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⁺.     

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.     

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.     

A rhythm in the flowering response of photoperiodically-induced Pharbitis nil to agents affecting cytosolic calcium and pH

Previous work with modulators of Ca²⁺ and pH has indicated that elevated levels of cytoplasmic Ca²⁺ and pH are required during the first 4-5 h of the dark period for successful floral induction in Pharbitis nil Chois cv. Violet. In the present study we further examined the effect of modulators of Ca²⁺ and pH by supplying them at various times prior to the inductive dark period. Peaks of inhibition by the Ca²⁺ chelator, EGTA, were observed in seedings treated 8, 18-and 34 h before the start of the dark period. When seedlings of slightly different ages (within one diurnal cycle) were treated with EGTA, maximum inhibition was always obtained in plants treated 8 h before the start of the dark period. Peaks of inhibition by the acidifying agents, salicylic acid and Na-propionate, were observed at -2 to -6 h and at -10 to - 14 h. Treatment with the alkalizing agent, trisodium citrate, enhanced the flowering response with maximum enhancement at -6 to -8 h and at - 18 to - 20 h. We hypothesize that treatment with modulators starts an oscillation in endogenous levels of Ca²⁺ and pH. The levels of Ca²⁺ and pH prevailing at the commencement of the inductive dark period will influence the ability of the plant to perceive or to respond to the photoperiodic induction.     

Ethylene and ABA interactions in the regulation of flower induction in Pharbitis nil

Hormones are included in the essential elements that control the induction of flowering. Ethylene is thought to be a strong inhibitor of flowering in short day plants (SDPs), whereas the involvement of abscisic acid (ABA) in the regulation of flowering of plants is not well understood. The dual role of ABA in the photoperiodic flower induction of the SDP Pharbitis nil and the interaction between ABA and ethylene were examined in the present experiments. Application of ABA on the cotyledons during the inductive 16-h-long night inhibited flowering. However, ABA application on the cotyledons or the shoot apices during the subinductive 12-h-long night resulted in slight stimulation of flowering. Application of ABA also resulted in enhanced ethylene production. Whereas nordihydroguaiaretic acid (NDGA) - an ABA biosynthesis inhibitor - applied on the cotyledons of 5-d-old seedlings during the inductive night inhibited both the formation of axillary and of terminal flower buds, application of 2-aminoethoxyvinylglycine (AVG) and 2,5-norbornadiene (NBD) - inhibitors of ethylene action - reversed the inhibitory effect of ABA on flowering. ABA levels in the cotyledons of seedlings exposed to a 16-h-long inductive night markedly increased. Such an effect was not observed when the inductive night was interrupted with a 15-min-long red light pulse or when seedlings were treated at the same time with gaseous ethylene during the dark period. Lower levels of ABA were observed in seedlings treated with NDGA during the inductive night. These results may suggest that ABA plays an important role in the photoperiodic induction of flowering in P. nil seedlings, and that the inhibitory effect of ethylene on P. nil flowering inhibition may depend on its influence on the ABA level. A reversal of the inhibitory effect of ethylene on flower induction through a simultaneous treatment of induced seedlings with both ethylene and ABA strongly supports this hypothesis.     

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.     

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.     

On the role of calcium in the mechanism of ADH action

With an increased influx of Ca2+ in the cytoplasm, the response of cells to ADH in the urinary bladder of the frog was lowered by addition of ionophore A23187 from the side of the basolateral cell membrane, but inhibited when it was added from the apical cell membrane. The removal of calcium by EGTA from the serosal surface was accompanied by a sharp increase of osmotic permeability not only to water, but also to inulin; while when calcium was removed from the mucosal surface of the urinary bladder, osmotic permeability was not changed. After being added to the Ringer solution from the outer surface of the apical cell membrane, the inhibitors of Ca2+ channels (verapamil, Ni2+, Mn2+, Co2+) decreased the effect of ADH. These data indicate that Ca2+ applied onto the outer surface of apical plasma membrane plays an important role in the action of ADH.     

Flowering response of Ipomoea batatas scions grafted onto Pharbitis nil stocks

Young seedlings of Ipomoea batatas (L.) Lam. cv. Big One did not form floral buds, but were induced to flower when grafted onto Pharbitis nil Chois. cv. Violet with its cotyledons exposed to a 16 h dark period (SD). Four SD were required to induce flowering in I. batatas scions when the grafted plants were first grown under an 8 h dark period (LD) for 18 days and then exposed to SD. Transmission of the flowering stimulus across the graft union required 4 days. It was also slow in the graft combination of P. nil and P. nil , but increased greatly when the graft union was established more completely. These results suggest that the flowering stimulus of P. nil may move symplastically and its life may be between 4 and 6 days. Although the leaves of I. batatas inhibited flowering, the flowering response of P. nil grafted onto I. batatas suggested that the involvement of a transmissible flowering-inhibitor was unlikely.     

The effects of elevation and depletion of intracellular free calcium on progesterone and prostaglandin production by the primate corpus luteum.

The role of the phosphatidylinositol second messenger system in luteal regulation has not been extensively studied, particularly in the primate. The objectives of this study were (1) to further characterize the response of the primate CL to the calcium ionophore A23187, in terms of intracellular free calcium concentrations ([Ca2+]i) and progesterone (P) production; and (2) to assess the effects of depleting, as well as elevating, available calcium on luteal P and prostaglandin (PG) production. The response to A23187, in terms of [Ca2+]i, was measured by fura-2 fluorescence microscopy of single small and large luteal cells. A23187 significantly increased [Ca2+]i in both cell types (p less than 0.01). P production (basal and hCG-stimulated) by dispersed primate luteal cells incubated for various times (1-8 h) with and without A23187 was measured. Treatment with A23187 rapidly (within 1-2 h) attenuated (p less than 0.05) the time-dependent increase in basal and hCG-stimulated P production. Luteal P and PG production following treatment with the calcium ionophore, ionomycin, alone or in combination with additional CaCl2, was also monitored. Treatment with ionomycin (p less than 0.01) and CaCl2 (p less than 0.01) inhibited luteal P production. In contrast, treatment with ionomycin stimulated (p less than 0.01) luteal PG production. To determine the effects of Ca2+ depletion on luteal function, P and PG production by cells incubated for 2 and 8 h in the absence and presence of the Ca(2+)-chelator EGTA was measured. Luteal production of both P and PG was inhibited by 8-h treatment with EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metabolism of inhibitor of flowering and prostaglandin biosynthesis, acetylsalicylic acid, in Pharbitis nil cotyledons

Acetylsalicylic acid, which applied to cotyledons of the short day plant Pharbitis nil prior to an inductive 16-h dark period inhibits flowering by 90 %, is converted to salicylic acid and to a lesser extent to gentisic acid in the cotyledons during this 16-h dark period. Our results confirmed that salicylic acid and gentisic acid are responsible for the inhibition of flowering. They also inhibit prostaglandin biosynthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flowering Response of Pharbitis nil to Agents Affecting Cytoplasmic pH

Permeant weak acids and auxins have been shown to reduce the cytosplasmic pH in several systems. Lactic, citric, formic, butyric, salicylic, parahydroxybenzoic, propionic acid, and sodium propionate inhibited the flowering response of Pharbitis nil seedlings when applied immediately before an inductive dark period. The acidic auxins IAA, indolebutyric, and α-naphtaleneacetic acid, as well as the nonacidic auxin α-naphtaleneaceteamid, also inhibited the flowering response. Inhibition was generally more pronounced with a 12-hour than with a 16-hour dark period. Salicylic acid and sodium propionate shifted the response curve of the dark period by about 2 hours. Salicyclic acid, sodium propionate, and indolebutyric acid were inhibitory when applied during the first few hours of the dark period. The permeant weak bases NH₄Cl, procaine, and trisodium citrate enhanced the flowering response. NH₄Cl reduced the length of the critical dark period. The inhibition of flowering by acids and auxins as well as the promotion of flowering by bases was obtained even when only the cotyledons had been treated. The inhibition of floral induction by auxins may not be dependent on their effect on the cytoplasmic pH.     

Seed and Embryo Germination in Syringa vulgaris and S. reflexa as Affected by Temperature during Seed Development

A nitrogen source was needed for the flowering of Lemna gibba L., a long-day plant, and L. perpusilla Torr., a shortday plant. The level of endogenous amino acids analyzed by an Amino Acid Analyzer, rose during the first few inductive cycles, but was reduced during later stages of the flowering process. Serine and threonine levels increased during the light period and decreased during the dark period in L. perpusilla. Exogenous serine and threonine added to the culture medium at 10^?6M increased the rate of flowering by more than 35% over the controls. Cysteine inhibited flowering, while other amino acids had little or no promotive effect on flowering. Serine and threonine increased flowering rate in L. perpusilla only when added during a dark period of the inductive cycle. The addition of amino acids during a light period not followed by a dark period had no effect on flowering.     

Calcium-activated hydrolysis of phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate in guinea-pig synaptosomes

1. Addition of the bivalent ionophore A23187 to synaptosomes isolated from guinea-pig brain cortex and labelled with [(32)P]phosphate in vitro or in vivo caused a marked loss of radioactivity from phosphatidyl-myo-inositol 4-phosphate (diphosphoinositide) and phosphatidyl-myo-inositol 4,5-bisphosphate (triphosphoinositide) and stimulated labelling of phosphatidate. No change occurred in the labelling of other phospholipids. 2. In conditions that minimized changes in internal Mg(2+) concentrations, the effect of ionophore A23187 on labelling of synaptosomal di- and tri-phosphoinositide was dependent on Ca(2+) and was apparent at Ca(2+) concentrations in the medium as low as 10(-5)m. 3. An increase in internal Mg(2+) concentration stimulated incorporation of [(32)P]phosphate into di- and tri-phosphoinositide, whereas lowering internal Mg(2+) decreased labelling. 4. Increased labelling of phosphatidate was independent of medium Mg(2+) concentration and apparently only partly dependent on medium Ca(2+) concentration. 5. The loss of label from di- and tri-phosphoinositide caused by ionophore A23187 was accompanied by losses in the amounts of both lipids. 6. Addition of excess of EGTA to synaptosomes treated with ionophore A23187 in the presence of Ca(2+) caused a rapid resynthesis of di- and tri-phosphoinositide and a further stimulation of phosphatidate labelling. 7. Addition of ionophore A23187 to synaptosomes labelled in vivo with [(3)H]inositol caused a significant loss of label from di- and tri-phosphoinositide, but not from phosphatidylinositol. There was a considerable rise in labelling of inositol diphosphate, a small increase in that of inositol phosphate, but no significant production of inositol triphosphate. 8. (32)P-labelled di- and tri-phosphoinositides appeared to be located in the synaptosomal plasma membrane. 9. The results indicate that increased Ca(2+) influx into synaptosomes markedly activates triphosphoinositide phosphatase and diphosphoinositide phosphodiesterase, but has little or no effect on phosphatidylinositol phosphodiesterase.     

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.     

Cellular and morphological changes at the terminal shoot apex of the short-day plant Pharbitis nil during the transition to flowering

Changes in morphology and measurements of cell doubling time were recorded for the first time in the terminal shoot apex of the short-day plant, Pharbitis nil Chois. ( Ipomea nil L.) cv. Violet, undergoing the floral transition. A treatment comprising 48 h darkness given to 4-day-old plants resulted in 100% flowering at the shoot terminal meristem. An inhibitory treatment comprising two 5 min red night-breaks during the 48 h dark period was used to discriminate between events essential for flowering, and those changes resulting from shifts from light to darkness and vice versa. Morphology was studied using both light microscopy and scanning electron microscopy. Cell doubling times were measured using the colchicine accumulation of metaphases method. An increase in the rate of primordial initiation, a change in the divergence angle and a change in phyllotaxis occurred during the floral transition. Moreover, the apex widened and flattened following the inductive dark treatment; the cell doubling time decreased in the peripheral zone and increased in the central zone of these pre-floral meristems.     

The semidian rhythm in flowering response of Pharbitis nil in relation to dark period time measurement and to a circadian rhythm

When seedlings of Pharbitis nil Choisy, cv. Violet, are exposed to a single inductive dark period at 27°C, brief interruptions with red light (R) can be promotive after 2–3 h of darkness but increasingly inhibitory to flowering up to the 8–9th h of darkness. This rhythmic response to R interruptions can be advanced in phase by > 1 h when the preceding light period is interrupted with far-red (FR) 2 h before darkness (FR -2 h) or with FR – 15 h, whereas FR –8 h or FR–22 h retard the rhythm. These shifts in the R interruption rhythm are paralleled by equal shifts in the length of the dark period required for flowering. Brief FR interruptions of darkness displayed a similar rhythm which was also advanced by FR –2 h and retarded by FR –8 h. We conclude therefore that the semidian rhythm in the light, which we have previously described, continues through at least the first 12 h of darkness, is manifested in the R interruption rhythm, and determines the critical night length. A circadian rhythm with a marked effect on flowering was also identified, but several lines of evidence suggest that the circadian and semidian rhythms have independent additive effects on flowering and do not appear to show phase interaction.     

Time-Measuring Processes Involved in the Photoperiodic Response of Lemna paucicostata 441

Lemna paucicostata 441 exposed to a single dark period of variouslengths showed a rhythmic flowering response with a 22- to 24-hperiod, even when the dark period was preceded by continuouslight. The critical night length (about 12 h) was scarcely influencedby pretreatment with 8D–4L (8 h of darkness followed by4 h of light), 8D–8L or 8D–12L. However, the rhythmof the response in the second cycle was markedly damped by thepretreatment with 8D–4L or 8D–12L, and was slightlyamplified by 8D–8L. The flowering response to a red-light interruption given atdifferent times in the inductive dark period also showed circadianrhythmicity even when the dark period was preceded by continuouslight, and this rhythmicity was scarcely influenced by a dark-lighttreatment given prior to the inductive dark period. A red-lightinterruption given at the 6th or 14th hour of the dark periodmarkedly shifted the phase of the rhythm of the response tothe length of the following dark period (the former delayedand the latter advanced), but that given at the same phase markedlyweakened and disturbed the rhythmicity of the response to ared-light interruption given in the following dark period. (Received March 21, 1992; Accepted June 12, 1992)     

枸杞体细胞胚发生中外源Ca2+的作用

脱分化的枸杞叶片外植体愈伤组织转入含有2,4-D的MS培养基上分化培养后有大量胚性细胞的分化和体细胞胚发生;加入一定量的外源Ca2 或45Ca2 ,明显地提高了胚性愈伤组织中体细胞胚发生的频率;加入Ca2 的鳌合剂EGTA则显著降低了体细胞胚发生频率;胚性愈伤组织中CaM的水平在多细胞原胚期和球形胚期显著升高,加入外源Ca2 后CaM含量几乎成倍增加;胚性愈伤组织中蛋白质组分与活性都远远多于或高于非胚性愈伤组织,加Ca2 后蛋白质组分种类也增加.