Induction of flowering in Lemna paucicostata,a short-day plant,by chelating agents and iron

Summary Lemna paucicostata is a short-day plant which normally flowers only in a medium supplemented with EDTA or EDDHA. On a molar basis EDDHA is more effective for induction of flowering. The chelating agent can be replaced by high concentrations of ferric citrate in the medium. Simultaneous supply of both EDDHA and a high level of ferric citrate results in flowering even under long days.     

Induction and promotion of flowering by heat-treated catecholamines in Lemna paucicostata

Alkali- and heat-treated norepinephrine, a catecholamine, induced flowering of short-day (SD) plant Lemna paucicostata 151 even under long-day (LD) conditions. Flowering induced with a lower concentration of heat-treated norepinephrine was promoted under SD conditions but inhibited by a night break. The related compounds L-dopa and dopamine also promoted flowering under SD conditions when they were heat-treated.     

Induction of flowering by nitrogen deficiency in Lemna paucicostata 441

Lemna paucicostata 441, a short-day plant, flowered even undercontinuous light in nitrogen-deficient, half-strength Hutner’smedium, when the endogenous level of nitrogen was decreasedto 1.4µg/mg fr wt or lower, but no flowering occurredin nitrogen-deficient, modified Hoagland medium when the endogenouslevel of nitrogen was similarly reduced. The failure to flowerin this medium can be ascribed to the presence of high concentrationsof calcium, the absence of EDTA, and low pH. Daylength-independent flowering induced by nitrogen deficiencywas greatly enhanced by the addition to the growth medium ofmicronutrients and EDTA at high concentrations. Among the micronutrients,zinc seemed to be the most important. (Received May 30, 1988; Accepted September 22, 1988)     

Induction of flowering by cytokinins in a short-day plant, Lemna paucicostata

Lemna paucicostata, normally a short-day plant, can be inducedto flower under long-day conditions by providing a cytokininin a medium containing a high level of ferric citrate (5 x 10^–4M).Interestingly, when a cytokinin and EDDHA are present togetherin the medium, flowering is induced even at low levels of iron(10^–5 and 5 x 10^–5M ferric citrate). However, inthe absence of a cytokinin, flowering takes place only undershort days. (Received September 30, 1968; )     

Spectral sensitivity of the flowering response in green and etiolated Lemna paucicostata T-101

Light sensitivity in the reversal of the far-red inhibitionof flowering in etiolated Lemna paucicostata T-101 was 10- (612nm) to 33-fold (660 nm) higher than that of green plants. Theaction spectrum of this response for etiolated plants showeda peak near 660 nm, but for green plants it was near 612 nm.Thus, the effect of light on flowering is influenced greatlyby the screening effect of photosynthetic pigments. (Received July 24, 1980; )     

Spectral sensitivity of the flowering response in green and etiolated Lemna paucicostata T-101

Light sensitivity in the reversal of the far-red inhibitionof flowering in etiolated Lemna paucicostata T-101 was 10- (612nm) to 33-fold (660 nm) higher than that of green plants. Theaction spectrum of this response for etiolated plants showeda peak near 660 nm, but for green plants it was near 612 nm.Thus, the effect of light on flowering is influenced greatlyby the screening effect of photosynthetic pigments. (Received July 24, 1980; )     

Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata

The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering.     

Effect of nitrate concentration in the medium on the flowering of Lemna paucicostata 6746

Addition of copper or tungstate to or exclusion of molybdenumfrom M-sucrose medium induced long-day flowering in Lemna paucicostata6746 provided the medium contained sufficient nitrate. By contrast,ferricyanide, cyanide or silver induced long day flowering evenin nitrate-deficient, M-sucrose medium. (Received August 26, 1977; )     

Aspartokinase of Lemna paucicostata Hegelm. 6746

A sensitive and specific method was developed for assay of aspartokinase (EC 2.7.2.4) in crude extracts of Lemna paucicostata. Lysine inhibited approximately 93%, and threonine approximately 6%; together, these amino acids inhibited 99%. Inhibition by lysine was synergistically increased by S-adenosylmethionine, which by itself had no effect on activity. Essentially complete inhibition of threonine-resistant activity was obtained with lysine, and of lysine-resistant activity with threonine. Inhibition by lysine and threonine was additive, with no indication of concerted inhibition. Aspartate concentration had no effect on the relative proportions of lysine- and threonine-sensitive activities. Aspartokinase activity was in large excess of that reported by other workers, the maximum capacity (V_max) far exceeding the in vivo requirements. Estimations of rates of aspartokinase in vivo suggest that the step catalyzed by this enzyme may not be the overall `rate-limiting' one for entry of 4-carbon units into the aspartate family of amino acids, and that feedback inhibition of this enzyme by lysine and threonine may not be a major factor in regulating flux through this step.     

Investigations on the effects of metal ions and chelating agents on growth and flowering of Lemna gibba G 3

The effect of different chelating agents on growth and floweringof Lemna gibba G 3 was studied in M and HUTNER'S media. Theincorporation of EDDHA and Fe-EDDHA in the media resulted inprofuse flowering and gibbous character of the fronds. However,EDTA was relatively less effective. It was demonstrated thatthe metal which influenced flowering in L. gibba G 3 was mostlikely copper. (Received August 19, 1970; )     

Threonine Synthase of Lemna paucicostata Hegelm. 6746

Threonine synthase (TS) was purified approximately 40-fold from Lemna paucicostata, and some of its properties determined by use of a sensitive and specific assay. During the course of its purification, TS was separated from cystathionine γ-synthase, establishing the separate identity of these enzymes. Compared to cystathionine γ-synthase, TS is relatively insensitive to irreversible inhibition by propargylglycine (both in vitro and in vivo) and to gabaculine, vinylglycine, or cysteine in vitro. TS is highly specific for O-phospho-l-homoserine (OPH) and water (hydroxyl ion). Nucleophilic attack by hydroxyl ion is restricted to carbon-3 of OPH and proceeds sterospecifically to form threonine rather than allo-threonine. The K_m for OPH, determined at saturating S-adenosylmethionine (AdoMet), is 2.2 to 6.9 micromolar, two orders of magnitude less than values reported for TS from other plant tissues. AdoMet markedly stimulates the enzyme in a reversible and cooperative manner, consistent with its proposed role in regulation of methionine biosynthesis. Cysteine (1 millimolar) caused a slight (26%) reversible inhibition of the enzyme. Activities of TS isolated from Lemna were inversely related to the methionine nutrition of the plants. Down-regulation of TS by methionine may help to limit the overproduction of threonine that could result from allosteric stimulation of the enzyme by AdoMet.     

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 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.     

Some Characteristics of Protein Kinases in Lemna paucicostata

The three protein kinases of Lemna paucicostata that are separableby DEAE-Sephacel chromatography have been designated PI, PIIand PIII [Kato et al. (1983) Plant & Cell Physiol. 24: 841].The optimum pH for the PI and PII enzymes was 7.5 and for thePHI enzyme 7.0. The activities of these enzymes were stimulatedby divalent cations, the maximum stimulation being producedby 5 nw Mg^{2 $} for PI, by 3 mM Co^{2 $} for PII and by 1 mM Mn^2$ for PIII. The cytokinins; benzyladenine, kinetin and zeatin,inhibited the activity of the PIII enzyme. The molecular weightsof the PI and PII enzymes did not change after incubation withcAMP even though their activities were regulated by this compound. (Received October 17, 1983; )     

Biosynthesis and Metabolism of NAD in Lemna paucicostata 151

We have already reported that NiA and related compounds had plant-growth-promoting and flower-inducing activities in duckweed, Lemna paucicostata 151. These effects may be concerned with the biosynthesis and metabolism of NAD. So, for the first step, the various enzyme activities related to them were investigated in this study to obtain some fundamental information on the action mechanism of these compounds and metabolites. Extremely high enzyme activity of nicotinamidase and very high enzyme activity of N AD glycohydrolase were found. The enzyme activities of nicotinate phosphoribo- syltransferase, quinolinate phosphoribosyltransferase, and nicotinate methyltransferase were easily detected. In contrast, nicotinamide phosphoribosyltransferase and ADP-ribosyltransferase activities were very low and nicotinamide methyltransferase activity was not detectable. NiA and NAm administered to the plant were rapidly incorporated into N AD and metabolized to several compounds. Postulation of the action mechanism is discussed.     

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)     

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)     

Uptake of Choline and Ethanolamine by Lemna paucicostata Hegelm. 6746

Lemna paucicostata Hegelm. 6746 possesses specific systems for uptake of choline and ethanolamine. Each is distinct from the six other systems for uptake of organic compounds so far identified in this plant. Both systems show biphasic kinetics, so that uptake by them can be described as the composite result of two Michaelis-Menten processes. Inhibitor studies are reported which indicate the very strict structural specificity of each system. The kinetic constants of choline uptake are such that, at an external concentration of 0.65 micromolar, the total requirement of the plant for this compound would be met, 41% via the high affinity system and 59% via the lower. At an external concentration of 2.4 micromolar ethanolamine, an amount of this compound sufficient to form the total choline of the plant would be supplied, 59% via the high affinity system and 41% via the lower. These, and other observations, strongly support the physiological importance of these systems under natural conditions.     

Flower-Promoting Effects of Iron and EDDHA in Lemna paucicostata 151

Lemna paucicostata 151 cultured in 1/10 strength M medium containing50 µM FeCl₃ easily flowered in response to short days,although it scarcely flowered under any photoperiod when themedium contained the standard amount of iron (2 µM FeCl₃).The flowering response was accomparied by an increase in theiron content of the plants, which was maximal at pH 5.0. Instandard M medium containing 50 µM FeCl³, this plant didnot flower even though it had a high iron content. Ethylenediamine-di (o-hydroxyphenylacetic acid) (EDDHA) inducedflowering of this strain under continuous light even in theabsence of iron and copper, and its effect was slightly loweredby the presence of iron in the medium. Thus the flower-inducingactivity of EDDHA could not be attributed to the action of ironor copper. EDTA inhibited both the iron uptake and floweringin Fe-rich medium under short-day conditions. (Received May 16, 1986; Accepted July 25, 1986)     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.