Role of Catecholamines in Promotion of Flowering in a Short-Day Duckweed, Lemna paucicostata 6746

l-Epinephrine, l-norepinephrine, and l-isoproterenol substantially promote flowering under a photoperiodic regime of 8 hours light and 16 hours darkness in Lemna paucicostata 6746 when grown on the modified Bonner-Devirian medium devoid of ethylenediaminetetraacetic acid. If catecholamines are provided to plants at 10⁻⁴ molar level prior to transferring them to the short-day regime, they not only induce more floral primordia but also significantly improve flower development and sustain the flowers for a longer period. Propranolol (10⁻⁴ molar), a β-adrenergic blocking agent, partially suppresses flowering and the inhibition of flowering is relieved by catecholamines.     

Floral Induction in a Photoperiodically Insensitive Duckweed, Lemna paucicostata LP6 : Role of Glutamate, Aspartate, and Other Amino Acids and Amides

The effects of 20 amino acids and two amides were studied on the flowering of a photoperiodically insensitive duckweed, Lemna paucicostata LP6. Alanine, asparagine, aspartate, cystine, glutamate, glutamine, glycine, lysine, methionine, proline, serine, and threonine induced flowering under a photoperiodic regime of 16 hours light and 8 hours darkness. Among these, glutamate and aspartate were found to be the most effective for flower induction. These acids could initiate flowering even at 5 × 10⁻⁷ molar level, though maximal flowering (about 80%) was obtained at 10⁻⁵ molar. Change in the photoperiodic schedule or the pH of the nutrient medium did not influence glutamate- or aspartate-induced flowering. The low concentrations at which glutamate and aspartate are effective suggests that they may have a regulatory role rather than simply acting as metabolites.     

Effects of Agitating Culture Condition on the Growth,Metabolic and Carotenoid Profiles of <Emphasis Type="Italic">Lemna paucicostata</Emphasis>

The effects of agitating culture conditions on the growth, metabolic and carotenoid profiles of Lemna paucicostata were investigated in this study using gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry. Lemna paucicostata cultivated under static and agitating conditions showed no significant difference in total plant growth. Various alcohols, amino acids, fatty acids, organic acids, sugars, and phenolic compounds were identified in the sample. The higher relative yields of ferulic acid and GABA were obtained under the agitating condition on day 28, whereas that of coumaric acid was higher under the static condition on day 42. Significantly higher relative yields of antheraxanthin and lutein were achieved under agitation condition than static condition on day 42. In addition, fucoxanthinol was identified for the first time in L. paucicostata culture. The results of this study suggest that static and agitating culture conditions, as well as optimal harvest timing, should be selected according to the target products including ferulic acid, GABA, coumaric acid, antheraxanthin and lutein in L. paucicostata culture.     

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)     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Epinephrine, Propranolol, and the Sucrose-Ammonium Inhibition of Flowering in Lemna paucicostata 6746

The sucrose-ammonium inhibition of flowering Lemna paucicostata 6746 in continuous blue light or in short days was partially overcome by epinephrine. This reversal was prevented by propranolol, an antagonist of epinephrine in animals. In ammonium-free medium, propranolol inhibited flowering, and this inhibition was completely overcome by epinephrine. Increased levels of Ca²⁺, Pi and nitrate partially reversed the inhibition by propranolol. Concentrations of cAMP, adenine, and adenosine that partially overcame the sucrose-ammonium inhibition did not affect flowering in cultures treated with propranolol. The possibility is discussed that the effects on flowering of sucrose-ammonium, propranolol, and epinephrine were due to altered intracellular levels of cAMP or of a cAMP-like compound.     

Flower-Inducing Activity of Water Extracts of Various Plant Species, in Particular Pharbitis nil

The crude water extracts of leaves of many plant species belongingto Spermatophyta and some belonging to Bryophyta induced floweringof Lemna paucicostata 151 (PI51) under continuous light, atthe concentrations equivalent to 0.1 to 10 mg fr wt leaf per10 ml culture medium (mg fr wt/10 ml). The extract of Salvinia(Pterydophyta) added together with the extract of Lemna at aconcentration lower than that necessary to cause flowering alsoinduced flowering. The activity of the water extracts of someplants varied considerably from experiment to experiment dueto unknown factors, but the extracts of Pharbitis nil strainViolet, a sensitive short-day plant, always showed a high activity,as did the extracts of Lemna paucicostata reported previously. The extract of Pharbitis cotyledons induced flowering of P151even at 0.3 mg fr wt/10 ml, and significantly promoted floweringof L. paucicostata 441 and 6746 at 1–3 mg fr wt/10 ml.Ex-udate from the cuttings of the seedlings was also active.However, neither the activity of the water extract nor thatof the exudate could be correlated with photoperiodic floralinduction. On the other hand, the extract of leaves or cotyledonshad higher activity (on a fr wt basis) than that of other organs,and the activity of the extract of cotyledons changed with theirage roughly in parallel with their photoperiodic sensitivity. (Received April 17, 1989; Accepted August 10, 1989)     

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)     

Homocysteine Biosynthesis in Green Plants: Physiological Importance of the Transsulfuration Pathway in Lemna paucicostata

To permit an assessment of the relative contributions of the transsulfuration and the direct sulfhydration pathways for homocysteine biosynthesis, the time course of incorporation of ³⁵S from ³⁵SO₄²⁻ into various sulfur-containing compounds in Lemna paucicostata has been determined. Plants were grown with either low (4.5 micromolar) or ample (1,000 micromolar) sulfate in the medium. At the shortest labeling times, ³⁵S-cystathionine was the predominant ³⁵S-containing organic sulfur compound. The flux of sulfur into cystathionine was sufficient to sustain the known rate of methionine biosynthesis. It was calculated that transsulfuration accounted for at least 90 and 85% of the total homocysteine synthesis in low and ample sulfate-grown plants, respectively (and may have accounted for 100%). No marked rise in the ³⁵S-soluble cysteine:³⁵S-homocysteine ratio was observed even at the shortest labeling times, but it is argued that this may be due to (a) the observed compartmentation of soluble cysteine, and (b) the impracticality of using labeling times shorter than 17 seconds. Additional evidence supporting the importance of transsulfuration in Lemna is briefly described.     

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)     

Nickel and the metabolism of urea by Lemna paucicostata Hegelm. 6746

With urea as sole nitrogen source, the addition of 5×10^-5 M nickel sulfate to axenic cultures of Lemna paucicostata 6746 approximately doubles the rate of vegetative growth. Under a standard light-dark schedule, Ni²⁺ changes the daily pattern of respiratory CO₂ output on urea from one having a single daily peak to one with two daily peaks which resembles that on ammonium or nitrate as sole nitrogen source. It also increases CO₂ output by as much as 3-fold on a fresh-weight basis. These data represent the first confirmation in an intact higher plant of proposals, based on enzymology and tissue culture responses, for a role of Ni²⁺ in urea metabolism. Further, they indicate the possible existence of two distinct pathways of urea utilization.     

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.     

C14-Oxylipin glucosides isolated from Lemna paucicostata

Oxylipin glucosides (2-4) were isolated from Lemna paucicostata with their structures and absolute configurations elucidated by spectroscopic and chemical methods. Compounds 2-4 were glucosides of C₁₄ oxylipin which were synthesized from α-linolenic acid via the 9-lipoxygenase pathway.     

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)     

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)     

Phytochrome-mediated changes in the membrane potential of subepidermal cells of Lemna paucicostata 6746

Light-stimulated transmembrane potential changes have been measured continuously after implantation of microelectrodes into subepidermal cells of the short-day plant Lemna paucicostata 6746. Irradiation for 5 min with white or red light caused a transient hyperpolarization. These potential changes could be suppressed with 10^-6 M DCMU. Irradiation of DCMU-inhibited plants with far-red light for 5 min hyperpolarized the membrane potential, which thereafter was not changed by further far-red application. Consecutive red light irradiation for 5 min depolarized the membrane potential. The red/far-red reversibility of the potential changes (which could be repeated several times with a single plant) suggests the participation of phytochrome.Abbreviations EDTA ethylenediaminetetraacetate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - P_r, (P_fr) red- (far-red-) absorbing form of phytochrome     

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 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)     

Growth and flowering of Lemna paucicostata I. General aspects and role of chelating agents in flowering

Lemna paucicostata HEGELM. is normally a short-day plant andflowers only in the presence of a chelating agent (EDTA or EDDHA)in the medium. The plant can be induced to flower even by asingle long night treatment; the flowering percentage, however,increases with further inductive cycles. The length of the criticaldark period depends upon the chelating agent employed in themedium. It is between 10 and 12 hr in the medium containingEDTA and about 8 hr in the EDDHA-supplemented medium. Red lightinterruption in the middle of the dark period—even fora minute—is inhibitory for flowering. Attempts to identify the metal ion(s) chelated reveal that thechelating agents affect flowering by facilitating iron uptake.This is also supported by the fact that the requirement of achelating agent for flowering can be overcome with an excessof iron in the medium. Interestingly, provision of EDDHA andexcess of ferric citrate, together, can bring about floweringeven under long days. ¹Originally HEGELMAIER (1) designated L. paucicostata as a separatespecies; however, THOMPSON (2) and DAUBS (3) have treated itsynonymous to L. perpusilla. More recently, based on physiologicaland chemotaxonomic studies, the distinctiveness of L. paucicostatafrom L. perpusilla has been favoured (4, 5). (Received September 8, 1969; )