Accumulation of Phenylpropanoids in Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Poor Nutrition

Flowering of Pharbitis nil strain Violet is induced in continuouslight under poor nutritional conditions. High-performance liquidchromatography of extracts of the cotyledons revealed that twocompounds in addition to chlorogenic acid accumulate under suchconditions. The compounds were identified as pinoresinol glucosideand p-coumaroylquinic acid. The endogenous levels of these phenylpropanoidswere correlated with the flowering response when nutrition waspoor. However, activation of phenylpropanoid biosynthesis seemednot to be essential for the induction of flowering. (Received May 17, 1993; Accepted July 26, 1993)     

Dihydrokaempferol Glucoside from Cotyledons Promotes Flowering in Pharbitis nil

An extract of cotyledons of Pharbitis nil, which had been exposedto short-day conditions, was tested for flower-promoting activityin a shoot-tip assay system in vitro. The crude extract hadno flower-promoting activity, however, after partitioning ofthe crude extract with dichloromethane, the resulting aqueousfraction had flower-promoting activity. This activity was separatedinto two fractions by column chromatography on Toyopearl HW-40.One active fraction was identified as dihydrokaempferol-7-O-rß-D-glucoside(DHK-glc). This compound exhibited flower-promoting activityat the extremely low concentration of 4.4x10^-9. (Received April 25, 1995; Accepted August 11, 1995)     

The Inhibition of Flowering by Non-Induced Cotyledons of Pharbitis nil

Inhibitory effects on flowering of a non-induced cotyledon havebeen examined in Pharbitis nil seedlings. The photoperiodicinduction of one cotyledon was accomplished by wrapping it inaluminium foil for 13 to 15 h while the seedling remained inthe light. The presence of the other cotyledon in the lightblocked this inductive stimulus. The timing of its inhibitoryeffect suggested that its action was to block the expressionof the inductive stimulus, presumably at the shoot apex. Byvarying the area of the non-induced cotyledon parallel inhibitoryeffects were shown on export of stimulus and of ¹⁴C-labelledassimilate to the apex from the induced cotyledon. Thus, partof the inhibition was by interference with assimilate/stimulusco-transport in the phloem. However, an additional inhibitoryeffect was also evident and for this second component therewas no relationship between assimilate and stimulus transport.This latter inhibition was generated by brief light interruptionsof darkness given to one cotyledon only whilst the other waswrapped. The control treatment, removal of the unwrapped cotyledon,did not alter flowering compared to seedlings with intact, darkenedcotyledons. Thus, these studies show that the brief night interruptionsacted to trigger a photoperiodically sensitive inhibitor notto block induction. The implications of these findings are discussedin relation to models of time measurement in the photoperiodiccontrol of flowering. (Received March 20, 1989; Accepted November 16, 1989)     

Flower-inducing and -inhibiting activities of Phloem Exudate from Cotyledons of Pharbitis Seedlings

The flower-inducing and -inhibiting activities of phloem exudate (PE) prepared from cotyledons of Pharbitis seedlings were examined, using apex cultures in vitro from Pharbitis as a bioassay system.The PE was prepared from photoperiodically-induced cotyledons (SD-PE). The SD-PE was subjected to the following fractionations: When the SD-PE was extracted with CHCl₃ and then ethyl acetate, the inducing activity was located in the final aqueous fraction. The activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). The diffusate was fractionated by ion exchange chromatography, and flower-inducing activity was found in the fraction adsorbed onto anion exchange resin. When the fraction was applied to a Sep-Pak C18 cartridge, the activity eluted with 25% MeOH. As a result of the above fractionation, activity was increased about 30-fold.The nature of the flower-inhibiting activity of the PE taken from cotyledons exposed to continuous-light conditions was examined (CL-PE). The inhibiting activity was decreased as the cotyledons were exposed to longer dark periods; it appeared to be heat-stable. The CL-PE also inhibited flowering in Lemna. The CL-PE was subjected to the following fractionations: When the CL-PE was extracted with CHCl₃ and ethyl acetate, activity was located in the final aqueous fraction. Activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). When the diffusate was fractionated by ion exchange chromatography, the activity was found in the flow-through fraction. When the fraction was applied to a hydroxyapatite cartridge, the activity eluted with 25 mM sodium phosphate buffer. When the fraction was re-dialyzed (molecular weight cut off was 1,000), the diffusate contained the activity. As a result of the above fractionation, activity was increased about 10-fold.     

Accumulation of Phenylpropanoids in the Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Low Temperature Treatment, and the Effect of Precedent Exposure to High-Intensity Light

Extracts from the cotyledons of seedlings of Pharbitis nil strain‘Violet’ cultured at low temperature, which inducestheir flowering even in continuous light, with or without precedentexposure to high-intensity light, which shortens the periodof low temperature required for flowering, were analyzed byHPLC for substances correlating with the flower-inducing process.The content of two phenylpropanoids were found to increase duringthe low-temperature, and were identified as 3-O-feruloylquinicacid and dehydrodiconiferyl alcohol-13-O-ß-D-glucoside.The increase was more rapid in the cotyledons exposed to high-intensitylight before the low-temperature. This suggests that the accumulationof these compounds is correlated to the promotive effect ofhigh-intensity light on the flower-induction by low temperature. (Received March 7, 1994; Accepted April 2, 1994)     

Correlation between Long-Day Flowering and Root Elongation in Pharbitis nil, Sfrain Kidachi

Long-day flowering of Pharbitis nil, dwarf strain Kidachi, at20?C was greatly influenced by the size of the culture vesseland the number of plants per vessel. The smaller the vessel,the greater the flowering response. The volume of nutrient solutionper plant was not decisive for long-day flowering. For instance,plants cultured singly in 200-ml beakers flowered, but thosecultured in 5,000-ml vessels (33?26?11.5 cm, 48 plants per vessel)did not, even though there was only about 100 ml of nutrientsolution per plant. Long-day flowering was always accompaniedby the suppression of root elongation, but not by a decreasein the dry weight of roots or shoots, or in the rate at whichthe leaf primordia appeared (plastochrone). Aeration of thenutrient solution or culture in vermiculite promoted root elongationeven in small vessels, thereby inhibiting long-day flowering.Thus the suppression of root elongation seems to be necessaryfor long-day flowering. Removal of the roots or cotyledons;however, suppressed long-day flowering even when root elongationwas inhibited by culture in small vessels. When plants werecultured at 24?C, suppression of root elongation (culture ina small vessel) did not induce long-day flowering; but, short-daytreatment induced flowering without suppressing root elongation. (Received April 19, 1982; Accepted June 24, 1982)     

Enhancement of Flowering by Acetic Acid Bacteria-oxidized Products in Pharbitis Seedlings

When seedlings of Pharbitis nil Chois., strain Shifukurin weregrown in a medium containing acetic acid bacteria-oxidized ethanol(AABOE) or acetic acid bacteria-oxidized methanol (AABOM), 100%flowering was induced by exposure to a single dark period of9.5 to 10.5 hr, while this regime induced little or no floweringin control plants. When the plants were grown in water containingthe freeze-dried substance from an AABOE solution, floweringwas also enhanced. Vegetative growth was considerably suppressedby these fermented alcohols and by the freeze-dried substancefrom an AABOE solution. However, the freeze-dried substancefrom the AABOM solution had no effect on either flowering orvegetative growth. (Received May 13, 1981; Accepted August 21, 1981)     

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.     

The Relationship Between Flowering Induction and the Level of Adenosine Triphosphate in Pharbitis nil

A study has been made on the changes of ATP and protein content in cotyledons and apices of Pharbitis nil after flowering induction. Protein content of the cotyledons which have just got through the induction is 68% higher than that of the control, but the difference trends to disappear there after. The. difference of protein content between the induced and uninduced apices is not so obvious in the first three days after induction, but quite evident on the fourth day (30% higher in the induced apices) suggesting that there is some relationship between protein metabolism and flowering induction both in the cotyledons and in the apices. Just after the seedlings have been induced, ATP content of the cotyledons is getting much (134%) higher than that of the control and the tendency is retained towards the fourth day after induction. Generally ATP content in apices is one order of magnitude higher than that in cotyledons. Although ATP content in the apices is only slightly higher than that of the control soon after induction, it gains quite a lot in the second day until the fifth day the end of our experiment. In the third day after induction ATP level in the apices reaehs to the maximum (20.6×10-2 μmol/g, apices) which is 37% higher than that of the control. The results show that flowering induction is bound to be followed by increase of proteins and ATP both in apices and in cotyledoms. It also. shows both formation of the stimulus in induced cotyledons and evocation in the apices might be all concerned in expression of some genes and synthesis of new RNA and protein. According to the maximum peak of ATP in the apices and cotyledons appeared in 3rd to 4th day after induction, it seems that the inductive effect both in the cotyledons and apices might continue for some time under the following uninduced condition.     

Phase Shifting Effects in the Photoperiodic Rhythm of Flowering in Dark-Grown Seedlings of Pharbitis nil Choisy

Dark-grown seedlings of Pharbitis nil Choisy received an initialsaturating fluence of red (R) light (R₁), followed at intervalsby further R pulses (R₂ and R₃). R₂ was given at different timesafter R₁. R₂ was used to scan the subsequent 72 h period. The initial exposure to R (R₁) initiated a circadian rhythmin the flowering response to the scanning R exposure (R₂). Thephase of the rhythm was shifted by the second exposure to R(R₂) and the sensitivity of the phase-shifting response variedwith the time of giving the R₂ pulse. The direct response toR₂ (i.e., the magnitude of flowering produced in the absenceof a scanning R₂ exposure) also varied in sensitivity. WhenR² was given 4h after R₁, the phase-shift was achieved by anexposure of 20 s (sufficient to establish 20–25% Pfr/P)but more than 80 s was required to saturate the direct floweringresponse at this time. When given 16 h after R₁, 80 s of R₂(sufficient to establish 55% Pfr/P) was required for the phase-shift,whereas the maximum promotion of flowering was produced by only5 s R. These differences in fluence-response relationships indicatethat the direct flowering response to a dark interruption withR and the effect of such an interruption to phase-shift theunderlying rhythm are distinct processes. (Received April 30, 1986; Accepted November 11, 1986)     

Correlation between Biosynthesis of Chlorogenic Acid and Flowering in Asparagus Seedlings Induced by Carbamate Compounds

The flowering of Asparagus seedlings induced by carbamate compoundswe had developed was triggered when the chemicals were appliedin such a way that they were active during the period of shootdifferentiation, i.e., 4 to 10 days after seeding. The rateof flowering was closely correlated to the decrease in the chlorogenicacid content of the bud primordium caused by the carbamate treatment.Cytokinins stimulated metabolism in the buds and decreased theinhibitory effect of the carbamates on it. The site of actionof the chemicals appears to be somewhere on the metabolic pathwaythat leads to the synthesis of chlorogenic acid. (Received August 2, 1990; Accepted February 14, 1991)     

Carbon Dioxide and Flowering in Pharbitis nil Choisy

The effects of photoperiod on floral and vegetative development of Pharbitis nil were modified by atmospheric CO₂ concentrations maintained during plant growth. Short day (SD) photoperiods caused rapid flowering in Pharbitis plants growing in 0.03 or 0.1% CO₂, while plants in long day (LD) conditions remained vegetative. At 1 or 5% CO₂, however, flower buds were developed under both the SD and LD photoperiods. Flowering was earliest in the plants exposed to SD at low CO₂ concentrations which formed floral buds at stem node 3 or 4. At high CO₂ concentrations, floral buds did not form until stem node 6 or 7. Both high CO₂ concentrations and LD photoperiods tended to enhance stem elongation and leaf formation.     

Studies on the Photoreceptors for the Promotion and Inhibition of Flowering in Dark-Grown Seedlings of Pharbitis nil Choisy

Three-day-old etiolated seedlings of Pharbitis nil were exposedto red light for 10 min and sprayed with N⁶-benzyladenine beforetransfer to a 48-h inductive dark period, after which they weregrown under continuous white light. A second red irradiationpromoted flowering when given at the 5 and 24th hour of theinductive dark period but inhibited flowering at the 10 and15th hour. Far-red light inhibited flowering when given at anytime during the first 24 h of the dark period. Red/far-red reversibilitywas clearly observed at the 0, 5, 10 and 24th hour, but notat the 15th hour when both red and far-red lights completelyinhibited flowering. The action spectrum for the inhibition of flowering at the 15thhour of the inductive dark period had a sharply defined peakat 660 nm and closely resembled the absorption spectrum of theP_R form of phytochrome. The photoreceptors involved in thesephotoreactions are discussed. (Received June 10, 1983; Accepted July 6, 1983)     

Accumulation of Ascorbic Acid in the Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Low-Temperature Treatment and the Effect of Prior Exposure to High-Intensity Light

Seedlings of Pharbitis nil strain ‘Violet’ werecultured at a low temperature, which induces their floweringeven in continuous light, with or without prior exposure tohigh-intensity light, which enhances the flower-inducing effectof the exposure to low temperature. Analysis by HPLC of extractsof cotyledons showed that the level of an unstable compoundincreased during these treatments, in addition to the increasein levels of phenylpropanoids reported previously. The compoundwas identified as ascorbic acid from the spectroscopic data.The change in the concentration of ascorbic acid at low temperaturewas correlated with the increase in the induction of floweringand the increase in levels of the phenylpropanoids. The rapidincrease in level of ascorbic acid after exposure to high-intensitylight reflected the promotive effect of high-intensity lighton the induction of flowering at low temperature. However, levelsof ascorbic acid also increased in seedlings of P. nil strain‘Kidachi’ that were cultured in high-intensity light,a treatment that does not induce flowering in this strain. Thus,ascorbic acid cannot be associated with the induction of floweringby high-intensity light alone. Ascorbic acid increased the rateof formation of caffeic acid from p-coumaric acid in vitro,a result that suggests that ascorbic acid might be involvedin the increases in levels of phenylpropanoids in the seedlings. (Received April 17, 1995; Accepted August 1, 1995)     

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.     

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.

     

Flowering in seedlings of Pharbitis nil induced by benzyladenine applied under a non-inductive daylength

Benzyladenine (BA) induced flowering when applied at a highconcentration (10_–3M) to the cotyledons of seedlings ofPharbitis nil held in a non-inductive photoperiod (continuousirradiation). The transition to flowering occurred rapidly (within4 to 5 days) following as few as two days of application ofBA. There was no flowering after 20 days in control seedlings. Induction of flowering by BA was sometimes, but not invariably,associated with (i) greater retention of dry matter in the cotyledonsand (ii) inhibition of shoot elongation and (iii) a slower rateof increase in root and shoot dry weight. Slowing stem elongationby application of growth inhibitors or by removal of one orboth cotyledons did not induce flowering. Various cytokininsother than BA were active but so was triacanthine a structurally-related,non-cytokinin compound. Non-hormonal action of these compoundsis implied. Possible direct effects of BA on processes of floweringare discussed. ¹Present address:Faculty of Agriculture, Mie University, TsuCity, Mie Prefecture 514-22, Japan. (Received July 14, 1980; )     

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil

Flowering and dwarfism induced by 5‐azacytidine and zebularine, which both cause DNA demethylation, were studied in a short‐day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non‐inductive long‐day (LD) conditions. The flower‐inducing effect of 5‐azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non‐inductive photoperiodic conditions. These results suggest that the flowering‐related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A₃ but not by t‐zeatin or kinetin. The change in the genome‐wide methylation state was examined by methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD‐specific fragments were extracted from the gel of the MS‐AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.     

Gibberellins in Relation to Growth and Flowering in Pharbitis nil Chois

Flowering can be modified by gibberellins (GAs) in Pharbitis nil Chois. in a complex fashion depending on GA type, dosage, and the timing of treatment relative to a single inductive dark period. Promotion of flowering occurs when GAs are applied 11 to 17 hours before a single inductive dark period. When applied 24 hours later the same GA dosage is inhibitory. Thus, depending on their activity and the timing of application there is an optimum dose for promotion of flowering by any GA, with an excessive dose resulting in inhibition. Those GAs highly promotory for flowering at low doses are also most effective for stem elongation (2,2-dimethyl GA₄ GA₃₂ > GA₃ > GA₅ > GA₇ > GA₄). However, the effect of GAs on stem elongation contrasts markedly with that on flowering. A 10- to 50-fold greater dose is required for maximum promotion of stem elongation, and the response is not influenced by time of application relative to the inductive dark period. These differing responses of flowering and stem elongation raise questions about the use of relatively stable, highly bioactive GAs such as GA₃ to probe the flowering response. It is proposed that the `ideal' GAs for promoting flowering may be highly bioactive but with only a short lifetime in the plant and, hence, will have little or no effect on stem elongation.