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)     

Flower-Inducing Activity of Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Deficient Medium

Lemna paucicostata 151, a weakly responsive short-day plant,flowers even under continuous illumination when cultured onnitrogen-free medium for more than 72 hours with subsequentculture on nitrogen-rich medium. During the nitrogen-free culture,the protease activity and protein content of the plant increasedand decreased, respectively. The plant contained a protein(s)that induced flowering of the plant when added to the medium.The level of this protein(s) also decreased during the nitrogen-freeculture. The total amount of free amino acids in plants culturedon nitrogen-free medium for 96 hours decreased to about 15%of that in plants at the start of nitrogen-free culture, butlevels of some amino acids increased. These amino acids wereexamined for their effects on flowering of plants cultured onnitrogen-deficient or nitrogen-free medium. Most of the aminoacids had no effect on flowering. However, profuse floweringwas induced when lysine was added to the medium. Lysine promotedthe flower-inductive process(es) rather than the developmentof flower buds. These results suggest that nitrogen deficiency-induced floweringof the plants is induced by lysine, which is generated froma specific protein(s) by proteolysis. (Received May 11, 1992; Accepted July 30, 1992)     

Inhibition of Flower Induction by Some Inhibitors of Protease in the Short-day Plant Lemna paucicostata 6746 and the Long-day Plant Lemna gibba G3

Four inhibitors of proteases, namely, bestatin, diisopropylfluorophosphate, elastatinal and p-toluenesulfonyl-L-lysinechloromethyl ketone hydrochloride, were examined for their effectson flowering of a short-day plant Lemna paucicostata 6746 anda long-day plant Lemna gibba G3. Each of the inhibitors greatlyinhibited the flowering of Lemna paucicostata 6746 that is normallyinduced by nitrogen deficiency. Bestatin or elastatinal givenonly during the first half of the culture period inhibited theflowering more clearly than when each was given during the latterhalf, suggesting that they inhibited the inductive process(es)involved in flowering rather than development of flower buds.Bestatin or elastatinal greatly inhibited the flowering of Lemnapaucicostata 6746 induced by photoperiodic stimulus, ferricyanideand continuous far-red light. Simultaneous application of thesetwo inhibitors was more effective in the inhibition of photoperiodicallyinduced and ferricyanide-induced flowering than was each inhibitoralone. They also completely inhibited the photoperiodic floweringof Lemna gibba G3. These results suggest that the inductionor activation of some proteases, probably followed by the degradationof some protein(s), is necessary for the induction of floweringin both these plants. (Received November 21, 1989; Accepted February 19, 1990)     

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-Inducing Activity of Exogenous Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Rich Medium

Flower-inducing activity of lysine was examined in Lemna paucicostata151, a weakly responsive short-day plant, cultured on nitrogen-richmedium under long-day conditions (continuous light). Lemna paucicostata151 was homogenized in a solution of lysine and the homogenatewas centrifuged. The supernatant (lysine-containing extract)was added to nitrogen-rich medium after passage through a membranefilter to give various concentrations of lysine in the medium.Flowering was induced in plants grown for six days on mediumthat contained lysine at concentrations above 0.25 µM.In plants grown on medium that contained 1 µM lysine,a significant flowering response was observed on the fourthday of culture. However, the flower-inducing activity of lysinedisappeared when the lysine-containing extract was added tothe medium and the medium was then autoclaved, suggesting thatthe active principle is unstable to autoclaving. Among derivativesof lysine tested, lysine hydroxamate had the highest flower-inducingactivity and lysyl lysine had almost same activity as that oflysine. When added to the medium without homogenization withplant material, lysine and lysyl lysine had flower-inducingactivity but lysine hydroxamate did not induce flowering. (Received April 26, 1993; Accepted November 8, 1993)     

Regulation of the formation of protease inBacillus megaterium

Protease is synthesized by the cultures growing in a glucose-containing mineral medium. However, it is formed even during incubation of the washed cells in a nitrogen free medium. The enzyme synthesis is decreased substantially by the addition of the individual amino acids or their mixture. Threonine, isoleucine, leucine and valine are the most inhibitory. Arginine, cysteine, glycine, lysine and tryptophan in concentrations of 10³ m do not inhibit the production of protease. The growth of the culture is also somewhat inhibited by threonine and isoleucine, the repression of protease being, however, much higher. Concentrations of 10³ m inhibit its synthesis by 80–90%. However, the enzyme activity is not influenced. The inhibition is caused byl,-isomers. Repression of the enzyme synthesis after the addition of threonine into the medium is much greater in a growing culture than in a culture starving in a nitrogen-free medium. However the level of free threonine in the pool is roughly the same in both growing and non-growing cultures. A mixture of 13 amino acids, which themselves are little inhibitory, suppresses the synthesis of protease much more than threonine or isoleucine. The inhibitory effect of the individual amino acids on the enzyme formation is apparently additive.     

Regulation of the formation of protease byBacillus megaterium

The formation of protease takes place in washed cells ofBacillus megaterium incubated in a nitrogen-free medium. The rate of enzyme synthesis is decreased much less than that of cell proteins as compared with growing cells. The synthesis of protease in a nitrogen-free medium requires the presence of glucose. The omission of glucose results in stopping of the enzyme formation and substantial decrease of the rate of protein synthesis. Protease is not synthesized when the washed cells are incubated in a phosphate, free medium. The incubation of the cells in a nitrogen-free medium results in a decrease of the concentration of amino acids in the pool. In a phosphate-free medium the content of free amino acids increases temporarily and decreases again later. When the culture grown in the medium containing threonine or threonine and isoleucine in addition to NH₄ ions is transferred into the medium without amino acids, no protease formation is found during derepression of enzymes synthesizing both amino acids. The cells grown in a medium containing casamino acids begin to form the enzyme after a short lag period when transferred into the medium containing NH₄ as a sole nitrogen source or into a nitrogen-free medium.     

Inhibition of Flowering in the Long-Day Plant Lemna gibba G3 by Hutner's Medium and its Reversal by Medium Modification

The long-day plant Lemna gibba G3 flowers normally in E medium(Hoagland-type medium plus 30 µM EDTA) but in 0.5 H mediumthere is no flowering. Ammonium is present in 0.5 H medium andis known to inhibit flowering in L. gibba G3, but even in NH₄⁺-free0.5 H medium there is virtually no flowering under continuouslight. Increasing the phosphate concentration of the NH₄⁺-free0.5 H medium from 1.15 ITIM to 12 or 16 mM results in substantialflowering. Decreasing the EDTA concentration from 850 µIMto 250 µM, or raising the nitrate concentration from 4mM to 12 mM, results in only a small increase in flowering.If the decrease in EDTA and increase in nitrate are combinedwith the increase in phosphate, however, the flowering responseis nearly as good as that obtained using E medium. Thus, withthese three changes the inhibitory effect of NH₄⁺free 0.5 Hmedium for flowering in L. gibba G3 is almost completely reversed In the above studies flowering was not limited by daylength.When plants were grown on E medium under an 11 hour daylengthwhere flowering is limited by daylength, decreasing the phosphateconcentration in the medium reduced flowering, but increasingthe phosphate concentration in the medium did not stimulateflowering. Thus, when flowering is limited by daylength, highphosphate will not cause flowering, but a certain level of phosphateappears to be necessary for the expression of photoinductionunder long days. (Received January 14, 1986; Accepted June 24, 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)     

Flower Induction by Suppression of Nitrate Assimilation in Lemna paucicostata 6746

In vitro activity of nitrate reductase was studied in Lemnapaucicostata 6746 grown on modified Hoagland medium supplementedwith 1% sucrose, containing various inhibitors. Copper, silver,tungstate or cyanide which induces daylength-independent flowering,inhibited the nitrate reductase activity, but azide which doesnot induce daylength-independent flowering did not. Molybdate-deficientmedium induced flowering, and inhibited nitrate reductase activity.Lowering of nitrate level of the medium also induced daylength-independentflowering. These results suggest that the suppression of nitrate assimilationcauses daylength independent flowering in Lemna paucicostata6746, and that one of the flower-inducing actions of the copper,silver, tungstate, cyanide or the deletion of molybdate is tosuppress the nitrate assimilation. (Received June 26, 1985; Accepted October 30, 1985)     

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)     

Effects of IAA, Zeatin, Ammonium Nitrate and Sucrose on the Initiation and Development of Floral Buds in Torenia Stem Segments Cultured In Vitro

In Torenia stem segments cultured on a defined medium from whichammonium nitrate and growth regulators were omitted, adventitiousbuds were readily formed from epidermal tissue, with subsequentdifferentiation of floral buds. Using this plant material, thecorrelation between the time of application of various chemicalsand the time-course of floral bud differentiation was investigated.Histological examination showed that adventitious buds werevegetative during the first two weeks of the culture, and floralprimordia appeared after about three to four weeks of culture.We divided the flowering process in Torenia stem segments intothe following 3 phases: the first phase (first 2 weeks) duringwhich adventitious buds are formed, the second phase (3rd and4th weeks) during which floral buds are initiated and the thirdphase (5th to 12th weeks) during which floral buds develop.Then we added IAA, zeatin, ammonium nitrate or a high concentrationof sucrose to the medium during one, two or three of these phases.Ammonium nitrate added during the third phase suppressed floralbud development, but the high concentration of sucrose givenduring this phase stimulated it. These two chemicals influencedonly the development of floral buds previously initiated. Theapplication of IAA during the first phase promoted both theinitiation and development of floral buds. However, its applicationafter 2 weeks of culture failed to promote floral bud formation.Zeatin inhibited floral bud formation in a manner similar toammonium nitrate, but if it was added to the medium only duringthe first phase, it slightly promoted the initiation and developmentof floral buds. (Received July 7, 1981; Accepted October 12, 1981)     

Sucrose and Hexose Release by Excised Stem Segments of Vicia faba L.: The Sucrose-Specific Stimulating Influence of Cuscuta on Sugar Release and the Activity of Acid Invertase

By washing out solutes in 0.5 mM CaSO⁴at 25 °C during aperiod of 5–6 h, the release of sugars by excised stemsegments of Vicia faba L. was measured. The stem parasite Cuscutaeuropaea strongly stimulated the release of sucrose into theefflux medium; this effect was most marked during the last hoursof each experiment but this stimulating effect of the parasitecould not be detected for glucose and fructose. The fact thatparasitized stem segments released higher than normal hexoseamounts during the last hours of several experiments, couldbe explained as the result of extracellular hydrolysis of sucroseby free space invertase. A high free space acid invertase activitywas present in young stem segments of Vicia faba and in tissuesof Cuscuta. The stimulating influence of Cuscuta on sugar releaseby cells of stem segments appears sucrose-specific, supportingthe idea that the stimulating influence of Cuscuta on sugarrelease is restricted to the sieve-tube system. When metabolic inhibitors were added to the washing solutionor when segments were incubated at low temperature, no cleareffect of the parasite could be observed and for all segments(parasitized and non-parasitized) a strongly enhanced releaseof sucrose into the efflux medium was found during the lasthours of an experiment. These data support the idea that anintensive resorption of sucrose occurs within stem segments,after its release into the apoplast. Key words: Cuscuta europaea, Parasitic relationship, Phloem unloading     

Flowering of the long-day plant, Lemna gibba, under short-day schedules composed of red and far-red light

Flowering in Lemna gibba, a long-day duckweed, can be inducedunder a short-day condition when the photoperiodic regimes areR₇FR₃ (7 hr red followed by 3 hr far-red), R₅FR₅ and R₃FR₇.This indicates the necessity of a proper balance between redand far-red effects for flowering. The flowering induced bythese regimes is inhibited by a brief exposure to red givenat the start of darkness and this inhibition is reversed bysubsequent exposure to far-red. Thus, the red/far-red reversibleeffect is found only at the beginning of darkness for floweringof L. gibba. However, flowering of L. gibba is promoted by a red light breakgiven near the middle of a 14 hr dark period. The promotiveeffect is not reversed by subsequent exposure to far-red, i.e.,the effect of the red break converts from inhibition to promotionas when given later in the dark period, which suggests the involvementof a timing mechanism. (Received July 21, 1973; )     

Reduction of active elastase concentration by means of immobilized inhibitors: a novel therapeutic approach

The inhibitory power of three different active Nylon membranes, separately loaded with three different protease inhibitors, was studied with the aim of reducing the increased elastase concentration occurring during hemodialysis or extracorporeal blood circulation in patients undergoing cardiopulmonary bypass. Chemical grafting was carried out to make the inert Nylon membrane suitable for the immobilization of the inhibitors. The behavior of immobilized alpha(1)-antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), or elastatinal was separately studied. alpha(1)-Antitrypsin and BPTI were covalently immobilized by means of a diazotization process, whereas elastatinal was covalently attached via a condensation process mediated by glutaraldehyde. The inhibitory power of each membrane type was studied as a function of the amount of immobilized inhibitor and temperature. All active membranes have shown good inhibitory power. The most efficient membrane was that loaded with alpha(1)-antitrypsin, the less efficient that with BPTI.     

Effects of antimetabolites on flowering in Arabidopsis

Effects of five kinds of antimetabolites on flowering in Arabidopsiswere studied. 8-Azaguanine, acriflavine, ethidium bromide andstreptomycin accelerated flowering but cycloheximide did not.The accelerating effect of 8-azaguanine was reversed by guanine. (Received April 30, 1971; )     

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)     

Flowering Response in Relation to C and N Contents of Pharbitis nil Plants Cultured in Nitrogen-Poor Media

Flowering of seedlings of Pharbitis nil, strains Violet andTendan, cultured in modified White's medium, was promoted bymedium dilution, the critical dark period being shortened byabout 15 min. Dilution of the N source alone was enough to causethe medium-dilution effect. Dilution of the culture medium duringthe day before and on the day of exposure to the dark-period(a total of two days) caused the maximum dilution effect. TheC and N contents of the cotyledons and of the shoot apices changedrapidly in response to medium dilution. In 1/2-strength White'smedium with 1/1,000 strength NO₃^– which was most effectivefor flower promotion, the C-N ratio was highest. In 1/2-strengthmodified White's medium, in which flowering was lowest withthe longest critical dark period, the C-N ratio was lowest.Thus, there is a close relation between flowering response andthe C-N ratio in cotyledons or shoot apices of Pharbitis nil. (Received September 14, 1984; Accepted January 26, 1985)     

Long-Day Flowering of Perilla Plants Cultured in Nitrogen-Poor Media

Green-leaved and red-leaved Perilla plants (short-day plants)cultured aseptically on diluted modified White's medium or onfull strength White's medium (W) containing reduced concentrationsof nitrogen sources, initiated flower buds under continuouslight (2,000–2,200 lux fluorescent lamps) at 24–26?C.The addition of sucrose to the medium promoted flower formation;the optimum concentration was 2% in 1/10?W medium. The plantscultured on unfertilized vermiculite also developed flower budsreadily, unlike those on fertilized vermiculite. High-intensity light (8,000 lux fluorescent lamps) given duringthe first 30 days of culture promoted flowering. This effectwas also produced to a lesser degree by the addition of sucroseto the medium, instead. On the other hand, high-intensity lightgiven during the second 30 days or throughout the culture periodinhibited flowering, irrespective of the presence of sucrosein the medium. (Received February 4, 1982; Accepted June 14, 1982)     

Gametogesis in Chlamydomonas I. Effect of light on the induction of sexuality

When each mating strain of Chlamydomonas sp. was incubated ina nitrogen-free medium, its sexuality was separately inducedby light. Studies were made on the mechanism of this inductionwith the following results. (1) At 20?C, both mating strainsrequired light for the induction; no sexuality being observedin the dark. (2) DCMU did not appreciably inhibit inductionand aeration with CO²-free air stimulated it. Thus, this inductionis not related to the process of photosynthesis. (3) At 4?C,incubation in the dark in a nitrogen-free medium for 24 hr inducedsexuality. Prolonged incubation at a low temperature in thedark can replace the effect of light physiologically. (Received March 7, 1973; )