Cytochrome P450 monooxygenases as reporters for circadian-regulated pathways

Abstract

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.

Figure 1.

P450 loci showing circadian phasing for at least one of the four array conditions. The Venn diagram shows the numbers of genes overlapping among the four circadian conditions.

Figure 2.

Phenylpropanoid pathway. This map adapted from the AraCyc Pathway shows the genes responsible for steps in the phenylpropanoid pathway, with black boxes indicating genes that are circadian regulated and underlines indicating P450 loci. [See online article for color version of this figure.]

Figure 3.

Circadian-regulated transcripts in known pathways. A to E, RNAs from the LL_LLHC and LL_LDHC conditions were analyzed on RT-PCR gel blots for the transcripts listed above each panel or set of panels. The RT-PCR products for P450s in each sample were background corrected, normalized against RT-PCR products for constitutive UBQ10 in each sample, and recorded relative to the 0-h signal in each time course. The UBQ10 normalizations for B to E are shown in A. F to J, These P450 transcript levels were quantified and plotted relative to the 0-h signal for each time course. K to O, RNAs from the LL_LLHC and LL_LDHC conditions were analyzed on microarrays, normalized using gcRMA, and plotted relative to the 0-h signals. Values on the x axis indicate hours after each time-course sampling was initiated. White and gray bars on the x axis indicate subjective day and subjective night during the time course; blank and grid bars indicate high (22°C) and low (12°C) temperatures maintained during the time course. [See online article for color version of this figure.]

Figure 4.

Maps of four circadian-regulated pathways. These maps adapted from AraCyc Pathway correspond to the carotenoid (A), oxylipin (B), glucosinolate (C), and brassinosteroid (D) pathways. Black boxes indicate genes that are circadian regulated, and underlines indicate P450 loci. [See online article for color version of this figure.]

Figure 5.

Phasing in the carotenoid and glucosinolate pathways. A, Normalized microarray circadian gene expression data of LL_LDHH for genes in the carotenoid pathway include PSY (phytoene synthase; At5g17230), PDS (phytoene dehydrogenase; At4g14210), ZDS (ζ-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 (ɛ-ring hydroxylase on carotenes; CYP97C1; At3g53130). The carotenoid intermediate pathway is shown in blue, the zeaxanthin branch is shown in black, and the lutein branch is shown in red. B, Normalized microarray time-course data plotted every 4 h, with light and temperature conditions indicated below the data, are shown for genes in the aliphatic branch of glucosinolate synthesis and include three P450s, CYP79F1 (At1g16400), CYP79F2 (At1g16410), and CYP83A1 (At4g13770), as well as SUR1 (alkylthiohydroximate C-S lyase; At2g20610), UGT (UDP-glycosyltransferase; At1g24100), ST1 (sulfotransferases; At1g18590), ST2 (At1g74090), AOP2 (2-oxoglutarate-dependent dioxygenases; At4g03060), and AOP3 (At4g03050). Circadian data are also shown for genes in the indole branch of glucosinolate synthesis and include three P450s, CYP79B2 (At4g39950), CYP79B3 (At2g22330), and CYP83B1 (At4g31500), as well as SUR1, UGT, and DST (desulfoglucosinolate sulfotransferase; At1g74100). Values on the x axis indicate hours after each time-course sampling was initiated. White and gray bars on the x axis indicate subjective day and subjective night during the time course; blank and grid bars indicate high (22°C) and low (12°C) temperature maintained during the time course.

Figure 6.

The positions of elements in the 2-kb promoter sequences of genes of different pathways. A, Positions of overrepresented EE in the promoters of genes in the flavonol branch. B, Positions of overrepresented G-BOX [LRE] in the promoters of genes in the intermediate flavonoid pathway and the downstream flavonol and anthocyanin branches. C, Positions of overrepresented GL-MET2 in the promoters of genes in the flavonol branch. D, Positions of overrepresented JA1 in the promoters of genes in the oxylipin pathway. E and F, Spacing of elements overrepresented in the CYP97A3 and CYP97C1 promoters in the lutein branch of the carotenoid pathway. The numbers of each element found in these promoters are shown in parentheses behind each motif name.

Figure 7.

Phenylpropanoid subnetwork. Genes networked in their expression profiles are shown with nodes corresponding to CHS (chalcone synthase; At5g13930), TT5 (chalcone isomerase; At3g55120), CHI (chalcone isomerase; At5g05270), F3H (naringenin 3-dioxygenase; At3g51240), TT7 (flavonoid 3′-monooxygenase; CYP75B1; At5g07990), F3OG2 (flavonol 3-O-glucosyltransferases; At4g01070), F3OG3 (At5g54060), and DFR (dihydroflavonol 4-reductase; At5g42800). FS (flavonol synthase; At3g50210) is not connected to these. A, Genes at nodes in this network that contain G-BOX [LRE] have thick red circles; associated genes that also contain G-BOX [LRE] have thin red circles. B, Phasing of genes at nodes in this network. Values on the x axis indicate hours after each time-course sampling was initiated. White and gray bars on the x axis indicate subjective day and subjective night during the time course; blank and grid bars indicate high (22°C) and low (12°C) temperature maintained during the time course.

Figure 8.

Circadian regulation of P450s in the phenylpropanoid pathway in wild-type and PAP1-overexpressing (pap1-D) mutant seedlings. RT-PCR gel blots for constitutive UBQ10 (A), PAP1 (B), CYP73A5 (C), CYP75B1 (D), CYP98A3 (E), and CYP84A1 (F) were compared in wild-type and pap1-D seedlings over a 48-h period starting at 7 d of growth under LL_LDHH conditions (collected and analyzed in the Schuler laboratory). UBQ10 expression levels (A) under LL_LDHH were used for normalization, and each normalized transcript level is shown above each lane. The gcRMA-normalized microarray data for PAP1 and these four phenylpropanoid P450s on the LL_LDHH arrays performed with wild-type seedling RNAs (collected and analyzed by the Millar laboratory) are shown in G. Values on the x axis indicate hours after each time-course sampling was initiated. White and gray bars on the x axis indicate subjective day and subjective night during the time course at continuous 22°C. [See online article for color version of this figure.]