EXPERIMENTAL CONTROL OF FLOWERING IN LEMNA. III. A RELATIONSHIP BETWEEN MEDIUM COMPOSITION AND THE OPPOSITE PHOTOPERIODIC RESPONSES OF L. PERPUSILLA 6746 AND L. GIBBA G3

Hillman , William S. (Yale U., New Haven, Conn.) Experimental control of flowering in Lemna. III. A relationship between medium composition and the opposite photoperiodic responses of L. perpusilla 6746 and L. gibba G3. Amer. Jour. Bot. 48(5): 413–419. Illus. 1961.—In a simple Hoagland-type medium, L. perpusilla 6746 flowers, irrespective of daylength; L. gibba G3 does not flower under any daylength, as long as the medium is changed frequently. Ethylenediaminetetraacetic acid (EDTA) prevents the flowering of L. perpusilla in long days but not in short, and brings about the flowering of L. gibba in long days but not in short. The same results are obtained with medium “aged” by the growth in it of L. gibba for several weeks (in any photoperiod) as well as with tartaric acid. The effectiveness of both EDTA and “aged” medium is greater at pH levels near 5 than at 4 or below, probably reflecting action through metal-chelation. These results are most easily interpreted as effects on photoperiodic sensitivity, and suggest a central role of metals in photoperiodism.     

Comparison of the Ability of Salicylic Acid and Ferricyanide to Induce Flowering in the Long-day Plant, Lemna Gibba G3

Both salicylic acid and ferricyanide induce flowering in the long-day plant Lemna gibba L., strain G3 under 8- and 9-hour short days. In both cases the effect is daylength-dependent. Salicylic acid is ineffective on daylengths less than 8 hours and ferricyanide is ineffective on daylengths less than 5 hours. When both substances are given together a striking synergistic interaction is observed, and some flowering is obtained on daylengths as short as 3 hours. However, even with the optimal combinations the flower-inducing effect remains daylength-dependent.     

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)     

The Role of Benzoic Acid and Plant Hormones in Flowering of Lemna gibba G3

The level of benzoic acid was measured in Lemna gibba G3 grownon M and E media under inductive and non-inductive daylengths.Benzoic acid was slightly higher in plants grown on M mediumbut there was no difference in the benzoic acid levels in floweringand vegetative plants. When L. gibba G3 was grown under continuouslight on 1/10 M medium or 1/2 H medium there was virtually noflowering, but addition of benzoic acid to either medium ledto a substantial flowering response. In both cases this floweringresponse was inhibited by the plant hormones IAA, GA₃, ABA andzeatin, with IAA and GA₃ being the least inhibitory and ABAbeing the most inhibitory. This same pattern of inhibition wasseen when L. gibba G3 was grown on M medium under continuouslight, conditions that lead to photoinduction of flowering.These results leave open the possibility that endogenous benzoicacid may interact with other factors to influence the floweringresponse in L. gibba G3. (Received November 13, 1984; Accepted February 27, 1985)     

The Influence of Nicotinic Acid and Plant Hormones on Flowering in Lemna

Nicotinic acid induces flowering in Lemna paucicostata 151 and381 and Lemna gibba G3 when they are grown in one tenth-strengthM medium under continuous light. For L. paucicostata 151 and381, the simultaneous addition of IAA, GA₃ or ABA to the mediumleads to an inhibition of the flower-inducing effect of nicotinicacid, while zeatin leads to a further stimulation of floweringabove that obtained by nicotinic acid alone. By contrast, inL. gibba G3 all four plant hormones inhibit the nicotinic acid-inducedstimulation of flowering. The effect of nicotinic acid on flowering in all three plantsis strongly daylength dependent when the plants are grown inhalf-strength Hutner's medium. Thus, nicotinic acid causes floweringin L. gibba G3 on continuous light but not on 9L:15D or 10L:14Dregimes. In L. paucicostata 381 nicotinic acid has a small effecton 12L:12D regime, a large effect on a 13L:11D regime and noeffect with daylengths longer than 14 hours, and in L. paucicostata151 nicotinic acid is only effective on daylengths shorter thanabout 11 hours. However, in L. paucicostata 151 and 381 treatmentwith both nicotinic acid and zeatin results in flowering undercontinuous light on half-strength Hutner's medium. Nicotinic acid is present in different Lemna but its concentrationdoes not appear to be influenced by changes in daylength. Thus,flowering clearly cannot be controlled by nicotinic acid actingalone, but the results of this study indicate that nicotinicacid could interact with other factors, possibly including oneor more of the known plant hormones, to influence the floweringprocess in Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

Identification of the Flower-inducing Factor Isolated from Aphid Honeydew as being Salicylic Acid

Honeydew produced by the aphid Dactynotus ambrosiae when feeding on flowering or vegetative plants of the short day plant Xanthium strumarium contains an active substance capable of inducing flowering in the long day plant Lemna gibba G3. In the present study, this active material has been identified as salicylic acid through the use of gas-liquid chromatography and mass, infrared, and ultraviolet spectrometry. Authentic salicylic acid induces flowering in L. gibba G3 under strict short day conditions with an optimal response at about 5.6 μm. The possible significance of salicylic acid for the control of flowering in Xanthium or L. gibba G3 is discussed.     

Uptake and Metabolism of [C]Salicylic Acid in Lemna gibba G3

When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH₄⁺-free 0.5 H medium) there is virtually no flowering, but addition of 10 micromolar salicylic acid (SA) to the medium results in substantial flowering. Using this system, the uptake and metabolism of [¹⁴C]SA in L. gibba G3 has been examined. SA uptake is rapid and linear for at least the first 24 hours. After 30 minutes, nearly 90% of the radioactivity in the plants is present as free SA. Part of this is rapidly converted to one or more bound forms of SA that appear either in the acidic butanol fraction or in the aqueous residue, and after 12 hours an equilibrium is reached between the free and bound forms of SA. When plants receive SA for 6 days and then are switched to control medium, both the free and bound SA remain nearly constant for at least 5 days. However, there is virtually no transfer of SA from mother fronds to daughter fronds, indicating that the SA is apparently sequestered within the cell. Cell fractionation studies show that nearly 95% of the SA remains in the supernatant even after a 2-hour centrifugation at 300,000 g. Thus, it is unlikely that SA is confined within a specific organelle, but rather is probably secreted into the vacuole.     

Comparison of the flowering behavior of the long-day plant Lemna gibba G3 from different laboratories

In an effort to determine the cause for the wide discrepanciesin the level of flowering response reported for the long-dayplant Lemna gibba L., strain G3, cultures of L. gibba G3 wereobtained from the laboratories of W. S. Hillman (G3-H), R. Kandeler(G3-K), Y. Oota (G3-O) and and A. Pieterse (G3-P) and comparedto the L. gibba G3 (G3-C) from this laboratory. Under continuouslight all cultures gave FL% values of 77 or above, and on a9L:15D short-day treatment, all cultures were completely vegetative.However, on daylengths of 10 to 12 hr, small but statisticallysignificant differences were obtained for the different cultures.The critical daylength curves for G3-G, which showed the shortestcritical daylength, and G3-K, which showed the longest criticaldaylength, differed by approximately one hour. Salicylic acidtreatment caused flower promotion in each culture, but statisticallysignificant differences were obtained between some of the culturesin their response to salicylic acid. It is concluded that the large discrepancies in the floweringresponses of L. gibba G3 that have been reported are due primarilyto differences in culturing methods and counting proceduresin the different laboratories. However, the results also indicatethat there may be distinct cultures of L. gibba G3 that exhibitsmall physiological and/or genetic differences that would makeprecise quantitative comparison between different laboratoriesvery difficult. (Received January 23, 1979; )     

Influence of Giving Salicylic Acid for Different Time Periods on Flowering and Growth in the Long-Day Plant Lemna gibba G3

When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH₄⁺-free 0.5 H medium) growth is excellent, but flowering is severely inhibited and often is zero. Addition of 10 micromolar salicylic acid (SA) to NH₄⁺-free 0.5 H medium quickly reverses this inhibition and leads to optimal flowering. The SA treatment also leads to a considerable reduction in the growth rate and increase in frond gibbosity. Removal of SA from the medium quickly leads to an increase in the growth rate and a large decrease in flowering. Thus, for maximal effectiveness SA must be present in the medium for the entire experiment, and the effect of SA is clearly not inductive.     

Flowering Responses of the Long-day Plant Lemna gibba G3

Lemna gibba L., strain G3, exhibits a qualitative long-day flowering response with a critical daylength on a 24-hour cycle of about 10 hours. Evidence is presented that the onset of daughter frond formation in a given frond inhibits the activity of the flowering meristem. Consequently, flower induction can only occur in fronds smaller than about 0.05 to 0.07 mm long. Although a minimum of 1 long day seems to be sufficient to induce the formation of flower primordia, at least 6 long days are required to obtain mature flowers since long days are also required for the early stages of flower development. The critical night length on 24, 48 and 72-hour cycles is respectively 14, 16, and 18 to 22 hours. The close similarity between the critical night length for the different cycle lengths is explained in terms of an inhibitory effect of darkness both on flower initiation and flower development. A 10-hour dark period is more inhibitory to flowering on a 36-hour cycle than on 24, 48, 60 or 72-hour cycles. It is suggested that darkness inhibits flowering through the formation of a light-labile flower inhibitor which acts to inhibit the functioning of the flowering stimulus.     

EXPERIMENTAL CONTROL OF FLOWERING IN LEMNA. IV. INHIBITION OF PHOTOPERIODIC SENSITIVITY BY COPPER

Hillman , William S. (Brookhaven Nat'l. Lab., Upton, N. Y.) Experimental control of flowering in Lemna. IV. Inhibition of photoperiodic sensitivity by copper. Amer. Jour. Bot. 49(8): 892–897. Illus. 1962.—Lemna perpusilla 6746 and L. gibba G3 are both photoperiodi c in a purified Hoaglandtype medium; purification thus imitates the effects of high levels of chelating agents previously reported. On the addition of 0.5–10.0μM/liter cupric ion (optimum about 2.0μM/liter) L. perpusilla, instead of responding as a short-day plant, flowers in both long and short days, while L. gibba, formerly a long-day plant, loses its ability to flower. A similar, but less distinct, effect of Hg can be attributed to a “copper-sparing” action. No other ion tested, including Cd, Co, Cr, Mn, Ni, Pb or Zn, had a similar effect. Certain macronutrient ions, probably Ca and phosphate, modify but do not obscure the effects of copper. The action of copper can be interpreted as an inhibition of photoperiodic sensitivity, since it makes both plants respond as if under short days regardless of the actual conditions. Some possible mechanisms, including disturbance of the photoperiodic pigment system, are discussed.     

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)     

Effect of Low-intensity Red and Far-red Light and High-intensity White Light on the Flowering Response of the Long-day Plant Lemna gibba G3

The long-day plant Lemna gibba L., strain G3 exhibits a relatively low sensitivity to short, white-light interruptions given during the dark period of a short-day cycle. However, the plants are fairly sensitive to low-intensity red light treatments given during a 15-hour dark period on the third day of a 2LD-(9L:15D)-2LD-7SD schedule. Far-red light is almost as effective as red light, and attempts to reverse the red light response with subsequent far-red light treatments have not been successful. Blue light proved to be without effect. When plants were grown on a 48-hour cycle with 15 minutes of red light every 4 hours during the dark period, the critical daylength was reduced from about 32 hours to slightly less than 12 hours.

Continuous red light induced a fairly good flowering response. However, as little as 1 hour of white light each day gave a significant improvement in the flowering response over that of the continuous red light control. White light of 600 to 700 ft-c was more effective than white light of 60 to 70 ft-c. The white light was much more effective when divided into 2 equal exposures given 8 to 12 hours apart. These results suggest an increase in light sensitivity with regard to flower induction about 8 to 10 hours after the start of the light period.

     

Influence of day length and temperature on number of main stem leaves and time to flowering in lupin

A growth chamber experiment was carried out to investigate the influence of day length and temperature on the development of flowering in eight varieties of the three grain lupin species Lupinus albus (Wat and C3396), L. angustifolius (Gungurru, Polonez and W26) and L. luteus, (Juno, Radames and Teo). The plants were grown at two temperatures, 10°C and 18°C, in combination with five daylength regimes: 10, 14, 18, 24 h day at full light intensity and 10 h full light extended with 8 h low intensity light. Increased daylength decreased days from sowing to flowering in all varieties, but had little effect on thermal time to flowering in most varieties. However, C3396, W26 and Radames had a significantly longer thermal time to flowering at high, non‐vernalising temperature (18°C) at short daylengths. Low light intensity daylength extension did not significantly influence thermal time to flowering. For flower initiation, measured as number of leaves on the main stem three types of response were found. All varieties formed fewer leaves on the main stem at 10°C than at 18°C, although the two thermo‐neutral varieties of L. luteus, Juno and Teo, gave only a small response to temperature and daylength. In Polonez, Gungurru and Wat, low temperature decreased leaf number, but there was only a small response to changes in daylength. Three varieties, C3396, W26 and Radames, showed longer thermal time to flowering at 18°C with short daylengths. This could be explained by a greater number of main stem leaves formed at short daylength at non‐vernalising temperatures. Increased daylength decreased leaf number in these varieties, but never to a smaller number than for plants grown at 10°C. In these varieties, low intensity extension of the daylength had a similar (W26, Radames) or decreased (C3396) effect compared to full light extension. The hastening of time to flowering by long days could be separated into two effects: a high light energy effect hastened development by increasing the rate of leaf appearance in all varieties, while low light energy in thermo‐sensitive varieties was able to substitute for vernalisation by decreasing leaf number.     

Effect of Heat-Treated Noradrenaline on Flowering in Lemna

In a previous study, heat-treated noradrenaline induced flowering of the short-day plant Lemna paucicostata Hegelmaier 151. In the present study, we found that heat-treated noradrenaline also had flower-inducing activity in short-day L. paucicostata strains 441 and 6746 and in long-day L. gibba strain G3. The flower-inducing activity in these plants was enhanced by water homogenates of eggplant (Solanum melongena L.).     

Gibberellin and CCC Effects on Flowering and Growth in the Long-day Plant Lemna gibba G3

The application of gibberellic acid (GA₃) to the non-rosette long-day plant Lemna gibba G3 at concentrations from 0.1 to 100 mg/l did not induce flowering on short days and inhibited flowering on long days at concentrations of 1 mg/l and higher. On both short and long days GA₃ concentrations above 1 mg/l caused a decrease in frond size and fresh and dry weight, but an increase in the rate of frond production and thus an increase in the # VF (number of vegetative fronds). Identical results were obtained when gibberellin A₇ was used instead of GA₃.     

Investigations on the effects of metal ions and chelating agents on growth and flowering of Lemna gibba G 3

The effect of different chelating agents on growth and floweringof Lemna gibba G 3 was studied in M and HUTNER'S media. Theincorporation of EDDHA and Fe-EDDHA in the media resulted inprofuse flowering and gibbous character of the fronds. However,EDTA was relatively less effective. It was demonstrated thatthe metal which influenced flowering in L. gibba G 3 was mostlikely copper. (Received August 19, 1970; )     

Early effects of floral induction on cell division in the shoot apex of Silene

The changes in cell number, the relative proportions of interphase nuclei with different amounts of DNA, mitotic index and labelling index have been investigated in the shoot apex of Silene coeli-rosa L. (a long-day plant) during the first long day of photoinduction, and compared with the corresponding changes in plants in short days. 3 h after the start of induction the proportion of nuclei in the G2 phase of the cell cycle had increased, the mitotic index tended to be higher, and the labelling index was lower than in plants in short days. 8–9 h later the values for plants in the long day had become similar to those for plants in short days. No evidence was obtained for a synchronisation of cells in one phase of the cell cycle as a result of photoinduction. The results obtained were consistent with a temporary shortening of the cell cycle in the induced apices over the first long day which resulted in a greater increase in cell number by the end of the first day of photoinduction than in plants in short days.Abbreviations LD long day - SD short day     

FLOWERING OF DICHONDRA IN RESPONSE TO TEMPERATURE AND DAYLENGTH

Experiments using controlled environment facilities showed that flowering of Dichondra repens was promoted by chilling plants at 10 C or below. Optimum length of the chilling period was 5–6 weeks. Unchilled plants did not flower. The flowering stimulus resulting from chilling was destroyed by temperatures above 21 C. Rate of flowering was increased by short days during chilling, but short days could not substitute for the chilling requirement. Optimum daylength for flower initiation following chilling was approximately 14 hr and the optimum temperature was approximately 15 C. Flower buds developed in leaf axils of primary stems and laterals, but stem apices remained vegetative. When the chilling requirement was met flowering continued indefinitely as the plants grew.     

Flowering responses of Lemna perpusilla and L. gibba in relation to nitrate concentration in the culture medium

Flowering responses of Lemna perpusilla strain 6746, a short-dayplant, and L. gibba strain G3, a long-day plant, to nitrateconcentration in Hoagland's type medium with or without EDTA,were compared. Maximum flowering of L. perpusilla under SD occurredat higher nitrate concentrations than did colony proliferation.Even under CL, L. perpusilla grown at sub-optimal nitrate concentrationsfor colony proliferation, flowered irrespective of the presenceof EDTA which reduces flowering. Unlike L. perpusilla, L. gibba failed to flower under SD atany nitrate concentration whether or not EDTA was added. UnderCL, however, L. gibba flowered at almost any nitrate concentrationwith or without EDTA. Double optima for nitrate concentrationwas exhibited in the presence of EDTA; optimal concentrationfor colony proliferation came between the two optima for flowering. We concluded that the nitrogen level of the medium is importantin regulating flowering of duckweeds, and that the effect ofEDTA, if any, may primarily be on colony proliferation and onlysecondarily or antagonistically on flowering. ¹ Present address: Institute for Agricultural Research, TohokuUniversity, Sendai 980, Japan. (Received September 25, 1971; )