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.

     

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)     

Role of the Photoperiod Preceding a Flower-Inductive Dark Period in Dark-Grown Seedlings of Pharbitis nil Choisy

Flowering responses to a single photoperiod, of various durationsand irradiances, followed by an inductive dark period were investigatedwith dark-grown seedlings of Pharbitis nil Choisy. The numberof flower buds induced in each plant (NFB) increased with theincrease of both duration and irradiance of the photoperiod.Reciprocity did not hold for this photoresponse within the rangeof 0-16 h and 2.5-10 W-m^-2, NFB depending on the duration ratherthan the irradiance. With lengthening of the dark period followinga photoperiod of 8 h or less, two different phases alternatelyappeared so that NFB sharply increased at 20-24 h and 40-43h after the onset of the photoperiod, then gradually decreased.When the photoperiod was longer than 8 h, NFB sharply increasedat 12–16 h after the end of the photoperiod and remainedaround the saturated value with longer dark periods. Far-redlight given immediately after the photoperiod inhibited flowering,the inhibitory effect being stronger the shorter the photoperiod.This far-red effect is mediated by phytochrome and P_FR seemsto be required during the inductive dark period following ashort photoperiod for floral induction. (Received December 23, 1983; Accepted April 12, 1984)     

Site of Perception of the Far-Red Inhibition of Flowering in Pharbitis nil Choisy

The site of perception of the inhibition of flowering by far-redlight (729 nm) was investigated in the short-day plant Pharbitisnil, using a specially designed optical fibre apparatus withinterference filters. The cotyledon was found to be the primarysite of perception and no inhibition was obtained when the apexalone was irradiated. However, when the apex and cotyledon weresimultaneously irradiated there was a reduction in the numberof terminal flower buds compared with the cotyledon alone. Forwhole plants, far-red light centred at 729 nm (10 nm bandwidth)was considerably more inhibitory than at 719 nm. ¹Present address: Department of Biology, Guru Nanak Dev University,Amritsar—143005, Panjab, India. (Received April 28, 1986; Accepted June 23, 1986)     

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.     

Evidence for the Involvement of Calcium Ions in the Photoperiodic Induction of Flowering in Pharbitis nil

The effects of ethyleneglycol-bis-(ß-aminoethyl ether)N,N,N',N'-tetraacetic acid, a specific chelator of Ca²⁺ ions;lanthanum chloride, a calcium channel blocker; and chlorpromazine,a calmodulin antagonist, on the photoperiodic induction of floweringin Pharbitis nil were studied by perfusing the plants with aqueoussolutions of the various compounds. All these compounds inhibitedflowering when applied before an inductive 16-h dark periodbut they did not inhibit flowering when applied after the inductivedark period. The results imply that Ca²⁺ ions are involved inthe photoperiodic induction of flowering in P. nil. (Received August 14, 1992; Accepted November 24, 1992)     

牵牛全草的化学成分研究

从牵牛全草(Pharbitis nilChoisy)乙酸乙酯提取物中分离得到十个化合物。据理化性质和光谱分析鉴定其结构为:顺式阿魏酸酰对羟基苯乙胺(1),对羟基桂皮酸酰对羟基苯乙胺(2),阿魏酸酰对羟基苯乙胺(3),顺式对羟基桂皮酸酰对羟基苯乙胺(4),桂皮酸酰对羟基苯乙胺(5),对羟基苯乙胺(6),7-羟基香豆素(7),6-甲氧基-7-羟基香豆素(8),尿嘧啶(9),β-谷甾醇葡萄糖苷(10)。化合物1～9均为首次从该植物中得到。     

Effects of Aminooxyacetic Acid and Its Derivatives on Flowering in Pharbitis nil

Aminooxyacetic acid (AOA) inhibited photoperiodically inducedflowering in Pharbitis nil. The application of AOA to the plumulejust after an inductive period was the most effective in inhibitingflowering. A correlation between inhibition of flowering andinhibition of glutamic-oxalacetic transaminase activity wasobserved with fifteen aminooxy derivatives. (Received April 18, 1992; Accepted June 25, 1992)     

Involvement of Calcium in the Photoperiodic Flower Induction Process of Pharbitis nil

EGTA, a specific Ca(2+) chelator, inhibited the flowering response of Pharbitis nil when applied to the cotyledons immediately before the inductive dark period. Calcium sprayed 30 minutes after the EGTA blocked the effect of EGTA. The length of the critical dark period was increased both by EGTA and by LaCl(3). The calmodulin antagonists W-7 and chlorpromazine also reduced the flowering response. On the other hand, A23187, a calcium ionophore, increased the flowering response. Both EGTA and A23187 were effective at certain times of the photoperiod but had almost no effect when applied at other times. The results indicate that the level of endogenous Ca(2+) may be limiting for floral induction in Ph. nil. Ca(2+) seems to play a role during the early stages of the inductive dark period.     

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)     

Electrophoretic Analysis of Florally-Evoked Meristems of Pharbitis nil Choisy cv. Violet

Seedlings of Pharbitis nil Choisy cv. Violet were subjectedto a single SD (floral induction) or NB (inhibition of induction)treatment. Soluble proteins were extracted from the apical regionof seedlings just before the treatment or at various times afterthe treatment and were analyzed by two-dimensional polyacrylamidegel electrophoresis (2-D PAGE) to identify the changes specificallyinvolved in floral induction. The patterns of polypeptides werealmost identical between apices of short day-treated plants(SD-plants) and night break-treated plants (NB-plants), andquantitative differences were found in five polypeptide spots.Intensities of three of these polypeptide spots increased graduallyor transiently after SD treatment. The remaining two spots increasedin intensities after NB treatment. A fairly consistent resultswere obtained by a similar analysis of proteins labelled invivo. These differences were detected at relatively early stagesof flower formation, and some of them were retained or evenmagnified at a later stage. These findings suggest that thereare remarkably subtle differences between the complements ofproteins in the evoked and non-evoked apical meristems, andthat a few consistent quantitative differences are clearly apparentat the relatively early stages of formation of flowers. (Received September 20, 1989; Accepted November 16, 1989)     

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.     

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)     

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.     

Metabolism and Translocation of Gibberellins in the Seedlings of Pharbitis nil (II). Photoperiodic Effects on Metabolism and Translocation of Gibberellins Applied to Cotyledons

We investigated the metabolism and translocation of two gibberellins(GAs), [³H]GA₂₀ and [³H]GA₁, which were applied at low concentrationto the cotyledons of Pharbitis nil (cv. Violet). Seedlings weregrown under three different photoperiodic conditions: continuouslight (CL-CL), continous light followed by short day conditions(CL-DT) and long day conditions followed by short day conditions(DT-DT). Translocation of the applied [³H]GAs from cotyledonsto hypocotyls was promoted by DT for all GAs examined. Whilethe conversion of the translocated [³H]GA₁ to [³H]GA₈ and itsconjugates was rapid in hypocotyl, the conversion of translocated[³H]GA₂₀ to [³H]GA₂₉ was slow. Radioactivity in epicotyls wasdetected much more rapidly on application of [³H]GA₂₀ than of[³H]GA₁, [³H]GA₈ and [³H]GA₂₉ and their conjugates. The conversionof [³H]GA₂₀ to [³H]GA₁ in the epicotyl was more rapid underCL-CL conditions. This result in consistent with the higherlevel of endogenous GA₁ existing in epicotyls under CL-DT thanDT-DT conditions. However, when [³H]GA₁ was applied to the cotyledon,only small amounts of [³H]GA₈ and its conjugates were detectedin the epicotyl regardless of the photoperiodic conditions.This result may suggest that the translocation and metabolismof [³H]GA₂₀ from cotyledons to epicotyl was faster under CL-CLthan DT-DT conditions and may correlate with the increased epicotylelongation of GA₂₀ treated plants under CL-DT than DT-DT conditions. (Received June 28, 1995; Accepted November 2, 1995)     

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.     

Transient Increase in the Level of mRNA for a Germin-Like Protein in Leaves of the Short-Day Plant Pharbitis nil during the Photoperiodic Induction of Flowering

A cDNA corresponding to an in vivo labeled protein whose levelincreased during flower-inductive darkness in the cotyledonof the short-day plant Pharbitis nil Choisy cv. Violet was isolatedand characterized. The deduced amino-acid sequence of the protein(designated PnGLP; P. nil germin-like protein) showed homologyto that of a GLP of Sinapis alba, a leaf protein, the mRNA accumulationof which showing circadian oscillations. PnGLP mRNA was detectedspecifically in the cotyledon and leaf, in particular, in theyoung expanded cotyledon and leaf. Accumulation of PnGLP mRNAincreased transiently during flower-inductive darkness and peakedat a time that corresponded approximately to the critical nightlength. This mRNA peak was reduced by a brief exposure to redlight at the 8th hour of darkness. The level of PnGLP mRNA peakedabout 10 h from the beginning of the dark period, whereas itwas reported that the level of mRNA for GLP of a long-day plantS. alba increased about 14 h from the beginning of the lightperiod. Thus, the different time courses of accumulation ofthe mRNAs for leaf-specific GLPs appear to reflect the differencesin photoperiodic responses of each plant. (Received May 16, 1996; Accepted July 5, 1996)