Original Paper Environ. Control Biol., 52 (1), 21
27, 2014 DOI: 10.2525/ecb.52.21 Characterization of Circadian Rhythms Through a Bioluminescence Reporter Assay in Lactuca sativa L.

1 1 1 2 1 Takanobu HIGASHI ,AkikoKAMITAMARI ,NobuyaOKAMURA ,KazuyaUKAI , Kenichi OKAMURA , 1 2 Takahiro TEZUKA and Hirokazu FUKUDA

1 Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai 599
8531, Japan 2 Department of Mechanical Engineering, Graduate School of Engineering, Osaka Prefecture University, Sakai 599
8531, Japan

(Received April 21, 2013; Accepted December 7, 2013)

Circadian rhythms are observed in many physiological events in plant, and clock genes orchestrate the rhythms of ex- pression of many genes. Precise environmental control of circadian rhythms provides a key technology for enhancing plant growth in artificial environments. In this study, we investigated the basic properties of a circadian rhythm for establishment of its control engineering in lettuce (Lactuca sativa L.). Bioluminescence of transgenic lettuce carrying an AtCCA1::LUC construct as a reporter of circadian gene expression was measured in young lettuce seedlings. We observed three basic prop- erties; free-running circadian rhythms under constant conditions without day-night cycles, entrainment to red and blue light cycles with 12 h light-12 h dark period, and temperature compensation of a free-running period in three lettuce cultivars (Cisco, Cos, Greenwave). In addition, a light-quality dependence of the free-running period and sensitivity to blue-light cy- cles with small amplitude (20% of variance in average light intensity) and non-24 h period were also observed. The results in this study indicated that it is possible to control lettuce circadian rhythms by non-24 h period light cycles in LED illumi- nation. It will play an important role in the research of control engineering for the circadian clock of lettuce in closed plant factories with artificial lighting. Keywords : bioluminescence assay, entrainment, light emitting diode, plant factory, synchronization, temperature compen- sation

REGULATORs)andTOC1 (TIMING OF CAB INTRODUCTION EXPRESSION 1), are generated by transcriptional feedback loops in each cell (Alabadí et al., 2001; Nakamichi et al., Plants synchronize internal physiological events with 2004, 2010, 2012; Takase et al., 2013). This endogenous diurnal environmental changes using the endogenous rhythm is called a circadian rhythm, and it has three basic circadian clock, which is an oscillation generator with a pe- properties. First, it has a self-sustained rhythm with about riod of approximately 24 h (Michael and McClung, 2003; a 24 h period, even if there is no external period change Montaigu et al., 2010). Expression of crucial genes such as such as day-night cycles. The period of self-sustained those involved in photosynthesis, sucrose metabolism, and rhythm is called the natural period or free-running period abiotic stress responses are orchestrated by the circadian (Harmer, 2009). Second, it entrains (synchronizes) to envi- clock (Harmer et al., 2000; Graf et al., 2010; Farré, 2012). ronmental periods via light or temperature cycles, even if In addition, the matching of the circadian rhythm to diurnal their periods are not equal to that of the circadian rhythm. environmental cycles affects plant growth and yield, Finally, the third key feature of circadian rhythms is tem- namely circadian resonance (Dodd et al., 2005; Fukuda et perature compensation. The rate of a typical chemical reac- al., 2011). Therefore, control of the circadian rhythm tion doubles with a 10°C increase in temperature

would be useful for the development of closed plant culti- (temperature coefficient Q10
2) (McClung, 2006), while vation systems (plant factories with artificial light) the period of the circadian rhythm of CCA1 expression in

(Giddings et al., 2000; Fukuda et al., 2011). Arabidopsis exhibits a Q10
0.86 (Nakamichi et al., 2004). In Arabidopsis thaliana, a model plant in molecular These three basic properties are utilized as the defining at- biology and physiology, the circadian clock has been well tributes of circadian rhythms for all living organisms. studied (Mizoguchi et al., 2002; McClung, 2006; Harmer, In studies characterizing circadian rhythms, biolumi- 2009; Jose et al., 2010). Periodic expression of the prior nescent reporter systems using firefly luciferase (luc)for genes circadian clock, for example, CCA1 (CIRCADIAN clock genes have been successfully used in plants, animals CLOCK ASSOCIATED 1), LHY (LATE ELONGATED and cyanobacteria for detection of circadian rhythms in liv- HYPOCOTYL), PRRs (PSEUDO-RESPONSE ing cells (Millar et al., 1995; Welsh and Kay, 2005). For

Corresponding author : Hirokazu Fukuda, fax: 81
72
254
7916, e-mail : [email protected]

Vol. 52, No. 1 (2014) 
 T. HIGASHI ET AL.

example, an Arabidopsis CCA1 (AtCCA1) promoter-luc About1dbeforestarting to monitor bioluminescence, construct was recently used as a circadian expression 1 mM luciferin solution (500
l) dissolved in water con- marker (Nakamichi et al., 2004). Using this construct, the taining 0.002% Triton X-100 was injected into the medium free-running period, entrainment and temperature compen- using a micropipette (final luciferin concentration, about sation attributes of circadian rhythms have been clarified in 0.1 mM). Before bioluminescence assay, plants grew to the Arabidopsis and Lemna (Miwa et al., 2006). In particular, stage with the second pair of leaves in aseptic dish. the Lemna circadian system has been characterized by Bioluminescence assay semi-transient bioluminescence monitoring using a particle Bioluminescence from each individual plant was as- bombardment method. In addition, bioluminescent reporter sayed with a monitoring system developed by Kondo et al. systems have been used to investigate complex (1993). Using this system, bioluminescence was detected spatiotemporal dynamics in leaves and roots generated by from each plate by a photomultiplier tube (Hamamatsu the interaction of cellular circadian rhythms (Fukuda et al., H7360-01MOD; Hamamatsu Photonics KK, Japan) en- 2007, 2012; Wenden et al., 2012). In our previous study, closed in a light-tight box. Each plate was on a turntable the phase wave in leaves and striped waves in roots have that was rotated under the photomultiplier tube sequentially been unraveled in Arabidopsis and Lactuca sativa L. (Ukai every 20 min under control of a computer. Therefore, the et al., 2012). Moreover, a control method for the circadian plant in each plate was exposed to the dark period for 4 min rhythm has been studied theoretically on the basis of the every 20 min (1.5 min or more of darkness allows chloro- experiments with AtCCA1 promoter-luc bioluminescence phyll fluorescence to decay). The bioluminescence moni- (Fukuda et al., 2008, 2013). Thus, a bioluminescence assay toring system was in a temperature-controlled chamber with an AtCCA1 promoter-luc construct has been utilized (MIR-553, Sanyo Electric Co., Ltd.) at 22.00.5°C except widely in several studies for characterization of diverse for temperature compensation experiments. In experiments plant species and for development of control engineering of investigating temperature compensation, the temperature circadian rhythms. However, control engineering and/or its was differently set in three independent experiments (18°C, basic studies for crop plants have not been studied. 22°C or 26°C). In this study, we aimed to address the three basic Light conditions during measurement of biolumines- properties of circadian rhythm in lettuce (L. sativa L.) using cence the AtCCA1 promoter-luc bioluminescence assay. Lettuce We used several different light conditions. In experi- is a leafy vegetable and a commercially important crop in ments investigating the free-running period, seedlings were closed plant factories because of its suitability for measured under continuous dark (DD) or continuous light

hydroponic cultivation, requirement for only low illumina- (LL) using red LED (p 660 nm, 20 nm, EPITEX

tion, and non-diurnal cultivation conditions (i.e., continu- INC., Kyoto, Japan) and blue LED (p470 nm, 25 ous light and constant temperature). To monitor the nm, TOYODA GOSEI CO., LTD., Aichi, Japan) mixtures: circadian rhythm of lettuce, we used the transgenic lettuce red 100
mol m
2 s
1 and blue 0
mol m
2 s
1 (R100/B0); cultivars reported in Ukai et al., 2012, in which an AtCCA1 red 80
mol m
2 s
1 and blue 20
mol m
2 s
1 (R80/B20); promoter-luc construct was introduced (Nakamichi et al., red 50
mol m
2 s
1 and blue 50
mol m
2 s
1 (R50/B50); 2004) via Agrobacterium-mediated transformation. In ad- or red 0
mol m
2 s
1 and blue 100
mol m
2 s
1 (R0/ dition, we used red and blue light emitting diodes (LEDs) B100). In experiments on entrainment to light-dark cycles, as for plant growth illumination to investigate the light bioluminescence was measured under 12-h light: 12-h dark quality dependence of circadian rhythms. cycles using 100
mol m
2 s
1 red LED light or blue LED light. On the other hand, in experiments for entrainment to MATERIALS AND METHODS sinusoidal blue LEDs, bioluminescence was measured under continuous red LED light (80
mol m
2 s
1) with pe- Plant material and growing conditions riodic blue LED light, in which the waveform of periodic Experiments were carried out using transgenic lettuce blue light was sinusoidal with an amplitude of 20
mol (Lactuca sativa L. cv. Cisco, Cos, and Greenwave; the m
2 s
1. The period of sinusoidal blue light decreased or fixed lines of lettuce cultivars from TAKII & Co., Ltd., increased over time (as shown in Fig. 3a and 3b). In ex- Kyoto, Japan) AtCCA1::LUC with the clock gene AtCCA1 periments investigating temperature compensation, seed- fused to a modified firefly luciferase gene (Nakamichi et lings were under continuous light (LL) using 100
mol al., 2004; Ukai et al., 2012). AtCCA1 promoter-luc, pABH- m
2 s
1 red LED light. CCA1::LUC-C cassette (Nakamichi et al., 2004) was trans- formed into the lettuce plants via an Agrobacterium RESULTS AND DISCUSSION tumefaciens-mediated method (Bechtold et al., 1993). AtCCA1::LUC plants were grown on gellan gum- Free-running rhythms solidified Murashige and Skoog plant salt mixture medium Bioluminescence showed a circadian rhythm in the (Wako Co., Ltd., Osaka, Japan) with 2% (w/v) sucrose in three lettuce cultivars under DD, as reported in Ukai et al. each aseptic dish (40 mm in diameter) under 12-h light: (2012). The circadian rhythm could be observed in almost dark cycles using 100
mol m
2 s
1 of fluorescent white all cells of the seedlings. Under LL conditions using red or light (FL15D-B; Hitachi, Ltd., Tokyo, Japan) at 221°C blue LED illumination, bioluminescence also showed a for 14 d except for temperature compensation experiments. clear circadian rhythm (Fig. 1a
1c). Although the

 Environ. Control Biol. CIRCADIAN RHYTHMS IN LETTUCE

Fig. 1 Circadian rhythms of bioluminescence in transgenic lettuce AtCCA1::LUC under continuous conditions. (a-c) Free-running rhythms of bioluminescence in Cisco (a), Cos (b) and Greenwave (c) under red () and blue ( ) LED illumination. Each data point is the average bioluminescence of 10 seedlings, in which the bioluminescence of each seedling was normalized to the first peak of bio- luminescence and the average then calculated. (d, e) Free-running period of seedlings of T1 (d) and T2 (e) generations under several ratios of combined red and blue light, e.g. R100/B0, R80/B20, R50/B50, R0/B100, and R0/B0 (dark). Error bars indicate SE. The numbers indicate the average period of 5
7 seedlings in (d) and 10 seedlings in (e). In each cultivar, means followed by the same letters are not significantly different (P 0.05, Tukey-Kramer test).

amplitude of circadian rhythm decreased over time under light intensity decreases, the period is lengthened (Song LL conditions, the rhythm was maintained with a diurnal and Noh, 2007). In Arabidopsis, the free-running period of (circadian) period. The rhythmic period under LL using cab2::luc expression in wild type changed from about 30 h red LEDs tended to be shorter than under LL using blue for 1
mol m
2 s
1 to 24 h for 100
mol m
2 s
1 using red LEDs in all three cultivars (Fig. 1a
1c). Figure 1d shows light (Covington et al., 2001). Based on studies using the average period under four types of light quality and DD phytochrome (PHY) mutants, PHYs play a role in control- conditions in the T1 generation of the three cultivars. ling the period length of the circadian clock (Song and Cisco seedlings under red LL conditions (R100/B0) Noh, 2007). It has been suggested that PHYA is specifi- showed the shortest period, 19.9 h. Increasing the ratio of cally involved in the regulation of period length under low- blue tended to lengthen the rhythmic period, i.e., 21.6 h for intensity red and blue light, while PHYB, -D, and -E (R80/B20), 23.2 h for (R50/B50), and 23.6 h for mediate the perception of high-intensity red light. (R0/B100). Such a dependence of period on light quality Cryptochromes, the blue-light photoreceptors, also play a was observed in the other two cultivars, Cos and role in controlling the period length of the circadian clock. Greenwave, although the variance with respect to light The influence of multiple photoreceptors in controlling pe- quality depended on cultivar. In addition, the light quality riod length seems to be complex. In generally, PHYs and dependence of the period was maintained in T2 seedlings CRYs in diverse plants play in the same manner as in (Fig. 1e), although the period was different between T1 and Arabidopsis (Lariguet and Dunand, 2005). Thus, the light- T2 seedlings. As shown in Fig. 1d and 1e, there was a dif- quality dependence of period length in lettuce may be ference in the period length between T1 and T2 genera- caused by the similar mechanism in Arabidopsis tions, in particular for R100/B0, R0/B100 conditions in (Yamaguchi and Kamiya, 2002). Cisco, indicating that genetic variation in period length Entrainment to light-dark cycles regulation exists in the original commercial plant Bioluminescent rhythms in the three cultivars were en- (McClung, 2006). For that reason, the T1 generation might trained in light-dark (LD) cycles using red or blue LED il- better maintain the properties of the circadian clock in the lumination (Fig. 2). Under red LD cycles with a 24 h original commercial seeds than the T2 generation. period, the peak bioluminescent rhythm appeared at t3, For diverse living organisms, the free-running period 27, 50.7 h; that is, the peak appeared about 3 h after the end of circadian rhythm generally depends on light intensity of night. Under blue LD cycles with a 24 h period, the and light quality, an effect known as Aschoff’s rule peak also appeared about 3 h after the end of night. After (Aschoff, 1960); as the light intensity increases, the period applying LD cycles for entrainment (t72 h), the plants of free-running rhythms is diminished; in contrast, as the showed a circadian rhythm with their inherent free-running

Vol. 52, No. 1 (2014) 
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Fig. 2 Entrained bioluminescence to light-dark cycles in transgenic lettuce AtCCA1::LUC under red (a) and blue (b) LED illuminations (100
mol m2 s1). After 3 d for entrainment, LD light cycle was changed to continuous light. Each data point is the average bio- luminescence of 10 seedlings, in which bioluminescence of each seedling was normalized to the first peak of bioluminescence and the average then calculated.

period. The amplitude of the free-running circadian depends on cultivar, which might be due to genetic differ- rhythms decreased over time (t
72 h), while the rhythm ences. was amplified by entrainment under LD cycles (0 ht72 The method of entrainment by a weak sinusoidal sig- h). Under LD conditions, the average amplitude of the nal has been studied theoretically in physics (Kuramoto, circadian rhythm was about 0.8, and under LL conditions, 1984; Harada et al., 2010). The general result from theo- it was about 0.3. These results show that the circadian retical analysis is that autonomous oscillators can adjust clock in lettuce is strongly entrained and is amplified by 12 their frequencies to that of external cycles above critical h light-12 h dark cycles of either red or blue LEDs. forcing amplitude. In forcing amplitude versus forcing fre- Next, we demonstrated another entrainment phenome- quency diagram, there are long vertical entrainment regions non induced by weak light cycles with non-24 h period. called Arnold tongues (Pikovsky et al., 2001). Therefore, Although the light-dark cycle is the strongest stimulus for by increasing the amplitude of sinusoidal signal, the entrainment of circadian rhythm, entrainment can occur entrainment in Greenwave under the period-increasing con- even in response to weak environmental signals. Figure 3a dition will occur as a result due to proceed to the and 3b show hybrid LED lights with constant red light (80 entrainment regions.
mol m2 s1) and sinusoidal blue light (with the light in- Temperature compensation tensity changed periodically from 0 to 40
mol m2 s1). AtCCA1 gene expression rhythms were observed in The period of the sinusoidal blue lights was decreased or LL using red LED at three different temperatures (18°C, increased every two cycles, as shown in Fig. 3a and 3b. 22°C, 26°C). The periods of such free-running rhythms Figure 3c shows the bioluminescence rhythms under the were statistically estimated. The periods of the rhythms at period-decreasing conditions. In all three cultivars, the different temperatures showed no significant differences peaks of the bioluminescence rhythm were delayed relative (Fig. 4). A large difference in temperature resulted in no to that of the sinusoidal light by about /2 rad. Figure 3d significant difference in periods, suggesting that the proper- shows that the period of bioluminescence rhythms de- ties of rhythms generated in lettuce seedlings appear to ful- creased along with the period of sinusoidal blue light. fill the diagnostic criteria of a circadian rhythm, namely Thus, the three cultivars were entrained to the sinusoidal temperature compensation. To confirm the temperature blue light within a period ranging from 24 h to 18 h. Figure compensation quantitatively, we used the temperature coef-

3e shows the bioluminescence rhythms and Fig. 3f shows ficient Q10 as follows: its period under the period-increasing condition. Cisco and 10  Cos seedlings were also entrained to sinusoidal blue light
T2 t2 t1 Q10   within a period ranging between 24 h and 30 h, while T1

Greenwave seedlings were not entrained under these condi- where T1 and T2 are the periods at low t1 (°C) and high t2

tions. In Cisco and Cos seedlings, the circadian rhythm (°C) temperature, respectively. Q10 for the difference be- was advanced by about /2 rad relative to sinusoidal light. tween 18°C and 26°C was 1.08 in Cisco, 1.09 in Cos, and In all cases, the bioluminescent rhythm showed strongly 1.08 in Greenwave, indicating that lettuce circadian damped oscillation under sinusoidal blue light conditions. rhythms possess strong temperature compensation. In a Our results revealed that even a sinusoidal signal with previous study of Arabidopsis, the measured period of the small amplitude (20% of variance in average light inten- rhythms at high temperature (26°C) was considerably sity) is able to entrain the circadian rhythm in lettuce. It in- shorter than at low temperature (18°C) (Nakamichi et al., dicates that lettuce circadian rhythm can be controlled by 2004). This difference between lettuce and Arabidopsis the small photic signals under continuous lighting without might be caused by several differences in culture and nights. In addition, the range of entrainment with this weak growth conditions, such as light intensity and quality. sinusoidal light covers a wide range of period, from 18 to In this study, the waveform and the three basic proper- 30 h with the exception of Greenwave. The different re- ties of AtCCA1::LUC bioluminescence rhythms in lettuce sponse of Greenwave means that the ability for entrainment were similar to that of Arabidopsis. This suggests that an

 Environ. Control Biol. CIRCADIAN RHYTHMS IN LETTUCE

Fig. 3 Entrainment to hybrid LED illumination combining constant red light and sinusoidal blue light. (a, b) Schematic diagram of light conditions. Broken straight line indicates the light intensity of red LED illumination and the sinusoidal line indicates that of blue LED illumination. (c, d) Bioluminescence rhythms (c) and their periods (d) under period-decreasing conditions. (e, f) Bioluminescence rhythms (e) and their periods (f) under period-increasing conditions. Bioluminescence data (c, e) are the average bioluminescence of 10 seedlings, in which bioluminescence of each seedling was normalized to the first peak of bioluminescence and the average then calculated. Bold line indicates bioluminescence rhythms and thin sinusoidal line indicate the light intensity of blue LED illumination. In (d, f), the average period was obtained from 5 seedlings under each set of conditions, and the horizontal lines indicate the period of blue cycles. The error bars indicate SE.

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 T. HIGASHI ET AL.

Fig. 4 Temperature compensation. The free-running period was measured at a different temperature (18°C, 22°C, 26°C) under continuous red light. The average period was obtained from 10 seedlings under each set of conditions in Cisco (a), Cos (b) and Greenwave (c). In each cultivar, means followed by the same letters are not significantly different (P0.05, Tukey-Kramer test). The error bars indicate SE.

AtCCA1 promoter produces similar results in lettuce plants Aschoff, J. 1960. Exogenous and endogenous components in and Arabidopsis; i.e., transcriptional cis-elements for circadian rhythms. Cold Spring Harb. Symp. Quant. Biol. 25

circadian expression in the AtCCA1 promoter can be simi- 11 28. Bechtold, N., Ellis, J., Pelletier, G. 1993. In planta larly used by clock-related transcription factors in lettuce. Agrobacterium mediated gene transfer by infiltration of adult Our experiments revealed that the properties of rhythms in Arabidopsis thaliana plants. C. R. Acad. Sci. Paris Life Sci. lettuce consistently fulfilled the diagnostic criteria of the 316: 1194
1199. circadian rhythm, namely, they were free-running and Covington, M. F., Panda, S., Liu, X. L., Strayer, C. A., Wagner, D. showed entrainment and temperature compensation. R., Kay, S. A. 2001 ELF3 modulates resetting of the Arabidopsis clock gene homologs are also rhythmic in crop circadian clock in arabidopsis. Plant Cell 13: 1305
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clock system is conserved in higher plants, including crops. circadian clock. Plant Biol. 14: 401 410. Filichkin, S. A., Breton, G., Priest, H. D., Dharmawardhana, P., The significant results from this study are the light Jaiswal, P., Fox, S. E., Michael, T. P., Chory, J., Kay, S. A., quality dependence of the free-running period and the Mockler, T. C. 2011. Global profiling of rice and poplar entrainment to weak sinusoidal signals using blue LED illu- transcriptomes highlights key conserved circadian-controlled mination. These results suggest that the oscillation fre- pathways and cis-regulatory modules. PLoS ONE 6: e16907. quency of the circadian clock of lettuce can be controlled Fukuda, H., Nakamichi, N., Hisatsune, M., Murase, H., Mizuno, T. by light quality, e.g., acceleration of circadian frequency by 2007. Synchronization of plant circadian oscillators with a red illumination, and precision control by periodic alterna- phase delay effect of the vein network. Phys. Rev. Lett. 99: tion of light quality with small amplitude. It is also ex- 098102. Fukuda, H., Uchida, Y., Nakamichi, N. 2008. Effect of a dark pected that optimal cultivation under a non-24 h period pulse under continuous red light on the Arabidopsis thaliana using LED illumination in closed plant factories can be de- circadian rhythm. Environ. Control Biol. 46:123
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