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Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple are CYP736A Proteins

Biphenyl 4-Hydroxylases Involved in Aucuparin Biosynthesis in Rowan and Apple are CYP736A Proteins
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  Biphenyl 4-Hydroxylases Involved in AucuparinBiosynthesis in Rowan and Apple Are CytochromeP450 736A Proteins 1[OPEN] Debabrata Sircar 2,3  , Mariam M. Gaid 2  , Cornelia Chizzali, Dennis Reckwell, David Kaufholdt, Till Beuerle,Giovanni A.L. Broggini, Henryk Flachowsky, Benye Liu, Robert Hänsch, and Ludger Beerhues* Institute of Pharmaceutical Biology (D.S., M.M.G., D.R., T.B., B.L., L.B.) and Institute of Plant Biology (D.K.,R.H.), Technische Universität Braunschweig, 38106 Braunschweig, Germany; Plant Pathology, Institute of Integrative Biology, Eidgenössische Technische Hochschule Zürich, 8092 Zurich, Switzerland (C.C., G.A.L.B.);and Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Breeding Research onHorticultural and Fruit Crops, 01326 Dresden, Germany (H.F.) ORCID IDs: 0000-0002-2788-119X (D.S.); 0000-0002-7290-0628 (M.M.G.); 0000-0001-9337-4527 (C.C.); 0000-0001-5449-0170 (D.R.); 0000-0001- 5037-6653 (T.B.); 0000-0001-8527-565X (B.L.); 0000-0002-2147-2731 (L.B.). Upon pathogen attack, fruit trees such as apple (  Malus  spp.) and pear ( Pyrus  spp.) accumulate biphenyl and dibenzofuranphytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenylsynthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan ( Sorbusaucuparia ) was isolated and functionally expressed in yeast ( Saccharomyces cerevisiae ). SaB4H was named CYP736A107. Nocatalytic function of CYP736 was known previously. SaB4H exhibited absolute speci fi city for 3-hydroxy-5-methoxybiphenyl.In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanineammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter geneconstruct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembranedomain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (  Malus  3 domestica ) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the  fi re blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hswere closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehydeand related compounds were not converted by SaB4H. Rosaceous species include a number of valuable fruittrees, which are widespread in the temperate regions of the world. Economically important fruits are the pomefruits, such as apple (  Malus  spp.) and pear ( Pyrus  spp.),the stone fruits, including cherry, peach, plum, apricot,and almond (all  Prunus  spp.), and the berry fruits,such as strawberry ( Fragaria  spp.) and blackberry andraspberry (both  Rubus  spp.). All these fruits are widelyconsumed for their tastes, nutritional values, and con-tents of health-promoting metabolites. In addition, theRosaceae include a number of widespread ornamentalspecies. A large variety of rose ( Rosa  spp.) cultivars were bred, and hawthorn ( Crataegus  spp.) and rowan ( Sorbus spp.) are common as ornamental trees.Over the centuries, cultivars with favorable proper-ties were selected; however, vegetative propagation bygrafting led to genetic uniformity and decreased re-sistance to pests and diseases (Norelli et al., 2003;Gessler and Patocchi, 2007). The defense potential of plants consists of multiple strategies, one of which isthe accumulation of phytoalexins (Dixon, 2001). Plantshave elaborated an amazing array of more than200,000 natural products, many of which confer se-lective advantage against herbivore and pathogen at-tacks (Dixon, 2001; Hartmann, 2007). Two closelyrelated classes of phytoalexins are biphenyls and di- benzofurans, which are formed by apple, pear, andrelated fruit trees. This group of pome-bearing specieshas long been known as Rosaceae subfamily Maloi-deae but recently was reclassi fi ed as the subtribeMalinae of the subfamily Amygdaloideae (temporarily 1 This work was supported by the Deutsche Forschungsgemein-schaft (grant no. BE 1174/9 – 2). 2 These authors contributed equally to the article. 3 Present address: Biotechnology Department, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India.* Address correspondence to author responsible for distribution of materials integral to the fi ndings presented in this article in accordance with the policy de-scribed in the Instructions for Authors ( is:Ludger Beerhues (, M.M.G., R.H., and L.B. designed the research; D.S., M.M.G.,C.C., D.R., D.K., T.B., H.F., B.L., and R.H. performed the research;D.S., M.M.G., C.C., T.B., G.A.L.B., R.H., and L.B. analyzed the data;D.S., M.M.G., and L.B. wrote the article. [OPEN] Articles can be viewed without a 428  Plant Physiology  ,  June 2015, Vol. 168, pp. 428 – 442,    2015 American Society of Plant Biologists. All Rights Reserved.  www.plant.orgon June 8, 2015 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2015 American Society of Plant Biologists. All rights reserved.  Pyrinae of the Spiraeoideae; Campbell et al., 2007;Potter et al., 2007).Two decades ago, Kokubun and Harborne (1995)identi fi ed biphenyls and dibenzofurans as the phyto-alexins of the Malinae. So far, 10 biphenyls and 17dibenzofurans have been detected, which are formedde novo in response to biotic and abiotic stress (ChizzaliandBeerhues,2012).Theoccurrenceofpathogen-induced biphenyl and dibenzofuran compounds is con fi ned tothe Malinae (Kokubun and Harborne, 1995). However,some taxa other than the Malinae contain biphenylsand dibenzofurans as preformed constituents, whichcontribute to the constitutive defense barrier (Hüttneret al., 2010). The majority of the biphenyl and dibenzo-furan phytoalexins were found in response to fungalattack, either natural infection or arti fi cial inoculation(Chizzali and Beerhues, 2012). A single article reports theaccumulation of biphenyls and dibenzofurans after bac-terial challenge (Chizzali et al., 2012b). The phytoalexinsinhibit spore germination, germ tube development, andmycelial growth as well as bacterial propagation, al-though the underlying mechanisms of antimicrobial ac-tion are still unknown (Chizzali and Beerhues, 2012).The most common biphenyl phytoalexin is aucu-parin, which was detected in eight Malinae speciesafter pathogen attack (Chizzali and Beerhues, 2012).The scaffold of this compound is formed by biphenylsynthase (BIS), a type III polyketide synthase, whichcatalyzes the condensation of the unusual starter sub-strate benzoyl-CoA with three molecules of malonyl-CoA to yield 3,5-dihydroxybiphenyl (Fig. 1; Liu et al.,2004, 2007). The benzoyl moiety of the starter substratestems from  L -Phe, whose carbon skeleton is channeledinto the phytoalexin biosynthetic pathway by the ac-tion of phenylalanine ammonia lyase (PAL; Hansonand Havir, 1981). Little is known about the conversionof the PAL product, trans-cinnamic acid, to benzoyl-CoA in the Malinae. Benzaldehyde dehydrogenasewas detected in chitosan-treated rowan ( Sorbus aucu- paria ) cell cultures, which serve as an in vitro systemfor studying biphenyl and dibenzofuran metabolism inthe Malinae (Gaid et al., 2009; Hüttner et al., 2010). The fi rst complementary DNA (cDNA) encoding BIS wasisolated from these cell cultures after elicitor treatment(Liu et al., 2007). The elicitors that ef  fi ciently inducephytoalexin formation in rowan cell cultures includepreparations from the fungus  Venturia inaequalis , thecausal agent of scab, and the bacterium  Erwinia amy-lovora , the causal agent of   fi re blight (Hüttner et al.,2010). These infections are among the most devastatingdiseases encountered in apple orchards (MacHardy,1996; Vanneste, 2000). Recently, the downstream en-zymes that metabolize the BIS product, 3,5-dihydrox-ybiphenyl, have been studied in cell-free extracts andmicrosomes from rowan cell cultures after the additionof an aqueous elicitor preparation from  V. inaequalis (Khalil et al., 2013). Interestingly, the sequence of the biosynthetic reactions leading from 3,5-dihydroxybiphenylto aucuparin was  O -methylation → 4-hydroxylation → O -methylation(Fig.1).Thealkylationstepswerecatalyzed by two distinct  O -methyltransferases, and the hydro-xylation reaction was catalyzed by a cytochrome P450(CYP), which was named biphenyl 4-hydroxylase (B4H;Khalil et al., 2013).Here, we report the isolation of the SaB4H codingsequence using a subtracted cDNA library of rowanand the recently published genome sequence of apple(  Malus  3  domestica  ‘ Golden Delicious ’ ). In addition,the MdB4H coding sequences of cv Golden Deliciousand cv Holsteiner Cox were cloned. The expression of  SaB4H   and  MdB4H   was analyzed in rowan cell culturesafter treatment with elicitor from the scab fungus and inapple stems after infection with the  fi re blight bacte-rium, respectively. Besides functional and phylogeneticanalyses, the subcellular localization of SaB4H reporterfusions was studied in  Nicotiana benthamiana . RESULTScDNA Cloning of SaB4H Starting with a SubtractedRowan cDNA Library Pools of mRNA were isolated from chitosan-treatedand nontreated rowan cell cultures and used to construct Figure 1.  Aucuparin biosynthesis in  S. aucuparia  cell cultures treatedwith  V. inaequalis   (Khalil et al., 2013). Plant Physiol. Vol. 168, 2015 429 Biphenyl 4-Hydroxylases Are CYP736A Proteins  www.plant.orgon June 8, 2015 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2015 American Society of Plant Biologists. All rights reserved.  a suppression subtraction hybridization (SSH) cDNA li- brary, which consisted of differentially expressed tran-scripts. A total of 2,000 cDNA clones were sequencedand bioinformatically processed (Supplemental Fig. S1),leading to the detection of 15 CYP contigs. The individ-ual sequences were used to search the apple genomesequence, which is available via the Rosaceae GenomeDatabase (GDR). Apple sequences sharing a high degreeof similarity with the rowan contigs were  fi ltered againstCYP sequences in the data bank related to the mod-i fi cation of products of the shikimate pathway. Thesein silico analyses, details of which are listed inSupplemental Table S1, resulted in the selection of sixapple unigenes that shared high sequence identity(87.5% – 97%) with the rowan contigs 62, 120, and 162.Gene-speci fi c primers were derived and led to theampli fi cation of a transcript from  V. inaequalis -treatedrowan cell cultures. After 5 9  and 3 9  extensions, theresulting full-length cDNA was reampli fi ed using aproofreading polymerase. The cDNA consisted of a1,500-bp coding sequence (cds), a 188-bp 5 9  untrans-lated region (UTR), a 260-bp 3 9 UTR, and a 32-bp poly(A) tail.The cds encoded a 499-amino acid protein with apredicted molecular mass of 56.9 kD and a pI of 6.82.The protein was named CYP736A107, based on thestandardized CYP nomenclature system (Nelson, 2009).Its structural analysis revealed the presence of motifsthat are characteristic of plant CYPs (Fig. 2). The highlyconserved heme-binding domain FxxGxRxCxG (Chapple,1998) was present as FGSGRRRCAG. The N-terminalmembrane anchor comprising 19 amino acids wasfollowed by the Pro-rich region PPGPRGFP [consensus(P/I)PGPx(G/P)xP; Schalk et al., 1999]. In addition, theKETLR(K) helix and the PERFE motif were present (Baket al., 2011). The amino acid sequence of CYP736A107shared 39.6% and 40.1% identities with functionallyestablished  fl avonoid 3 9 -hydroxylases from  Epimediumsagittatum  (ADE80941) and  Brassica napus  (ABC58722),respectively. cDNA Cloning of Class II CPR from  V. inaequalis -TreatedRowan Cell Cultures NADPH-cytochrome P450 reductase (CPR) is theelectron-transfer partner for CYPs (Jensen and Møller,2010). The apple unigene MDP0000167955 shared highsequence similarity with  CPR  genes in the data bankand the only CPR transcript fragment present in theSSH cDNA library. Using gene-speci fi c primers and aproofreading polymerase, an SaCPR transcript wascloned from  V. inaequalis -treated rowan cell cultures.The SaCPR cDNA contained a 2,139-bp cds encoding a712-amino acid polypeptide with a predicted molecu-lar mass of 78.8 kD. The protein shared 81% sequenceidentity each with  Gossypium hirsutum  CPR (ACN54324)andpoplar( Populus trichocarpa )CPR3(AAK15261)aswellas 75.5% and 65.5% identities with Arabidopsis (  Arabi-dopsis thaliana ) CPR2 (AAK17169) and CPR1 (CAA46814),respectively.Theconservedbindingdomainsforcofactorsand substrates (FMN, FAD, NADPH, and CYP) and themembrane anchor near the N terminus were present(Supplemental Fig. S2). The extended N-terminal se-quence upstream of the membrane anchor identi fi edthe rowan enzyme as a class II CPR (Ro et al., 2002). Functional Characterization of CYP736A107 The CYP736A107 cds and the CPR cds were clonedin the same expression vector and heterologouslyexpressed in yeast ( Saccharomyces cerevisiae ), from Figure 2.  Alignment of B4H and con-iferaldehyde 5-hydroxylase (CA5H) aminoacid sequences with common motifs high-lighted. SaB4H, B4H from  S. aucuparia (CYP736A107); Md(GD)B4H, B4H fromcv Golden Delicious (CYP736A163); Md(HC)B4H, B4H from cv Holsteiner Cox(CYP736A163); CmCA5H, CA5H from Chrysanthemum  3 morifolium   (BAM66576).Gaps introduced to maximize the alignmentare indicated by dashes. 430 Plant Physiol. Vol. 168, 2015 Sircar et al.  www.plant.orgon June 8, 2015 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2015 American Society of Plant Biologists. All rights reserved.  which microsomes containing the two proteins wereisolated. Based on the carbon monoxide differencespectrum (Supplemental Fig. S3), the CYP expressionlevel was 286 nmol g 2 1 microsomal protein. Microsomeswere incubated with 3-hydroxy-5-methoxybiphenyl,which was recently found as a B4H substrate usingmicrosomes from elicitor-treated rowan cell cul-tures (Khalil et al., 2013). In the presence of NADPH,3-hydroxy-5-methoxybiphenyl was 4-hydroxylatedto yield noraucuparin, as demonstrated by gaschromatography-mass spectrometry (GC-MS) in com-parison with an authentic reference compound (Fig. 3).In addition, identical retention time values and UVspectra were observed in HPLC-diode array detection(DAD) analysis (Supplemental Fig. S4). No productformation was found in assays that contained heat-denatured protein or microsomes from yeast cellsharboring the empty vector. Nor was CYP736A107activity detected when the rowan CPR cds was omittedfrom the vector, indicating the lack of interactionwith yeast CPR. Substrates structurally related to3-hydroxy-5-methoxybiphenyl were converted neitherunder standard assay conditions nor at varying pHvalues (5.5 – 10.5), except for 3,5-dihydroxybiphenyl.However, the relative activity was less than 2%, leadingto the formation of only trace amounts of 3,4,5-trihydroxybiphenyl (Table I). Notably, 2-hydroxy-4-methoxydibenzofuran, which has the same substitutionpattern as 3-hydroxy-5-methoxybiphenyl, was not con-verted. Furthermore, stilbenes and one-ring compoundswere not substrates. Nor did the enzyme accept cinnamicacid derivatives, such as ferulic acid and coniferaldehyde.Thus, CYP736A107 was identi fi ed as SaB4H.Enzyme activity was strictly dependent on NADPH,whose replacement with NADH reduced the activityto approximately 15%. In the presence of both NADPHand NADH, the activity was increased by approxi-mately 15%. The pH and temperature optima were 7.5and 30°C, respectively. B4H activity was linear withtime up to 45 min and with increasing CYP concen-trations in the standard assay up to 15 pmol, as de-termined by carbon monoxide difference spectroscopy.The  K  m  values for 3-hydroxy-5-methoxybiphenyl andNADPH were 2.4  6  0.07 and 72.3  6  7.5  m M , respec-tively (Table II). Electron transfer from NADPH wasexclusively due to rowan CPR, because yeast CPRalone failed to accomplish detectable SaB4H acti-vity. The turnover number ( k  cat ) and the catalytic ef- fi ciency ( k  cat / K  m ) with 3-hydroxy-5-methoxybiphenylwere 0.342 min 2 1 and 2,375  M 2 1 s 2 1 , respectively(Table II). When SaCPR was incubated with cyto-chrome  c  as an arti fi cial electron acceptor (Urbanet al., 1994), the  K  m  value for NADPH was 51  6  5.4 m M . No appreciable loss of SaB4H activity was ob-served upon storage of microsomal preparations inTris-EDTA-glycerol buffer at 2 80°C for 6 months. At4°C, the enzyme activity decreased to about 20% to30% within 24 h. Expression of  SaPAL  ,  SaBIS1  , and  SaB4H   in V. inaequalis -Treated Rowan Cell Cultures Transcript levels in rowan cell cultures after theaddition of an aqueous extract from the scab-causing fungus were analyzed by reverse transcription Figure 3.  GC-MS analysis of SaB4H assays. The enzymic product (noraucuparin) was analyzed as a trimethylsilyl derivativeresulting from  N  -methyl- N  -(trimethylsilyl) trifluoroacetamide treatment. The control incubation contained heat-denaturedmicrosomes. Plant Physiol. Vol. 168, 2015 431 Biphenyl 4-Hydroxylases Are CYP736A Proteins  www.plant.orgon June 8, 2015 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2015 American Society of Plant Biologists. All rights reserved.  (RT)-PCR (Fig. 4). The gene-speci fi c primer pairs usedled to the ampli fi cation of 407-, 882-, 379-, and 300-bpfragments of SaPAL, SaBIS1, SaB4H, and SaActintranscripts, respectively. Actin mRNA served as acontrol for equal template amounts. SaB4H transcriptswere detectable as early as 0.5 h after the onset of elic-itation. The mRNA level peaked at 4 h and decreasedslowly thereafter. Similar changes were found for theSaBIS1 transcript level, which, however, increased moreslowly before reaching the maximum at 4 h. A signi fi -cantly faster increase was observed for the SaPALmRNA level, which already peaked after 1 to 2 h andreturned rapidly to the basal  SaPAL  expression levelthat was present at the onset of elicitation. Table I.  Substrate specificity of SaB4H  Substrate Formula Activity Substrate Formula Activity % %  3-Hydroxy-5-methoxybiphenyl 100 Resorcinol 03,5-Dihydroxy-biphenyl  , 2 Orcinol monomethyl ether 03,5-Dimethoxy-biphenyl 0 Orcinol 02’,3,5-Trihydroxy-biphenyl 0 3,5-Dihydroxy-benzoic acid 02,5-Dihydroxy-biphenyl 0 Benzoic acid 02-Hydroxy-4-methoxydibenzofuran 0 Ferulic acid02,4-Dihydroxy-dibenzofuran 0 Coniferaldehyde 0Pinosylvin 0 4-Coumaric acid 0Resorcinol monomethyl ether 0 Cinnamic acid 0 432 Plant Physiol. Vol. 168, 2015 Sircar et al.  www.plant.orgon June 8, 2015 - Published by www.plantphysiol.orgDownloaded from  Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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