Comparison of the Ability of Salicylic Acid and Ferricyanide to Induce Flowering in the Long-day Plant, Lemna Gibba G3

Both salicylic acid and ferricyanide induce flowering in the long-day plant Lemna gibba L., strain G3 under 8- and 9-hour short days. In both cases the effect is daylength-dependent. Salicylic acid is ineffective on daylengths less than 8 hours and ferricyanide is ineffective on daylengths less than 5 hours. When both substances are given together a striking synergistic interaction is observed, and some flowering is obtained on daylengths as short as 3 hours. However, even with the optimal combinations the flower-inducing effect remains daylength-dependent.     

Chemical control of flowering in the long-day plantLemna gibba G3

Lemna gibba G3 is an ideal system for studying the chemical control of flowering in a photoperiodic plant due to its small size and aquatic growth habit which allow substances to be taken up continuously and rapidly distributed throughout the plant. Each of the known plant growth regulators has been tested onL. gibba G3 and only the gibberellins appear to be important for flowering, although they are not the limiting factor for flowering on short days. Salicylic acid (SA) and ferricyanide will both induce flowering inL. gibba G3 with ferricyanide being most effective on short days where flowering is daylength limited and SA most effective where flowering is limited by factors other than daylength. The ferricyanide action is probably due to HCN and it may act during photoperception or photoinduction. SA is most effective when reversing the inhibition caused by various parameters including copper and agar, and its effect is always strongly daylength dependent. It is postulated that SA may interact with the flowering stimulus to promote flowering and thus that SA acts at some point following photoinduction and the formation of the flowering stimulus.     

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)     

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; )     

Experimental control of flowering in Pistia stratiotes L.

GA₃, salicylic acid and EDDHA induced flowering in Pistia stratiotesin vitro under short days and conditions of continuous light.It has been hypothesized that EDDHA and salicylic acid bringabout the same effect on flowering in Pistia and the duckweedspecies Lemma gibba G3 whereas the effect of GA₃ on the floweringmechanism of these species is basically different. (Received March 14, 1978; )     

Uptake and Metabolism of [C]Salicylic Acid in Lemna gibba G3

When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH₄⁺-free 0.5 H medium) there is virtually no flowering, but addition of 10 micromolar salicylic acid (SA) to the medium results in substantial flowering. Using this system, the uptake and metabolism of [¹⁴C]SA in L. gibba G3 has been examined. SA uptake is rapid and linear for at least the first 24 hours. After 30 minutes, nearly 90% of the radioactivity in the plants is present as free SA. Part of this is rapidly converted to one or more bound forms of SA that appear either in the acidic butanol fraction or in the aqueous residue, and after 12 hours an equilibrium is reached between the free and bound forms of SA. When plants receive SA for 6 days and then are switched to control medium, both the free and bound SA remain nearly constant for at least 5 days. However, there is virtually no transfer of SA from mother fronds to daughter fronds, indicating that the SA is apparently sequestered within the cell. Cell fractionation studies show that nearly 95% of the SA remains in the supernatant even after a 2-hour centrifugation at 300,000 g. Thus, it is unlikely that SA is confined within a specific organelle, but rather is probably secreted into the vacuole.     

Influence of Giving Salicylic Acid for Different Time Periods on Flowering and Growth in the Long-Day Plant Lemna gibba G3

When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH₄⁺-free 0.5 H medium) growth is excellent, but flowering is severely inhibited and often is zero. Addition of 10 micromolar salicylic acid (SA) to NH₄⁺-free 0.5 H medium quickly reverses this inhibition and leads to optimal flowering. The SA treatment also leads to a considerable reduction in the growth rate and increase in frond gibbosity. Removal of SA from the medium quickly leads to an increase in the growth rate and a large decrease in flowering. Thus, for maximal effectiveness SA must be present in the medium for the entire experiment, and the effect of SA is clearly not inductive.     

Isolation of Flower-inducing and Flower-inhibitory Factors from Aphid Honeydew

The aphid Dactynotus ambrosiae Thomas has been allowed to feed on vegetative or flowering plants of the short-day plant Xanthium strumarium L., and the honeydew which they produce is extracted and tested for an effect on flowering using the long-day plant Lemna gibba L., strain G3 for the bioassay. One zone of flower-inducing activity and at least two zones of flower-inhibitory activity are consistently obtained from the honeydew extracts. The levels of flower-inducing and flower-inhibitory activity are not demonstrably different in vegetative and flowering honeydew. The honeydew extracts are inactive on Xanthium but do give some flower induction with the short-day plant Lemna perpusilla Torr., strain 6746. The flower-inducing activity is clearly of plant origin and is present in the phloem since the same active material can be obtained from vegetative or flowering Xanthium by methanol extraction, and honeydew produced by aphids feeding on a chemically defined synthetic diet is completely without flower-inducing activity. This is the first report of successful flower induction in the long-day plant L. gibba G3 by some means other than long-day treatment.     

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)     

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; )     

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)     

EXPERIMENTAL CONTROL OF FLOWERING IN LEMNA. III. A RELATIONSHIP BETWEEN MEDIUM COMPOSITION AND THE OPPOSITE PHOTOPERIODIC RESPONSES OF L. PERPUSILLA 6746 AND L. GIBBA G3

Hillman , William S. (Yale U., New Haven, Conn.) Experimental control of flowering in Lemna. III. A relationship between medium composition and the opposite photoperiodic responses of L. perpusilla 6746 and L. gibba G3. Amer. Jour. Bot. 48(5): 413–419. Illus. 1961.—In a simple Hoagland-type medium, L. perpusilla 6746 flowers, irrespective of daylength; L. gibba G3 does not flower under any daylength, as long as the medium is changed frequently. Ethylenediaminetetraacetic acid (EDTA) prevents the flowering of L. perpusilla in long days but not in short, and brings about the flowering of L. gibba in long days but not in short. The same results are obtained with medium “aged” by the growth in it of L. gibba for several weeks (in any photoperiod) as well as with tartaric acid. The effectiveness of both EDTA and “aged” medium is greater at pH levels near 5 than at 4 or below, probably reflecting action through metal-chelation. These results are most easily interpreted as effects on photoperiodic sensitivity, and suggest a central role of metals in photoperiodism.     

Gibberellin and CCC Effects on Flowering and Growth in the Long-day Plant Lemna gibba G3

The application of gibberellic acid (GA₃) to the non-rosette long-day plant Lemna gibba G3 at concentrations from 0.1 to 100 mg/l did not induce flowering on short days and inhibited flowering on long days at concentrations of 1 mg/l and higher. On both short and long days GA₃ concentrations above 1 mg/l caused a decrease in frond size and fresh and dry weight, but an increase in the rate of frond production and thus an increase in the # VF (number of vegetative fronds). Identical results were obtained when gibberellin A₇ was used instead of GA₃.     

Flowering responses of Lemna perpusilla and L. gibba in relation to nitrate concentration in the culture medium

Flowering responses of Lemna perpusilla strain 6746, a short-dayplant, and L. gibba strain G3, a long-day plant, to nitrateconcentration in Hoagland's type medium with or without EDTA,were compared. Maximum flowering of L. perpusilla under SD occurredat higher nitrate concentrations than did colony proliferation.Even under CL, L. perpusilla grown at sub-optimal nitrate concentrationsfor colony proliferation, flowered irrespective of the presenceof EDTA which reduces flowering. Unlike L. perpusilla, L. gibba failed to flower under SD atany nitrate concentration whether or not EDTA was added. UnderCL, however, L. gibba flowered at almost any nitrate concentrationwith or without EDTA. Double optima for nitrate concentrationwas exhibited in the presence of EDTA; optimal concentrationfor colony proliferation came between the two optima for flowering. We concluded that the nitrogen level of the medium is importantin regulating flowering of duckweeds, and that the effect ofEDTA, if any, may primarily be on colony proliferation and onlysecondarily or antagonistically on flowering. ¹ Present address: Institute for Agricultural Research, TohokuUniversity, Sendai 980, Japan. (Received September 25, 1971; )     

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)     

Specific interactions in the physiology of flowering and gibbosity of Lemna gibba G3

Fronds of Lemna gibba G3 became conspicuously gibbous when ethrel,an ethylenereleasing compound, was added to the nutrient medium.Maximal gibbosity was obtained at ethrel concentrations of 1µg/ml and higher. Unlike the chelating agent, EDDHA, whichcauses profuse flowering and markedly gibbous fronds under long-dayconditions, ethrel did not affect flowering. In the presenceof an optimal concentration of EDDHA (10 µ/ml), ethreleven significantly inhibited flowering and caused developmentof excessively gibbous fronds. Autoclaved gibberellic acid specifically negated the ethreleffect as it does that of EDDHA. Three decomposition productsof GA₃, allogibberic acid, epiallogibberic acid and gibbericacid, also nullified flowering and gibbosity in the presenceof EDDHA. A fourth decomposition product of GA₃, epigibbericacid, inhibited gibbosity but hardly affected flowering. Salicylic acid was confirmed to affect flowering and gibbosityin L. gibba G3. However, contrary to an earlier report, it didnot induce flowering under short-day conditions. (Received January 10, 1976; )     

Induction of flowering in Lemna gibba G3 under short-day conditions

In the presence of the chelating agent EDDHA, long-day duckweedLemna gibba G3 was induced to flower under a short-day scheduleof 9 hr of light and 15 hr of darkness in a 24-hr cycle. Weconcluded that EDDHA creates effects very similar to those ofsalicylic acid. When EDDHA or salicylic acid was added to thenutrient medium in combination with BA, flowering was inducedeven under conditions of 8 hr of light and 16 hr of darkness.Under a photoperiod of 9 hr, BA markedly enhanced the effectof EDDHA as well as salicylic acid. On the other hand, BA alonewas ineffective as far as flowering was concerned. By quantitativeinteractions, BA seems to complement the modifying effect ofEDDHA or salicylic acid on flowering in this duckweed strain. (Received June 25, 1976; )     

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)     

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)     

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)