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⁺.     

Photoperiodically independent flowering of Pharbitis nil plants regenerated from flower buds

Summary Flower buds and anthers of the short-day plant Pharbitis nil were treated either with thermic shock (7 or 35°C) or osmotic/trophic shock (12% sucrose) for 24 h. Explants were transferred either to Murashige and Skoog medium (MS) with addition of 6-benzylaminopurine (BA; 4.4μM) and 6% sucrose or to the same growth medium containing 22 μM BA and 3% sucrose. Both media were supplemented with α-naphthaleneacetic acid (NAA; 0.55 μM). Osmotic/trophic shock stimulated the occurrence of shoots on flower buds grown on medium containing 22 μM BA. Thermic shock (7 and 35°C) inhibited this process on both types of explants. Regenerated plantlets were transferred to MS medium supplemented with 6% sucrose, gibberellic acid (GA₃; 1.44μM), NAA (0.55 μM) and Ca²⁺ (0.66 mgl⁻¹). After 3–4 wk they were able to produce flowers without photoperiodic induction.     

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.     

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.     

Inhibition of stem growth and flower formation in Pharbitis nil with N,N-dimethylaminosuccinamic acid (B 995)

Summary In the short-day plant Pharbitis nil, strain Violet, flower formation is inhibited by application of the growth retardant N,N-dimethylaminosuccinamic acid (B 995) via the roots for a period of 24 hours prior to one inductive long night. Terminal flower bud formation is suppressed by a B 995 concentration of 100 mg/l, but for complete suppression of all axillary flower buds 2000 mg/l is required. Inhibition of flower formation is also caused by B 995 application via plumules or cotyledons, even if made at the end of the inductive night. B 995 treatment always results in short, thick internodes and dark-green leaves.Transport of ¹⁴C-labeled B 995 from cotyledons to plumules and roots takes place during a 16-hour dark period. However, very little label moves from a treated to an untreated cotyledon. Application of B 995 to one of the two cotyledons results in flower inhibition, although the untreated cotyledon produces sufficient flower hormone to induce optimal flower formation. It is concluded therefore that in the short-day plant Pharbitis B 995 does not affect flower hormone production, but rather inhibits floral initiation by interfering with the action of the hormone in the shoot apex.Inhibition of flower formation by B 995 can be completely overcome by application of gibbrellin A₃ to the plumulus before the long nigh. A dose of 0.01 g GA₃/apex is sufficient to re-establish flowering, but much more GA₃ is required to restore internode length equal to that of the control. Indole-3-acetic acid and naphthalene acetic acid are totally inactive in overcoming B 995 inhibition of flower formation and growth.The growth rate of Pharbitis plants treated with B 995 and continuously grown in long-day conditions is initially low, but reaches the same level as in untreated plants approximately 25 days after treatment. ¹⁴C-labeled B 995 applied to cotyledons accumulates to a high degree in roots and in the basal part of the shoots. ¹⁴C-B 995 is metabolized very slowly and persists therefore in Pharbitis plants for prolonged periods of time.     

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.     

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)     

Neolignan and monoterpene glycoside from the seeds of Pharbitis nil

Phytochemical investigation of an EtOH extract of Pharbitis nil seeds (Convolvulaceae) resulted in the isolation and identification of a new neolignan, 7R,8S-threo-dihydroxydehydrodiconiferyl alcohol (1), and a new monoterpene glycoside, (3Z,7S)-7-hydroxy-3,7-dimethyl-3,8-octadienyl-β-d-glucopyranoside (2), together with a known compound, ethyl α-l-arabinofuranoside (3). The chemical structures of these compounds were unambiguously determined using physical data, HR-ESI–MS and spectroscopic evidence, including 1D and 2D NMR experiments. The anti-inflammatory activities of the isolates were evaluated by estimating the inhibition of nitric oxide (NO) production. Compounds 1 and 2 reduced NO levels in lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells. In addition, compound 2 showed weak cytotoxicity against the HCT-15 cell line with an IC₅₀ value of 28.6 μM.     

牵牛复合体（旋花科）的分类学研究

采用解剖镜和扫描电镜对牵牛复合体[牵牛Pharbitis nil（L．）Choisy、裂叶牵牛P.hederacea（L．）Choisy和圆叶牵牛P.purpurea（L．）Voigt]的叶形、萼片、果实和花粉形态进行了观察,同时对其叶绿体DNA trnL—F和核基因ITS进行了分析。结果表明,牵牛、裂叶牵牛与圆叶牵牛的外部形态,在叶形、萼片的形状及毛被、果实形态上的差异明显;花粉形态的差别也较为明显;在trnL-F和ITS序列分别构建的系统树中,牵牛、裂叶牵牛和圆叶牵牛聚为一个分支,牵牛和裂叶牵牛聚为一个小分支,ITS序列系统树的支持率为70％,trnL—F序列分子树的支持率仅为63％。根据以上不同,本研究认为牵牛、裂叶牵牛和圆叶牵牛都应作为种级处理。     

Chloride absorption and distribution in chlorine-deficient Pharbitis nil

Determination and distribution of radioactive chloride in Pharbitis nil was investigated by a bio-imaging analyzer. When leaves that contained various amounts of ³⁶Cl were analyzed with the imaging analyzer and then each sample was homogenized and its radioactivity measured in a liquid scintillation counter, radioactive levels recorded by the analyzer were directly proportional to the radioactivity determined by the counter. When plants that had been grown in full nutrient solution were incubated in [³⁶Cl]-containing solution, more activity was found in young leaves than in mature leaves, while little radioactivity was detected in shrivelled leaves and the nonsymptomatic cusp of young leaves of plants that had been grown in chlorine-deficient solution.     

Phytochrome in Pharbitis nil during and after de-etiolation

Phytochrome (P) was measured by in vivo spectrophotometry in the cotyledons of Pharbitis nil Choisy cv. Violet. In etiolated plants exposure to red light (R) results in a rapid fall in P content by destruction of the far-red absorbing form of phytochrome (Pfr). In continuous R or white light the P content falls as a result of destruction to a stable 3% of that originally present. In Norflurazon-treated white light de-etiolated plants, Pfr undergoes reversion in darkness to the red absorbing form of phytochrome (Pr) with a half life of 4 h at 19°C, while total P remains constant. Synthesis of Pr after de-etiolation occurs at a slow rate and is enhanced by terminating the de-etiolation light treatment with far-red light. The results are discussed in relation to the P control of flowering.     

The role of gibberellins in the growth of floral organs of Pharbitis nil

Evidence that the synthesis of GA₃ is involved in the growthof floral orga'ns of Pharbitis nil is presented. GAs in floralorgans at different developmental stages were surveyed usingTLC followed by the bioassay with two dwarf rice seedlings,‘Tanginbozu’ and ‘Waito-C’. The amountof GAs in the petal and stamen increased rapidly after the petalemerged from calyx, reached a maximum 12 hr before anthesis,then declined markedly thereafter. The GA content in the calyxremained unchanged before and after anthesis, and that in thepistil increased after anthesis. Pharbitis flowers containedat least two active GAs, one of which was probably GA₃, theother appeared to be GA₁₉. GA₃ was detected in relatively largeamounts in both the petal and stamen during their rapid elongation.In the calyx, which showed little increase in fresh weight duringrapid flower growth, GA₉ was the dominant GA. Exogenously suppliedGA₃ promoted elongation of sections in excised young filaments.Sucrose was necessary for definite growth promotion by GA₃.GA₁₉ had little effect on filament elongation, and IAA was ratherinhibitive. (Received July 29, 1972; )     

RNA containing poly-A sequence in Pharbitis nil cotyledons and its template activity in vitro

RNA containing poly-A sequence was separated from the totalnucleic acid of morning glory cotyledons by cellulose columnchromatography. The RNA was preferentially retained on poly-Uimmobilized filter and had high template activity in a bacterialcell-free protein synthesizing system. Electrophoretic analysissuggested that this poly-A RNA is 10 S RNA and the size of thepoly-A sequence is about 3.5 S. Sub-cellular distribution ofthe poly-A RNA was also investigated. (Received December 7, 1973; )     

Endogenous alpha-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil

Alpha-ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, that is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] is a signal compound found in Lemna paucicostata after exposure to stress, such as drought, heat or osmotic stress. KODA reacts with catecholamines to generate products that strongly induce flowering, although KODA itself is inactive [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201]. We examined the role of KODA in the flower-induction process of Pharbitis nil (violet). KODA was identified for the first time in seedlings of P. nil grown under a flower-inductive condition (16-h dark exposure), by means of LC-SIM and LC-MS/MS. In addition, the changes in endogenous KODA levels (evaluated after esterification of KODA with 9-anthryldiazomethane) during the flower-inductive phase in short day-induced cotyledons were closely related to flower induction. The KODA concentration sharply increased in seedlings during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. The increase of KODA level occurred in cotyledonal blades, but not in other parts (petiole, hypocotyls and shoot tip). When the 16-h dark period was interrupted with a 10-min light exposure at the 8th h, flower induction was blocked and KODA level also failed to increase. The degree of elevation of KODA concentration in response to 16-h dark exposure was the highest when the cotyledons had just unfolded, and gradually decreased in seedlings grown under continuous light for longer periods, reaching the basal level at the 3rd day after unfolding. Flower-inducing ability also decreased in a similar manner. These results suggest that KODA may be involved in flower induction in P. nil.