Cerium and Lanthanum Promote Floral Initiation and Reproductive Growth of Arabidopsis Thaliana

Plant Science 159 (2000) 117–124 www.elsevier.com/locate/plantsci

Cerium and lanthanum promote ﬂoral initiation and reproductive growth of Arabidopsis thaliana

Ya-Wen He, Chiang-Shiong Loh *

Department of Biological Sciences, National Uni6ersity of Singapore, 14 Science Dri6e 4, Singapore 117543, Singapore

Received 1 December 1999; received in revised form 2 June 2000; accepted 5 July 2000

Abstract

The effects of cerium and lanthanum on the vegetative growth, floral initiation and reproductive growth of Arabidopsis thaliana were studied. Addition of cerium nitrate (0.5–10 mM) or lanthanum nitrate (0.5–50 mM) to the culture medium significantly increased the lengths of primary roots, but had no significant effects on the number of rosette leaves produced per plant, plant heights and dry weights during the vegetative growth stage (17 days after seed germination). The percentage of plants bolted was significantly increased with the addition of 0.5–10.0 mM cerium nitrate or lanthanum nitrate. The combination of 0.5 mM cerium nitrate and 0.5 mM lanthanum nitrate was found to be most effective on the induction of floral initiation. The height, dry weight and average number of flower numbers of 35-day-old plants growing in media containing cerium nitrate or/and lanthanum nitrate (0.5–10.0 mM) were found to be significantly higher than those in the control medium. The endogenous levels of cytokinins (zeatin riboside, dihydrozeatin riboside and isopentenyl adenosine) and carbohydrates (sucrose, glucose and fructose) in leaf and root tissues of plants growing in the medium supplemented with 0.5 mM cerium nitrate and 0.5 mM lanthanum nitrate were not significantly different from those of plants in the control medium. Application of 0.5 mM cerium nitrate and 0.5 mM lanthanum nitrate enhanced the effects of 10−6 M IPA on root growth, plant height and flowering. The role of cerium and lanthanum in promoting floral initiation and reproductive growth and the possibility of developing non-hormonal flowering promoting agents are discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Arabidopsis thaliana; Cerium; Lanthanum; Cytokinin; Flowering

1. Introduction REEs makes it imperative that more detailed information on the effects of REEs on biological The rare earth elements (REEs) comprise a systems be available. group of 15 trivalent metallic elements with similar The effects of REEs, especially lanthanum, in chemical properties. They normally occur as basic different animal tissues have been studied exten- oxides and phosphate complexes in nature [1,2]. sively [1]. Most of the work carried out on REEs Since the introduction of ion exchange techniques, in animal systems was based on the use of REE the separation of the rare earth elements from ion as a substitute or antagonist for Ca2+ to their ores and from one another has become prac- monitor the movement of calcium and water, and tical and many new uses of REEs have been to investigate the role of calcium in muscle and developed [3]. The widespread industrial usage of nerve activity [4].

Abbre6iations: BA, benzyladenine; Ce, Cerium; DHZR, dihy- The results from limited studies on the effect of drozeatin riboside; IPA, isopentenyl adenosine; La, Lanthanum; MS REEs on plant growth are conﬂicting. Early re- medium Murashige and Skoog (1962) medium; Nd, Neodymium; ports indicated that the REE were inhibitory to REEs, rare earth elements; ZR, zeatin riboside. 3+ 3+ * Corresponding author. Tel.: +65-874-2916; fax: +65-779-5671. plant growth. For example, La and Nd were E-mail address: [email protected] (C.-S. Loh). found to inhibit elongation of oat coleoptile sec-

tions [5]. Colloidal lanthanum caused an almost drate (La(NO3)3·6H2O, Sigma) were dissolved in complete inhibition of cell division and root elon- Mili-Q water and sterilized by membrane filtra- gation in the root tips of barley plants [6]. La3+ tion (Millipore, 0.45 mm) and stored at room had been shown to inhibit root elongation of temperature in the dark. Stock solutions of wheat [7,8]. More recent reports, however, cerium nitrate and lanthanum nitrate were added demonstrated some positive effects of REEs on to the autoclaved basal medium prior to dispens- plant growth. Diatloff et al. [9] reported that ing into Magenta GA7® containers (Magenta corn root growth increased significantly with the Corp., USA). Seeds were germinated in the dark applications of cerium (0.63 mM) and lanthanum and 2-day-old seedlings were placed under 16 h (0.63 mM). Applications of lanthanum and photoperiod (54 mmol−1m−2s−1 provided by cerium were also reported to increase spike pro- Cool White fluorescent lamps) at 2592°C. duction in wheat [10]. In pot trials, applications of cerium sulphate (up to 100 mg/kg) enhanced 2.2. Growth measurements root and shoot growth of Phaseolus radiatus and Brassica pekinensis [11]. Lengths of primary roots were scored 10 days Results from field trials were also inconsistent. after seed germination. The number of leaves The increase in crop yield reported by workers produced per plant was scored 17 days after seed from China ranged between 8 and 50%, with the germination. Plant heights and dry weights were common response being of the order of 8–15% scored 17 and 35 days after seed germination, [12,13]. However, no response was found by respectively. Dry weights were taken by drying spraying and seed dressing of a summer fodder 100 plants in an oven (55°C) for 1 week. Floral crop (Brassica sp.) with REEs in a field trial initiation was recorded when the plant bolted carried out in Australia (cited in [4]). In view of with at least 1 cm long inflorescence stalk. this, it is essential to study and elucidate the effects of REE on essential stages of growth and 2.3. Extraction and determination of endogenous development of a model plant species such as cytokinins and carbohydrates Arabidopsis thaliana. In this report, we investigated the effects of cerium (Ce) and lanthanum Approximately 1 g fresh weight of tissues was (La) on vegetative and reproductive growth of A. homogenized in 4 ml of 80% ethanol followed by thaliana and correlated some of the responses to 1 h incubation at 4°C. After centrifugation at increased sensitivity of cell to plant growth regu- 1670×g for 3 min, the supernatant was trans- lators. ferred to another centrifuge tube. The tissues were re-extracted with 2 ml of 80% ethanol, and the supernatant was pooled together after centrifugation. The extracts were vacuum evaporated 2. Materials and methods at 4°C (Eppendorf Concentrator 5301) to 1/4 volume and then stored at −20°C after filter- 2.1. Plant materials, culture media and growth sterilization (Milipore, 0.2 mm). The analysis of conditions zeatin riboside (ZR), dihydrozeatin riboside (DHZR), and isopentenyl adenosine (IPA) were Seeds of A. thaliana L. Heynh cv. Columbia performed by immunoassay detection kits (Sigma (LEHLE SEEDS, USA) were surface sterilized Chemical Company) according to the protocols by soaking in 75% alcohol for 30 s and followed provided by the manufacturer. All hormonal lev- by 15% Clorox® for 15 min. The seeds were then els were expressed in terms of pmol per gram rinsed five times in sterilized water prior to cul- fresh weight (pmol/g.f.wt). The analysis of su- ture. The 1/4 strength Murashige and Skoog crose, glucose and fructose were performed by medium [14] was used for seed germination and assay kits (Sigma Chemical Company) according as basal medium. The pH of the medium was to the protocols provided by the manufacturer. adjusted to 5.8 before agar (Difco, 0.8%) was The levels of sucrose were expressed in term of added. All media were autoclaved for 20 min at mg per gram fresh weight (mg/g.f.wt). The levels

121°C. Cerium nitrate hexahydrate (Ce(NO3)3 of glucose and fructose were expressed in term of m m ·6H2O, Sigma) and lanthanum nitrate hexahy- g per g fresh weight ( g/g.f.wt). Y.-W. He, C.-S. Loh / Plant Science 159 (2000) 117–124 119

3. Results tions of cerium nitrate or lanthanum nitrate (0.5– 2.5 mM) significantly increased the percentages of 3.1. Effects on 6egetati6e growth plants bolted on days 20 and 23. For example, 69.3% of the plants growing in the medium with The vegetative and reproductive growth of A. 2.5 mM cerium nitrate bolted on day 23 whereas thaliana plant was separated. Prior to bolting, an only 39.4% bolted in basal medium. About 65.7% A. thaliana plant consisted of a rosette of small of the plants in the medium containing 0.5 mM leaves with a main hypocotyl. In the present lanthanum nitrate bolted on day 23 (Table 2). study, results showed that additions of 0.5–50 mM The percentages of plants bolted on day 27 were cerium or lanthanum had no significant effects on not significantly different between media with or the vegetative growth in terms of height and dry without cerium nitrate or lanthanum nitrate weight. Cerium and lanthanum also had no sig- (Table 2). nificant effect on the average number of rosette None of the plants growing in basal medium leaves 17 days after seed germination (data not bolted on day 19. Additions of certain combina- shown). Additions of higher concentrations of tions of cerium nitrate and lanthanum nitrate (e.g. cerium or lanthanum inhibited the vegetative 0.5+0.5 mM, 0.5+1.0 mM, 1.0+0.5 mM, 1.0+ growth (data not shown). 1.0 mM) resulted in early flowering; 4.6–8.3% of the plants bolted as early as day 19. The combina- 3.2. Effects on root growth tion of 0.5 mM cerium nitrate and 0.5 mM lanthanum nitrate was most effective in induction of A. thaliana plants were observed to produce flowering. Nearly 90% of the plants bolted on day only primary roots 10 days after seed germina- 23 in the medium containing 0.5 mM cerium ni- tion. In REE-free medium, the length of primary trate and 0.5 mM lanthanum nitrate compared to roots was about 2.0 cm. Addition of 0.5–10.0 mM only 39.4% bolted in the basal medium. On day cerium nitrate to the culture medium significantly 27, no significant difference in the percentage of increased the lengths of primary roots with plants bolted was observed in the media with or longest root (2.84 cm) observed in the medium without REE (Table 2). containing 10.0 mM cerium nitrate (Table 1). Sim- In order to assess the effects of additional ni- ilarly, addition of lanthanum (0.5–50.0 mM) trates from lanthanum nitrate and cerium nitrate singly or in combination with cerium nitrate sig- on floral initiation, we replaced these two chemi- nificantly increased the lengths of roots (Table 1). cals with potassium nitrate. The effects of additional nitrates (0.3 or 1.0% of the total nitrate 3.3. Effects on floral initiation level in the 1/4 strength MS medium) on floral initiation were investigated. The percentages of In basal medium, the first appearance of bolting plants bolted in the media supplemented with the (8.9%) was observed on day 20. About 39.4% of additional nitrate were not significantly different the plants bolted on day 23 and by day 27 more from those in the basal medium (data not shown). than 98% of the plants bolted (Table 2). Addi- Thus any effects of cerium nitrate and lanthanum

Table 1 Effects of cerium nitrate and lanthanum nitrate on the growth of primary root of A. thaliana 10 days after seed germination

Average root length (cm) a

Concentration (mM) Cerium nitrate Lanthanum nitrate Cerium nitrate and lanthanum nitrate 0.0 2.0490.06a 2.0490.06a 1.9690.04a 0.5 2.6290.05b 2.5890.04c 2.6590.03b 1.0 2.6390.04b 2.6590.04c 2.5890.04b 10.0 2.8490.04c 2.4790.05b 2.5490.05b 50.0 1.9290.04a 2.4090.04b 1.8190.03c

a Values were expressed as mean9SE. There were 81–95 plants per treatment. Means within the same column followed by the same letter were not signiﬁcantly different according to Tukey’s pairwise comparisons (P=0.05). 120 Y.-W. He, C.-S. Loh / Plant Science 159 (2000) 117–124

Table 2 Effects of cerium or/and lanthanum on ﬂoral initiation of A. thaliana

Cerium nitrate (mM)Lanthanum nitrate (mM) Percentage of plants bolteda

Days after seed germination

19 20 23 27

0.0 0.0 0.0 8.993.2 39.497.9 97.791.4 0.5 0.0 14.592.5 59.499.9 96.491.0 2.5 0.0 17.493.7 69.3910.4 95.191.1 10.0 0.0 4.391.9 51.295.3 97.790.7 100.0 0.0 0.0 20.592.7 94.991.5 0.50.0 15.692.6 65.797.8 96.490.9 2.5 0.0 13.391.1 62.395.4 98.791.9 10.00.0 7.491.7 57.895.5 94.992.0 100.0 0.0 0.0 25.794.2 95.192.2 0.1 0.10.0 12.992.2 61.197.1 94.992.7 0.5 0.10.0 20.293.2 80.199.7 96.792.1 0.5 0.5 7.892.4 25.795.0 89.596.6 98.790.9 0.5 1.07.492.1 23.794.4 82.598.7 97.690.5 1.0 0.5 4.691.3 19.294.2 80.6911 95.891.6 1.0 1.08.391.7 18.392.9 72.099.8 97.591.3 2.5 2.5 0.0 17.893.4 60.397.5 98.490.9

a Values were expressed as mean9SE. There were 94–97 plants per treatment. Plants were grown under 16 h photoperiod and at 2592°C. nitrate on flowering were due to cerium or from 10.3 to 11.9 per plant (Fig. 3). Plants grow- lanthanum. ing in media with cerium nitrate and lanthanum nitrate (0.5–10.0 mM) produced about 10.7 to 14.7 3.4. Effects on reproducti6e growth flowers. Medium containing combination of 2.5 mM cerium nitrate and 2.5 mM lanthanum nitrate Thirty-five day-old plants growing in media was observed most effective in flower production with 0.5–10.0 mM cerium nitrate were significantly (Fig. 2 and Fig. 3). taller than those growing in basal medium (Fig. 1a). With 0.5 mM cerium nitrate, plant heights reached 13.0 cm compared to 7.9 cm in basal 3.5. Effects on the endogenous le6els of ZR, DHZR medium. Combinations of cerium nitrate and lan- and IPA thanum nitrate did not show cumulative effects on plant heights (Fig. 1a). Changes in endogenous levels of ZR, DHZR The average dry weight of 35 day-old plants and IPA in leaf and root tissues of plants growing growing in media with 0.5 or 2.5 mM cerium in the basal medium and in the medium containing nitrate (350 and 379 mg respectively) were signifi- 0.5 mM cerium nitrate and 0.5 mM lanthanum cantly higher than those growing in basal medium nitrate from day 12 to 18 were investigated. There (221 mg) (Fig. 1b). Dry weights of plants were also was no significant difference in endogenous level significantly higher with the addition of lanthanum of ZR, DHZR and IPA between the tissues in the nitrate or both (0.5–2.5 mM) (Fig. 1b and Fig. 2). basal medium and in REE-supplemented medium Plants growing in basal medium produced an (data not shown). average of 7.4 flowers (Fig. 3). The average num- However, we noticed that the endogenous levels ber of flowers per plant in media containing of ZR or DHZR in root tissues of plants in the cerium nitrate (0.5–10.0 mM) ranged from 10.4 to basal medium declined from about 218 or 249 12.3. In media containing 0.5–10.0 mM lanthanum pmol/g.f.wt. on day 12 to 39.9 pmol/g.f.wt. on day nitrate, the average number of flowers ranged 18. The endogenous levels of IPA in root tissues Y.-W. He, C.-S. Loh / Plant Science 159 (2000) 117–124 121

increased from 969 to 1351 pmol/g.f.wt. on day 12 to 4057–4419 pmol/g.f.wt. on day 16. Subse- quently, a signiﬁcant decrease in the level of IPA to 1120–1437 pmol/g.f.wt on day 18 was observed.

3.6. Effects on the endogenous le6els of carbohydrate in leaf tissues

There was no signiﬁcant difference in endogenous levels of sucrose, glucose and fructose between the leaf tissues (12–18 days after seed germination) in the basal medium and in REE- supplemented medium (data not shown). The levels of sucrose (0.7–1.1 mg/g.f.wt) remained stable 12–16 days after germination but decreased to about 0.1 mg/g.f.wt. on day 18. In the control and REE-treated leaf tissues, the levels of glucose and fructose on days 16 and 18 were signiﬁcantly higher than that on days 12 and 14 (data not shown).

3.7. Effects of 10−6 M IPA on root growth, plant height and ﬂowering in the presence of 0.5 vM cerium nitrate and 0.5 vM lanthanum nitrate

IPA was found to be most effective on root growth and flowering among six naturally occur- ring cytokinins tested (data not shown). In order to further study the mechanism of action of REEs Fig. 1. Effects of cerium nitrate and lanthanum nitrate on on flowering, the effects of exogenous IPA with or height (a) and dry weight (b) of A. thaliana 35 days after seed without REEs on root growth and floral initiation germination. There were 100 plants per treatment. Bars indi- were investigated (Table 3). Nine days after plants cate SE. at 2-leaf stage (12 days after seed germination) were transferred to medium supplemented with 10−6 M IPA, root growth was observed significantly increased. On day 20 after seed germination, 32% of the plants in the medium containing 10−6 M IPA were observed to produce flower buds compared to none in the control medium. However, addition of 10−6 M IPA significantly reduced plant height (Table 3). Nine days after plants at 2-leaf stage were transplanted to the medium with 10−6 M IPA, 0.5 mM cerium nitrate and 0.5 mM lanthanum nitrate, no beneficial effects on root growth were observed. Plant height and the percentage of plants with flower buds on day 20 were further reduced (Fig. 4, Table 3). The Fig. 2. Effects of 2.5 mM cerium nitrate and 2.5 mM lan- effects of 10−6 M IPA in the presence of 0.5 mM thanum nitrate on height and flower production of A. thaliana cerium nitrate and 0.5 mM lanthanum nitrate were 35 days after seed germination. Plants were grown under 16 h similar to that of 10−5 M IPA in terms of root photoperiod and at 2592°C. Scale bar=2.0 cm. (A) Control plants. (B) Treated plants. growth and plant height. 122 Y.-W. He, C.-S. Loh / Plant Science 159 (2000) 117–124

Fig. 3. Effects of cerium nitrate and lanthanum nitrate on the number of ﬂowers produced 35 days after seed germination. There were 100 plants per treatment. Bars indicate SE.

Fig. 4. Effects of 10−6 M IPA on root growth and flowering 4. Discussion in the presence of 0.5 mM cerium nitrate and 0.5 mM lanthanum nitrate. Plants were grown under 16 h photoperiod Cerium and lanthanum are two important ele- and at 2592°C. Scale bar=1.5cm. (A) 10−6 M IPA-treated ments of the REEs. The effects of REE on biolog- plant. (B) 10−6 M IPA-treated plant in the presence of 0.5 m m ical systems had attracted much attention since the M cerium nitrate and 0.5 M lanthanum nitrate. 1970s. Regrettably, results from field trials, pot amount of REEs can both be accurately con- trials and laboratory study on the effects of REEs trolled. The growth conditions of all plants are the on plant growth and development are inconsistent same. In addition, among the limited reports re- [4]. This is partly attributed to the fact that ab- garding REEs, investigations were only conducted sorption of REEs could be influenced by factors on one or two stages of plant growth and develop- such as soil pH, soil chelates and the available ment, such as root elongation [5,9,15], leaf and levels of fertilizers [4,15]. In view of this, the stem growth [11], spike production [10], and crop present investigation was conducted using a yield [12]. In the present study, the effects of REEs defined medium and under aseptic conditions. In on root growth, vegetative and reproductive this system, the composition of medium and the growth were all studied.

Table 3 Effects of IPA on root growth, plant height and ﬂowering in the presence of 0.5 mM cerium nitrate (Ce) and 0.5 mM lanthanum nitrate (La)

TreatmentAverage fresh weight of Average plant % of plants with ﬂower Average number of roots from 50 plants (mg)a height (cm)a buds 20 days after seed ﬂowers per planta germinationa

Control 71.399.4a 9.190.4a 0a 8.890.5a 10−6 M IPA104.5914.1b 4.690.2b 32.295.5b 4.190.2b 10−6 M IPA+0.5 mM 70.3917.2a 3.290.5c 21.493.7c 3.690.6b Ce+0.5 mMLa 10−5 M IPA52.497.9c 1.690.2d 0a 3.890.5b

a Values were expressed as mean9SE. There were 50 plants per treatment. The average root fresh weight was scored 21 days after seed germination. The plant height and the number of flowers per plant were scored 35 days after seed germination. For the experiment of IPA treatment, plants were first grown in 1/4 strength MS medium. Plants at two-leaf stage (12 days after seed germination) were transplanted to IPA-supplemented media for further growth. Means within the same column followed by the same letter were not significantly different according to Tukey’s pairwise comparisons (P=0.05). Y.-W. He, C.-S. Loh / Plant Science 159 (2000) 117–124 123

Experiments with corn and mungbean revealed carbohydrates were two of the most important that applications of lanthanum had no beneficial physiological signals that induced flowering. Ap- effect on the total dry matter yield [9]. In a field plication of 0.5 mM cerium and 0.5 mM lanthanum trial carried out in Australia, no response was on Arabidopsis could induce early flowering (Table found by spraying and seed dressing of a summer 2), but had no effects on the endogenous levels of fodder crop (Brassica sp.) with REEs (cited in [4]). ZR, DHZR, IPA, sucrose, glucose and fructose in In the present study, addition of cerium and lan- leaf and root tissues (data not shown). These thanum was found to have no beneficial effects on results implied that the increased reproductive the vegetative growth of Arabidopsis (Fig. 1a and growth and flowering induced by REEs was not Fig. 3a). However, REEs were found to have related to changes in the endogenous levels of beneficial effects on reproductive growth. Applica- cytokinins and carbohydrates. tion of low concentrations of cerium and lan- On the other hand, addition of 0.5 mM cerium thanum significantly promoted reproductive nitrate and 0.5 mM lanthanum significantly en- growth in Arabidopsis in terms of height of bolt- hanced the effects of 10−6 M IPA on root growth ing, earliness of flowering and the number of and floral initiation (Fig. 4, Table 3). Lanthanum flowers produced per plant (Tables 2 and 3, Fig. had been reported to increase greatly the binding 1). The beneficial effects of REEs on reproductive of auxin to A6ena coleoptile segments [17]. The growth were also reported in Day lily and narcis- action of cytokinin in stimulating the adventitious sus (cited in [4]). Meehan et al. [10] reported that bud initiation in Torenia stem segments was inhib- addition of REE promoted spike production in ited by 0.3 mM LaCl3 [18]. These results suggested wheat. that addition of REEs could increase the sensitiv- In the present study, addition of 0.5–10.0 mM ity of cell to plant growth regulators. However, cerium nitrate or lanthanum nitrate significantly there was very little information available on how increased root growth. For example, in the pres- REEs increased the sensitivity of cell to plant ence of 1.0 mM lanthanum nitrate the average root growth regulators. We suggested that REEs might length was 2.65 cm, about 32% increase over the affect the sensitivity of cell through their effects on control (Table 1). Similar findings were reported in the membrane fluidity and membrane binding of corn and mungbean [9,15]. For example, addition hormones. Firstly, La3+, by virtue of an ionic of lanthanum at 0.63 mM increased the root radius similar to Ca2+ and a valence higher than growth of corn by 36% and 0.19 mM lanthanum Ca2+, could bind at superficially located Ca2+ increased mungbean root by 21% relative to the absorption sites in a less reversible manner than controls [9]. How cerium and/or lanthanum pro- does Ca2+ itself. Membrane stabilization takes moted root growth remains unclear. Roots of place when an agent, such as La3+ or Ca2+, corns and mungbean were found to be able to causes protein and protein/liquid complexes to accumulate high levels of lanthanum in solutions become less fluid [19]. Some fluidity is essential to containing lanthanum [9]. Diatloff et al. [9], how- maintain optimum functions and destabilization ever, suspected that the enhanced root growth of may result in an improved membrane selectivity corn in solutions containing cerium or lanthanum and overall function [4]. Secondly, it had been might be due to amelioration of H+ toxicity. It suggested that hormones acting through an attach- was also suggested that, at low levels, the rare ment to site of action on a membrane might be earths acted as micronutrients, with toxicity occur- subjected to the effects of solutes (especially La3+) ring at high concentrations [11]. In the present that alter membrane characteristics [20]. Low con- study, the basal medium contained micronutrients centrations of cerium nitrate and lanthanum ni- and all media were prepared with the same pH. trate can be expected to be particularly significant Thus it was possible that the increased root in this respect because of its potency as membrane growth after REE application was not due to the stabilizer [4]. effects of micronutrients or amelioration of H+ Non-hormonal agents that promote only repro- toxicity. ductive growth and flowering but not vegetative There was very little information available on growth are useful for many crop species. For the mechanism of action of REEs on flowering. example, in grain crops or floricultural crops, it is According to Bernier et al. [16], cytokinins and undesirable to produce excessive number of leaves 124 Y.-W. He, C.-S. Loh / Plant Science 159 (2000) 117–124 or shoots. In certain breeding programs, earlier [6] R.F.M. Van Steveninck, M.E. Van Steveninck, D. 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