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LEAFY homologous gene
Plant Science 176 (2009) 643–649
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Gibberellin promotes flowering of chrysanthemum by upregulating CmFL, a chrysanthemum FLORICAULA/LEAFY homologous gene Katsuhiko Sumitomo, Tuoping Li 1, Tamotsu Hisamatsu * National Institute of Floricultural Science (NIFS), National Agriculture and Food Research Organization (NARO), 2-1 Fujimoto, Tsukuba, Ibaraki 305-8519, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 May 2008 Received in revised form 27 January 2009 Accepted 3 February 2009 Available online 11 February 2009
The plant hormone gibberellin (GA) induces flower formation in several long-day (LD) plants, and exogenous GA can partly substitute for chilling treatment in cold-dependent plants. Both chilling and GA are required to promote flowering of a short-day (SD) plant chrysanthemum as observed in many plants. Chilling and GA requirement for flowering of four cultivars were examined, and genetic variation in them was shown: those that required GA also required chilling for flowering, but those that did not require GA showed no chilling requirement. With regard to LEAFY in Arabidopsis thaliana, GA promoted the expression of CmFL, a FLORICAULA/LEAFY homologous gene from chrysanthemum, and the upregulation of CmFL required GA in cultivars with a chilling requirement. Therefore, this GA requirement can be principally attributed to the chilling requirement for flowering. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chilling requirement Chrysanthemum FLORICAULA/LEAFY homologous gene Flowering Gibberellin
1. Introduction Chrysanthemums (Chrysanthemum morifolium Ramat.) are important ornamental plants around the world. This short-day (SD) herbaceous perennial shows seasonal changes in extension growth and flowering, and this is an adaptation to a temperate climate ([1,2], Hisamatsu, unpublished). During winter, plants cease elongation and flowering, show slow expansion of leaf, and form rosettes to avoid cold damage. These plants have a large chilling requirement for the subsequent resumption of elongation. In spring, they start extension growth as temperature rises. In autumn, buds flower, while the remaining buds enter dormancy under SD and relative low temperature conditions. The effects of environmental and physiological factors on this growth habit remain poorly understood, but such knowledge is essential for regulating their growth and flowering in order to produce plants with uniform quality in commercial production. Treatment with the plant hormone gibberellin (GA) induces flower formation in a number of species grown under noninductive conditions [3,4]. In the long-day (LD) plant Lolium temulentum, GAs act as a floral signal along with the product of the gene FLOWERING LOCUS T [5,6]. In Arabidopsis thaliana, another LD plant, the use of several GA biosynthesis and signalling mutants has confirmed that
* Corresponding author. Tel.: +81 29 838 6801; fax: +81 29 838 6842. E-mail address: [email protected] (T. Hisamatsu). 1 Present address: Department of Food Science, Tianjing Agricultural University, 300384 Tianjin, China. 0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.02.003
endogenous GA promotes flowering and that severe reduction of endogenous GAs delays flowering in LD and prevents flowering in SD [7–9]. At the molecular level, GA promotion of flowering involves increased expression of LEAFY (LFY), a floral meristem identity gene, by activating its LFY promoter [10]. In LD and chilling-requiring plants, GA may also regulate their flowering. Application of GA can partly substitute for their chilling treatment [4]. Exposure to low temperature may increase their GA biosynthesis after return to higher temperatures as compared to those without chilling treatment [11–14], and low temperatures may enhance the sensitivity or responsiveness of plants to GAs [15–19]. The requirement of chilling for both extension growth and flowering of chrysanthemum [20] suggests the possibility of a common GA regulation. For flowering of chrysanthemum, GA can substitute for the chilling requirement in photoperiod-insensitive cultivars [21]. However, in SD cultivars under non-inductive LD, GA does not induce and hasten flowering [21,22]. This indicates that GA does not substitute for SD in the floral transition. Moreover, this is consistent with the evidence that GA synthesis only increases in LD [4] and that GA is required in LD-responsive plants [6,8,23]. To investigate the relationship between the responsiveness of flowering to chilling and GA and the role of GA in flowering of chrysanthemum, we used chilling-dependent and chilling-independent cultivars and examined the effect of GA on a floral meristem identity gene, CmFL (GenBank accession no. AB451217), which is a FLORICAULA/LFY homologue. FLORICAULA (FLO) and LFY play important roles in the floral transition. FLO (GenBank accession no. M55525) is a floral meristem identity gene from Antirrhinum majus, and LFY (GenBank accession no. DQ447103) is
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its Arabidopsis homologue [24,25]. Here, we confirm that GA is involved in the chilling requirement of these plants, and show that GA upregulates CmFL as a part of the floral transition of chillingdependent chrysanthemums. 2. Materials and methods 2.1. Effects of GA and chilling on flowering Rooted cuttings of chrysanthemum cultivars ‘City’, ‘Naganoqueen’ and ‘Reagan’ and line 94-4008 were planted in 7.5-cm plastic pots containing a commercial horticultural soil (KurehaEngei-Baido, Kureha Chemical Co. Ltd., Tochigi, Japan). Flowering of chrysanthemum is inhibited by an exposure to light given during the night (night break). Plants were grown in a glasshouse (heated when the temperature decreased below 15 8C and ventilated when it increased above 25 8C) under natural photoperiod conditions with a 5-h night break from 10 p.m. to 3 a.m. with incandescent lamps. Uniconazole-P (UCZ, Sumitomo Chemical Co. Ltd., Tokyo, Japan), an inhibitor of endogenous GA biosynthesis [26], was used to remove the effect of endogenous GA before GA3 treatments to examine GA3 sensitivity. Ten days after planting, UCZ was applied to the soil in a 10-ml water solution containing 50 mg l1 of active ingredient. At the same time and 1 week later, all aerial parts were sprayed to runoff with a solution containing 25 mg l1 of UCZ. After these UCZ treatments, half of the plants were transferred into a growth chamber that was controlled at 208/15 8C (light/dark) with a floral-inducing 12-h SD photoperiod. The other plants were given a chilling treatment in another growth chamber (3 8C, 12-h light period with a 5-h night break) for 8 weeks, sprayed one more time with UCZ and transferred into a growth chamber with the same 12-h SD and 208/15 8C conditions as mentioned above to test the effect of chilling on flowering. To examine the effect of GA on flowering, UCZ-treated plants of both unchilled and chilled population were given GA3 treatments. GA3 (Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan) was prepared as a 20% aqueous ethanol solution (v/v, containing 0.05% Tween 20). Ten microliters of GA3 solution (0, 0.0025, 0.025, or 0.25 mg ml1) was applied four times to shoot tips on days 0, 2, 4 and 6 from transfer of the plants into the SD growth chamber. Total dose of applied GA3 was 0, 0.1, 1, or 10 mg per plant, respectively. Plants given neither UCZ nor GA3 in both unchilled and chilled population were also used in the experiment. We recorded the date when flower buds became visible, and the percentage of flowering plants on 56 d after transfer into the SD growth chamber. Plants that had no visible buds were dissected under a binocular microscope to examine whether the apices had initiated flower buds. Stem length was measured twice: on transfer into the SD growth chamber and at the end of the experiment. 2.2. The effect of GA on the expression of the CmFL gene Rooted cuttings of ‘Reagan’ and line 94-4008 were planted in 7.5-cm plastic pots and grown in a glasshouse with a night break as mentioned above. Plants were treated with UCZ 10 d after planting and then given an 8-week chilling treatment after UCZ treatments as described above. All plants were transferred into a growth chamber that was controlled at 208/17.5 8C (light/dark) with a floral-inducing 12-h SD photoperiod, and 10 ml of GA3 solution (0 or 0.25 mg ml1) was applied four times to shoot tips of UCZtreated plants on days 0, 2, 4 and 6 from transfer of the plants into the SD growth chamber. Total dose of GA3 was 0 or 10 mg per plant. Flowering and stem length were determined 28 d after transfer into the SD growth chamber as described above. For studies of the expression of the CmFL gene, 5-mm-long shoot tips were collected on 0, 7, 14, 21 and 28 d after transfer into
the SD growth chamber. Total RNA was extracted with an RNeasy Plant Mini Kit (Qiagen K.K., Tokyo, Japan) and treated with RNasefree DNase (Qiagen K.K.) according to the manufacturer’s instructions. For each sample, 500 ng of total RNA was reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics K.K., Tokyo, Japan) according to the manufacturer’s instructions. The cDNA was diluted to 4% of its original concentration, and 5 ml was used in a 15-ml quantitative realtime polymerase chain reaction (Q-PCR) reaction mixture with SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan) on a LightCycler system (Roche Diagnostics K.K.). A chrysanthemum CmACTIN cDNA (GenBank accession no. AB205087) was identified by reverse-transcription PCR and used as a constitutive control. QPCR analysis of these genes was performed with the primers CmACTIN-forward (50 -GATGACGCAGATCATGTTCG-30 ) and CmACTIN-reverse (50 -AGCATGTGGAAGTGCATACC-30 ), CmFL-forward (50 CATTGATGCCATATTTAACTC-30 ) and CmFL-reverse (50 -ACACGGATCATTCATTGTATA-30 ). PCR reactions were performed with an initial denaturing step of 95 8C for 20 s, followed by 40 cycles of 95 8C for 5 s, 60 8C for 20 s, and 72 8C for 15 s. Transcript level of CmFL in the sample were compared directly after normalization against a CmACTIN loading standard. The calibrator sample was designated as the most highly expressed time point for CmFL, with an expression of 1. 3. Results 3.1. Effects of GA and chilling on flowering and growth Stem elongation of non-UCZ- and non-GA3-treated plants with chilling was 3.5-fold and 6.8-fold larger than those without chilling in ‘City’ and line 94-4008, respectively (Figs. 1 and 2). Chilling promoted stem elongation of these cultivars. On the other hand, chilling had no effect on stem elongation in non-UCZ- and non-GA3-treated plants of ‘Reagan’ and promoted slight elongation in those of ‘Nagano-queen’. UCZ treatment completely inhibited stem elongation, and plants formed a rosette in all cultivars. GA3 reversed this cessation of stem elongation, and stem length increased as GA3 dose increased from 0.1 to 10 mg per plant. In ‘City’ and line 94-4008, stem length of the chilled plants treated with 1 and 10 mg GA3 were larger than the unchilled plants treated with 1 and 10 mg GA3. Chilling clearly enhanced responsiveness to exogenous GA3 in stem elongation of ‘City’ and line 94-4008 plants. In ‘City’ and line 94-4008, non-UCZ- and non-GA3-treated plants without chilling did not show flowering (Figs. 2 and 3). On the other hand, non-UCZ- and non-GA3-treated plants with chilling showed flower bud initiation. ‘City’ and line 94-4008 plants showed an obligate requirement for chilling for flowering. UCZ application blocked this flowering. This inhibition was reversed by 1 and 10 mg GA3 applications, and the percentage of flowering plant was higher as GA3 dose increased. Only 7.1 and 21.4% of plants without chilling showed flower bud initiation at the highest dose of GA3 in ‘City’ and line 94-4008, respectively. On the other hand, 84.6 and 92.9% of plants with chilling showed flower bud initiation at 1 mg GA3 in ‘City’ and line 944008, respectively. Further, all plants showed flower bud initiation at 10 mg GA3. Chilling strongly enhanced responsiveness to exogenous GA3 in flowering and stem elongation of ‘City’ and line 94-4008 plants. In ‘Reagan’ and ‘Nagano-queen’, all plants initiated flower buds regardless of any chemical application. UCZ did not block flowering although ‘Reagan’ and ‘Nagano-queen’ plants showed the same inhibition of stem elongation by UCZ and the same reversion of stem elongation by GA3 as ‘City’ and line 94-4008 plants (Figs. 1–3). ‘Reagan’ and ‘Nagano-queen’ plants also showed no chilling requirement for
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Fig. 1. Effect of GA3 and chilling on stem elongation in chrysanthemum (A) ‘City’, (B) line 94-4008, (C)‘Reagan’ and (D) ‘Nagano-queen’. Shaded bars represent unchilled plants, open bars represent 8-week chilled plants. Error bars represent S.E. (n = 14). GA3 (0, 0.1, 1 or 10 mg per plant in total) was applied over 6 d after UCZ application. No chemicals, non-UCZ- and non-GA3-treated plants.
flowering. Therefore, ‘Reagan’ and ‘Nagano-queen’ serve as de facto controls for flowering. 3.2. The effect of GA on the expression of the CmFL gene In confirmation of the data of plants with chilling in Fig. 3, UCZ completely inhibited flowering of line 94-4008 plants for more than 90 d, and GA3 reversed this inhibition (Table 1). Days to visible flower buds of plants treated with 10 mg GA3 after UCZ application (UCZ + GA plants) was the same as those of non-UCZ- and nonGA3-treated plants. UCZ inhibited stem elongation. GA3 reversed this cessation of stem elongation. ‘Reagan’ plants showed parallel reduction in stem elongation by UCZ, but all plants flowered. Days to visible flower buds increased in plants treated with UCZ (UCZ plants) and UCZ + GA plants compared to those in non-UCZ- and non-GA3-treated plants. The effect of GA3 on the expression of the CmFL gene was examined. The coding region of CmFL is 1,239 bp, and encodes a putative protein of 413 amino acids, which is 97% identical to DFL (GenBank accession no. AY559245), a FLO/LFY homologous gene from the wild species of chrysanthemum, Dendranthema lavandufolium [27]. Comparison of an amino acid sequence alignment containing CmFL and other FLO/LFY proteins showed the presence of several conserved regions (see Supplementary Fig. S1). The putative amino acid sequence of CmFL showed 60% homology with LFY of A. thaliana, 68% with FLO of A. majus, 66% with FALSIFLORA of Solanum lycopersicum (GenBank accession no. AF197934), 67% with ALF of Petunia x hybrida (GenBank accession no. AF030171), and 66% with UNIFOLIATA of Pisum sativum (GenBank accession no. AF010190). The abundance of transcripts of CmFL in shoot tips
was determined by using Q-PCR while the floral transition was occurring (Li, unpublished). CmFL showed threshold cycle number response at 18–28 cycles. In addition, transgenic Arabidopsis plants constitutively expressing CmFL flowered earlier than wild-type
Table 1 Effect of GA3 on flowering and extension growth in chrysanthemum line 94-4008 and ‘Reagan’. Significant differences determined at P < 0.01 using Tukey’s test are indicated by different letters in each line. Values are mean S.E. (n = 12). No chemicals, non-UCZ- and non-GA3-treated plants. UCZ, an application to soil (0.5 mg/ pot) and spray treatments of UCZ (25 mg l1). UCZ + GA, GA3 (10 mg per plant in total) was applied over 6 d after UCZ application. Treatments
UCZ GA3
No chemicals
UCZ
UCZ + GA
+
+ +
Line and cultivar
Treatments No chemicals
UCZ
UCZ + GA
26.7 1.8 b 23.4 1.4 b
0.6 0.1 a 0.7 0.1 a
31.0 0.6 b 27.9 0.7 c
Percentage of flowering plants 94-4008 100 Reagan 100
0 100
100 100
Days to visible flower buds 94-4008 23.8 0.7 a Reagan 18.6 0.1 a
NBa (>90) 27.8 0.1 c
22.3 0.6 a 20.1 0.3 b
Stem elongation (cm) 94-4008 Reagan
a
NB, no visible flower buds.
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Fig. 2. Plants grown in SD with or without the application of GA3. (A) Unchilled line 94-4008. (B) Chilled line 94-4008. (C) Unchilled ‘Reagan’. (D) Chilled ‘Reagan’. GA3 (0, 0.1, 1 or 10 mg per plant in total) was applied over 6 d after UCZ application. No chemicals, non-UCZ- and non-GA3-treated plants.
Col plants under 9-h SD photoperiod at 20 8C (Fig. 4, and see Supplementary Table S1). The shoot tip transcript level of CmFL was low at transfer into the SD growth chamber (Fig. 5). By day 14, CmFL expression began to increase in non-UCZ- and non-GA3-treated plants and UCZ + GA plants. In UCZ plants of line 94-4008, CmFL expression was blocked in parallel with the blocking of flowering. In contrast, in ‘Reagan’ plants, UCZ did not block flowering and had little effect on CmFL expression. 4. Discussion Chrysanthemum shows large seasonal difference in extension growth and flowering. Extension growth and flowering depend on the combination of temperature and day length. In our studies, temperature is significant, and temperature of the previous season is an important factor in determining the capacity for growth and flowering in the subsequent season. Exposure to summer heat reduces this capacity. This decrease narrows the temperature range over which extension growth and flowering are possible, and plants show slower extension growth and flowering in autumn
than in spring at the same growing temperature [2]. For example, at growing temperatures of 158/10 8C (light/dark), chrysanthemum plants showed rapid extension growth and flowering in spring but showed neither in autumn after exposure to high temperatures [2]. Here, the dependence of extension growth on exogenous GA shows that GA promotes the capacity for extension growth (Fig. 1). Chilling is also involved because it increased the responsiveness of stem elongation to exogenous GA3. The link between the seasonality of extension capacity and responsiveness to GA follows from our use of UCZ to reduce endogenous GA levels in chrysanthemum [28]. Consistent with this claim, endogenous GA level in bioassays increases rapidly in chrysanthemum in association with high extension growth rates after chilling [29]. Chilling also promotes flowering [20], and this is evident in our experiments. Potential genetic variation in the chilling requirement for the promotion of flowering is also evident for the cultivars studied here. ‘City’ and line 94-4008 have a distinct seasonality of flowering associated with their chilling requirement, and they rarely flower after autumn, as shown in a further study (Hisamatsu, unpublished). In contrast, ‘Reagan’ and ‘Nagano-queen’ with their lack of a chilling requirement showed a stable flowering response in our study ([2], Hisamatsu, unpublished). Annual commercial production requires the selection of novel adaptive flowering traits suited to the greenhouse environment, where chrysanthemums are never exposed to chilling thus no flowers could be harvested if the plants required chilling for flowering. ‘Reagan’ and ‘Naganoqueen’ meet this requirement since they have lost the chilling requirement for flowering during the selection in breeding for stable flowering. Because ‘City’ and line 94-4008 require chilling for the floral transition, they are more similar to the progenitor wild chrysanthemums in Japan, which require chilling for the floral transition (Kawata, unpublished), although the ultimate progenitor of the current chrysanthemum cultivars is yet to be identified [30]. The GA requirement for the floral transition varied from an absence in ‘Reagan’ and ‘Nagano-queen’ to a need for GA in ‘City’ and line 94-4008, where UCZ inhibited flowering and GA reversed it (Fig. 3). We suggest that GA is involved in the floral transition as an early informational signal because the UCZ-treated plants did not show floral morphogenesis in ‘City’ and line 94-4008 by the end of the experiment, i.e., 56 d after transfer to SD conditions. Especially, GA enhanced the expression of the CmFL gene (Fig. 5), a floral meristem identity gene, which in Arabidopsis responds directly to GA [10]. It is yet to be determined whether there is a common signal pathway linking chilling and GA to extension growth and flowering, but since there is no seasonal chilling requirement for flowering of ‘Reagan’ and ‘Nagano-queen’, their endogenous GA level may be non-limiting. There is a dominant requirement for SD exposure for flowering of chrysanthemum, and GA does not induce flowering of plants maintained in LD [21,22]. Compared with plants transferred to SD, which by 28 d had flowered (Table 1) and which showed high levels of CmFL expression (Fig. 5), gene expression was negligible in plants maintained in LD at 28 d, and these plants had still not flowered by 50 d (Li, unpublished). A failure of GA to substitute for SD for flowering is consistent with the extensive evidence that at least for LD species, it is LD that increases GA biosynthesis in association with their flowering [4]. In contrast, chilling-induced flowering can be replaced by application of GA to UCZ-treated plants (Fig. 3) and even to unchilled photoperiod-insensitive cultivars [21]. A partial explanation for these findings is that chilling promotes GA biosynthesis in chrysanthemum, as suggested by GA bioassays [29]. However, there must be some additional changes in the cold since GA response was greater after chilling (Figs. 1 and 3). No molecular biological approach has been developed to study flowering in chrysanthemum, although they
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Fig. 3. Effect of GA3 and chilling on flowering in chrysanthemum (A) ‘City’, (B) line 94-4008, (C) ‘Reagan’ and (D) ‘Nagano-queen’. Percentage of flowering plants of unchilled (closed circles, broken line) or 8-week chilled plants (open squares, solid line). Data were collected 56 days after transfer into the SD growth chamber. Error bars represent S.E. (n = 14). GA3 (0, 0.1, 1 or 10 mg per plant in total) was applied over 6 d after UCZ applications. No chemicals, non-UCZ- and non-GA3-treated plants.
are important ornamental crops and growers adopt precision regulation of flowering in commercial production. At the molecular level, our results suggest that GA may regulate flowering through upregulation of CmFL expression, as demonstrated here for chrysanthemum and previously for Arabidopsis [10]. However, because of the clear parallels between chilling-induced flowering and vernalization responses as studied in Arabidopsis, wheat and barley, there could also be important changes in the expression of a whole suite of vernalization-responsive genes related to FLOWERING LOCUS C (FLC) or VERNALIZATION2, which are considered as negative regulators whose level is depressed by chilling [31–34].
GA could compete with the repressor in chrysanthemum. The smaller dose of GA required for flowering in the chilled plants indicates that chilling treatment reduced the effect of the repressor, as suggested by the results that the extent of chilling quantitatively determines the level of FLC in Arabidopsis [35]. The chilled plants still showed GA dependence for flowering and upregulation of CmFL since the effect of repressor might remain after 8 weeks of chilling [35,36]. GA may be less important for flowering in ‘Reagan’ and ‘Nagano-queen’. Considering these plants to be similar to mutants containing a deletion in the flowering repressor that chilling and GA repress can explain the
Fig. 4. CmFL induces early flowering in Arabidopsis thaliana. Wild-type Col plant (A) and 35S::CmFL overexpression plant (B) grown under 9-h SD photoperiod at 20 8C. The terminal flower is visible (arrow).
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Fig. 5. Effect of GA3 on the expression of CmFL in chrysanthemum (A) ‘Reagan’ and (B) line 94-4008. Changes in the gene expression are shown as calculated value relative to the maximum value in the same Q-PCR assay. Error bars represent SE (n = 4). No chemicals (closed circles and solid line), non-UCZ- and non-GA3-treated plants. UCZ (closed triangles and broken line), an application to soil (0.5 mg per pot) and spray treatments of UCZ (25 mg l1). UCZ + GA (open circles and solid line), GA3 (10 mg per plant in total) was applied over 6 d after UCZ application.
independence of GA for flowering and upregulation of CmFL in them. Genetic variation in the GA requirement for the floral transition can be principally attributed to the chilling requirement for flowering. Overall, our preferred model of the regulation of flowering of chrysanthemum involves a chilling response that includes both GA and an unknown separate input. The latter might be considered as negative regulators, as shown in the vernalization response for Arabidopsis and cereals. In our ongoing studies, the cultivar differences that we have reported here for extension growth and flowering of chrysanthemum might allow us to understand such complex environmental regulation, however, this will first require identification of gene homologous to those involved in a chilling response as in Arabidopsis and cereals. Acknowledgments We thank Dr. Rod W. King (Commonwealth Scientific and Industrial Research Organisation, Canberra, Australia) for his valuable comments on the manuscript. This work was supported by grants from National Agriculture and Food Research Organization (NARO), Japan.
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K. Sumitomo et al. / Plant Science 176 (2009) 643–649 [29] P.B. Tompsett, W.W. Schwabe, Growth hormone changes in chrysanthemum moliforium. Effects of environmental factors controlling flowering, Ann. Bot. 38 (1974) 269–285. [30] S. Kishimoto, R. Aida, M. Shibata, Identification of chloroplast DNA variations by PCR-RFLP analysis in Dendranthema, J. Jpn. Soc. Hortic. Sci. 72 (2003) 197–204. [31] S.D. Michaels, R.M. Amasino, FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering, Plant Cell 11 (1999) 949–956. [32] B. Trevaskis, D.J. Bagnall, M.H. Ellis, W.J. Peacock, E.S. Dennis, MADS box genes control vernalization-induced flowering in cereals, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 13099–13104.
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Gibberellin promotes flowering of chrysanthemum by upregulating CmFL, a chrysanthemum FLORICAULA/LEAFY homologous gene Katsuhiko Sumitomo, Tuoping Li 1, Tamotsu Hisamatsu * National Institute of Floricultural Science (NIFS), National Agriculture and Food Research Organization (NARO), 2-1 Fujimoto, Tsukuba, Ibaraki 305-8519, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 May 2008 Received in revised form 27 January 2009 Accepted 3 February 2009 Available online 11 February 2009
The plant hormone gibberellin (GA) induces flower formation in several long-day (LD) plants, and exogenous GA can partly substitute for chilling treatment in cold-dependent plants. Both chilling and GA are required to promote flowering of a short-day (SD) plant chrysanthemum as observed in many plants. Chilling and GA requirement for flowering of four cultivars were examined, and genetic variation in them was shown: those that required GA also required chilling for flowering, but those that did not require GA showed no chilling requirement. With regard to LEAFY in Arabidopsis thaliana, GA promoted the expression of CmFL, a FLORICAULA/LEAFY homologous gene from chrysanthemum, and the upregulation of CmFL required GA in cultivars with a chilling requirement. Therefore, this GA requirement can be principally attributed to the chilling requirement for flowering. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chilling requirement Chrysanthemum FLORICAULA/LEAFY homologous gene Flowering Gibberellin
1. Introduction Chrysanthemums (Chrysanthemum morifolium Ramat.) are important ornamental plants around the world. This short-day (SD) herbaceous perennial shows seasonal changes in extension growth and flowering, and this is an adaptation to a temperate climate ([1,2], Hisamatsu, unpublished). During winter, plants cease elongation and flowering, show slow expansion of leaf, and form rosettes to avoid cold damage. These plants have a large chilling requirement for the subsequent resumption of elongation. In spring, they start extension growth as temperature rises. In autumn, buds flower, while the remaining buds enter dormancy under SD and relative low temperature conditions. The effects of environmental and physiological factors on this growth habit remain poorly understood, but such knowledge is essential for regulating their growth and flowering in order to produce plants with uniform quality in commercial production. Treatment with the plant hormone gibberellin (GA) induces flower formation in a number of species grown under noninductive conditions [3,4]. In the long-day (LD) plant Lolium temulentum, GAs act as a floral signal along with the product of the gene FLOWERING LOCUS T [5,6]. In Arabidopsis thaliana, another LD plant, the use of several GA biosynthesis and signalling mutants has confirmed that
* Corresponding author. Tel.: +81 29 838 6801; fax: +81 29 838 6842. E-mail address: [email protected] (T. Hisamatsu). 1 Present address: Department of Food Science, Tianjing Agricultural University, 300384 Tianjin, China. 0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.02.003
endogenous GA promotes flowering and that severe reduction of endogenous GAs delays flowering in LD and prevents flowering in SD [7–9]. At the molecular level, GA promotion of flowering involves increased expression of LEAFY (LFY), a floral meristem identity gene, by activating its LFY promoter [10]. In LD and chilling-requiring plants, GA may also regulate their flowering. Application of GA can partly substitute for their chilling treatment [4]. Exposure to low temperature may increase their GA biosynthesis after return to higher temperatures as compared to those without chilling treatment [11–14], and low temperatures may enhance the sensitivity or responsiveness of plants to GAs [15–19]. The requirement of chilling for both extension growth and flowering of chrysanthemum [20] suggests the possibility of a common GA regulation. For flowering of chrysanthemum, GA can substitute for the chilling requirement in photoperiod-insensitive cultivars [21]. However, in SD cultivars under non-inductive LD, GA does not induce and hasten flowering [21,22]. This indicates that GA does not substitute for SD in the floral transition. Moreover, this is consistent with the evidence that GA synthesis only increases in LD [4] and that GA is required in LD-responsive plants [6,8,23]. To investigate the relationship between the responsiveness of flowering to chilling and GA and the role of GA in flowering of chrysanthemum, we used chilling-dependent and chilling-independent cultivars and examined the effect of GA on a floral meristem identity gene, CmFL (GenBank accession no. AB451217), which is a FLORICAULA/LFY homologue. FLORICAULA (FLO) and LFY play important roles in the floral transition. FLO (GenBank accession no. M55525) is a floral meristem identity gene from Antirrhinum majus, and LFY (GenBank accession no. DQ447103) is
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its Arabidopsis homologue [24,25]. Here, we confirm that GA is involved in the chilling requirement of these plants, and show that GA upregulates CmFL as a part of the floral transition of chillingdependent chrysanthemums. 2. Materials and methods 2.1. Effects of GA and chilling on flowering Rooted cuttings of chrysanthemum cultivars ‘City’, ‘Naganoqueen’ and ‘Reagan’ and line 94-4008 were planted in 7.5-cm plastic pots containing a commercial horticultural soil (KurehaEngei-Baido, Kureha Chemical Co. Ltd., Tochigi, Japan). Flowering of chrysanthemum is inhibited by an exposure to light given during the night (night break). Plants were grown in a glasshouse (heated when the temperature decreased below 15 8C and ventilated when it increased above 25 8C) under natural photoperiod conditions with a 5-h night break from 10 p.m. to 3 a.m. with incandescent lamps. Uniconazole-P (UCZ, Sumitomo Chemical Co. Ltd., Tokyo, Japan), an inhibitor of endogenous GA biosynthesis [26], was used to remove the effect of endogenous GA before GA3 treatments to examine GA3 sensitivity. Ten days after planting, UCZ was applied to the soil in a 10-ml water solution containing 50 mg l1 of active ingredient. At the same time and 1 week later, all aerial parts were sprayed to runoff with a solution containing 25 mg l1 of UCZ. After these UCZ treatments, half of the plants were transferred into a growth chamber that was controlled at 208/15 8C (light/dark) with a floral-inducing 12-h SD photoperiod. The other plants were given a chilling treatment in another growth chamber (3 8C, 12-h light period with a 5-h night break) for 8 weeks, sprayed one more time with UCZ and transferred into a growth chamber with the same 12-h SD and 208/15 8C conditions as mentioned above to test the effect of chilling on flowering. To examine the effect of GA on flowering, UCZ-treated plants of both unchilled and chilled population were given GA3 treatments. GA3 (Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan) was prepared as a 20% aqueous ethanol solution (v/v, containing 0.05% Tween 20). Ten microliters of GA3 solution (0, 0.0025, 0.025, or 0.25 mg ml1) was applied four times to shoot tips on days 0, 2, 4 and 6 from transfer of the plants into the SD growth chamber. Total dose of applied GA3 was 0, 0.1, 1, or 10 mg per plant, respectively. Plants given neither UCZ nor GA3 in both unchilled and chilled population were also used in the experiment. We recorded the date when flower buds became visible, and the percentage of flowering plants on 56 d after transfer into the SD growth chamber. Plants that had no visible buds were dissected under a binocular microscope to examine whether the apices had initiated flower buds. Stem length was measured twice: on transfer into the SD growth chamber and at the end of the experiment. 2.2. The effect of GA on the expression of the CmFL gene Rooted cuttings of ‘Reagan’ and line 94-4008 were planted in 7.5-cm plastic pots and grown in a glasshouse with a night break as mentioned above. Plants were treated with UCZ 10 d after planting and then given an 8-week chilling treatment after UCZ treatments as described above. All plants were transferred into a growth chamber that was controlled at 208/17.5 8C (light/dark) with a floral-inducing 12-h SD photoperiod, and 10 ml of GA3 solution (0 or 0.25 mg ml1) was applied four times to shoot tips of UCZtreated plants on days 0, 2, 4 and 6 from transfer of the plants into the SD growth chamber. Total dose of GA3 was 0 or 10 mg per plant. Flowering and stem length were determined 28 d after transfer into the SD growth chamber as described above. For studies of the expression of the CmFL gene, 5-mm-long shoot tips were collected on 0, 7, 14, 21 and 28 d after transfer into
the SD growth chamber. Total RNA was extracted with an RNeasy Plant Mini Kit (Qiagen K.K., Tokyo, Japan) and treated with RNasefree DNase (Qiagen K.K.) according to the manufacturer’s instructions. For each sample, 500 ng of total RNA was reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics K.K., Tokyo, Japan) according to the manufacturer’s instructions. The cDNA was diluted to 4% of its original concentration, and 5 ml was used in a 15-ml quantitative realtime polymerase chain reaction (Q-PCR) reaction mixture with SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan) on a LightCycler system (Roche Diagnostics K.K.). A chrysanthemum CmACTIN cDNA (GenBank accession no. AB205087) was identified by reverse-transcription PCR and used as a constitutive control. QPCR analysis of these genes was performed with the primers CmACTIN-forward (50 -GATGACGCAGATCATGTTCG-30 ) and CmACTIN-reverse (50 -AGCATGTGGAAGTGCATACC-30 ), CmFL-forward (50 CATTGATGCCATATTTAACTC-30 ) and CmFL-reverse (50 -ACACGGATCATTCATTGTATA-30 ). PCR reactions were performed with an initial denaturing step of 95 8C for 20 s, followed by 40 cycles of 95 8C for 5 s, 60 8C for 20 s, and 72 8C for 15 s. Transcript level of CmFL in the sample were compared directly after normalization against a CmACTIN loading standard. The calibrator sample was designated as the most highly expressed time point for CmFL, with an expression of 1. 3. Results 3.1. Effects of GA and chilling on flowering and growth Stem elongation of non-UCZ- and non-GA3-treated plants with chilling was 3.5-fold and 6.8-fold larger than those without chilling in ‘City’ and line 94-4008, respectively (Figs. 1 and 2). Chilling promoted stem elongation of these cultivars. On the other hand, chilling had no effect on stem elongation in non-UCZ- and non-GA3-treated plants of ‘Reagan’ and promoted slight elongation in those of ‘Nagano-queen’. UCZ treatment completely inhibited stem elongation, and plants formed a rosette in all cultivars. GA3 reversed this cessation of stem elongation, and stem length increased as GA3 dose increased from 0.1 to 10 mg per plant. In ‘City’ and line 94-4008, stem length of the chilled plants treated with 1 and 10 mg GA3 were larger than the unchilled plants treated with 1 and 10 mg GA3. Chilling clearly enhanced responsiveness to exogenous GA3 in stem elongation of ‘City’ and line 94-4008 plants. In ‘City’ and line 94-4008, non-UCZ- and non-GA3-treated plants without chilling did not show flowering (Figs. 2 and 3). On the other hand, non-UCZ- and non-GA3-treated plants with chilling showed flower bud initiation. ‘City’ and line 94-4008 plants showed an obligate requirement for chilling for flowering. UCZ application blocked this flowering. This inhibition was reversed by 1 and 10 mg GA3 applications, and the percentage of flowering plant was higher as GA3 dose increased. Only 7.1 and 21.4% of plants without chilling showed flower bud initiation at the highest dose of GA3 in ‘City’ and line 94-4008, respectively. On the other hand, 84.6 and 92.9% of plants with chilling showed flower bud initiation at 1 mg GA3 in ‘City’ and line 944008, respectively. Further, all plants showed flower bud initiation at 10 mg GA3. Chilling strongly enhanced responsiveness to exogenous GA3 in flowering and stem elongation of ‘City’ and line 94-4008 plants. In ‘Reagan’ and ‘Nagano-queen’, all plants initiated flower buds regardless of any chemical application. UCZ did not block flowering although ‘Reagan’ and ‘Nagano-queen’ plants showed the same inhibition of stem elongation by UCZ and the same reversion of stem elongation by GA3 as ‘City’ and line 94-4008 plants (Figs. 1–3). ‘Reagan’ and ‘Nagano-queen’ plants also showed no chilling requirement for
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Fig. 1. Effect of GA3 and chilling on stem elongation in chrysanthemum (A) ‘City’, (B) line 94-4008, (C)‘Reagan’ and (D) ‘Nagano-queen’. Shaded bars represent unchilled plants, open bars represent 8-week chilled plants. Error bars represent S.E. (n = 14). GA3 (0, 0.1, 1 or 10 mg per plant in total) was applied over 6 d after UCZ application. No chemicals, non-UCZ- and non-GA3-treated plants.
flowering. Therefore, ‘Reagan’ and ‘Nagano-queen’ serve as de facto controls for flowering. 3.2. The effect of GA on the expression of the CmFL gene In confirmation of the data of plants with chilling in Fig. 3, UCZ completely inhibited flowering of line 94-4008 plants for more than 90 d, and GA3 reversed this inhibition (Table 1). Days to visible flower buds of plants treated with 10 mg GA3 after UCZ application (UCZ + GA plants) was the same as those of non-UCZ- and nonGA3-treated plants. UCZ inhibited stem elongation. GA3 reversed this cessation of stem elongation. ‘Reagan’ plants showed parallel reduction in stem elongation by UCZ, but all plants flowered. Days to visible flower buds increased in plants treated with UCZ (UCZ plants) and UCZ + GA plants compared to those in non-UCZ- and non-GA3-treated plants. The effect of GA3 on the expression of the CmFL gene was examined. The coding region of CmFL is 1,239 bp, and encodes a putative protein of 413 amino acids, which is 97% identical to DFL (GenBank accession no. AY559245), a FLO/LFY homologous gene from the wild species of chrysanthemum, Dendranthema lavandufolium [27]. Comparison of an amino acid sequence alignment containing CmFL and other FLO/LFY proteins showed the presence of several conserved regions (see Supplementary Fig. S1). The putative amino acid sequence of CmFL showed 60% homology with LFY of A. thaliana, 68% with FLO of A. majus, 66% with FALSIFLORA of Solanum lycopersicum (GenBank accession no. AF197934), 67% with ALF of Petunia x hybrida (GenBank accession no. AF030171), and 66% with UNIFOLIATA of Pisum sativum (GenBank accession no. AF010190). The abundance of transcripts of CmFL in shoot tips
was determined by using Q-PCR while the floral transition was occurring (Li, unpublished). CmFL showed threshold cycle number response at 18–28 cycles. In addition, transgenic Arabidopsis plants constitutively expressing CmFL flowered earlier than wild-type
Table 1 Effect of GA3 on flowering and extension growth in chrysanthemum line 94-4008 and ‘Reagan’. Significant differences determined at P < 0.01 using Tukey’s test are indicated by different letters in each line. Values are mean S.E. (n = 12). No chemicals, non-UCZ- and non-GA3-treated plants. UCZ, an application to soil (0.5 mg/ pot) and spray treatments of UCZ (25 mg l1). UCZ + GA, GA3 (10 mg per plant in total) was applied over 6 d after UCZ application. Treatments
UCZ GA3
No chemicals
UCZ
UCZ + GA
+
+ +
Line and cultivar
Treatments No chemicals
UCZ
UCZ + GA
26.7 1.8 b 23.4 1.4 b
0.6 0.1 a 0.7 0.1 a
31.0 0.6 b 27.9 0.7 c
Percentage of flowering plants 94-4008 100 Reagan 100
0 100
100 100
Days to visible flower buds 94-4008 23.8 0.7 a Reagan 18.6 0.1 a
NBa (>90) 27.8 0.1 c
22.3 0.6 a 20.1 0.3 b
Stem elongation (cm) 94-4008 Reagan
a
NB, no visible flower buds.
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Fig. 2. Plants grown in SD with or without the application of GA3. (A) Unchilled line 94-4008. (B) Chilled line 94-4008. (C) Unchilled ‘Reagan’. (D) Chilled ‘Reagan’. GA3 (0, 0.1, 1 or 10 mg per plant in total) was applied over 6 d after UCZ application. No chemicals, non-UCZ- and non-GA3-treated plants.
Col plants under 9-h SD photoperiod at 20 8C (Fig. 4, and see Supplementary Table S1). The shoot tip transcript level of CmFL was low at transfer into the SD growth chamber (Fig. 5). By day 14, CmFL expression began to increase in non-UCZ- and non-GA3-treated plants and UCZ + GA plants. In UCZ plants of line 94-4008, CmFL expression was blocked in parallel with the blocking of flowering. In contrast, in ‘Reagan’ plants, UCZ did not block flowering and had little effect on CmFL expression. 4. Discussion Chrysanthemum shows large seasonal difference in extension growth and flowering. Extension growth and flowering depend on the combination of temperature and day length. In our studies, temperature is significant, and temperature of the previous season is an important factor in determining the capacity for growth and flowering in the subsequent season. Exposure to summer heat reduces this capacity. This decrease narrows the temperature range over which extension growth and flowering are possible, and plants show slower extension growth and flowering in autumn
than in spring at the same growing temperature [2]. For example, at growing temperatures of 158/10 8C (light/dark), chrysanthemum plants showed rapid extension growth and flowering in spring but showed neither in autumn after exposure to high temperatures [2]. Here, the dependence of extension growth on exogenous GA shows that GA promotes the capacity for extension growth (Fig. 1). Chilling is also involved because it increased the responsiveness of stem elongation to exogenous GA3. The link between the seasonality of extension capacity and responsiveness to GA follows from our use of UCZ to reduce endogenous GA levels in chrysanthemum [28]. Consistent with this claim, endogenous GA level in bioassays increases rapidly in chrysanthemum in association with high extension growth rates after chilling [29]. Chilling also promotes flowering [20], and this is evident in our experiments. Potential genetic variation in the chilling requirement for the promotion of flowering is also evident for the cultivars studied here. ‘City’ and line 94-4008 have a distinct seasonality of flowering associated with their chilling requirement, and they rarely flower after autumn, as shown in a further study (Hisamatsu, unpublished). In contrast, ‘Reagan’ and ‘Nagano-queen’ with their lack of a chilling requirement showed a stable flowering response in our study ([2], Hisamatsu, unpublished). Annual commercial production requires the selection of novel adaptive flowering traits suited to the greenhouse environment, where chrysanthemums are never exposed to chilling thus no flowers could be harvested if the plants required chilling for flowering. ‘Reagan’ and ‘Naganoqueen’ meet this requirement since they have lost the chilling requirement for flowering during the selection in breeding for stable flowering. Because ‘City’ and line 94-4008 require chilling for the floral transition, they are more similar to the progenitor wild chrysanthemums in Japan, which require chilling for the floral transition (Kawata, unpublished), although the ultimate progenitor of the current chrysanthemum cultivars is yet to be identified [30]. The GA requirement for the floral transition varied from an absence in ‘Reagan’ and ‘Nagano-queen’ to a need for GA in ‘City’ and line 94-4008, where UCZ inhibited flowering and GA reversed it (Fig. 3). We suggest that GA is involved in the floral transition as an early informational signal because the UCZ-treated plants did not show floral morphogenesis in ‘City’ and line 94-4008 by the end of the experiment, i.e., 56 d after transfer to SD conditions. Especially, GA enhanced the expression of the CmFL gene (Fig. 5), a floral meristem identity gene, which in Arabidopsis responds directly to GA [10]. It is yet to be determined whether there is a common signal pathway linking chilling and GA to extension growth and flowering, but since there is no seasonal chilling requirement for flowering of ‘Reagan’ and ‘Nagano-queen’, their endogenous GA level may be non-limiting. There is a dominant requirement for SD exposure for flowering of chrysanthemum, and GA does not induce flowering of plants maintained in LD [21,22]. Compared with plants transferred to SD, which by 28 d had flowered (Table 1) and which showed high levels of CmFL expression (Fig. 5), gene expression was negligible in plants maintained in LD at 28 d, and these plants had still not flowered by 50 d (Li, unpublished). A failure of GA to substitute for SD for flowering is consistent with the extensive evidence that at least for LD species, it is LD that increases GA biosynthesis in association with their flowering [4]. In contrast, chilling-induced flowering can be replaced by application of GA to UCZ-treated plants (Fig. 3) and even to unchilled photoperiod-insensitive cultivars [21]. A partial explanation for these findings is that chilling promotes GA biosynthesis in chrysanthemum, as suggested by GA bioassays [29]. However, there must be some additional changes in the cold since GA response was greater after chilling (Figs. 1 and 3). No molecular biological approach has been developed to study flowering in chrysanthemum, although they
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Fig. 3. Effect of GA3 and chilling on flowering in chrysanthemum (A) ‘City’, (B) line 94-4008, (C) ‘Reagan’ and (D) ‘Nagano-queen’. Percentage of flowering plants of unchilled (closed circles, broken line) or 8-week chilled plants (open squares, solid line). Data were collected 56 days after transfer into the SD growth chamber. Error bars represent S.E. (n = 14). GA3 (0, 0.1, 1 or 10 mg per plant in total) was applied over 6 d after UCZ applications. No chemicals, non-UCZ- and non-GA3-treated plants.
are important ornamental crops and growers adopt precision regulation of flowering in commercial production. At the molecular level, our results suggest that GA may regulate flowering through upregulation of CmFL expression, as demonstrated here for chrysanthemum and previously for Arabidopsis [10]. However, because of the clear parallels between chilling-induced flowering and vernalization responses as studied in Arabidopsis, wheat and barley, there could also be important changes in the expression of a whole suite of vernalization-responsive genes related to FLOWERING LOCUS C (FLC) or VERNALIZATION2, which are considered as negative regulators whose level is depressed by chilling [31–34].
GA could compete with the repressor in chrysanthemum. The smaller dose of GA required for flowering in the chilled plants indicates that chilling treatment reduced the effect of the repressor, as suggested by the results that the extent of chilling quantitatively determines the level of FLC in Arabidopsis [35]. The chilled plants still showed GA dependence for flowering and upregulation of CmFL since the effect of repressor might remain after 8 weeks of chilling [35,36]. GA may be less important for flowering in ‘Reagan’ and ‘Nagano-queen’. Considering these plants to be similar to mutants containing a deletion in the flowering repressor that chilling and GA repress can explain the
Fig. 4. CmFL induces early flowering in Arabidopsis thaliana. Wild-type Col plant (A) and 35S::CmFL overexpression plant (B) grown under 9-h SD photoperiod at 20 8C. The terminal flower is visible (arrow).
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Fig. 5. Effect of GA3 on the expression of CmFL in chrysanthemum (A) ‘Reagan’ and (B) line 94-4008. Changes in the gene expression are shown as calculated value relative to the maximum value in the same Q-PCR assay. Error bars represent SE (n = 4). No chemicals (closed circles and solid line), non-UCZ- and non-GA3-treated plants. UCZ (closed triangles and broken line), an application to soil (0.5 mg per pot) and spray treatments of UCZ (25 mg l1). UCZ + GA (open circles and solid line), GA3 (10 mg per plant in total) was applied over 6 d after UCZ application.
independence of GA for flowering and upregulation of CmFL in them. Genetic variation in the GA requirement for the floral transition can be principally attributed to the chilling requirement for flowering. Overall, our preferred model of the regulation of flowering of chrysanthemum involves a chilling response that includes both GA and an unknown separate input. The latter might be considered as negative regulators, as shown in the vernalization response for Arabidopsis and cereals. In our ongoing studies, the cultivar differences that we have reported here for extension growth and flowering of chrysanthemum might allow us to understand such complex environmental regulation, however, this will first require identification of gene homologous to those involved in a chilling response as in Arabidopsis and cereals. Acknowledgments We thank Dr. Rod W. King (Commonwealth Scientific and Industrial Research Organisation, Canberra, Australia) for his valuable comments on the manuscript. This work was supported by grants from National Agriculture and Food Research Organization (NARO), Japan.
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