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)     

Ferricyanide Induces Flowering by Suppression of Nitrate Assimilation in Lemna paucicostata 6746

Lemna paucicostata 6746, a short-day plant, produced flowerbuds even under continuous light when cultured for 3 days inferricyanide containing ammonium-free medium followed by cultureon nitrogen-rich medium (either nitrate or ammonium). Dailytreatment with ferricyanide in the absence of ammonium for morethan 8 hours, which completely inhibited nitrate reductase activitywithin 6 hours after the addition to the medium, induced daylength-independentflowering even when the ammonium-rich medium was given duringthe remaining hours. The presence of ammonium for 1 hour atthe middle of the 14-h ferricyanide treatment almost completelysuppressed floral induction. (Received March 6, 1986; Accepted June 3, 1986)     

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)     

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.     

Promotion of Flowering in Lemna paucicostata 6746 (a Short-Day Duckweed) by Cytokinins

Lemna paucicostata 6746 is a short-day plant and flowers inresponse to a single photoinductive cycle. Flowering in thisduckweed could be promoted, under short days, in the presenceof a cytokinin in the nutrient medium. 6-Benzyladenine (BA)was the most effective for promotion of flowering, followedby zeatin and kinetin. Since EDTA itself is promotive for floweringin this plant, the cytokinin effect is best observed in mediumdevoid of EDTA. Nevertheless, in their combined presence floweringwas more as compared to when either of these was individuallypresent. This additive effect on flowering was especially prominentwhen the plants were kept in near-critical photoperiods. Besidesthe increase in percentage of the flowering fronds, floweringis also sustained a little longer in the cytokinin-treated plants.BA, however, did not alter the critical dark period requirement,either in the presence or absence of EDTA. (Received March 26, 1983; Accepted May 6, 1983)     

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 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)     

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 Inhibition in Lemna paucicostata 6746 Due to Night Interruption

The floral response to various 24-h photoperiodic cycles ofthe short-day plant, Lemna paucicostata 6746 was investigated.No day that had a main photoperiod longer than about 14 h wasable to induce flowers, evidence that the critical day lengthwas ca.14 h. Flowering in the 12-, 9- or 6-h day was inhibitedcompletely by a light pulse inserted daily in the ‘inhibitionzone’ that ranged from about 14 h after the precedingdawn to about 14 h before the next dusk. In the 3- and 1-h days,only the pulse applied 14 h after the dawn completely inhibitedflowering. These results suggest that the daily night interruption prohibitedflowering only when it was linked to either the preceding orthe subsequent main photoperiod to form a skeleton photoperiodwhose length was equal to, or longer than, the critical daylength. Analysis of the floral response to skeleton schedules11:13 and 13:11 on Pittendrigh's model of the photoperiodicclock indicated that light-on circadian oscillation probablyis involved in the day length measurement. ¹ Dedicated to the memory of Dr. Joji Ashida. (Received July 13, 1982; Accepted January 17, 1983)     

pH Dependence of the Copper Effect on Flowering, Growth and Chlorophyll Content in Lemna paucicostata 6746

The short-day plant Lemna paucicostata 6746 took up the sameamount of copper from the medium whether the pH of the mediumwas 4.1 or 5.1. At pH 4.1, an addition of copper to the mediumresulted in an unchanged chlorophyll content, a somewhat reducedgrowth rate and a substantial induction of long-day flowering.By contrast, at pH 5.1 the same copper concentration causeda reduction in the chlorophyll content and strong inhibitionof growth, but it did not induce any long-day flowering. (Received June 14, 1982; Accepted October 14, 1982)     

Influence of Ammonium on the Ability of Salicylic Acid to Induce Flowering in the Short-Day Plant Lemna paucicostata 6746

When the short-day plant Lemna paucicostata 6746 is grown inhalf-strength Hutner's medium, which contains 1.25 mM NH4NO3,salicylic acid does not induce flowering on daylengths of 16hr or longer. By contrast, in M (Hoagland-type medium), Pirson-Seidelor ammonium-free half-strength Hutner's media, none of whichcontain ammonium, salicylic acid is able to induce some floweringeven under continuous light. Neverthless, in each of these threemedia the effect of salicylic acid is strongly daylength dependentbecause there is a sharp drop in the flowering response to salicylicacid between the 12 and 16 hr daylengths, and the floweringresponse is nearly constant from the 16 hr daylength to continuouslight. Ammonium has the opposite effect and at 50 to 75 µMis able to overcome the salicylic acid effect completely andprevent any flowering on daylength of 16 hr or longer. (Received December 3, 1980; Accepted March 5, 1981)     

Effect of Ferricyanide, Ferrocyanide and KCN on Growth and Flowering in the Short-Day Plant Lemna paucicostata 6746

Flowering in the short-day plant Lemna paucicostata 6746 canbe induced under continuous light by the addition of ferricyanie,ferrocyanide or KCN to M-sucrose medium. Each substance is nearly10 times more effective when the flasks are covered by glassbeakers than when cotton plugs are used. By contrast, when floweringis induced under continuous light by copper or by short-daytreatment, neither flowering nor growth are affected by whetherglass beakers or cotton plugs are used. Ferricyanide, ferrocyanideand KCN are also able to induce long-day flowering when theplants are grown on Msucrose medium in small beakers that areplaced in a covered storage dish that also contains a solutionof one of these compounds. Addition of a KOH trap to the storagedish completely blocks the flowering induced by these compounds.If [¹⁴C]ferrocyanide is added to the storage dish both the M-sucrosemedium and the plants contain significant amounts of radioactivity,the amount of radioactivity being proportional to the floweringresponse. These results indicate that ferricyanide, ferrocyanideand KCN break down to release HCN and that it is the HCN whichis responsible for inducing flowering in L. paucicostata 6746under continuous light. ¹Present address: Department of Biology, Osaka Kyoiku University,Ikeda, Osaka 563, Japan. ²Present address: Institute of Horticulture, The Volcani Center,P. O. B. 6, Bet-Dagan, Israel. (Received January 17, 1983; Accepted March 24, 1983)     

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)     

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 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)     

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)     

Flower-promoting effect of some amino acids and amides in Lemna paucicostata 6746

Cultures of Lemna paucicostata 6746 were exposed to a single96-hr dark period followed by continuous illumination at 24?1?C.Flowering percentage increased to a maximum 3 days after theend of the dark period and then fell off to 0% on the 5th day.Among 20 amino acids and 2 amides tested, addition of asparagine,aspartate, glutamate, -alanine, glycine and serine clearly increasedthe flowering percentages and retarded the regression of floralbuds by 2–3 days. These substances given after the endof the long dark period were more effective than those givenduring the dark period, suggesting that they favored the flower-producingprocess following the inductive dark process. On the other hand,if the above amino acids or amide were applied under repeatedlight-dark cycles, they shortened the critical dark period by1–2 hr and almost completely nullified the light-breakeffect. They seem to promote the flower-inductive dark process,too. Glutamate, for instance, was effective even at 5 µM, whilethis amino acid is found in the plant body in large quantities.The mechanism of flower promotion by these amino acids and amideremains unknown. (Received June 3, 1976; )     

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; )     

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)     

Flowering response of Lemna perpusilla 6746 to a single dark period

Lemna perpusilla 6746 is induced to flower by a single longdark period, but the floral buds once formed disappear afterseveral days under 5000 lux/25?C. Such regression of floralbuds is prevented by lowering the light intensity or temperature,but if the light intensity and/or temperature are lowered beyondcritical levels, new floral buds form. If the cultures are subjectedto 100 lux/20?C, neither regression nor new formation of floralbuds occurs. Under such conditions, the number of floral frondsreaches maximum about 6 days after the inductive dark periodand reamins unchanged for at least 10 days, while the percentageof floral fronds rapidly decreases thereafter, owing to thedilution by newly developed vegetative fronds. When the cultures are subjected to various lengths of a singledark period (25?C) followed by 100 lux/20?C, flowering responsesrepresented by the number of floral fronds per flask show rhythmicfluctuation with a cycle length of about 24 hr. Similar rhythmicresponse is observed when a brief light interruption is givenat different times during a single long dark period. (Received December 2, 1974; )