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.     

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.     

Changes in nucleic acid synthesis in cotyledons and apical buds of chenopodium rubrum l. associated with photoperiodic induction

The nucleic acid (NA) fractions were analyzed in cotyledons and apical buds ofChenopodium rubrum plants by means of acrylamide electrophoresis at the end of the dark period of a different number of photoperiodic cycles or after transfer of the plants to light for 4 h subsequent to the termination of the dark period. The plants were labelled with³²P three hours prior to sampling. The uptake of³²P into the cotyledons was higher in light than in darkness in all cases, however, it was not in correlation with³²P incorporation into the NA fractions. After one dark period lasting 8 or 16 h NA synthesis in light did not increase in comparison with darkness. After two or more photoperiodic cycles NA synthesis was higher in light than in darkness irrespective of whether the dark period lasted 8 or 16 h. NA synthesis was distinctly highest after two inductive cycles lasting 16 h. In buds NA synthesis was slightly shifted in favour of ribosomal RNA as compared with cotyledons. In the cotyledons the increase in light was mainly duo to a raise of rRNA synthesis whereas in the buds synthesis of sRNA and DNA increased, as well.     

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     

The incorporation of uridine-3H into the shoot apices of photoperiodically induced and non-induced plants ofChenopodium rubrum L.

The influence of photoperiodic induction on the incorporation of uridine-³H into the shoot apices ofChenopodium rubrum was studied using the technique of autoradiography. No increase in uridine incorporation was detected either during induction lasting three days or immediately after its termination. Pyroninophylia likewise did not rise. However, changes in uridine incorporation related to morphogenetic activity during leaf formation and later during differentiation of inflorescences were well marked. The distribution of label in the nucleus immediately after three inductive cycles shows the ratio of extranucleolar to nucleolar incorporation to be higher in non-induced control plants than in induced ones. Data from literature pointing to an activation of RNA synthesis during transition to flowering are discussed and compared with other systems where ontogenetic changes are accompanied by marked changes in RNA synthesis. It is assumed that the activation of RNA synthesis after induction is connected mainly with the activation of growth. However, inChenopodium rubrum photoperiodic induction proceeds together with limited growth and without activation of RNA synthesis.     

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.     

Gibberellins in the control of photoperiodic flower transition in Pharbitis nil

The role of gibberellins in the photoperiodic flower induction of short-day plant Pharbitis nil has been investigated. It has been found that the endogenous content of gibberellins in the cotyledons of P. nil is low before and after a 16-h-long inductive dark period. During the inductive night the content of gibberellins is high at the beginning of darkness and about the middle of the dark period. Exogenous GA₃ when applied to the cotyledons of non-induced plants does not replace the effect of the inductive night but it can stimulate the intensity of flowering in plants cultivated on suboptimal photoperiods. GA₃ could also reverse the inhibitory effect of end-of-day far-red light irradiation on P. nil flowering. 2-Chloroethyltri-methylammonium chloride (CCC) applied to the cotyledons during the inductive night also inhibited flowering. GA₃ could reverse the inhibitory effect of CCC. The obtained results strongly suggest that gibberellins are involved in the phytochrome controlled transition of P. nil to flowering. Their effect could be additive to that of photoperiodic induction.     

Changes in Cotyledon mRNA during Ethylene Inhibition of Floral Induction in Pharbitis nil Strain Violet

Floral induction in seedlings of Pharbitis nil strain Violet, with one cotyledon removed, was manipulated by applying various ethylene treatments to the remaining cotyledon during a 16 hour inductive dark period. Exposure of cotyledons to ethylene (100 microliters per liter) for 4 hours at different times during the dark period inhibited flowering to some extent, with inhibition being greater towards the end of the dark period. RNA from cotyledons given a 16 hour dark period (induced) or exposed to 100 microliters per liter ethylene throughout the dark period, which completely inhibited flowering, was examined. The poly(A)⁺RNA was translated in vitro using a wheat germ system, and the resulting translation products were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were substantial qualitative and quantitative differences between the poly(A)⁺RNA extracted from induced cotyledons and that from those exposed to ethylene throughout the dark period. Some of these changes are similar to those observed when flowering was inhibited by photoperiodic treatments (M Lay-Yee, RM Sachs, MS Reid 1987 Planta. In press). The significance of these findings to our understanding of the molecular control of flower induction is discussed.     

Variation in the Photoperiodic Control of Flowering of Two Cultivars of Antirrhinum majus L.

The susceptibility to photoperiodic induction of an early (PinkIce) and a late (Orchid Rocket) flowering variety of Antirrhinummajus was investigated. At various times during developmentplants of both varieties were subjected to 1, 2, 3 or 4 long-daycycles. The early variety became increasingly more responsiveto long-days while the late variety showed no positive responsethroughout the course of the experiment. One hour light-breaksgiven midway through the 16-h dark period evoked a decreasein the leaf number of Pink Ice plants. The leaf areas of bothvarieties increased to levels equivalent to long-day grown plants.The net CO₂ uptake of plants grown in light-breaks increasedin proportion to the increase in leaf area. However the specificuptake of CO₂ per unit area was similar for light-break andshort-day grown plants but was substantially lower than thelong-day plants. Suggestions are made to explain the differencesin photoperiodic response of the two varieties in terms of variationin assimilation rate.     

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.     

Involvement of signal transduction pathway components in photoperiodic flower induction in Pharbitis nil

The possible participation of several major components of the signal transduction pathway in photoperiodic flower induction was examined in Pharbitis cotyledons. Exogenous applications of GTP-γ-S (1–10 μ M ) or of the phorbol ester, phorbol 12-myristate-13-acetate (PMA, 0.1–5.0 μ M ) to Pharbitis plants held under a marginal inductive period (11.5 h dark) significantly increased their flowering response. Membrane lipid fluidity, GTP-binding and protein kinase activity were increased following a single flowering-inducing dark period of 16 h; however, a light-break of 10 min that abolished flower induction failed to reverse the dark-induced increase in these processes. Photo-inductive dark conditions significantly increased the content of diacylglycerol (DAG) and phosphoinositides in the cotyledon membranes, together with the activities of their kinases, and a light break decreased them to control levels and below. In addition, a single spraying with GTP-γ-S or PMA at 1 μ M significantly increased both the lipid content and the kinase activities. These compounds also enhanced the kinase activities in vitro. It is concluded that DAG and phosphoinositide metabolism play a role in the linking of the photoperiodic induction of the phytochrome with the flowering response in Pharbitis nil .     

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.     

The role of indol-3-ylacetic acid in regulation of juvenility inXanthium strumarium L.

Cotyledons ofXanthium strumarium, organs with low sensitivity to photoperiodic treatment show a higher free indol-3-ylacetic acid level (by about 35 %) than the first pair leaves, organs with high sensitivity to photoperiodic treatment. This was seen in plants of three different age groups : A. with the first pair of leaves of 15–20 mm in length; B. with the first pair of leaves having finished their growth and C. with the third leaf of 30–40 mm in length. Changes in free IAA level during the inductive dark period were similar in both cotyledons and leaves of the first pair. The level of IAA rose in the first half of the dark period, began to decrease in the latter half, reaching nearly initial level at its end. Application of IAA (10⁻⁴ – 10⁻²M) to the cotyledons reduced their already low photoperiodic sensitivity resulting in inhibition of flowering (almost 70 % using 10⁻⁴M IAA). Elevated free IAA level is assumed to be one of the causal factors of low photoperiodic sensitivity of cotyledons.     

SPECIFIC RNA FROM PHOTOPERIODICALLY INDUCED COTYLEDONS OF PHARBITIS NIL

The nucleotide ratio of several RNA species from cotyledonsof Pharbitis nil subjected to a single 16 hr night with or withouta 15 min light-break, or to continuous light was investigated.RNA species examined were RNAs from nuclear, mitochondrial,microsomal, and supernatant fractions separated by differentialcentrifugation, and s-, r-, and m-RNAs fractionated by methylatedalbumin column chromatography. Of the RNAs examined, m-RNA alone was found to change its nucleotideratio with photoperiod applied. Thus as compared with m-RNAfrom non-induced cotyledons (exposed to continuous light oran interrupted night), m-RNA from cotyledons induced by an uninterruptednight contained significantly reduced guanylic and cytidylicacids on molar ratio basis. A working hypothesis was proposed that floral stimulus productionin cotyledons may be directed by gene DNA derepressed photoperiodically. (Received October 18, 1966; )     

[14C]-Assimilate partitioning in photoperiodically induced seedlings of Pharbitis nil. The effect of benzyladenine

Partitioning of [¹⁴C]-labeled assimilates was studied in relation to photoperiodic floral induction and evocation in one-week-old Pharbitis nil Choisy cv. 'Violet' seedlings. In plants kept under 16 h photoperiods, one 15 h night induced 100% axillary flowering whereas a 24 h night induced both terminal and axillary flowering. A 15 min night break of red light given 8 h after the beginning of the dark period inhibited flowering. Total [¹⁴C]-assimilate distribution among major sinks (plumules + epicotyl and roots + hypocotyl) from a single source cotyledon was unchanged by one inductive night; however, import of [¹⁴C]-assimilates into shoot apices was increased in induced plants compared to vegegative controls. This increase was several-fold in plants subjected to a 24 h night. N⁶-Benzyladenine (BA) application to cotyledons or plumules under non-saturating night lengths increased the number of floral buds per plant without affecting the position of the first floral bud (i.e. the speed of induction). The same treatment caused increased label accumulation in induced apices, while it only slightly affected non-induced ones. The mode of action of BA on flowering through growth stimulation and resulting assimilate mobilization is discussed.     

Changes in organ growth ofChenopodium rubrum due to suboptimal and multiple photoperiodic cycles with and without flowering effect

The growth changes of cotyledons, leaves, hypocotyls and roots due to photoperiodic induction in short day plantChenopodium rubrum were investigated in relation to flowering. Six-day old plants were induced by photoperiods with a different number of dark hours. We found that the degree of inhibition which occurred during induction in the growth of leaves, cotyledons and roots similarly as the stimulation of hypocotyl is proportional to the length of dark period. The photoperiods with 12, 16 and 20 dark hours bring about marked inhibition of growth and at the same time induce flowering in terminal and axillary meristems. The inhibitory effect of critical period for flowering,i.e. 8 dark hours, is not apparent in all criteria used and even the flower differentiation is retarded. The photoperiods of 4 and 6 dark hours did not affect growth and were ineffective in inducing flowering even if their number has been increased. The experiments with inductive photoperiod interrupted by light break have clearly shown that growth pattern characteristic for induced plants can be evoked in purely vegetative ones. Such statement did not exclude the possible importance of growth inhibition as a modifying factor of flower differentiation. We demonstrated that the early events of flower bud differentiation are accompanied by stimulation of leaf growth. The evaluation of growth and development of axillary buds at different nodes of insertion enabled us to quantify the photoperiodic effect and to detect the effects due to differences in dark period length not exceeding 2 hours.     

DIFFERENTIAL RNA SYNTHESIS IN THE SHOOT APEX OF PHARBITIS NIL DURING FLORAL EVOCATION

Early events in the evocation of the flower in Pharbitis nil Chois seedlings were investigated by following the incorporation of tritiated uridine into the shoot apex. The uridine was applied to the expanded cotyledons of seedlings at 8 hr into the inductive dark period. The shoot tips were fixed at 20 hr (a 12-hr labeling period). After the 12-hr labeling period there was considerable label throughout the shoot tips of both control plants (dark period interrupted with 5 min of red light at 8 hr) and plants induced to flower. Both RNase and acid hydrolysis removed the nonexchangeable label and the Azure B staining, thus leading to the conclusion that the uridine was incorporated into RNA. Induction in the cotyledons was followed quickly by an increased synthesis of RNA in the rib meristem region of the receptor bud at the time when the floral stimulus is assumed to be arriving. The increase in RNA synthesis is revealed by an increase in the rib meristem/central zone ratio of counts due to the incorporation of tritiated uridine. A comparison of counts in each of the two regions revealed that the change in ratio was due to an increase in the rib meristem and not due to a decrease in the central zone in induced shoot apices. The initial activation of the rib meristem probably occurred by 16 hr from the beginning of the dark period. Tendencies in the literature to disregard the role of the rib meristem in giving rise to part of the flower are discussed.     

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.     

Exogenously applied melatonin (N-acetyl-5-methoxytryptamine) affects flowering of the short-day plant Chenopodium rubrum

Melatonin ( N -acetyl-5-methoxytryptamine) is an animal hormone synthesized predominantly at night. It often serves as a signal of darkness that regulates circadian rhythmicity and photoperiodism. Melatonin has also been found in algae and higher plants, including the short-day flowering plant Chenopodium rubrum . To test its involvement in plant photoperiodism, melatonin solutions were applied to the cotyledons and plumules of 5-day-old-seedlings of Chenopodium rubrum L., ecotype 374. ³H-labelled melatonin was readily taken up by the plants and was very stable for a period of 37 h from application. Treatment with 100 and 500 µ M melatonin significantly reduced flowering of plants exposed to a single inductive 12-h darkness. Melatonin was efficient only when applied before lights off or during the first half of the dark period. This indicates that melatonin affects some early steps of the transition to flowering. However, it had no effect on the period or phase of a circadian rhythm in photoperiodic time measurement. Melatonin agonists (2-I-melatonin, 6-Cl-melatonin, CGP 52608) and 5-hydroxytryptamine also reduced flowering, whereas 5-methoxytryptamine did not. The results demonstrate that exogenous melatonin is able to influence the early stages of photoperiodic flower induction and/or flower development in a higher plant. Possible mechanisms for this effect are discussed.