Induction of flowering by cytokinins in a short-day plant, Lemna paucicostata

Lemna paucicostata, normally a short-day plant, can be inducedto flower under long-day conditions by providing a cytokininin a medium containing a high level of ferric citrate (5 x 10^–4M).Interestingly, when a cytokinin and EDDHA are present togetherin the medium, flowering is induced even at low levels of iron(10^–5 and 5 x 10^–5M ferric citrate). However, inthe absence of a cytokinin, flowering takes place only undershort days. (Received September 30, 1968; )     

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)     

Growth and flowering of Lemna paucicostata II. Role of growth regulators

Lemna paucicostata HEGELM., a short-day plant, can be inducedto flower under long days by provision of cytokinins in themedium. Of several cytokinins tested, zeatin and 6-benzylaminopurineare the most potent. They are effective when added in a mediumcontaining excess of iron and thus can bring about the sameaffect as EDDHA. Interestingly, when both EDDHA and a cytokininare provided together, flowering occurs even in the medium containingnormal level of iron. Other cytokinins such as SD 8339, kinetinand 6-(,-dimethylallylamino)-purine are also effective in mediumcontaining a slightly higher level of ferric citrate. In contrastto cytokinins, indole-3-acetic acid and gibberellic acid arenot only ineffective by themselves, but even nullify the inductiveeffect of cytokinins on flowering. Growth retardants such asCCC and abscisic acid have been found to inhibit flowering athigh levels. (Received September 8, 1969; )     

Growth and flowering of Lemna paucicostata I. General aspects and role of chelating agents in flowering

Lemna paucicostata HEGELM. is normally a short-day plant andflowers only in the presence of a chelating agent (EDTA or EDDHA)in the medium. The plant can be induced to flower even by asingle long night treatment; the flowering percentage, however,increases with further inductive cycles. The length of the criticaldark period depends upon the chelating agent employed in themedium. It is between 10 and 12 hr in the medium containingEDTA and about 8 hr in the EDDHA-supplemented medium. Red lightinterruption in the middle of the dark period—even fora minute—is inhibitory for flowering. Attempts to identify the metal ion(s) chelated reveal that thechelating agents affect flowering by facilitating iron uptake.This is also supported by the fact that the requirement of achelating agent for flowering can be overcome with an excessof iron in the medium. Interestingly, provision of EDDHA andexcess of ferric citrate, together, can bring about floweringeven under long days. ¹Originally HEGELMAIER (1) designated L. paucicostata as a separatespecies; however, THOMPSON (2) and DAUBS (3) have treated itsynonymous to L. perpusilla. More recently, based on physiologicaland chemotaxonomic studies, the distinctiveness of L. paucicostatafrom L. perpusilla has been favoured (4, 5). (Received September 8, 1969; )     

Studies on the growth and flowering of a short-day plant,Wolffia microscopica

Summary As found earlier, supply of EDTA was obligatory for both flowering and satisfactory vegetative growth in Wolffia microscopica. It is now shown that the metal affecting growth and flowering is most probably iron. Omission of Fe but not of Cu, Zn, Mn and B from the medium markedly affects vegetative growth. There exists also a strong interaction between EDTA and Fe, one being largely inactive in the absence of the other. When Fe-EDDHA is substituted for Fe-citrate and EDTA in the medium, no great effect is seen in vegetative growth, but flowering takes place even under continuous light. Studies with ⁵⁹Fe show that, in the medium containing Fe-EDDHA, Fe uptake is stimulated several-fold; this is apparently associated with the flowering condition.Abbreviations EDTA ethylenediaminetetraacetic acid - EDDHA ethylenediamine-di-o-hydroxyphenylacetic acid     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

Specific interactions in the physiology of flowering and gibbosity of Lemna gibba G3

Fronds of Lemna gibba G3 became conspicuously gibbous when ethrel,an ethylenereleasing compound, was added to the nutrient medium.Maximal gibbosity was obtained at ethrel concentrations of 1µg/ml and higher. Unlike the chelating agent, EDDHA, whichcauses profuse flowering and markedly gibbous fronds under long-dayconditions, ethrel did not affect flowering. In the presenceof an optimal concentration of EDDHA (10 µ/ml), ethreleven significantly inhibited flowering and caused developmentof excessively gibbous fronds. Autoclaved gibberellic acid specifically negated the ethreleffect as it does that of EDDHA. Three decomposition productsof GA₃, allogibberic acid, epiallogibberic acid and gibbericacid, also nullified flowering and gibbosity in the presenceof EDDHA. A fourth decomposition product of GA₃, epigibbericacid, inhibited gibbosity but hardly affected flowering. Salicylic acid was confirmed to affect flowering and gibbosityin L. gibba G3. However, contrary to an earlier report, it didnot induce flowering under short-day conditions. (Received January 10, 1976; )     

Induction of flowering in Lemna gibba G3 under short-day conditions

In the presence of the chelating agent EDDHA, long-day duckweedLemna gibba G3 was induced to flower under a short-day scheduleof 9 hr of light and 15 hr of darkness in a 24-hr cycle. Weconcluded that EDDHA creates effects very similar to those ofsalicylic acid. When EDDHA or salicylic acid was added to thenutrient medium in combination with BA, flowering was inducedeven under conditions of 8 hr of light and 16 hr of darkness.Under a photoperiod of 9 hr, BA markedly enhanced the effectof EDDHA as well as salicylic acid. On the other hand, BA alonewas ineffective as far as flowering was concerned. By quantitativeinteractions, BA seems to complement the modifying effect ofEDDHA or salicylic acid on flowering in this duckweed strain. (Received June 25, 1976; )     

Effect of chelating agents on bud induction and accumulation of iron and copper by the moss Bartramidula bartramioides

The chelating agents, EDDHA, its iron salt, EDTA, and salicylic acid enhance bud formation in Bartramidula bartramioides (Griff.) Wijk & Marg. Salicylic acid elicits optimal response at 10^–4 M , whereas the other substances do so at 10^–7 M . Increased concentration of ferric citrate and cupric sulphate also stimulate bud induction. The accumulation of Fe³⁺ and Cu²⁺ is facilitated by chelators. The endogenous iron content is maximum at 10^–7 M EDDHA or EDTA, which is also the concentration optimal for bud induction.     

Evaluation of different iron compounds in chlorotic Italian lemon trees (Citrus lemon).

The severe deficiency of iron or ferric chlorosis is a serious problem of most citrus trees established in calcareous soils, as a result of the low availability of iron in these soils and the poor uptake and limited transport of this nutrient in trees. The objective of this study was to evaluate the response of chlorotic Italian lemon trees (Citrus lemon) to the application of iron compounds to roots and stems. On comparing the effects of aqueous solutions of ferric citrate, ferrous sulphate and FeEDDHA chelate, applied to 20% of the roots grown in soil and sand, of trees that were planted in pots containing calcareous soil, it was observed that the chelate fully corrected ferric chlorosis, while citrate and sulphate did not solve the problem. EDDHA induced the root uptake of iron as well as the movement of the nutrient up to the leaves. With the use of injections of ferric solutions into the secondary stem of adult trees, ferric citrate corrected chlorosis but ferrous sulphate did not. The citrate ion expanded the mobility of iron within the plant, from the injection points up to the leaves, whereas the sulphate ion did not sufficiently improve the movement of iron towards the leaf mesophyll.     

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate ⁵⁵Fe³⁺ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of ⁵⁵Fe³⁺ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.     

Mechanisms of EDDHA effects on the promotion of floral induction in the long-day plant Lemna minor (L.)

EDDHA added in an optimal concentration (20.5 mumol.L-1) to a modified Pirson-Seidel nutrient solution induces flowering in some clones of the species Lemna minor, Lemna gibba and Spirodela polyrrhiza, which in the absence of EDDHA in the same nutrient solution do not flower. By adding EDDHA (20.5 mumol.L-1), floral induction under LD conditions is optimally promoted in the long-day (LD) species Lemna minor. After adding EDDHA to the nutrient solution, before floral induction and during flowering, Zn, Mn and Cu content is significantly increased in plants. Zn-EDDHA (0.86 mumol.L-1), Mn-EDDHA (1.51 mumol.L-1) and Cu-EDDHA (0.12 mumol.L-1), when used individually, greatly promote flowering under LD conditions as compared to flowering in the same nutrient solution with an equivalent quantity of Zn, Mn or Cu in the nonchelate form. If, on the other hand, Zn-EDDHA and Mn-EDDHA are added to the nutrient solution together (instead of Zn and Mn in nonchelate form), their effect on the promotion of flowering is less than in the case of their individual use. This shows that there is antagonism between Zn-EDDHA and Mn-EDDHA that is eliminated by adding EDDHA to the nutrient solution. We obtained the highest percentage of flowering plants (i.e. 74%) if we added EDDHA (20.5 mumol.L-1) to the nutrient solution containing Mn, Zn and Cu in chelate form. 74% of flowering plants actually means that flowering was achieved in all physiologically mature plants. Our results show that EDDHA promotes floral induction in Lemna minor under LD conditions, especially through chelating Zn, Mn and Cu, and, in addition, through eliminating the antagonism between Mn and Zn chelates EDDHA. Zn-EDDHA (0.86 mumol.L-1) also promote floral differentiation, especially cell division of microspore mother cells into dyads and those into microspore tetrads, which can be seen in microphotographs. When investigating possible pathways through which Mn-EDDHA, Zn-EDDHA and Cu-EDDHA promote flowering, we studied the effects of various concentrations of IAA and sucrose added to the nutrient solution as well. The results support the hypothesis that one of the possible pathways in which Mn-EDDHA promotes floral induction is through auxin oxidase, whereas Zn-EDDHA and Cu-EDDHA probably promote it through the enhancement of the photosynthesis and synthesis of sucrose.     

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)     

活性污泥中Fe(Ⅲ)还原微生物的分离、鉴定及还原特性

采用平板分离法和柠檬酸铁还原实验法相结合,从城市污水处理厂活性污泥中分离获得Fe(Ⅲ)还原菌F7,经形态观察、生理生化和16S rDNA序列分析及同源性比对鉴定为恶臭假单胞菌(Pseudomonas putida).在不同柠檬酸铁浓度和不同pH条件下的实验表明,柠檬酸铁浓度为0.32g/L时,菌株生长情况较好,柠檬酸铁浓度为0.16g/L时,Fe(Ⅲ)异化还原比例较高;pH6.5时,菌株生长情况较好,Fe(Ⅲ)异化还原量较多.     

REQUIREMENTS FOR THE MAINTENANCE AND GROWTH OF APHANIZOMENON FLOS-AQUAE IN CULTURE1,2

Aphanizomenon flos-aquae has been introduced into unialgal culture in a completely defined medium and maintained for more than 3 years in the colonial flake form found in nature. The color and size of the flake and general appearance of the cells are substantially improved over those found in the original material at the time of collection from Upper Klamath Lake, Klamath County, Oregon. The alga readily forms both akinetes and heterocysts in culture Bacteria-free cultures have not been obtained. A precipitate-free medium designated ASM No. 8a has been developed from the ASM of McLachlan and Gorham. Their medium was altered by modifying the concentration of certain elements and by the addition of sodium bicarbonate, a 1/25 dilution of Arnon's trace element solutions B₇ and C₁₃, hydrogen ferric ethylenediamine di-o-hydroxyphenylacetate (EDDHA), and disodium ethylene dinitrilotetra-acetate (Na₂EDTA).     

Experimental control of flowering in Pistia stratiotes L.

GA₃, salicylic acid and EDDHA induced flowering in Pistia stratiotesin vitro under short days and conditions of continuous light.It has been hypothesized that EDDHA and salicylic acid bringabout the same effect on flowering in Pistia and the duckweedspecies Lemma gibba G3 whereas the effect of GA₃ on the floweringmechanism of these species is basically different. (Received March 14, 1978; )     

Obligate Requirement of Salicylic Acid for Short-Day Induction of Flowering in a New Duckweed, Wolffiella hyalina 7378

Wolffiella hyalina 7378, a member of the Lemnaceae, does notflower in the basal media of Bonner-Devirian, Hoagland, Hutner(1/2 strength), M medium of Hillman or Pirson-Seidel under eitherlong or short day photoperiodic regimes. Well-known chelatingagents, such as EDTA, EDDHA and 8-hydroxyquinoline, which havebeen shown to significantly influence flowering in other membersof the Lemnaceae, also do not show any effect in this strain.It is, however, noteworthy that W. hyalina 7378 flowers profuselyunder a short-day photoperiodic schedule of 8 h light and 16h darkness provided that 10^–5 M salicylic acid is supplementedto modified Bonner and Devirian medium. The critical daylengthof W. hyalina 7378 is 13 h. Further, at least two inductivephotocycles are required for initiation of flowering. As tothe mechanism of SA action, it appears that SA effect on floweringis hormonal in nature rather than due to chelation of metalions. (Received August 25, 1986; Accepted January 9, 1987)     

Changes in sugar beet leaf plasma membrane Fe(III)-chelate reductase activities mediated by Fe-deficiency, assay buffer composition, anaerobiosis and the presence of flavins

Summary Different assay conditions induce changes in the ferric chelate reductase activities of leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet. With an apoplasttype assay medium the ferric chelate reductase activities did not change significantly when Fe(III)-EDTA was the substrate. However, with ferric citrate as substrate, the effect depended on the citrateto-Fe ratio. When the citrate-to-Fe ratio was 20 1, the effects were practically unappreciable. However, with a lower citrate-to-Fe ratio of 5 1 the activities were significantly lower with the apoplast-type medium than with the standard assay medium. Our data also indicate that anaerobiosis during the assay facilitates the reduction of ferric malate and Fe(III)-EDTA by plasma membrane preparations. Anaerobiosis increased by approximately 50% the plasma membrane ferric chelate reductase activities when Fe(III)-EDTA was the substrate. With ferric malate anaerobiosis increased activities by 70–90% over the values obtained in aerobic conditions. However, with ferric citrate the increase in activity by anaerobiosis was not significant. We have also tested the effect of riboflavin, flavin adenine dinucleotide, and flavin mononucleotide on the plasma membrane ferric chelate reductase activities. The presence of flavins generally increased activities in plasma membrane preparations from control and Fe-deficient plants. Increases in activity were generally moderate (lower than twofold). These increases occurred with Fe(III)-EDTA and Fe(III)-citrate as substrates.Abbreviations BPDS bathophenantroline disulfonate - FC ferric chelate - FC-R ferric chelate reductase - PM plasma membrane     

Significance of Polyamines for Flowering in Spirodela punctata

Spirodela punctata strain O 5, a quantitative long-day plant,flowers only when ethylene-diamine-di-o-hydroxyphenylaceticacid (EDDHA) or salicylic acid was added to the nutrient medium[Scharfetter et al. (1978) Z. Pflanzenphysiol. 87: 445]. Undersuch conditions, cyclohexylamine and methylglyoxal-bis(guanylhydrazone)(both blockers of polyamine synthesis) inhibited flowering withoutsignificant effects on vegetative growth. Supply of spermidineabolished completely the inhibitor effects, but cannot replacethe EDDHA effect on flowering. ¹Dedicated to Prof. O. H. Volk, Wurzburg, on his 85th birthday. ²Present address: Facultad de Ciencias Forestales, UniversidadAutónoma de Nuevo León. Ap. Post. 41, 67700 Linares,Nuevo León, México. (Received October 19, 1988; Accepted February 3, 1989)