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The Biochemical Characterization of Two Carotenoid Cleavage Enzymes from Arabidopsis Indicates That a Carotenoid-derived Compound Inhibits Lateral Branching

The Biochemical Characterization of Two Carotenoid Cleavage Enzymes from Arabidopsis Indicates... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 45, Issue of November 5, pp. 46940–46945, 2004 Printed in U.S.A. The Biochemical Characterization of Two Carotenoid Cleavage Enzymes from Arabidopsis Indicates That a Carotenoid-derived □ S Compound Inhibits Lateral Branching* Received for publication, August 6, 2004, and in revised form, August 31, 2004 Published, JBC Papers in Press, September 1, 2004, DOI 10.1074/jbc.M409004200 Steven H. Schwartz‡§, Xiaoqiong Qin‡, and Michele C. Loewen From the ‡Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 and National Research Council Plant Biotechnology Institute, Saskatoon, Saskatchewan S7N 0W9, Canada organisms. Vitamin A, for example, is required for vision and Enzymes that are able to oxidatively cleave carote- noids at specific positions have been identified in ani- development in animals. In plants, abscisic acid is necessary mals and plants. The first such enzyme to be identified for seed development and adaptation to various environmental was a nine-cis-epoxy carotenoid dioxygenase from stresses. The synthesis of these apocarotenoids and others is maize, which catalyzes the rate-limiting step of abscisic catalyzed by a class of oxygenases that cleave specific double acid biosynthesis. Similar enzymes are necessary for the bonds resulting in two products with carbonyls at the site of synthesis of vitamin A in animals and other carotenoid- cleavage. For many years it was believed that the enzymes that derived molecules in plants. In the model plant, Arabi- catalyze these reactions were dioxygenases. In a recent study, dopsis, there are nine hypothetical proteins that share it was found that oxygen in one of the products comes from some degree of sequence similarity to the nine-cis-epoxy water (1), indicating that these enzymes may be monooxygen- carotenoid dioxygenases. Five of these proteins appear ases. For simplicity, all enzymes will be referred to the name to be involved in abscisic acid biosynthesis. The remain- given when originally described. ing four proteins are expected to catalyze other carote- In plants, nine-cis-epoxy-carotenoid dioxygenases (NCEDs) noid cleavage reactions and have been named carote- catalyze the rate-limiting step in abscisic acid biosynthesis. An noid cleavage dioxygenases (CCDs). The hypothetical NCED from maize was the first carotenoid cleavage enzyme to proteins, AtCCD7 and AtCCD8, are the most disparate be cloned and characterized (2, 3). A number of NCEDs (4–7) members of this protein family in Arabidopsis. The max3 and similar enzymes that are necessary for the synthesis of and max4 mutants in Arabidopsis result from lesions in other apocarotenoids have since been identified in a variety of AtCCD7 and AtCCD8. Both mutants display a dramatic plants (8–10). The enzymes that cleave -carotene to form two increase in lateral branching and are believed to be molecules of retinal in animals also belong to this family impaired in the synthesis of an unidentified compound (11–14). that inhibits axillary meristem development. To deter- mine the biochemical function of AtCCD7, the protein Within the genome sequence of the model plant, Arabidopsis, was expressed in carotenoid-accumulating strains of there are nine hypothetical proteins that share some degree of Escherichia coli. The activity of AtCCD7 was also tested sequence similarity to the NCEDs. Five of these proteins are in vitro with several of the most common plant carote- believed to be involved in abscisic acid synthesis (15). Four noids. It was shown that the recombinant AtCCD7 pro- members of this protein family in Arabidopsis do not cluster tein catalyzes a specific 9–10 cleavage of -carotene to with previously characterized NCEDs and are considered un- produce the 10-apo--carotenal (C ) and -ionone (C ). 27 13 likely to be abscisic acid biosynthetic enzymes. These proteins When AtCCD7 and AtCCD8 were co-expressed in a -car- may, however, catalyze other carotenoid cleavage reactions and otene-producing strain of E. coli, the 13-apo--carote- are more appropriately referred to as carotenoid cleavage di- none (C ) was produced. The C product appears to 18 18 oxygenases (CCDs). The AtCCD1 protein, for example, cata- result from a secondary cleavage of the AtCCD7-derived lyzes the symmetric 9–10, 9–10 cleavage of various carote- C product. The sequential cleavages of -carotene by noids (8). The specific biochemical functions for the remaining AtCCD7 and AtCCD8 are likely the initial steps in the three CCDs from Arabidopsis (AtCCD4, -7, and -8) have not yet synthesis of a carotenoid-derived signaling molecule been reported. that is necessary for the regulation lateral branching. The max4 mutant in Arabidopsis and the rms1 mutant in pea result from a lesion in AtCCD8 and a pea ortholog (16). Both mutants display an increase in lateral branching and are Apocarotenoids are a diverse class of compounds that are believed to be impaired in the synthesis of an unknown com- derived from the oxidative cleavage of carotenoids. These com- pound that inhibits axillary meristem development or bud out- pounds serve important biological functions in a variety of growth. The max3 mutant also displays an increase in lateral branching that results from a lesion in the AtCCD7 gene (17), indicating that AtCCD7 is also involved in the synthesis of this * This work was supported by United States National Science Foun- dation Grant IBN-0115004 and United States Department of Energy inhibitor. The biochemical characterization of AtCCD7 and Grant DE-FG02-91ER20021. The costs of publication of this article AtCCD8 is an important step in identifying the biologically were defrayed in part by the payment of page charges. This article must active compound. It has been reported that the expression of therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. The abbreviations used are: NCED, nine-cis-epoxy-carotenoid di- § To whom correspondence should be addressed. Tel.: 517-353-0697; oxygenase; CCD, carotenoid cleavage dioxygenase; HPLC, high per- Fax: 517-353-9168; E-mail: [email protected]. formance liquid chromatography; ORF, open reading frame. 46940 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Biochemical Characterization of AtCCD7 and AtCCD8 46941 TABLE I Plasmids used in this study Source or Plasmid Derivation and function reference pACLYC Biosynthetic genes from Erwinia herbicola for the synthesis of lycopene chm 30 pACBETA Biosynthetic genes from E. herbicola for the synthesis of ,-carotene chm 19 pACZEAX Biosynthetic genes from E. herbicola for the synthesis of zeaxanthin chm 30 pBHS1 A full-length clone of AtCCD7 was amplified with the following primer pair: 5-ATGTCTCTCCCTATCCCGC-3 (primer 1) and 5-TCAGTCGCTAGCCCATAAAC-3 (primer 2). The fragment was subcloned into pGEM-T easy vector from Promega (Madison, WI) amp pBHS2 A truncated fragment resulting in a 31-amino acid N-terminal deletion was amplified with primer 3: 5-GCCGCA- ATATCAATATCTATACC-3 and primer 2 (above). The fragment was subcloned into pGEM-T easy vector amp pBHS3 A NotI fragment from pBHS1 was subcloned into the NotI site of pGEX 5x-3 from Amersham Biosciences. For expression of AtCCD7 protein as a glutathione S-transferase fusion protein amp pBHS4 A NotI fragment from pBHS2 was subcloned into the NotI site of pGEX 5x-3. For expression of a truncated AtCCD7 protein as a glutathione S-transferase fusion protein amp pBHS5 A cDNA for a -carotene 9–10 cleavage dioxygenase (-Diox II) from mouse was obtained from the ResGen™ clone collection (clone 2536812). The -Diox II gene was amplified with a gene-specific primer (5- ATGTTGGGACCGAAGCAAAG-3) and a vector primer (5-CGACCTGCAGCTCGAGCACA-3). The fragment was cloned into pGEM T-easy amp pBHS6 By partial digestion of pBHS5 with EcoRI, the -Diox II gene was isolated and subcloned into pGEX 5x-3 vector for protein expression as a glutathione S-transferase fusion protein amp pHB3-His6 UPS expression vector for histidine-tagged fusions (Arabidopsis stock center no. CD3–595) 31 pAT1 With Cre-lox site-specific recombination, the AtCCD8 ORF was placed into pHB3-His for expression as a histidine-tagged protein under the control of a T7 promoter amp pJHS1 With T7 promoter and terminator primers, the promoter and the AtCCD8 ORF from pAT1 was amplified and subcloned into the ZraI site of pGEX 5x-3 amp pJHS2 With T7 promoter and terminator primers, the promoter and the AtCCD8 ORF from pAT1 was amplified and subcloned into the ZraI site of pBHS5 amp were washed with water, and the diethyl ether layer was retained. The AtCCD7 in carotenoid-producing strains of Escherichia coli volume of the diethyl ether was reduced under nitrogen, and a solution results in a reduced accumulation of carotenoids and the pro- of 10% KOH in methanol was added to saponify the samples. After 30 duction of some apocarotenoids (17). However, the specific re- min, the samples were partitioned into diethyl ether and washed with action catalyzed by AtCCD7 has not yet been established. It is water. The diethyl ether layer was dried under a gentle stream of demonstrated here that the recombinant AtCCD7 protein cat- nitrogen, and the samples were stored at 80 °C until HPLC analysis. alyzes a 9–10 cleavage of -carotene to produce the 10-apo-- The E. coli extracts were analyzed on a Waters 600 HPLC (Milford, MA) equipped with a Waters 996 photodiode array detector. Samples carotenal (C ) and -ionone (C ). The AtCCD8 protein is able 27 13 were injected on a 5-mC Adsorbosil column from Alltech (Deerfield, to catalyze a secondary cleavage of the 10-apo--carotenal at 18 IL) and eluted with 50% acetonitrile and water at 1 ml min for 4 min the 13–14 position to produce the 13-apo--carotenone (C ). followed by a linear gradient to 100% acetonitrile over 16 min. The gradient was then shifted to 100% acetone over 12 min and left at 100% EXPERIMENTAL PROCEDURES acetone for an additional 5 min. Cloning of AtCCD7 and AtCCD8—A cDNA clone of AtCCD7 was In Vitro Assays with Recombinant AtCCD7—For protein expression, obtained by reverse transcription-PCR with RNA isolated from 1-week- 5 ml of an overnight culture was used to inoculate a 100-ml culture in old Arabidopsis seedlings. Total RNA was reverse transcribed with 2 YT medium (per liter: 16 g of tryptone, 10 g of yeast extract, and 5 g Superscript from Invitrogen and an 18-mer oligo (dT) primer. The of NaCl). Cultures were grown at 37 °C until an A of 0.7 was reached. AtCCD7 gene was then amplified with platinum Taq polymerase from Expression of proteins was induced by the addition of 0.2 mM isopropyl Invitrogen and the primers listed in Table I. The sequence of the cloned TM -D-thiogalactopyranoside, and the cultures were grown at 28 °C for an cDNA was identical with the sequence in GenBank (NM_130064). additional 5 h. The E. coli cells were harvested by centrifugation and The AtCCD8 ORF in the pUNI51 vector was produced by the Salk/ resuspended in 4 ml of lysis buffer (40 mM Tris, pH 7.5, 20 mM NaCl, 2 Stanford/PGEC consortium (18) and obtained from the Arabidopsis mM MgCl , 100 g lysozyme, and 100 units of endonuclease). The cells stock center (stock number U19580). Subsequent subcloning of AtCCD7 were left on ice for 20 min and then frozen in liquid nitrogen. After cells and AtCCD8 for recombinant protein expression is described in Table I. were thawed, Triton X-100 was added to a final concentration of 0.25%, Expression and Analysis of AtCCD7 in Carotenoid-accumulating and the cells were shaken on ice for 30 min. The recombinant protein Strains of E. coli—Most proteins in this study were expressed with an was bound to glutathione-agarose from Sigma, washed three times with N-terminal glutathione S-transferase tag (Table I). For expression in Tris-buffered saline, and then released by cleavage with Factor Xa from carotenoid-accumulating strains of E. coli (19), 2-ml cultures were Novagen (Madison, WI) for7hat4 °C.The carotenoid substrates were grown overnight in LB medium with 100 gml ampicillin and 35 g extracted from plant tissues and purified by HPLC as described previ- ml chloramphenicol. The overnight cultures were used to inoculate a ously (20). Assays contained 0.1% Triton X-100, 0.5 mM FeSO ,5mM 30-ml culture of LB with the same antibiotics. After 24 h at 28 °C, 0.1 4 ascorbate, and the appropriate carotenoid substrate in 100 mM Tris, pH mM isopropyl -D-thiogalactopyranoside was added, and the cultures 7.0. The assay products were partitioned into ethyl acetate, dried under were left at room temperature for an additional 48 h. At the same time N , and analyzed by HPLC or thin-layer chromatography. To determine the recombinant protein was expressed, ferrous sulfate was added to a 2 the K and V values, the Michaelis-Menten equation was solved by final concentration of 10 mg liter . m max non-linear regression using Sigma Plot 4.01 from Jandel Scientific (San For quantitative analysis of carotenoid accumulation, 1 ml of culture Rafael, CA). was centrifuged, and the medium was discarded. The cell pellet was The Co-expression of AtCCD7 and AtCCD8 in Carotenoid-accumulat- resuspended in 100 l of formaldehyde, and then 1 ml of the ethanol ing Strains of E. coli—For expression of AtCCD8 or co-expression of was added. The tubes were placed at 4 °C for 3 h before the cell debris AtCCD7 and AtCCD8, the BL21 (AI) strain from Invitrogen was used, was removed by centrifugation. For -carotene- and zeaxanthin-accu- and cultures were grown as described above. For induction of the tac mulating strains of E. coli, absorbance was measured with a spectro- promoter expressing AtCCD7, isopropyl -D-thiogalactopyranoside was photometer at 453 nm. For the lycopene-accumulating strains, absorb- added to a final concentration of 0.1 mM. Induction of the AtCCD8 ance was measured at 472 nm. The carotenoid content was calculated with known extinction coefficients. protein, under the control of a T7 promoter, was achieved by the For analysis of apocarotenoid products, a culture was centrifuged, addition of 0.02% arabinose. The cells and media were extracted and and the cell pellet was extracted sequentially with formaldehyde, meth- analyzed as described above. anol, and diethyl ether. The media were partitioned into an equal Characterization of Apocarotenoid Products—The apocarotenoid volume of diethyl ether. Both the cell extract and the medium partition products were purified by reverse phase HPLC (described above), re- 46942 Biochemical Characterization of AtCCD7 and AtCCD8 FIG.2. HPLC analysis of carotenoid-accumulating strains that are expressing AtCCD7. Contour plots, which allow for a range of wavelengths to be monitored simultaneously, are presented in supple- mental Fig. 1. A, extracted chromatogram (absorbance at 310 nm) of the pACBETA strain with the -ionone peak indicated. B, extracted chro- matogram (absorbance at 400 nm) of the pACBETA strain with the 10-apo--carotenol indicated. Inset, on-line spectrum of the indicated peak. C, extracted chromatogram (absorbance at 422 nm) of pACLYC FIG.1. A, the expression of AtCCD7 or an empty vector control strain with the 10-apo-lycopenol indicated. Inset, on-line spectrum of (pGEX)in E. coli strains that accumulate lycopene (pACLYC), -caro- the indicated peak. The -carotene or lycopene peaks are indicated by tene (pACBETA), or zeaxanthin (pACZEAX). B, quantitative analysis of asterisks. carotenoid accumulation in liquid-grown cultures. were extracted and analyzed by reverse phase HPLC. Two duced with NaBH , and further purified by normal phase HPLC on an major compounds were detected in the pACBETA/AtCCD7 analytical porasil column from Waters Corporation (Milford, MA) that was equilibrated with 97:3 (hexane:ethyl acetate) at 2 ml min . The strain that were not detected in the pACBETA strain with an column was eluted with a linear gradient to 50% ethyl acetate over 12 empty vector. One product had a UV-visible spectrum and min. Fractions were collected, dried, and dissolved in hexane to deter- retention time that was identical to -ionone (C ) (Fig. 2A). mine their absorption spectra. The C product of AtCCD7, 10-apo-- The second product had a UV-visible spectrum that was carotenal, and the NaBH reduced form were also analyzed by positive consistent with the 10-apo--carotenol (C ) (Fig. 2B) (22, ion fast atom bombardment. Samples were introduced by direct inser- 23). Several smaller peaks, which had similar retention times tion probe with 3-nitrobenzyl alcohol as the matrix. The molecular ions (M and M ) for the 10-apo--carotenal and carotenol were apparent. and absorption spectra, are most likely cis isomers of the C A high-resolution spectrum for the M of the 10-apo--carotenol was product. These apocarotenoids would result from the 9–10 also obtained. The trimethylsilyl derivative of the 13-apo--carotenol cleavage of -carotene (Fig. 3). The initial cleavage product was analyzed by gas chromatography-mass spectrometry with a would be an aldehyde, but it is subsequently reduced to the DB5-MS column (30 m with an inner diameter of 0.32 mm, 0.25-m corresponding alcohol by E. coli. The reduction of the alde- film, J&W Scientific) and the following temperature program: 100 °C hyde cleavage products to alcohols by E. coli has been re- for 1 min, 100–230 °C at 40 °C/min, 230–280 °C at 8 °C/min, and 280– 300 °C at 20 °C/min. ported previously (8, 13). When AtCCD7 was expressed in a lycopene-producing strain of E. coli, a small amount of a RESULTS compound with a UV-visible spectrum similar to the10-apo- Expression of ATCCD7 in Carotenoid-accumulating Strains lycopenol was detected (Fig. 2C). No apocarotenoid products of E. coli—To determine the reactions catalyzed by AtCCD7, a were detected when AtCCD7 was expressed in a zeaxanthin- glutathione S-transferase fusion protein was expressed in sev- accumulating strain. eral carotenoid-accumulating strains of E. coli. In previous In Vitro Assays with Recombinant AtCCD7—To further ex- studies, the expression of a functional carotenoid cleavage en- plore the specificity of AtCCD7 and delimit the endogenous zyme in these strains resulted in a reduced or altered color substrates, in vitro assays were performed with the affinity- development (8–10, 13, 21). A moderate level of AtCCD7 ex- purified protein and several common plant carotenoids. These pression in a lycopene-producing strain (pACLYC) or zeaxan- assays were analyzed by thin-layer chromatography (Fig. 4) thin-producing strain (pACZEAX) had little effect on color de- and HPLC (supplemental Fig. 2). No products were detected velopment (Fig. 1, A and B). Conversely, expression of AtCCD7 with lycopene, lutein, zeaxanthin, violaxanthin, or neoxanthin in a -carotene-producing strain (pACBETA) had a significant as substrates. A single product was apparent in assays with effect on the accumulation of this carotenoid (Fig. 1, A and B). -carotene. The UV-visible spectra of the enzyme product and The cells and media from the AtCCD7 expression strains of the NaBH reduced form were very similar to published 4 Biochemical Characterization of AtCCD7 and AtCCD8 46943 FIG.3. The 9–10 cleavage of -carotene catalyzed by the re- combinant AtCCD7 protein and the subsequent reduction of the 10-apo--carotenal to the corresponding alcohol by E. coli or NaBH . FIG.4. Thin-layer chromatography analysis of assays with the recombinant AtCCD7 protein and some common plant carote- noids. Enzyme assay products were separated on a thin-layer silica plate that was developed in hexane and 2-propanol (90:10). Following chromatography, the plate was sprayed with 2,4-dinitrophenylhy- drazine to detect aldehydes and ketones. The 10-apo--carotenal is FIG.5. A–C, HPLC analysis of pACBETA strains with a construct for the expression of AtCCD8 (A), the co-expression of AtCCD7 and indicated by an arrow. HPLC analysis of in vitro assays with lycopene and -carotene are presented in supplemental Fig. 2. AtCCD8 (B), or the co-expression of -Diox II and AtCCD8 (C). Contour plots for the expression of AtCCD8 and the co-expression of AtCCD7 and AtCCD8 are presented in supplemental Fig. 1, C and D. D, the UV-visible spectra of 13-apo--carotenone (C ) and the reduced prod- spectra for the 10-apo--carotenal and the 10-apo--carotenol, uct, 13-apo--carotenol, in hexane. respectively (supplemental Fig. 2B) (23). The [M] and [M  H] ions for the 10-apo--carotenal and 10-apo--carotenol were detected by fast atom bombardment-mass spectroscopy, and a Co-expression of AtCCD7 and AtCCD8 in Carotenoid-accu- high-resolution spectrum of the reduced product matched the mulating Strains—No apocarotenoids were detected with the molecular formula for the 10-apo--carotenol, C H O. The expression of AtCCD8 in the carotenoid-accumulating strains 27 38 calculated mass of the apocarotenol is 378.29230, whereas the of E. coli (data not shown). Because mutations in AtCCD7 and experimentally determined mass of the compound was AtCCD8 result in the same phenotype, it is likely that the two 378.2928 (an error of 1.3 ppm from the calculated). gene products function in the same pathway. So, a construct for The AtCCD7 protein contains a probable chloroplast tar- the co-expression of AtCCD7 and AtCCD8 was transformed geting sequence of 31 amino acids (TargetP V1.01), which is into the carotenoid-accumulating strains. In addition to the consistent with a role in carotenoid metabolism. A truncated AtCCD7 cleavage products, -ionone and the 10-apo--carote- AtCCD7 protein (587 amino acids) lacking the N-terminal nol, a third product was identified in the -carotene co-expres- targeting sequence was also able to catalyze the 9–10 cleav- sion strain (Fig. 5A and supplemental Fig. 1D). The absorption age of -carotene (data not shown). The addition of water- spectrum of the product in hexane before and after reduction miscible organic solvents has been shown to enhance the with NaBH (Fig. 5B) is similar to the 13-apo--carotenone and activity of lignostilbene dioxygenases (24), which share se- carotenol (C ) (25). A trimethylsilyl derivative of the reduced quence similarity to the CCDs and catalyze a similar double product was analyzed by gas chromatography-mass spectrom- bond cleavage reaction. The addition of methanol to in vitro etry (Fig. 6). The observed molecular ion of 332 is the expected assays with AtCCD7 had a stimulatory effect on activity at mass for the trimethylsilyl derivative of the 13-apo--carotenol. concentrations up to 25% (data not shown). The kinetic val- An authentic standard of 13-apo--carotenone was produced ues for the standard reaction were: K  15.2 M and V  according to a synthesis described previously (26). The chroma- m max 4.5 pmol/mg of protein/min. With the addition of 25% meth- tography, UV-visible spectra, and mass spectra of the E. coli anol, the K and V values increased to 20.0 M and 10.1 product and the synthetic product were the same. m max pmol/mg of protein/min. The absence of the 13-apo--carotenone when AtCCD8 is 46944 Biochemical Characterization of AtCCD7 and AtCCD8 FIG.7. The proposed cleavage of the 10-apo--carotenal cata- lyzed by AtCCD8 to form the 13-apo--carotenone and a C dialdehyde. In plants, the cyclization of lycopene is a major branch point in carotenoid biosynthesis. The introduction of two -rings produces -carotene, which may subsequently be converted to zeaxanthin, violaxanthin, and neoxanthin. The introduction of a -ring and an -ring produces -carotene, which is converted primarily to lutein in most tissues. The lut2 mutant in Arabi- dopsis is unable to produce -carotene and lutein (30) and has FIG.6. Mass spectrum of the trimethylsilyl derivative of 13- apo--carotenol isolated from a -carotene-accumulating strain no branching phenotype. Therefore, it is unlikely that either of of E. coli that is co-expressing AtCCD7 and AtCCD8. these carotenoids is a precursor of the lateral branch inhibitor. By the same rationale, mutants impaired in epoxy-carotenoid expressed alone in the -carotene strain indicates that synthesis (20) indicate that violaxanthin and neoxanthin are ATCCD8 cleaves the C product, the 10-apo--carotenal or not the precursors of the lateral branching inhibitor. The most carotenol (Fig. 7). A C product may also result from this likely precursor of the branching inhibitor would then be zea- cleavage reaction, but it has not yet been identified. To deter- xanthin, -carotene, or an acyclic precursor. mine whether the synthesis of the 13-apo--carotenone re- The recombinant AtCCD7 protein was able to catalyze the quired a specific interaction between the AtCCD7 and AtCCD8 9–10 cleavage of -carotene to produce the 10-apo--carotenol proteins, a construct was made for the co-expression of -Diox (C ) and -ionone (C ). The AtCCD1 protein and homologs 27 13 II from mouse and AtCCD8. The -Diox II also catalyzes a 9–10 from other plants also catalyze a 9–10 cleavage reaction (8, 10). cleavage of -carotene (22), but it shares only 13% identity and There are, however, several key distinctions between the reac- 23% similarity with AtCCD7. Co-expression of -Diox II and tions catalyzed by AtCCD1 and AtCCD7. The recombinant AtCCD8 also resulted in the production of 13-apo--carotenone AtCCD1 protein cleaves various carotenoids symmetrically at (Fig. 5C). both the 9–10 and 9–10 positions. Therefore, AtCCD1 does DISCUSSION not produce the 10-apo--carotenal. The more axillary branching mutants, max3 and max4, re- Because AtCCD7 and AtCCD8 have both been implicated in sult from lesions in the AtCCD7 and AtCCD8 genes, respec- the synthesis of a lateral branching inhibitor, the two proteins tively (16, 17). Because of an inability to repress the outgrowth were co-expressed in the carotenoid-accumulating strains of E. of axillary buds, the max mutants display an increase in lateral coli. When co-expressed in the -carotene-accumulating strain, branching and have a bushy appearance. In many plants, the the C and C products of AtCCD7 were detected. A third 27 13 outgrowth of axillary buds is inhibited by the shoot apex. This product was also produced by this strain and identified as the phenomenon is often referred to as apical dominance and is 13-apo--carotenone (C ). Because this product was not de- regulated in large part by the plant hormone, auxin. Auxin is tected when AtCCD8 was expressed by itself, it most likely synthesized in the shoot apex and transported to the base of the results from a secondary cleavage of the C product of plant where it inhibits lateral bud outgrowth. If the shoot apex AtCCD7. A C product may also result from this cleavage is damaged, auxin levels are reduced and lateral buds may be reaction, but it has not yet been identified. The lignostilbene released from dormancy. There are, however, several lines of dioxygenases, which share sequence similarity with the CCDs evidence to indicate that auxin does not inhibit the outgrowth and catalyze a similar cleavage reaction, have been shown to of axillary buds directly. Grafting experiments with branching function as homodimers or heterodimers (32). Direct analysis of mutants in various species have provided strong evidence for AtCCD7 and AtCCD8 dimer formation was complicated be- the existence of another long-distance signal that inhibits the cause the majority of the recombinant AtCCD8 protein was outgrowth of axillary buds. The phenotype of several branching insoluble (data not shown). The C compound was also pro- mutants in pea (rms1, -2, and -5), the max1, 3, and 4 mutants duced when AtCCD8 was co-expressing with a 9–10 cleavage in Arabidopsis, and the decreased apical dominance mutant in enzyme from mouse (-Diox II), providing indirect evidence petunia (dad1-1) can be rescued by grafting to a wild type that dimerization is not essential for the activity of AtCCD8. rootstock (16, 27–29). These results indicate that a compound Formation of an AtCCD7-AtCCD8 heterodimer could, however, that is synthesized in the root is capable of inhibiting axillary increase the rate of the second cleavage reaction. bud outgrowth. Because the compound moves acropetally from The phenotype of the max3/atccd7 and max4/atccd8 mutants the root to the shoot, it is most likely transported through the and the biochemical evidence presented here suggest that xylem. Considering that the AtCCD7 and AtCCD8 proteins are AtCCD7 and AtCCD8 are necessary for the synthesis of an similar to carotenoid cleavage dioxygenases characterized pre- apocarotenoid that inhibits axillary bud outgrowth. The cleav- viously, it is likely that the inhibitor of axillary bud outgrowth age of -carotene by AtCCD7 to a C product is likely the first is a carotenoid-derived molecule (2, 8, 9, 10). committed step in the biosynthetic pathway of this inhibitor. Biochemical Characterization of AtCCD7 and AtCCD8 46945 15. Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T., Tabata, S., The C product may subsequently be cleaved by AtCCD8 to Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2001) Plant J. form the 13-apo--carotenone (C )andaC product. Either of 18 9 27, 325–333 16. Sorefan, K., Booker, J., Haurogne, K., Goussot, M., Bainbridge, K., Foo, E., the AtCCD8 cleavage products could give rise to the biologi- Chatfield, S., Ward, S., Beveridge, C., Rameau, C., and Leyser, O. (2003) cally active inhibitor. Grafting experiments with the max1 Genes Dev. 17, 1469–1474 mutant in Arabidopsis indicate that this mutant is also im- 17. Booker, J., Auldridge, M., Wills, S., McCarty, D., Klee, H., and Leyser, O. (2004) Curr. Biol. 14, 1232–1238 paired in the synthesis of the branching inhibitor (29). There- 18. Yamada, K., Lim, J., Dale, J. M., Chen, H., Shinn, P., Palm, C. J., Southwick, fore, it is likely that there is one additional step in the pathway. A. M., Wu, H. C., Kim, C., Nguyen, M., Pham, P., Cheuk, R., Karlin- Newmann, G., Liu, S. X., Lam, B., Sakano, H., Wu, T., Yu, G., Miranda, M., The identification of the biologically active compound will re- Quach, H. L., Tripp, M., Chang, C. H., Lee, J. 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The Biochemical Characterization of Two Carotenoid Cleavage Enzymes from Arabidopsis Indicates That a Carotenoid-derived Compound Inhibits Lateral Branching

Schwartz, Steven H.; Qin, Xiaoqiong; Loewen, Michele C.
Journal of Biological Chemistry – Nov 1, 2004
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 45, Issue of November 5, pp. 46940–46945, 2004 Printed in U.S.A. The Biochemical Characterization of Two Carotenoid Cleavage Enzymes from Arabidopsis Indicates That a Carotenoid-derived □ S Compound Inhibits Lateral Branching* Received for publication, August 6, 2004, and in revised form, August 31, 2004 Published, JBC Papers in Press, September 1, 2004, DOI 10.1074/jbc.M409004200 Steven H. Schwartz‡§, Xiaoqiong Qin‡, and Michele C. Loewen From the ‡Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 and National Research Council Plant Biotechnology Institute, Saskatoon, Saskatchewan S7N 0W9, Canada organisms. Vitamin A, for example, is required for vision and Enzymes that are able to oxidatively cleave carote- noids at specific positions have been identified in ani- development in animals. In plants, abscisic acid is necessary mals and plants. The first such enzyme to be identified for seed development and adaptation to various environmental was a nine-cis-epoxy carotenoid dioxygenase from stresses. The synthesis of these apocarotenoids and others is maize, which catalyzes the rate-limiting step of abscisic catalyzed by a class of oxygenases that cleave specific double acid biosynthesis. Similar enzymes are necessary for the bonds resulting in two products with carbonyls at the site of synthesis of vitamin A in animals and other carotenoid- cleavage. For many years it was believed that the enzymes that derived molecules in plants. In the model plant, Arabi- catalyze these reactions were dioxygenases. In a recent study, dopsis, there are nine hypothetical proteins that share it was found that oxygen in one of the products comes from some degree of sequence similarity to the nine-cis-epoxy water (1), indicating that these enzymes may be monooxygen- carotenoid dioxygenases. Five of these proteins appear ases. For simplicity, all enzymes will be referred to the name to be involved in abscisic acid biosynthesis. The remain- given when originally described. ing four proteins are expected to catalyze other carote- In plants, nine-cis-epoxy-carotenoid dioxygenases (NCEDs) noid cleavage reactions and have been named carote- catalyze the rate-limiting step in abscisic acid biosynthesis. An noid cleavage dioxygenases (CCDs). The hypothetical NCED from maize was the first carotenoid cleavage enzyme to proteins, AtCCD7 and AtCCD8, are the most disparate be cloned and characterized (2, 3). A number of NCEDs (4–7) members of this protein family in Arabidopsis. The max3 and similar enzymes that are necessary for the synthesis of and max4 mutants in Arabidopsis result from lesions in other apocarotenoids have since been identified in a variety of AtCCD7 and AtCCD8. Both mutants display a dramatic plants (8–10). The enzymes that cleave -carotene to form two increase in lateral branching and are believed to be molecules of retinal in animals also belong to this family impaired in the synthesis of an unidentified compound (11–14). that inhibits axillary meristem development. To deter- mine the biochemical function of AtCCD7, the protein Within the genome sequence of the model plant, Arabidopsis, was expressed in carotenoid-accumulating strains of there are nine hypothetical proteins that share some degree of Escherichia coli. The activity of AtCCD7 was also tested sequence similarity to the NCEDs. Five of these proteins are in vitro with several of the most common plant carote- believed to be involved in abscisic acid synthesis (15). Four noids. It was shown that the recombinant AtCCD7 pro- members of this protein family in Arabidopsis do not cluster tein catalyzes a specific 9–10 cleavage of -carotene to with previously characterized NCEDs and are considered un- produce the 10-apo--carotenal (C ) and -ionone (C ). 27 13 likely to be abscisic acid biosynthetic enzymes. These proteins When AtCCD7 and AtCCD8 were co-expressed in a -car- may, however, catalyze other carotenoid cleavage reactions and otene-producing strain of E. coli, the 13-apo--carote- are more appropriately referred to as carotenoid cleavage di- none (C ) was produced. The C product appears to 18 18 oxygenases (CCDs). The AtCCD1 protein, for example, cata- result from a secondary cleavage of the AtCCD7-derived lyzes the symmetric 9–10, 9–10 cleavage of various carote- C product. The sequential cleavages of -carotene by noids (8). The specific biochemical functions for the remaining AtCCD7 and AtCCD8 are likely the initial steps in the three CCDs from Arabidopsis (AtCCD4, -7, and -8) have not yet synthesis of a carotenoid-derived signaling molecule been reported. that is necessary for the regulation lateral branching. The max4 mutant in Arabidopsis and the rms1 mutant in pea result from a lesion in AtCCD8 and a pea ortholog (16). Both mutants display an increase in lateral branching and are Apocarotenoids are a diverse class of compounds that are believed to be impaired in the synthesis of an unknown com- derived from the oxidative cleavage of carotenoids. These com- pound that inhibits axillary meristem development or bud out- pounds serve important biological functions in a variety of growth. The max3 mutant also displays an increase in lateral branching that results from a lesion in the AtCCD7 gene (17), indicating that AtCCD7 is also involved in the synthesis of this * This work was supported by United States National Science Foun- dation Grant IBN-0115004 and United States Department of Energy inhibitor. The biochemical characterization of AtCCD7 and Grant DE-FG02-91ER20021. The costs of publication of this article AtCCD8 is an important step in identifying the biologically were defrayed in part by the payment of page charges. This article must active compound. It has been reported that the expression of therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. The abbreviations used are: NCED, nine-cis-epoxy-carotenoid di- § To whom correspondence should be addressed. Tel.: 517-353-0697; oxygenase; CCD, carotenoid cleavage dioxygenase; HPLC, high per- Fax: 517-353-9168; E-mail: [email protected]. formance liquid chromatography; ORF, open reading frame. 46940 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Biochemical Characterization of AtCCD7 and AtCCD8 46941 TABLE I Plasmids used in this study Source or Plasmid Derivation and function reference pACLYC Biosynthetic genes from Erwinia herbicola for the synthesis of lycopene chm 30 pACBETA Biosynthetic genes from E. herbicola for the synthesis of ,-carotene chm 19 pACZEAX Biosynthetic genes from E. herbicola for the synthesis of zeaxanthin chm 30 pBHS1 A full-length clone of AtCCD7 was amplified with the following primer pair: 5-ATGTCTCTCCCTATCCCGC-3 (primer 1) and 5-TCAGTCGCTAGCCCATAAAC-3 (primer 2). The fragment was subcloned into pGEM-T easy vector from Promega (Madison, WI) amp pBHS2 A truncated fragment resulting in a 31-amino acid N-terminal deletion was amplified with primer 3: 5-GCCGCA- ATATCAATATCTATACC-3 and primer 2 (above). The fragment was subcloned into pGEM-T easy vector amp pBHS3 A NotI fragment from pBHS1 was subcloned into the NotI site of pGEX 5x-3 from Amersham Biosciences. For expression of AtCCD7 protein as a glutathione S-transferase fusion protein amp pBHS4 A NotI fragment from pBHS2 was subcloned into the NotI site of pGEX 5x-3. For expression of a truncated AtCCD7 protein as a glutathione S-transferase fusion protein amp pBHS5 A cDNA for a -carotene 9–10 cleavage dioxygenase (-Diox II) from mouse was obtained from the ResGen™ clone collection (clone 2536812). The -Diox II gene was amplified with a gene-specific primer (5- ATGTTGGGACCGAAGCAAAG-3) and a vector primer (5-CGACCTGCAGCTCGAGCACA-3). The fragment was cloned into pGEM T-easy amp pBHS6 By partial digestion of pBHS5 with EcoRI, the -Diox II gene was isolated and subcloned into pGEX 5x-3 vector for protein expression as a glutathione S-transferase fusion protein amp pHB3-His6 UPS expression vector for histidine-tagged fusions (Arabidopsis stock center no. CD3–595) 31 pAT1 With Cre-lox site-specific recombination, the AtCCD8 ORF was placed into pHB3-His for expression as a histidine-tagged protein under the control of a T7 promoter amp pJHS1 With T7 promoter and terminator primers, the promoter and the AtCCD8 ORF from pAT1 was amplified and subcloned into the ZraI site of pGEX 5x-3 amp pJHS2 With T7 promoter and terminator primers, the promoter and the AtCCD8 ORF from pAT1 was amplified and subcloned into the ZraI site of pBHS5 amp were washed with water, and the diethyl ether layer was retained. The AtCCD7 in carotenoid-producing strains of Escherichia coli volume of the diethyl ether was reduced under nitrogen, and a solution results in a reduced accumulation of carotenoids and the pro- of 10% KOH in methanol was added to saponify the samples. After 30 duction of some apocarotenoids (17). However, the specific re- min, the samples were partitioned into diethyl ether and washed with action catalyzed by AtCCD7 has not yet been established. It is water. The diethyl ether layer was dried under a gentle stream of demonstrated here that the recombinant AtCCD7 protein cat- nitrogen, and the samples were stored at 80 °C until HPLC analysis. alyzes a 9–10 cleavage of -carotene to produce the 10-apo-- The E. coli extracts were analyzed on a Waters 600 HPLC (Milford, MA) equipped with a Waters 996 photodiode array detector. Samples carotenal (C ) and -ionone (C ). The AtCCD8 protein is able 27 13 were injected on a 5-mC Adsorbosil column from Alltech (Deerfield, to catalyze a secondary cleavage of the 10-apo--carotenal at 18 IL) and eluted with 50% acetonitrile and water at 1 ml min for 4 min the 13–14 position to produce the 13-apo--carotenone (C ). followed by a linear gradient to 100% acetonitrile over 16 min. The gradient was then shifted to 100% acetone over 12 min and left at 100% EXPERIMENTAL PROCEDURES acetone for an additional 5 min. Cloning of AtCCD7 and AtCCD8—A cDNA clone of AtCCD7 was In Vitro Assays with Recombinant AtCCD7—For protein expression, obtained by reverse transcription-PCR with RNA isolated from 1-week- 5 ml of an overnight culture was used to inoculate a 100-ml culture in old Arabidopsis seedlings. Total RNA was reverse transcribed with 2 YT medium (per liter: 16 g of tryptone, 10 g of yeast extract, and 5 g Superscript from Invitrogen and an 18-mer oligo (dT) primer. The of NaCl). Cultures were grown at 37 °C until an A of 0.7 was reached. AtCCD7 gene was then amplified with platinum Taq polymerase from Expression of proteins was induced by the addition of 0.2 mM isopropyl Invitrogen and the primers listed in Table I. The sequence of the cloned TM -D-thiogalactopyranoside, and the cultures were grown at 28 °C for an cDNA was identical with the sequence in GenBank (NM_130064). additional 5 h. The E. coli cells were harvested by centrifugation and The AtCCD8 ORF in the pUNI51 vector was produced by the Salk/ resuspended in 4 ml of lysis buffer (40 mM Tris, pH 7.5, 20 mM NaCl, 2 Stanford/PGEC consortium (18) and obtained from the Arabidopsis mM MgCl , 100 g lysozyme, and 100 units of endonuclease). The cells stock center (stock number U19580). Subsequent subcloning of AtCCD7 were left on ice for 20 min and then frozen in liquid nitrogen. After cells and AtCCD8 for recombinant protein expression is described in Table I. were thawed, Triton X-100 was added to a final concentration of 0.25%, Expression and Analysis of AtCCD7 in Carotenoid-accumulating and the cells were shaken on ice for 30 min. The recombinant protein Strains of E. coli—Most proteins in this study were expressed with an was bound to glutathione-agarose from Sigma, washed three times with N-terminal glutathione S-transferase tag (Table I). For expression in Tris-buffered saline, and then released by cleavage with Factor Xa from carotenoid-accumulating strains of E. coli (19), 2-ml cultures were Novagen (Madison, WI) for7hat4 °C.The carotenoid substrates were grown overnight in LB medium with 100 gml ampicillin and 35 g extracted from plant tissues and purified by HPLC as described previ- ml chloramphenicol. The overnight cultures were used to inoculate a ously (20). Assays contained 0.1% Triton X-100, 0.5 mM FeSO ,5mM 30-ml culture of LB with the same antibiotics. After 24 h at 28 °C, 0.1 4 ascorbate, and the appropriate carotenoid substrate in 100 mM Tris, pH mM isopropyl -D-thiogalactopyranoside was added, and the cultures 7.0. The assay products were partitioned into ethyl acetate, dried under were left at room temperature for an additional 48 h. At the same time N , and analyzed by HPLC or thin-layer chromatography. To determine the recombinant protein was expressed, ferrous sulfate was added to a 2 the K and V values, the Michaelis-Menten equation was solved by final concentration of 10 mg liter . m max non-linear regression using Sigma Plot 4.01 from Jandel Scientific (San For quantitative analysis of carotenoid accumulation, 1 ml of culture Rafael, CA). was centrifuged, and the medium was discarded. The cell pellet was The Co-expression of AtCCD7 and AtCCD8 in Carotenoid-accumulat- resuspended in 100 l of formaldehyde, and then 1 ml of the ethanol ing Strains of E. coli—For expression of AtCCD8 or co-expression of was added. The tubes were placed at 4 °C for 3 h before the cell debris AtCCD7 and AtCCD8, the BL21 (AI) strain from Invitrogen was used, was removed by centrifugation. For -carotene- and zeaxanthin-accu- and cultures were grown as described above. For induction of the tac mulating strains of E. coli, absorbance was measured with a spectro- promoter expressing AtCCD7, isopropyl -D-thiogalactopyranoside was photometer at 453 nm. For the lycopene-accumulating strains, absorb- added to a final concentration of 0.1 mM. Induction of the AtCCD8 ance was measured at 472 nm. The carotenoid content was calculated with known extinction coefficients. protein, under the control of a T7 promoter, was achieved by the For analysis of apocarotenoid products, a culture was centrifuged, addition of 0.02% arabinose. The cells and media were extracted and and the cell pellet was extracted sequentially with formaldehyde, meth- analyzed as described above. anol, and diethyl ether. The media were partitioned into an equal Characterization of Apocarotenoid Products—The apocarotenoid volume of diethyl ether. Both the cell extract and the medium partition products were purified by reverse phase HPLC (described above), re- 46942 Biochemical Characterization of AtCCD7 and AtCCD8 FIG.2. HPLC analysis of carotenoid-accumulating strains that are expressing AtCCD7. Contour plots, which allow for a range of wavelengths to be monitored simultaneously, are presented in supple- mental Fig. 1. A, extracted chromatogram (absorbance at 310 nm) of the pACBETA strain with the -ionone peak indicated. B, extracted chro- matogram (absorbance at 400 nm) of the pACBETA strain with the 10-apo--carotenol indicated. Inset, on-line spectrum of the indicated peak. C, extracted chromatogram (absorbance at 422 nm) of pACLYC FIG.1. A, the expression of AtCCD7 or an empty vector control strain with the 10-apo-lycopenol indicated. Inset, on-line spectrum of (pGEX)in E. coli strains that accumulate lycopene (pACLYC), -caro- the indicated peak. The -carotene or lycopene peaks are indicated by tene (pACBETA), or zeaxanthin (pACZEAX). B, quantitative analysis of asterisks. carotenoid accumulation in liquid-grown cultures. were extracted and analyzed by reverse phase HPLC. Two duced with NaBH , and further purified by normal phase HPLC on an major compounds were detected in the pACBETA/AtCCD7 analytical porasil column from Waters Corporation (Milford, MA) that was equilibrated with 97:3 (hexane:ethyl acetate) at 2 ml min . The strain that were not detected in the pACBETA strain with an column was eluted with a linear gradient to 50% ethyl acetate over 12 empty vector. One product had a UV-visible spectrum and min. Fractions were collected, dried, and dissolved in hexane to deter- retention time that was identical to -ionone (C ) (Fig. 2A). mine their absorption spectra. The C product of AtCCD7, 10-apo-- The second product had a UV-visible spectrum that was carotenal, and the NaBH reduced form were also analyzed by positive consistent with the 10-apo--carotenol (C ) (Fig. 2B) (22, ion fast atom bombardment. Samples were introduced by direct inser- 23). Several smaller peaks, which had similar retention times tion probe with 3-nitrobenzyl alcohol as the matrix. The molecular ions (M and M ) for the 10-apo--carotenal and carotenol were apparent. and absorption spectra, are most likely cis isomers of the C A high-resolution spectrum for the M of the 10-apo--carotenol was product. These apocarotenoids would result from the 9–10 also obtained. The trimethylsilyl derivative of the 13-apo--carotenol cleavage of -carotene (Fig. 3). The initial cleavage product was analyzed by gas chromatography-mass spectrometry with a would be an aldehyde, but it is subsequently reduced to the DB5-MS column (30 m with an inner diameter of 0.32 mm, 0.25-m corresponding alcohol by E. coli. The reduction of the alde- film, J&W Scientific) and the following temperature program: 100 °C hyde cleavage products to alcohols by E. coli has been re- for 1 min, 100–230 °C at 40 °C/min, 230–280 °C at 8 °C/min, and 280– 300 °C at 20 °C/min. ported previously (8, 13). When AtCCD7 was expressed in a lycopene-producing strain of E. coli, a small amount of a RESULTS compound with a UV-visible spectrum similar to the10-apo- Expression of ATCCD7 in Carotenoid-accumulating Strains lycopenol was detected (Fig. 2C). No apocarotenoid products of E. coli—To determine the reactions catalyzed by AtCCD7, a were detected when AtCCD7 was expressed in a zeaxanthin- glutathione S-transferase fusion protein was expressed in sev- accumulating strain. eral carotenoid-accumulating strains of E. coli. In previous In Vitro Assays with Recombinant AtCCD7—To further ex- studies, the expression of a functional carotenoid cleavage en- plore the specificity of AtCCD7 and delimit the endogenous zyme in these strains resulted in a reduced or altered color substrates, in vitro assays were performed with the affinity- development (8–10, 13, 21). A moderate level of AtCCD7 ex- purified protein and several common plant carotenoids. These pression in a lycopene-producing strain (pACLYC) or zeaxan- assays were analyzed by thin-layer chromatography (Fig. 4) thin-producing strain (pACZEAX) had little effect on color de- and HPLC (supplemental Fig. 2). No products were detected velopment (Fig. 1, A and B). Conversely, expression of AtCCD7 with lycopene, lutein, zeaxanthin, violaxanthin, or neoxanthin in a -carotene-producing strain (pACBETA) had a significant as substrates. A single product was apparent in assays with effect on the accumulation of this carotenoid (Fig. 1, A and B). -carotene. The UV-visible spectra of the enzyme product and The cells and media from the AtCCD7 expression strains of the NaBH reduced form were very similar to published 4 Biochemical Characterization of AtCCD7 and AtCCD8 46943 FIG.3. The 9–10 cleavage of -carotene catalyzed by the re- combinant AtCCD7 protein and the subsequent reduction of the 10-apo--carotenal to the corresponding alcohol by E. coli or NaBH . FIG.4. Thin-layer chromatography analysis of assays with the recombinant AtCCD7 protein and some common plant carote- noids. Enzyme assay products were separated on a thin-layer silica plate that was developed in hexane and 2-propanol (90:10). Following chromatography, the plate was sprayed with 2,4-dinitrophenylhy- drazine to detect aldehydes and ketones. The 10-apo--carotenal is FIG.5. A–C, HPLC analysis of pACBETA strains with a construct for the expression of AtCCD8 (A), the co-expression of AtCCD7 and indicated by an arrow. HPLC analysis of in vitro assays with lycopene and -carotene are presented in supplemental Fig. 2. AtCCD8 (B), or the co-expression of -Diox II and AtCCD8 (C). Contour plots for the expression of AtCCD8 and the co-expression of AtCCD7 and AtCCD8 are presented in supplemental Fig. 1, C and D. D, the UV-visible spectra of 13-apo--carotenone (C ) and the reduced prod- spectra for the 10-apo--carotenal and the 10-apo--carotenol, uct, 13-apo--carotenol, in hexane. respectively (supplemental Fig. 2B) (23). The [M] and [M  H] ions for the 10-apo--carotenal and 10-apo--carotenol were detected by fast atom bombardment-mass spectroscopy, and a Co-expression of AtCCD7 and AtCCD8 in Carotenoid-accu- high-resolution spectrum of the reduced product matched the mulating Strains—No apocarotenoids were detected with the molecular formula for the 10-apo--carotenol, C H O. The expression of AtCCD8 in the carotenoid-accumulating strains 27 38 calculated mass of the apocarotenol is 378.29230, whereas the of E. coli (data not shown). Because mutations in AtCCD7 and experimentally determined mass of the compound was AtCCD8 result in the same phenotype, it is likely that the two 378.2928 (an error of 1.3 ppm from the calculated). gene products function in the same pathway. So, a construct for The AtCCD7 protein contains a probable chloroplast tar- the co-expression of AtCCD7 and AtCCD8 was transformed geting sequence of 31 amino acids (TargetP V1.01), which is into the carotenoid-accumulating strains. In addition to the consistent with a role in carotenoid metabolism. A truncated AtCCD7 cleavage products, -ionone and the 10-apo--carote- AtCCD7 protein (587 amino acids) lacking the N-terminal nol, a third product was identified in the -carotene co-expres- targeting sequence was also able to catalyze the 9–10 cleav- sion strain (Fig. 5A and supplemental Fig. 1D). The absorption age of -carotene (data not shown). The addition of water- spectrum of the product in hexane before and after reduction miscible organic solvents has been shown to enhance the with NaBH (Fig. 5B) is similar to the 13-apo--carotenone and activity of lignostilbene dioxygenases (24), which share se- carotenol (C ) (25). A trimethylsilyl derivative of the reduced quence similarity to the CCDs and catalyze a similar double product was analyzed by gas chromatography-mass spectrom- bond cleavage reaction. The addition of methanol to in vitro etry (Fig. 6). The observed molecular ion of 332 is the expected assays with AtCCD7 had a stimulatory effect on activity at mass for the trimethylsilyl derivative of the 13-apo--carotenol. concentrations up to 25% (data not shown). The kinetic val- An authentic standard of 13-apo--carotenone was produced ues for the standard reaction were: K  15.2 M and V  according to a synthesis described previously (26). The chroma- m max 4.5 pmol/mg of protein/min. With the addition of 25% meth- tography, UV-visible spectra, and mass spectra of the E. coli anol, the K and V values increased to 20.0 M and 10.1 product and the synthetic product were the same. m max pmol/mg of protein/min. The absence of the 13-apo--carotenone when AtCCD8 is 46944 Biochemical Characterization of AtCCD7 and AtCCD8 FIG.7. The proposed cleavage of the 10-apo--carotenal cata- lyzed by AtCCD8 to form the 13-apo--carotenone and a C dialdehyde. In plants, the cyclization of lycopene is a major branch point in carotenoid biosynthesis. The introduction of two -rings produces -carotene, which may subsequently be converted to zeaxanthin, violaxanthin, and neoxanthin. The introduction of a -ring and an -ring produces -carotene, which is converted primarily to lutein in most tissues. The lut2 mutant in Arabi- dopsis is unable to produce -carotene and lutein (30) and has FIG.6. Mass spectrum of the trimethylsilyl derivative of 13- apo--carotenol isolated from a -carotene-accumulating strain no branching phenotype. Therefore, it is unlikely that either of of E. coli that is co-expressing AtCCD7 and AtCCD8. these carotenoids is a precursor of the lateral branch inhibitor. By the same rationale, mutants impaired in epoxy-carotenoid expressed alone in the -carotene strain indicates that synthesis (20) indicate that violaxanthin and neoxanthin are ATCCD8 cleaves the C product, the 10-apo--carotenal or not the precursors of the lateral branching inhibitor. The most carotenol (Fig. 7). A C product may also result from this likely precursor of the branching inhibitor would then be zea- cleavage reaction, but it has not yet been identified. To deter- xanthin, -carotene, or an acyclic precursor. mine whether the synthesis of the 13-apo--carotenone re- The recombinant AtCCD7 protein was able to catalyze the quired a specific interaction between the AtCCD7 and AtCCD8 9–10 cleavage of -carotene to produce the 10-apo--carotenol proteins, a construct was made for the co-expression of -Diox (C ) and -ionone (C ). The AtCCD1 protein and homologs 27 13 II from mouse and AtCCD8. The -Diox II also catalyzes a 9–10 from other plants also catalyze a 9–10 cleavage reaction (8, 10). cleavage of -carotene (22), but it shares only 13% identity and There are, however, several key distinctions between the reac- 23% similarity with AtCCD7. Co-expression of -Diox II and tions catalyzed by AtCCD1 and AtCCD7. The recombinant AtCCD8 also resulted in the production of 13-apo--carotenone AtCCD1 protein cleaves various carotenoids symmetrically at (Fig. 5C). both the 9–10 and 9–10 positions. Therefore, AtCCD1 does DISCUSSION not produce the 10-apo--carotenal. The more axillary branching mutants, max3 and max4, re- Because AtCCD7 and AtCCD8 have both been implicated in sult from lesions in the AtCCD7 and AtCCD8 genes, respec- the synthesis of a lateral branching inhibitor, the two proteins tively (16, 17). Because of an inability to repress the outgrowth were co-expressed in the carotenoid-accumulating strains of E. of axillary buds, the max mutants display an increase in lateral coli. When co-expressed in the -carotene-accumulating strain, branching and have a bushy appearance. In many plants, the the C and C products of AtCCD7 were detected. A third 27 13 outgrowth of axillary buds is inhibited by the shoot apex. This product was also produced by this strain and identified as the phenomenon is often referred to as apical dominance and is 13-apo--carotenone (C ). Because this product was not de- regulated in large part by the plant hormone, auxin. Auxin is tected when AtCCD8 was expressed by itself, it most likely synthesized in the shoot apex and transported to the base of the results from a secondary cleavage of the C product of plant where it inhibits lateral bud outgrowth. If the shoot apex AtCCD7. A C product may also result from this cleavage is damaged, auxin levels are reduced and lateral buds may be reaction, but it has not yet been identified. The lignostilbene released from dormancy. There are, however, several lines of dioxygenases, which share sequence similarity with the CCDs evidence to indicate that auxin does not inhibit the outgrowth and catalyze a similar cleavage reaction, have been shown to of axillary buds directly. Grafting experiments with branching function as homodimers or heterodimers (32). Direct analysis of mutants in various species have provided strong evidence for AtCCD7 and AtCCD8 dimer formation was complicated be- the existence of another long-distance signal that inhibits the cause the majority of the recombinant AtCCD8 protein was outgrowth of axillary buds. The phenotype of several branching insoluble (data not shown). The C compound was also pro- mutants in pea (rms1, -2, and -5), the max1, 3, and 4 mutants duced when AtCCD8 was co-expressing with a 9–10 cleavage in Arabidopsis, and the decreased apical dominance mutant in enzyme from mouse (-Diox II), providing indirect evidence petunia (dad1-1) can be rescued by grafting to a wild type that dimerization is not essential for the activity of AtCCD8. rootstock (16, 27–29). These results indicate that a compound Formation of an AtCCD7-AtCCD8 heterodimer could, however, that is synthesized in the root is capable of inhibiting axillary increase the rate of the second cleavage reaction. bud outgrowth. Because the compound moves acropetally from The phenotype of the max3/atccd7 and max4/atccd8 mutants the root to the shoot, it is most likely transported through the and the biochemical evidence presented here suggest that xylem. Considering that the AtCCD7 and AtCCD8 proteins are AtCCD7 and AtCCD8 are necessary for the synthesis of an similar to carotenoid cleavage dioxygenases characterized pre- apocarotenoid that inhibits axillary bud outgrowth. The cleav- viously, it is likely that the inhibitor of axillary bud outgrowth age of -carotene by AtCCD7 to a C product is likely the first is a carotenoid-derived molecule (2, 8, 9, 10). committed step in the biosynthetic pathway of this inhibitor. Biochemical Characterization of AtCCD7 and AtCCD8 46945 15. Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T., Tabata, S., The C product may subsequently be cleaved by AtCCD8 to Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2001) Plant J. form the 13-apo--carotenone (C )andaC product. Either of 18 9 27, 325–333 16. Sorefan, K., Booker, J., Haurogne, K., Goussot, M., Bainbridge, K., Foo, E., the AtCCD8 cleavage products could give rise to the biologi- Chatfield, S., Ward, S., Beveridge, C., Rameau, C., and Leyser, O. (2003) cally active inhibitor. Grafting experiments with the max1 Genes Dev. 17, 1469–1474 mutant in Arabidopsis indicate that this mutant is also im- 17. Booker, J., Auldridge, M., Wills, S., McCarty, D., Klee, H., and Leyser, O. (2004) Curr. Biol. 14, 1232–1238 paired in the synthesis of the branching inhibitor (29). There- 18. Yamada, K., Lim, J., Dale, J. M., Chen, H., Shinn, P., Palm, C. J., Southwick, fore, it is likely that there is one additional step in the pathway. A. M., Wu, H. C., Kim, C., Nguyen, M., Pham, P., Cheuk, R., Karlin- Newmann, G., Liu, S. X., Lam, B., Sakano, H., Wu, T., Yu, G., Miranda, M., The identification of the biologically active compound will re- Quach, H. L., Tripp, M., Chang, C. H., Lee, J. 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Published: Nov 1, 2004

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