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First published online March 10, 2006; 10.1104/pp.106.079145 Plant Physiology 141:299-309 (2006) © 2006 American Society of Plant Biologists Diurnal Regulation of the Brassinosteroid-Biosynthetic CPD Gene in Arabidopsis1,[W]Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H–6701 Szeged, Hungary (S.B., A.-M.S., L.K.-B., F.N., M.S.); Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DD, United Kingdom (J.C., G.J.B.); Division of Biology, Imperial College London, Wye Campus, Wye TN25 5AH, United Kingdom (G.J.B.); and Department of Biosciences, Teikyo University, Utsunomiya 320–8551, Japan (K.S., T.Y.)
Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.
Plant development is determined by complex interaction between endogenous programs and environmental signals, with light being one of the most important environmental cues. Light controls essential biological processes, such as germination, shade avoidance, de-etiolation, phototropism, chloroplast movement, stomatal opening, circadian entrainment, and flowering. The plasticity of plant responses to light is ensured by multiple photoreceptors having different wavelength specificities and sensitivities, and activating target genes through either distinct or integrated signaling mechanisms. In Arabidopsis (Arabidopsis thaliana), red/far-red signals are perceived by five phytochromes, PHYA to PHYE, whereas two types of cryptochromes and phototropins function as blue and UVA light receptors (for review, see Sullivan and Deng, 2003  ; Chen et al., 2004). Light regulation is crucial for coordinating the metabolic and physiological functions of plants with the alternating light and dark periods during the day. In addition to direct, light-dependent diurnal regulation of gene activities, light signals are crucial for entrainment of the endogenous circadian clock that ensures cyclic expression of circadian-controlled genes with a roughly 24-h period length, thereby contributing to the precise timing/phasing of numerous biochemical and physiological processes during the day. On the other hand, a hallmark of circadian rhythms is that they persist for several days under constant environmental conditions (McClung, 2001). The importance of these processes can be gauged by the fact that approximately 11% of the genes in Arabidopsis show diurnal patterns of expression and that about 25% of these are also under circadian regulation (Schaeffer et al., 2001).
Whereas the perception of light signals is an intracellular process, responses at the tissue and organ levels are mediated and coordinated through the action of phytohormones. Several lines of evidence suggest that almost all hormone groups participate in the control of photomorphogenic processes and that this involves complex cross talk between the signaling routes of the different hormones. Light can control the effect of phytohormones by influencing their biosynthesis, metabolism, transport, and/or perception (Nemhauser and Chory, 2002
One hormone group that has been implicated in photobiological responses is brassinosteroids (BRs). In addition to the role of these steroidal phytohormones in promoting growth, fertility, and stress resistance, they also regulate photomorphogenesis, and seedlings of some of the first characterized BR-deficient mutants were shown to have short hypocotyls, open cotyledons, and elevated expression of light-regulated nuclear genes when grown in the dark (Chory et al., 1991
BRs are synthesized from phytosterols through multiple, mostly oxidative reactions leading to brassinolide (BL), the biologically most active BR. In vivo conversion studies revealed two alternative reaction routes, the early and late C-6 oxidation pathways that utilize 6-oxo or 6-deoxo intermediates, respectively (Choi et al., 1997
The severe BR-deficient phenotype caused by brassinazole, a specific inhibitor of BR biosynthesis, suggests that de novo synthesis is a key factor in determining the level of the bioactive hormone (Asami et al., 2000
Homeostatic regulation, ensuring the adjustment of optimal BR concentrations under various physiological conditions, also requires temporal or permanent inactivation of excess hormone. In Arabidopsis, mutants overexpressing the BAS1/CYP734A1 and CHI2/SHK1/SOB7/CYP72C1 genes were shown to reduce the level of active BRs (Neff et al., 1999
Expression studies of the Arabidopsis CYP85 and CYP90 genes by reverse transcription (RT)-PCR provided valuable clues regarding the timing and spatial distribution of BR synthesis (Bancos et al., 2002
Diurnal Changes in the Expression of BR-Biosynthetic Genes Fusion constructs with firefly LUC reporter offer a flexible, noninvasive system for studying the in vivo activity of BR-biosynthetic gene promoters. Initial characterization of Arabidopsis lines carrying CPD promoter-driven LUC revealed that the luminescence levels detected by a CCD camera system vary depending on the time of the day. In time-course experiments, CPD:LUC transgenic seedlings that were raised under alternating 12-h-light/12-h-dark photoperiods (LD) showed a characteristic diurnal pattern of LUC activity (Fig. 1A ). Following lights-on, a sudden increase of luminescence intensity resulted in a maximum, then values gradually decreased, reaching a minimum around midday, then increased to a second maximum at lights-off. Thereafter, the detected luminescence decreased again throughout the night until a second minimum at the end of the dark phase.
To see if other BR-biosynthetic genes can show similar diurnal changes of activity, we also generated LUC fusion with the promoter of CYP85A2. This gene encodes a BR C-6 oxidase catalyzing the last conversion steps of BL synthesis in Arabidopsis (Kim et al., 2005 ; Nomura et al., 2005), and its organ-specific expression pattern has been shown to be similar to that of CPD (Bancos et al., 2002; Castle et al., 2005). Although the luminescence emitted by CYP85A2:LUC transgenic seedlings was about 10-fold lower than that of the CPD:LUC-carrying ones, it also exhibited a diurnal profile with maxima and minima at similar time points as seen in the case of CPD (Fig. 1B). But, compared to CPD, CYP85A2 promoter activity showed a broader morning peak, and its daily changes had higher amplitude. To ascertain that the expression data obtained with the CPD:LUC construct faithfully reflect the activity of the CPD promoter, in 1-week-old seedlings we determined the steady-state levels of the CPD mRNA during the LD cycle using semiquantitative RT-PCR assay (Fig. 1C). The amount of the transcript was found lowest before lights-on and highest before lights-off, whereas intermediate values were observed at midday and in the middle of the night. These data are in good agreement with the luminescence levels of the CPD:LUC transgenic line (Fig. 1B).
Under LD conditions, the sudden changes of CPD expression at lights-on and lights-off suggested a regulatory role of light in control of transcriptional activity. To determine the temporal profile of CPD activity in the absence of light signals, we measured the luminescence levels of CPD:LUC seedlings in both continuous white light (LL) and continuous dark (DD), following a 7-d LD entrainment period. In LL, the LUC activity showed a free-running circadian oscillation that was maintained for several days. The expression cycles had roughly 24-h period lengths and approximately 2-fold rhythmic amplitude, with minimum points at subjective midday and maxima in the subjective night (Fig. 2A ). Circadian activity of the CPD promoter could also be observed in DD, but under these conditions the cycles became attenuated after 3 d, at which point the expression level decreased to about 10% of the initial value (Fig. 2B).
Compared to the LL activity of CPD, the diurnal expression curve shows a strong increase at lights-on and a sudden decrease following lights-off, suggesting a positive role of light in the regulation of CPD (Fig. 1A). An activity minimum in the middle of the light phase shows temporal coincidence with the one observed at the subjective light period of the free-running LL cycle (Fig. 2A), and a shoulder in the second half of the dark phase seems to correspond to the postmaximum part of the LL curve measured at the same time of the subjective dark period (Fig. 2A). These similarities between the LD and LL profiles indicate that CPD expression is under dual control, with a light induction superimposed upon the circadian oscillation of the transcriptional activity.
To clarify which light signaling pathway(s) is involved in the induction of CPD expression, we measured the activity of the CPD:LUC transgene in LD using light sources of different spectral characteristics. Three samples of seedlings were grown to 1 week old under LD photoperiods, and then their LUC activity was measured in LD cycles with the same light source, or by replacing white illumination with monochromatic red or blue light periods (RD and BD conditions). As shown in Figure 3A , diurnal cycling of CPD activity was maintained in RD, and the expression values were very similar to those seen in LD. By contrast, BD regimes caused a decrease in the expression level and severe dampening of the diurnal oscillation, resulting in an activity profile resembling the one detected in DD (Fig. 2B). Because red light was sufficient for enabling diurnal cycles, whereas blue light of the same intensity was not, these data suggest a major role for phytochrome photoreceptors in the light induction of CPD.
To verify the importance of phytochrome signaling in the light response, we measured the diurnal expression of CPD:LUC in transgenic seedlings lacking functional PHYA and PHYB, the two most abundant phytochromes. In these seedlings, the luminescence intensity in LD was only about 10% of that measured in wild-type background, and activity changes at lights-on and lights-off were barely recognizable (Fig. 3B). Instead, the LD expression detected in phyAphyB plants indicates the dominance of circadian regulation (Fig. 3C). The experiments showing diminished transgene induction by blue light or in phytochrome-deficient background reveal the primary role of phytochrome signaling in the diurnal regulation of CPD activity, and also in determining the level of expression.
The transcription of CPD is stringently controlled by the level of biologically active BRs, and in turn the expression of CPD can influence BR accumulation. Therefore, it was important to clarify whether the hormonal feedback regulation is involved in the light and/or circadian control of CPD expression. To address this question, we measured the expression of the CPD:LUC transgene in the BR-insensitive bri1 mutant that had been shown to express CPD at an elevated level (Bancos et al., 2002
We also measured the LL and DD expression of CPD:LUC in bri1 seedlings to find out whether the circadian expression is dependent on BR signaling. In LL a similar oscillation pattern was observed as in the wild-type background, although the amplitude was lower in both absolute and relative terms (Fig. 4B). By contrast, the activities measured in DD were quite different from those of the wild-type control. Although a gradual dampening of the cycle amplitudes was also observed in bri1 plants, this was not accompanied by a rapid decrease of the expression level, which remained about as high as under LL conditions (Fig. 4C). These results show that the circadian regulation of CPD activity is maintained in the absence of BR signaling, whereas its repression in DD is caused primarily by hormonal feedback regulation.
Diurnal oscillation in the expression of BR-biosynthetic genes suggests that the rate of BR synthesis varies during the day/night cycle. To determine whether daily changes of CPD and CYP85A2 expression correlate with the steroid hormone content, we performed quantitative gas chromatography (GC)-mass spectrometry (MS) analysis of the endogenous BRs in 1-week-old wild-type seedlings during the LD cycle at 6-h intervals (Table I ). The early C-6 oxidation intermediates cathasterone, teasterone, and 3-dehydro-6-deoxoteasterone were not detectable in the samples taken in the morning just before lights-on, at midday, in the evening before lights-off, and at midnight (0, 6, 12, and 18 h from lights-on, respectively). Levels of the late C-6 oxidation BRs and castasterone showed only minor differences, without any apparent diurnal variation. But, in contrast to the steadiness of the intermediate pools, at midday the seedlings accumulated large amounts of BL, the highly active BR that remained below the detection limit in samples collected at the other three time points of the day. These data show a remarkable increase of the active hormone content at the middle of the light period and also that the synthesis of BL does not appreciably affect the pools of BR intermediates.
Because in DD the expression level of CPD decreased rapidly in the wild type but not in the bri1 background, we also wanted to find out the effect of prolonged dark growth on the endogenous BR content. Wild-type seedlings were grown to 1 week old in LD, then BRs were analyzed in samples that were either placed in darkness for 48 h, starting from the end of the eighth light period, or grown further for the same time in LD. As shown in Table II , the amounts of BR intermediates were only slightly influenced by extended dark treatment, and the level of the bioactive castasterone remained essentially unchanged. These measurements indicate that the repression of CPD activity in DD does not the result from an increase of the endogenous BR content.
Feedback Repression of CPD Reveals Enhanced BR Responsiveness in the Dark Our finding that BR-dependent down-regulation of CPD expression in DD was not accompanied by an increase of BR levels suggested that light conditions might influence the efficiency of feedback regulation. To find out whether dark repression of CPD is controlled by the BZR1 transcription factor, we measured how DD affected the activity of a modified CPD promoter (mCPD) that contains a point mutation within the BZR1-binding sequence. Seedlings harboring the mCPD:LUC fusion accumulated about 10-fold higher amount of the LUC mRNA than those with the CPD:LUC control, and the transcript level decreased only to 92% of the initial value, opposed to the 8% of the control, upon treatment with 100 nM BL (Fig. 5A ). CCD camera measurements revealed that the luminescence profiles of mCPD:LUC seedlings in LD and subsequent DD are very similar to those of CPD:LUC in the bri1 background (Fig. 5B). This result shows that depletion of the CPD transcript in DD is primarily the consequence of BZR1-mediated feedback regulation. Because in seedlings GC-MS analyses did not detect an increase of the BR content in DD, our data strongly suggest that the feedback response is a result of enhanced BR susceptibility in the dark.
De novo synthesis plays an important role in setting the endogenous levels of bioactive BRs. In transgenic Arabidopsis, BR biosynthesis could be increased by overexpression of DWF4/CYP90B1, one of the rate-limiting enzymes (Choe et al., 2001 ), and parallel changes in the amounts of CPD: -glucuronidase and native CPD transcripts revealed that the mRNA level of CPD is controlled primarily at the level of transcription (Mathur et al., 1998). Accordingly, reporter fusions with promoters of BR-biosynthetic genes, such as those of CPD, CYP85A1, and CYP85A2, could be used successfully to determine developmental and temporal expression patterns, as well as active sites of BR synthesis (Mathur et al., 1998; Castle et al., 2005). Whereas the crucial role and relatively high transcription level of CPD make its promoter ideal for in vivo expression assays using LUC reporter, such studies require faithful correspondence between the activities of endogenous CPD and the LUC reporter. During the diurnal cycle, we found good agreement between the levels of the CPD transcript and CPD:LUC-derived luminescence intensity, showing that this reporter construct is a reliable indicator of CPD expression.
Our data on the daily changes of CPD transcript levels correspond to those detected by a microarray analysis designed to identify diurnally expressed Arabidopsis genes (supplemental data to Schaeffer et al., 2001
In CPD:LUC-carrying seedlings, LUC activity increased rapidly following lights-on. Whereas the observed light induction is consistent with the concomitant accumulation of CPD mRNA, the initial increase can be slightly exaggerated by a weak, nonspecific, and transient enhancement of LUC activity, perhaps resulting form ATP pool expansion, in response to light (Millar et al., 1992
Red light-specific induction of CPD:LUC, as well as its severely reduced activity and light response in the phyAphyB mutant, suggest a primary role for phytochrome signaling in the light regulation of CPD. Although in wild-type background we could also observe a slight increase of LUC activity following blue light exposure, which was used within the photobiologically active fluence range (Devlin and Kay, 2000
An increasing amount of evidence indicates the involvement of phytohormones in various light-dependent physiological processes, and in some cases the role of de novo hormone synthesis has been clearly demonstrated. For instance, light was shown to promote the germination of Arabidopsis and lettuce seeds by increasing their GA content via the induction GA 3
So far only a few studies have addressed the role of light in influencing the biosynthesis and removal of BRs. In Arabidopsis, higher BR levels were detected in light-grown plants than in dark-grown ones, though the plants used in these analyses were not of the same age (Choe et al., 2001
Recently, it has been reported that the expression of Arabidopsis CYP734A1/CYP72B1 and CYP72C1, both encoding BR-catabolizing P450s, are light repressed (Turk et al., 2003
The rate of hypocotyl elongation in Arabidopsis shows circadian cycling, with maxima at 4 to 6 h after subjective midday (Dowson-Day and Millar, 1999
Earlier studies revealed that all P450 genes of BR biosynthesis are feedback regulated by bioactive BRs (Bancos et al., 2002 The physiological benefits of diurnally controlled BR synthesis are yet to be elucidated. This complex regulation may ensure optimal hormone levels in accordance with the daily changes in growth and metabolic functions. It may also be important in maintaining BR homeostasis by coordinating oppositely regulated biosynthesis and catabolism during the light and dark periods of the day. Elevated CPD and CYP85A2 expression, as well as BL accumulation, are in apparent contradiction with the reduced hypocotyl elongation during the light periods. This anomaly is likely caused by the observed light-dependent decrease in BR responsiveness. Our data and earlier reports indicate concerted regulation of BR levels and sensitivity, while flexibility of this dual control may be ensured by developmental and organ-specific differences between these two effects. Although the impact of de novo BR synthesis is not clear, the complex transcriptional regulation of the biosynthetic enzymes indicates its apparent physiological importance.
An interesting possibility is that, by controlling the accumulation of BL during the light phases, diurnal regulation of BR-biosynthetic genes may influence flowering time in Arabidopsis. Recently, it has been shown that intense synthesis of bioactive BRs is associated with reproductive development (Montoya et al., 2005
Plant Material Seeds of transgenic and wild-type Arabidopsis (Arabidopsis thaliana; ecotype Columbia) were surface sterilized and sown on Murashige and Skoog medium (Duchefa) supplemented with 1% (w/v) Suc and 0.2% (w/v) Phytagel (Sigma). Seedlings were grown at 22°C in controlled-environment chambers (SANYO Electronic) under alternating regimes of 12 h white fluorescent light (50–60 µmol m–2 s–1) and 12 h dark (LD). LL and DD were provided using the same conditions as in the corresponding phases of LD. Blue and red light emitted by LED panels at wavelengths 470 ± 5 and 660 ± 5 nm, respectively, were used at a fluence rate of approximately 10 µmol m–2 s–1, approximately equal to their spectral proportions in the white light.
The CPD:LUC+ reporter construct was generated by cloning the –968 to –5 segment (relative to the translation start) of the CPD promoter (Mathur et al., 1998
Seven-day-old LD-grown transgenic seedlings harboring LUC reporter constructs were transferred to plastic plates with fresh medium. Patches of 100 seedlings were then sprayed twice with filter-sterilized 25 mM D-luciferin (Biosynth A.G.) and 0.01% (v/v) Triton X-100 solution in 5 mM Tris-phosphate buffer, pH 8.0. The last spraying was done 12 h before zero time (the onset of the eighth light period), 24 h before the start of the LUC activity measurements. Alternatively, following surface sterilization, seeds were sown on Murashige and Skoog medium supplemented with 0.5 mM luciferin, placed to 4°C for 12 h, then germinated and raised in DD. Bioluminescence imaging was done at 22°C with a liquid nitrogen-cooled backilluminated digital CCD camera (Princeton Instruments), taking 25-min exposures every second hour. Luminescence intensities were evaluated using Metamorph imaging software (Meta Series 4.5; Universal Imaging). All measurements were repeated at least four times. Since the data were highly reproducible (see Supplemental Fig. 2), in each case we show the result of a representative experiment.
For determining endogenous BR levels during the diurnal cycle, wild-type seedlings were grown in LD, then, starting from the onset of the eighth light period, harvested at 6-h intervals. In three experiments, the plant samples collected at 0 h (subjective morning), 6 h (subjective noon), 12 h (subjective evening), and 18 h (subjective midnight) were immediately frozen in liquid nitrogen, and then subjected to lyophilization. For measuring the BR content in DD, LD-entrained wild-type seedlings were transferred to DD or kept further in LD (control) from the end of the eighth light period (0 h, subjective evening). In three experiments, dark-treated samples and their LD controls were harvested after 48 h (subjective evening), as described above. During and at the end of the dark periods, seedlings were harvested under green safe light.
BR extraction, purification, and analysis were carried out as described by Nomura et al. (2001)
Total RNA was isolated using TRI reagent (Sigma) as recommended by the manufacturer. For semiquantitative RT-PCR assay, RNA was obtained from the same plant batches that were used for BR analysis during the diurnal cycle. Each cDNA sample was prepared from 5 µg of RNA using Ready-To-Go T-primed first-strand kit (Pharmacia Biotech). Primers, PCR conditions, and detection of the amplified products derived from the CPD and UBQ10 (constitutive control) transcripts were as described by Bancos et al. (2002)
We thank Katalin Jószai (Institute of Plant Biology, BRC HAS) for her technical support, Yumiko Yamada (Department of Biosciences, Teikyo University) for the purification of plant extracts, Jianming Li (Department of Cellular and Developmental Biology, University of Michigan) for providing the bri1-101 mutant, and Suguru Takatsuto (Department of Chemistry, Joetsu University of Education) for supplying deuterated BR standards. Received February 13, 2006; returned for revision February 13, 2006; accepted February 28, 2006.
1 This work was supported by the Hungarian Scientific Research Fund (grant nos. T 42639 to M.S. and F 47013 to L.K.-B.), an International Joint Project grant of the Royal Society (to G.J.B. and M.S.), research grants from the Biotechnology and Biological Sciences Research Council (to G.J.B.) and the Human Frontiers Science Program (RG00162–2000 to T.Y. and G.J.B), and a Grant-in-Aid for Scientific Research (B13460050 to T.Y.) from the Ministry of Education, Science, Sports and Culture of Japan. 
2 These authors contributed equally to the paper.
3 Present address: David H. Smith Center for Vaccine Biology and Immunology, Aab Institute for Biomedical Sciences, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Miklós Szekeres (szekeres{at}nucleus.szbk.u-szeged.hu).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.079145. * Corresponding author; e-mail szekeres{at}nucleus.szbk.u-szeged.hu; fax 36–62–433434.
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-hydroxylation steps in brassinosteroid biosynthesis. Plant Cell 10: 231–243