyledons resembled those in etiolated bean leaves in their absorption, fluorescence, and phototransformability. A different pattern of protochlorophyll accumulation was observed during the dark cycles of photoperiodic greening. The short wavelength species appeared within 24 hours after sowing. Subsequently, the long wavelength form accumulated and disappeared. The long wavelength to short wavelength protochlorophyll emission intensity ratio reached a maximum (~3:1) during the second dark cycle, then declined during subsequent dark cycles. Short wavelength species were continuously present in the light and dark. Primary corn and bean leaves exhibited a similar pattern of protochlorophyll accumulation. In cucumber cotyledons, both the short and long wavelengths species appeared to be directly phototransformable at all stages of photoperiodic development. It thus appears that whereas the long wavelength protochlorophyll species is the major chlorophyll precursor during primary photoconversion in older etiolated tissues, both long wavelength and short wavelength species seem to contribute to chlorophyll formation during greening under natural photoperiodic conditions.