Flower-inducing and -inhibiting activities of Phloem Exudate from Cotyledons of Pharbitis Seedlings

The flower-inducing and -inhibiting activities of phloem exudate (PE) prepared from cotyledons of Pharbitis seedlings were examined, using apex cultures in vitro from Pharbitis as a bioassay system.The PE was prepared from photoperiodically-induced cotyledons (SD-PE). The SD-PE was subjected to the following fractionations: When the SD-PE was extracted with CHCl₃ and then ethyl acetate, the inducing activity was located in the final aqueous fraction. The activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). The diffusate was fractionated by ion exchange chromatography, and flower-inducing activity was found in the fraction adsorbed onto anion exchange resin. When the fraction was applied to a Sep-Pak C18 cartridge, the activity eluted with 25% MeOH. As a result of the above fractionation, activity was increased about 30-fold.The nature of the flower-inhibiting activity of the PE taken from cotyledons exposed to continuous-light conditions was examined (CL-PE). The inhibiting activity was decreased as the cotyledons were exposed to longer dark periods; it appeared to be heat-stable. The CL-PE also inhibited flowering in Lemna. The CL-PE was subjected to the following fractionations: When the CL-PE was extracted with CHCl₃ and ethyl acetate, activity was located in the final aqueous fraction. Activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). When the diffusate was fractionated by ion exchange chromatography, the activity was found in the flow-through fraction. When the fraction was applied to a hydroxyapatite cartridge, the activity eluted with 25 mM sodium phosphate buffer. When the fraction was re-dialyzed (molecular weight cut off was 1,000), the diffusate contained the activity. As a result of the above fractionation, activity was increased about 10-fold.     

Flower-Inducing Activity of Phloem Exudates from Pharbitis cotyledons Exposed to Various Photoperiods

The phloem exudate prepared from the cotyledons of Pharbitisseedlings that had been exposed to a single dark period (oflonger than 10 h) induced flowering in cultured apices excisedfrom non-induced seedlings. The flower-inducing activity ofthe exudate increased as the seedlings were exposed to longerperiods of darkness. The highest activity was associated withthe exudate taken from cotyledons exposed to a single 16-h darkperiod. The activity of the exudate taken from cotyledons exposedto an inductive dark period was clearly reduced by interruptionof the dark period. The addition of exudate taken from threecotyledons to 10 ml of medium resulted in the highest flower-inducingactivity. About 50% of cultured apex explants formed floralbuds, even when the concentration of the exudate was reducedto 0.1 cotyledon equivalents per 10 ml of medium. The flower-inducingactivity of the exudate appeared to be heat-stable. (Received December 13, 1991; )     

The metabolic pathway of salicylic acid rather than of chlorogenic acid is involved in the stress-induced flowering of Pharbitis nil

We examined the involvement of chlorogenic acid (CGA) and salicylic acid (SA) in the stress-induced flowering of Pharbitis nil (synonym Ipomoea nil). The incorporation efficiency of exogenously applied CGA and the deactivation rate of incorporated CGA were determined in cotyledons by high-performance liquid chromatography. The assay plants could not incorporate a sufficient amount of CGA via roots. The perfusion technique by which the assay solution was forced into the plant from the cut end of the hypocotyl improved the efficiency of CGA incorporation. However, no flower-inducing activity was detected, indicating that CGA was not involved in flowering. It was concluded that the close correlation between CGA content and flowering response is merely coincidence or a parallelism. Flowering under long-day conditions induced by low-temperature stress was completely inhibited by aminooxyacetic acid (AOA), an inhibitor of phenylalanine ammonialyase. The flower-inhibiting effect of AOA was nullified by co-applied t-cinnamic acid and by benzoic acid. This indicates that the metabolic pathway from t-cinnamic acid to SA via benzoic acid is involved in the stress-induced flowering. The results indicate that the metabolic pathway of SA is involved in the stress-induced flowering of P. nil not the metabolic pathway of CGA.     

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.     

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.     

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.     

Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation

The flower-inducing effect of 5-azacytidine, a DNA demethylating reagent, was examined in several plant species with a stable or unstable photoperiodically induced flowering state under non-inductive photoperiodic conditions. The long day plant Silene armeria , whose flowering state is stable and the short day plant Pharbitis nil , whose flowering state is unstable were induced to flower by 5-azacytidine under a non-inductive condition. Thus, the replacement of photoinduction by 5-azacytidine treatment is not specific to Perilla frutescens . On the other hand, 5-azacytidine did not induce flowering in Xanthium strumarium whose flowering state is stable and Lemna paucicostata whose flowering state is unstable. Thus, epigenetics caused by DNA demethylation may be involved in the regulation of photoperiodic flowering irrespective of the stability of the photoperiodically induced flowering state.     

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.     

Leaf-produced floral signals

Florigen is the hypothetical leaf-produced signal that induces floral initiation at the shoot apex. The nature of florigen has remained elusive for more than 70 years. But recent progress toward understanding the regulatory network for flowering in Arabidopsis has led to the suggestion that FLOWERING LOCUS T (FT) or its product is the mobile flower-inducing signal that moves from an induced leaf through the phloem to the shoot apex. In the past year, physical and chemical evidence has shown that it is FT protein, and not FT mRNA, that moves from induced leaves to the apical meristem. These results have established that FT is the main, if not the only, component of the universal florigen.     

Flower-Inducing Activity of Phloem Exudate in Cultured Apices from Pharbitis Seedlings

Phloem exudate from cotyledons of photoperiodically inducedPharbitis plants induced flowering in apices excised from non-inducedseedlings and cultured in vitro. The exudate also stimulatedflowering in apices excised from photo-induced seedlings andcultured under long-day conditions. The application of benzoicacid had similar effects. Both the exudate from non-inducedplants and gallic acid suppressed flowering in apices from photo-inducedseedlings. It appears that the phloem exudate contains flower-inducingor flower-suppressing substance(s), depending on the plant materialsfrom which it was collected. (Received August 15, 1989; Accepted May 14, 1990)     

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.     

Comparison of gibberellin-like substances from cotyledons and phloem exudate collected from cotyledons of Pharbitis nil in relation to photoperiodic flowering

Gibberellin (GA)-like substances were analyzed in extracts from cotyledons and phloem exudate collected from cotyledons in photoinduced and vegetative seedlings of the short-day plant Pharbitis nil Chois. var. Violet, using high performance liquid chromatography (HPLC) and the dwarf rice bioassay, to see whether any specific GA-like substances were transported from the photoinduced cotyledons via phloem. Cotyledon extracts exhibited five peaks of free GA-like activity in HPLC, whereas only one or two active peaks were detected in phloem exudate extracts. The level of free GA-like activity was considerably lower in phloem exudate than in the cotyledons. In five out of six analyses of cotyledons and phloem exudate, there were substantially higher levels of free GA-like substances in photoinduced plants. Conjugated GA-like substances were present in much higher levels than free GA-like substances in the cotyledon extracts but the levels were not influenced by daylength. In phloem exudate extracts there was no conjugated GA-like substances. The free GA-like substances that are transported via phloem cochromatographed with GA_5/20 and GA₁₉ on HPLC. These were significantly higher in photoinduced plants and thus could have some influence on the photoperiodically-induced flowering in P. nil.     

Flower Induction by Suppression of Nitrate Assimilation in Lemna paucicostata 6746

In vitro activity of nitrate reductase was studied in Lemnapaucicostata 6746 grown on modified Hoagland medium supplementedwith 1% sucrose, containing various inhibitors. Copper, silver,tungstate or cyanide which induces daylength-independent flowering,inhibited the nitrate reductase activity, but azide which doesnot induce daylength-independent flowering did not. Molybdate-deficientmedium induced flowering, and inhibited nitrate reductase activity.Lowering of nitrate level of the medium also induced daylength-independentflowering. These results suggest that the suppression of nitrate assimilationcauses daylength independent flowering in Lemna paucicostata6746, and that one of the flower-inducing actions of the copper,silver, tungstate, cyanide or the deletion of molybdate is tosuppress the nitrate assimilation. (Received June 26, 1985; Accepted October 30, 1985)     

长日照处理对牵牛开花抑制作用研究 Ⅰ．不同时间长日照处理对花芽分化和子叶蛋白质的影响

日本紫花牵牛（Ｐｈａｒｂｉｌｉｓｎｉｌｃｖ．Ｖｉｏｌｅｔ）子叶完全展开后,短日照诱导前、诱导后和两个短日照间的长日照处理对植株的花芽分化都有一定的抑制作用。双向凝胶电泳分析表明,长日照处理的牵牛子叶内存在着短日照处理子叶内没有的两种蛋白质（ｐＩ４．１,ＭＷ１６．５ｋＤ;ｐＩ４．２,ＭＷ１６．５ｋＤ）。这些蛋白质可能与长日照抑制牵牛植株的花芽分化有一定关系。     

Isolation and Identification of Nicotinic Acid as a Flower-Inducing Factor in Lemna

Extracts of flowering plants of the long-day plant Lemna gibbaG3 and the short-day plants Lemna paucicostata 151 and 381 weretested on L. paucicostata 151 for flower-inducing activity.Crude extracts failed to show any activity but after severalpurification steps three fractions with flower-inducing activitywere obtained. One fraction obtained from all three plants wasshown to contain nicotinic acid by mass spectroscopic and NMRspectroscopic analyses. These results raise the possibilitythat nicotinic acid may act to influence the flowering processin Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

长日照处理对牵牛开花抑制作用研究 Ⅱ.光周期处理与牵牛花芽分化及基因活动的关系

单个光周期暗期长度短于12h时,牵牛植株营养生长旺盛,开花受到抑制,并且出现了诱导光周期处理（ISD）子叶中没有的二种蛋白质或多肽（pI4.1,MW16.5kD;pI4.2,MW16．5kD）。连续光照处理（ICL）子叶内出现了短日照处理（ISD）子叶内没有的体外翻译蛋白质分子量为17．4kD的Poly（A~ ）mRNA。牵牛子叶内的这些变化可能与抑制牵牛花芽分化有一定的关系。     

Cyclic GMP stimulates flower induction of <Emphasis Type="Italic">Pharbitis nil</Emphasis> via its influence on cGMP regulated protein kinase

It was revealed that cGMP is involved in the control of photoperiodic flower induction. Further insight into the signalling function of cGMP is likely to be obtained by analysis of its effectors. Therefore, in the present study, we used various agents that cause changes in cGMP-dependent kinase (PKG) activity and examined their effects on the activity of kinase isolated from Pharbitis nil and flower induction. It was found that exogenous applications of PKG activators (cGMP, 8-pCPT-cGMP, 8-Br-cGMP, 8-pCPT-PET-cGMP) to cotyledons which were exposed to a 12-h-long subinductive night significantly increased flowering response. From among the many antagonists of cGMP-dependent protein kinase Rp-8-Br-PET-cGMPS, Rp-8-pCPT-cGMP and the synthetic heptapeptide inhibitor of PKG were used for our analysis. When Rp-8-Br-PET-cGMPS and Rp-8-pCPT-cGMP were applied during a 16-h-long inductive night, significant reduction in the number of flower buds was observed, whereas synthetic heptapeptide did not change the intensity of flowering. The influence of the analysed chemicals on protein kinase activity was also examined in vitro. With the exception of synthetic heptapeptide, which seems ineffective, the enzyme activity was stimulated by all agonists and significantly reduced by all antagonists. The activity of protein kinase was assayed in P. nil soluble protein fractions from plants grown under flower-inducing and non-inducing conditions. In vitro phosphorylation was slightly greater in the soluble fraction obtained from plants grown under the flower-inducing condition, reaching 1.05 nmol/min/mg protein, when compared to the control 0.81 nmol/min/mg protein. In relation to the results described above, we can conclude that cGMP as a mediator participating in photoperiodic flower induction may govern this process by the phosphorylation mechanism via its influence on cGMP-dependent protein kinase activity.     

Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.