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First published online November 6, 2003; 10.1104/pp.103.030882 Plant Physiology 133:1643-1653 (2003) © 2003 American Society of Plant Biologists CYP72B1 Inactivates Brassinosteroid Hormones: An Intersection between Photomorphogenesis and Plant Steroid Signal Transduction1Department of Biology, Washington University, Campus Box 1137, One Brookings Drive, St. Louis, Missouri 63130 (E.M.T., M.A.D., Q.I.T., M.M.N.); RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351–0198, Japan (S.F., H.S., Y.S., S.Y.); and Department of Chemistry, Joetsu University of Education, Joetsu-shi, Niigata 943–8512, Japan (S.T.)
Active brassinosteroids, such as brassinolide (BL) and castasterone, are growth promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase encoded by CYP72B1 has been implicated in brassinosteroid catabolism as well as photomorphogenesis. We expressed CYP72B1 in yeast, coupled with brassinosteroid feeding, and established the biochemical function to be the hydroxylation of BL and castasterone, to give 26-hydroxybrassinolide and 26-hydroxycastasterone, respectively. Brassinosteroid feeding experiments with wild-type Arabidopsis, a CYP72B1 null mutant, and a CYP72B1 overexpression line demonstrated that carbon 26 hydroxylation of active brassinosteroids is an endogenous function of CYP72B1. Seedling growth assays demonstrated that 26-hydroxybrassinolide is an inactive brassinosteroid. Genetic and physiological analysis of the hypocotyl response to exogenous BL and varying intensities of white and monochromatic light suggested that CYP72B1 modulates photomorphogenesis primarily through far-red light and to a lesser extent through blue- and red-light pathways. CYP72B1 transcript accumulation in dark-grown seedlings was organ specific and down-regulated after 1 h of illumination in dim white, red, and blue light, but not far-red light. CYP72B1 translational fusions with the  -glucuronidase reporter gene demonstrated that protein levels increased in the hypocotyl elongation zone when shifted from the dark to far-red light, but not blue or red light. We propose a model in which Arabidopsis seedling development switches from dark-grown development (skotomorphogenesis) to light-grown development (photomorphogenesis) in part by rapid modulation of brassinosteroid sensitivity and levels. CYP72B1 provides an intersection between the light and brassinosteroid pathways mainly by far-red-light-dependent modulation of brassinosteroid levels.
Brassinolide (BL) is the most active brassinosteroid, a class of polyhydroxylated plant-specific steroids. The isolation of BL in 1979 identified the structure to be a cholestane derivative (Grove et al., 1979  ). Animal steroids are likewise cholestane derivates (Mussig and Altmann, 2001). Analysis of BL biosynthetic mutants (det2 and cpd) in Arabidopsis revealed similarities between animal and plant steroids. Rescue of the det2 and cpd pleiotropic phenotypes by exogenous BL established the commonality of steroids as fundamental hormones in both animal and plant development (Li et al., 1996; Szekeres et al., 1996). Cloning of the CPD gene from Arabidopsis demonstrated that animals and plants each use cytochrome P450 monooxygenases (CYP450s) for steroid biosynthesis (Szekeres et al., 1996). Analysis of the human steroid 5 -reductase (hS5R) and its Arabidopsis ortholog, DET2, demonstrated a common mechanism of steroid hormone activation between animals and plants (Li et al., 1996). Both human isoenzymes of hS5R reduce testosterone to dihydrotestosterone to amplify a weak hormone signal, whereas DET2 reduces BL precursors (Fujioka et al., 2002). Ecdysone, an insect steroid hormone, is structurally similar to BL and inactivated by carbon 26 hydroxylation via the action of an unidentified CYP450 (Williams et al., 2000). An analogous inactivation of BL was suggested when overexpression of BAS1/CYP72B1, a putative Arabidopsis CYP450, resulted in Arabidopsis (bas1-D) and tobacco plants with BL-deficient-like phenotypes and heightened accumulation of 26-hydroxybrassinolide (26-OHBL; Neff et al., 1999).
Although many animal steroid hormones have been firmly established as components of endocrine systems with well-characterized roles in animal development, the role of brassinosteroids in plant development is less clear. BL can affect cell elongation, division, and differentiation, thus defects in brassinosteroid synthesis or sensing are likely to affect many plant development pathways (Friedrichsen and Chory, 2001
Brassinosteroid-deficient or -insensitive mutants as well as Arabidopsis seedlings treated with a brassinosteroid biosynthesis inhibitor (Brz) have light-grown phenotypes when grown in the dark (Fankhauser and Chory, 1997
A previous genetic characterization using overexpression and antisense lines suggested that BAS1/CYP72B1 acts as a point of cross-talk between brassinosteroids and photomorphogenesis. Though present in wild-type adult plants, BL was undetectable in adult bas1-D mutants. Levels of CS were reduced in bas1-D plants compared with the wild type. When labeled BL was fed to bas1-D seedlings, a heightened accumulation of 26-OHBL was detected relative to the wild type. Together, these observations lead to the hypothesis that, in Arabidopsis, active brassinosteroids can be inactivated via CYP72B1-mediated hydroxylation at carbon 26 (Neff et al., 1999
CYP72B1 Is a Brassinosteroid Carbon 26 Hydroxylase
We took two complementary approaches to test the hypothesis that CYP72B1 converts BL to 26-OHBL and to examine the possibility that CS is also a substrate for this enzyme. First, we expressed CYP72B1 cDNA in the WAT11 yeast strain using the pYeDP60 yeast-expression vector (Pompon et al., 1996
To test the endogenous function of CYP72B1 in Arabidopsis, seedlings of a null mutant in this gene (cyp72b1-1), the wild type (Wassilewskija 2 [Ws-2]), and a transgenic line overexpressing CYP72B1 (cyp72b1-ox8) were fed deuterium-labeled BL and assayed for conversion to deuterium-labeled 26-OHBL by GC-MS (Table I). A reduced level of deuterium-labeled 26-OHBL was detected in the null mutant, and an increased level was detected in the cyp72b1-ox8 line compared with Ws-2. Feeding experiments with unlabeled BL gave qualitatively similar results. CS feeding resulted in conversion to 26-OHCS in a manner similar to BL feeding (Table I). Statistical analysis (Student's t test, paired, one-tailed) indicated that the wild type (Ws-2) converts each substrate to its 26-hydroxylated product more so than the cyp72b1-1 null mutant (P = 0.02) and less than the CYP72B1 overexpression line, cyp72b1-ox8 (P = 0.0003). The small amount of 26-OHBL detected in the cyp72b1-1 null mutant could be due to the action of a nonspecific or partially redundant CYP450. These genetic results confirmed the biochemical function of CYP72B1 established by the yeast functional assay and demonstrated that carbon-26 hydroxylation of the active brassinosteroids, BL and CS, is an endogenous biochemical function of CYP72B1 in Arabidopsis.
Bioassays with synthetic 26-OHBL demonstrate that the CYP72B1 product is an inactive brassinosteroid in rice (Seto et al., 1999
RNA null and overexpression lines were used to test the hypothesis that CYP72B1 is involved in photomorphogenesis, and more specifically, deetiolation (transition from growth in the dark to growth in the light; Fig. 3A). Hypocotyl elongation inhibition in response to light is well characterized and often used as a phenotypic marker for deetiolation. We first assayed the seedling hypocotyl length of Ws-2 and the cyp72b1-1 null mutant in response to increasing concentrations of exogenous BL when grown in the dark or bright-white light (Fig. 3B). There was essentially no difference between Ws-2 and cyp72b1-1 in response to BL in the dark. The only exception was a slightly shorter cyp72b1-1 hypocotyl at a concentration of 10 nM. In contrast, cyp72b1-1 was slightly more responsive to increasing concentrations of exogenous BL when grown in white light (Fig. 3B). This light-dependent phenotype suggested that CYP72B1 is involved in de-etiolation.
To further test the hypothesis that BL antagonizes de-etiolation in Arabidopsis, we assayed hypocotyl growth inhibition for Ws-2 seedlings grown at various combinations of white-light fluence rates and BL concentrations (Fig. 3C, Ws-2). The combination with the greatest response (i.e. shortest hypocotyls) had the highest white-light fluence rate and the lowest concentration of BL. In contrast, the combination with the least response had the lowest white-light fluence rate and the highest concentration of BL. A gradation between these two extremes was produced based on the combination of the relative amounts of light and BL. These data demonstrated that light and BL are antagonistic with respect to hypocotyl growth and suggested that active brassinosteroids have a negative regulatory role in Arabidopsis de-etiolation. If active brassinosteroids are negative regulators of de-etiolation, then brassinosteroid inactivation should act as a positive modulator of de-etiolation via the removal of such a negative regulator. The cyp72b1-1 null mutant and the cyp72b1-ox8 line were used to test this hypothesis (Fig. 3). Loss of CYP72B1 expression resulted in reduced responsiveness to light (removal of a positive modulator) and increased responsiveness to BL (removal of an inactivating enzyme) at every condition tested, which resulted in a graph with the same basic pattern as Ws-2, but raised (Fig. 3C, cyp72b1-1). Analysis of the cyp72b1-ox8 line showed the opposite response (Fig. 3C, cyp72b1-ox8). These results are consistent with a model in which CYP72B1 is modulating de-etiolation via BL inactivation.
To begin to examine the role of CYP72B1 in the various photoreceptor response pathways, fluence rate-response analysis of cyp72b1-1 was compared with Ws-2 in white and monochromatic light (Fig. 4). Arabidopsis uses five red/far-red absorbing photoreceptors (phytochromes A–E) and two blue/UVA photoreceptors (cryptochromes1 and 2). Reduction in responsiveness of a null mutant to white light indicates a genetic role in light responsiveness, whereas reduction in responsiveness to a particular color of light is a first step in examining a genetic role in a specific photoreceptor response pathway. All conditions resulted in no difference in the dark and a slightly taller hypocotyl in low fluence rate conditions (Fig. 4). This growth response to light is similar to the previously described CYP72B1 antisense lines (Neff et al., 1999
The CYP72B1 null and overexpression lines were assayed for responsiveness to exogenous BL at a single concentration (10 nM) and to light at a single fluence rate (Fig. 5). In the dark without BL supplementation, cyp72b1-1 and Ws-2 were the same height, but in each light condition except blue, cyp72b1-1 was significantly taller. In far-red light without BL supplementation, cyp72b1-1 was 30% taller than Ws-2. In far-red light with BL supplementation, cyp72b1-1 was 55% taller than Ws-2. A similar change in response to exogenous BL was seen in white light, but not in blue or red light, which suggests that far-red light may be important to the role of CYP72B1 in photomorphogenesis (Fig. 5A).
Unlike cyp72b1-1, cyp72b1-ox8 was shorter (33%) than Ws-2 in the dark without BL supplementation, and the difference was smaller on BL-containing plates (21%). This pattern of response to BL in the dark was also seen in blue- and red-light conditions (Fig. 5B). In far-red light, cyp72b1-ox8 was 31% shorter than Ws-2 when the medium was not supplemented with BL, and the difference was larger on BL-containing plates (45%). The results from analysis of a CYP72B1 overexpression line are therefore consistent with the results from a CYP72B1 null line. In this assay, loss of CYP72B1 caused an apparent increase in BL sensitivity in far-red light and overexpression of CYP72B1 had the opposite result.
Arabidopsis seedlings were assayed for differential CYP72B1 RNA transcript accumulation by real time reverse transcriptase (RT)-PCR (Fig. 6). CYP72B1 and internal reference RNA (UBQ10) were accurately and reproducibly measured from a 2-fold serial dilution of total cDNA by this method (Fig. 6, A and B). The ratio of CYP72B1 to UBQ10 RNA level was then compared between various light conditions and organs (Fig. 6, C and D).
After 1 h of exposure to far-red light, there was no detectable change in CYP72B1 transcript accumulation (Fig. 6C). In contrast, 1 h of exposure to red, blue, and white light resulted in an approximate 4-fold reduction. The presence of a single null allele of PHYA, PHYB, or CRY1 was unable to prevent the reduction of CYP72B1 transcript accumulation in white light (Fig. 6C). This suggests that multiple photoreceptor pathways interact to modulate CYP72B1 transcript accumulation. After 1 h of exposure to white light, the level of CYP72B1 transcript in the root was unchanged (Fig. 6D). The apex and the hypocotyl, on the other hand, showed a decrease in CYP72B1 RNA abundance similar to whole seedlings, although the hypocotyl had more CYP72B1 RNA in both conditions.
The entire CYP72B1 gene from 1.5-kb upstream of the start codon to the last base before the stop codon was cloned in frame to the uidA gene, which encodes the
In all light conditions, there was an obvious GUS stain in the root at the root/hypocotyl transition zone (Fig. 7, F–J). In far-red light, there was a clear GUS stain in the elongation zone of the hypocotyl just below and often including the apical hook (Fig. 7B). CYP72B1 protein accumulation appeared unchanged between dark, red-, blue-, and white-light conditions (Fig. 7, A and C–E).
Biochemistry of CYP72B1
A previous genetic characterization using overexpression and antisense lines suggested that the CYP72B1 enzyme inactivates brassinosteroids (Neff et al., 1999
In this study, we demonstrated that CYP72B1 expressed in yeast can convert BL as well as CS to the 26-hydroxy derivatives (Table I). These data confirm the biochemistry of the CYP72B1 enzyme and suggest that CYP72B1 can have broad substrate specificity for active brassinosteroids. This broad substrate specificity is analogous to the Arabidopsis DWARF/CYP85 enzyme, a CYP450 involved in brassinosteroid biosynthesis (Shimada et al., 2001
Furthermore, we showed that feeding brassinosteroids to the cyp72b1-1 null mutant resulted in relatively less accumulation of the 26-hydroxy derivatives, whereas a transgenic line overexpressing CYP72B1 resulted in more (Table I). The detection of a small amount of 26-OHBL in the cyp72b1-1 null mutant suggests the presence of a redundant CYP450. CYP72C1 is a candidate for providing the redundant 26-OHBL activity. The dwarf phenotypes of chibi2, an activation-tagged Arabidopsis mutant with high CYP72C1 transcript levels, are similar to those of bas1-D (see Bishop and Yokota, 2001
Relative to wild-type controls, bioassays with synthetic 26-OHBL supplemented to the growth medium also supported the notion that CYP72B1 enzymatic activity results in an inactive brassinosteroid (Fig. 2). Bioassays with synthetic 28-OHBL resulted in typical brassinosteroid responses, which demonstrate that addition of one hydroxyl group to the BL side chain does not impair uptake of the resulting molecule (Seto et al., 1999
Taken together, these results support a mechanism of brassinosteroid hormone inactivation in plants, which is analogous to the inactivation of the insect molting hormone ecdysone. Namely, the CYP450-mediated hydroxylation of an active hormone at the carbon-26 position resulting in an inactive congener. Interestingly, ecdysone is the non-plant steroid that is structurally the most similar to BL (Voigt et al., 2001
We also tested the hypothesis that CYP72B1 is involved in photomorphogenesis. This developmental pathway can be assayed by measuring the response of Arabidopsis hypocotyl elongation to white or monochromatic light. A simple genetic test is to determine whether a line that is defective in the gene of interest is also less responsive to these conditions. The cyp72b1-1 hypocotyls were less responsive to all light conditions tested, suggesting that CYP72B1-mediated inactivation of brassinosteroids is involved in each of the pathways that make up photomorphogenesis (Fig. 4). This simple test does not rule out the possibility that CYP72B1/brassinosteroids act on hypocotyl elongation in a pathway that is parallel to light inhibition of hypocotyl elongation. For example, an attractive hypothesis is that light simultaneously activates growth-inhibiting and -promoting pathways so that the rate of hypocotyl elongation in any condition is the resultant or balance between two opposing influences of light (Parks et al., 2001 Mutants defective in brassinosteroid biosynthesis (det2/cpd) or perception (bri1) are short in the dark as well as in the light. This demonstrates that brassinosteroids are required for the hypocotyl to elongate. In fact, addition of BL to the growth medium has a stimulatory affect on hypocotyl elongation of light-grown seedlings. A seemingly contradictory phenomenon is the ability of exogenous BL to inhibit hypocotyl elongation in the dark at concentrations that have a stimulatory effect in the light (Fig. 3B). This suggests that the BL response is nearly saturated in the dark, and as a consequence, increases in concentration of this growth-promoting hormone act to inhibit or "poison" the response. This response dichotomy may be due to differential BL sensitivity between the two environments.
An analogous mechanism is the dichotomy of response to auxin between the shoot and the root in response to gravity (Ottenschlager et al., 2003
Loss of CYP72B1 had only a minor effect on the BL dose response of dark-grown seedlings. At 10 nM BL, the dark-grown seedlings are shorter/more-responsive in the cyp72b1-1 background compared with Ws-2 (Figs. 3B and 5A). Although in the absence of exogenous BL, cyp72b1-1 is the same height as Ws-2 in the dark. This suggests that CYP72B1 does not play a role in skotomorphogenesis but may play a role in the inactivation of exogenous BL in the dark. Recent microarray data have shown that CYP72B1 transcript accumulation increases in response to exogenous BL (Goda et al., 2002
We present data that investigate the interaction between light and CYP72B1 at the genetic, RNA, and protein level. In all cases the connection was strongest with far-red light. Furthermore, double mutant analysis of bas1-D and a phytochrome A null allele (phyA-201) demonstrated that the increased responsiveness of the overexpressor to far-red light is dependent on the photoreceptor phyA (Neff et al., 1999
Exogenous BL can also elicit increases in CYP72B1 RNA abundance (Goda et al., 2002
The original hypothesis that CYP72B1 is a BL carbon-26 hydroxylase has been confirmed using yeast and Arabidopsis with altered CYP72B1 expression and has been extended to include carbon-26 hydroxylation of CS. One of the products of this enzymes activity, 26-OHBL, has also been shown to be a functionally inactive brassinosteroid during seedling development. The brassinosteroid sensitivity phenotypes in farred light by the CYP72B1 null and overexpression lines, coupled with the observation that CYP72B1 transcript levels are not decreased and the protein accumulation is increased by brief exposure to farred light, suggests that far-red light modulates brassinosteroid levels in part by the action of CYP72B1. If far-red light also modulates seedling sensitivity to brassinosteroids, then the effect of CYP72B1, and brassinosteroid inactivation in general may be amplified. The lack of blue- and red-light brassinosteroid sensitivity phenotypes in the CYP72B1 null, coupled with the lack of detectable GUS protein reporter and decreased RNA in theses conditions, suggests that red and blue light may provide only weak modulation of brassinosteroid levels through the action of CYP72B1.
Plant Material Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes. Arabidopsis ecotype Ws-2 was used as the wild-type starting material for the generation of a null allele of CYP72B1 (cyp72b1-1) and the CYP72B1 overexpression line (cyp72b1-ox8). A null allele line of DET2 (det2-1), ecotype Col-0, was used for analysis of 26-OHBL activity. The photoreceptor null alleles used in the quantitative RT-PCR are in the Col-0 ecotype: CRY1 (cry1-102), PHYA (phyA-211), and PHYB (phyB-9). The T-DNA insertion line (cyp72b1-1) came from the Wisconsin Knockout Facility (Madison; http://www.biotech.wisc.edu/Arabidopsis/) and was identified at the University of Arizona (Tucson; http://Ag.Arizona.Edu/p450/). The original cyp72b1-1 line was back-crossed to Ws-2, and F2 seeds were collected from kanamycin-resistant (Kanr) F1 plants. Each F2 population segregated approximately 15 Kanr to 1 kanamycin-sensitive (Kans) seedling, indicating that the original line contains two unlinked loci of T-DNA insertion. F3 seeds from Kanr F2 parents were assayed for Kanr, and those with a 3:1 ratio (Kanr:Kans) were analyzed by PCR for the presence of the T-DNA in the CYP72B1 open reading frame. All F4 seed populations from an F3 line containing the T-DNA in CYP72B1 were either homozygous for Kanr or segregated 3:1 (Kanr:Kans). PCR analysis showed that those populations homozygous for Kanr were also homozygous for the T-DNA insertion in CYP72B1. Sequencing of the T-DNA insertion site showed the insertion to occur within the second exon, at 1,412 bp downstream of the ATG.
For this study, pMN17 (CYP72B1 gene and promoter driven by 4 x 35S enhancer elements) was randomly inserted into the genome of Arabidopsis ecotype Ws-2 via Agrobacterium tumefaciens-mediated transformation to create a CYP72B1 overexpression series. Plasmid pMN17 is described by Neff et al. (1999 Fifteen T2 seedlings from each of the 16 lines were transplanted to soil and grew to maturity in greenhouse/long-day conditions. Each T2 plant was harvested individually, and an aliquot of each of the T3 seed pools was surface sterilized and sprinkle plated onto gentamycin-containing plates as above. Each T3 line was scored for the ratio of gentamycin resistance versus sensitivity and uniformity of seedling phenotype. Eight lines with good seed set and greatest degree of seedling uniformity were kept. All homozygous resistant T3 seedlings from each of the eight remaining lines were transferred from plates to soil (3-inch pots, four seedlings per pot). The cyp72b1-ox8 line is a weak CYP72B1 overexpressor (based on severity of the adult dwarfism phenotype), having a uniform phenotype between siblings and good seed set. The transgenic lines cyp72b1-ox7 and cyp72b1-ox9 also have a uniform phenotype and good seed set, but they both have a greater degree of dwarfism (marker for high expression).
Seeds were surface sterilized by 5 min of agitation in 70% (v/v) ethanol with 0.05% (v/v) Triton X-100, followed by 5 min of agitation in 95% (v/v) ethanol with 0.05% (v/v) Triton X-100, and 5 min of agitation in 95% (v/v) ethanol. A disposable Pasteur pipette was used to transfer the seed/ethanol solution to a sterilized 90-mm filter paper placed inside a sterile petri dish. Seeds were allowed to air dry in a sterile laminar-flow hood and then sown immediately onto growth medium or sealed in the petri dish with parafilm and stored at 4°C.
Surface-sterilized seeds were sown onto 90-mm square petri dishes containing 1% (w/v) phytagel (Sigma, St. Louis), 1.5% (w/v) Suc, and one-half Murashige and Skoog salts (catalog no. 11118-023, Invitrogen, Carlsbad, CA) in a sterile laminar flow hood. Seeds were sown individually and spaced approximately 10 mm apart in a triangular pattern using a sterile 200-µL pipette tip. Sown petri dishes were then wrapped in aluminum foil and incubated upside down in a 4°C cold room for 5 d to stratify seeds for uniform germination. Hormone plates contained BL dissolved in 95% (v/v) ethanol (CIDtech Research Inc., Missisuaga, ON, Canada), 26-OHBL dissolved in 95% (v/v) ethanol (see Neff et al., 1999
After 5 d of incubation at 4°C, plates were unwrapped and incubated for 30 min in a growth chamber containing red-light-emitting diodes (model E-30B, Percival Scientific, Perry, IA) to induce uniform germination. Plates were then incubated in the dark for 23.5 h and then incubated in a similar growth chamber, but with white light, for 72 h. All chamber temperatures were at 24°C. White light of 130 µmol m-2 s-1 (measured by a LI-250 Light Meter [LI-COR, Lincoln, NE]) was derived from eight fluorescent tubes (F17T8/TL741 17WATT) from Philips (Eindhoven, The Netherlands) and two incandescent tubes (PC: 13487 25T10/F CD 25WATTS 120 V) from GE (Fairfield, CT). One to six layers of gray, fiberglass window screening were used to vary the fluence rate.
After 4 d of growth, plates were moved to 4°C cold room for storage until measured. Seedlings were gently lifted with forceps from plates onto acetate sheets and digitized with a flat-bed scanner at a resolution of 1,200 dpi. Seedling scans were measured using ImageJ 1.29j software (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/java1.3.1).
The entire CYP72B1 gene from just 5' of the stop codon to 1.5 kb 5' of the translational start codon was amplified from BAC F18A8 using the following primers: forward 5'-AGT TAC GGT CGA CAC TCT TGC CTT GGT TAG A, reverse 5'-GGA TTG CTT CTC CAT GGA ATC CTC ATG ATT GGT CA. The resulting product was then ligated into pCAMBIA1301 after digestion with the restriction endonucleases BamHI and NcoI. Amplification and sequencing was performed as below with pED8.
Whole seedlings were transferred from plates to a 1.5-mL centrifuge tube containing 1 mL of GUS staining solution (1 mM 5-bromo-4-chloro-3-indolyl-
The coding region of CYP72B1 cDNA was cloned into the pYeDP60 yeast expression vector (Pompon et al., 1996
Yeast expression of the CYP72B1 gene was performed as described previously (Bishop et al., 1999
GC-MS analysis was carried out on a mass spectrometer (JMS-AM SUN200, JEOL, Tokyo) connected to a gas chromatograph (6890A, Agilent Technologies, Wilmington, DE) with a capillary column DB-5 (0.25 mm x 15 m, 0.25-µm film thickness; J&W Scientific, Folsom, CA). Analysis was conducted under the following conditions: ionization, EI (70 eV); carrier gas, helium at a flow rate of 1 mL min-1; injection temperature, 280°C; column temperature, 80°C for 1 min, elevated to 320°C at 30°C min-1, and then maintained at 320°C.
For metabolic experiments, Arabidopsis seeds were surface sterilized and placed on petri plates containing one-half-strength Murashige and Skoog medium supplemented with 1% (w/v) Suc and 0.8% (w/v) agar at 22°C in the light. Seven days after sowing, seedlings (Ws-2, cyp72b1-1: 15 seedlings, and cyp72b1-ox8: 30 seedlings) were transferred to 200-mL flasks containing 30 mL of one-half-strength Murashige and Skoog medium supplemented with 1% (w/v) Suc. The seedlings were grown at 22°C in the light on a shaker (120 rpm). Nine days after transfer, an ethanol solution (10 µg 10 µL-1) of BL, CS, [2H6]-BL, or [2H6]-CS was added to each flask. The seedlings were allowed to grow for another day under the same growth conditions and then extracted with methanol. The methanol extract was partitioned between ethyl acetate and water. The ethyl acetate-soluble fraction was purified and analyzed by the methods described above.
CYP72B1 transcript accumulation was measured as the ratio of CYP72B1 RNA level to ubiquitin10 (UBQ10) RNA level (internal reference). Each RNA level was measured as the cycle threshold above background fluorescence using a Smart Cycler real time PCR machine from Cepheid (Sunnyvale, CA). The CYP72B1 PCR product is 6-carboxy-fluorescein labeled, and UBQ10 is 6-carboxy-4',5'-dichloro-2',7'-dimethyoxy-fluoresein labeled due to incorporation of light upon extension primers from Invitrogen (Carlsbad, CA). The CYP72B1 forward primer is labeled on the third base from the 3' end: 5'-CAA CAA GCG GCG AAG GAT TTG TTG. The CYP72B1 reverse primer is unlabeled: 5'-CAC TCC TCC ACC AAT GTC CTG AA. The UBQ10 forward primer is labeled on the third base from the 3' end: 5'-GTA CGT CGC AGC TTG AGG ATG GAC GTA C. The UBQ10 reverse primer is unlabeled: 5'-ACG AAG ACG CAG GAC CAA GT. Total RNA was isolated from 18 x 4-d-old seedlings using the RNeasy Plant Mini Kit from Qiagen (Valencia, CA). Total cDNA was produced using the SuperScriptIII First-Strand Synthesis System for RT-PCR from Invitrogen of which 0.5 µL was used in a 25-µL PCR reaction using the Platinum Quantitative SuperMix-UDG from Invitrogen. CYP72B1 PCR cycle parameters: 50°C/120 s; 95°C/120 s; 45 repeats, 95°C/15 s, 68°C/30 s. UBQ10 PCR cycle parameters: 50°C/120 s; 95°C/120 s; 45 repeats, 95°C/60 s, 64°C/60 s.
We thank Joe Chappell, Emily Gamelin, Makoto Kobayashi, Garish Murthy, Masayo Sekimoto, Shunji Takahashi, Kathleen Tang, and Anna Zeide for their technical assistance; Rene Feyereisen, Frans Tax, and Tiegang Lu for identifying and supplying the cyp72b1-1 mutant; and Denis Pompon and Philippe Urban for the WAT 11 yeast strain. We appreciate the critical review of this manuscript by Tuan-hua Ho, Eric Richards, Ian Street, and Jason Ward. We thank Danielle Turk for assisting with graphic design and layout. Received July 24, 2003; returned for revision August 28, 2003; accepted September 15, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.030882.
1 This work was supported by the National Science Foundation (grant no. 0114726 to M.M.N.). Q.I.T. and M.A.D. were supported in part by the National Science Foundation Research Experience for Undergraduates (grant no. 0114726). Q.I.T. was supported in part by a WU/HHMI Summer Undergraduate Research Fellowship funded by an Undergraduate Biological Sciences Education Program grant from the Howard Hughes Medical Institute to Washington University. A research prize to E.M.T., from the Jean Lowenhaupt Botany Fund, defrayed publication costs. * Corresponding author; e-mail mneff{at}biology2.wustl.edu; fax 314–935–4432.
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