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.     

Physiological Structure of the Critical Photoperiod of Lemna paucicostata 6746

The critical day length or the length of the critical photoperiodfor the short-day duckweed, Lemna paucicostata 6746 is about14 h (Oota 1983). With the min-SD method, I found that not thewhole critical photoperiod but only its initial and terminalbrief fractions, called respectively the LI- and L2-phases,need be illuminated for a given day to be a noninductive day.Inversely, the darkened LI- and/or L2-phase makes the day inductive.The rest of the day can be either darkened or illuminated withoutmodifying the inductive or noninductive property of the day. Thus, the physiological structure of the critical photoperiodfor L. paucicostata 6746 closely resembles that of the criticalphotoperiod for the long-day duckweed, L. gibba G3 (Oota 1981). (Received May 24, 1983; Accepted September 21, 1983)     

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.     

Cystathionine Synthesis in Yeast: an Alternative Pathway for Homocysteine Biosynthesis

Cystathionine synthesis from O-acetylhomoserine and cysteine has been demonstrated in yeast extracts for the first time. The activity is less than that of O-acetylhomoserine sulfhydrylase, but it is higher than that reported for homoserine O-transacetylase and therefore should not be growth limiting. Cystathionine synthase seems to share the regulatory properties of the sulfhydrylase, and both activities are missing from the methionine auxotroph Saccharomyces cerevisiae EY9, suggesting that both reactions are catalyzed by the same enzyme. However, cystathionine synthase activity was lost during purification of the sulfhydrylase, suggesting that the two reactions may be catalyzed by separate enzymes. Since previous studies have shown that yeast extracts can catalyze the cleavage of cystathionine to homocysteine, our results show the existence of two complete alternate pathways for homocysteine biosynthesis in yeast. Which of these is the major physiological pathway remains to be determined.     

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; )     

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 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)     

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.     

Physiological Function of Night Interruption in Lemna paucicostata 6746: Action of Light as a Phaser on the Photoperiodic Clock

Flowering of Lemna paucicostata 6746 under short-day conditionsis completely inhibited by daily night interruption given tothe "inhibition zone" that starts at Zeitgeber time (ZT) 14,i.e., 14 h after dawn, and ends 14 h before the next dusk [Oota(1983a) Plant & Cell Physiol. 24: 327]. With a modifiedmin-SD method, most of these night interruptions were foundto signal the false dawn (false ZT 0) after one entraining cycle.Thus, on and after day 2 the interruption was associated withthe next main photoperiod to form a noninductive skeleton photoperiod.However, a light pulse applied at the start of the inhibitionzone, caused no phase shift in the photoperiodic clock, andformed a noninductive skeleton photoperiod in association withthe preceding main photoperiod. The complete floral inhibition due to the night interruptionwas ascribed to the illumination of both the LI-phase (realor false ZT 0) and L2-phase (real or false ZT 14), or the twolight-sensitive fractions of the original or shifted criticalphotoperiod, by the thus formed skeleton photoperiod, just aswas the case for the floral inhibition by complete photoperiodslonger than the critical daylength, 14 h [Oota (1983a), Oota(1983b) Plant & Cell Physiol. 24: 1503]. (Received October 20, 1983; Accepted January 7, 1984)     

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.     

Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata

The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the path-way for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: serine → ethanolamine → phosphoethanolamine → phosphatidylethanolamine. A possible alternative is: serine → phosphatidylserine → phosphatidylethanolamine → ethanolamine → phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO₂ from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives.     

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.     

Time-Measuring Processes Involved in the Photoperiodic Response of Lemna paucicostata 441

Lemna paucicostata 441 exposed to a single dark period of variouslengths showed a rhythmic flowering response with a 22- to 24-hperiod, even when the dark period was preceded by continuouslight. The critical night length (about 12 h) was scarcely influencedby pretreatment with 8D–4L (8 h of darkness followed by4 h of light), 8D–8L or 8D–12L. However, the rhythmof the response in the second cycle was markedly damped by thepretreatment with 8D–4L or 8D–12L, and was slightlyamplified by 8D–8L. The flowering response to a red-light interruption given atdifferent times in the inductive dark period also showed circadianrhythmicity even when the dark period was preceded by continuouslight, and this rhythmicity was scarcely influenced by a dark-lighttreatment given prior to the inductive dark period. A red-lightinterruption given at the 6th or 14th hour of the dark periodmarkedly shifted the phase of the rhythm of the response tothe length of the following dark period (the former delayedand the latter advanced), but that given at the same phase markedlyweakened and disturbed the rhythmicity of the response to ared-light interruption given in the following dark period. (Received March 21, 1992; Accepted June 12, 1992)     

植物体内甾醇的合成和生理作用

植物甾醇属于甾类化合物,以自由状态,与复杂脂类或和某些化合物结合成复合体的形式,存在于高等植物的细胞膜,细胞质和细胞器内,细胞膜内的甾醇和磷脂相互作用能够保持膜结构的稳定和调节细胞膜透性。植物体内甾醇的合成是从乙酰辅酶A（acetyl-CoA)开始,并有其种类特异性和特有的酶催化系统。甾醇参与植物对外界的胁迫反应,因此,获得甾醇突变体植株是抗病虫育种的有效途径。     

Importance of the Isovalerate Carboxylation Pathway of Leucine Biosynthesis in the Rumen

Certain anaerobic ruminal bacteria synthesize the leucine carbon skeleton by use of a pathway different from that described in other microorganisms. These organisms carboxylate the intact carbon skeleton of isovalerate, synthesizing leucine-2-C(14) from isovalerate-1-C(14). Strains of Bacteroides ruminicola and Peptostreptococcus elsdenii were like Ruminococcus flavefaciens in that they incorporated appreciable amounts of C(14) from isovalerate-1-C(14) into cellular protein and in that the only labeled amino acid found was leucine. The specific activity of beta-isopropylmalate dehydrogenase in extracts from R. flavefaciens and from the mixed bacterial population from the rumen was very low as compared with the specific activity of this enzyme in extracts from Escherichia coli. This suggests that the pathway of leucine biosynthesis that operates in many aerobic and facultative microorganisms is not the major pathway in rumen bacteria. This was supported by the finding that after fermentation of whole rumen contents with acetate-2-C(14), leucine from the bacterial cells had a specific activity lower than one would expect if acetate was incorporated directly into carbons 1 and 2 of leucine.     

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)     

Old-Culture Flowering of Lemna paucicostata 6746 in Aged Hutner's Medium

Lemna paucicostata 6746, a short-day plant, flowers in agedHutner's medium even under continuous light, when the endogenousnitrogen level decreases to below 1.6 µmg fr wt. At thesenitrogen levels, daylength-independent flowering of the plantcan be induced even in fresh Hutner's medium. Thus, old-cultureflowering in Hutner's medium is due to nitrogen deficiency inthe plants. ¹Present address: Biological Institute, Faculty of Science,Shizuoka University, Shizuoka 422, Japan. (Received February 12, 1987; Accepted August 28, 1987)     

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)     

Diurnal Fluctuations in the Activity of the Enzyme Nitrate Reductase in Lemna paucicostata

Diurnal fluctuations were observed in nitrate reducta.se activity in Lemna paucicostatu Hegelm, grown under 16-h photoperiod. The enzyme activity showed a single peak in the middle of the photo period, i.e. 8 h after the onset of light. The activity decreased during the latter half of the photoperiod and reached a minimum level in the middle of the dark period. The peak in enzyme activity persists in continuous light as well as continuous dark, at least for a couple of days, suggesting the existence of endogenous rhythmicity