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Modifications in Aster response to long-day conditions caused by overexpression of phytochrome A or B
Plant Science 163 (2002) 439 /447 www.elsevier.com/locate/plantsci
Modifications in Aster response to long-day conditions caused by overexpression of phytochrome A or B Israela Wallerstein a,*, Izhack Wallerstein b, Dayana Libman a, Boris Machnic a, Garry C. Whitelam c,1 b
a Department of Ornamental Horticulture, Agricultural Research Organisation, The Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel Department of Crop, Garden and Natural Resources, Agricultural Research Organisation, The Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel c Department of Biology, University of Leicester, Leicester LE1 7RH, UK
Received 18 February 2002; received in revised form 19 April 2002; accepted 14 May 2002
Abstract We studied the role of phytochromes A and B in the regulation of critical day length, night-break signal and the quantitative effects of sunlight on inflorescence development, a long-day induced process, in Aster . We used transgenic plants overexpressing either PHY A or PHY B, different natural daylight conditions, and day extension and night break treatments by low-fluence-rate fluorescent or incandescent lighting with a red to far-red ratio of 7.3 and 0.6, respectively. Wild-type plants had a higher sensitivity to day extension with fluorescent than incandescent lighting and a quantitative response to natural daylight conditions. Their response to a day extension with either incandescent or fluorescent lighting and to a night break with incandescent, but not fluorescent, lighting was subject to the quantitative effect of daylight conditions during the natural short day. Overexpression of PHY A shortened the critical day length for inflorescence development from 14 to 8 h. Overexpression of PHY B shortened the length of the night break needed to induce inflorescence development from 2 h to 15 min. Overexpression of either PHY A or B reduced the quantitative effect of natural daylight. For Aster plants grown under commercial conditions, from autumn through spring, the overexpression of either PHYA or PHYB substantially increased the yield of flowering shoots. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phytochrome A; Phytochrome B; Flowering time; Aster ; Day length; Night break
1. Introduction Long-day plants are adapted to respond to the duration, irradiance and quality of daylight. The perception of long days, which are inductive for flowering, is a complicated process involving photoreceptors, the endogenous circadian clock and several floweringtime genes [1]. Light interacts, via the photoreceptors, with the operation of the endogenous clock and the expression of the flowering-time genes [2]. The role of light has both qualitative and quantitative aspects. The qualitative aspect includes its importance for critical day
* Corresponding author. Tel.: /972-3-968-3765; fax: /972-3-9660589 E-mail addresses: [email protected] (I. Wallerstein), [email protected] (G.C. Whitelam). 1 Tel.: /44-116-252-3396; fax: /44-116-252-2791.
length and night-break effects, and the quantitative aspect includes increased response with increased fluence rate. The characteristics of critical day length and night break in long-day plants differ from those in shortday plants. In the former, the daylight spectrum and high fluence rates can shorten the critical day length and relatively long exposure to light during the night break is needed to induce flowering [3]. Several photoreceptors play roles in the transduction of light to the endogenous clock and flowering-time genes, each being activated by specific qualities and quantities of light and they probably interact with each other. However, their aforementioned role in the different components of long-day inductive conditions needs further physiological confirmation. The perception of light inductive to the flowering process is a time-dependent rhythmic process [3]. As with other endogenous rhythms, the circadian clock
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genes control the onset and period of this rhythm [1,4 / 6]. Light affects flowering in long-day plants via the clock and/or after the clock. Input of light signals to the clock affects genes that are part of the clock mechanism and acts as a reset and light-on signal for the circadian rhythm [1,3,7,8]. Long application of light affects the expression period and amplitude of flowering-time genes involved in the regulation of flowering in long day plants [9 /11]. Two time-dependent, long-day effects on flowering are the result of the circadian clock: the night-break (light break during the night) effect and the critical day length needed to induce flowering. Critical day length is a result of the rhythm, period and amplitude in flowering-time gene expression whereas the night-break treatment can act either as a light on signal to the clock or as a regulator of flowering-time genes after the clock [12]. These two different modes of action may have different interactions with the natural day: light on starts the photoperiod whereas the effect of a long photoperiod on flowering-time gene expression occurs toward the end of the photoperiod [2]. The phytochrome photoreceptors play an important role in the phototransduction of light for induction of flowering. This is reflected by the specific effects of the red and far-red light spectra in this process. During the second half of the photoperiod, long-day plants respond positively, in early flowering, to light that is rich in the far-red region of the spectrum, and to fluence rate [3,13/15]. Both, the red and far-red regions are active in the night-break induction of flowering. The efficiency of red compared with far-red in the night-break effect differs among plants. The actions of red or far-red light in night break were not reversible suggesting the high irradiance response (HIR) for the phytochrome mode of action during this treatment [3]. Phytochromes A and B participate in the phototransduction of light signal to the circadian clock and to the flowering-time genes [1,4,5,10,16,17]. The two phytochromes can shorten the period in the rhythm of clockcontrolled gene expression [17,18] resulting, for flowering, in a shorter critical day length. A shorter critical day length in long-day plants means flowering under shorter photoperiods. Phytochrome A mediates the flowering response of Arabidopsis and pea to day length under short [19,20] as well as long photoperiods, including night-break treatments [19 /23]. Except in the case of tissue culturegrown plants [19], this information is based on null mutants and at least in one case [20] it shows an interaction between phytochromes A and B in the perception of day-length signal for flowering. The effect of PHY A overexpression on the perception of day length signal to flowering may help to provide more information on its role in this process.
Phytochrome B has been identified as a flowering inhibitor in long-day plants, because of its negative interaction with the blue-light photoreceptor CRY2 and because of the early-flowering phenotype of the phyB mutant [10,11,24,25]. However, phytochrome B does not interact directly with flowering-time genes downstream of the long-day induction of flowering [26,27] and the phyB mutant flowers early, independent of day length [22,26/29]. The information on phytochrome B as a flowering inhibitor is based on null-mutant responses, in which the flowering response to day length is severely reduced, however, the null-mutant response may also indicate the importance of phytochrome B to the daylength controlling mechanism. In this respect, the effect of PHYB overexpression on the day-length controlling mechanism may provide important information. Except in the case of tissue culture grown plants [19], where overexpression of PHYB did not inhibit flowering, there is no information on the effect of its overexpression on flowering in long-day plants under short- or long-day conditions. Phytochromes A and B act as the photoreceptors in several photomorphogenic processes. Overexpression of either phytochrome A or B amplifies the responses to light in these processes [30]. Their overexpression also amplified the response to light of CAB 2, a clockcontrolled gene [18]. In a fluence rate-dependent process such as flowering in long-day plants, this quantitative effect of phytochrome overexpression is expected. We used natural daylight and Aster ‘Sun Karlo’ plants overexpressing either PHYA or PHYB to study the participation of these phytochromes in the qualitative and quantitative aspects of critical-day-length and night-break induction of the flowering response. By using the sun as the light source we hoped to trigger all possible interactions that normally occur under natural conditions. Aster (Asteraceae) is a long-short-day plant in terms of its flowering response [31]. It has a rosette growth form under short-day conditions whereas long days induce the transition from rosette to inflorescence shoot, the capitulum initiates under intermediate day length followed by flower initiation under short-day conditions. Following the transition from rosette to inflorescence, further elongation and branching of this shoot depends on the inhibition of capitulum initiation caused by long-day conditions.
2. Materials and methods 2.1. Plant material We used oat PHYA (in pRFY1 plasmid vector carried by Agrobacterium tumefaciens strain 2260) or Arabidopsis PHYB (in pROKB plasmid vector carried
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by A. tumefaciens strain LBA4404) described by Halliday et al. [32] to transform Aster ‘Sun Karlo’ plants according to the method described by Draper et al. [33] for Nicotiana tabacum . The transformation was confirmed using PCR and RT-PCR. Because Aster cultivars are not homozygous and are commercially propagated vegetatively the transformed plants were also vegetatively propagated from rosette-shoot cuttings. Cuttings were taken following three growth cycles, each time at the end of a growth cycle that started as a rooted rosette shoot and ended in flowering. During each growth cycle, the transformation effect on the response to day-length conditions was followed. At the end of the fourth cycle, transformation was confirmed by RT-PCR and the rooted cuttings were used in the experiments. The RT-PCR procedure included total RNA extraction with TRI reagent (MRC Inc.) followed by precipitation of residual DNA in 3 M sodium acetate, mRNA reverse transcription using the reverse transcriptase SuperScript II (GibcoBRL), Oligo (dT) [12 /18] primer (GibcoBRL) or 18S rDNA anti-sense primer, and finally PCR in the presence of the PHY s or 18S rDNA primers. To be sure that DNA was not present in the total RNA extraction, samples were checked in PCR before the RT procedure. The results of the RT-PCR (Fig. 6) indicate quantitative differences in transgenic mRNA among the different lines. 2.2. Light and day-length conditions We used the sun as the light source during the shortday period and artificial light for day-extension and night-break treatments. Under phytotron conditions (day/night temperatures of 20/12 8C) the short-day period included 8 h exposure to daylight (400 mmol m 2 s 1 PAR) and day extensions of 2 h were applied either, by daylight or by lowintensity artificial light. Artificial light during day extension was from incandescent plus fluorescent lamps with a fluence rate of 20 mmol m 2 s 1 PAR, 8.1 mmol m 2 s 1 red, 8.0 mmol m 2 s 1 far-red light and a red to far-red ratio of 1.02. Night break, in the middle of the 16 h dark period, was for 15 or 30 min from incandescent lamps, 1.4 mmol m 2 s 1 PAR, 0.9 mmol m 2 s 1 red, 1.5 mmol m 2 s 1 far-red and red to farred ratio of 0.6, or from fluorescent lamps, 2.4 mmol m 2 s 1 PAR, 1.1 mmol m 2 s 1 red, 0.15 mmol m 2 s 1 far-red and red to far-red ratio of 7.3. The plants grew through their entire growth cycle (from rosette to flowering) under a constant photoperiod. In the greenhouse, differences in natural day length within the short photoperiod regime and in fluence rate were used to determined quantitative effects of light. The natural short days from autumn through spring were either shortened to 10 h or extended by an artificial low-light intensity of 0.5 mmol m 2 s 1 PAR from
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either incandescent or fluorescent lamps. The same artificial light was used for a 2 h night-break at the middle of the natural night. The natural daylight effect was studied during two sequential growth cycles under a constant temperature regime of 24/14 8C, day/night. The natural day length and fluence rate during the periods of inflorescence development in the two growth cycles is presented in Table 1. The growth cycle included inflorescence development under long-day conditions and flower development under short natural-day conditions, except where the entire growth cycle was under short days of 10 h exposure to daylight. The transfer from long- to short-day conditions was considered to have occurred when 90% of the plant inflorescences had reached 55 cm in length. Two transgenic lines overexpressing PHYA and two overexpressing PHYB were tested for their potential commercial value during the period of commercial production from August through May in Israel. Rosette-shoot cuttings were rooted and planted on August 15, and day extensions started 3 weeks later using incandescent lighting (0.5 mmol m 2 s 1 PAR). The photoperiod treatments included natural daylength or natural day extension to photoperiods of 14 or 16 h. Three growth cycles were performed. At the end of each growth cycle, in November, February and May, the number and quality of the cut flowering shoots were determined. During the production period, the natural daylength varied between 13 h 40 min and 10 h (middle of winter), and the sun irradiance between 1500 and 300 mmol m 2 s 1 PAR, respectively.
3. Results 3.1. Critical day length A day length of 10 h was short for wild-type plants (cv. Sun Karlo, Table 2 and Fig. 5): their inflorescences did not elongate and as a consequence, they did not flower (as will be shown later its critical day-length is 14 h). Similarly, a day length of 10 h was short for PHYB overexpressing plants (Fig. 5). Overexpression of PHYA shortened the critical day length to 8 h (or less) in line A4-7 and to 10 h in line A17-3 (Table 2 and Fig. 5). The difference in critical day length between the two PHYATable 1 Natural day length and irradiance conditions included in the artificial long-day period of inflorescence shoot elongation during two growth cycles of transformed Aster cv. Sun Karlo plants in the greenhouse Daylength (h:min)
Irradiance (mmol m 2 s 1 PAR)
First growth cycle start: 12:00, end: 10:40 start: 1000, end: 500 Second growth cycle start: 10:06, end: 11:00 start: 300, end: 500
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Table 2 Day length and irradiance effects on inflorescence shoot and flower development in Aster cv. Sun Karlo plants transformed with PHYA Transgenic line
Light source for day extension
A 4 /7 A 4 /7 A 4 /7 A 17 /3 A 17 /3 A 17 /3 Sun Karlo Sun Karlo Sun Karlo
None Lamps Sun None Lamps Sun None Lamps Sun
Time to flowering (days) 7290.8 7692.0 7790.4 8893.7 9594.5
Rosette leaves at flowering (number) 6.890.5 5.290.4 4.890.4 12.291.0 6.590.7 3.890.5 1591.2 14.891.8 15.790.9
Plants were exposed to 8 h daylight extended to 10 h by daylight or artificial lighting in the phytotron. Data from 20 plants were used for means and standard error calculation. All inflorescent shoots were longer than 60 cm.
overexpressing lines (Table 2), and between the transgenic and wild-type lines, suggests that overexpression of phytochrome A does not eliminate the requirement for long days but shortens the critical day length needed for the long-day effect. The number of rosette leaves produced prior to shoot elongation indicated the physiological time of transition from rosette to inflorescence elongation (the beginning of the flowering process), a typical response of Aster to long-day conditions. The lower number of rosette leaves produced by PHYA line A4-7 when day length was extended from 8 to 10 h indicates the retained sensitivity to day length in this line. The sensitivity to fluence rate at the end of the photoperiod was also retained in PHYA -overexpressing plants, as indicated by comparing the number of rosette leaves produced by A17-3 under day extension by sunlight or artificial light, both with similar red to far-red ratio. The PHYA line A4-7 with the shortest critical day length did not respond to the difference in fluence rate during day extension. 3.2. Night-break effect Following 8 h exposure to daylight, short night-break treatments of 30 min using either incandescent or fluorescent lighting did not induce inflorescence development in wild-type cv. Sun Karlo plants (Tables 3 and 4). The PHYB lines B3-1 and B12-6, with critical day lengths longer than 10 h, showed high sensitivity to night breaks with either incandescent or fluorescent lighting. Under both types of light, they responded to a 15 min night break with inflorescence development. A difference between the two PHYB lines was found in their quantitative response to the increase in night-break duration, under longer night break B12-6 produced less rosette leaves independent of light source, whereas B3-1 exhibited a weak response only to longer fluorescent lighting indicating that overexpression of PHYB increased the sensitivity to night break but did not eliminate its quantitative effect. Following 8 h exposure to daylight, the PHY A line A17-3 (with critical day
length of 10 h) developed inflorescence only when the night break was applied for 30 min with light from incandescent lamps. The quantitative effect of nightbreak duration on PHYA plants was reflected in the higher number of rosette leaves produced by A4-7 under the shorter night break, independent of light source. 3.3. The effect of natural daylight in the response to day extensions with artificial lighting To test the effect of natural daylight on the rate of inflorescence development under the different photoperiod treatments, plants were grown for two sequential growth cycles under different natural day lengths and irradiances included in an artificially constant photoperiod, in the greenhouse. During the first growth cycle, the natural day length was longer and the daylight fluence rate higher than during the second growth cycle (Table 1). The sensitivity of each Aster line to natural daylight conditions is represented by the difference in time needed to reach the same developmental stage between the two growth cycles. When exposed to 10 h daylight during the entire two growth cycles in the greenhouse, the PHYA lines differed in their response to the daylight fluence rate, as indicated by the difference in length between the two growth cycles of each line (Fig. 1). Wild-type cv. Sun Karlo plants produced inflorescences only under day extension treatments. Under natural days extended to 16 h by incandescent lighting the wild type showed high sensitivity to natural daylight: longer natural days with higher fluence rates during the period of inflorescence development in the first growth cycle shortened the length of their entire growth cycle (Fig. 1). Under the same day-extension conditions, the PHYA lines differed in their response to the difference in natural daylight conditions during the two growth periods: A4-7 had the lowest and A17-3 the highest response (Fig. 1). Most PHYA lines had a lower response to natural daylight than the wild-type plants. This might be the result of shorter critical day length and/or higher efficiency in light transduction.
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Table 3 Inflorescent shoot and flower development on Aster cv. Sun Karlo plants transformed with PHYA or PHYB Transgenic lines
Night break duration (min)
A 4 /7 A 4 /0.7 A 17 /3 A 17 /3 B 12 /6 B 12 /6 B 3 /1 B 3 /1 Sun Karlo Sun Karlo
15 30 15 30 15 30 15 30 15 30
Time to flowering (days) 7691.0 7791.5 / 9892.0 9994.6 9992.6 10191.0 10092.6 / /
Rosette leaves at flowering (number) 5.090.4 3.890.2 10.791.1 5.590.6 8.690.8 5.590.6 4.890.8 4.890.2 12.390.9 13.092.1
Plants were treated with 8 h exposure to daylight followed by a short night break with incandescent lighting in the phytotron. Data from 20 plants were used for means and standard error calculation. All inflorescent shoots were longer than 60 cm.
Table 4 Inflorescent shoot and flower development on Aster cv. Sun Karlo plants transformed with PHYA or PHYB Transgenic lines
Night break duration (min)
A 4 /7 A 4 /.7 A 17 /3 A 17 /3 B 12 /6 B 12 /6 B 3 /1 B 3 /1 Sun Karlo Sun Karlo
15 30 15 30 15 30 15 30 15 30
Time to flowering (days) 8091.3 6791.6 / / 8292.6 6491.6 7791.7 5792.1 / /
Rosette leaves at flowering (number) 6.490.6 4.790.2 12.890.4 13.990.4 6.590.5 4.990.5 7.490.8 6.090.4 15.590.7 16.891.2
Plants were treated with 8 h exposure to daylight followed by a short night break with fluorescent lighting in the phytotron. Data from 20 plants were used for means and standard error calculation. All inflorescent shoots were longer than 50 cm.
Fig. 1. The length of the growth-cycle period of Aster plants cv. Sun Karlo and transformed PHYA lines, under the effect of photoperiod and natural day extension with incandescent lighting. Day length treatments of 10 h exposure to natural daylight or extension of natural daylight to 16 h by incandescent lighting (0.5 mmol m 2 s 1 PAR) were applied in the greenhouse. The time needed for inflorescence shoot elongation and flowering was measured during two sequential growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively. Data from 20 plants were used for means and standard error calculation.
The wild-type plants had a critical day length of nearly 14 h and in this photoperiod, they responded to natural-day extension with fluorescent but not incandescent lighting whereas the PHYB lines also responded to the incandescent lighting (Figs. 2 and 3), indicating a shorter critical day length and/or more efficient light transduction. This shorter critical day length was associated with a reduced effect of natural daylight conditions under day extensions to 16 h. Under the two photoperiods, 14 and 16 h, the PHYA lines showed almost no difference in their response to photoperiod and to the difference in natural daylight conditions under each photoperiod. Extending the natural day to 14 or 16 h with fluorescent, instead of incandescent lighting slightly delayed inflorescence development in the PHYA lines, even though their critical day length was 10 h or less (Figs. 2 and 3). 3.4. The effect of natural daylight conditions on the response to night break with artificial lighting A 2 h night-break with low-intensity incandescent or fluorescent lighting induced inflorescence development
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Fig. 2. Time needed for inflorescence shoot elongation in Aster cv. Sun Karlo plants and transformed PHYA and PHY B lines under natural-day extension with incandescent lighting. Plants were grown in the greenhouse under natural day extensions to 14 or 16 h by incandescent lighting, 0.5 mmol m 2 s 1 PAR. The time needed for inflorescence shoot elongation to 55 cm in 90% out of 30 plants was measured during two sequential growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively.
Fig. 3. Time needed for inflorescence shoot elongation in Aster cv. Sun Karlo plants and transformed PHYA and PHYB lines under the effect of natural day extension with fluorescent lighting. Plants were grown in the greenhouse under natural-day extensions to 14 or 16 h by fluorescent lighting (0.5 mmol m 2 s 1 PAR). Time needed for inflorescence shoot elongation to 55 cm in 90% out of 30 plants was measured during two growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively.
in ‘Sun Karlo’ plants (Fig. 4). The developmental rate of these plants when treated with incandescent lighting was highly influenced by daylight conditions, whereas treatment by fluorescent lighting almost overcame the daylight effect (almost no difference between the developmental rate during the two growth cycles). This difference in daylight effect between the response to the two qualities of light was due to the relatively higher efficiency of incandescent lighting following the longer natural day (first growth cycle) and of fluorescent lighting following the shorter natural day (second growth cycle). The difference between light qualities in their daylight-dependent effects during night break
Fig. 4. Time needed for inflorescence shoot elongation in Aster cv. Sun Karlo plants and transformed PHYA lines and PHYB lines under natural day conditions and night break with incandescent or fluorescent lighting. Under greenhouse conditions, night-break treatments were applied in the middle of the night for 2 h with either fluorescent or incandescent lighting (0.5 mmol m 2 s 1 PAR). Time needed for inflorescence shoot elongation to 55 cm in 90% out of 30 plants was measured during two sequential growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively.
Fig. 5. An illustration of transgenic Aster plants grown under 10 h exposure to natural daylight. The wild type, ‘Sun Karlo’ (C) and PHYB -overexpressing lines (B12-6 and B3-1) remained in the rosette growth stage, whereas the PHYA -overexpressing lines (A4-7 and A28) developed inflorescence shoots and flowers.
indicates different modes of action. The effect of PHYA or PHYB overexpression on reducing the daylightdependent response under all photoperiod treatments almost eliminated the difference between their response to fluorescent or incandescent lighting during the night break. 3.5. Commercial production of flowering shoots The commercial quality of the transgenic flowering shoots, as reflected by their length and weight, was similar to that of wild-type, and nearly the same for all treatments; however, the number of flowering shoots was greatly affected by the expression of transgenic
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4. Discussion
Fig. 6. Oat PHY A and Arabidopsis PHY B mRNA in transformed lines of Aster plants. The photograph includes the results of RT-PCR carried in the presence (/) or absence ( /) of the reverse transcriptase SuperScript II. Total RNA in the extract is represented by 18S rRNA.
PHY genes. Overexpression of either PHYA or PHYB increased the number of flowering shoots (mainly in the second and third growth cycles, data not shown, Table 5). PHYA overexpression in line A2-8 increased the total shoot yield by 52% without day extensions and by 115% with minimum day extensions. PHY B overexpression increased the total shoot yield by 94% in lines B3-1 and B12-6 under day extensions to 14 and 16 h, respectively. Table 5 Yield of flowering shoots (cut-flowers) produced by wild-type Aster cv. Sun Karlo and four transgenic lines overexpressing either PHYA or PHYB Transgenic line Photo-period Total shoot yield Total shoots yield (h) (no/m 2) (%) Sun-Karlo A2 /8 A2 /8 A4 /7 B3 /1 B12 /6
16 Natural 14 Natural 14 16
17297.6 261910.3 371915.8 215913.1 336918.2 335914.5
100 151 215 125 195 195
Plants were grown under commercial conditions from autumn through spring. Natural daylength changed during the production period between 13 h:40 min and 10 h. Day extension was by incandescent lighting. During this period, three cuttings were performed at the end of each growth cycle. Flowering shoots were collected from three replicates of 2 m2 each, counted and expressed as percent from wild type yield.
Using the sun as the source for the main light period within the diurnal light/dark cycle, we exposed the plants to the light spectrum and fluence rate, and to the diurnal cycle, to which they were naturally adapted. Under these conditions, the phytochromes may play their regulatory role in the control of flowering under natural conditions. In our study, the treatments were designed to manipulate the position of daylight within the photoperiod and to affect the light spectrum and fluence rate close to and during the marginal zone of critical day length. In this way, we demonstrated the importance of light quality and quantity during this period, the specific role of PHY A overexpression in determining the timing of the critical day length, and the specific role of PHY B overexpression in the night-break signal. Critical day length in many long-day plants has a marginal zone, which varies according to the quality and quantity of light applied close to, and during, this part of the photoperiod [3]. Light applied after the critical day length quantitatively accelerates the flowering response. In this respect, inflorescence development in Aster is a typical long-day process. Long photoperiods, as well as longer exposure to daylight within a constant artificial photoperiod, accelerated inflorescence development. Overexpression of either PHYA or PHYB in Aster caused inflorescence development under a day length that was short for wild-type plants. The PHYA line A4-7 developed inflorescence shoots under a photoperiod of 8 h whereas the PHYB lines B12-6 and AB3-1 did so under natural days extended to 14 h with incandescent lighting, both short photoperiods for wild-type plants. The effects of phytochrome overexpression can be attributed to an increase in light transduction during the marginal zone of the critical day length or to a shortening of the critical day length. Overexpression of either PHYA or PHYB improved the perception of sunlight and reduced the quantitative effect of light on the rate of inflorescence development. The difference in critical day length found between the PHYA -overexpressing line A4-7 (8 h) and wild-type plants (14-h) is about 6 h, a period that is too long to be included in the marginal zone of critical day length. The other possibility, which needs further study, is that PHYA overexpression shortened the critical day length by affecting the clock-controlled period in the rhythm of flowering-time gene expression. This possibility would explain our finding of PHY A inducing flowering under short-day conditions as well as lowering the sensitivity to daylight conditions under longer photoperiods. However, the importance of fluence rate close to the critical day length was preserved in the PHYA -overexpressing plants, as indicated by A17-3 (critical day-
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length of 10 h) response to fluence rate during day extension from 8 to 10 h. In wild-type Aster, fluorescent rather than incandescent lighting (red: far-red /7.3 vs. red: far-red /0.6, respectively), was more efficient at causing inflorescence shoot development when applied close to the critical day length (14 h), indicating a positive role for the type II phytochromes, to which phytochrome B belongs. In addition, overexpression of PHYB in Aster had a positive effect on the long-day process of inflorescence development. These findings do not support the conclusion-drawn from the response of the Arabidopsis phyB mutant to both short and long days [22,26 /29,34] that phytochrome B acts as an inhibitor of flowering. In our study, PHYB overexpression had a specific effect on the perception of the night-break signal, and an accelerating effect on the response to day extension under relatively low fluence rate conditions. In wild-type Aster plants, the effects of a night break with incandescent lighting and a day extension with either incandescent or fluorescent lighting were subject to the influence of daylight conditions. In contrast, reduced sensitivity to the same daylight conditions appeared in the response to night-break with fluorescent lighting. One possible explanation for this difference in sensitivity to daylight conditions is a difference in the timing of the light-on signal associated with differences in daylight conditions during the second half of the photoperiod. Following dawn (the light-on signal) the second half of the photoperiod included daylight and artificial light applied as a day extension or night break with incandescent lighting. The proportion between daylight and artificial light during this period varies with the variation in natural day-length. However, following the light-on signal applied during a night break with fluorescent lighting, the second half of the photoperiod overlapped the natural day with its high fluence rate, daylight conditions. The pronounced effect of PHY B overexpression on the perception of nightbreak signal in terms of the flowering process is in accordance with PHY B role in the transduction of redlight signal to the endogenous clock [17]. Under our experimental conditions, the pronounced effect of PHY A on the critical day length interfered with our ability to identify its possible effect on night break perception. In another study [35], the role of PHY A as a transducer of light to the circadian clock was demonstrated when light was applied before and in continuity with dawn (indicating an HIR): this possibility was not included in our experiments. The negative role of phytochrome B in the regulation of flowering in Arabidopsis was deduced from early flowering of the null mutant [22,26 /29] and from its effect on CRY2 activity under continuous light, where light on or light off signals do not exist [10,25]. In other studies, phytochrome B has been found to be the
primary high-intensity red-light photoreceptor for the input of light signal to the circadian clock [17] and a closely related circadian-clock gene [36]. The similarity between phyB and elf3 plants in their phenotype and function in the flowering process, together with the role of ELF3 in the transduction of light to the clock [37 /39] further supports the idea that phytochrome B is either a component of the circadian clock or an important light transducer for the clock’s entrainment. A role for phytochrome B in the clock’s entrainment could explain why the null-mutant flowering response does not necessarily indicate an inhibitory effect of phytochrome B in the flowering process induced by long days. In our study, phytochrome B played a significant role in the night-break signal, suggesting a role in the light-on signal to the circadian clock. Eliminating this signal may interfere with the clock-controlled rhythm and cause early flowering independent of day length, as is the case in elf3 [40]. According to this role overexpression of PHYB increased the transduction of light to the clock, and therefore, the efficiency of the light-on signal during night break. Our study describes the initiation of the flowering response in Aster , i.e. the transition from rosette to inflorescence shoot and the further development of this shoot. Although inflorescence shoot development in Aster is a prerequisite for flower initiation, the latter process occurs under short-day conditions. Further study is needed to demonstrate a direct effect of phytochrome overexpression on flower initiation under long-day conditions. Nevertheless, in Aster , overexpression of either PHYA or PHYB had a pronounced effect on increasing the commercial yield of flowering shoots (‘cut flowers’) and on decreasing the need for supplemental lighting.
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[25] T.C. Muckler, H. Guo, H. Yang, H. Duong, C. Lin, Antagonistic action of Arabidopsis cryptochromes and phytochrome B in the regulation of floral induction, Development 126 (1999) 2073 / 2082. [26] M.H. Blazquez, D. Weigel, Independent regulation of flowering by phytochrome B and gibberellins in Arabidopsis , Plant Physiol. 120 (1999) 1025 /1032. [27] K.J. Halliday, M. Koornneef, G.C. Whitelam, Phytochrome B and at least one other phytochrome mediate the flowering response of Arabidopsis thaliana to low red/far red ratio, Plant Physiol. 104 (1994) 1311 /1315. [28] N. Goto, T. Kumagai, M. Koornneef, Flowering responses to light breaks in photomorphogenic mutants of Arabidopsis thaliana , a long day plant, Physiol. Plant 83 (1991) 209 /215. [29] J.W. Reed, P. Nagpal, D.S. Poole, M. Furuyas, J. Chory, Mutation in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development , Plant Cell 5 (1993) 147 /157. [30] G.C. Whitelam, P.F. Devlin, Roles of different phytochromes in Arabidopsis Photomorphogenesis, Plant Cell Environ. 20 (1997) 752 /759. [31] W.W. Schwabe, Aster novi-belgi , in: A.H. Halevy (Ed.), Handbook of Flowering, vol. 5, CRC Press, Boca Raton FL, 1985, pp. 29 /41. [32] K.J. Halliday, B. Thomas, G.C. Whitelam, Expression of heterologous phytochrome A, B, or C in transgenic tobacco plants alters vegetative development and flowering time, Plant J. 12 (1997) 1079 /1090. [33] J. Draper, R. Scott, J. Hamil, Transformation of dicotyledonous plant cells using the Ti plasmid of Agrobacterium tumefaciens and the Ri plasmid of A. rhizogenes , in: J. Draper, R. Scott, P. Armitage, R. Waldon (Eds.), Plant Genetic Transformation and Gene Expression. A Laboratory Manual, Blackwell Sci Pub, Oxford, 1988, pp. 69 /160. [34] J. Chory, Light modulation of vegetative development, Plant Cell 9 (1997) 1225 /1234. [35] M.J. Yanovsky, M. Izaguirre, J.A. Wagmaister, C. Gatz, S.D. Jackson, B. Thomas, J.J. Casal, Phytochrome A resets the circadian clock and delays tuber formation under long days in potato, Plant J. 23 (2000) 223 /232. [36] L. Kozma-Bognar, A. Hall, E. Adam, S.C. Thain, F. Nagay, A.J. Millar, The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B, Proc. Natl. Acad. Sci. USA 96 (1999) 14652 /14657. [37] K.A. Hicks, A.J. Millar, I.A. Carre, D.E. Somers, M. Straume, D.R. Meeks-Wagner, S.A. Kay, Conditional circadian dysfunction of the Arabidopsis early-flowering 3 mutant, Science 274 (1996) 790 /792. [38] H.G. McWatters, R.M. Bastow, A. Hall, A.J. Millar, The ELF3 zeitnehmer regulates light signalling to the circadian clock, Nature 408 (2000) 716 /720. [39] J.W. Reed, P. Nagpal, R.M. Bastow, K.S. Solomon, M.J. Dowson-Day, R.P. Elumalai, A.J. Millar, Independent action of ELF3 and phy B to control hypocotyl elongation and flowering time, Plant Physiol. 122 (2000) 1149 /1160. [40] M.T. Zagotta, K.A. Hicks, C.I. Jacobs, J.C. Young, R.P. Hangarter, D.R. Meeks-Wagner, The Arabidopsis ELF3 gene regulates vegetative photomorphogenesis and the photoperiodic induction of flowering, Plant J. 10 (1996) (1996) 691 /702.
Modifications in Aster response to long-day conditions caused by overexpression of phytochrome A or B Israela Wallerstein a,*, Izhack Wallerstein b, Dayana Libman a, Boris Machnic a, Garry C. Whitelam c,1 b
a Department of Ornamental Horticulture, Agricultural Research Organisation, The Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel Department of Crop, Garden and Natural Resources, Agricultural Research Organisation, The Volcani Center, P.O. Box 6, Bet-Dagan 50-250, Israel c Department of Biology, University of Leicester, Leicester LE1 7RH, UK
Received 18 February 2002; received in revised form 19 April 2002; accepted 14 May 2002
Abstract We studied the role of phytochromes A and B in the regulation of critical day length, night-break signal and the quantitative effects of sunlight on inflorescence development, a long-day induced process, in Aster . We used transgenic plants overexpressing either PHY A or PHY B, different natural daylight conditions, and day extension and night break treatments by low-fluence-rate fluorescent or incandescent lighting with a red to far-red ratio of 7.3 and 0.6, respectively. Wild-type plants had a higher sensitivity to day extension with fluorescent than incandescent lighting and a quantitative response to natural daylight conditions. Their response to a day extension with either incandescent or fluorescent lighting and to a night break with incandescent, but not fluorescent, lighting was subject to the quantitative effect of daylight conditions during the natural short day. Overexpression of PHY A shortened the critical day length for inflorescence development from 14 to 8 h. Overexpression of PHY B shortened the length of the night break needed to induce inflorescence development from 2 h to 15 min. Overexpression of either PHY A or B reduced the quantitative effect of natural daylight. For Aster plants grown under commercial conditions, from autumn through spring, the overexpression of either PHYA or PHYB substantially increased the yield of flowering shoots. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phytochrome A; Phytochrome B; Flowering time; Aster ; Day length; Night break
1. Introduction Long-day plants are adapted to respond to the duration, irradiance and quality of daylight. The perception of long days, which are inductive for flowering, is a complicated process involving photoreceptors, the endogenous circadian clock and several floweringtime genes [1]. Light interacts, via the photoreceptors, with the operation of the endogenous clock and the expression of the flowering-time genes [2]. The role of light has both qualitative and quantitative aspects. The qualitative aspect includes its importance for critical day
* Corresponding author. Tel.: /972-3-968-3765; fax: /972-3-9660589 E-mail addresses: [email protected] (I. Wallerstein), [email protected] (G.C. Whitelam). 1 Tel.: /44-116-252-3396; fax: /44-116-252-2791.
length and night-break effects, and the quantitative aspect includes increased response with increased fluence rate. The characteristics of critical day length and night break in long-day plants differ from those in shortday plants. In the former, the daylight spectrum and high fluence rates can shorten the critical day length and relatively long exposure to light during the night break is needed to induce flowering [3]. Several photoreceptors play roles in the transduction of light to the endogenous clock and flowering-time genes, each being activated by specific qualities and quantities of light and they probably interact with each other. However, their aforementioned role in the different components of long-day inductive conditions needs further physiological confirmation. The perception of light inductive to the flowering process is a time-dependent rhythmic process [3]. As with other endogenous rhythms, the circadian clock
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 1 4 1 - 3
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genes control the onset and period of this rhythm [1,4 / 6]. Light affects flowering in long-day plants via the clock and/or after the clock. Input of light signals to the clock affects genes that are part of the clock mechanism and acts as a reset and light-on signal for the circadian rhythm [1,3,7,8]. Long application of light affects the expression period and amplitude of flowering-time genes involved in the regulation of flowering in long day plants [9 /11]. Two time-dependent, long-day effects on flowering are the result of the circadian clock: the night-break (light break during the night) effect and the critical day length needed to induce flowering. Critical day length is a result of the rhythm, period and amplitude in flowering-time gene expression whereas the night-break treatment can act either as a light on signal to the clock or as a regulator of flowering-time genes after the clock [12]. These two different modes of action may have different interactions with the natural day: light on starts the photoperiod whereas the effect of a long photoperiod on flowering-time gene expression occurs toward the end of the photoperiod [2]. The phytochrome photoreceptors play an important role in the phototransduction of light for induction of flowering. This is reflected by the specific effects of the red and far-red light spectra in this process. During the second half of the photoperiod, long-day plants respond positively, in early flowering, to light that is rich in the far-red region of the spectrum, and to fluence rate [3,13/15]. Both, the red and far-red regions are active in the night-break induction of flowering. The efficiency of red compared with far-red in the night-break effect differs among plants. The actions of red or far-red light in night break were not reversible suggesting the high irradiance response (HIR) for the phytochrome mode of action during this treatment [3]. Phytochromes A and B participate in the phototransduction of light signal to the circadian clock and to the flowering-time genes [1,4,5,10,16,17]. The two phytochromes can shorten the period in the rhythm of clockcontrolled gene expression [17,18] resulting, for flowering, in a shorter critical day length. A shorter critical day length in long-day plants means flowering under shorter photoperiods. Phytochrome A mediates the flowering response of Arabidopsis and pea to day length under short [19,20] as well as long photoperiods, including night-break treatments [19 /23]. Except in the case of tissue culturegrown plants [19], this information is based on null mutants and at least in one case [20] it shows an interaction between phytochromes A and B in the perception of day-length signal for flowering. The effect of PHY A overexpression on the perception of day length signal to flowering may help to provide more information on its role in this process.
Phytochrome B has been identified as a flowering inhibitor in long-day plants, because of its negative interaction with the blue-light photoreceptor CRY2 and because of the early-flowering phenotype of the phyB mutant [10,11,24,25]. However, phytochrome B does not interact directly with flowering-time genes downstream of the long-day induction of flowering [26,27] and the phyB mutant flowers early, independent of day length [22,26/29]. The information on phytochrome B as a flowering inhibitor is based on null-mutant responses, in which the flowering response to day length is severely reduced, however, the null-mutant response may also indicate the importance of phytochrome B to the daylength controlling mechanism. In this respect, the effect of PHYB overexpression on the day-length controlling mechanism may provide important information. Except in the case of tissue culture grown plants [19], where overexpression of PHYB did not inhibit flowering, there is no information on the effect of its overexpression on flowering in long-day plants under short- or long-day conditions. Phytochromes A and B act as the photoreceptors in several photomorphogenic processes. Overexpression of either phytochrome A or B amplifies the responses to light in these processes [30]. Their overexpression also amplified the response to light of CAB 2, a clockcontrolled gene [18]. In a fluence rate-dependent process such as flowering in long-day plants, this quantitative effect of phytochrome overexpression is expected. We used natural daylight and Aster ‘Sun Karlo’ plants overexpressing either PHYA or PHYB to study the participation of these phytochromes in the qualitative and quantitative aspects of critical-day-length and night-break induction of the flowering response. By using the sun as the light source we hoped to trigger all possible interactions that normally occur under natural conditions. Aster (Asteraceae) is a long-short-day plant in terms of its flowering response [31]. It has a rosette growth form under short-day conditions whereas long days induce the transition from rosette to inflorescence shoot, the capitulum initiates under intermediate day length followed by flower initiation under short-day conditions. Following the transition from rosette to inflorescence, further elongation and branching of this shoot depends on the inhibition of capitulum initiation caused by long-day conditions.
2. Materials and methods 2.1. Plant material We used oat PHYA (in pRFY1 plasmid vector carried by Agrobacterium tumefaciens strain 2260) or Arabidopsis PHYB (in pROKB plasmid vector carried
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by A. tumefaciens strain LBA4404) described by Halliday et al. [32] to transform Aster ‘Sun Karlo’ plants according to the method described by Draper et al. [33] for Nicotiana tabacum . The transformation was confirmed using PCR and RT-PCR. Because Aster cultivars are not homozygous and are commercially propagated vegetatively the transformed plants were also vegetatively propagated from rosette-shoot cuttings. Cuttings were taken following three growth cycles, each time at the end of a growth cycle that started as a rooted rosette shoot and ended in flowering. During each growth cycle, the transformation effect on the response to day-length conditions was followed. At the end of the fourth cycle, transformation was confirmed by RT-PCR and the rooted cuttings were used in the experiments. The RT-PCR procedure included total RNA extraction with TRI reagent (MRC Inc.) followed by precipitation of residual DNA in 3 M sodium acetate, mRNA reverse transcription using the reverse transcriptase SuperScript II (GibcoBRL), Oligo (dT) [12 /18] primer (GibcoBRL) or 18S rDNA anti-sense primer, and finally PCR in the presence of the PHY s or 18S rDNA primers. To be sure that DNA was not present in the total RNA extraction, samples were checked in PCR before the RT procedure. The results of the RT-PCR (Fig. 6) indicate quantitative differences in transgenic mRNA among the different lines. 2.2. Light and day-length conditions We used the sun as the light source during the shortday period and artificial light for day-extension and night-break treatments. Under phytotron conditions (day/night temperatures of 20/12 8C) the short-day period included 8 h exposure to daylight (400 mmol m 2 s 1 PAR) and day extensions of 2 h were applied either, by daylight or by lowintensity artificial light. Artificial light during day extension was from incandescent plus fluorescent lamps with a fluence rate of 20 mmol m 2 s 1 PAR, 8.1 mmol m 2 s 1 red, 8.0 mmol m 2 s 1 far-red light and a red to far-red ratio of 1.02. Night break, in the middle of the 16 h dark period, was for 15 or 30 min from incandescent lamps, 1.4 mmol m 2 s 1 PAR, 0.9 mmol m 2 s 1 red, 1.5 mmol m 2 s 1 far-red and red to farred ratio of 0.6, or from fluorescent lamps, 2.4 mmol m 2 s 1 PAR, 1.1 mmol m 2 s 1 red, 0.15 mmol m 2 s 1 far-red and red to far-red ratio of 7.3. The plants grew through their entire growth cycle (from rosette to flowering) under a constant photoperiod. In the greenhouse, differences in natural day length within the short photoperiod regime and in fluence rate were used to determined quantitative effects of light. The natural short days from autumn through spring were either shortened to 10 h or extended by an artificial low-light intensity of 0.5 mmol m 2 s 1 PAR from
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either incandescent or fluorescent lamps. The same artificial light was used for a 2 h night-break at the middle of the natural night. The natural daylight effect was studied during two sequential growth cycles under a constant temperature regime of 24/14 8C, day/night. The natural day length and fluence rate during the periods of inflorescence development in the two growth cycles is presented in Table 1. The growth cycle included inflorescence development under long-day conditions and flower development under short natural-day conditions, except where the entire growth cycle was under short days of 10 h exposure to daylight. The transfer from long- to short-day conditions was considered to have occurred when 90% of the plant inflorescences had reached 55 cm in length. Two transgenic lines overexpressing PHYA and two overexpressing PHYB were tested for their potential commercial value during the period of commercial production from August through May in Israel. Rosette-shoot cuttings were rooted and planted on August 15, and day extensions started 3 weeks later using incandescent lighting (0.5 mmol m 2 s 1 PAR). The photoperiod treatments included natural daylength or natural day extension to photoperiods of 14 or 16 h. Three growth cycles were performed. At the end of each growth cycle, in November, February and May, the number and quality of the cut flowering shoots were determined. During the production period, the natural daylength varied between 13 h 40 min and 10 h (middle of winter), and the sun irradiance between 1500 and 300 mmol m 2 s 1 PAR, respectively.
3. Results 3.1. Critical day length A day length of 10 h was short for wild-type plants (cv. Sun Karlo, Table 2 and Fig. 5): their inflorescences did not elongate and as a consequence, they did not flower (as will be shown later its critical day-length is 14 h). Similarly, a day length of 10 h was short for PHYB overexpressing plants (Fig. 5). Overexpression of PHYA shortened the critical day length to 8 h (or less) in line A4-7 and to 10 h in line A17-3 (Table 2 and Fig. 5). The difference in critical day length between the two PHYATable 1 Natural day length and irradiance conditions included in the artificial long-day period of inflorescence shoot elongation during two growth cycles of transformed Aster cv. Sun Karlo plants in the greenhouse Daylength (h:min)
Irradiance (mmol m 2 s 1 PAR)
First growth cycle start: 12:00, end: 10:40 start: 1000, end: 500 Second growth cycle start: 10:06, end: 11:00 start: 300, end: 500
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Table 2 Day length and irradiance effects on inflorescence shoot and flower development in Aster cv. Sun Karlo plants transformed with PHYA Transgenic line
Light source for day extension
A 4 /7 A 4 /7 A 4 /7 A 17 /3 A 17 /3 A 17 /3 Sun Karlo Sun Karlo Sun Karlo
None Lamps Sun None Lamps Sun None Lamps Sun
Time to flowering (days) 7290.8 7692.0 7790.4 8893.7 9594.5
Rosette leaves at flowering (number) 6.890.5 5.290.4 4.890.4 12.291.0 6.590.7 3.890.5 1591.2 14.891.8 15.790.9
Plants were exposed to 8 h daylight extended to 10 h by daylight or artificial lighting in the phytotron. Data from 20 plants were used for means and standard error calculation. All inflorescent shoots were longer than 60 cm.
overexpressing lines (Table 2), and between the transgenic and wild-type lines, suggests that overexpression of phytochrome A does not eliminate the requirement for long days but shortens the critical day length needed for the long-day effect. The number of rosette leaves produced prior to shoot elongation indicated the physiological time of transition from rosette to inflorescence elongation (the beginning of the flowering process), a typical response of Aster to long-day conditions. The lower number of rosette leaves produced by PHYA line A4-7 when day length was extended from 8 to 10 h indicates the retained sensitivity to day length in this line. The sensitivity to fluence rate at the end of the photoperiod was also retained in PHYA -overexpressing plants, as indicated by comparing the number of rosette leaves produced by A17-3 under day extension by sunlight or artificial light, both with similar red to far-red ratio. The PHYA line A4-7 with the shortest critical day length did not respond to the difference in fluence rate during day extension. 3.2. Night-break effect Following 8 h exposure to daylight, short night-break treatments of 30 min using either incandescent or fluorescent lighting did not induce inflorescence development in wild-type cv. Sun Karlo plants (Tables 3 and 4). The PHYB lines B3-1 and B12-6, with critical day lengths longer than 10 h, showed high sensitivity to night breaks with either incandescent or fluorescent lighting. Under both types of light, they responded to a 15 min night break with inflorescence development. A difference between the two PHYB lines was found in their quantitative response to the increase in night-break duration, under longer night break B12-6 produced less rosette leaves independent of light source, whereas B3-1 exhibited a weak response only to longer fluorescent lighting indicating that overexpression of PHYB increased the sensitivity to night break but did not eliminate its quantitative effect. Following 8 h exposure to daylight, the PHY A line A17-3 (with critical day
length of 10 h) developed inflorescence only when the night break was applied for 30 min with light from incandescent lamps. The quantitative effect of nightbreak duration on PHYA plants was reflected in the higher number of rosette leaves produced by A4-7 under the shorter night break, independent of light source. 3.3. The effect of natural daylight in the response to day extensions with artificial lighting To test the effect of natural daylight on the rate of inflorescence development under the different photoperiod treatments, plants were grown for two sequential growth cycles under different natural day lengths and irradiances included in an artificially constant photoperiod, in the greenhouse. During the first growth cycle, the natural day length was longer and the daylight fluence rate higher than during the second growth cycle (Table 1). The sensitivity of each Aster line to natural daylight conditions is represented by the difference in time needed to reach the same developmental stage between the two growth cycles. When exposed to 10 h daylight during the entire two growth cycles in the greenhouse, the PHYA lines differed in their response to the daylight fluence rate, as indicated by the difference in length between the two growth cycles of each line (Fig. 1). Wild-type cv. Sun Karlo plants produced inflorescences only under day extension treatments. Under natural days extended to 16 h by incandescent lighting the wild type showed high sensitivity to natural daylight: longer natural days with higher fluence rates during the period of inflorescence development in the first growth cycle shortened the length of their entire growth cycle (Fig. 1). Under the same day-extension conditions, the PHYA lines differed in their response to the difference in natural daylight conditions during the two growth periods: A4-7 had the lowest and A17-3 the highest response (Fig. 1). Most PHYA lines had a lower response to natural daylight than the wild-type plants. This might be the result of shorter critical day length and/or higher efficiency in light transduction.
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Table 3 Inflorescent shoot and flower development on Aster cv. Sun Karlo plants transformed with PHYA or PHYB Transgenic lines
Night break duration (min)
A 4 /7 A 4 /0.7 A 17 /3 A 17 /3 B 12 /6 B 12 /6 B 3 /1 B 3 /1 Sun Karlo Sun Karlo
15 30 15 30 15 30 15 30 15 30
Time to flowering (days) 7691.0 7791.5 / 9892.0 9994.6 9992.6 10191.0 10092.6 / /
Rosette leaves at flowering (number) 5.090.4 3.890.2 10.791.1 5.590.6 8.690.8 5.590.6 4.890.8 4.890.2 12.390.9 13.092.1
Plants were treated with 8 h exposure to daylight followed by a short night break with incandescent lighting in the phytotron. Data from 20 plants were used for means and standard error calculation. All inflorescent shoots were longer than 60 cm.
Table 4 Inflorescent shoot and flower development on Aster cv. Sun Karlo plants transformed with PHYA or PHYB Transgenic lines
Night break duration (min)
A 4 /7 A 4 /.7 A 17 /3 A 17 /3 B 12 /6 B 12 /6 B 3 /1 B 3 /1 Sun Karlo Sun Karlo
15 30 15 30 15 30 15 30 15 30
Time to flowering (days) 8091.3 6791.6 / / 8292.6 6491.6 7791.7 5792.1 / /
Rosette leaves at flowering (number) 6.490.6 4.790.2 12.890.4 13.990.4 6.590.5 4.990.5 7.490.8 6.090.4 15.590.7 16.891.2
Plants were treated with 8 h exposure to daylight followed by a short night break with fluorescent lighting in the phytotron. Data from 20 plants were used for means and standard error calculation. All inflorescent shoots were longer than 50 cm.
Fig. 1. The length of the growth-cycle period of Aster plants cv. Sun Karlo and transformed PHYA lines, under the effect of photoperiod and natural day extension with incandescent lighting. Day length treatments of 10 h exposure to natural daylight or extension of natural daylight to 16 h by incandescent lighting (0.5 mmol m 2 s 1 PAR) were applied in the greenhouse. The time needed for inflorescence shoot elongation and flowering was measured during two sequential growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively. Data from 20 plants were used for means and standard error calculation.
The wild-type plants had a critical day length of nearly 14 h and in this photoperiod, they responded to natural-day extension with fluorescent but not incandescent lighting whereas the PHYB lines also responded to the incandescent lighting (Figs. 2 and 3), indicating a shorter critical day length and/or more efficient light transduction. This shorter critical day length was associated with a reduced effect of natural daylight conditions under day extensions to 16 h. Under the two photoperiods, 14 and 16 h, the PHYA lines showed almost no difference in their response to photoperiod and to the difference in natural daylight conditions under each photoperiod. Extending the natural day to 14 or 16 h with fluorescent, instead of incandescent lighting slightly delayed inflorescence development in the PHYA lines, even though their critical day length was 10 h or less (Figs. 2 and 3). 3.4. The effect of natural daylight conditions on the response to night break with artificial lighting A 2 h night-break with low-intensity incandescent or fluorescent lighting induced inflorescence development
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Fig. 2. Time needed for inflorescence shoot elongation in Aster cv. Sun Karlo plants and transformed PHYA and PHY B lines under natural-day extension with incandescent lighting. Plants were grown in the greenhouse under natural day extensions to 14 or 16 h by incandescent lighting, 0.5 mmol m 2 s 1 PAR. The time needed for inflorescence shoot elongation to 55 cm in 90% out of 30 plants was measured during two sequential growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively.
Fig. 3. Time needed for inflorescence shoot elongation in Aster cv. Sun Karlo plants and transformed PHYA and PHYB lines under the effect of natural day extension with fluorescent lighting. Plants were grown in the greenhouse under natural-day extensions to 14 or 16 h by fluorescent lighting (0.5 mmol m 2 s 1 PAR). Time needed for inflorescence shoot elongation to 55 cm in 90% out of 30 plants was measured during two growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively.
in ‘Sun Karlo’ plants (Fig. 4). The developmental rate of these plants when treated with incandescent lighting was highly influenced by daylight conditions, whereas treatment by fluorescent lighting almost overcame the daylight effect (almost no difference between the developmental rate during the two growth cycles). This difference in daylight effect between the response to the two qualities of light was due to the relatively higher efficiency of incandescent lighting following the longer natural day (first growth cycle) and of fluorescent lighting following the shorter natural day (second growth cycle). The difference between light qualities in their daylight-dependent effects during night break
Fig. 4. Time needed for inflorescence shoot elongation in Aster cv. Sun Karlo plants and transformed PHYA lines and PHYB lines under natural day conditions and night break with incandescent or fluorescent lighting. Under greenhouse conditions, night-break treatments were applied in the middle of the night for 2 h with either fluorescent or incandescent lighting (0.5 mmol m 2 s 1 PAR). Time needed for inflorescence shoot elongation to 55 cm in 90% out of 30 plants was measured during two sequential growth cycles under different natural day conditions (Table 1). Grey and black columns indicate the first and second growth cycle, respectively.
Fig. 5. An illustration of transgenic Aster plants grown under 10 h exposure to natural daylight. The wild type, ‘Sun Karlo’ (C) and PHYB -overexpressing lines (B12-6 and B3-1) remained in the rosette growth stage, whereas the PHYA -overexpressing lines (A4-7 and A28) developed inflorescence shoots and flowers.
indicates different modes of action. The effect of PHYA or PHYB overexpression on reducing the daylightdependent response under all photoperiod treatments almost eliminated the difference between their response to fluorescent or incandescent lighting during the night break. 3.5. Commercial production of flowering shoots The commercial quality of the transgenic flowering shoots, as reflected by their length and weight, was similar to that of wild-type, and nearly the same for all treatments; however, the number of flowering shoots was greatly affected by the expression of transgenic
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4. Discussion
Fig. 6. Oat PHY A and Arabidopsis PHY B mRNA in transformed lines of Aster plants. The photograph includes the results of RT-PCR carried in the presence (/) or absence ( /) of the reverse transcriptase SuperScript II. Total RNA in the extract is represented by 18S rRNA.
PHY genes. Overexpression of either PHYA or PHYB increased the number of flowering shoots (mainly in the second and third growth cycles, data not shown, Table 5). PHYA overexpression in line A2-8 increased the total shoot yield by 52% without day extensions and by 115% with minimum day extensions. PHY B overexpression increased the total shoot yield by 94% in lines B3-1 and B12-6 under day extensions to 14 and 16 h, respectively. Table 5 Yield of flowering shoots (cut-flowers) produced by wild-type Aster cv. Sun Karlo and four transgenic lines overexpressing either PHYA or PHYB Transgenic line Photo-period Total shoot yield Total shoots yield (h) (no/m 2) (%) Sun-Karlo A2 /8 A2 /8 A4 /7 B3 /1 B12 /6
16 Natural 14 Natural 14 16
17297.6 261910.3 371915.8 215913.1 336918.2 335914.5
100 151 215 125 195 195
Plants were grown under commercial conditions from autumn through spring. Natural daylength changed during the production period between 13 h:40 min and 10 h. Day extension was by incandescent lighting. During this period, three cuttings were performed at the end of each growth cycle. Flowering shoots were collected from three replicates of 2 m2 each, counted and expressed as percent from wild type yield.
Using the sun as the source for the main light period within the diurnal light/dark cycle, we exposed the plants to the light spectrum and fluence rate, and to the diurnal cycle, to which they were naturally adapted. Under these conditions, the phytochromes may play their regulatory role in the control of flowering under natural conditions. In our study, the treatments were designed to manipulate the position of daylight within the photoperiod and to affect the light spectrum and fluence rate close to and during the marginal zone of critical day length. In this way, we demonstrated the importance of light quality and quantity during this period, the specific role of PHY A overexpression in determining the timing of the critical day length, and the specific role of PHY B overexpression in the night-break signal. Critical day length in many long-day plants has a marginal zone, which varies according to the quality and quantity of light applied close to, and during, this part of the photoperiod [3]. Light applied after the critical day length quantitatively accelerates the flowering response. In this respect, inflorescence development in Aster is a typical long-day process. Long photoperiods, as well as longer exposure to daylight within a constant artificial photoperiod, accelerated inflorescence development. Overexpression of either PHYA or PHYB in Aster caused inflorescence development under a day length that was short for wild-type plants. The PHYA line A4-7 developed inflorescence shoots under a photoperiod of 8 h whereas the PHYB lines B12-6 and AB3-1 did so under natural days extended to 14 h with incandescent lighting, both short photoperiods for wild-type plants. The effects of phytochrome overexpression can be attributed to an increase in light transduction during the marginal zone of the critical day length or to a shortening of the critical day length. Overexpression of either PHYA or PHYB improved the perception of sunlight and reduced the quantitative effect of light on the rate of inflorescence development. The difference in critical day length found between the PHYA -overexpressing line A4-7 (8 h) and wild-type plants (14-h) is about 6 h, a period that is too long to be included in the marginal zone of critical day length. The other possibility, which needs further study, is that PHYA overexpression shortened the critical day length by affecting the clock-controlled period in the rhythm of flowering-time gene expression. This possibility would explain our finding of PHY A inducing flowering under short-day conditions as well as lowering the sensitivity to daylight conditions under longer photoperiods. However, the importance of fluence rate close to the critical day length was preserved in the PHYA -overexpressing plants, as indicated by A17-3 (critical day-
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length of 10 h) response to fluence rate during day extension from 8 to 10 h. In wild-type Aster, fluorescent rather than incandescent lighting (red: far-red /7.3 vs. red: far-red /0.6, respectively), was more efficient at causing inflorescence shoot development when applied close to the critical day length (14 h), indicating a positive role for the type II phytochromes, to which phytochrome B belongs. In addition, overexpression of PHYB in Aster had a positive effect on the long-day process of inflorescence development. These findings do not support the conclusion-drawn from the response of the Arabidopsis phyB mutant to both short and long days [22,26 /29,34] that phytochrome B acts as an inhibitor of flowering. In our study, PHYB overexpression had a specific effect on the perception of the night-break signal, and an accelerating effect on the response to day extension under relatively low fluence rate conditions. In wild-type Aster plants, the effects of a night break with incandescent lighting and a day extension with either incandescent or fluorescent lighting were subject to the influence of daylight conditions. In contrast, reduced sensitivity to the same daylight conditions appeared in the response to night-break with fluorescent lighting. One possible explanation for this difference in sensitivity to daylight conditions is a difference in the timing of the light-on signal associated with differences in daylight conditions during the second half of the photoperiod. Following dawn (the light-on signal) the second half of the photoperiod included daylight and artificial light applied as a day extension or night break with incandescent lighting. The proportion between daylight and artificial light during this period varies with the variation in natural day-length. However, following the light-on signal applied during a night break with fluorescent lighting, the second half of the photoperiod overlapped the natural day with its high fluence rate, daylight conditions. The pronounced effect of PHY B overexpression on the perception of nightbreak signal in terms of the flowering process is in accordance with PHY B role in the transduction of redlight signal to the endogenous clock [17]. Under our experimental conditions, the pronounced effect of PHY A on the critical day length interfered with our ability to identify its possible effect on night break perception. In another study [35], the role of PHY A as a transducer of light to the circadian clock was demonstrated when light was applied before and in continuity with dawn (indicating an HIR): this possibility was not included in our experiments. The negative role of phytochrome B in the regulation of flowering in Arabidopsis was deduced from early flowering of the null mutant [22,26 /29] and from its effect on CRY2 activity under continuous light, where light on or light off signals do not exist [10,25]. In other studies, phytochrome B has been found to be the
primary high-intensity red-light photoreceptor for the input of light signal to the circadian clock [17] and a closely related circadian-clock gene [36]. The similarity between phyB and elf3 plants in their phenotype and function in the flowering process, together with the role of ELF3 in the transduction of light to the clock [37 /39] further supports the idea that phytochrome B is either a component of the circadian clock or an important light transducer for the clock’s entrainment. A role for phytochrome B in the clock’s entrainment could explain why the null-mutant flowering response does not necessarily indicate an inhibitory effect of phytochrome B in the flowering process induced by long days. In our study, phytochrome B played a significant role in the night-break signal, suggesting a role in the light-on signal to the circadian clock. Eliminating this signal may interfere with the clock-controlled rhythm and cause early flowering independent of day length, as is the case in elf3 [40]. According to this role overexpression of PHYB increased the transduction of light to the clock, and therefore, the efficiency of the light-on signal during night break. Our study describes the initiation of the flowering response in Aster , i.e. the transition from rosette to inflorescence shoot and the further development of this shoot. Although inflorescence shoot development in Aster is a prerequisite for flower initiation, the latter process occurs under short-day conditions. Further study is needed to demonstrate a direct effect of phytochrome overexpression on flower initiation under long-day conditions. Nevertheless, in Aster , overexpression of either PHYA or PHYB had a pronounced effect on increasing the commercial yield of flowering shoots (‘cut flowers’) and on decreasing the need for supplemental lighting.
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