Role of Salicylic Acid and Benzoic Acid in Flowering of a Photoperiod-Insensitive Strain, Lemna paucicostata LP6

Lemna paucicostata LP6 does not normally flower when grown on basal Bonner-Devirian medium, but substantial flowering is obtained when 10 μm salicylic acid (SA) or benzoic acid is added to the medium. Benzoic acid is somewhat more effective than SA, and the threshold level of both SA and benzoic acid required for flower initiation is reduced as the pH of the medium is lowered to 4.0. SA- or benzoic acid-induced flowering is enhanced in the simultaneous presence of 6-benzylaminopurine (BAP), although BAP per se does not influence flowering in strain LP6. Continuous presence of SA or benzoic acid in the culture medium is essential to obtain maximal flowering. A short-term treatment of the plants (for first 24 h) with 10 μm SA or benzoic acid, followed by culture in the basal medium containing 1 μm BAP can, however, stimulate profuse flowering. Benzoic acid is more effective than SA, and the effect is more pronounced at pH 4 than at 5.5. Thus, under these conditions, flowering is of an inductive nature. Experiments with [¹⁴C]SA and [¹⁴C]benzoic acid have provided evidence that at pH 4 there is relatively more uptake of benzoic acid than SA, thus leading to an increased flowering response. The data obtained from the experiments designed to study the mobility of [¹⁴C]SA and [¹⁴C]-benzoic acid from mother to daughter fronds indicate that there is virtually no mobility of SA or benzoic acid between fronds.     

Floral Induction in a Photoperiodically Neutral Duckweed, Lemna paucicostata Strain LP6: Role of Chelating Agents and Iron

Lemna paucicostata, strain LP₆, does not ordinarily flower underany photoperiodic schedule, when grown in the modified Bonner-Devirianmedium supplemented with 10^–4 M EDTA and 1% sucrose (thisis a medium which is otherwise satisfactory for short day inductionof flowering of other strains of this duckweed). However, when the ferric citrate concentration in the culturemedium containing EDTA was raised 10-fold over that in the normalmedium, a low but significant flowering could be initiated inthis duckweed, irrespective of the length of the photoperiod.A similar flowering response was obtained when ferric citratewas replaced by ferrous sulphate or ferric chloride, therebyindicating that the inductive effect of higher level of ferriccitrate on flowering in strain LP₆ is due to its iron component. Some flowering in this strain could be induced even in mediumcontaining normal level of iron, provided the level of EDTA,itself, was raised to 5?10^–4 (5% flowering) or to 10^–3M (12% flowering), but replacement of EDTA by EDDHA led to trulyremarkable effects. With EDDHA, flowering could be induced at very high levels (90%)under all photoperiodic regimes tried. Floral initiation couldbe obtained even with 10^–6 M EDDHA, though the optimallevel ranged from 5?10^–6 to 10^–5 M. (Received October 4, 1985; Accepted June 26, 1986)     

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)     

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)     

Floral Induction in Short-Day Lemna paucicostata 6746 by 8-Hydroxyquinoline, under Long Days

Lemna paucicostata 6746 is a short-day duckweed and flowersin response to a single photoinductive cycle. Its critical darkperiod requirement is ca. 10 h. Flowering in this duckweed couldbe induced by 8-hydroxyquinoline (8-HQ) under an otherwise non-inductivelong-day regime of 16 h light and 8 h darkness; the criticaldark period requirement for initiation of flowering was thusreduced by at least 2 h. However, 8-HQ was ineffective in initiatingflowering in strain 6746 under continuous illumination. Atomicabsorption analysis of the plant material revealed that thecontent of both iron and copper is markedly higher in the plantstreated with 8-HQ. A comparison of the effects of 8-HQ and thoseof EDTA and ethylenediamine-di(o-hydroxyphenylacetic acid),on flowering of strain 6746, has also been made. (Received August 23, 1983; Accepted October 18, 1983)     

Flowering and Endogenous Levels of Plant Hormones in Lemna Species

The occurrence and endogenous level of various plant hormoneswere measured for the short-day plants Lemna paucicostata 151and 381 and the long-day plant Lemna gibba G3 to determine whetherany of them are involved in the photoperiodic control of flowering.ABA, IAA, GA₁, GA₂₉, GA₃₄, GA₅₃, trans- and cis-zeatin, trans-and cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenine and N⁶-(²-isopentenyl)adenosine were definitely detected in each species, while GA₄was only detected in L. gibba G3 and GA₂₀ was only detectedin L. paucicostata 151. The endogenous levels of ABA and IAAwere in the range of 1–7 ng/g fr wt and were not significantlydifferent in vegetative and flowering plants. The endogenousgibberellin levels were generally higher in Lemna grown underlong-day rather than short-day conditions. The endogenous cytokininlevels were almost the same in both flowering and vegetativeplants of L. paucicostata 151 and 381. In L. gibba G3, however,the level of cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenineand N⁶-(²-sopentenyl) adenosine were higher in vegetative thanin flowering plants. These results indicate that there is not necessarily a directrelation between endogenous plant hormone levels and flowering,and that the chemical basis for the photoperiodic control offlowering cannot be explained solely by changes in hormone levels.The possibility remains, however, that one or more of the planthormones has some influence of secondary importance on the floweringprocess in Lemna. (Received January 29, 1986; Accepted July 12, 1986)     

Flowering and Endogenous Levels of Benzoic Acid in Lemna Species

The occurrence of benzoic acid, a flower-inducing factor inLemna species, in L. paucicostata strains 151, 381, 321 andL. gibba G3 was established by several purification steps andfinal use of gas liquid chromatography-selected ion monitoring.Quantitative analyses of benzoic acid were made in non-floweringand flowering Lemna to determine differences in levels. Theendogenous level of benzoic acid was shown to vary dependingon culture conditions, but no positive correlation could befound between the endogenous level of benzoic acid and floweringof Lemna. (Received October 6, 1982; Accepted December 23, 1982)     

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.     

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)     

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.     

Induction of Flowering in Lemna paucicostata by Dicoumarol and 4-Hydroxycoumarin

Dicoumarol, an antagonist of vitamin K, not only promoted theflower-inducing activity of vitamin K₅, but also induced floweringin Lemna paucicostata when added alone to the medium. The flower-inducingactivity of dicoumarol was comparable to that of benzoic acidand could be greatly intensified by simultaneous applicationof benzyladenine as was the case with benzoic acid. Warfarin,another antagonist of vitamin K, did not induce flowering. 4-Hydroxycoumarin, a component of dicoumarol, was also active,but coumarin and 7-hydroxycoumarin were not. Dicoumarol hadonly a slight flower-inducing activity for strains 441 and 6746under continuous light, but had a strong flower-promoting effectunder a near critical photoperiod. That is, the effect of dicoumarolwas daylength-dependent. (Received April 22, 1985; Accepted August 21, 1985)     

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)     

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-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)     

Effects of Exogenous Amino Acids on Iron Uptake in Relation to their Effects on Photoperiodic Flowering in Lemna paucicostata 6746

The flowering of Lemna paucicostata 6746 grown on 14-h photoperiodwas enhanced by the addition of high concentrations of ironto the medium, which also increased the endogenous iron concentration.The addition of asparagine, aspartate, glutamate, -alanine,glycine or serine to the medium also increased the endogenousiron level, resulting in the promotion of flowering. In contrast,the addition of cysteine, cystine, glutamine, arginine, threonineor phenylalanine lowered the endogenous iron level, resultingin the inhibition of flowering. Glycine and asparagine added to the medium during an inductive96-h dark period did not promote iron uptake and had no effecton flowering, but when added during the subsequent 120-h lightperiod, they promoted both iron uptake and flowering response.The increase in the endogenous iron level seems to favor floraldevelopment rather than induction of photoperiodic floweringof Lemna paucicostata 6746. (Received September 8, 1986; Accepted March 31, 1987)     

Phototactic Chloroplast Displacement in the Photosynthetic Mutant, Lemna paucicostata Strain 1073

The photosynthetic mutant, strain 1073, of Lemna paucicostataTorr. (L. perpusilla Hegelm.) which has a block in the electrontransport chain between plastoquinone and cytochrome f is capableof light-induced chloroplast displacement movements. At 8000–14000 lx, chloroplasts of the mutant move from their positionadjacent to the inner periclinal wall of the mesophyll cellsto the anticlinal walls, i.e. along those walls parallel tothe direction of the light. Light does not appear to enhancerespiration of the photosynthetic mutant or of the wild typestrain (6746). These and other results support the idea thatchloroplast displacement in light is not solely the result oflight effects on photosynthesis and respiration. Lemna paucicostata Torr., photosynthetic mutant, phototaxis, chloroplast displacement     

Inhibitory Action of Blue and Far-Red Light in the Flowering of Lemna paucicostata

In a new strain of short-day duckweed (Lemna paucicostata T-101), blue and far-red light-induced inhibition of flowering was investigated. Flowering of this strain failed to be induced under a short-day photoperiod of blue and far-red light, although it responded as a typical short-day plant in red and white light. When the short-day photoperiod of blue or far-red light was terminated by a 15 min red light pulse, flowering recovered completely. This inducing effect of red light was reversed by subsequent exposure to far-red light. Furthermore, it could be demonstrated that 30 min of blue light completely reversed the flowering inductive effect of 5 min red light and vice versa. Evidence is presented suggesting that the inhibitory action of blue and far red light may be due to the lowering of phytochrome P_fr levels below those required to start the dark reactions which lead to flowering. These results are discussed in relation to the time measurement system of photoperiodism.     

Conversion of Lysine to L-Pipecolic Acid Induces Flowering in Lemna paucicostata 151

The natural occurrence of L-pipecolic acid and conversion oflysine to L-pipecolic acid in Lemna paucicostata 151 were demonstrateddefinitively by GC-MS. The strong flower-inducing activity ofL-pipecolic acid has already been demonstrated. Thus, the presentstudy indicates that the effect of lysine on flowering is mediatedby L-pipecolic acid. (Received June 30, 1997; Accepted August 22, 1997)     

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)     

A Comparative Study on the Short-Day- and the Benzoic Acid-Induced Flowering in Lemna paucicostata

The addition of a high concentration of FeCl₃ to the medium(1/10 strength M medium) slightly inhibited the benzoic acid(BA)-induced flowering of Lemna paucicostata 151 in continuouslight, although it promoted the flowering induced by short-day(SD) conditions. SD treatment had no significant effect on BA-inducedflowering in the medium with a standard concentration of iron,in which this plant hardly responds to SD, but greatly promotedit in the medium containing iron at a high concentration, inwhich this plant clearly responds to SD. The effect of BA seemsto be independent of but additive to the photoperiodic stimulus. In photosensitive strains 6746 and 441, a low concentrationof BA slightly lengthened the critical photoperiod but had noflower-inducing effect under continuous light. Since an optimalconcentration of BA induced flowering even under continuouslight in these strains, this was considered to be due to photoperiod-independentpromotion of flowering rather than shortening of the time-measuringprocess in the photoperiodic reaction. (Received August 19, 1986; Accepted February 21, 1987)