Effect of L-Pipecolic Acid on Flowering in Lemna paucicostata and Lemna gibba

L-Pipecolic acid was found to be effective in inducing floweringof Lemna paucicostata 151, 381, 441 and 6746, and of Lemna gibbaG3. When the plants were grown on half-strength Hutner's medium,L-pipecolic acid caused profuse flowering of L. paucicostata151 maintained under 9 and 10 h of light daily. In L. paucicostata441 and 6746, L-pipecolic acid had a strong flower-promotingeffect under a near critical photoperiod. In L. paucicostata381, by contrast, L-pipecolic acid had only a very small effecton flowering. In L. gibba G3 substantial promotion of floweringwas observed under continuous light. When one-twentieth-strengthHutner's medium was used as the basic medium, L-pipecolic acidstimulated flowering in all strains of Lemna examined, evenunder continuous light. When L. paucicostata 151 was grown on one-tenth-strength M mediumor one-twentieth-strength Hutner's medium, the flower-inducingactivity of L-pipecolic acid was greatly enhanced by cytokininunder continuous light. However, when this strain was grownwith 9 h of illumination daily, this synergistic effect of cytokininwas only slight. A short-term (even 1-h) treatment with L-pipecolicacid resulted in flowering, suggesting that L-pipecolic acidis involved in the induction of flowering, rather than its evocation.D-Pipecolic acid also had flower-inducing activity, but itsactivity was 50 times lower than that of the L-isomer. (Received January 23, 1992; Accepted March 9, 1992)     

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)     

Flowering and Endogenous Levels of Plant Hormones in Lemna Species

The occurrence and endogenous level of various plant hormoneswere measured for the short-day plants Lemna paucicostata 151and 381 and the long-day plant Lemna gibba G3 to determine whetherany of them are involved in the photoperiodic control of flowering.ABA, IAA, GA₁, GA₂₉, GA₃₄, GA₅₃, trans- and cis-zeatin, trans-and cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenine and N⁶-(²-isopentenyl)adenosine were definitely detected in each species, while GA₄was only detected in L. gibba G3 and GA₂₀ was only detectedin L. paucicostata 151. The endogenous levels of ABA and IAAwere in the range of 1–7 ng/g fr wt and were not significantlydifferent in vegetative and flowering plants. The endogenousgibberellin levels were generally higher in Lemna grown underlong-day rather than short-day conditions. The endogenous cytokininlevels were almost the same in both flowering and vegetativeplants of L. paucicostata 151 and 381. In L. gibba G3, however,the level of cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenineand N⁶-(²-sopentenyl) adenosine were higher in vegetative thanin flowering plants. These results indicate that there is not necessarily a directrelation between endogenous plant hormone levels and flowering,and that the chemical basis for the photoperiodic control offlowering cannot be explained solely by changes in hormone levels.The possibility remains, however, that one or more of the planthormones has some influence of secondary importance on the floweringprocess in Lemna. (Received January 29, 1986; Accepted July 12, 1986)     

Flowering and Endogenous Levels of Benzoic Acid in Lemna Species

The occurrence of benzoic acid, a flower-inducing factor inLemna species, in L. paucicostata strains 151, 381, 321 andL. gibba G3 was established by several purification steps andfinal use of gas liquid chromatography-selected ion monitoring.Quantitative analyses of benzoic acid were made in non-floweringand flowering Lemna to determine differences in levels. Theendogenous level of benzoic acid was shown to vary dependingon culture conditions, but no positive correlation could befound between the endogenous level of benzoic acid and floweringof Lemna. (Received October 6, 1982; Accepted December 23, 1982)     

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)     

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)     

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)     

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)     

The Role of Benzoic Acid and Plant Hormones in Flowering of Lemna gibba G3

The level of benzoic acid was measured in Lemna gibba G3 grownon M and E media under inductive and non-inductive daylengths.Benzoic acid was slightly higher in plants grown on M mediumbut there was no difference in the benzoic acid levels in floweringand vegetative plants. When L. gibba G3 was grown under continuouslight on 1/10 M medium or 1/2 H medium there was virtually noflowering, but addition of benzoic acid to either medium ledto a substantial flowering response. In both cases this floweringresponse was inhibited by the plant hormones IAA, GA₃, ABA andzeatin, with IAA and GA₃ being the least inhibitory and ABAbeing the most inhibitory. This same pattern of inhibition wasseen when L. gibba G3 was grown on M medium under continuouslight, conditions that lead to photoinduction of flowering.These results leave open the possibility that endogenous benzoicacid may interact with other factors to influence the floweringresponse in L. gibba G3. (Received November 13, 1984; Accepted February 27, 1985)     

Inhibition of Flowering in the Long-Day Plant Lemna gibba G3 by Hutner's Medium and its Reversal by Medium Modification

The long-day plant Lemna gibba G3 flowers normally in E medium(Hoagland-type medium plus 30 µM EDTA) but in 0.5 H mediumthere is no flowering. Ammonium is present in 0.5 H medium andis known to inhibit flowering in L. gibba G3, but even in NH₄⁺-free0.5 H medium there is virtually no flowering under continuouslight. Increasing the phosphate concentration of the NH₄⁺-free0.5 H medium from 1.15 ITIM to 12 or 16 mM results in substantialflowering. Decreasing the EDTA concentration from 850 µIMto 250 µM, or raising the nitrate concentration from 4mM to 12 mM, results in only a small increase in flowering.If the decrease in EDTA and increase in nitrate are combinedwith the increase in phosphate, however, the flowering responseis nearly as good as that obtained using E medium. Thus, withthese three changes the inhibitory effect of NH₄⁺free 0.5 Hmedium for flowering in L. gibba G3 is almost completely reversed In the above studies flowering was not limited by daylength.When plants were grown on E medium under an 11 hour daylengthwhere flowering is limited by daylength, decreasing the phosphateconcentration in the medium reduced flowering, but increasingthe phosphate concentration in the medium did not stimulateflowering. Thus, when flowering is limited by daylength, highphosphate will not cause flowering, but a certain level of phosphateappears to be necessary for the expression of photoinductionunder long days. (Received January 14, 1986; Accepted June 24, 1986)     

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)     

The Role of Plant Hormones and Benzoic Acid in Flowering of Lemna paucicostata 151 and 381

The effects of plant hormones on flowering of Lemna paucicostata151 and 381 were investigated when exogenously applied in combinationwith benzoic acid. These strains are very sensitive to benzoicacid and flower readily on application of benzoic acid. Theflower-inducing effect of benzoic acid was strongly modifiedby plant hormones: gibberellins, indole-3-acetic acid and abscisicacid were inhibitory, while cytokinins were promotive (synergistic),suggesting that the balance between endogenous levels of benzoicacid and plant hormones contributes to the regulation of floweringin Lemna. (Received October 6, 1982; Accepted December 23, 1982)     

Comparison of the flowering behavior of the long-day plant Lemna gibba G3 from different laboratories

In an effort to determine the cause for the wide discrepanciesin the level of flowering response reported for the long-dayplant Lemna gibba L., strain G3, cultures of L. gibba G3 wereobtained from the laboratories of W. S. Hillman (G3-H), R. Kandeler(G3-K), Y. Oota (G3-O) and and A. Pieterse (G3-P) and comparedto the L. gibba G3 (G3-C) from this laboratory. Under continuouslight all cultures gave FL% values of 77 or above, and on a9L:15D short-day treatment, all cultures were completely vegetative.However, on daylengths of 10 to 12 hr, small but statisticallysignificant differences were obtained for the different cultures.The critical daylength curves for G3-G, which showed the shortestcritical daylength, and G3-K, which showed the longest criticaldaylength, differed by approximately one hour. Salicylic acidtreatment caused flower promotion in each culture, but statisticallysignificant differences were obtained between some of the culturesin their response to salicylic acid. It is concluded that the large discrepancies in the floweringresponses of L. gibba G3 that have been reported are due primarilyto differences in culturing methods and counting proceduresin the different laboratories. However, the results also indicatethat there may be distinct cultures of L. gibba G3 that exhibitsmall physiological and/or genetic differences that would makeprecise quantitative comparison between different laboratoriesvery difficult. (Received January 23, 1979; )     

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.     

Effects of some metabolic inhibitors on flowering of Lemna gibba G3, a long-day duckweed

Flowering of Lemna gibba G₃, a long-day duckweed, was inhibitedby adding CuSO₄, AgNO₃, HgCl₂, Na₂WO₄ or iodoacetamide to themedium at the concentrations inducing long-day flowering inLemna paucicostata 6746, a short-day duckweed. This suggeststhat these metabolic inhibitors affected the photoperiodic sensitivityrather than directly affecting flower initiation. Ferricyanidepromoted flowering in both of these short-day and long-day duckweeds. (Received July 7, 1977; )     

A Flower-Inducing Substance Derived from Norepinephrine upon Contact with Intact Lemna Plants

A norepinephrine solution in which intact plants of Lemna paucicostatahad been immersed for 30 min or on which intact Lemna plantshad been placed for 24 h had strong flower-inducing activityin L. paucicostata 151, but norepinephrine added to the distilledwater in which Lemna plants had been immersed had no activity. (Received May 24, 1991; Accepted July 5, 1991)     

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; )     

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 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)