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)     

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)     

Role of Salicylic Acid and Benzoic Acid in Flowering of a Photoperiod-Insensitive Strain, Lemna paucicostata LP6

Lemna paucicostata LP6 does not normally flower when grown on basal Bonner-Devirian medium, but substantial flowering is obtained when 10 μm salicylic acid (SA) or benzoic acid is added to the medium. Benzoic acid is somewhat more effective than SA, and the threshold level of both SA and benzoic acid required for flower initiation is reduced as the pH of the medium is lowered to 4.0. SA- or benzoic acid-induced flowering is enhanced in the simultaneous presence of 6-benzylaminopurine (BAP), although BAP per se does not influence flowering in strain LP6. Continuous presence of SA or benzoic acid in the culture medium is essential to obtain maximal flowering. A short-term treatment of the plants (for first 24 h) with 10 μm SA or benzoic acid, followed by culture in the basal medium containing 1 μm BAP can, however, stimulate profuse flowering. Benzoic acid is more effective than SA, and the effect is more pronounced at pH 4 than at 5.5. Thus, under these conditions, flowering is of an inductive nature. Experiments with [¹⁴C]SA and [¹⁴C]benzoic acid have provided evidence that at pH 4 there is relatively more uptake of benzoic acid than SA, thus leading to an increased flowering response. The data obtained from the experiments designed to study the mobility of [¹⁴C]SA and [¹⁴C]-benzoic acid from mother to daughter fronds indicate that there is virtually no mobility of SA or benzoic acid between fronds.     

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)     

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)     

Measurement of Endogenous Phosphorus Levels in Relation Flowering in the Long-Day Plant Lemna gibba G3

When Lemna gibba G3 was grown on E medium, a decrease in thephosphate concentration caused a suppression of flowering andlead to a decrease in the phosphorus concentration in the plants.Addition of salicylic acid reversed this inhibition withoutcausing an increase in the phosphorus concentration, while additionof copper or ammonium to Hoagland-type medium inhibited flowering,also without affecting the phosphorus concentration. Plantsgrown under 8 h or 24 h daylengths exhibited a FL% of 0 or about50, respectively, but showed no difference in their phosphorusconcentrations. These results indicate that a high phosphorusconcentration is not always required for flowering in Lemnagibba G3. When plants were grown on a modified E medium in which 1/50strength Hutner’s medium micronutrients were substitutedfor the normal E medium micronutrients, the suppression of floweringcaused by reduced phosphate was completely eliminated. Addingeach micronutrient individually at the normal concentrationto the modified E medium demonstrated that manganese inhibitedflowering in modified E medium with a low phosphate concentration. (Received January 31, 1986; Accepted July 4, 1986)     

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)     

Flower-Inducing Activity of Benzoic Acid Derivatives for Lemna minor

Lemna minor, strain M601, scarcely flowers in full or 1/10 strengthM medium supplemented with 1% sucrose either in long-day orshort-day conditions, but easily flowers when some benzoic acidderivatives are added to 1/10 strength M-sucrose medium. Thepresence of cytokinin in the medium improves the activity ofbenzoic acid but not significantly that of salicylic acid. Thestructure-activity relationships for the flower-inducing activityof substituted benzoic acid derivatives in the presence of cytokininwere analyzed quantitatively in terms of physicochemical substituentparameters. In the presence of cytokinin, the flower-inducingactivity is determined by electronic and steric effects of thesubstituent. The higher the electron withdrawing ability andthe lower the bulkiness of the substituent, the higher is theactivity. In the absence of cytokinin, the activity is enhancedby higher electron withdrawing ability of the substituent, butthe steric effect is insignificant. In both conditions, thehydroxyl group in the ortho position exhibits an additionaleffect to enhance activity, and the molecular form responsiblefor the activity seems to be undissociated neutral species.The structure-activity relationship in L. minor M601 is basicallysimilar to that for L. paucicostata 151. (Received February 2, 1983; Accepted May 2, 1983)     

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)     

Effect of 8-Hydroxyquinoline on Flowering and Endogenous Levels of Iron and Copper in Lemna paucicostata, Strain LP6

Lemna paucicostata LP₆, a strain of duckweed isolated locally,does not flower under any photoperiodic schedule when grownin Bonner and Devirian or other media routinely employed invarious laboratories for studies on flowering in Lemnaceae.Flowering in this strain could be induced, however, by 8-hydroxyquinoline(8-HQ)—a well-known copper chelating agent—irrespectiveof the length of the photoperiod. To our knowledge, this isthe first report where a direct induction of flowering in aduckweed by 8-HQ has been observed. Atomic absorption analysisof the plant material revealed that the endogenous level ofcopper is significantly higher in the plants treated with 8-HQ.This is contrary to the general assumption that chelating agentsinfluence flowering of duckweeds by causing a reduction in theuptake of copper ions and making them less available to theplants. (Received May 23, 1983; Accepted July 22, 1983)     

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.     

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)     

Relationship between Structure and Flower-inducing Activity of Benzoic Acid Derivatives in Lemna paucicostata 151

Lemna paucicostata, strain 151, does not flower when grown in1/10 strength M medium containing 1% sucrose, but easily flowerswhen benzoic acid or its substituted derivatives are added tothe medium. The structure-activity relationship of benzoic acidderivatives are quantitatively analyzable by means of physicochemicalparameters. Aromatic substituent constants a (electronic parameter)and Vw (van der Waals volume steric parameter) delineate thevariations in the flower-inducing activity, i.e., the higherthe electron-withdrawing ability as well as the less the bulkinessof the substituents, the higher the activity. The exceptionsare the derivatives substituted with NO₂ at any position andwith OH at meta and para positions; they exhibit an activitylower than expected from their substituent parameters. 2-Hydroxybenzoicacid (salicylic acid) exhibits activity higher than the calculatedvalue, suggesting participation of the chelating effect in theflower-inducing activity besides electronic and steric effects.The molecular form responsible for the activity is suggestedto be an undissociated neutral species. (Received May 21, 1981; Accepted September 29, 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)     

Relationship between Flower Induction by Benzoic Acid and its Metabolism in Lemna paucicostata 151

The flower-inducing activities in Lemna paucicostata 151 offour major metabolites of benzoic acid (N-benzoyl aspartate,benzyl 6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranoside,O-benzoyl isocitrate and O-benzoyl malate) were measured, andthe effects on the uptake and metabolism of benzoic acid dueto change in the level of the benzoic acid concentration orto the addition of plant hormones were investigated. N-Benzoylaspartate had weak activity, and O-benzoyl isocitrate and malatehad fairly strong activities, while benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosideshowed no activity. As the concentration of benzoic acid rose,the ratio of N-benzoyl aspartate increased and that of benzyl6-O-ß-D-apiofuranosyl-O-ß-D-glucopyranosidedecreased. GA₃ and IAA, inhibitors of flower induction by benzoicacid, seemed to promote conversion to N-benzoyl aspartate insteadof to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranoside.The conversion to N-benzoyl aspartate was considered to be adetoxification process and that to benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucopyranosidemay be directly related to flower induction in Lemna. (Received November 2, 1987; Accepted January 23, 1988)     

Biological Conversion of Benzoic Acid in Lemna paucicostata 151 and its Relation to Flower Induction

Benzoic acid, known to induce flowering in Lemnaceae, was shownto be converted to four major compunds in Lemna paucicostata151. These compounds were isolated and determined to be N-benzoylaspartate, benzyl 6-O-ßdD-apiofuranosyl-ß-D-glucopyranoside,O-benzoyl isocitrate and O-benzoyl malate. (Received November 2, 1987; Accepted January 23, 1988)     

Seed Development in the Recalcitrant Species Quercus robur L: Water Status and Endogenous Abscisic Acid Levels

Water status and endogenous ABA levels were measured duringthe development of the embryonic axis and cotyledons in fruitsof Quercus robur L. As dry matter accumulated in the embryoduring development, both moisture content and osmotic potential(     

Studies on the Flowering Mechanism in Lemna

The dark reaction of the short day plant Lemna perpusilla was investigated. It was found that 3-phosphoglycerate and pyruvate (10^?6M) increased the flowering rate in the presence of nitrates. Pyruvate-2-¹⁴C was added to the culture solution during two hours of the dark reaction and ¹⁴C was incorporated into serine, aspartate and glutamate. It was postulated that pyruvate reacted with a nitrogen source forming an intermediate, possibly aspartate, which was further converted into serine. L. perpusilla failed to flower when the dark period was interrupted with red light and as a result endogenous serine accumulated in a high concentration. The dark reaction of L. perpusilla, in which serine was involved, required (1) oxygen, (2) ATP, (3) moderate temperature, and (4) an enzyme system.     

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.).