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CYP79D enzymes contribute to jasmonic acid-induced formation of aldoximes and other nitrogenous volatiles… Luck, Katrin; Jirschitzka, Jan; Irmisch, Sandra; Huber, Meret; Gershenzon, Jonathan; Köllner, Tobias G Oct 4, 2016

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RESEARCH ARTICLE Open AccessCYP79D enzymes contribute to jasmonicacid-induced formation of aldoximes andother nitrogenous volatiles in twoErythroxylum speciesKatrin Luck1, Jan Jirschitzka1,2, Sandra Irmisch1,3, Meret Huber1, Jonathan Gershenzon1 and Tobias G. Köllner1*AbstractBackground: Amino acid-derived aldoximes and nitriles play important roles in plant defence. They are well-knownas precursors for constitutive defence compounds such as cyanogenic glucosides and glucosinolates, but are alsoreleased as volatiles after insect feeding. Cytochrome P450 monooxygenases (CYP) of the CYP79 family catalyze theformation of aldoximes from the corresponding amino acids. However, the majority of CYP79s characterized so farare involved in cyanogenic glucoside or glucosinolate biosynthesis and only a few have been reported to beresponsible for nitrogenous volatile production.Results: In this study we analysed and compared the jasmonic acid-induced volatile blends of two Erythroxylum species,the cultivated South American crop species E. coca and the African wild species E. fischeri. Both species produced differentnitrogenous compounds including aliphatic aldoximes and an aromatic nitrile. Four isolated CYP79 genes (two from eachspecies) were heterologously expressed in yeast and biochemically characterized. CYP79D62 from E. coca and CYP79D61and CYP79D60 from E. fischeri showed broad substrate specificity in vitro and converted L-phenylalanine, L-isoleucine,L-leucine, L-tryptophan, and L-tyrosine into the respective aldoximes. In contrast, recombinant CYP79D63 from E. cocaexclusively accepted L-tryptophan as substrate. Quantitative real-time PCR revealed that CYP79D60, CYP79D61, andCYP79D62 were significantly upregulated in jasmonic acid-treated Erythroxylum leaves.Conclusions: The kinetic parameters of the enzymes expressed in vitro coupled with the expression patterns of thecorresponding genes and the accumulation and emission of (E/Z)-phenylacetaldoxime, (E/Z)-indole-3-acetaldoxime,(E/Z)-3-methylbutyraldoxime, and (E/Z)-2-methylbutyraldoxime in jasmonic acid-treated leaves suggest that CYP79D60,CYP79D61, and CYP79D62 accept L-phenylalanine, L-leucine, L-isoleucine, and L-tryptophan as substrates in vivo andcontribute to the production of volatile and semi-volatile nitrogenous defence compounds in E. coca and E. fischeri.Keywords: Erythroxylum, Cytochrome P450 monooxygenase, CYP79, Aldoxime, VolatilesBackgroundPlant volatiles play diverse roles in the interactionsbetween plants and their environment. Flower volatiles,for example, can attract pollinators while vegetative vola-tiles are involved in plant defence, either directly by repel-ling the attacker or indirectly by e.g. attracting herbivoreenemies [1–5]. The formation and emission of vegetativevolatiles is often induced by chewing or sucking herbi-vores and the resulting volatile blends usually containdozens of substances from diverse classes of natural com-pounds [6–9]. Herbivore-induced volatile blends are ingeneral dominated by terpenes and green leaf volatiles(GLVs, C6 aldehydes, alcohols and esters derived fromfatty acid cleavage), but comprise also aromatic com-pounds, alcohols, and nitrogen-containing amino acidderivatives [10]. While the formation and biological rolesof terpenes and GLVs have been extensively studied in the* Correspondence: koellner@ice.mpg.de1Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745Jena, GermanyFull list of author information is available at the end of the article© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Luck et al. BMC Plant Biology  (2016) 16:215 DOI 10.1186/s12870-016-0910-5past, our knowledge about the other components ofherbivore-induced volatile blends is still limited.Nitrogen-containing vegetative volatiles such as aldox-imes, nitriles and nitro compounds are widely distrib-uted among the angiosperms and have been reportedfrom e.g. the Salicaceae, Fabaceae, Solanaceae, Cucurbi-taceae, Rutaceae, Rosaceae, and Poaceae [11]. Poplars(Salicaceae), for example, release a complex mixture ofaliphatic aldoximes, aliphatic and aromatic nitriles, andan aromatic nitro compound in response to herbivory bygypsy moth (Lymantria dispar) larvae [7, 11, 12].Although these nitrogen-containing volatiles are minorcomponents of the total blend, they likely play im-portant roles in indirect and direct poplar defence.Electrophysiological recordings and olfactometer bio-assays revealed that volatile aldoximes were more at-tractive for a gypsy moth parasitoid than the majorterpenes and GLVs [12]. Moreover, poplar nitrileswere shown to be repellent for gypsy moth caterpil-lars, while volatile and semi-volatile aldoximes hadtoxic effects on these larvae [11, 13].Aldoximes and nitriles are produced from amino acidsthrough the action of cytochrome P450 monooxygenases(CYP) of the CYP79 and CYP71/736 families (recentlyreviewed in [14]). CYP79 enzymes accept amino acids assubstrates and catalyse the formation of aldoximes bytwo successive N-hydroxylations, a dehydration and adecarboxylation reaction [15, 16]. The aldoximes formedcan then serve as substrates for CYP71 enzymes, whichconvert them into the corresponding nitriles [13]. Thefirst characterized CYP79 enzyme, CYP79A1 from Sor-ghum bicolor, was identified and characterized in 1995by Sibbesen and co-workers [16]. It catalyses the reac-tion from L-tyrosine to p-hydroxyphenylacetaldoxime,which is further converted into the cyanogenic glucosidedhurrin in sorghum [16]. While most of the CYP79enzymes characterized so far produce aldoximes asprecursors for cyanogenic glucosides, glucosinolates, andother non-volatile nitrogen-containing defence com-pounds, a few CYP79s from two different poplar species(CYP79D6v3 and CYP79D7v2 from Populus trichocarpaand CYP79D6v4 from P. nigra) have been reported to beresponsible for herbivore-induced volatile production[11, 12, 17]. CYP79s involved in cyanogenic glucosideand glucosinolate formation usually possess high sub-strate specificity, thus determining the specificity of theentire pathway [18–21]. In contrast, poplar CYP79D6and CYP79D7 have broader substrate specificity andproduce complex mixtures of volatile and semi-volatilealdoximes [11, 12, 17].To expand our knowledge about the formation ofvolatile aldoximes and nitriles, we have now begun to in-vestigate and compare their biosynthesis in the genusErythroxylum. Two species with different geographicalorigins and cultivation histories were chosen for thisanalysis. Erythroxylum coca is an economically andpharmacologically important crop cultivated on the east-ern slopes of the Andes since more than 8000 years. E.fischerii, in contrast, is a wild species native to the trop-ical forests in Africa. Both species are members of theErythroxylaceae, which belong, like poplars, to the di-verse order Malpighiales. Since it has been shown thatthe formation of volatiles can be induced by artificialtreatments with the plant hormone jasmonic acid (JA)(e.g. [12, 22]), we measured and compared volatile emis-sion in response to JA treatment in E. coca and E.fischeri and detected numerous nitrogen-containingcompounds. Candidate CYP79 genes isolated from bothspecies were then heterologously expressed in yeast, andenzyme characterization and gene expression analysis in-dicated a potential function of individual ErythroxylumCYP79 proteins in volatile aldoxime formation.ResultsJasmonic acid induces the emission of nitrogenousvolatiles in Erythroxylum coca and E. fischeriMany plant species respond to herbivory with an in-creased JA accumulation that induces the biosynthesis ofdiverse plant defence compounds including nitrogen-containing volatiles [23]. Hence to study the formation ofnitrogenous volatiles in Erythroxylum species, we collectedand compared the volatile blends of untreated and JA-treated twigs of E. coca and E. fischeri. Although both spe-cies emitted volatiles from untreated twigs, JA-treatmentsignificantly increased volatile emission (Table 1). Theblends from control and JA-treated twigs of E. coca and E.fischeri were dominated by monoterpenes (e.g. (E)-β-oci-mene, mentha-1,5,8-triene, and linalool), sesquiterpenes(e.g. β-elemene and (E,E)-α-farnesene) and the homoter-penes (3E)-4,8-dimethylnona-1,3,7-triene (DMNT) and(3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT).In addition, both species produced significant amounts ofnitrogenous volatiles such as (E/Z)-2-methylbutyraldoxime,(E/Z)-3-methylbutyraldoxime, benzyl cyanide, phenylni-troethane, an unidentified nitro compound, and indole inresponse to JA treatment (Table 1). As typical herbivore-induced vegetative volatiles, green leaf volatiles were alsopresent in JA-induced E. coca and E. fischeri blends. Not-ably, the qualitative compositions of the JA-induced volatileblends of both species were nearly identical and the totalamounts of released volatiles were in the same range. How-ever, there were major quantitative differences in the emis-sion of single volatiles between E. coca and E. fischeri(Table 1). While E. coca, for instance, emitted β-elemene asmajor sesquiterpene and produced minor amounts of(E,E)-α-farnesene, E. fischerii released large amounts of(E,E)-α-farnesene and produced only traces of β-elemene.Another remarkable difference was found for indole, whichLuck et al. BMC Plant Biology  (2016) 16:215 Page 2 of 15Table 1 Volatile compounds of Erythroxylum coca and E. fischeri released from untreated twigs (control) and jasmonic acid-treatedtwigs (JA treatment)Erythroxylum coca Erythroxylum fischeriCompound Control JA treatment P-value Control JA treatment P-value(Mean ± SE) (Mean ± SE) (Mean ± SE) (Mean ± SE)N-containing volatiles(E)-3-methylbutyraldoxime* 0.6 ± 0.4 40.1 ± 9.6 0.020 0 + 0 23.7 + 7.4 0.037(E)-2-methylbutyraldoxime* 0.4 ± 0.4 51.5 ± 9.7 0.018 0 + 0 16.1 + 10.6 0.037(Z)-2-methylbutyraldoxime* 0.2 ± 0.2 9.5 ± 2.2 0.018 0 + 0 4.4 + 1.3 0.037(Z)-3-methylbutyraldoxime* 0 ± 0 29.7 ± 6.6 0.014 0 + 0 17.1 + 7.2 0.037benzyl cyanide* 0.1 ± 0.1 315.6 ± 76.5 0.018 2.1 + 1.2 241 + 199.7 0.050phenylnitroethane 3.7 ± 0.7 99 ± 26.4 0.021 7.7 + 4.6 62.4 + 39.8 0.050unidentified nitro compound 0.1 ± 0.1 3.1 ± 0.9 0.018 0.8 + 0.8 21 + 11.8 0.046indole* 4.5 ± 2 256.2 ± 50.3 0.021 2.1 + 1 13.2 + 2.3 0.050monoterpenoidsmyrcene* 4.7 ± 0.8 29.2 ± 3.5 0.021 5.7 + 1.8 36.1 + 4.7 0.050(Z)-ocimene* 4.2 ± 0.5 107.7 ± 19.9 0.021 4.2 + 1.5 83.4 + 9.8 0.050(E)-ocimene* 289.1 ± 54.1 14685.7 ± 4017.2 0.021 281.3 + 105.8 9257 + 847.3 0.050allo-ocimene 0 ± 0 15.6 ± 2 0.014 0 + 0 1.7 + 0.4 0.037mentha-1,5,8-triene 1.1 ± 0.2 25.6 ± 3.3 0.021 10.7 + 6.2 572.7 + 252.6 0.050(Z)-linalool oxide 6.7 ± 1.6 130.7 ± 30.8 0.021 2.6 + 0.9 16.8 + 3 0.050(E)-epoxy-ocimene 3.5 ± 0.6 780.2 ± 152.6 0.021 1.4 + 0.4 105.6 + 43.6 0.050linalool* 64.4 ± 20.1 1709.8 ± 555.1 0.021 56 + 23.3 178.6 + 16.8 0.050unidentified monoterpene 1 0 ± 0 6.5 ± 0.7 0.014 0.9 + 0.4 19.8 + 6.6 0.050unidentified monoterpene oxide 1 0.1 ± 0.1 9 ± 1.9 0.018 0 + 0 1.9 + 1.4 0.121unidentified monoterpene oxide 2 0.3 ± 0.2 11.5 ± 1.9 0.020 0 + 0 6 + 0.3 0.037sesquiterpenoidsβ-elemene* 133.6 ± 32.4 208.2 ± 48.7 0.248 0 + 0 1 + 0 0.037(E)-β-caryophyllene* 79.4 ± 17.1 72.9 ± 9.8 0.564 0.2 + 0.2 5.3 + 0.3 0.046(Z,E)-α-farnesene 1.8 ± 1.8 16.2 ± 2.6 0.018 0 + 0 3.1 + 1.5 0.037(E,E)-α-farnesene 17.5 ± 5.2 76.5 ± 14.6 0.021 240.4 + 176.9 1505.1 + 698.4 0.127unidentified sesquiterpene 1 10.7 ± 4.9 26.6 ± 3.8 0.083 0.6 + 0.3 6.4 + 0.6 0.050unidentified sesquiterpene 2 9.8 ± 4.4 20.9 ± 2.8 0.083 0.1 + 0.1 3.3 + 0.3 0.046unidentified sesquiterpene 3 13.6 ± 5.8 24.7 ± 3.7 0.083 0 + 0 0.3 + 0.3 0.317unidentified sesquiterpene 4 12.8 ± 7.5 46.5 ± 13.5 0.083 8.8 + 6.3 42.2 + 14.9 0.127unidentified sesquiterpene 5 4.1 ± 2.4 19.8 ± 4.5 0.042 0.5 + 0.3 1.4 + 0.2 0.050unidentified sesquiterpene 6 12 ± 3.3 23.6 ± 4 0.043 0 + 0 0.6 + 0.6 0.317homoterpenesDMNT* 91.2 ± 20.2 112.1 ± 25.7 0.773 14.8 + 6.3 51.6 + 25.4 0.127TMTT* 524.6 ± 138.9 1009.9 ± 218.1 0.043 187.6 + 91.2 630.5 + 159.4 0.050diterpenesgeranyl linalool* 3.8 ± 1 19.2 ± 3.9 0.021 0 + 0 0 + 0 NAGLVs and ester(Z)-3-hexenyl acetate* 9.5 ± 3.8 15.7 ± 2.6 0.149 4.6 + 2.3 80.5 + 32.6 0.050(E)-2-hexenyl acetate 0 ± 0 0 ± 0 NA 4.6 + 1 21.3 + 9 0.275(Z)-3-hexenol* 0.1 ± 0.1 0 ± 0 0.317 0 + 0 11.3 + 0.3 0.037Luck et al. BMC Plant Biology  (2016) 16:215 Page 3 of 15was one of the dominant nitrogen-containing volatiles in E.coca but was a minor compound in E. fischeri.Identification of CYP79 enzymes from E. coca and E.fischeriTo identify putative Erythroxylum CYP79 genes, aTBLASTN search against an in-house 454 cDNAsequencing database of E. coca young leaf tissue [24, 25]was conducted using the amino acid sequence ofCYP79D6v3 from Populus trichocarpa [11] as inputsequence. One sequence representing a putative P450enzyme of the CYP79 family was identified. Amplifica-tion of this gene resulted in two highly homologoussequences that were designated as CYP79D62 andCYP79D63 according to the general P450 nomenclature(D.R. Nelson, P450 Nomenclature Committee). PCRwith cDNA made from JA-treated E. fischeri leaves usingthe primer pair designed for amplification of E. cocasequences revealed an additional gene (CYP79D60). Toidentify further potential CYP79D candidates, primersspecific to conserved regions among the obtained geneswere designed and PCR was performed with cDNAmade from JA-treated Erythroxylum leaves. While mostof the resulting amplicons were identical to CYP79D62,CYP79D63, and CYP79D60, one fragment amplifiedfrom E. fischeri cDNA showed sequence divergence andthe isolated full-length clone was designated asCYP79D61.Motifs reported to be conserved in nearly all P450enzymes, such as the ProProxxPro motif at the N-terminus, the heme binding site ProPheGlyxGlyAr-gArgxCysxGly, and the ProGluArgPhe motif, could beidentified in the obtained Erythroxylum CYP79 se-quences (Fig. 1). In comparison to the general P450consensus sequences [26], Erythroxylum CYP79 motifsshowed substitutions characteristic for the CYP79family. Moreover, a CYP79-specific AsnPro motif in oneof the proposed substrate binding sites [26] was also foundin the Erythroxylum sequences (Fig. 1). A dendrogramanalysis showed that Erythroxylum CYP79 enzymesTable 1 Volatile compounds of Erythroxylum coca and E. fischeri released from untreated twigs (control) and jasmonic acid-treatedtwigs (JA treatment) (Continued)(Z)-3-hexenyl propionate 13.1 ± 3.1 81.5 ± 14.8 0.021 1.9 + 0.6 14.8 + 7.9 0.050(Z)-3-hexenyl isobutyrate 2.3 ± 0.2 91.5 ± 17.5 0.014 2.5 + 0.5 55 + 14.2 0.037(Z)-3-hexenyl 2-methylbutanoate 0 ± 0 65.2 ± 24.7 0.047 0 + 0 8.2 + 4.5 0.121(E)-3-hexenyl hexanoate 2.2 ± 0.5 36.7 ± 5.7 0.021 0.7 + 0.2 4.7 + 0.9 0.050(Z)-3-hexenyl tiglate 35.4 ± 8.2 132.3 ± 18.1 0.021 1.2 + 0.3 6.2 + 1.5 0.050unidentified ester 0 ± 0 6.1 ± 1 0.021 0 + 0 1.8 + 0.2 0.050alcoholsbenzyl alcohol* 4.9 ± 1.4 53.1 ± 7 0.021 0.1 + 0.1 3.4 + 3.1 0.2462-phenylethanol* 1.3 ± 0.3 104.3 ± 24.2 0.021 1 + 0.8 36.4 + 33.9 0.127unidentified compoundsunidentified compound 1 3.6 ± 0.7 39.5 ± 4.5 0.021 3.1 + 1.1 40.2 + 5.5 0.050unidentified compound 2 0 ± 0 24 ± 4.6 0.014 0 + 0 5.5 + 2.1 0.037unidentified compound 3 3.8 ± 0.6 35.7 ± 6.8 0.021 2.6 + 0.8 32.9 + 12.7 0.050unidentified compound 4 0.1 ± 0.1 16.7 ± 3.2 0.018 0 + 0 14.7 + 5.9 0.037unidentified compound 5 1.4 ± 0.9 31.4 ± 9.7 0.020 0 + 0 0 + 0 NAunidentified compound 6 1.3 ± 1.3 85 ± 22.8 0.018 0 + 0 1.8 + 0.3 0.037unidentified compound 7 56.4 ± 12.5 71 ± 7.2 0.386 21 + 10.7 41 + 12.1 0.275unidentified compound 8 0 ± 0 12.9 ± 2.3 0.014 0 + 0 2.9 + 0.7 0.037unidentified compound 9 0.5 ± 0.2 10.1 ± 1.1 0.021 0.5 + 0.3 9.7 + 1.7 0.050unidentified compound 10 0.5 ± 0.2 18.8 ± 0.9 0.021 0.6 + 0.4 9.5 + 0.3 0.050unidentified compound 11 1.9 ± 0.3 13.8 ± 0.6 0.021 0.6 + 0.2 7.2 + 0.8 0.050unidentified compound 12 0 ± 0 10.7 ± 0.4 0.014 0 + 0 5.2 + 0.5 0.037unidentified compound 13 0 ± 0 27.6 ± 5.6 0.014 0 + 0 7.1 + 7.1 0.317total volatiles 1508 ± 322 21101 ± 4605 0.021 874 ± 402 13351 ± 458 0.050Emission rates are displayed as means ± SE in ng g-1 fresh weight h-1 (E. coca, n = 4; E. fischeri, n = 3). P-values are based on the results from Kruskal-Wallis ranksum tests between the control and the JA-treatment. P-values ≤ 0.05 indicate significant differences and are shown in bold. Compounds identified using authenticstandards are marked with asterisks (*). Unmarked compounds were identified by comparison of their mass spectra with those of reference librariesLuck et al. BMC Plant Biology  (2016) 16:215 Page 4 of 15grouped together with CYP79D6v3, CYP79D7v2, andCYP79D6v4 from poplar and CYP79D enzymes fromother plants (Fig. 2).To test the enzymatic activity of the identified Erythroxy-lum CYP79s, genes were heterologously expressed inSaccharomyces cerevisiae (WAT11) and microsomesharbouring recombinant protein were incubated with thepotential amino acid substrates L-phenylalanine, L-tyrosine,L-tryptophan, L-leucine, and L-isoleucine in the presenceof NADPH as cosubstrate. CYP79D63 showed narrowsubstrate specificity and was only able to accept tryptophanas substrate, converting it into (E/Z)-indole-3-acetaldoxime(Fig. 3). In contrast, CYP79D62, CYP79D60, andCYP79D61 accepted all tested amino acids and produced(E/Z)-phenylacetaldoxime, (E/Z)-p-hydroxyphenylacetal-doxime, (E/Z)-indole-3-acetaldoxime, (E/Z)-3-methylbutyr-aldoxime, and (E/Z)-2-methylbutyraldoxime, respectively,from the amino acids listed above (Figs. 3 and 4). Assaysusing microsomes from yeast cells expressing the emptyvector, assays without NADPH and assays with boiledproteins showed no activity (data not shown).Km values for the different substrates of CYP79D60,CYP79D62, and CYP79D63 are given in Table 2. Sincemeasurements of carbon monoxide difference spectrafailed, we were not able to determine the proteinconcentrations in the microsomes and thus to calculatethe turnover numbers for the different substrates.Instead, the relative product formation with 1 mM ofthe respective amino acid substrate was measured(Table 2). For CYP79D60 and CYP79D62, the combin-ation of relatively low Km values for L-Phe and L-Leucombined with a high rate of product formation suggestthat these amino acids are the preferred substrates inplanta. Although CYP79D63, which only accepted L-TrpFig. 1 Amino acid sequence alignment of Erythroxylum CYP79s with CYP79A1 from Sorghum bicolor and CYP79D6v3 and CYP79D7v2 fromPopulus trichocarpa. Black boxes mark conserved residues and grey boxes mark residues with similar physicochemical properties. The conservedmotifs are labeled and ‘NP’ indicates the exchange of the generally conserved CYP motif, Thr-(Thr/Ser), with the Asn-Pro motif typical of theCYP79 familyLuck et al. BMC Plant Biology  (2016) 16:215 Page 5 of 15as a substrate, had a lower Km value for this amino acidthan CYP79D60 and CYP79D61 (0.48 ± 0.05 mM versus2.74 ± 0.11 mM and 1.09 ± 0.04 mM, respectively), therate of product formation indicates a low turn-overnumber for this enzyme.Gene expression analysis of Erythroxylum CYP79 genesQuantitative real-time PCR (qRT-PCR) was used tocompare transcript accumulation of CYP79 genes be-tween untreated and JA-treated twigs in E. coca and E.fischeri. To identify reference genes with stable expres-sion under our experimental conditions, we analysedtranscript accumulation of a set of nine potential E. cocaqRT-PCR reference genes [27] in untreated and JA-treated leaves of E. coca and E. fischeri (Additional file 1:Tables S1 and S2). Expressed protein Ec6409 and theclathrin adaptor complex subunit Ec11142 were chosenas reference genes for qRT-PCR analysis of CYP79 genesin E. coca and E. fischeri, respectively, based on their lowCt value variability between the different treatments(Additional file 1: Tables S1 and S2). In E. coca,CYP79D62 showed a significantly upregulated gene ex-pression in JA-treated twigs in comparison to untreatedcontrols (Fig. 5a). In contrast, transcript accumulation ofCYP79D63 was not influenced by the treatment (Fig. 5a).In E. fischeri, CYP79D60 and CYP79D61 were both signifi-cantly upregulated after JA treatment (Fig. 5b), but the aver-age Cq value for CYP79D61 was higher than the averageCq value for CYP79D60 in JA-treated leaves (27.4 versus20.5), suggesting higher gene expression for CYP79D60 incomparison to CYP79D61 after JA treatment.Accumulation of aldoximes, indole-3-acetic acid, andamino acids in JA-treated Erythroxylum plantsTo test whether CYP79 products accumulate in JA-treated and untreated leaves of E. coca and E. fischeri,we analysed leaf methanol extracts using liquidchromatography-tandem mass spectrometry. (E/Z)-Phenylacetaldoxime, (E/Z)-indole-3-acetaldoxime, (E/Z)-2-methylbutyraldoxime, and (E/Z)-3-methylbutyraldox-ime showed significantly increased accumulation in bothspecies after JA-treatment in comparison to untreatedcontrols (Fig. 6). Only trace amounts of these aldoximescould be detected in untreated leaves. Notably, the in-duced accumulation of (E/Z)-2-methylbutyraldoximeand (E/Z)-3-methylbutyraldoxime corresponded wellwith the emission of these compounds from JA-treatedleaves (Table 1). The absence of the aromatic aldoximes(E/Z)-phenylacetaldoxime and (E/Z)-indole-3-acetaldox-ime in the volatile blends (Table 1) is most likely due toFig. 2 Rooted phylogenetic tree of Erythroxylum CYP79D proteins and characterized CYP79 proteins from other plants. The tree was inferred byusing the neighbor joining method and n = 1000 replicates for bootstrapping. Bootstrap values are shown next to each node. CYP71E1 was usedas the outgroup. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Enzymes described in thisstudy are shown in bold. Accession numbers: CYP71E1, AF029858.1; CYP79F1, NM_101507.2; CYP79F2, AF275259.1; CYP79B2, NM_120158.2;CYP79B1, AF069494.1; CYP79B3, NM_127798.3; CYP79A61, KP297890.1; CYP79A1, U32624.1; CYP79D2, AY834390.1; CYP79D1, AY834391.1; CYP79E2,AF140610.1; CYP79E1, AF140609.1; CYP79A2, AF245302.1; CYP79D4, AY599896.1; CYP79D3, AY599895.1; CYP79D16, AB920488.1; CYP79D7v2,KF562516.1; CYP79D6v3, KF562515.1; CYP79D6v4, KF870998.1Luck et al. BMC Plant Biology  (2016) 16:215 Page 6 of 15their low volatility in comparison to the aliphaticaldoximes. In contrast to the aldoximes, indole-3-acetic acid (IAA), a potential conversion product of(E/Z)-indole-3-acetaldoxime, was constitutively pro-duced in untreated and JA-treated leaves of both E.coca and E. fischeri (Fig. 6).The analysis of amino acids as potential CYP79substrates in JA-treated leaves vs. untreated controlleaves revealed a significant induction for L-Ala,L-Val, L-Thr, L-Leu, L-Ile, L-His, L-Phe, L-Trp, andL-Tyr in E. coca and for L-Ala, L-Asp, and L-Gln inE. fischeri (Additional file 1: Table S3).DiscussionThe production of volatiles in response to insect herbiv-ory appears to be a widespread part of plant defence.Herbivore-induced volatiles can influence the feeding oroviposition behaviour of herbivores and are described toattract herbivore enemies such as parasitic wasps, preda-tory arthropods, and insectivorous birds [1]. Jasmonicacid, a phytohormone known to be involved in severalphysiological processes, plays an important role intriggering different plant defence reactions includingvolatile formation [23, 28, 29]. Thus, pure JA or itsderivatives and mimics are often used as artificialelicitors for the induction of vegetative volatile emission[22, 30, 31]. In this study we showed that JA also inducedthe emission of complex volatile blends in E. coca andE. fischeri. The blends were dominated by terpenes andGLVs, but also possessed nitrogen-containing compoundssuch as the nitrile benzyl cyanide, phenylnitroethane,and some aliphatic aldoximes (Table 1). The roles ofherbivore-induced nitrogenous volatiles in direct andindirect plant defense have recently been investigated inpoplar. Olfactometer experiments showed that benzylcyanide and two other volatile nitriles had a strong repel-lant activity against gypsy moth caterpillars, a generalistherbivore known to feed on poplar [13]. Volatile aliphaticaldoximes were found to be attractive for a parasitoid ofgypsy moth larvae in laboratory as well as field experi-ments [12], and the semi-volatile (E,Z)-phenylacetaldox-ime, which accumulated after herbivory in poplar leaves,decreased survival and weight gain of gypsy moth larvaein feeding experiments [11]. Since in the present studyvolatile aliphatic and aromatic aldoximes, nitriles andnitro compounds and the semi volatile (E,Z)-phenylacetal-doxime were found to be emitted from or accumulated inthe two investigated Erythroxylum species after treatmentwith JA in the same order of magnitude as that previouslyreported for herbivore-damaged poplar leaves [11–13],these compounds might play similar roles in plantdefence against natural Erythroxylum herbivores suchFig. 3 Biochemical characterization of Erythroxylum coca CYP79D62 and CYP79D63. The genes were heterologously expressed in Saccharomycescerevisae and microsome preparations containing the recombinant proteins were incubated with the potential amino acid substrates L-Phe, L-Tyr,L-Trp, L-Leu, and L-Ile. The respective reaction products of each substrate are depicted sequentially next to their LC-MS/MS tracesLuck et al. BMC Plant Biology  (2016) 16:215 Page 7 of 15as Eloria noyesi and Eucleodora cocae, two caterpillarpests, or the leaf cutting ant Acromyrmex spp. [32].Using homology-based searches, four genes with simi-larity to CYP79s from other plants could be identified inE. coca and E. fischeri. CYP79D60 and CYP79D61 fromE. fischeri and CYP79D62 from E. coca were significantlyupregulated after JA treatment (Fig. 4) and the encodedenzymes had broad substrate specificity (Figs. 2 and 3).The kinetic parameters of CYP79D60 and CYP79D62were in the range reported for those of previouslycharacterized poplar CYP79 enzymes [11]. Although theKm values were relatively high, it has been suggested thatthe low substrate affinity of CYP79 enzymes has evolvedto avoid possible depletion of free amino acid pools inplants [19]. Considering both the Km and maximalvelocity values for the conversion of the different substrates(Table 2), it is likely that CYP79D60 and CYP79D62 acceptL-phenylalanine, L-leucine, L-isoleucine, and L-tryptophanas substrates in planta. Moreover, the accumulationand emission of their aldoxime products after JAtreatment (Table 1; Fig. 5) coupled with the JA-inducedexpression of their genes (Fig. 4) indicate that CYP79D62Fig. 4 Biochemical characterization of Erythroxylum fischeri CYP79D60 and CYP79D61. The genes were heterologously expressed in Saccharomycescerevisae and microsome preparations containing the recombinant proteins were incubated with the potential amino acid substrates L-Phe, L-Tyr,L-Trp, L-Leu, and L-Ile. The names of the respective reaction products are listed sequentially next to their LC-MS/MS tracesTable 2 Kinetic parameters for CYP79D60, CYP79D62, and CYP79D63. The maximal velocities were measured in the presence of 1mM substrate. CYP79D63 showed no activity with L-Phe, L-Leu, L-Ile, and L-TyrCYP79D60 CYP79D62 CYP79D63substrate Km(mM)Maximal velocity(ng*h-1*assay-1)Km(mM)Maximal velocity(ng*h-1*assay-1)Km(mM)Maximal velocity(ng*h-1*assay-1)L-Phe 0.58 ± 0.05 160.44 ± 4.33 0.67 ± 0.07 424.00 ± 15.24 - -L-Leu 0.23 ± 0.08 68.58 ± 2.49 0.59 ± 0.08 256.90 ± 7.45 - -L-Ile 1.28 ± 0.25 32.62 ± 5.16 3.27 ± 0.42 42.55 ± 3.49 - -L-Trp 2.74 ± 0.11 26.50 ± 0.32 1.09 ± 0.04 16.36 ± 0.31 0.48 ± 0.05 2.92 ± 0.14L-Tyr 6.09 ± 1.32 4.66 ± 0.43 4.99 ± 0.46 73.18 ± 1.93 - -Luck et al. BMC Plant Biology  (2016) 16:215 Page 8 of 15and CYP79D60 contribute to herbivore-induced aldoximeformation in E. coca and E. fischeri, respectively. TheJA-induced production of aldoximes might be furtherpromoted by the increased accumulation of the respectiveamino acid substrates in JA-treated leaves (Additional file1: Table S1). Since CYP79D60 and CYP79D61 are highlysimilar to each other (93 % amino acid identity; Fig. 1) andshowed no remarkable differences in in vitro assays(Fig. 3), the kinetic parameters of CYP79D61 werenot determined in this study. Although it is likely thatCYP79D61 has similar kinetic constants toCYP79D60, the lower expression level of CYP79D61in JA-treated leaves in comparison to CYP79D60 sug-gests only a minor role for this enzyme in aldoximeproduction in E. fischeri.While CYP79D60, CYP79D61, and CYP79D62 are likelyinvolved in plant defense, the biological function ofCYP79D63 remains unclear. In contrast to the otherthree enzymes, CYP79D63 accepted exclusively L-tryptophan as substrate (Fig. 2). The affinity of CYP79D63Fig. 5 Trancript abundance of CYP79D genes in jasmonic acid-treated and untreated control leaves of Erythroxylum coca (a) and E. fischeri (b).Twigs were cut and placed in either tap water (ctr) or jasmonic acid (200 μM) for 18 h. Gene expression was determined by qRT-PCR. Means andstandard errors are shown (E. coca, n = 4; E. fischeri, n = 3). The Kruskal-Wallis rank sum test was used to test for statistical significance. P-values≤0.05 indicate significant difference between the treatments. ctr, control treatment; JA, jasmonic acid treatmentFig. 6 The accumulation of different aldoximes and indole-3-acetic acid (IAA) in jasmonic acid-treated and untreated control leaves of Erythroxylumcoca and E. fischeri. Twigs were cut and placed in either tap water (ctr) or jasmonic acid (200 μM) for 18 h. Aldoximes and IAA were extracted withmethanol and analyzed using LC-MS/MS. Means and standard errors are shown (E. coca, n = 4; E. fischeri, n = 3). The Kruskal-Wallis rank sum test wasused to test for statistical significance. P-values≤ 0.05 indicate significant difference between the treatments. ctr, control treatment; JA, jasmonicacid treatmentLuck et al. BMC Plant Biology  (2016) 16:215 Page 9 of 15for L-tryptophan was higher in comparison to CYP79D60,CYP79D61, and CYP79D62 (Table 2); however, the lowrelative product formation indicates a low turnover num-ber for this enzyme. Since gene expression was not influ-enced by JA treatment, it is unlikely that CYP79D63contributes to herbivore-induced accumulation of (E,Z)-indole-3-acetaldoxime. In many plants, the conversion of(E,Z)-indole-3-acetaldoxime into the corresponding acidis thought to serve as an alternative route for the forma-tion of auxin [33–36] and thus it is conceivable thatCYP79D63 might produce (E,Z)-indole-3-acetaldoxime asprecursor for constitutive auxin formation in leaves orother growing plant parts of E. coca. A comprehensivecorrelation between CYP79D63 gene expression and theaccumulation of auxin in different plant organs and differ-ent developmental stages might help to elucidate the po-tential role of CYP79D63 in auxin formation.As a result of domestication, many crop plants showaltered levels of secondary compounds in comparison totheir wild relatives [37]. E. coca, for example, has beencultivated for thousands of years and has been selectedfor high-level production of the pharmacologically activetropane alkaloid cocaine [38]. The cultivated speciescontains 20-100 times more cocaine in its leaves thenclosely related wild species [39]. While such selection forhigh-level production of useful compounds or for low-level production of undesired compounds is controlled bythe breeder, domestication can also have unrecognizedand unwanted side effects. The accumulation of an in-active allele of (E)-β-caryophyllene synthase during breed-ing of North American maize, for instance, led to the lossof (E)-β-caryophyllene production in most of these lines[40]. (E)-β-Caryophyllene is usually released as volatilefrom herbivore-damaged maize leaves and roots and hasbeen shown to be involved in different indirect defence re-actions above and below ground [40–43]. In this studywe showed that E. coca and E. fischeri accumulate andrelease the same aldoximes, nitriles, and nitro com-pounds after JA-treatment in comparable amounts,suggesting that domestication did not alter these plantdefence responses in cultivated E. coca. Whether thequantitative differences between other single com-pounds in the JA-induced volatile bouquets of E. cocaand E. fischeri are species specific or are the result ofthe breeding of E. coca, is still unclear.ConclusionsHerbivore-induced volatile blends are in general verycomplex and contain dozens of substances. However, theenzymatic machinery behind this complexity is oftenastonishingly simple, comprising only a handful ofenzymes with broad substrate and/or product specificity.Terpene synthases, the key enzymes in terpene biosyn-thesis, for instance, can produce mixtures of up to50 compounds from one substrate [44]. Moreover,methyltransferases and acyltransferases involved in theformation of volatile esters have been reported to acceptmultiple substrates [31, 45, 46]. Such promiscuity inthe substrate and/or product specificity of volatile-producing enzymes seems to be a general phenomenonthat allows plants to efficiently produce a large mixtureof different volatiles with only a limited number ofenzymes. Mixtures may have specific advantages in plantdefense [47]. Recently we showed that two poplarCYP79s involved in volatile aldoxime formation alsoexhibit broad substrate specificity in contrast to all otherpreviously described CYP79s [11]. The Erythroxylumenzymes characterized in this study represent the secondexample for CYP79s having broad substrate specificityand it is thus tempting to speculate that such promiscu-ity might be a general feature for CYP79s formingherbivore-induced volatiles. However, further researchon volatile aldoxime-producing CYP79 enzymes fromdiverse plant families is still needed to substantiate thisassertion and to understand the evolutionary and structuralcauses of broad substrate specificity in this enzyme class.MethodsPlant material and plant treatmentSeeds of Erythroxylum coca var coca were obtained fromthe botanical garden Bonn, Germany, and were germi-nated in sterilized potting soil. Live plants of E. fischeriwere collected in Kenya and shipped to the MPICE. Plantswere grown in a growth chamber set at 22 °C under a 12h/12 h light/dark cycle, with humidity of 65 % and 70 %,respectively, and were fertilized once a week with Ferty 3(15-10-15) and Wuxal Top N (Planta Düngemittel,Regenstauf, Germany). The cultivation of E. coca wasauthorized by the Bundesinstitut für Arzneimittel undMedizinprodukte (BfArM) (permit number, BtM 4515971).For jasmonic acid (JA) treatment, JA (100 mg/ml etha-nol) was diluted in tap water to a final concentration of200 μM. Ethanol was diluted in tap water in the sameway and used as control. From each E. coca plant (n = 4),four twigs of about 15–20 cm in length were cut andimmediately placed in glass beakers containing either JA(two twigs) or control solution (the other two twigs). ForE. fischeri, two twigs of about 20 cm in length were cutfrom each plant (n = 3) and only one twig was used pertreatment. Twigs were left in JA or control solutionovernight for 18 h before the volatile collection.Volatile collection and analysisVolatile collections were performed in a growth chamberunder conditions as described above. Glass beakers con-taining the Erythroxylum twigs were separately placed in3 l glass desiccators which were tightly closed. Purifiedair pumped into the desiccator at a rate of 0.5 l min-1Luck et al. BMC Plant Biology  (2016) 16:215 Page 10 of 15came into contact with the plant and left the vesselthrough a filter packed with 30 mg Super-Q (ARS,Inc., Gainesville, FL, USA). Volatiles were collectedfor 5 h (9 am–2 pm). After the collection, the plantmaterial was immediately frozen in liquid nitrogen forfurther analysis. The volatile compounds were des-orbed from the filters by eluting the filter twice with100 μl dichloromethane containing nonyl acetate as aninternal standard (10 ng μl-1).Qualitative and quantitative volatile analysis was con-ducted using an Agilent 6890 Series gas chromatograph(Agilent Technologies GmbH, Waldbronn, Germany)coupled to an Agilent 5973 quadrupole mass selectivedetector (interface temp, 270 °C; quadrupole temp, 150 °C; source temp, 230 °C; electron energy, 70 eV) or aflame ionization detector (FID) operated at 300 °C, re-spectively. The constituents of the volatile bouquetwere separated using a ZB-WAX column (Phenom-enex, Aschaffenburg, Germany, 60 m × 0.25 mm ×0.15 μm) and He (MS) or H2 (FID) as carrier gas.The sample (1 μL) was injected without split at aninitial oven temperature of 40 °C. The temperaturewas held for 2 min and then increased to 225 °C witha gradient of 5 °C min-1, held for another 2 min, andthen further increased to 250 °C with 100 °C min-1and a hold for 1 min.Compounds were identified by comparison of reten-tion times and mass spectra to those of authenticstandards obtained from Fluka (Seelze, Germany),Roth (Karlsruhe, Germany), Sigma (St. Louis, MO,USA), and Bedoukian (Danbury, CT, USA) or by ref-erence spectra in the Wiley and National Institute ofStandards and Technology libraries. The absoluteamount of all compounds was determined based ontheir FID peak area in relation to the area of the in-ternal standard.Plant tissue sampling, RNA extraction, and reversetranscriptionErythroxylum leaf material was harvested immediatelyafter the volatile collection, flash-frozen with liquid ni-trogen, and stored at -80 °C until further processing.After grinding the frozen leaf material in liquid nitro-gen to a fine powder, total RNA was isolated usingan InviTrap Spin Plant RNA kit (Stratec, Berlin,Germany) according to manufacturer’s instructions.RNA concentration, purity, and quality were assessedusing a spectrophotometer (NanoDrop 2000c, ThermoScientific, Wilmington, DE, USA) and an Agilent2100 Bioanalyzer. RNA was treated with TurboDNase(ThermoFisher Scientific, https://www.thermofisher.com) prior to cDNA synthesis. Single-stranded cDNAwas prepared from 1 μg of DNase-treated RNA usingSuperScriptTM III reverse transcriptase and oligo(dT12-18) primers (Invitrogen, Carlsbad, CA, USA).Identification and heterologous expression of CYP79genesA TBLASTN search against an in-house 454 cDNA se-quencing database of E. coca young leaf tissue withCYP79D6v3 from Populus trichocarpa (GenBankAHF20912.1) as input sequence revealed one sequencewith similarity to plant CYP79s. The full-length genewas designated as CYP79D62 according to the generalP450 nomenclature (D.R. Nelson, P450 NomenclatureCommittee) and could be amplified from cDNA attainedfrom JA-treated leaves of E. coca. The PCR product wascloned into the sequencing vector pCR®-Blunt II-TOPO®(Invitrogen) and both strands were fully sequenced usingthe Sanger method. Sequencing of several clones re-vealed a second CYP79 gene that was designated asCYP79D63. Using the primers designed for amplificationof E. coca CYP79 genes, a CYP79 sequence could beamplified from cDNA made from JA-treated E. fischerileaves (CYP79D60). To identify further potentialCYP79D candidates, primers specific to conservedregions among the obtained genes were designed andPCR was performed with cDNA made from JA-treatedErythroxylum leaves. While most of the resulting ampli-cons were identical to CYP79D62, CYP79D63, andCYP79D60, one fragment amplified from E. fischericDNA showed sequence divergence. RacePCR was per-formed to obtain the full-length clone, which was desig-nated as CYP79D61. Primer sequence information isgiven in Additional file 1: Table S4.For heterologous expression in Saccharomyces cerevi-siae, the complete open reading frames of CYP79D62,CYP79D63, CYP79D61, and CYP79D60 were cloned intothe pESC-Leu2d vector [48] as NotI/SacI fragments. Theresulting constructs were transferred into the S. cerevi-siae strain WAT11 [49] and single yeast colonies werepicked to inoculate starting cultures containing 30 mLSC minimal medium lacking leucine (6.7 g L-1 yeastnitrogen base without amino acids, but with ammoniumsulfate). Other components: 100 mg L-1 of L-adenine, L-arginine, L-cysteine, L-lysine, L-threonine, L-tryptophanand uracil; 50 mg L-1 of the amino acids L-aspartic acid,L-histidine, L-isoleucine, L-methionine, L-phenylalanine,L-proline, L-serine, L-tyrosine, L-valine; 20 g L-1 D-glucose. The cultures were grown overnight at 28 °C and180 rpm. One OD of the starting cultures (approx. 2 ×107 cells mL-1) was used to inoculate 100 mL YPGA fullmedium cultures (10 g L-1 yeast extract, 20 g L-1 bacto-peptone, 74 mg L-1 adenine hemisulfate, 20 g L-1 D-glucose) which were grown for 32–35 h (until OD about5), induced by the addition of galactose and cultured foranother 15–18 h. The cultures were centrifuged (7500g,Luck et al. BMC Plant Biology  (2016) 16:215 Page 11 of 1510 min, 4 °C), the supernatant was decanted, and the cellpellets were resuspended in 30 mL TEK buffer (50 mMTris-HCl pH 7.5, 1 mM EDTA, 100 mM KCl) andcentrifuged again. Then, the pellets were carefully resus-pended in 2 mL of TES buffer (50 mM Tris-HCl pH 7.5, 1mM EDTA, 600 mM sorbitol, 10 g L-1 bovine serum frac-tion V protein and 1.5 mM β-mercaptoethanol) andtransferred to a 50 mL conical tube. Glass beads (0.45–0.50 mm diameter, Sigma-Aldrich Chemicals, Steinheim,Germany) were added so that they filled the full volume ofthe cell suspension. Yeast cell walls were disrupted by 5cycles of 1 min shaking by hand and subsequent coolingdown on ice for 1 min. The crude extracts were recoveredby washing the glass beads 4 times with 5 mL TES. Thecombined washing fractions were centrifuged (7500g, 10min, 4 °C), and the supernatant was transferred to anothertube and centrifuged again (100,000g, 60 min, 4 °C). Theresulting microsomal protein fractions were homogenizedin 2 mL TEG buffer (50 mM Tris-HCl, 1 mM EDTA, 30% w/v glycerol) using a glass homogenizer (Potter-Elveh-jem, Fisher Scientific, Schwerte, Germany). Aliquots werestored at -20 °C.Analysis of recombinant CYP79To determine the substrate specificity of ErythroxylumCYP79 enzymes, yeast microsomes harboring recombin-ant protein were incubated for 30 min at 25 °C and 300rpm individually with the potential substrates L-Phe, L-Val, L-Leu, L-Ile, L-Tyr, and L-Trp in glass vials containing300 μL of the reaction mixture (75 mM sodium phosphatebuffer (pH 7.0), 1 mM substrate (concentration was vari-able for Km determination), 1 mM NADPH, and 10 μL ofthe prepared microsomes). Reaction products were ana-lyzed using LC-MS/MS as described below.For the determination of the Km values, assays werecarried out in triplicate and stopped by placing the sam-ples on ice after 300 μL MeOH were added. Enzymeconcentrations and incubation times were chosen so thatthe reaction velocity was linear during the incubationtime period.qRT-PCR analysiscDNA was prepared as described above and diluted 1:10with water. For the amplification of CYP79D genefragments with a length of about 100–150 bp, primerpairs were designed having a Tm ≥ 60 °C, a GC contentbetween 40–55 %, and a primer length in the range of20–25 nt (see Additional file 1: Table S4 for primer in-formation). Primer specificity was confirmed by agarosegel electrophoresis, melting curve analysis, and standardcurve analysis and by sequence verification of clonedPCR amplicons. Primer pair efficiency was determinedusing the standard curve method with fivefold serialdilution of cDNA and was found to be between 97 and104 %. Samples were run in triplicate using the Brilliant®III SYBR® Green QPCR Master Mix (Stratagene, Carls-bad, CA, USA). The following PCR conditions wereapplied for all reactions: Initial incubation at 95 °C for 3min followed by 40 cycles of amplification (95 °C for 5 s,60 °C for 10 s). Plate reads were taken during theannealing and the extension steps of each cycle. Data forthe melting curves were recorded at the end of cyclingfrom 60 °C to 95 °C.All samples were run on the same PCR machine(Bio–Rad CFX Manager 3.1, Bio-Rad Laboratory,Hercules, CA, USA) in an optical 96-well plate. Three(E. fischeri) or four (E. coca) biological replicates wereanalyzed as triplicates in the qRT-PCR for each of thethree treatments.LC-MS/MS analysis of aldoximes, amino acids, and auxinFor determining amino acid and aldoxime concentra-tion, 100 mg of plant powder was extracted with 1 mLMeOH. For the measurement of amino acids, the MeOHextract was diluted 1:10 with water and spiked with 13C,15N labeled amino acids (algal amino acids 13C,15N, Iso-tec, Miamisburg, OH, USA) at a concentration of 10 μgof the mix per mL. Amino acids in the diluted MeOHextract were directly analyzed by LC-MS/MS as recentlydescribed [11].Aldoximes were measured from MeOH extractsusing an Agilent 1200 HPLC system coupled to anAPI 5000 tandem mass spectrometer (Applied Biosys-tems, Darmstadt, Germany). Formic acid (0.2 %) inwater and acetonitrile were employed as mobilephases A and B, respectively, on a Zorbax EclipseXDB-C18 column (50 × 4.6 mm, 1.8 μm, AgilentTechnologies). The elution profile was: 0–4 min, 10–70 % B; 4–4.1 min, 70–100 % B; 4.1–5 min 100 % Band 5.1–7 min 10 % B at a flow rate of 1.1 mL min–1. The API 5000 tandem mass spectrometer was oper-ated in positive ionization mode (ionspray voltage,5500 eV; turbo gas temp, 700 °C; nebulizing gas, 60psi; curtain gas, 30 psi; heating gas, 50 psi; collisiongas, 6 psi). MRM was used to monitor precursorion→ product ion reactions for each analyte as fol-lows: m/z 136.0→ 119.0 (collision energy (CE), 17 V;declustering potential (DP), 56 V) for phenylacetal-doxime; m/z 102.0→ 69.0 (CE, 13 V; DP, 31 V) for 2-methylbutyraldoxime; m/z 102.0→ 46.0 (CE, 15 V;DP, 31 V) for 3-methylbutyraldoxime; m/z 175.0→158.0 (CE, 17 V; DP, 56 V) for indole-3-acetaldoximeand m/z 152.0→ 107.0 (CE, 27 V; DP, 100 V) forp-hydroxyphenylacetaldoxime. The concentration ofaldoximes was determined using external standardcurves made with authentic standards synthesized asdescribed in the literature [11].Luck et al. BMC Plant Biology  (2016) 16:215 Page 12 of 15Indole-3-acetic acid (IAA) was analyzed as follows:100 mg of plant powder were extracted with 300 μLMeOH, and 200 μL of the extract was diluted 1:10with water containing 0.1 % formic acid and loadedonto equilibrated Chromabond® HR-X polypropylenecolumns (45 μm, Macherey Nagel, Düren, Germany).The columns were washed with acidified water. The fractioncontaining the auxins was eluted with 1 mL acetonitrile,which was then dried under a stream of nitrogen gas. Thesamples were redissolved in 30 μL MeOH and subsequentlyanalyzed by the same LC-MS/MS system as describedabove. Separations were performed on an Agilent XDB-C18column (50 mm × 4.6, 1.8 μm). Eluents A and B were watercontaining 0.05 % formic acid and acetonitrile, respectively.The elution profile was: 0–0.5 min, 5 % B in A; 0.5–4.0 min,5–50 % B; 4.1–4.5 min 100 % B and 4.6–7 min 5 % B. Theflow rate was set to 1.1 mL min-1. The API 5000 tandemmass spectrometer was operated in positive ionization mode(ion spray voltage, 5500 eV; turbo gas temp, 700 °C; nebuliz-ing gas, 60 psi; curtain gas, 30 psi; heating gas, 50 psi;collision gas, 6 psi). The MRM transition and parametersettings for IAA were as follows: m/z 176→ 130 (CE, 19 V;DP, 31 V). IAA concentration was determined by spikingthe plant extracts with known amounts of 2H5-IAA(OlChemIm Ltd., Olomouc, Czech Republic).Sequence analysis and phylogenetic tree constructionAn alignment of Erythroxylum CYP79 genes and charac-terized CYP79 genes from other plants was constructedusing the MUSCLE (codon) algorithm (gap open, -2.9;gap extend, 0; hydrophobicity multiplier, 1.5; clusteringmethod, upgmb) implemented in MEGA6 [50]. Based onthe translated MUSCLE codon alignment, a tree wasreconstructed with MEGA6 using a neighbor joiningalgorithm (model/method, JTT model; substitutions type,amino acids; rates among sites, uniform rates; gaps/miss-ing data treatment, partial deletion; site coverage cutoff,80 %). A bootstrap resampling analysis with 1000 repli-cates was performed to evaluate the tree topology.Statistical analysisDifferences in gene expression, volatile emission, and theaccumulation of aldoximes, auxin, and amino acidsbetween jasmonic acid-induced and untreated controlplants were analyzed with Kruskal-Wallis rank sum testsfor E. coca and E. fischeri separately in R version 3.1.1 [51].Accession numbersSequence data for genes in this article can be found inthe GenBank under the following identifiers: CYP79D60(KX344462), CYP79D61 (KX344460), CYP79D62 (KX344463),CYP79D63 (KX344461).Additional fileAdditional file 1: Table S1. Expression levels of potential house-keeping genes in jasmonic acid-treated (JA) and untreated control (ctr)leaves of Erythroxylum fischeri. Table S2. Expression levels of potentialhouse-keeping genes in jasmonic acid-treated (JA) and untreated control(ctr) leaves of Erythroxylum coca. Table S3. Amino acid concentrations inuntreated (control) and jasmonic acid-treated (JA treatment) leaves ofErythroxylum coca and E. fischeri. Table S4. Oligonucleotides used forisolation and qRT-PCR analysis of Erythroxylum coca and E. fischeri CYP79Dgenes. (DOCX 38 kb)AbbreviationsCYP: Cytochrome P450 monooxygenase; GLV: Green leaf volatile;JA: Jasmonic acid; qRT-PCR: Quantitative real-time PCR; DMNT: (3E)-4,8-dimethylnona-1,3,7-triene; TMTT: (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraeneAcknowledgementsWe thank Tamara Krügel and all the MPI-CE gardeners for their help with rearingthe Erythroxylum plants. Furthermore, we thank Patrick Chalo Mutiso from theUniversity of Nairobi, Kenya, for collecting and shipping Erythroxylum fischeri plants.FundingThe research was funded by the Max-Planck Society.Availability of data and materialsAll supporting data are included as additional files. Constructs described inthis work and datasets analysed during the current study are available fromthe corresponding author upon request. Sequences were deposited inGenBank (see Methods section).Authors’ contributionsJJ, SI, JG, and TGK designed research. KL, JJ, SI, and TGK carried out theexperimental work. MH and TGK analysed data. TGK wrote the manuscript.All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable.Ethics approval and consent to participateNot applicable.Author details1Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, D-07745Jena, Germany. 2Present address: Fraunhofer Institute for Molecular Biologyand Applied Ecology IME, Forckenbeckstrasse 6, D-52074 Aachen, Germany.3Present address: Michael Smith Laboratories, University of British Columbia,Vancouver, Canada.Received: 6 August 2016 Accepted: 27 September 2016References1. Unsicker SB, Kunert G, Gershenzon J. Protective perfumes: the role ofvegetative volatiles in plant defense against herbivores. Curr Opin Plant Biol.2009;12(4):479–85.2. De Moraes CM, Mescher MC, Tumlinson JH. Caterpillar-induced nocturnalplant volatiles repel conspecific females. Nature. 2001;410:577–80.3. Turlings TCJ, Tumlinson JH, Lewis WJ. 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