Inhibition of photoperiodic floral induction in Pharbitis nil by ethylene

Application of ethylene at 100 ppm or higher completely inhibitedflowering in Pharbitis nil when made during an inductive darkperiod. Exposing plants to ethylene before or after the inductivedark period produced only slight or almost no inhibition. Ethylenewas effective when it was applied only to a cotyledon, but wasineffective when applied only to a receptor bud. Ethylene hadno effect on translocation of the floral stimulus. Ethylene-treatedcotyledon did not transfer any flower inhibiting entity. Thus,ethylene is considered to inhibit the induction process(es)in cotyledons. Except for an initial temporary cotyledon epinasty, ethylenetreatment had no effect on the subsequent growth and vigor ofplants. This temporary cotyledon epinasty disappeared withinthe next 24 hr. (Received May 4, 1972; )     

Calcium and photoperiodic flower induction in Pharbitis nil

The relationship between phytochrome-mediated induction of flowering, Ca²⁺ transport and metabolism in Pharbitis nil Chois cv. Violet seedlings has been investigated. Ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA), a specific Ca⁺ chelator, caused a 30–40% inhibition of flowering in Pharbitis subjected to complete photoperiodic induction. It was most effective when applied during the light period preceding along inductive dark period. The agonist of calcium channels. Bay K-8644, did not affect flowering, while Nifedipine, Verapamil and La³⁺ (antagonists of calcium channels) only slightly inhibited this process. A similar small effect has been found when the plants were treated with Li⁺ (inhibitor of the membrane phospholipids pathway), and with chlorpromazine (a camodulin inhibitor). Except for EGTA, the effect of the other substances did not depend on the timing of their application. The results of the present study suggest that the effect of all the substances applied was not specific, and flowering is not directly dependent on transport and intracellular metabolism of Ca²⁺.     

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     

RNA metabolism in apices of Pharbitis nil daring floral induction

RNA metabolism was studied in apices of Pharbitis nil duringand after floral induction. In continuous light ³H-uridine accumulatedin RNA at a constant rate over an 18 hr period. In darkness,however, the rate of accumulation of label into RNA was constantuntil the 10th hour at which time a rapid burst of accumulationoccurred, peaking at the 14th hour of darkness and followedby a net loss of label. The RNA involved in this burst is probablymRNA due to its size and poly(A) content. This phenomenon doesnot seem to be associated with floral induction, since the siteof perception is the apex, and it also occurs under conditionswhere floral initiation is inhibited by a brief light interruptionof the dark period. Immediately after floral induction by a16-hr dark period the rate of RNA synthesis was suppressed about14%. This suppression lasts for about 12 hr and was followedby a twofold increase in the rate of RNA synthesis, comparedto non-induced apices, at 64 hr after the beginning of the inductivedark period. These post-induction changes were found to occurin all RNA fractions. ¹Present address: Department of Radiation Biology and Biophysics,University of Rochester School of Medicine and Dentistry, Rochester,N.Y. 14642, U.S.A. (Received March 15, 1976; )     

Gibberellins are not essential for photoperiodic flower induction of Pharbitis nil

The influence on photoperiodic flowering of (2-chloroethyl)trimethylmmonium chloride (CCC), an inhibitor of gibberellin (GA) biosynthesis, was studied in the short-day plant Pharbitis nil cv. Violet. The cotyledons contained high levels of endogenous bioactive gibberellins, whereas in the plumules and first leaves the levels were low or undetectable. The first leaf responded to a single'dark treatment by inducing flowering when it was 10 mm or wider. Similar seedlings, but without cotyledons, were used as the assay plants to study the effect of CCC on photoperiodic flowering. Treatment with CCC had no effect on flowering of seedlings without cotyledons, although stem elongation was inhibited. By contrast. CCC inhibited flowering of the intact seedlings with cotyledons. Gibberellic acid applied to the shoot apex or to the first leaf promoted flowering in the CCC-treated seedlings without cotyledons. The results indicate thai gibberellins are not essential for the flower induction process in leaves, but that they promote flower initiation and/or later processes in the shoot apices.     

The involvement of cyclic GMP in the photoperiodic flower induction of Pharbitis nil

The involvement of cGMP in the regulation of the flowering of Pharbitis nil was investigated through exogenous applications of cGMP and chemicals that are able to change the cGMP level and analyses of endogenous cGMP level. Exogenous applications of cGMP and 8-pCPT-cGMP (a cyclic GMP non hydrolyzed analog) to P. nil plants, which were exposed to a 12-h-long subinductive night, significantly increased flowering response. NS-2028 (guanylyl cyclase inhibitor) inhibited flowering when that compound was applied during a 16-h-long inductive night, whereas SNP (guanylyl cyclase activator) increased the flowering when plants were subjected to a 12-h-long subinductive night. The inhibitors of cyclic nucleotides phosphodiesterase (isobutyl-methylxanthine and dipyridamole), which increase the cytosolic cGMP level, promoted the flowering and allowed the length of the dark period necessary for induction of flowering to be reduced. The endogenous cGMP level was also measured after the treatment of P. nil seedlings with those chemicals. Results have clearly shown that compounds that were used in physiological experiments modulated endogenous cGMP level. There was a significant difference in the cyclic GMP level between 16-h-long night conditions and a long night with a night-break. During a long inductive night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, 14 h, whereas a 10 min flash of red light in the middle of the night was able to modify these rhythmical changes in the second half of the long night. These results have shown that there are oscillations in the concentration of cGMP in the night and the biosynthesis and/or deactivation of cGMP is affected by light treatment and therefore it may be involved in the regulation of photoinduction processes in cotyledons. From these combined results, we propose a hypothesis that cGMP is involved in the control of photoperiodic flower induction in Pharbitis nil.     

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 .     

Immediate intraplumular distribution of macromolecular syntheses following floral induction in Pharbitis nil

Uridine and thymidine incorporation into the peripheral vs.central zone of the Japanese Morning Glory apex, as well asinto the apical leaves, was measured a few hours before andbeyond the end point of the inducing long night. The enhancedincorporation was greatest in the peripheral zone and in theapical leaves suggesting that immediate pre-prefloral effectsare mainly expressed "vegetatively". (Received December 11, 1974; )     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbitis nil Chois

For dark-grown seedlings of Pharbitis nil capacity to flower in response to a single inductive dark period was established by 24 h white, far-red (FR) or ruby-red (BCJ) light and by a skeleton photoperiod of 10 min red (R)-24 h dark-10 min R. FR alone was ineffective without a brief terminal (R) irradiation, confirming that the form of phytochrome immediately prior to darkness is a crucial factor for flowering in Pharbitis. The magnitude of the flowering response was significantly greater after 24 h FR or white light (WL) (at 18° C and 27° C) than after two brief skeleton R irradiations, but the increased flowering response was not attributable to photosynthetic CO₂ uptake because this could not be detected in seedlings exposed to 24 h WL at 18° C. Photophosphorylation could have contributed to the increased flowering response as photosystem I fluorescence was detectable in plants exposed to FR, BCJ, or WL, but there were large differences between flowering response and photosystem I capacity as indicated by fluorescence. We conclude that phytochrome plays a major role in photoresponses regulating flowering. There was no simple correlation between developmental changes, such as cotyledon expansion and chlorophyll formation during the 24-h irradiation period, and the capacity to flower in response to a following inductive dark period. Changes in plastid ultrastructure were considerable in light from fluorescent lamps and there was complete breakdown of the prolamellar body with or without lamellar stacking at 27 or 18° C, respectively, but plastid reorganization was minimal in FR-irradiated seedlings.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing from of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

Changes in ATP in relation to floral induction and initiation in Pharbitis nil

No changes in metabolism of adenosine phosphates as a function of short day induction were detected in cotyledons of Pharbitis nil Chois strain Violet. A gradual increase in ATP level was detected throughout the dark period in plumules. A rapid decline of ATP pool size was observed in induced plumules shortly after floral induction. The decline occurred close to the 14th hour of the dark period, 1 to 1.5 h after the dark period length required for a 90% flowering response, which is thought to be the minimum time required for transport of the floral stimulus (and assimilates) from the induced cotyledons to the plumule. Transport of the major adenylates from the cotyledons was verified using [¹⁴C]-adenine. Estimates of the amount, and rate, of adenylate transport suggest that the cotyledons could be an important source of adenylates to re-establish the ATP pool size in evoked plumules.     

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.     

Qualitative analysis by isoelectric focusing of the protein content of Pharbitis nil apices and cotyledons during floral induction

The protein content of apices and cotyledons in Morally inducedor vegetative plants of Pharbitis nilwas examined using isoelectricfocusing. No differences were found in the protein patternsproduced by apical tissue with and without floral induction.Cotyledons, however, repeatedly showed the distinct loss ofa single protein band on floral induction. ¹Current address: Department of Radiation Biology and Biophysics,The University of Rochester School of Medicine and Dentristry,Rochester, N.Y. 14642, U.S.A. (Received March 29, 1976; )     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Abscisic acid both promotes and inhibits photoperiodic flowering of Pharbitis nil

Abscisic acid (ABA) has been reported to have diverse effects on photoperiodic flowering. Activity of a natural ABA, (+)-( S )-abscisic acid (S-ABA), was recently suggested to be somewhat different from that of racemic ABA, which has been used in previous work. Use of S-ABA might enable clarification of the role of ABA in flowering. S-ABA inhibited flowering of the short-day plant Pharbitis nil (cv. Violet) when given before or 4 h after the start of a 14-h inductive dark period, and promoted flowering when given 12 h after the start of the dark period or later. The flower-promoting effect was observed when ABA was applied to the shoot apex. These results indicate that ABA has a dual effect on photoperiodic flowering of P. nil : it may inhibit the time-measuring process as well as promote some processes that proceed after generation of the flowering stimulus.     

Biochemical evidence for a calcium-dependent protein kinase from Pharbitis nil and its involvement in photoperiodic flower induction

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

EGTA inhibits floral induction of Pharbitis nil via its influence on gas exchange properties of stomata

The purpose of the study was to determine inhibitory effect of calcium chelator; ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) on flowering of a short-day (SD) plant Pharbitis nil. It was found that 20 mM solution of EGTA applied on cotyledons of 5-d-old P. nil seedlings four hours before the start of 16-h-long induction night decreased the flowering response by 55% compared to the control plants not treated with this Ca²⁺ chelator. It also caused a very significant decrease of photosynthesis rate, transpiration rate and stomatal conductance both in light and darkness conditions. The results of this study confirm earlier hypothesis suggesting the effect of Ca²⁺ and its modulators on P. nil flowering is due to their influence on the stomata.     

Phaseshifting of the photoperiodic flowering response rhythm in Pharbitis nil by red-light pulses

The control by light of the flowering response rhythm in the short-day plant Pharbitis nil Choisy cv. Violet was examined by giving a single pulse of light at various times between 1 and 6 h after a 24-h light period. When the first circadian cycle of the rhythm was monitored, it was found that a pulse of red light given at 1, 2 or 3 h into a 72-dark period caused a 1-h delay of the phase of the response rhythm, while a pulse at 6 h caused a 2-h delay. These results support the hypothesis that, when red-light pulses are given at hourly intervals, they are as effective as continuous light in preventing the onset of dark timing because they repeatedly return the rhythm to the circadian time at which it is apparently suspended in continuous light. The perception of and response to continuous light and red-light pulses are also briefly discussed.