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Red:far-red light ratio and far-red light integral promote or retard growth and flowering in Eustoma grandiflorum (Raf.) Shinn
Scientia Horticulturae 120 (2009) 101–106
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Red:far-red light ratio and far-red light integral promote or retard growth and flowering in Eustoma grandiflorum (Raf.) Shinn§ Asuka Yamada *, Takahiro Tanigawa, Takuro Suyama, Takatoshi Matsuno, Toshihiro Kunitake Fukuoka Agricultural Research Center, Chikushino 818-8549, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2008 Received in revised form 15 August 2008 Accepted 5 September 2008
Night break treatment was applied to Eustoma grandiflorum ‘Nail Peach Neo’ using light sources with different red (R: 660 30 nm): far-red (FR: 730 30 nm) ratios or FR light intensities in order to investigate growth and flowering responses. Flower initiation and induction were promoted by night break treatment with a low R:FR light source, but was delayed by a high R:FR ratio. The promotion or delay of flower bud formation was accompanied by a decrease or an increase, respectively, in the number of nodes on the main stem at anthesis to the first floret. The difference between date of visible bud with plants under night break treatment and that of the control was approximated with high accuracy by a sigmoid function of the logarithms of R:FR ratio. The threshold R:FR ratio demarcating the promotion and delay of date of visible bud was about 5.3 under the experimental conditions used. The critical R:FR ratios for promotion or delay of visible bud would be about 0.5 and 50.0, respectively. In addition, the time from planting to visible bud was approximated with an exponential function of FR light intensity. The maximum acceleration of date of visible bud by long-day treatment would be 20 days, and the critical FR light intensity would be 2.0 mmol m2 s1. It is concluded that growth and flowering of E. grandiflorum can be regulated by long-day treatment using light sources with different R:FR ratios or FR light intensities. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Light quality Lisianthus Long-day treatment Photomorphogenesis Red:far-red ratio
1. Introduction Eustoma grandiflorum (Raf.) Shinn. cultivars are quantitative long-day plants and thus their flowering is promoted by long days (Ohkawa, 2003; Tsukada et al., 1982; Yamada et al., 2008b; Zaccai and Edri, 2002). Islam et al. (2005) reported that plants grown under a high light integral flowered earlier than those under a low light integral of the same day length. Presently, incandescent lamps are recommended to irradiate this long-day species to promote flowering. However, growth and flowering of Eustoma plants are either promoted or delayed by night break treatment using different types of light sources. Far-red fluorescent lamp, a plant growth fluorescent lamp and an incandescent lamp each with a low R:FR ratio (0.01, 0.43, and 0.65, respectively) promoted growth and flowering, whereas a daylight-type fluorescent lamp with a high R:FR ratio (5.0) delayed growth and flowering (Yamada et al., 2008a). Many studies have investigated the effects of specific light wavelengths, such as R (600–700 nm), FR (700–800 nm) or blue (B)
§ Part of this paper was presented at the 2007 Autumn Meeting of the Japanese Society for Horticultural Science. * Corresponding author. E-mail address: [email protected] (A. Yamada).
0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.09.009
light (400–500 nm), on growth and flowering in long-day plants. Brown and Klein (1971) reported that delayed floral induction of Arabidopsis plants occurred under continuous incandescent light lacking far-red wavelengths. However, Goto et al. (1991) reported that the night break with either FR, R, B and white light was effective at promoting flowering in Arabidopsis plants, of which FR light was the most effective. In Lemna gibba L., FR light was almost as effective as R light, B light had no effect, and continuous R light induced flowering response (Cleland and Briggs, 1968). Consequently, the effects of illumination types with different light qualities on floral induction in long-day plants depend on the species in question. In this paper, the effects of a range of R:FR ratios or FR light intensities on promotion or retardation of growth and flowering of E. grandiflorum were investigated. The relationship between the light quality and light intensity on growth and flowering is discussed. 2. Materials and methods 2.1. Method of raising seedlings Seeds of E. grandiflorum ‘Nail Peach Neo’ (Miyoshi Co., Yamanashi, Japan) were sown on 11 July 2006 in plastic
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germination trays with 288 cells (21 mL/cell) filled with Metromix 350 (Sun Gro Horticulture Distribution Inc., USA). The medium was adequately moistened and the seeded trays were kept for 4 weeks in a dark cool-room at 10 8C. The trays were transferred to a greenhouse and grown under a constant day (6:00–18:00)/night (18:00–6:00) temperature regime of 25/15 8C for 7 weeks to promote bolting and flowering of the plants in autumn and winter. 2.2. General planting and growing methods Seedlings were planted on 26 September in a plant boxes (60 cm long 18 cm wide 15 cm deep) filled with a medium comprising a 5:2:2:1 ratio of paddy soils (Chikushino, Fukuoka, Japan), Metro-mix 350, leaf molds (Hayashi Engei Shizai Co., Okayama, Japan), and volcanic soils (Bora-sands, Midori Sangyo Co., Fukuoka, Japan), respectively. The mix in each plant box contained 30 g slow-release fertilizer (N:P2O5:K2O = 13:16:10, Long Total Kaki 1-gou, 100-day-type, Chisso Asahi Hiryo Co., Tokyo, Japan) and were watered once every morning by hand. The seedlings were grown in a greenhouse, which was ventilated when the air temperature exceeded 25 8C and the night temperature was 15 8C. After planting, the seedlings were irradiated with night break treatment from 22:00 to 3:00 until anthesis to the first floret of all plants. There were 10 plants per experimental treatment which was replicated three times. The photon flux density at red (R, 660 30 nm) and far-red (FR, 730 30 nm) wavelengths of each light source was measured with a data logger (LI-1400, LI-COR, Inc., USA) fitted with a Skye sensor (SKR 110, Llandrindod Wells, Powys, UK). The blue light (B, 430 30 nm) intensity of each light source was calculated from the spectrum distribution characteristics. The control, plants were grown under ambient day length. Date of visible flower buds, date of flowering, number of nodes on the main stem and cut flower length at anthesis to the first floret, and length of each internode on the main stem were recorded. 2.3. Day length and solar radiation during the experimental period The day length at planting (26 September) and at flowering of the plants (late December to mid-January) ranged from about 12.5 to 11 h (Fig. 1). The mean solar radiation was high (15– 20 MJ m2 day1) in late September and early October, and declined gradually from mid-October to January (5–8 MJ m2 day1). 2.4. Experiment 1: effects of nigh break treatment using light sources with different R:FR ratios on growth and flowering characteristics Night break treatment was applied to the seedlings using a combination, a far-red-light fluorescent lamp (FL20SFR-74,
Fig. 1. Day length and solar radiation during the experimental period. (*) Day length and (*) solar radiation.
Toshiba Lighting and Technology Co., Kanagawa, Japan) and a red-light fluorescent lamp (FL20SR/20, Matsushita Denco, Osaka, Japan). The spectrum distribution characteristics of the far-redlight (A) and red-light (B) fluorescent lamps are shown in Fig. 2. The seedlings were irradiated by a combination of these two light sources with a R (660 30 nm): FR (730 30 nm) ratio of either 0.5, 1.0, 2.0, 3.0, 5.0 or 10.0. The photosynthetic photon flux density (PPFD) at the top of the seedlings was set at 4.5 mmol m2 s1 in each treatment. 2.5. Experiment 2: effects of night break treatment with different FR light intensities on growth and flowering characteristics The seedlings were given night break treatment using incandescent lamps (DENS 100 V, 75 W, G80K, Toshiba Lighting and Technology Co., Kanagawa, Japan) with a R:FR ratio of 0.6. The seedlings were irradiated with FR (730 30 nm) light regimes of either 0.5, 1.0, 2.0 or 3.0 mmol m2 s1 of photon flux density. 3. Results 3.1. Experiment 1: effects of night break treatment using light sources with different R:FR ratios on growth and flowering characteristics The far-red-light fluorescent lamp had a high far-red light integral and also had stronger blue (430 30 nm) and green (550 30 nm) light integrals than those of the red-light fluorescent
Fig. 2. Spectrum distribution characteristics of the light sources used for night break treatment. (A) Far-red-light fluorescent lamp and (B) red-light fluorescent lamp.
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Table 1 Photon flux density (PFD) of blue (B), red (R) and far-red (FR) light at different R:FR ratio regimes measured at the top of the seedlings when the photosynthetic photon flux density (PPFD) at the top of the seedlings was 4.5 mmol m2 s1 in each treatment. PFD (mmol m2 s1) R:FR ratio
Blue (B) (430 30 nm)
Red (R) (660 30 nm)
Far-red (FR) (730 30nm)
0.5 1.0 2.0 3.0 5.0 10.0
1.07 0.79 0.58 0.49 0.41 0.34
1.04 1.35 1.59 1 69 1.78 1.85
2.08 1.35 0.80 0.56 0.36 0.18
lamp (Fig. 2). At a R:FR ratio of 0.5, the photon flux density (PFD) of R and FR light was 1.04 and 2.08 mmol m2 s1, respectively, and that of blue light (1.07 mmol m2 s1) was the highest of all the treatments (Table 1). With increasing R:FR ratio, the PFD of red light was high and that of FR and blue light was lower. The growth and flowering of Eustoma plants 112 days after planting are shown in Fig. 3. Mean dates of visible bud and flowering were earliest in both R:FR ratios of 0.5 and 1.0 (visible bud on 18th November, and flowering date on 1st January) (Table 2). The time from planting to visible bud with low R:FR ratios of 0.5, 1.0, 2.0 or 3.0 was significantly shorter than that in the control. In contrast, the time from planting to visible bud with a high R:FR ratio of 10.0 was 8 days longer than that of the control. Mean date of visible bud with a R:FR ratio of 5.0 was identical to that of the control. The mean flowering date among the treatments showed a similar trend to mean date of visible bud. The difference between date of visible bud with night break treatment and that in the control was directly related to the logarithm of the R:FR ratios (Fig. 4). The relationship was approximated with high accuracy (R2 = 0.97) by the sigmoid function (Boltzmann function) in the equation: Y ¼ A2 þ
ðA1 A2 Þ ð1 þ expððX X 0 Þ=dXÞÞ
where Y the difference in mean date of visible bud between night break treatment and that of the control, X the logarithm of the R:FR ratio, A1 = 12.4, A2 = 14.6, X0 = 0.73 and dX = 0.25. According to this function, night break treatment promoted or delayed date of visible bud by a maximum of 12.4 and 14.6 days, respectively, relative to the date of visible bud in the control during the experimental period. It is estimated that the critical R:FR ratios for promotion or delay of visible bud would be 0.5 or 50, respectively. As the constant X0 (0.73) was the inflection point of this function, the R:FR ratio representing the boundary between promotion and delay of visible bud was 5.34.
Fig. 3. Comparative growth and flowering of E. grandiflorum ‘Nail Peach Neo’ grown with night break treatment using light sources with different R:FR ratios 112 days after planting. The numerical value is the R:FR ratio of the light source. The control plants were grown in ambient daylight without night break treatment.
Fig. 4. Relationship between the logarithm of the R:FR ratio of the light sources and the difference in mean date of visible bud between night break treatments and that of the control. The relationship is approximated with the equation above the graph. R2 is the coefficient of determination. Vertical bars represent the S.E. (n = 3).
R:FR ratios less than 3.0 resulted in a decreased in the number of nodes on the main stem compared to the control plants (Table 2). The number of nodes was lowest (7.4) in response to a R:FR ratio of 0.5 or 1.0. The number of nodes with a R:FR ratio of 5.0 was equivalent to that of the control, whereas plants treated with a R:FR ratio of 10.0 produced the greatest number of nodes (10.5) of all the treatments. Mean internode length of the main stem from the base to the seventh node was shorter with R:FR ratios exceeding 5.0, whereas that with R:FR ratios less than 2.0 was
Table 2 Effects of night break treatment using light sources with different R:FR ratios on growth and flowering characteristics of E. grandiflorum ‘Nail Peach Neo’. R:FR ratio
Mean budding date (date-month)
Days from planting to budding
0.5 1.0 2.0 3.0 5.0 10.0
18-Nov 18-Nov 20-Nov 26-Nov 29-Nov 7-Dec
54 54 56 62 65 73
Non-treatment (control)
29-Nov
65 b
y z
ez e d c b a
Mean internode length of the main stem from the base to the seventh node. Mean separation within the column by Turkey’s HSD test, 5% level.
Mean flowering date (date-month)
Number of nodes on main stem
Mean internode lengthy (cm)
1-Jan 1-Jan 5-Jan 12-Jan 18-Jan 29-Jan
7.4 7.4 8.3 8.6 9.4 10.5
4.1 4.4 4.2 3.7 3.3 3.3
19-Jan
d d c c b a
9.4 b
b a ab c d d
3.8 c
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Table 3 Effects of night break treatment with different FR light intensities on growth and flowering characteristics of E. grandiflorum ‘Nail Peach Neo’. Photon flux density of FR (mmol m2 s1)
Mean budding date (date-month)
Days from planting to budding
0.5 1.0 2.0 3.0
22-Nov 16-Nov 11-Nov 10-Nov
57 52 47 46
29-Nov
65 a
Non-treatment (control) y z
bz c d d
Mean flowering date (date-month)
Number of nodes on main stem
Mean internode lengthy (cm)
7-Jan 30-Dec 22-Dec 22-Dec
8.3 7.7 6.9 6.9
4.4 4.4 4.9 5.1
19-Jan
9.4 a
b bc c c
b b a a
3.8 c
Mean internode length of the main stem from the base to the seventh node. Mean separation within the column by Turkey’s HSD test, 5% level.
longer compared to the control 3.8 cm. The longest mean internode length (4.4 cm) occurred with a R:FR ratio of 1.0. 3.2. Experiment 2: effects of night break treatment with different FR light intensities on growth and flowering characteristics The mean dates of visible bud and flowering of plants in every night break treatment with a FR light intensity from 0.5 to 3.0 mmol m2 s1 were earlier than those in the control of 29th November and 19th January, respectively (Table 3). Higher FR light intensities promoted mean dates of visible bud and flowering compared to lower ones, but there was no significant difference between those at FR light intensities of 2.0 and 3.0 mmol m2 s1. The difference between date of visible bud in the night break treatments and that in the control was directly related to FR light intensity (Fig. 5). The relationship was approximated with high accuracy (R2 = 0.99) by the exponential function in the equation: X Y ¼ A1 exp þ y0 t1 where Y the difference in mean date of visible bud between night break treatment and that of the control, X FR light intensity, y0 = 19.86, A1 = 21.77 and t1 = 0.83. According to this function, the maximum promotion of visible bud compared to natural day length would be about 20 days during the experimental period. The number of nodes on the main stem was lowest (6.9) with a FR light intensity of either 2.0 or 3.0 mmol m2 s1. FR light intensities of 0.5 and 1.0 also reduced the number of nodes compared to the controls. Mean internode length of the main stem from the base to the seventh node was longer than that in the
Fig. 5. Relationship between the photon flux density of far-red light of light sources and the difference in mean date of visible bud between night break treatments and that of the control. The relationship is approximated with the equation above the graph. R2 is the coefficient of determination. Vertical bars represent the S.E. (n = 3).
control with FR light intensities from 0.5 to 3.0 mmol m2 s1. The longest mean internode length (5.1 cm) was obtained with a FR light intensity of 3.0 mmol m2 s1. 4. Discussion The response of light quality on growth and development of horticultural crops, our study concentrated on the effects of longday treatments by using different types of light sources with specific light quality on growth and flowering in E. grandiflorum. Yamada et al. (2008b) reported that long-day treatment using incandescent lamps promoted flowering and reduced the time from planting to visible bud, but not the time from visible bud to flowering. As flowering was earlier, the number of nodes on the main stem at anthesis to the first floret was reduced. Tsukada et al. (1982) also reported that long-day length promoted flower bud initiation, following which the response of flower bud development to day length was neutral. From the results above, the influence of long-day treatment on flowering of E. grandiflorum should be estimable by recording the time from planting to visible bud and the number of nodes on the main stem at anthesis to the first floret. In the first experiment, the difference in date of visible bud with night break treatment relative to that of the control was approximated with high accuracy by the sigmoid function of the logarithm of R:FR ratios. This result indicates that floral induction in Eustoma plants might be directly affected by the R:FR ratio of the light source. The day length at planting (26th September) and at flower bud formation (mid-November to early December) ranged from 12.5 to 11 h during the experimental period. According to the function described above, the boundary between promotion and delaying of visible bud occurs with a R:FR ratio of about 5.3. Consequently, long-day treatment using light sources with a R:FR ratio above 5.3 would delay flowering and those with a R:FR ratio below 5.3 would promote it. The maximum promotion and delay in the date of visible bud might be 12.4 and 14.6 days, respectively, compared to that at ambient day length. It was also estimated that the critical R:FR ratio to promote or delay visible bud might be 0.5 and 50, respectively. The symmetry of the sigmoid relationship between the time to visible bud and logarithm of the R:FR ratio is of great interest as an orderly phenomenon of life in the same manner as, for example, the Fibonacci progression shown in the regular arrangement of florets in Helianthus annuus L. (Stewart, 1996). The above findings may provide effective guidelines for the regulation of flowering and improvement of cut flower quality in E. grandiflorum. When long-day treatment was applied using an incandescent lamp with a R:FR ratio of 0.6, the mean dates of visible bud and flowering are progressively earlier with the increasing ratio of FR light intensity. The time to visible bud was approximated with an exponential function of FR light intensity with high accuracy. According to this function, the maximum promotion of visible bud
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compared to the control was about 20 days and the critical FR light intensity might be 2.0 mmol m2 s1. Kadman-Zahavi and Gartenhause (1989) reported that flowering date in Gypsophila paniculata L. was directly related to the logarithm of the total 100 mmol m2 night1. It can be concluded that E. grandiflorum is very similar in this regard. Butler et al. (1959) discovered the chromoprotein phytochrome, which is widely present in plants and is sensitive to light in the R and FR regions of visible spectrum. Phytochrome exists as two interconvertible isomeric forms that preferentially absorb either R-light (Pr) or FR-light (Pfr). Phytochrome has a primary role in regulation of a diversity of photomorphogenetic processes. Deitzer et al. (1979) reported that flowering in Hordeum vulgare L. was promoted by supplementation of FR light in the main light portion of the photoperiod and this enhancement of flowering appeared to be mediated by the ‘high irradiance response’ of phytochrome. Matthiola incana (L.) R. Br. plants grown under a high R:FR photon flux film during the day and irradiated with red light at night flowered later than plants grown under ambient daylight (Yoshimura et al., 2002). Through molecular genetic studies of Arabidopsis thaliana L., a blue/UV-A photoreceptor termed cryptochrome (Ahmad and Cashmore, 1993) and a blue-light photoreceptor termed phototropin (Huala et al., 1997) were discovered. Cryptochrome is known to have important roles in response such as stem elongation, induction of flowering and regulation of circadian rhythms. Yamazaki et al. (2007) reported that cryptochromes, which absorb blue light, effectively acted on the induction of flowering, whereas phytochromes, which absorb red light, acted on the inhibition of flowering in Pharbitis nil L. However, the direct influence on flowering by differences of the R:FR ratio in the present study may indicate that phytochrome acts as the major photoreceptor for regulation of floral induction in E. grandiflorum. It is suggested that the phytochrome photoequilibrium changed depending on the R:FR ratio of the light source and that this led to promotion or delay of floral induction with low and high R:FR ratios, respectively. As a far-red-light fluorescent lamp emits comparatively more blue light than a red-light fluorescent lamp, blue light intensity in irradiation with a low R:FR ratio is higher than in that with a high R:FR. However, the experiments reported herein did not allow assessment of whether blue light has an additional effect on promotion of flowering under low R:FR ratio conditions. In the long-day plant Arabidopsis thaliana, photoreceptor mutations that affect flowering time have been extensively analyzed (Devlin and Kay, 2000; Izawa et al., 2002; Somers et al., 1998). There are genes encoding at least five phytocromes (PHYA, B, C, D and E) and two types of cryptchromes (CRY1, 2) in Arabidopsis. Thomas (2006) reported that light quality acted independently of the circadian system through PHYB, D, and E to regulate the floral inducer FLOWERRING LOCUS T (FT) (Kardailsky et al., 1999). Arabidopsis mutants deficient in PHYB flowered earlier than the wild type in both short-days and long-days, but retain sensitivity to day length (Reed et al., 1994). PHYB, along with PHYD and PHYE mediated early flowering in Arabidopsis in response to low red to far-red ratios (Franklin et al., 2003). From these results, in Eustoma plants, PHYB might mainly play an important physiological function. Concerning the influences of the R:FR ratio on stem elongation in E. grandiflorum, the mean internode length of the main stem was longer with a low R:FR ratio than that with a high R:FR ratio. Also, when the R:FR ratio remained constant (0.6), the mean internode length increased with increasing FR light intensity. Behringer et al. (1990) reported that stem elongation in Pisum sativum L. was affected by phytochrome activity in an experiment in which the R:FR ratio was changed. A linear relationship between phyto-
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chrome photoequilibrium and the logarithm of the stem elongation rate is observed (Wareing and Phyllips, 1989). Stem elongation of P. sativum L. was inhibited by blue and red light, and red light inhibited elongation mainly by reducing the cell wall yield coefficient (Kigel and Cosgrove, 1991). The Arabidopsis hypocotyl inhibition response in red or blue light was mediated by phytochrome and cryptochrome, respectively (Mockler et al., 1999). In the present study, the mean internode length with a R:FR ratio of 0.5 was significantly shorter than that with a R:FR ratio of 1.0. The reason for this may be that, as shown in Table 1, the PFD of blue light in the light source with a R:FR ratio of 0.5 was higher (1.07 mmol m2 s1) than in that with a R:FR ratio of 1.0 (0.79 mmol m2 s1). Our findings may allow the practical regulation of flowering time and improvement of cut flower quality of Eustoma cultivars by irradiating the plants using specific light sources. 5. Conclusion Flower bud initiation and development of E. grandiflorum was promoted or delayed by long-day treatment using light sources with different red (R): far-red (FR) ratios. The threshold R:FR ratio demarcating the promotion and delay of visible bud was about 5.3 in the experimental condition. Long-day treatment using light sources with a R:FR ratio above 5.3 might delay flowering and those with a R:FR ratio below 5.3 might promote it. When long-day treatment was applied to E. grandiflorum using light source with a R:FR ratio of 0.6, the date of visible bud was progressively earlier with increase in FR light intensity and the critical FR light intensity might be 2.0 mmol m2 s1. The growth and flowering of E. grandiflorum can be controlled by long-day treatment using specific light sources. References Ahmad, M., Cashmore, A.R., 1993. HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366, 162–166. Behringer, F.J., Davies, P.J., Reid, J.B., 1990. Genetic analysis of the role of gibberellin in the red light inhibition of stem elongation in etiolated seedlings. Plant Physiol. 94, 432–439. Brown, J.A.M., Klein, W.H., 1971. Photomorphogenesis in Arabidopsis thaliana (L) Heynh. Plant Physiol. 47, 393–399. Butler, W.L., Norris, H.H., Seigelman, H.W., Hendrecks, S.B., 1959. Detection, assay and preliminary purification of the pigment controlling photoresponsive development of plant. Proc. Nat. Acad. Sci. U.S.A. 25, 1703–1708. Cleland, C.F., Briggs, W.R., 1968. Effect of low-intensity red and far-red light and high-intensity white light on the flowering response of the long-day plant Lemna gibba G3. Plant Physiol. 43, 157–162. Deitzer, G.F., Hayes, R., Jabben, M., 1979. Kinetics and time dependece of the effect of far-red light on the photoperiodic induction of flowering in winter barley. Plant Physiol. 64, 1015–1021. Devlin, P.F., Kay, S.A., 2000. Cryptochromes are rerequired for phytochrome signaling to the circadian clock but not for rythmicity. Plant Cell 12, 2499–2509. Franklin, K.A., Praekelt, U., Stoddart, W.M., Billingham, O.E., Halliday, K.J., Whitelam, G.C., 2003. Phytocromes B, D, and E act redundantly to control multiple physiological responses in Arabidopsis. Plant Physiol. 131, 1340–1346. Goto, N., Kumagai, T., Koornneef, M., 1991. Flowering responses to light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant. Physiol. Plant 83, 209–215. Huala, E.P.W., Oeller, E., Liscum, I.-S., Han, E., Larsen, Briggs, W.R., 1997. Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278, 120–2123. Islam, N., Patil, G.G., Gislerød, H.R., 2005. Effect of photoperiod and light integral on flowering and growth of Eustoma gradiflorum (Raf) Shinn. Sci. Hortic. 103, 441– 445. Izawa, T., Oikawa, T., Sugiyama, N., Tanisaka, T., Yano, M., Shimamoto, K., 2002. Phytocrome mediates the external light signal to repress FT orthologs in photoperiodic flowering of rice. Genes Develop. 16, 2006–2020. Kadman-Zahavi, A., Gartenhause, M., 1989. The effects of light spectra fluence, duration and time of application on flowering of Gypsophila. Sci. Hortic. 40, 237– 245. Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen, S.K., Nguyen, J.T., Chory, J., Harrison, M.J., Weigel, D., 1999. Activation tagging of the floral inducer FT. Science 286, 1962–1965.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Red:far-red light ratio and far-red light integral promote or retard growth and flowering in Eustoma grandiflorum (Raf.) Shinn§ Asuka Yamada *, Takahiro Tanigawa, Takuro Suyama, Takatoshi Matsuno, Toshihiro Kunitake Fukuoka Agricultural Research Center, Chikushino 818-8549, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2008 Received in revised form 15 August 2008 Accepted 5 September 2008
Night break treatment was applied to Eustoma grandiflorum ‘Nail Peach Neo’ using light sources with different red (R: 660 30 nm): far-red (FR: 730 30 nm) ratios or FR light intensities in order to investigate growth and flowering responses. Flower initiation and induction were promoted by night break treatment with a low R:FR light source, but was delayed by a high R:FR ratio. The promotion or delay of flower bud formation was accompanied by a decrease or an increase, respectively, in the number of nodes on the main stem at anthesis to the first floret. The difference between date of visible bud with plants under night break treatment and that of the control was approximated with high accuracy by a sigmoid function of the logarithms of R:FR ratio. The threshold R:FR ratio demarcating the promotion and delay of date of visible bud was about 5.3 under the experimental conditions used. The critical R:FR ratios for promotion or delay of visible bud would be about 0.5 and 50.0, respectively. In addition, the time from planting to visible bud was approximated with an exponential function of FR light intensity. The maximum acceleration of date of visible bud by long-day treatment would be 20 days, and the critical FR light intensity would be 2.0 mmol m2 s1. It is concluded that growth and flowering of E. grandiflorum can be regulated by long-day treatment using light sources with different R:FR ratios or FR light intensities. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Light quality Lisianthus Long-day treatment Photomorphogenesis Red:far-red ratio
1. Introduction Eustoma grandiflorum (Raf.) Shinn. cultivars are quantitative long-day plants and thus their flowering is promoted by long days (Ohkawa, 2003; Tsukada et al., 1982; Yamada et al., 2008b; Zaccai and Edri, 2002). Islam et al. (2005) reported that plants grown under a high light integral flowered earlier than those under a low light integral of the same day length. Presently, incandescent lamps are recommended to irradiate this long-day species to promote flowering. However, growth and flowering of Eustoma plants are either promoted or delayed by night break treatment using different types of light sources. Far-red fluorescent lamp, a plant growth fluorescent lamp and an incandescent lamp each with a low R:FR ratio (0.01, 0.43, and 0.65, respectively) promoted growth and flowering, whereas a daylight-type fluorescent lamp with a high R:FR ratio (5.0) delayed growth and flowering (Yamada et al., 2008a). Many studies have investigated the effects of specific light wavelengths, such as R (600–700 nm), FR (700–800 nm) or blue (B)
§ Part of this paper was presented at the 2007 Autumn Meeting of the Japanese Society for Horticultural Science. * Corresponding author. E-mail address: [email protected] (A. Yamada).
0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.09.009
light (400–500 nm), on growth and flowering in long-day plants. Brown and Klein (1971) reported that delayed floral induction of Arabidopsis plants occurred under continuous incandescent light lacking far-red wavelengths. However, Goto et al. (1991) reported that the night break with either FR, R, B and white light was effective at promoting flowering in Arabidopsis plants, of which FR light was the most effective. In Lemna gibba L., FR light was almost as effective as R light, B light had no effect, and continuous R light induced flowering response (Cleland and Briggs, 1968). Consequently, the effects of illumination types with different light qualities on floral induction in long-day plants depend on the species in question. In this paper, the effects of a range of R:FR ratios or FR light intensities on promotion or retardation of growth and flowering of E. grandiflorum were investigated. The relationship between the light quality and light intensity on growth and flowering is discussed. 2. Materials and methods 2.1. Method of raising seedlings Seeds of E. grandiflorum ‘Nail Peach Neo’ (Miyoshi Co., Yamanashi, Japan) were sown on 11 July 2006 in plastic
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germination trays with 288 cells (21 mL/cell) filled with Metromix 350 (Sun Gro Horticulture Distribution Inc., USA). The medium was adequately moistened and the seeded trays were kept for 4 weeks in a dark cool-room at 10 8C. The trays were transferred to a greenhouse and grown under a constant day (6:00–18:00)/night (18:00–6:00) temperature regime of 25/15 8C for 7 weeks to promote bolting and flowering of the plants in autumn and winter. 2.2. General planting and growing methods Seedlings were planted on 26 September in a plant boxes (60 cm long 18 cm wide 15 cm deep) filled with a medium comprising a 5:2:2:1 ratio of paddy soils (Chikushino, Fukuoka, Japan), Metro-mix 350, leaf molds (Hayashi Engei Shizai Co., Okayama, Japan), and volcanic soils (Bora-sands, Midori Sangyo Co., Fukuoka, Japan), respectively. The mix in each plant box contained 30 g slow-release fertilizer (N:P2O5:K2O = 13:16:10, Long Total Kaki 1-gou, 100-day-type, Chisso Asahi Hiryo Co., Tokyo, Japan) and were watered once every morning by hand. The seedlings were grown in a greenhouse, which was ventilated when the air temperature exceeded 25 8C and the night temperature was 15 8C. After planting, the seedlings were irradiated with night break treatment from 22:00 to 3:00 until anthesis to the first floret of all plants. There were 10 plants per experimental treatment which was replicated three times. The photon flux density at red (R, 660 30 nm) and far-red (FR, 730 30 nm) wavelengths of each light source was measured with a data logger (LI-1400, LI-COR, Inc., USA) fitted with a Skye sensor (SKR 110, Llandrindod Wells, Powys, UK). The blue light (B, 430 30 nm) intensity of each light source was calculated from the spectrum distribution characteristics. The control, plants were grown under ambient day length. Date of visible flower buds, date of flowering, number of nodes on the main stem and cut flower length at anthesis to the first floret, and length of each internode on the main stem were recorded. 2.3. Day length and solar radiation during the experimental period The day length at planting (26 September) and at flowering of the plants (late December to mid-January) ranged from about 12.5 to 11 h (Fig. 1). The mean solar radiation was high (15– 20 MJ m2 day1) in late September and early October, and declined gradually from mid-October to January (5–8 MJ m2 day1). 2.4. Experiment 1: effects of nigh break treatment using light sources with different R:FR ratios on growth and flowering characteristics Night break treatment was applied to the seedlings using a combination, a far-red-light fluorescent lamp (FL20SFR-74,
Fig. 1. Day length and solar radiation during the experimental period. (*) Day length and (*) solar radiation.
Toshiba Lighting and Technology Co., Kanagawa, Japan) and a red-light fluorescent lamp (FL20SR/20, Matsushita Denco, Osaka, Japan). The spectrum distribution characteristics of the far-redlight (A) and red-light (B) fluorescent lamps are shown in Fig. 2. The seedlings were irradiated by a combination of these two light sources with a R (660 30 nm): FR (730 30 nm) ratio of either 0.5, 1.0, 2.0, 3.0, 5.0 or 10.0. The photosynthetic photon flux density (PPFD) at the top of the seedlings was set at 4.5 mmol m2 s1 in each treatment. 2.5. Experiment 2: effects of night break treatment with different FR light intensities on growth and flowering characteristics The seedlings were given night break treatment using incandescent lamps (DENS 100 V, 75 W, G80K, Toshiba Lighting and Technology Co., Kanagawa, Japan) with a R:FR ratio of 0.6. The seedlings were irradiated with FR (730 30 nm) light regimes of either 0.5, 1.0, 2.0 or 3.0 mmol m2 s1 of photon flux density. 3. Results 3.1. Experiment 1: effects of night break treatment using light sources with different R:FR ratios on growth and flowering characteristics The far-red-light fluorescent lamp had a high far-red light integral and also had stronger blue (430 30 nm) and green (550 30 nm) light integrals than those of the red-light fluorescent
Fig. 2. Spectrum distribution characteristics of the light sources used for night break treatment. (A) Far-red-light fluorescent lamp and (B) red-light fluorescent lamp.
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Table 1 Photon flux density (PFD) of blue (B), red (R) and far-red (FR) light at different R:FR ratio regimes measured at the top of the seedlings when the photosynthetic photon flux density (PPFD) at the top of the seedlings was 4.5 mmol m2 s1 in each treatment. PFD (mmol m2 s1) R:FR ratio
Blue (B) (430 30 nm)
Red (R) (660 30 nm)
Far-red (FR) (730 30nm)
0.5 1.0 2.0 3.0 5.0 10.0
1.07 0.79 0.58 0.49 0.41 0.34
1.04 1.35 1.59 1 69 1.78 1.85
2.08 1.35 0.80 0.56 0.36 0.18
lamp (Fig. 2). At a R:FR ratio of 0.5, the photon flux density (PFD) of R and FR light was 1.04 and 2.08 mmol m2 s1, respectively, and that of blue light (1.07 mmol m2 s1) was the highest of all the treatments (Table 1). With increasing R:FR ratio, the PFD of red light was high and that of FR and blue light was lower. The growth and flowering of Eustoma plants 112 days after planting are shown in Fig. 3. Mean dates of visible bud and flowering were earliest in both R:FR ratios of 0.5 and 1.0 (visible bud on 18th November, and flowering date on 1st January) (Table 2). The time from planting to visible bud with low R:FR ratios of 0.5, 1.0, 2.0 or 3.0 was significantly shorter than that in the control. In contrast, the time from planting to visible bud with a high R:FR ratio of 10.0 was 8 days longer than that of the control. Mean date of visible bud with a R:FR ratio of 5.0 was identical to that of the control. The mean flowering date among the treatments showed a similar trend to mean date of visible bud. The difference between date of visible bud with night break treatment and that in the control was directly related to the logarithm of the R:FR ratios (Fig. 4). The relationship was approximated with high accuracy (R2 = 0.97) by the sigmoid function (Boltzmann function) in the equation: Y ¼ A2 þ
ðA1 A2 Þ ð1 þ expððX X 0 Þ=dXÞÞ
where Y the difference in mean date of visible bud between night break treatment and that of the control, X the logarithm of the R:FR ratio, A1 = 12.4, A2 = 14.6, X0 = 0.73 and dX = 0.25. According to this function, night break treatment promoted or delayed date of visible bud by a maximum of 12.4 and 14.6 days, respectively, relative to the date of visible bud in the control during the experimental period. It is estimated that the critical R:FR ratios for promotion or delay of visible bud would be 0.5 or 50, respectively. As the constant X0 (0.73) was the inflection point of this function, the R:FR ratio representing the boundary between promotion and delay of visible bud was 5.34.
Fig. 3. Comparative growth and flowering of E. grandiflorum ‘Nail Peach Neo’ grown with night break treatment using light sources with different R:FR ratios 112 days after planting. The numerical value is the R:FR ratio of the light source. The control plants were grown in ambient daylight without night break treatment.
Fig. 4. Relationship between the logarithm of the R:FR ratio of the light sources and the difference in mean date of visible bud between night break treatments and that of the control. The relationship is approximated with the equation above the graph. R2 is the coefficient of determination. Vertical bars represent the S.E. (n = 3).
R:FR ratios less than 3.0 resulted in a decreased in the number of nodes on the main stem compared to the control plants (Table 2). The number of nodes was lowest (7.4) in response to a R:FR ratio of 0.5 or 1.0. The number of nodes with a R:FR ratio of 5.0 was equivalent to that of the control, whereas plants treated with a R:FR ratio of 10.0 produced the greatest number of nodes (10.5) of all the treatments. Mean internode length of the main stem from the base to the seventh node was shorter with R:FR ratios exceeding 5.0, whereas that with R:FR ratios less than 2.0 was
Table 2 Effects of night break treatment using light sources with different R:FR ratios on growth and flowering characteristics of E. grandiflorum ‘Nail Peach Neo’. R:FR ratio
Mean budding date (date-month)
Days from planting to budding
0.5 1.0 2.0 3.0 5.0 10.0
18-Nov 18-Nov 20-Nov 26-Nov 29-Nov 7-Dec
54 54 56 62 65 73
Non-treatment (control)
29-Nov
65 b
y z
ez e d c b a
Mean internode length of the main stem from the base to the seventh node. Mean separation within the column by Turkey’s HSD test, 5% level.
Mean flowering date (date-month)
Number of nodes on main stem
Mean internode lengthy (cm)
1-Jan 1-Jan 5-Jan 12-Jan 18-Jan 29-Jan
7.4 7.4 8.3 8.6 9.4 10.5
4.1 4.4 4.2 3.7 3.3 3.3
19-Jan
d d c c b a
9.4 b
b a ab c d d
3.8 c
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Table 3 Effects of night break treatment with different FR light intensities on growth and flowering characteristics of E. grandiflorum ‘Nail Peach Neo’. Photon flux density of FR (mmol m2 s1)
Mean budding date (date-month)
Days from planting to budding
0.5 1.0 2.0 3.0
22-Nov 16-Nov 11-Nov 10-Nov
57 52 47 46
29-Nov
65 a
Non-treatment (control) y z
bz c d d
Mean flowering date (date-month)
Number of nodes on main stem
Mean internode lengthy (cm)
7-Jan 30-Dec 22-Dec 22-Dec
8.3 7.7 6.9 6.9
4.4 4.4 4.9 5.1
19-Jan
9.4 a
b bc c c
b b a a
3.8 c
Mean internode length of the main stem from the base to the seventh node. Mean separation within the column by Turkey’s HSD test, 5% level.
longer compared to the control 3.8 cm. The longest mean internode length (4.4 cm) occurred with a R:FR ratio of 1.0. 3.2. Experiment 2: effects of night break treatment with different FR light intensities on growth and flowering characteristics The mean dates of visible bud and flowering of plants in every night break treatment with a FR light intensity from 0.5 to 3.0 mmol m2 s1 were earlier than those in the control of 29th November and 19th January, respectively (Table 3). Higher FR light intensities promoted mean dates of visible bud and flowering compared to lower ones, but there was no significant difference between those at FR light intensities of 2.0 and 3.0 mmol m2 s1. The difference between date of visible bud in the night break treatments and that in the control was directly related to FR light intensity (Fig. 5). The relationship was approximated with high accuracy (R2 = 0.99) by the exponential function in the equation: X Y ¼ A1 exp þ y0 t1 where Y the difference in mean date of visible bud between night break treatment and that of the control, X FR light intensity, y0 = 19.86, A1 = 21.77 and t1 = 0.83. According to this function, the maximum promotion of visible bud compared to natural day length would be about 20 days during the experimental period. The number of nodes on the main stem was lowest (6.9) with a FR light intensity of either 2.0 or 3.0 mmol m2 s1. FR light intensities of 0.5 and 1.0 also reduced the number of nodes compared to the controls. Mean internode length of the main stem from the base to the seventh node was longer than that in the
Fig. 5. Relationship between the photon flux density of far-red light of light sources and the difference in mean date of visible bud between night break treatments and that of the control. The relationship is approximated with the equation above the graph. R2 is the coefficient of determination. Vertical bars represent the S.E. (n = 3).
control with FR light intensities from 0.5 to 3.0 mmol m2 s1. The longest mean internode length (5.1 cm) was obtained with a FR light intensity of 3.0 mmol m2 s1. 4. Discussion The response of light quality on growth and development of horticultural crops, our study concentrated on the effects of longday treatments by using different types of light sources with specific light quality on growth and flowering in E. grandiflorum. Yamada et al. (2008b) reported that long-day treatment using incandescent lamps promoted flowering and reduced the time from planting to visible bud, but not the time from visible bud to flowering. As flowering was earlier, the number of nodes on the main stem at anthesis to the first floret was reduced. Tsukada et al. (1982) also reported that long-day length promoted flower bud initiation, following which the response of flower bud development to day length was neutral. From the results above, the influence of long-day treatment on flowering of E. grandiflorum should be estimable by recording the time from planting to visible bud and the number of nodes on the main stem at anthesis to the first floret. In the first experiment, the difference in date of visible bud with night break treatment relative to that of the control was approximated with high accuracy by the sigmoid function of the logarithm of R:FR ratios. This result indicates that floral induction in Eustoma plants might be directly affected by the R:FR ratio of the light source. The day length at planting (26th September) and at flower bud formation (mid-November to early December) ranged from 12.5 to 11 h during the experimental period. According to the function described above, the boundary between promotion and delaying of visible bud occurs with a R:FR ratio of about 5.3. Consequently, long-day treatment using light sources with a R:FR ratio above 5.3 would delay flowering and those with a R:FR ratio below 5.3 would promote it. The maximum promotion and delay in the date of visible bud might be 12.4 and 14.6 days, respectively, compared to that at ambient day length. It was also estimated that the critical R:FR ratio to promote or delay visible bud might be 0.5 and 50, respectively. The symmetry of the sigmoid relationship between the time to visible bud and logarithm of the R:FR ratio is of great interest as an orderly phenomenon of life in the same manner as, for example, the Fibonacci progression shown in the regular arrangement of florets in Helianthus annuus L. (Stewart, 1996). The above findings may provide effective guidelines for the regulation of flowering and improvement of cut flower quality in E. grandiflorum. When long-day treatment was applied using an incandescent lamp with a R:FR ratio of 0.6, the mean dates of visible bud and flowering are progressively earlier with the increasing ratio of FR light intensity. The time to visible bud was approximated with an exponential function of FR light intensity with high accuracy. According to this function, the maximum promotion of visible bud
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compared to the control was about 20 days and the critical FR light intensity might be 2.0 mmol m2 s1. Kadman-Zahavi and Gartenhause (1989) reported that flowering date in Gypsophila paniculata L. was directly related to the logarithm of the total 100 mmol m2 night1. It can be concluded that E. grandiflorum is very similar in this regard. Butler et al. (1959) discovered the chromoprotein phytochrome, which is widely present in plants and is sensitive to light in the R and FR regions of visible spectrum. Phytochrome exists as two interconvertible isomeric forms that preferentially absorb either R-light (Pr) or FR-light (Pfr). Phytochrome has a primary role in regulation of a diversity of photomorphogenetic processes. Deitzer et al. (1979) reported that flowering in Hordeum vulgare L. was promoted by supplementation of FR light in the main light portion of the photoperiod and this enhancement of flowering appeared to be mediated by the ‘high irradiance response’ of phytochrome. Matthiola incana (L.) R. Br. plants grown under a high R:FR photon flux film during the day and irradiated with red light at night flowered later than plants grown under ambient daylight (Yoshimura et al., 2002). Through molecular genetic studies of Arabidopsis thaliana L., a blue/UV-A photoreceptor termed cryptochrome (Ahmad and Cashmore, 1993) and a blue-light photoreceptor termed phototropin (Huala et al., 1997) were discovered. Cryptochrome is known to have important roles in response such as stem elongation, induction of flowering and regulation of circadian rhythms. Yamazaki et al. (2007) reported that cryptochromes, which absorb blue light, effectively acted on the induction of flowering, whereas phytochromes, which absorb red light, acted on the inhibition of flowering in Pharbitis nil L. However, the direct influence on flowering by differences of the R:FR ratio in the present study may indicate that phytochrome acts as the major photoreceptor for regulation of floral induction in E. grandiflorum. It is suggested that the phytochrome photoequilibrium changed depending on the R:FR ratio of the light source and that this led to promotion or delay of floral induction with low and high R:FR ratios, respectively. As a far-red-light fluorescent lamp emits comparatively more blue light than a red-light fluorescent lamp, blue light intensity in irradiation with a low R:FR ratio is higher than in that with a high R:FR. However, the experiments reported herein did not allow assessment of whether blue light has an additional effect on promotion of flowering under low R:FR ratio conditions. In the long-day plant Arabidopsis thaliana, photoreceptor mutations that affect flowering time have been extensively analyzed (Devlin and Kay, 2000; Izawa et al., 2002; Somers et al., 1998). There are genes encoding at least five phytocromes (PHYA, B, C, D and E) and two types of cryptchromes (CRY1, 2) in Arabidopsis. Thomas (2006) reported that light quality acted independently of the circadian system through PHYB, D, and E to regulate the floral inducer FLOWERRING LOCUS T (FT) (Kardailsky et al., 1999). Arabidopsis mutants deficient in PHYB flowered earlier than the wild type in both short-days and long-days, but retain sensitivity to day length (Reed et al., 1994). PHYB, along with PHYD and PHYE mediated early flowering in Arabidopsis in response to low red to far-red ratios (Franklin et al., 2003). From these results, in Eustoma plants, PHYB might mainly play an important physiological function. Concerning the influences of the R:FR ratio on stem elongation in E. grandiflorum, the mean internode length of the main stem was longer with a low R:FR ratio than that with a high R:FR ratio. Also, when the R:FR ratio remained constant (0.6), the mean internode length increased with increasing FR light intensity. Behringer et al. (1990) reported that stem elongation in Pisum sativum L. was affected by phytochrome activity in an experiment in which the R:FR ratio was changed. A linear relationship between phyto-
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chrome photoequilibrium and the logarithm of the stem elongation rate is observed (Wareing and Phyllips, 1989). Stem elongation of P. sativum L. was inhibited by blue and red light, and red light inhibited elongation mainly by reducing the cell wall yield coefficient (Kigel and Cosgrove, 1991). The Arabidopsis hypocotyl inhibition response in red or blue light was mediated by phytochrome and cryptochrome, respectively (Mockler et al., 1999). In the present study, the mean internode length with a R:FR ratio of 0.5 was significantly shorter than that with a R:FR ratio of 1.0. The reason for this may be that, as shown in Table 1, the PFD of blue light in the light source with a R:FR ratio of 0.5 was higher (1.07 mmol m2 s1) than in that with a R:FR ratio of 1.0 (0.79 mmol m2 s1). Our findings may allow the practical regulation of flowering time and improvement of cut flower quality of Eustoma cultivars by irradiating the plants using specific light sources. 5. Conclusion Flower bud initiation and development of E. grandiflorum was promoted or delayed by long-day treatment using light sources with different red (R): far-red (FR) ratios. The threshold R:FR ratio demarcating the promotion and delay of visible bud was about 5.3 in the experimental condition. Long-day treatment using light sources with a R:FR ratio above 5.3 might delay flowering and those with a R:FR ratio below 5.3 might promote it. When long-day treatment was applied to E. grandiflorum using light source with a R:FR ratio of 0.6, the date of visible bud was progressively earlier with increase in FR light intensity and the critical FR light intensity might be 2.0 mmol m2 s1. The growth and flowering of E. grandiflorum can be controlled by long-day treatment using specific light sources. References Ahmad, M., Cashmore, A.R., 1993. HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366, 162–166. Behringer, F.J., Davies, P.J., Reid, J.B., 1990. Genetic analysis of the role of gibberellin in the red light inhibition of stem elongation in etiolated seedlings. Plant Physiol. 94, 432–439. Brown, J.A.M., Klein, W.H., 1971. Photomorphogenesis in Arabidopsis thaliana (L) Heynh. Plant Physiol. 47, 393–399. Butler, W.L., Norris, H.H., Seigelman, H.W., Hendrecks, S.B., 1959. Detection, assay and preliminary purification of the pigment controlling photoresponsive development of plant. Proc. Nat. Acad. Sci. U.S.A. 25, 1703–1708. Cleland, C.F., Briggs, W.R., 1968. Effect of low-intensity red and far-red light and high-intensity white light on the flowering response of the long-day plant Lemna gibba G3. Plant Physiol. 43, 157–162. Deitzer, G.F., Hayes, R., Jabben, M., 1979. Kinetics and time dependece of the effect of far-red light on the photoperiodic induction of flowering in winter barley. Plant Physiol. 64, 1015–1021. Devlin, P.F., Kay, S.A., 2000. Cryptochromes are rerequired for phytochrome signaling to the circadian clock but not for rythmicity. Plant Cell 12, 2499–2509. Franklin, K.A., Praekelt, U., Stoddart, W.M., Billingham, O.E., Halliday, K.J., Whitelam, G.C., 2003. Phytocromes B, D, and E act redundantly to control multiple physiological responses in Arabidopsis. Plant Physiol. 131, 1340–1346. Goto, N., Kumagai, T., Koornneef, M., 1991. Flowering responses to light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant. Physiol. Plant 83, 209–215. Huala, E.P.W., Oeller, E., Liscum, I.-S., Han, E., Larsen, Briggs, W.R., 1997. Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278, 120–2123. Islam, N., Patil, G.G., Gislerød, H.R., 2005. Effect of photoperiod and light integral on flowering and growth of Eustoma gradiflorum (Raf) Shinn. Sci. Hortic. 103, 441– 445. Izawa, T., Oikawa, T., Sugiyama, N., Tanisaka, T., Yano, M., Shimamoto, K., 2002. Phytocrome mediates the external light signal to repress FT orthologs in photoperiodic flowering of rice. Genes Develop. 16, 2006–2020. Kadman-Zahavi, A., Gartenhause, M., 1989. The effects of light spectra fluence, duration and time of application on flowering of Gypsophila. Sci. Hortic. 40, 237– 245. Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen, S.K., Nguyen, J.T., Chory, J., Harrison, M.J., Weigel, D., 1999. Activation tagging of the floral inducer FT. Science 286, 1962–1965.
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