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First published online April 22, 2009; 10.1104/pp.108.130757 Plant Physiology 150:858-878 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Cytochrome P450 Monooxygenases as Reporters for Circadian-Regulated Pathways1,[C],[W],[OA]Department of Cell and Developmental Biology, University of Illinois, Urbana, Illinois 61801 (Y.P., M.A.S.); Department of Plant Biology and Pathology, Waksman Institute for Microbiology, Rutgers University, Piscataway, New Jersey 08854 (T.P.M.); Department of Crop Sciences, University of Illinois, Urbana, Illinois 61801 (M.E.H.); Section of Cell and Developmental Biology, University of California, La Jolla, California 92093 (S.A.K.); and Plant Biology Laboratory and Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, California 92037 (J.C.)
Cytochrome P450 monooxygenases (P450s) play important roles in the synthesis of diverse secondary compounds in Arabidopsis (Arabidopsis thaliana). Comparison of four data sets analyzing seedlings harvested over a 2-d period of constant conditions after growth with varying photoperiods and thermocycles recorded a total of 98 P450 loci as circadian regulated for at least one of the four conditions. Here, we further describe the circadian-regulated pathways using, as reporters, individual P450 loci that are likely to be rate limiting in secondary metabolic pathways. Reverse transcription-polymerase chain reaction gel blot analyses have confirmed circadian regulation of P450s in phenylpropanoid, carotenoid, oxylipin, glucosinolate, and brassinosteroid biosyntheses and have shown that both P450 and non-P450 genes in the many branches of the phenylpropanoid pathway have similar circadian patterns of expression. In silico analyses of the subsets of coregulated promoters have identified overrepresented promoter elements in various biosynthetic pathway genes, including MYB and MYB4 elements that are significantly more abundant in promoters for the core and lignin sections of phenylpropanoid metabolism. Interactions with these elements important for circadian regulation do not involve the MYB transcription factor PAP1, as previously proposed, since the expression patterns of circadian-regulated P450s are the same in pap1-D mutant seedlings as in wild-type seedlings. Further analysis of circadian-regulated promoters in other biochemical pathways provides us with the opportunity to identify novel promoter motifs that might be important in P450 circadian regulation.
The biological clock controls many processes in organisms as diverse as cyanobacteria and humans. In higher plants, circadian rhythms regulate physiological events including growth and development, photosynthesis, metabolic adaptation, protein synthesis, carbohydrate transport and storage, leaf and cotyledon movements, and hormone signaling responses (Harmer et al., 2000  ; Covington and Harmer, 2007; Michael et al., 2008). Microarray and enhancer-trapping experiments have estimated that between 15% and 36% of the Arabidopsis (Arabidopsis thaliana) genome is under circadian regulation at the transcriptional level (Michael and McClung, 2003; Edwards et al., 2006; Michael et al., 2008). Promoter analysis using these large microarray data sets has identified several promoter elements involved in phase-specific light and circadian regulation of expression, including the Morning Element (ME), CCA1-Binding Site (CBS), GATA, Evening Element (EE), and Midnight Module (PBX/TBX/SBX; Wang et al., 1997; Harmer et al., 2000; Hudson and Quail, 2003; Harmer and Kay, 2005; Michael et al., 2008). However, it remains unclear how specific pathways downstream of the core circadian clock are regulated at specific times of the day.
Cytochrome P450 monooxygenases (P450s) play critical roles in the synthesis of lignin, pigments, defense compounds, fatty acids, hormones, and signaling molecules in all plant species (Schuler, 1996
Carotenoids are the pigments responsible for many fruit and flower colors and some components of the light-harvesting complexes in photosynthesis (Bartley and Scolnick, 1995
Oxylipins are acyclic or cyclic oxidation products derived from polyunsaturated fatty acids that regulate many defense and developmental pathways in plants (Creelman and Mullet, 1997
Glucosinolates are a class of naturally occurring thioglucosides responsible for some of the unique tastes of many condiments. Many P450s exist in the pathways branching to the production of these compounds, with CYP79F1 and CYP79F2 mediating distinct functions in the conversion of short- and long-chain Met derivatives to oximes (Hansen et al., 2001
Brassinosteroids are steroidal plant hormones essential for many plant processes, including cell expansion and elongation, xylem differentiation, and pollen tube growth (Müssig et al., 2002
Phenylpropanoid synthesis represents one of the best-characterized pathways because it generates a wide variety of products found in most plants, including flavonoids that act as signaling molecules, protectants against UV light damage and microorganisms, lignins that are structural components of cell walls, and anthocyanins that act as floral pigments and attractants to insect pollinators (Dixon and Paiva, 1995 The analyses presented here of P450 expression patterns in four data sets, varying with respect to the thermal and photoperiod cycles used for entrainment, indicate that different combinations of these P450s display coordinated in-phase expression in the different entrainment conditions. The characterization of P450 responses to circadian regulation has potential to identify nodes that globally coordinate transcript abundance of many pathways to specific times of the day. Until now, the activities of specific pathways have, for the most part, only been inferred from analysis of genome-wide patterns. To better understand the coordination of the downstream synthetic and catabolic pathways conferring time-of-day-specific activities, we have utilized P450s as reporters for different nodes in the network emerging from the central circadian clock.
Circadian Variations in P450 Transcripts
Given the importance of P450s in many metabolic pathways, it is clear that they can serve as global reporters for cellular responses to internal and external cues. To analyze the extent to which P450s might be regulated by the circadian clock, four previously published circadian time courses (Michael et al., 2008
Across the four circadian conditions, 233 of the 246 full-length P450 genes and 26 P450 pseudogenes in Arabidopsis were detected on Affymetrix ATH1 arrays, with 11 of these array elements representing closely related P450 genes. Using a 0.8 correlation cutoff for predicting cycling loci (all four time courses analyzed together; P = 0.05, FDR = 5%; Michael et al., 2008 ), 98 P450 loci listed in Table II showed statistically significant circadian phasing for at least one of the four array conditions. Between 4% and 22% of the 250 P450 transcripts were circadian regulated under any one of the four conditions, with 39% of the gene list overlapping with one of the other three conditions (Fig. 1). Between 33% and 42% of the genes were specifically circadian regulated under only one condition. The fewest genes displaying circadian rhythms were detected under DD_DDHC conditions, consistent with the fact that generally fewer genes cycle under this condition (Michael et al., 2008). The greatest numbers of genes showing circadian rhythms were detected under LL_LDHH and LL_LDHC conditions rather than LL_LLHC conditions, suggesting that entrainment by light/dark cycles plays a role in P450 expression.
Reverse transcription (RT)-PCR gel blot analyses of samples from the LL_LLHC and LL_LDHC time courses performed with gene-specific primers and probes confirmed the cycling of P450 transcripts distributed in many different pathways, including CYP73A5, CYP75B1, CYP98A3, and CYP84A1 in phenylpropanoid synthesis (nos. 1–4 in Table II; highlighted in red in Fig. 2A online), CYP97C1 in carotenoid synthesis (no. 7 in Table II), CYP74A1 in jasmonate synthesis (no. 9 in Table II), CYP79B3 in glucosinolate synthesis (no. 15 in Table II), and CYP90A1 in brassinosteroid synthesis (no. 17 in Table II). Comparison of the UBQ10-normalized RNA levels shown in the left panels of Figure 3 with the gcRMA-normalized ATH1 array data shown in the right panels of Figure 3 indicates strong correlations for most of the transcripts analyzed. Analysis using the model-based pattern-matching HAYSTACK algorithm (Fig. 3K) indicates that the four P450s in the phenylpropanoid pathway are phased with a maximum at 22 h under the LL_LLHC conditions. Complementary analysis using semiquantitative RT-PCR gel blots on these same RNA samples (Fig. 3, A and F) indicates that these four P450s are phased at around 24 h. Comparisons between these two analytic methods indicate that, in some instances, the RT-PCR gel blots show obvious cycling variations and the ATH1 array data show much lower levels of variation. This is the case for CYP75B1 and CYP84A1 in the LL_LLHC time course (Fig. 3, F and K) as well as for CYP98A3 in the LL_LLHC time course, where RT-PCR gel blot signals show as much as a 10-fold difference between the initial time point and each day's minimum and microarray data show only a 2.5-fold difference. This is also the case for CYP90A1 in the LL_LDHC time course, where RT-PCR gel blot signals decrease more dramatically than the microarray data to an extreme minimum at 16 to 20 h (Fig. 3, I and N). Differences in the magnitude of these variations likely arise from the greater sensitivity of the RT-PCR gel blots. It is well known that microarray data, especially oligonucleotide microarray data, suffer from poor dynamic range. Although the results from these two methods differ in magnitude, both demonstrate circadian regulation of the P450s analyzed, and within each method, the circadian expression pattern of each gene appears to be highly reproducible.
Circadian Regulation of P450 Transcripts in Different Secondary Pathways
With P450s occurring at important nodes in many secondary pathways displaying circadian cycling, variations in their activities can impact an array of downstream synthetic and catabolic pathways and alter physiological functions over the course of a day. Comparison of the circadian phasing of the four P450 transcripts in the core and various branches of the phenylpropanoid pathway (CYP73A5, CYP75B1, CYP98A3, and CYP84A1; Fig. 2) indicates that the LL_LDHH arrays show similar phasing just before subjective dawn (20–21 h maxima) for three of these P450s, with the most profound increase for CYP75B1 in anthocyanin synthesis and slightly later phasing for CYP84A1 (1 h maximum; Table II). Two of these also cycle with similar normalized profiles in the LL_LDHC arrays with peaks before subjective dawn (Fig. 3A). In addition, CYP711A1 (MAX1; no. 5 in Table II), which is reported to be a positive regulator of the flavonoid pathway (Lazar and Goodman, 2006
In carotenoid synthesis (Fig. 4A), CYP97A3 and CYP97C1 occur in the lutein branch. Both of these (nos. 6 and 7 in Table II) show circadian phasing in the LL_LLHC arrays (19–20 h maxima) and the LL_LDHH arrays (13–14 h maxima; Fig. 5A). CYP97B3 (no. 8 in Table II), whose protein shares 45% to 46% amino acid identity with CYP97A3 and CYP97C1, shows circadian phasing at 12 h in the LL_LDHH arrays (below the cutoff used for Table II) and at 16 h in the LL_LDHC arrays, which is similar to the phasing of CYP97A3 and CYP97C1 on the arrays. The similarity of this phasing suggests that CYP97B3 may be under the same transcriptional regulation as the other CYP97 family genes. CYP711A1, which was previously mentioned as acting downstream of carotenoid cleavage dioxygenases (Booker et al., 2005
In oxylipin synthesis (Fig. 4B), CYP74A1 in JA synthesis and CYP74B2 in C6-volatile production are circadian regulated, with slightly different phasings in the LL_LLHC arrays (22 and 17 h maxima; nos. 9 and 10 in Table II). With many other loci mediating steps in JA synthesis, at least one locus at each step in the pathway is circadian regulated in the LL_LLHC and LL_LDHC arrays, with phasings between 13 and 18 h, at times slightly prior to the phasings seen for CYP74A1 and CYP74B2; among multiple loci coding for the same enzyme, those that are circadian regulated are indicated with boxes in Figure 4B. The only loci with noticeably different phasing from others in the JA synthetic pathway are one lipoxygenase (LOX1; no. 59 in Table III) mediating the synthesis of 9-hydroperoxides and not the 13-hydroperoxides needed for jasmonate production (Royo et al., 1996 ; Blee, 2002), one undefined 12-oxophytodienoate reductase (OPR; no. 66 in Table III), and S-adenosyl-L-Met:jasmonic acid carboxyl methyltransferase (JMT; no. 68 in Table III) catalyzing the last step in MeJ synthesis. As both LOX and OPR have many isoforms in Arabidopsis, it is likely that the individual members of these families are under different modes of transcriptional regulation. In the case of JMT, its phasing 2 to 11 h later than transcripts for previous steps in this pathway suggests that the proportions of JA and MeJ vary throughout these cycling periods.
In the aliphatic glucosinolate branch (Fig. 4C), CYP79F1, CYP79F2, and CYP83A1 (nos. 11–13 in Table II) are circadian regulated, with similar phasings under LL_LLHC (21–1 h maxima) as a number of other enzymes in this branched pathway (Fig. 5B, top). Not surprisingly, CYP79B2 and CYP79B3 in the indole glucosinolate branch (nos. 14 and 15 in Table II) are circadian regulated, with exactly the same phasings as most other enzymes in its branch (Fig. 5B, bottom). While below the cutoff used for Table II, CYP83B1 in this branch also cycles with this same phasing (Fig. 5B, bottom). The glucosinolate pathway has not previously been reported to have circadian rhythms. In brassinolide synthesis (Fig. 4D), four of the six synthetic P450s have different circadian phasings depending on the array conditions (nos. 16–19 in Table II). For example, the LL_LDHH arrays show similar phasing for CYP85A2 and CYP90C1 (ROT3; 1–3 h maxima) and a slightly later phasing for CYP90A1 (CPD; 5 h maximum), which is considered the initial rate-limiting enzyme in this pathway. The LL_LDHC arrays show significantly earlier phasing for CYP90A1 (8 h maximum) than for CYP90B1 (DWF4; 12 h maximum). The LL_LLHC arrays show slightly earlier phasing for CYP90C1 (6 h maximum) than for CYP90A1 and CYP90B1 (8–9 h maxima).
In the inactivation of plant hormones, the catabolism of brassinolide and other brassinosteroids is mediated by CYP734A1 (BAS1) and CYP72C1 (SOB1; Neff et al., 1999
To gain perspective on circadian controls over different pathways, circadian-regulated promoters in each branch of a pathway as well as in each overall pathway were searched for known elements that were overrepresented compared with their frequency in the 27,457 promoters of the Arabidopsis genome (annotated in the AGRIS sequence motif database [http://Arabidopsis.med.ohio-state.edu]; Davuluri et al., 2003
In promoters of genes for different branches of phenylpropanoid metabolism, many circadian-relevant elements are evident. The CBS (AAAAATCT) is overrepresented in the core pathway and the intermediate flavonoid branch. The ME (AACCAC) is frequent in the lignin and anthocyanin branches. The EE (AAAATATCT) reported as overrepresented in the phenylpropanoid pathway (Harmer et al., 2000
Analysis of genes associated with the CHS, TT5, CHI, F3H, TT7, FS, F3OG2, F3OG3, and DFR genes, whose promoters contain overrepresented G-BOX [LRE] motifs, was accomplished using a graphical Gaussian model to evaluate publicly available transcript profiling data (Ma et al., 2007 ). This analysis identified a network containing 97 nodes (genes) connected by 194 significant edges (interactions) with the gene pair Pearson correlation coefficient greater than 0.9 (Fig. 7A). MYB and MYB4 as well as the recently identified WLE1 (Hwang et al., 2008) are also significantly overrepresented in the core pathway and the lignin branch but not in the flavonol or anthocyanin branch. Among the novel elements identified in this pathway are GL-TRP2, GL-TRP5, GL-MET1, GL-MET3, and CAROT-A2 in the core pathway, GL-TRP3 and JA2 in the intermediate pathway, GL-MET3 in the lignin branch, and GL-MET2 in the flavonol branch (Fig. 6C); as their names indicate, most of these were first identified as overrepresented in the glucosinolate branched pathways utilizing Met and Trp. The promoter of CYP711A1, which has been reported to be a positive regulator of the flavonoid pathway (Lazar and Goodman, 2006) and shows the same phasing as phenylpropanoid P450s (20–22 h maxima in three of the array conditions), contains seven MYB4 and three additional MYB motifs (compared with 77,609 MYB4 and 20,163 MYB motifs in the 27,457 annotated Arabidopsis promoters) as well as G-BOX [LRE], GL-MET2, GL-MET3, GL-MET4, CAROT-A2, and MOTIF8. MYB4 (At4g38620), which negatively regulates this flavonoid pathway (Jin et al., 2000), is circadian regulated, with a 21 h maximum (LL_LLHC) that is the same as the phasing of CYP711A1, which positively regulates this pathway (data not shown).
In the lutein branch of the carotenoid pathway, DPBF1&2 is the only previously described element that appears to be overrepresented; in the carotenoid intermediate pathway, ABFS and T-BOX are overrepresented. With few known elements overrepresented in these carotenoid intermediate and branched pathways, AlignACE algorithms identified a number of novel overrepresented elements in the promoters of the zeaxanthin/abscisic acid branch (designated A), lutein branch (designated B), and core pathway (designated CO). These elements are identified with alphabetic and numerical designations and correspond to CAROT-A1 (AGAGA[AG][AG]), CAROT-A2 (CCAAAN[CA]A), CAROT-A3 (GAGA[AT]GA[AG]), CAROT-B1 ([CT]TTG[AG]AAG), CAROT-B2 ([GA][AG]AGAAGCT), CAROT-B3 (GAAGCT), and CAROT-CO (AGAAGA). Of these, CAROT-CO is overrepresented in all three parts of this pathway, and the others are more specific for promoters in branches of this pathway. Spacings of these elements in the CYP97A3 and CYP97C1 promoters are shown in Figure 6D. The promoter of CYP97B3, which codes for a P450 closely related to CYP97A3 and CYP97C1 in the lutein branch and shows intermediate phasing, contains CAROT-B1, CAROT-B2, and CAROT-CO as well as GL-MET2, GL-TRP3, CAROT-A3, and T-BOX. Notably, no EE exist in any of the carotenoid pathway promoters, and CBS exist in only two promoters. In the oxylipin pathway, circadian-regulated promoters in the AOS branch have overrepresented MYB4, MYB, RAV1-A, AG/AP1 BS, ATMYC2 in RD22, GL-TRP1, and MOTIF8 (ATTCANA), and five of the 10 promoters in this branch have EE. AlignACE analysis of this entire pathway identified three novel overrepresented elements, JA1 (ATGTGAAT), JA2 (AAGAA[GA]ANG), and JA3 (T[TC]GG[AG]CAA), that we had previously identified as overrepresented in MeJ-inducible promoters. Of these, JA2 is represented seven times in the AOC4 promoter, with six of these elements being present in short tandem direct repeats, indicating that its abundance is not uniform across promoters in the AOS branch. The circadian-regulated CYP74B2 promoter contains multiple ME, GATA [LRE], DPBF1&2, GL-TRP1, and MOTIF8, one each of the G-BOX [LRE], JA3, and CBS, and no EE; none of these, except possibly the ME, can be recorded as overrepresented, since this is the only locus in the HPL branch of oxylipin metabolism.
In the glucosinolate pathway, circadian-regulated promoters in the aliphatic glucosinolate branch contain MYB, MYB4, and I-BOX and the novel GL-MET3 (ANACCAAA), GL-TRP2 (ANNTTGAAA), GL-TRP4 (GTTGG[AT]G), and GL-TRP5 (ACCA[AG]CNA[AG]). Circadian-regulated promoters in the indole glucosinolate branch contain STRE (Marchler et al., 1993
Overrepresented elements in the collection of circadian-regulated brassinosteroid synthesis loci include the GATA [LRE] ([AT]GATA[GA]), ATMYB2 BS in RD22 (CTAACCA), MOTIF1 ([CT]GNTGATGTCA), MOTIF8, and novel BS1 (AAC[ACGT]CTTT) and BS2 (TATNTTAG). MOTIF1 was originally found to be overrepresented in the promoter sequences of Arabidopsis P450s induced by MeJ, SA, or BION and their combinations; MOTIF8 was originally found to be overrepresented in promoters of root-specific P450s. The only overrepresented element in the two circadian-regulated brassinosteroid degradation loci is ATMYB2 BS in RD22. EE are present once in the DWF1 promoter, twice in the CYP85A2 (BR6ox2) promoter, and twice in the CYP734A1 (BAS1) promoter. CBS are present in the 3-oxo-5
To better understand the relationships between these pathways and some of their predicted transcriptional regulators, we analyzed the expression patterns of PAP1, one MYB transcription factor that had been proposed to control the circadian regulation of the anthocyanin and lignin branches (Harmer et al., 2000
Our profilings of the four P450 transcripts responsible for rate-limiting steps in phenylpropanoid metabolism emphasize the similar circadian phasing of all transcripts in this pathway, even those in diverse branches (intermediate flavonoid branch, lignin branch, flavonol branch, and pelargonidin and cyanidin branches). Nearly all, including those needed in flavonoid and anthocyanin production, are expressed at their maximal levels just before subjective dawn, at a time when there is little light. Under some of these circadian regimes (LL_LDHC), the cycling of the CYP75B1 transcript is especially prominent, suggesting that its normal circadian cycling pattern is enhanced by exposure to light at subjective dawn. In Arabidopsis, regulation of the flavonoid and anthocyanin pathway transcripts such as CYP75B1 is controlled by a MYB and TTG1 complex with basic helix-loop-helix proteins (Dubos et al., 2008 ; Gonzalez et al., 2008). The light-dependent regulation of these genes by complex sets of regulators as well as circadian cycles highlights the complex regulatory mechanisms modulating each of the individual branches in this pathway.
One element likely to be involved in light induction of this promoter and others in the flavonoid/anthocyanin branch is the G-BOX [LRE]. This has previously been identified as a light-responsive element (Menkens and Cashmore, 1994 Our promoter analyses have also indicated that MYB and MYB4 are significantly overrepresented in the core and lignin branch of this phenylpropanoid pathway. One potential MYB transcription factor, PAP1, which was proposed to control circadian regulation of the anthocyanin and lignin branches, is indeed circadian regulated, with the same phasing as these branched pathways. However, direct analyses of phenylpropanoid pathway loci potentially targeted by this transcription factor in overexpressing pap-1D seedlings have indicated that the circadian-regulated FAH1 (CYP84A1) and REF8 (CYP98A3) loci in lignin synthesis and C4H (CYP73A5) in the core pathway are not modulated by PAP1. In contrast, the TT7 (CYP75B1) locus, which is directly involved in flavonoid and anthocyanin syntheses, shows some degree of overall enhanced accumulation in pap1-D seedlings, suggesting that PAP1 can modestly enhance expression of the flavonoid branch of this pathway. And, contrary to the suggestion that PAP1 regulates circadian phasing of phenylpropanoid transcripts, these increases in CYP75B1 transcripts fluctuate, with a circadian rhythm that is unaffected by the high PAP1 levels in this mutant, providing further evidence that PAP1 does not control circadian fluctuations of these loci and indicating that other transcription factors modulate circadian cycles in this pathway. How PAP1 expression and these branched pathways are controlled certainly requires further investigation. These results also dramatically demonstrate that the relationships between cycling genes and the cycling network cannot be inferred from time-of-day information and that additional experiments are required to dissect cascades of regulation.
Since it has been demonstrated that the coordination of daily activities confers fitness for specific environments (Michael et al., 2003
Recent research has indicated that multiple hormone responses are intertwined with circadian cycling, including abscisic acid, 1-aminocyclopropane-1-carboxylic acid, brassinolide, cytokinin, indole-3-acetic acid, MeJ, and SA (S.L. Harmer, unpublished data). Auxin synthesis has been reported to be gated by the circadian clock, allowing a plant to respond to auxin at restricted times of day (Covington and Harmer, 2007
Plant Materials and Growth Conditions Arabidopsis (Arabidopsis thaliana) Columbia ecotype and pap1-D mutant seeds were sterilized in 70% ethanol for 30 s and 15% bleach for 15 min and then rinsed in distilled water two to three times. One hundred to 200 seeds per time point were sown on half-strength Murashige and Skoog agar plates containing 0.8% agar without Suc and were kept in the dark at 4°C for 3 d before transfer to a growth chamber. Seedlings were entrained with 12-h-white-light/12-h-dark cycles at a continuous temperature (22°C) for 7 d prior to being released into continuous white light at 22°C (LL_LDHH conditions). After 1 d in continuous conditions, seedlings were harvested at subjective dawn and every 4 h over the course of the next 44 h.
The gene lists used for the analysis of different pathway promoters are derived from data available at The Arabidopsis Information Resource (http://www.arabidopsis.org). The circadian-regulated Arabidopsis P450 gene lists of four data sets are derived from analysis of Affymetrix Arabidopsis genome chips as described by Michael et al. (2008)
All microarray experiments were described previously (Mockler et al., 2007 Autoradiographs of the RT-PCR gel blots were scanned using an Epson Perfection 1250 scanner and quantified using ImageJ 1.41 software (http://rsbweb.nih.gov/ij/). RT-PCR signals for each sample were then background corrected and normalized against the RT-PCR signals for UBQ10 and reported relative to the RT-PCR signal for the first sample in each time course.
Approximately 100 seedlings per time point were frozen under liquid nitrogen and powdered, and total RNA was extracted using a beadbeater (Biospec Products), a Plant RNeasy kit, and on-column RNase-free DNase (Qiagen) according to the manufacturer's instructions. Quantitative RT-PCR gel-blot analysis of individual P450 transcripts was carried out by amplifying approximately 0.1 mg of total RNA from each sample in one-step RT-PCR containing 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 200 mM each dNTP, 200 mg mL–1 gelatin, 40 pmol of a 5' gene-specific primer, 80 pmol of a 3' oligo(dT) primer, 2 units of AMV reverse transcriptase (Promega), 8 units of RNasin (Promega), and 1 unit of GoTaq polymerase (Promega). First-strand cDNAs were synthesized for 30 min at 42°C and subsequently PCR amplified for 18 to 26 cycles, with each cycle consisting of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, followed by a final extension step of 72°C for 10 min. The numbers of PCR cycles used for each transcript (18 for CYP90A1 and UBQ10, 19 for CYP79B3, 20 for CYP73A5 and CYP84A1, 23 for CYP74A1, and 26 for CYP75B1 and CYP98A3) were determined to be within the linear PCR amplification range for each transcript. PCR products were fractionated on 1.5% agarose gels, transferred to Hybond-N (Amersham-Pharmacia Biotech), and probed with random hexamer 32P-labeled probes corresponding to approximately 150 nucleotides derived from the 3' untranslated region of each P450 locus or the Arabidopsis UBQ10 cDNA. The gene-specific primers used in this analysis were as follows: 73A5-5', 5'-TTGCACATCCTTAACCACTCC-3'; 75B1-5', 5'-GACTCGGGTCGGGTTAAAAT-3'; 84A1-5', 5'-GGGGTTTGGTATGGTGAAAA3'; 98A3-5', 5'-TTGACCGGATCTTAACCGAG-3'; 74A1-5', 5'-AGAAGAACCTCTCATCCATACATTTAGTC-3'; 90A1-5', 5'-CAGTTGGGTTCCTGCAGAGC-3'; 79B3-5', 5'-ACGTGTCGAGCTTATGGA-3'; PAP1-5', 5'-GACAACAGAAAAGGGGGACA-3'; oligo(dT), 5'-CGGAATTCTTTTTTTTTTTTTTTTT-3'; UBQ10-5', 5'-CTTGGTCCTGCGTCTTCGTGGTGGTTTC-3'; and UBQ10-3', 5'-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3'. Probe sequences for each of the P450s transcripts are as follows: CYP73A5, 51 nucleotides upstream to 86 nucleotides downstream from the stop codon of the At2g30490 locus; CYP75B1, 14 nucleotides upstream to 112 nucleotides downstream from the stop codon of the At5g07990 locus; CYP84A1, 45 nucleotides downstream to 149 nucleotides downstream from the stop codon of the At4g36220 locus; CYP98A3, 521 nucleotides upstream to 376 nucleotides upstream from the stop codon of the At2g40890 locus; CYP74A1, 766 nucleotides upstream to 572 nucleotides downstream from the stop codon of the At5g42650 locus; CYP90A1, 103 nucleotides upstream to 147 nucleotides downstream from the stop codon of the At5g05690 locus; CYP79B3, 103 nucleotides upstream to 390 nucleotides downstream from the stop codon of the At2g22330 locus; and PAP1, 82 nucleotides upstream to 38 nucleotides downstream from the stop codon of the At1g56650 locus.
Promoter searches for known cis-elements functional in Arabidopsis (annotated in the AGRIS sequence motif database; http://Arabidopsis.med.ohio-state.edu; Davuluri et al., 2003
For determination of overrepresented sequences, promoters within particular branched pathways listed in Table II (i.e. lignin and flavonol branches of phenylpropanoid synthesis) were analyzed for the frequency of each degenerate sequence motif compared with the frequency of each motif in the same size window upstream from the translation start sites of 27,457 genes annotated by the Arabidopsis Genome Initiative (ftp://ftp.arabidopsis.org/home/tair/Sequences/blast_datasets/). Pattern search methods and matching motifs are performed using scripts written in the programming language Perl 5.8.0 for i386-linux-thread-multi. P values for the probability of finding motifs in a subset of promoters are calculated by hypergeometric distribution, modeling sampling on a word-by-word basis. The significance cutoffs for P values are corrected for multiple testing according to the step-up procedure of Benjamini and Hochberg (1995)
The raw transcriptome data sets used for the analyses of circadian-regulated gene correlation were downloaded from Nottingham Arabidopsis Stock Centre arrays in the paper of Edwards et al. (2006)
The following materials are available in the online version of this article.
We thank Dr. Hui Duan for his technical assistance and care of plants and Dr. Shisong Ma for instruction in network analysis. Received October 3, 2008; accepted April 6, 2009; published April 22, 2009.
1 This work was supported by the National Science Foundation (grant no. MCB 0115068 to M.A.S.), by a Physiological and Molecular Plant Biology Program Fellowship to Y.P., by Waksman Institute startup funds to T.P.M., and by the National Institutes of Health (grant nos. GM52413 and GM62932 to J.C. and grant nos. GM56006 and GM67837 to S.A.K.).  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: Mary A. Schuler (maryschu{at}uiuc.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.130757 * Corresponding author; e-mail maryschu{at}uiuc.edu.
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Johns Hopkins University, Department of Biostatistics Working Papers, Baltimore, MD
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ABSTRACT
RESULTS
-carotenes (CYP97A3; LUT5) and subsequent 
-ring hydroxylations (CYP97C1; LUT1; Tian et al., 2004
-carotene desaturase; At3g04870), LYC (lycopene cyclase; At3g10230), B2 (β-carotene hydroxylases; At5g52570), B1 (At4g25700), ABA1 (zeaxanthin epoxidase; At5g67030), NPQ1 (violaxanthin deepoxidase precursor; At1g08550), LUT2 (lutein-deficient 2; At5g57030), LUT5 (β-ring hydroxylase on carotenes; CYP97A3; At1g31800), and LUT1 (
