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.     

Sulfate Uptake and Its Regulation in Lemna paucicostata Hegelm. 6746

The steady state concentrations of S-containing compounds formed in Lemna paucicostata Hegelm. 6746 in response to variations in source and concentrations of sulfur were measured. Neither growth rates nor protein accumulation were markedly affected by the various growth conditions. Ignoring complications due to possible compartmentation, the results are consistent with internal pools of both SO(4) (2-) and cyst(e)ine (or products of their metabolism), but not methionine, being effectors of regulation of high affinity SO(4) (2-) uptake. As SO(4) (2-) in the growth medium was increased to 10 mm, down-regulation of high affinity SO(4) (2-) uptake was more than compensated for by unregulated uptake via the "non-saturating" uptake system. Tissue inorganic SO(4) (2-) accumulated but formation of reduced sulfur remained constant. Some conversion of l-cystine sulfur to SO(4) (2-) occurred. Presence of l-cystine in the medium (a) down-regulated high affinity SO(4) (2-) uptake and (b) decreased the rate of SO(4) (2-) organification. The net results were decreased (7 mum l-cystine) or normal (14 mum l-cystine) total tissue SO(4) (2-) and dose-dependent accumulation of soluble cyst(e)ine and glutathione, but not of soluble methionine. l-Methionine was not metabolized to cyst(e)ine or its products. Presence of l-methionine in the medium led to increased total tissue sulfur, accounted for almost wholly by manyfold increases in soluble methionine, AdoMet, and S-methylmethionine sulfonium. Soluble cyst(e)ine increased slightly.     

In Vivo Regulation of Threonine and Isoleucine Biosynthesis in Lemna paucicostata Hegelm. 6746

Little, if any, regulation of threonine synthesis was observed in Lemna paucicostata Hegelm. 6746 supplemented with concentrations of threonine and/or isoleucine that allow for uptake of these amino acids in amounts sufficient for total plant requirements, and that increase tissue concentrations of soluble threonine manyfold. High tissue concentrations of soluble threonine generated endogenously in isoleucine-supplemented plants were no more effective in regulation than a similar concentration of threonine accumulated from the medium. These studies exclude also major regulation of threonine biosynthesis by bivalent repression by threonine plus isoleucine. Isoleucine biosynthesis was severely inhibited by supplementation with isoleucine, but not with threonine or methionine. The fivefold increase in soluble threonine in isoleucine-supplemented plants suggests that threonine dehydratase is a major locus for feedback regulation of isoleucine synthesis. It is concluded that regulation of threonine biosynthesis differs from that of the other amino acids of the aspartate family (isoleucine, methionine, and lysine), each of which strongly feedback regulates its own synthesis. Methionine supplementation had a negligible effect on the tissue concentration of soluble threonine, indicating that threonine is not important in balancing changes of flux into methionine by equivalent changes of flux through the step catalyzed by aspartokinase.     

The S-Methylmethionine Cycle in Lemna paucicostata

The metabolism of S-methylmethionine has been studied in cultures of plants of Lemna paucicostata and of cells of carrot (Daucus carota) and soybean (Glycine max). In each system, radiolabeled S-methylmethionine was rapidly formed from labeled l-methionine, consistent with the action of S-adenosyl-l-methionine:methionine S-methyltransferase, an enzyme which was demonstrated during these studies in Lemna homogenates. In Lemna plants and carrot cells radiolabel disappeared rapidly from S-methylmethionine during chase incubations in nonradioactive media. The results of pulse-chase experiments with Lemna strongly suggest that administered radiolabeled S-methylmethionine is metabolized initially to soluble methionine, then to the variety of compounds formed from soluble methionine. An enzyme catalyzing the transfer of a methyl group from S-methylmethionine to homocysteine to form methionine was demonstrated in homogenates of Lemna. The net result of these reactions, together with the hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine, is to convert S-adenosylmethionine to methionine and adenosine. A physiological advantage is postulated for this sequence in that it provides the plant with a means of sustaining the pool of soluble methionine even when overshoot occurs in the conversion of soluble methionine to S-adenosylmethionine. The facts that the pool of soluble methionine is normally very small relative to the flux into S-adenosylmethionine and that the demand for the latter compound may change very markedly under different growth conditions make it plausible that such overshoot may occur unless the rate of synthesis of S-adenosylmethionine is regulated with exquisite precision. The metabolic cost of this apparent safeguard is the consumption of ATP. This S-methylmethionine cycle may well function in plants other than Lemna, but further substantiating evidence is neeeded.     

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.     

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)     

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.     

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; )     

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.     

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)     

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.     

Adaptation of Lemna paucicostata to Sublethal Methionine Deprivation

During initial exposure to 40 nanomolar propargylglycine (PAG), Lemna paucicostata colonies undergo abnormal fragmentation and a lag in frond emergence, most severe at 24 to 48 hours. Thereafter, frond emergence resumes and the frond/colony ratio rises. Such `adapted' plants withstand subculture into the same concentration of PAG without fragmentation or decreases in frond emergence, and display enhanced tolerance to higher concentrations. Adaptation is not dependent upon outgrowth of a few preexisting especially tolerant plants. Exogenous methionine prevents these events and overcomes the PAG-induced lag in frond emergence even after it is underway. These changes in frond emergence are not reflected in the rates of protein and wet weight accumulation which decrease by about 25% during the first 24 hours and continue unchanged thereafter. Cystathionine γ-synthase activity rapidly decreases to 9% of control during the first 12 hours of exposure to 40 nanomolar PAG but thereafter climbs to 12% of control. Studies of the uptake and internal concentration of PAG during these events are reported.

Exposure to a combination of 36 micromolar lysine plus 3 micromolar threonine is an alternative means to bring about sublethal methionine deprivation. Thus exposed, Lemna undergoes an analogous sequence of effects on morphology and growth which are preventable by exogenous methionine and which lead to an adapted state. Cystathionine γ-synthase specific activity in plants adapted to 36 micromolar lysine plus 3 micromolar threonine is 1.8 times control. However, addition of PAG showed that under these conditions enzyme activity can be decreased to as little as 54% of control without affecting the growth rate. Together these results suggest that adaptation is related to methionine limitation and that the plants adjust, in part, by increasing the steady-state concentrations of cystathionine γ-synthase and other enzymes in the methionine pathway.

     

Flowering Induced by Nitrogen Deficiency in Lemna paucicostata 151

Lemna paucicostata 151 is a weakly responsive short-day plantwhen grown in non-aseptic ten-fold diluted E medium supplementedwith 1 µM 6-benzyladenine, but it flowered even underlong-day conditions (continuous light) when grown in this mediumfor more than 14 days. On the 14th day of culture, the levelof endogenous nitrogen in the plants decreased to about 60%of that in the plants inoculated at the start of the culture.The flowering obtained under long-day conditions was suppressedby raising the concentration of nitrogen in the medium, whileit was induced more rapidly by lowering the concentration ofnitrogen in the medium. Benzyladenine did not cause floweringby itself, but it was required for the flowering that was inducedby a reduction in the level of nitrogen in the medium. Thus,the flowering observed under long-day conditions is due to nitrogendeficiency in the plants. Two inhibitors of proteases, bestatin and elastatinal, clearlyinhibited the flowering induced by nitrogen deficiency. It appears,therefore, that the induction or activation of some protease(s)is involved in the flowering that is induced by nitrogen deficiency. (Received March 19, 1991; Accepted August 16, 1991)     

Flower Induction by Nitrogen Deficiency in Lemna paucicostata 6746

Lemna paucicostata 6746, a short-day plant, produced flowerbuds even under continuous light when cultured in nitrogen-deficientmodified Hoagland medium with 1% sucrose for 3 days or morefollowed by culture on nitrogen-rich medium (either nitrateor ammonium). Flowering was also induced by culture on mediumcontaining 20–100 µM nitrate as the sole nitrogensource for 10 days or more, but not on medium with a low ammoniumconcentration. However, if plants cultured on medium containing5–20 µM ammonium as the sole nitrogen source for10 days were grown in a nitrogen-rich medium for a further 4days, they produced flower buds. Thus, nitrogen deficiency caninduce day length-independent flowering in Lemna paucicoslata6746, but nitrogen is required for the manifestation of flowering. (Received January 31, 1986; Accepted April 24, 1986)     

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 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)