Floral Induction in a Photoperiodically Neutral Duckweed, Lemna paucicostata LP6: Interaction of Iron, EDTA and Cytokinins

Biochem. Physiol. Pflanzen 181, 559-564 (1986) VEB Gustav Fiseher Verlag J cna

Floral Induction in a Photoperiodically Neutral Duckweed, Lemna paucicostata LP6 : Interaction of Iron, EDTA and Cytokinins J. P. KHURANA and S. C. MAHESHWARI Unit for Plant Cell and .Molc('ular Biology and Dcpartment of Botany, University of Delhi, Delhi, India Key Term Index: Cytokinin-iron interaction, flowering (promotion), photoperiodically ncutral strain; Lemna paucicostata LP 6

Summary Lemna paucicostata LP6 is a photoperiodically neutral strain. Nevertheless, flowering in this duckweed occurs in the basal BONNER-DEVIRIAN medium (1939) only if the level of iron is raised 10-fold. Intensity of flowering is further increased in the presence of EDT A in the nutrient medium. But flowering could be enhanced to a truly remarkable degree if a cytokinin sueh as BA, zeatin, or kinetin, was supplied to plants, in the simultaneous presence of a high iron level (10- 4 }I). The maximal flowering was obtained when the cytokinin, employed, was added along with 10- 4 J1 Fecitrate and 10-4 M: EDTA (the optimal concentration for cytokinins was 100,ugjl). A point worthy of note, however, is that the cytokinins do not have any effed on flowering of this duckweed, whether in the presence or absence of EDTA, if only normal level of iron is supplied. The results obtained provide strong evidence for a role of cytokinins in flowering of LeI/lila paucicostata LP s in nature. Since cytokinins simulate the effeet of ehelating agents, the data are indicative of the possibility that their effed on flowering may also be exerted through a stimulative effect on uptake of iron; however this idea needs to be furthl'I confirmed.

Introduction

Naturally occurring plant growth substances, such as auxins, gibberellins, cytokinins, abscisic acid and ethylene, have been known to influence flowering in many higher plants (see BERNIER et al. 1981). Amongst these, cytokinins have proved to be of the greatest significance for induction and promotion of flowering so far as duckweeds (members of the family Lemnaceae) are concerned. Cytokinins have been shown to influence flowering in short day plants W olffia microscopica (MAHESHW ARI and VENKATARAMAN 1966; VENKATARAMAN et al. 1970), a strain of Lemna paucicostata (GnTA and MAHESHWARI 1969, 1970), L. paucicostata 6746 (KHURANA and MAHESHWARI 1983a), in long day plants Lemna gibba (OOTA and TSUDZUKI 1971; PIETERSE and MULLER 1977; see also CLELAND and TANAKA 1982), L. minor (KRAJNCIC 1982), in long-short-day plant Wolffia arrhiza (KRAJNCIC 1983) and in photoperiodically neutral plant Spirodela polyrrhiza (KRAJNCIC 1982). In L. paucicostata, strains 151 and A.bbre!:iatiolls: BA, 6-benzyladenine; EDDHA, ethylenediamine-di-o-hydroxyphenylacetie arid; EDTA, ethylenediaminetetraacetic acid; :\IR, multiplication rate

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381, FUJIOKA et al. (1983) reported that cytokinins act synergistically with benzoic acid and promote flowering. The present work pertains to Lemna paucicostata LP 6' a photoperiodically insensitive (neutral) strain, as 8 or 9 consecutive cycles of various photoperiods (ranging from 8 to 24 h, with corresponding hours of darkness in a 24 h schedule) have failed to evoke any flowering response (see KHURANA and MAHESHWARI 1983b). Earlier, it has been possible to induce flowering in it by salicylic acid and aspirin (KHURANA and MAHESHWARI 1978) and 8-hydroxyquinoline (KHURANA and MAHESHWARI 1983b), when the plants are grown in modified BONNER-DEVIRIAN medium. Flowering in strain LP 6 could also be induced when iron level was increased 10-fold (10-4 M) over that in the basal medium and to a still greater degree in the presence of 10- 4 M EDTA (details to be published elsewhere). In the present communication, promotion of flowering in L. paucicostata LP6 by cytokinins is being reported. It has been observed that for cytokinins to be effective, the requirement of a specially high level of Fe-citrate is nearly absolute and the presence of EDTA in the culture medium has a synergistic effect.

Material and Methods Aseptic cultures of Lemlla paucicostata, strain LPs (a local isolate), were maintained in the modified BONNER-DEVIRIAN medium comprising macronutrients of BONNER and DEVIRJAN (1939) medium and micronutrients of HELLER'S (1953) medium as per a standard practice in this laboratory. The medium was supplemented with 10-4 M EDTA and 1 % sucrose. For specific experiments, cultures were raised, depending upon the requirement, with or without EDT A and with basal or higher levels of Fe-citrate. The pH of the medium was adjusted to 5.5, before autoclaving at 1.08 kg . cm- 2 for 15 min. All ex perimental cultures were started with a single 3-frond colony in 250 ml Erlenmeyer flasks (each containing 100 ml nutrient solution). The temperature was main2 °C during day time and at 22 1 °C during darkness. Light was provided from a tainpcj at 26 mixed bank of ('001 daylight fluor escent tubes (Philips TL 65- 80 Wj54 RS, 6,800 OK) and incandescent lamps (Philips Argenta, 100 W). The fluence rate in the spectral range between 400 and 750 nm , measured with the help of Model SR spectroradiometer (Instrumentation Specialities Co., Lincoln, Nebraska), at plant level, varied from 10.0 to 10.5 W m- 2 • MR was calculated acrording to CLARK (1925) and flowering percentage was determined by dividing the number of flow ering fronds by total number of fronds and multiplying by 100. Three repli('ate cultures were employed for each treatment and experiments were repeated at least twice f or more details see KHl:RANA and ~fAHESHWARI 1984).

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Results

Effect of cytokinins in presence of normal level of iron 6-Benzyladenine, kinetin and zeatin were added at 1, 10, 100 and 1,000 ,Ltg/l levels to modified BONNER-DEVIRIAN medium, with or without EDTA. All the three cytokinins, at 1,000,Ltg/I, caused an increase in frond as well as colony size. However, MR was not affected significantly and flowering, too, could not be initiated by any of the three cytokinins employed, under either a photoperiodic schedule of 16 h light and 8 h dark or 8 h light and 16 h dark.

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Interaction of cytokinins and high level of iron An earlier study from this laboratory revealed that ferric citrate, EDDHA and cytokinins have a high degree of interaction on flowering in another strain of L. paucicostata (GUPTA and MAHESHWARI 1970). Since high Fe-citrate concentration also initiates flowering in L. paucicostata LP 6 (see Introduction), experiments were conducted to investigate whether it may have some interaction with cytokinins, in the presence or absence of EDTA (it should be noted here that interaction of ferric citrate and cytokinins was studied in the presence of EDTA only and not EDDHA because the latter itself is effective and induces flowering at low levels of iron; KHl'RAKA and MAHESHWARI, unpublished). Though the inclusion of a cytokinin in the medium did not significantly affect the MR, at 100 fig/l level a marked stimulative. effect on flowering was observed. Zeatin added to the medium containing excess of iron (10- 4 M Fe-citrate) was promotive for flowering, even at 1fig/l level, under a photoperiodic schedule of 16 h light and 8 h dark, irrespective of the presence of EDTA (Fig. 1). Like zeatin, BA and kinetin were also found to be effective in promoting flowering, in the presence as well as absence of EDTA (Fig. 1). However, of the three cytokinins, zeatin was the most effective at lower levels, e.g. 1 and 10 fig/I. At 100 and 1,000 fig/l all three were equally potent for flowering (Fig. 1). Simultaneously, in these experiments, a set of three replicate flasks for each level of cytokinins was subjected to three cycles of 8 h light and 16 h darkness with the first two and last three cycles of 16 h light and 8 h darkness, but no significant promotion in flowering was observed over those subjected to 16 h light and 8 h darkness. In experiments with cytokinins, flowering as well as non-flowering fronds became bigger in size. An unusual observation was made in this experiment - besides enlargement of flowers, occasionally flowers were observed in both the pouches of a frond.

Effect of adenine and adenosine As cytokinins had a definite promotive effect on flowering in L. paucicostata LP 6' attempts were made to determine whether their effect was specific or related compounds like adenine and adenosine could also elicit a similar response. Both compounds were added at 10-6 , 10-5 , 5 X 10-5 and 10-4 M levels, in modified BONNERDEVIRIAN medium supplemented with 10-4 M (10-fold higher than normal) level of Fe-citrate, in the presence of EDTA (10-4 M; normal level). In this experiment, neither adenine nor adenosine could promote flowering. As anticipated from earlier results, however, flowering in the control plants with high level of Fe-citrate and in the presence of EDTA was appreciable (ca. 30%). Discussion

It is clear from the data presented in this communication that L. paucicostata LP 6' employed in the present investigation, is photoperiodically neutral when grown in the modified BONNER-DEVIRIAN medium supplemented with EDTA and the normal level of iron. However, it flowers profusely, irrespective of the photoperiod, in the presence

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Fig. 1. Effect of 6-benzyladenine, zeatin and kinetin, on multiplication rate and flowering in Lemna paucicostata LP6' in the presence of 10-4 M (10-fold higher than basal level) ferric citrate. Experimental cultures were started with a single 3-frond colony (per flask) from an exponentially growing stock culture raised in the modified BONNER and DEVIRIAN medium, supplemented with EDTA (10- 4 M). All experimental plants, including the ones kept as control, were grown under photoperiods of 16 h light and 8 h darkness, in a 24 h cycle, in the absence or presence of EDTA in the modified BONNER and DEVIRL\N medium. A sample of nearly 60-100 fronds from each of the replicate flask was analysed for flowering after 9 d of inoculation. Each value plotted is the mean of three replicate cultures and vertical bars represent the extent of absolute variation in flowering percentage. Curves = perrentage of flowering fronds; Columns = multiplication rates.

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of anyone of the cytokinins, provided the level of iron is kept sufficiently high (10fold higher than normal). Incidentally, in all experiments conducted employing cytokinins, flowering was observed in the controls raised in "high iron" medium, without EDTA. The results obtained with strain LP6 conform to the ones reported earlier with a short day strain of L. paucicostata (GUPTA and MAHESHWARI 1970) where also cytokinins induced flowering under non-inductive long days, but only when iron was raised 10-fold to that in the basal medium. In L. paucicostata, employed by GUPTA and MAHESHWARI (1970), cytokinins were also effective, even at low levels of ferric citrate, provided EDDHA was present in the medium, instead of EDTA. In this regard, L. paucicostata LP6 is more responsive than the strain of L. paucicostata used for earlier investigations in this laboratory, as it can be directly induced to flower by EDDHA even at the normal level of iron (KHURANA and MAHESHWARI, unpublished) and also it responds to cytokinins. A very pertinent question that arises as a result of the present and earlier investigations on duckweeds is: Do cytokinins and chelating agents have a common mode of action? Some preliminary experiments in Wolffia microscopica (VENKATARAMAN and MAHESHWARI, unpublished) and L. paucicostata (GUPTA 1968; GUPTA and MAHESHw ARI 1970) indicated that cytokinins may stimulate uptake of iron. Although no new data have been obtained concerning endogenous levels of iron as affected by cytokinins, nevertheless, the experiments on a strain of L. paucicostata (GUPTA and MAHESHWARI 1970) and on L. paucicostata LP 6 in the present investigation - where cytokinins were effective only if iron level was increased lO-fold - do indicate that iron has a dose interaction with cytokinins. GUPTA and MAHESHWARI (1970) had speculated, on the basis of the data provided by SILLEN and MARTELL (1964), that cytokinins may directly chelate iron since adenine and adenosine, and other related compounds do have some metal chelating ability. Subsequently, some direct evidence in this regard has come from OOTA'S laboratory who on the basis of spectrophotometric data has established that not only cytokinins but also IAA and GA have affinity for metal ions like Fe3 + (ferric ions) and Cu 2+ (OOTA and TSUDZT!KI 1971). Strikingly, amongst these plant growth substances, cytokinins alone resemble EDTA-type chelates, as far as chelation of Fe2+ (ferrous ions) is concerned, because only kinetin, and not IAA or GA, binds to ferrous ions (OOTA and TSUDZUKI 1971). Whatever be the precise mode of action of cytokinins, i.e. whether the effect is direct as a "native" hormone or through chelation, it is interesting to note finally that in L. pallcicostata, employed earlier in this laboratory, the endogenous level of cytokinin - estimated on the basis of Nicotiana callus bioassay - increases significantly upon photoinduction (GUPTA 1968). Acknowledgement Financial assistance from the Department of Science and 'fe('hnology, Government of India is gratefully aeknowledged.

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References BERNIER, G., KINET, J.-l\I., and SACHS, R. )1.: The Physiology of Flowering, Vol. II. Transition to Reproductive Growth. CRC Press, Inc., Boca Raton, Florida 1981. BONNER, J., and DEVIRIAN, P. S.: Growth factor requirements of four species of isolated roots. Am. J. Bot. 26, 661-665 (1939). CLARK, N. A.: The rate of reproduction of Lemna major as a function of intensity and duration of light. J. Phys. Chern. 29, 935-951 (1925). CLELAND, C. F., and TANAKA, 0.: Influence of plant growth substances and salicylic acid on flowering and growth in the Lemnaceae (duckweeds). Aqua. Bot. 13, 3-20 (1982). FUJIOKA, S., YAMAGUCHI, I., MCROFCSHI, N., TAKAHASHI, N., K.UHARA, S., and TAKIMOTO, A.: The role of plant hormones and benzoic acid in flowering of Lemna paucicostata 151 and 38l. Plant Cell Physiol. 24, 241-246 (1983). GUPTA, S.: In vitro control of flowering in Lell1l1a paucicostata Hegelm. Ph. D. Thesis, University of Delhi, Delhi 1968. GUPTA, S., and MAHESHWARI, S. C.: Induction of flowering by cytokinins in a short-day plant, Lemna paucicostata. Plant Cell Physiol. 10, 231-233 (1969). GUPTA, S., andMAHESHWARI, S. C.: Growth and flowering of Lemnapaucicostata. II. Role of growth regulators. Plant Cell Physiol. 11, 97-106 (1970). HELLER, R.: Recherches sur la nutrition minerale des tissus vegetaux cultives in vitro. Ann. Sci. Nat. Bot. BioI. Veg. 14, 1-223 (1953). KHURANA, J. P., and MAHESHWARI, S. C.: Induction of flowering in Lemna paucicostata by salicylic acid. Plant Sci. Lett. 12, 127-131 (1978). KHURANA, J. P., and MAHESHWARI, S. C.: Promotion of flowering in Lemna paucicostata 6746 (a short-day duckweed) by cytokinins. Plant Cell Physioi. 24, 913-918 (1983 a). KHURANA, J. P., and MAHESHWARI, S. C.: Effect of 8-hydroxyquinoline on flowering and endogenous levels of iron and copper in Lemna paucicostata, strain LPs. Plant Cell Physiol. 24, 1251-1254 (1983b). KHUHANA, J. P., and MAHESHWARI, S. C.: Floral induction in short-day Lemna paucicostata 6746 by 8-hydroxyquinoline, under long days. Plant Cell Physiol. 20, 77-83 (1984). KRAJNCIC, B.: Effects of kinetin on floral induction and floral development in the species Lemna minor and Spirodela polyrrhiza. BioI. Vestn. 30, 85-104 (1982). KRAJNCrC, B.: The effects of cytokinins on flowering in the long-short-day plant Wolffia arrhiza (L.) Wimm. Z. Pflanzenphysioi. 112, 281-286 (1983). MAHESHWARI, S. C., and VENKATARAMAN, R.: Induction of flowering in a duckweed - Wolffia microscopica - by a new kinin, zeatin. Planta 70, 304-306 (1966). OOTA, Y., and TSUDZUKI, T.: Resemblance of growth substances to metal chelators with respect to their actions on duckweed growth. Plant Cell Physioi. 12, 619-631 (1971). PIETERSE, A. H., and MULLER, L. J.: Induction of flowering in Lemna gibba Ga under short-day conditions. Plant Cell Physioi. 18, 45-53 (1977). SILLEN, L. G., and MARTELL, A. E.: Stability constants of metal ion complexes. Chern. Soc. (special pUblication No. 17), London 1964. VENKATARAMAN, R., SETH, P. N., and MAHESHWARI, S. C.: Studies on the growth and flowering of a short-day plant, Wolffia microscopica. I. General aspects and induction of flowering by cytokinins. Z. Pflanzenphysiol. 62, 316-327 (1970). Received March 18, 1986; accepted April 21, 1986

Author's address: Prof. S. C. MAHESIIWARI, Programme Director, Unit for Plant Cell and Molecular Biology and Department of Botany, University of Delhi, Delhi - 110007, India.