Metabolism and Translocation of Gibberellins in Seedlings of Pharbitis nil. (I) Effect of Photoperiod on Stem Elongation and Endogenous Gibberellins in Cotyledons and Their Phloem Exudates

Gibberellin A₁, (GA₁), GA₁₉, and GA₂₀ in phloem exudates andcotyledons of seedlings of Pharbitis nil cv. Violet, grown underdifferent photoperiodic conditions, were qualitatively and semi-quantitativelyanalyzed by a combination of high performance-liquid chromatography(HPLC) and radioimmunoassays (RIA). The levels of GA₁₉ and GA₂₀were higher in cotyledons from plants grown under dark treatment(DT) conditons of 16 h-light/8 h-dark for 6 days followed by8 h-light/16 h-dark for 3 days than in those grown under continuouslight (CL) for 9 days. This relationship was also observed forthe GAs in phloem exudates, although the levels were much lowerthan in the cotyledons. When GAs were applied to the cotyledons,elongation of the epicotyl was promoted more by GA₂₀ than byGA₁ or GA₁₉, especially under the CL treatment. The relativeeffect of GA₁ and GA₂₀ on the epicotyl elongation was reversedwhen these GAs were applied to epicotyls pre-treated with prohexadione,an inhibitor of 2-oxoglutarate-dependent dioxygenases. ³Present address: Frontier Research Program, The Institute ofPhysical and Chemical Research (RIKEN), 2-1 Hirosawa, Wakoshi,Saitama, 351-01 Japan ⁴Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Nagoya, 464-01 Japan     

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.     

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)     

Gibberellins in Immature Seeds of Pharbitis nil

Two new gibberellins, gibberellins A₂₆ and A₂₇ were isolated from immature seeds of Japanese morning-glory (Pharbitis nil) and their structures were elucidated as I and IX.     

Gibberellins in Immature Seeds of Pharbitis nil

A new gibberellin, gibberellin A₂₀ (GA₂₀), was isolated from immature seeds of morning-glory (Pharbitis nil). Its structure was established as 4aα, 7α-dihydroxy-1β-methyl-8-methylenegibbane-1α, 10β-dicarboxylic acid-1→4a lactone (I) on the basis of its physicochemical analysis as well as chemical evidences. GA₂₀ shows marked growth promoting activities on dwarf maize d₂ and d₅ but weak activities on d₁, rice seedling and dwarf pea.     

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.     

Effect of Photoperiodic Induction on the Protein Synthesis of Cotyledons and the Morphology of Apical Meristem of Pharbitis nil Plant

After photoperiodic induction of Pharbits nil seedlings with two expanded cotyledons byshort day, the changes of protein in cotyledons or in shoot apex were investigated by polyacrylamide gel electrophoresis technique and electron, microscopy respectively Electron microscopicobservation shows that a kind of spherical electron-dense bodies appears in vacuoles of the apical meristem. Change of protein patterns also observed in the cotyledons. The number of basicprotein bands increased from eight in the untreated control to ten in the induced cotyledons, andthe number of, buffer-soluble protein bands increased from ten in the untreated control totwelve in the induced cotyledons. Authors suggest that the appearence of new protein bandsand electron-dense bodies is probably related to gene expression in the induction process ofphotoperiod of Pharbitis.     

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.     

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.     

Effect of Photoperiodic Floral Induction on the Metabolism of a Gibberellin Precursor, (-)-Kaurene, in Pharbitis nil

Pharbitis nil seedlings rapidly metabolized (-)-kaurene-17-¹⁴C administered to the cotyledons. Less than 20% of the radioactivity was recovered by extraction of the cotyledons on the following day. Of this the major metabolite was an unidentified acidic material which did not correspond chromatographically to any of the known gibberellins.     

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)     

The Involvement of Cyclic ADPR in Photoperiodic Flower Induction of Pharbitis nil

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from NAD⁺ by ADP-ribosyl cyclases described for several animal cells. Pharmacological studies suggest that cADPR is an endogenous modulator of Ca²⁺-induced Ca²⁺ release channels. There is also information about the sub-micromolar concentration of cADPR in plant cells. Whether cADPR can act as a Ca²⁺-mobilizing intracellular messenger in plant tissue is an unresolved question. Despite the obvious importance of monitoring cADPR cellular levels under various physiological conditions in plants, its measurement has been technically difficult and requires specialized reagents. In the present study a widely applicable sensitivity assay for cADPR is described. We show that Pharbitis nil tissue from cotyledons contains a certain cADPR level. To explain the possible roles of this second messenger in photoperiodic flower induction, some physiological experiments were also performed. The exogenous applications of cADPR to Pharbitis nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. Nevertheless 8-Br-cADPR inhibited flowering when these compounds were applied during a 16-h-long inductive night. The effect of ruthenium red, a calcium channel blocker and ryanodine, a calcium channel stimulator, on the photoperiodic induction of flowering was also studied. Ruthenium red, when applied before and during an inductive 16-h dark period, slightly inhibited flowering, whereas ryanodine, when applied before and during a 12-h long subinductive night, stimulated flower bud formation. We also confirmed evidence that Ca²⁺ ions are involved in the photoperiodic induction of flowering. Thus, the obtained results may suggest the involvement of cyclic ADPR-activated Ca²⁺ mobilization in the photoperiodic flower induction process in Pharbitis nil.     

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.

     

Effect of Light on Deamination and Oxidation of Adenylic Compounds in Cotyledons of Pharbitis nil

Since labelling of ureides from adenine-8-¹⁴C is higher in dark than in light, the influence of light on the deamination and the oxidation of adenylic compounds by cotyledon discs of Pharbitis nit was investigated. Among the three possible adenylic precursors for the deaminative step, adenine was found to be the best compound for the study of the deaminative rate, adenosine being easily hydrolyzed into adenine, and AMP undergoing an apparent complete hydrolysis before entering the cells. By analysis of adenine-8-¹⁴C metabolism for brief periods, it was determined that the rate of deamination of adenylic compounds was faster in light than in dark. In contrast, the activity of xanthine dehydrogenase was much higher in the dark than in light. The level of the activity of uricase was the same in both light and dark.     

The Role of Cotyledons in Flower Initiation of Pharbitis nil at Low Temperatures

Seedlings of Pharbitis nil, Strains Violet, Tendan and Kidachi,initiated floral buds under Continuous light when exposed totemperatures lower than 15, 15 and 21?C, respectively, throughoutthe experimental period, or to 13–14?C for a minimum durationof 10, 8 and 4 days, respectively. Cotyledons were necessaryfor floral initiation when the seedlings at the start of coldtreatment were 8 days old (10 days old for Kidachi) or younger,although neither cotyledons nor foliage leaves were necessarywhen the plants were older. When the cotyledons in young seedlingswere removed immediately after exposure to cold temperature(13–14?C) for 14 (Violet), 12 (Tendan) or 8 (Kidachi)days (cold treatment begun when the cotyledons had just unfolded),only a few plants initiated floral buds under continuous light.However, when the cotyledons were left attached for 2 more daysat 23?C, the plants produced as many flower buds as those withintact cotyledons, suggesting that cotyledons exposed to coldtemperature produce a floral stimulus which can be translocatedto buds even after the end of the cold treatment. (Received October 14, 1981; Accepted January 20, 1982)     

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)