The Influence of Nicotinic Acid and Plant Hormones on Flowering in Lemna

Nicotinic acid induces flowering in Lemna paucicostata 151 and381 and Lemna gibba G3 when they are grown in one tenth-strengthM medium under continuous light. For L. paucicostata 151 and381, the simultaneous addition of IAA, GA₃ or ABA to the mediumleads to an inhibition of the flower-inducing effect of nicotinicacid, while zeatin leads to a further stimulation of floweringabove that obtained by nicotinic acid alone. By contrast, inL. gibba G3 all four plant hormones inhibit the nicotinic acid-inducedstimulation of flowering. The effect of nicotinic acid on flowering in all three plantsis strongly daylength dependent when the plants are grown inhalf-strength Hutner's medium. Thus, nicotinic acid causes floweringin L. gibba G3 on continuous light but not on 9L:15D or 10L:14Dregimes. In L. paucicostata 381 nicotinic acid has a small effecton 12L:12D regime, a large effect on a 13L:11D regime and noeffect with daylengths longer than 14 hours, and in L. paucicostata151 nicotinic acid is only effective on daylengths shorter thanabout 11 hours. However, in L. paucicostata 151 and 381 treatmentwith both nicotinic acid and zeatin results in flowering undercontinuous light on half-strength Hutner's medium. Nicotinic acid is present in different Lemna but its concentrationdoes not appear to be influenced by changes in daylength. Thus,flowering clearly cannot be controlled by nicotinic acid actingalone, but the results of this study indicate that nicotinicacid could interact with other factors, possibly including oneor more of the known plant hormones, to influence the floweringprocess in Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

Isolation and Identification of L-Pipecolic Acid and Nicotinamide as Flower-Inducing Substances in Lemna

Flower-inducing factors in extracts of flowering Lemna gibbaG3 were investigated using Lemna paucicostata 151 as the bioassayplant. Fractions with flower-inducing activity were obtainedafter several purification steps. Two of the active substanceswere identified as L-pipecolic acid and nicotinamide by MS andNMR analyses. Both L-pipecolic acid and nicotinamide exhibited flower-inducingactivity in L. paucicostata 151 grown on one-tenth-strengthM medium containing benzyladenine, the former being ten timesas active as the latter. L-Pipecolic acid was active even at0.01 ppm (7.8 ? 10^–8 M). The effect of L-pipecolic acidon flowering strongly depended upon the presence of exogenouscytokinin. The coexistence of cytokinin seemed to be essentialfor L-pipecolic acid to exhibit flower-inducing activity. Incontrast, the effect of nicotinamide on flowering was basicallythe same as that of benzoic acid or nicotinic acid. (Received February 9, 1987; Accepted May 21, 1987)     

Flower-Inducing Activity of Water Extract of Lemna

Flowering of Lemna paucicostata 441 (P441), a sensitive short-dayplant (SDP), was promoted under a near critical photoperiodby the crude water extract of the same plant added to the medium.The extract induced flowering in L. paucicostata 151 (P151),a weakly responsive SDP, under continuous light. The activityfor P151 was greatly promoted by simultaneous application ofbenzyladenine, and the extract of only 0.3 mg fr wt plant addedto 10 ml of assay medium with 1 µM benzyladenine was active.Active substance(s) was similarly obtained from both flower-inducedand non-induced plants, and more or less from all species andstrains of Lemna tested, including P151. However, the extractof short-day strains was more active than that of L. gibba G3(G3), a long-day strain. G3 responded only slightly to the extractof either P441 or G3, whereas P151 responded far more stronglyto the extract of P441 than to that of G3. (Received April 17, 1989; Accepted August 10, 1989)     

Absorption of Copper by Lemna as Influenced by Some Factors Which Nullify the Copper Effect on Flowering and Growth

The effect of copper on flowering and growth of Lemna paucicostata6746 and Lemna gibba G3 in a copper-containing medium is nullifiedby the addition of EDTA, ammonium ions or salicylic acid tothe medium or a decrease in its nitrate concentration. Thesefactors were examined for their effects on the absorption ofcopper by the plants. The addition of EDTA to the medium completelyinhibited the absorption of copper in both species, thus eliminatingthe copper effect. Ammonium ions also inhibited copper absorption,their effectiveness rising with their concentration. Loweringthe nitrate concentration in the medium nullified the coppereffect on flowering in L. paucicostata 6746, and the additionof salicylic acid to the medium also nullified the copper effectin L. gibba G3, both without affecting the absorption of copper. (Received June 7, 1982; Accepted August 27, 1982)     

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)     

Old-Culture Flowering of Lemna paucicostata 6746 in Aged Hutner's Medium

Lemna paucicostata 6746, a short-day plant, flowers in agedHutner's medium even under continuous light, when the endogenousnitrogen level decreases to below 1.6 µmg fr wt. At thesenitrogen levels, daylength-independent flowering of the plantcan be induced even in fresh Hutner's medium. Thus, old-cultureflowering in Hutner's medium is due to nitrogen deficiency inthe plants. ¹Present address: Biological Institute, Faculty of Science,Shizuoka University, Shizuoka 422, Japan. (Received February 12, 1987; Accepted August 28, 1987)     

Flower-inducing Activity of Vitamin K in Lemna paucicostata

Vitamins K₁ K₃ and K₅ induced flowering in Lemna paucicostata151, a short-day plant, cultured in 1/10 strength M medium (1/10M medium) under continuous light, and their activity was greatlyintensified by simultaneous application of benzyladenine. Themost active of these was vitamin K₅ L. paucicostata 6746 ismore sensitive to vitamin K₅ than strain 151, but the effectof vitamin K₅ on strain 6746 was not intensified by benzyladenine.The flower-inducing activity of vitamin K₅ was intensified bythe addition of benzoic acid in both strains and by the additionof copper or ferricyanide in Strain 6746, when these chemicalswere added at such low concentrations that they would scarcelyinduce flowering. In strain 6746, vitamin K₅ added to 1/10 M had little effecton flowering under a subcritical photoperiod, while it clearlyinduced flowering under continuous light. In this strain, vitaminK₅ added to full strength M medium, in which this plant wasmore sensitive to short photoperiods than in 1/10 M medium,did not induce flowering even under continuous light, and wasrather inhibitory under short photoperiods. (Received August 14, 1984; Accepted October 16, 1984)     

Inhibition of Flower Induction by Some Inhibitors of Protease in the Short-day Plant Lemna paucicostata 6746 and the Long-day Plant Lemna gibba G3

Four inhibitors of proteases, namely, bestatin, diisopropylfluorophosphate, elastatinal and p-toluenesulfonyl-L-lysinechloromethyl ketone hydrochloride, were examined for their effectson flowering of a short-day plant Lemna paucicostata 6746 anda long-day plant Lemna gibba G3. Each of the inhibitors greatlyinhibited the flowering of Lemna paucicostata 6746 that is normallyinduced by nitrogen deficiency. Bestatin or elastatinal givenonly during the first half of the culture period inhibited theflowering more clearly than when each was given during the latterhalf, suggesting that they inhibited the inductive process(es)involved in flowering rather than development of flower buds.Bestatin or elastatinal greatly inhibited the flowering of Lemnapaucicostata 6746 induced by photoperiodic stimulus, ferricyanideand continuous far-red light. Simultaneous application of thesetwo inhibitors was more effective in the inhibition of photoperiodicallyinduced and ferricyanide-induced flowering than was each inhibitoralone. They also completely inhibited the photoperiodic floweringof Lemna gibba G3. These results suggest that the inductionor activation of some proteases, probably followed by the degradationof some protein(s), is necessary for the induction of floweringin both these plants. (Received November 21, 1989; Accepted February 19, 1990)     

Effect of Heat-Treated Noradrenaline on Flowering in Lemna

In a previous study, heat-treated noradrenaline induced flowering of the short-day plant Lemna paucicostata Hegelmaier 151. In the present study, we found that heat-treated noradrenaline also had flower-inducing activity in short-day L. paucicostata strains 441 and 6746 and in long-day L. gibba strain G3. The flower-inducing activity in these plants was enhanced by water homogenates of eggplant (Solanum melongena L.).     

Flower-Inducing Activity of Lysine in Lemna paucicostata 6746

Flowering of Lemna paucicostata 6746, a typical short-day plant,was induced by culture for 96 or 120 h in nitrogen-free mediumunder continuous illumination. To examine the effects of lysine,we homogenized entire plants of L. paucicostata 151 in a solutionof lysine and the supernatant obtained after centrifugationof the homogenate was added to the medium to give various concentrationsof lysine in the medium. Flowering of strain 6746 in nitrogen-freeor nitrogen-deficient culture medium was effectively promotedby the addition of a lysine-containing supernatant to the medium.The suppressive effect of elastatinal, a protease inhibitor,on the induction of flowering was almost completely reversedby the simultaneous application of a lysine-containing supernatantto the medium. During nitrogen-free culture, the level of endogenousfree lysine, expressed on the basis of the amount of total freeamino acids, increased. Lysine-containing supernatants alsoinduced flowering of plants in nitrogen-rich medium under continuousillumination. These findings suggest that endogenous lysineis involved in the induction of flowering in L. paucicostata6746 on nitrogen-free or nitrogen-deficient medium, as it isin the induction of flowering in L. paucicostata 151 (Received July 29, 1996; Accepted November 18, 1996)     

Influence of Ammonium on the Ability of Salicylic Acid to Induce Flowering in the Short-Day Plant Lemna paucicostata 6746

When the short-day plant Lemna paucicostata 6746 is grown inhalf-strength Hutner's medium, which contains 1.25 mM NH4NO3,salicylic acid does not induce flowering on daylengths of 16hr or longer. By contrast, in M (Hoagland-type medium), Pirson-Seidelor ammonium-free half-strength Hutner's media, none of whichcontain ammonium, salicylic acid is able to induce some floweringeven under continuous light. Neverthless, in each of these threemedia the effect of salicylic acid is strongly daylength dependentbecause there is a sharp drop in the flowering response to salicylicacid between the 12 and 16 hr daylengths, and the floweringresponse is nearly constant from the 16 hr daylength to continuouslight. Ammonium has the opposite effect and at 50 to 75 µMis able to overcome the salicylic acid effect completely andprevent any flowering on daylength of 16 hr or longer. (Received December 3, 1980; Accepted March 5, 1981)     

Conversion of Lysine to L-Pipecolic Acid Induces Flowering in Lemna paucicostata 151

The natural occurrence of L-pipecolic acid and conversion oflysine to L-pipecolic acid in Lemna paucicostata 151 were demonstrateddefinitively by GC-MS. The strong flower-inducing activity ofL-pipecolic acid has already been demonstrated. Thus, the presentstudy indicates that the effect of lysine on flowering is mediatedby L-pipecolic acid. (Received June 30, 1997; Accepted August 22, 1997)     

A Comparative Study on the Short-Day- and the Benzoic Acid-Induced Flowering in Lemna paucicostata

The addition of a high concentration of FeCl₃ to the medium(1/10 strength M medium) slightly inhibited the benzoic acid(BA)-induced flowering of Lemna paucicostata 151 in continuouslight, although it promoted the flowering induced by short-day(SD) conditions. SD treatment had no significant effect on BA-inducedflowering in the medium with a standard concentration of iron,in which this plant hardly responds to SD, but greatly promotedit in the medium containing iron at a high concentration, inwhich this plant clearly responds to SD. The effect of BA seemsto be independent of but additive to the photoperiodic stimulus. In photosensitive strains 6746 and 441, a low concentrationof BA slightly lengthened the critical photoperiod but had noflower-inducing effect under continuous light. Since an optimalconcentration of BA induced flowering even under continuouslight in these strains, this was considered to be due to photoperiod-independentpromotion of flowering rather than shortening of the time-measuringprocess in the photoperiodic reaction. (Received August 19, 1986; Accepted February 21, 1987)     

Inhibition of flowering in the long-day plant Lemna gibba G3 by Hutner's medium and its reversal by salicylic acid

Flowering in the long-day plant Lemna gibba L., strain G3 ispoor or absent in Hutner's medium even under continuous light,an effect generally ascribed to the ammonium content of themedium. However, flowering is also inhibited in ammonium-freemodifications of Hutner's medium, particularly in the presenceof sucrose, but is restored to high levels by the presence of10 µu salicylic acid. These results link two of the leastunderstood chemical effects in Lemnaceae flowering, and theyprovide a system in which large effects of salicylic acid canbe readily obtained. ²Present address: Lab. of Applied Bot., Fac. of Agric, KyotoUniv., Kyoto 606, Japan. (Received January 27, 1979; )     

Enhancement of long-day flowering by Mo-deficiency and application of some amino acids and asparagine in the short-day plant Lemna paucicostata 6746

The short-day plant Lemna paucicostata 6746 can be induced toflower on long days (continuous light) by the addition of copper,tungstate or ferricyanide to the medium, and in each case theeffect was greatly enhanced by deleting molybdate from the medium.Treatment with asparagine, aspartate, glutamate, -alanine, glycineorserine, all of which are known to increase the critical daylengthand almost nullify the light-break effect in L. paucicostata6746, enhanced the long-day flowering induced by copper, ferricyanideor Mo-deficiency. (Received October 13, 1978; )     

The Mode of Action of Benzoic Acid and Some Related Compounds on Flowering in Lemna paucicostata

Benzoic acid (BA) (10 µM) added to the medium during onlythe first 24 h of culture induced flowering in Lemna paucicostata151 even under continuous light at 24.5?C when 1/10 M medium(pH 4.0) containing 1 µM benzyladenine (BAd) was usedas the basic medium. Flower buds were produced on the 4th–5thday and almost all the fronds that developed during the subsequent3–4 days had flower buds. Even a 4-h treatment with BA(50 µM) followed by culture in the basic medium inducedflowering. This suggests that the effect of BA is inductive.A similar effect of BA was observed in strain 381, a sensitiveshort-day plant, but not in strain 441 or 6746. Even in the absence of BAd in the medium, a 24-h treatment withBA induced flowering, but the induced state disappeared rapidlyafter the 5th-6th day. BAd was effective when given after theBA treatment and had no significant effect when added duringthe BA treatment. BA given after a single inductive dark periodalso promoted flowering in strains 441 and 381. BAd seems towork to sustain the induced state or to promote the developmentof flower buds rather than inducing flowering. A short-term treatment with nicotinic acid (NA) at 200–500µM was as effective as 10µM BA, but that with salicylicacid (SA) was ineffective at all concentrations tested. 5-C1-SAand EDDHA were also effective, although not as effective asBA. (Received April 10, 1986; Accepted July 12, 1986)     

Isolation and Identification of Lutein from Lemna paucicostata 381 as an Inhibitor of Benzoic Acid-induced Flowering in Lemna paucicostata 151

Efforts were made to isolate flower-inhibitory substances from extracts of the short-day plant Lemna paucicostata 381. Lemna paucicostata 151, which was used in the bioassay, exhibits poor flowering in response to the photoperiod, but flowers profusely in response to benzoic acid. Therefore, only those substances that inhibit benzoic acid-induced flowering were studied. Several fractions obtained by silica gel column chromatography exhibited flower-inhibitory activity when tested on L. paucicostata 151. After several purification steps, one of the active principles was identified as lutein by MS, UV and NMR spectroscopic analyses. Lutein and its isomer zeaxanthin inhibited benzoic acid-induced flowering in both L. paucicostata 151 and 381.     

Flower-inducing Effect of Benzoic and Salicylic Acids in Various Strains of Lemna paucicostata and L. minor

The flower-inducing activities of benzoic and salicylic acidsadded to the medium differ with the species (Lemna paucicostataand L. minor), and even with the strains used. The type andpH of the medium used, full or 1/10 strength M medium at pH3.8, 4.4 or 5.1, or 1/2 or 1/20 strength NH₄⁺-free Hutner'smedium at pH 5.0, 6.0 or 7.0, also modify their activity. L.paucicostata, strain 151 is the most sensitive of the strainsused to both benzoic and salicylic acids followed by strain381. Such dramatic flowering responses were not obtained withthe other strains, but even strain 321, reportedly insensitiveto benzoic acid, could be induced to flower by adding benzoicacid to a modification of the medium. Benzoic acid is more effectivethan salicylic acid for all strains of L. paucicostata, butthe contrary is true for two L. minor strains tested. A higherpercentage of flowering is obtained in L. paucicostata in 1/2strength NH₄⁺-free Huter'sn medium than in M medium, exceptfor strain 151. When diluted, both media enhance flowering inall L. paucicostata strains. Generally, a lower concentrationof benzoic acid or salicylic acid is enough to induce floweringwhen the pH of the medium is lower. (Received March 30, 1981; Accepted May 16, 1981)     

Flower Induction by Nitrogen Deficiency in Lemna paucicostata 6746

Lemna paucicostata 6746, a short-day plant, produced flowerbuds even under continuous light when cultured in nitrogen-deficientmodified Hoagland medium with 1% sucrose for 3 days or morefollowed by culture on nitrogen-rich medium (either nitrateor ammonium). Flowering was also induced by culture on mediumcontaining 20–100 µM nitrate as the sole nitrogensource for 10 days or more, but not on medium with a low ammoniumconcentration. However, if plants cultured on medium containing5–20 µM ammonium as the sole nitrogen source for10 days were grown in a nitrogen-rich medium for a further 4days, they produced flower buds. Thus, nitrogen deficiency caninduce day length-independent flowering in Lemna paucicoslata6746, but nitrogen is required for the manifestation of flowering. (Received January 31, 1986; Accepted April 24, 1986)     

Effects of Ferrous and Phosphate Ions on Flowering in Lemna paucicostata 6746 in Diluted Hutner's Medium

In Lemna paucicostata 6746, P₂ (flower induction period) incontinuous darkness was largely extended by dilution of 1/2H1S medium (Hutner's medium supplemented with 1% sucrose) to1/10 strength. The dilution of 1/2 H1S medium to 1/10 strengthremoved completely the extension of P₁ (pre-flower inductionperiod) and P₂ due to a red light pulse given at the 7th h ofthe dark period, which was observed in 1/2 H1S medium. When iron and phosphate ions were added to 1/10 H1S medium upto the same concentration as in 1/2 H1S medium, the extensionof P₂ was removed completely. The red light pulse-induced extensionof P₁ was observed in 1/10 H1S medium only when iron ions wereadded. It is suggested that iron (Fe^{$ $}) and phosphate ionsare important in determining the rate of photoperiodic flowerinduction in L. paucicostata 6746. (Received December 23, 1983; Accepted June 6, 1984)