the flavonoid biosynthetic enzyme chalcone … flavonoid biosynthetic enzyme chalcone isomerase...

The Flavonoid Biosynthetic Enzyme Chalcone IsomeraseModulates Terpenoid Production in GlandularTrichomes of Tomato1[C][W][OPEN]

Jin-Ho Kang, John McRoberts, Feng Shi, Javier E. Moreno, A. Daniel Jones, and Gregg A. Howe*

Department of Energy-Plant Research Laboratory (J.-H.K., J.M., J.E.M., G.A.H.), Department of Chemistry(F.S., A.D.J.), and Department of Biochemistry and Molecular Biology (A.D.J., G.A.H.), Michigan StateUniversity, East Lansing, Michigan 48824

Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles inmediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant ofcultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiencywas associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstratethat Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes theconversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues oftomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af.Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our resultsestablish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production.Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands indynamic environments.

Flavonoids and terpenoids (isoprenoids) comprise twoof the largest groups of specialized metabolites in higherplants. They serve myriad and often complementaryroles in plant acclimation to changing environmentalconditions. For example, flavonoids and terpenoids di-rectly repel attack by insect herbivores and microbialpathogens and also mediate plant communication withsymbiotic bacteria, natural enemies of arthropod herbi-vores, and parasitic plants (Hirsch et al., 2003; Rasmannet al., 2005). Pigmented flavonoids and volatile terpenesprovide visual and olfactory cues, respectively, for theattraction of insect pollinators (Dudareva et al., 2006;Grotewold, 2006) and also protect against ultravioletradiation, elevated temperature, and oxidative stress (Liet al., 1993; Sharkey and Singsaas, 1995; Affek and Yakir,2002). Much of the scientific interest in flavonoids and

terpenoids reflects their importance in food quality,nutrition, and human health (Dillard and German, 2000;Middleton et al., 2000; Dixon et al., 2005; Bohlmann andKeeling, 2008).

Flavonoids are synthesized from Phe derivatives gen-erated via the shikimate and phenylpropanoid pathways(Tohge et al., 2013; Fig. 1). Chalcone synthase (CHS; EC2.3.1.74) condenses 4-coumaroyl-CoA and malonyl-CoAto form the open-chain flavonoid naringenin chalcone,which is converted to naringenin by chalcone isomerase(CHI; EC 5.5.1.6). Naringenin defines a key branch pointfor the synthesis of several major classes of flavonoids,including flavanones, flavonols, and anthocyanins. Fla-vonoid biosynthesis is controlled by transcription factorsthat coordinate the expression of multiple biosyntheticgenes in response to environmental and developmentalcues (Dixon and Paiva, 1995; Grotewold, 2006; Vogt,2010). Terpenoids originate from distinct metabolicpathways in which the C5 precursors dimethyl allyldiphosphate and isopentenyl diphosphate are sequen-tially combined to produce the building blocks for C10monoterpenes, C15 sesquiterpenes, and higher molecu-lar weight derivatives. Plastid-derived terpenoids aresynthesized by the methyl-erythritol phosphate (MEP)pathway (Fig. 1), whereas most extraplastidic terpe-noids are produced from the cytosolic mevalonic acidpathway (Bohlmann and Keeling, 2008). Relativelylittle is known about the regulatory mechanisms go-verning terpenoid biosynthesis in higher plants (Patraet al., 2013).

The terpenoid and flavonoid biosynthetic pathwaysare supplied with carbon skeletons generated in primary

1 This work was supported by the National Science Foundation(grant no. DBI–0604336) and the Chemical Sciences, Geosciences, andBiosciences Division, Office of Basic Energy Sciences, Office of Sci-ence, U.S. Department of Energy (grant no. DE–FG02–91ER20021 toG.A.H. for partial support for J.-H.K. and transgenic experiments).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Gregg A. Howe ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OPEN] Articles can be viewed online without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.113.233395

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metabolism but, otherwise, are thought to operate in-dependently of one another in most plant tissues (Fig. 1).Recent studies, however, are beginning to uncover met-abolic and regulatory connections between these twomajor branches of specialized metabolism. For example,dimethyl allyl diphosphate is used for the synthesis ofprenylated flavonoids and other terpenophenolics inglandular trichomes of some plant species (Nagel et al.,2008; Wang et al., 2008; Shen et al., 2012; Voo et al., 2012).There is also evidence that members of the MYB familyof transcription factors play a role in coordinating meta-bolic activity between the flavonoid and terpenoid bio-synthetic pathways (Bedon et al., 2010; Ben Zvi et al., 2012).

Glandular trichomes provide an excellent modelsystem in which to study the production of specializedmetabolites in a single, experimentally accessible celltype (Wagner, 1991; Lange et al., 2000; Schilmiller et al.,2008; Tissier, 2012; Voo et al., 2012). The type VI glan-dular trichome of tomato (Solanum lycopersicum) isparticularly well suited for the analysis of flavonoidand terpenoid synthesis, which dominates the met-abolic activity of this specialized epidermal structure(Duffey and Isman, 1981; Kennedy, 2003; van Schieet al., 2007; Schilmiller et al., 2010; Kang et al., 2010b).Mutants affected in the chemical composition, mor-phology, and density of type VI glands have providedinsight into the biological roles of glandular trichomesin resistance to insect herbivores (Li et al., 2004; Kanget al., 2010a, 2010b; Bleeker et al., 2012). Both the devel-opment and biosynthetic capacity of type VI trichomes

are responsive to environmental inputs and are regu-lated in part by the stress hormone jasmonate (Li et al.,2004; Boughton et al., 2005; van Schie et al., 2007; Peifferet al., 2009; Tian et al., 2012). The mechanisms that co-ordinate the developmental and metabolic plasticity ofglandular trichomes in tomato and other species arelargely unknown.

The anthocyanin free (af) mutant of tomato was identi-fied more than a half-century ago as an x-ray-inducedvariant that lacks anthocyanin production in all tissues(Burdick, 1958). In contrast to other anthocyanin-deficientmutants of tomato, Rick et al. (1976) showed that affoliage exhibits a lower density of type VI glandulartrichomes, reduced emission of leaf volatiles, and in-creased susceptibility to herbivorous flea beetles.These pleiotropic phenotypes led to the suggestion thataf may define a regulatory gene that controls both fla-vonoid production and trichome development (De Jonget al., 2004), but this hypothesis has remained untested.Here, we demonstrate that Af encodes a member(SlCHI1) of the CHI family of enzymes that catalyzesthe synthesis of a key branch point intermediate [2(S)-naringenin] in the flavonoid pathway. Transgeniccomplementation experiments showed that Af/CHI1 isrequired not only for the synthesis of anthocyaninsand other flavonoids but also plays a role in promotingthe accumulation of terpenoids in glandular trichomes.These findings suggest a role for CHI1 in coordinatingthe developmental plasticity and metabolic activity of anepidermal structure that shields against biotic stress.

Figure 1. Schematic overview of terpenoid andflavonoid biosynthetic pathways. The names ofcompounds and select enzymes (italicized) are asfollows: ANS, anthocyanidin synthase; CDP-ME,4-(cytidine 59-diphospho)-2-C-methyl-D-erythritol;CDP-MEP, 2-phospho-4-(cytidine 59-diphospho)-2-C-methyl-D-erythritol; CMK, 4-(cytidine 59-diphospho)-2-C-methyl-D-erythritol kinase; DAHP,3-deoxy-D-arabino-heptulosonate 7-phosphate;DFR, dihydroflavonol 4-reductase; DMAPP, dimeth-ylallyl diphosphate; DXP, 1-deoxy-D-xylulose5-phosphate; DXPS, DXP synthase; DXR, DXPreductoisomerase; F3H, flavanone 3-hydroxylase;FLS, flavonol synthase; FPP, farnesyl diphosphate;FPPS, FPP synthase; GPP, geranyl diphosphate;GPPS, GPP synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IPP, isopente-nyl diphosphate; MCT, 2-C-methyl-D-erythritol4-phosphate cytidylyltransferase; MTS, mono-terpene synthase; PAL, Phe ammonia lyase; PEP,phosphoenolpyruvate; STS, sesquiterpene syn-thase. Note that although some sesquiterpenesin tomato are synthesized via the MEP pathway(as shown for simplicity; Sallaud et al., 2009),most sesquiterpenes are derived from the cyto-solic mevalonate pathway. [See online articlefor color version of this figure.]

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RESULTS

The af Mutant Is Deficient in Flavonoid Accumulationin Type VI Glandular Trichomes

Under growth conditions in which wild-type plantsaccumulate relatively high anthocyanin levels, af mu-tant plants lacked visible anthocyanin pigmentation inall tissues, including leaves, stems, and hypocotyls(Fig. 2, A–D). This defect is associated with a reduceddensity of type VI glandular trichomes on leaves, asreported previously (Rick et al., 1976). We found thatthe density of type VI trichomes on the adaxial surfaceof af leaves (400 6 54 cm22) was approximately 30% ofthat on wild-type leaves (1,379 6 129 cm22). Light mi-croscopy and scanning electron microscopy confirmedthese findings and also showed that the size of type VIglands on af leaf and stem tissue is reduced in com-parison with the wild type (Fig. 2, E and F).Chlorogenic acid (caffeoylquinate) and rutin (quercetin-

3-O-rutinoside), which are both derived from 4-coumaroyl-CoA (Fig. 1), are two of the most abundant phenoliccompounds in tomato leaves and type VI glands, re-spectively (Duffey and Isman, 1981; Kang et al., 2010b).To further characterize the effect of af on phenolic

metabolism, we used liquid chromatography-massspectrometry (MS) to measure the levels of chlorogenicacid, rutin, and other nonvolatile secondary metabolitesin leaf-dip extracts obtained by brief immersion of ex-cised leaflets into acidified methanol. af leaves containedless than 10% of wild-type levels of rutin and otherflavonol derivatives, including kaempferol rhamnoside,a quercetin trisaccharide, and 3-O-methylmyricetin(Fig. 3A). Wild-type and af leaves contained similarlevels of chlorogenic acid and its precursor, quinic acid,as well as acyl sugars and glycoalkaloids (a-tomatineand dehydrotomatine). Metabolite analyses performedwith isolated type VI trichomes confirmed that themutant produces only trace levels of rutin in gland cells(Fig. 3B). Low levels of acyl sugars were found in typeVI extracts from both the wild type and af, whereaschlorogenic acid, quinic acid, and glycoalkaloids werenot detected in these extracts (Fig. 3B). These resultsindicate that af, in addition to blocking anthocyaninproduction in tissues throughout the plant, impairsthe flavonoid branch of phenylpropanoid metabolismin type VI trichome glands.

The af Mutant Exhibits Increased Susceptibilityto Herbivorous Beetles

The defense-related roles of glandular trichome-derived metabolites led us to test whether af plants areaffected in resistance to native populations of arthropodherbivores. Greenhouse-grown wild-type and af seed-lings were transplanted to the field and subsequentlymonitored for the presence of herbivores and associatedleaf damage. Potato flea beetle (Epitrix cucumeris) wasobserved much more frequently on the mutant than onneighboring wild-type plants (Fig. 4A). Moreover,af foliage received more herbivore damage than wild-type leaves, as determined by counting the number offlea beetle feeding sites (Fig. 4B). We also found that afplants were visited by two additional beetle species,Colorado potato beetle (Leptinotarsa decemlineata) andblister beetles (Epicauta funebris), which were not ob-served on wild-type plants (Fig. 4, C and D). These re-sults are consistent with previous field studies showingthat the afmutant is compromised in resistance to nativepopulations of the tobaccoflea beetle (Epitrix hirtipennis;Rick et al., 1976).

af Is a Null Mutation in CHI

TheAf locuswas previouslymapped to an interval onchromosome 5 that is defined by the Solanum pennelliiintrogression line IL5-2 (Tanksley et al., 1992; Eshed andZamir, 1995). Fine-mapping of the locus allowed us toposition Af to an approximately 200-kb region flankedby markers SGN-U571424 and CT93 (Fig. 5A). Amongthe approximately 31 hypothetical genes in this regionwere two tandemly duplicated genes predicted to en-code the flavonoid pathway enzyme CHI (Fig. 5B). The

Figure 2. Anthocyanin and trichome phenotypes of the tomato afmutant. A and B, Light microscopy images of the abaxial surface ofwild-type (A) and af (B) leaves. C and D, Light microscopy images ofwild-type (C) and af (D) stems. E and F, Scanning electron micrographsof the adaxial surface of wild-type (E) and af (F) leaves. Representativetype I and type VI trichomes are labeled I and VI, respectively. Three-week-old plants were used for all experiments. Bars = 1,000 mm(A and B), 500 mm (C and D), and 100 mm (E and F).

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well-established role of CHI in flavonoid biosynthesisindicated that these genes, which we designated CHI1(Solyc05g010320) and CHI2 (Solyc05g010310), weregood candidates forAf. Reverse transcription (RT)-PCRanalysis showed that CHI1 transcripts are expressed inboth wild-type and af leaves, whereas expression ofCHI2 was not detected in leaves of either genotype(Supplemental Fig. S1). This finding is consistent withpublicly available gene expression data for CHI1 andCHI2 (http://solgenomics.net). Sequencing of wild-type- and af-derived genomic clones containing CHI2did not reveal nucleotide polymorphisms at this locus.CHI1 genomic and complementary DNA (cDNA) clonesisolated from af, however, contained a six-nucleotidedeletion (preceded by a G-to-T transversion) within thelast exon. This polymorphism generates a prematurestop codon that truncates the C-terminal 18 amino acidsof the protein (Fig. 5C).

Based on an x-ray crystal structure of CHI (Jez et al.,2000), the C-terminal truncation of CHI1 in af is pre-dicted to affect an a-helix (a7) that does not participatedirectly in enzyme catalysis. Therefore, we performedexperiments to determine whether the mutant form ofSlCHI1 is enzymatically active. His-tagged derivativesof wild-type (CHI1WT) and mutant (CHI1af) CHI1 wereexpressed inEscherichia coli andpurified to homogeneity.

The amount of soluble CHI1af recovered from inducedEscherichia coli cellswas low in comparisonwithCHI1WT,and we found that this difference in expression resultedlargely from partitioning of CHI1af to the insoluble frac-tion (Supplemental Fig. S2A; data not shown). In vitroassays performed with affinity-purified proteins showedthatCHI1WT rapidlymetabolizes the substrate naringeninchalcone. In contrast, theactivityofCHI1afwasnotgreaterthan that of control reactions lacking enzyme (Fig. 5D).

We used immunoblot assays to determine whetherCHI1af accumulates in the af mutant. Polyclonal anti-bodies raised against CHI1WT reacted strongly andspecifically with a single polypeptide in wild-typeleaves (Fig. 5E). The apparent molecular mass of thisprotein, which was recovered in both supernatant andpellet fractions of crude leaf extracts, matched thepredicted size of CHI1 (24.6 kD). The anti-CHI1 anti-body did not specifically recognize proteins in thesoluble or particulate fraction from af leaves. Controlexperiments performed with recombinant enzymesshowed that the absence of immunoreactive CHI1 inaf did not result from a failure of the antibody to rec-ognize CHI1af (Supplemental Fig. S2B). These resultsindicate that the af mutation eliminates the enzymaticactivity of SlCHI1 and also prevents accumulation ofthe protein in planta.

Figure 3. af leaves are deficient in the productionof flavonoid-related metabolites. Levels of theindicated secondary metabolites were measuredin leaf-dip extracts (A) and isolated type VI glands(B). Each data point represents the mean 6 SE offour biological replicates from wild-type (WT)and af plants. Asterisks represent significant dif-ferences between wild-type and af plants (un-paired Student’s t test: *P , 0.05, **P , 0.01,***P , 0.001). nd, Not detected.


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We used chemical and genetic complementationapproaches to test whether the afmutant is impaired inCHI1-mediated conversion of naringenin chalcone tonaringenin. Consistent with a metabolic block at thisstep, supplementation (through the cut stem) of seed-lings with exogenous naringenin led to anthocyaninaccumulation in the major veins of naringenin-treatedbut not mock-treated af plants (Fig. 6A). In genetic ex-periments, we usedAgrobacterium tumefaciens-mediatedtransformation to introduce the wild-type CHI1 gene,expressed from the native CHI1 promoter, into the afmutant background. We recovered 16 independentprimary (T0) transformants that accumulated anthocy-anins in leaf and stem tissue following the transfer ofexplants from tissue culture medium to soil. Four rep-resentative anthocyanin-accumulating (AC+) lines (CHI1::CHI1-2, CHI1::CHI1-3, CHI1::CHI1-4, CHI1::CHI1-9)and one anthocyanin-deficient (AC2) line (CHI1::CHI1-20) were selected for further analysis (Fig. 6, B and C).All five of these transgenic lines tested positive by PCRfor the presence of the pCHI1::CHI1 transgene.Western-blot analysis confirmed that CHI1 protein accumulatedin the AC+ lines but not the AC2 line (Fig. 6C). Spec-trophotometric assays showed that the level of antho-cyanin in the AC+ lines was more than 5-fold that in the

af parental line and ranged from 68% to 113% of thatin wild-type leaves. The anthocyanin content ofCH1I::CHI1-20was not significantly different from thatin leaves of the af mutant. These collective findingsdemonstrate that the anthocyanin deficiency of the afmutant results from a loss of function of a single CHIisoform, SlCHI1.

SlCHI1 Modulates Terpenoid Production in Type VIGlandular Trichomes

Given the identification of af as a CHI1 mutant, wenext addressed the question of why af plants lack ofthe typical aroma of tomato foliage (Rick et al., 1976).Gas chromatography (GC)-MS was employed to ana-lyze the volatile composition of wild-type and af leavesand type VI trichomes. Analysis of leaf-dip extractsshowed that af produces less than 15% of wild-type levelsof several monoterpenes, including a-pinene, 2-carene,a-phellandrene, a-terpinene, and b-phellandrene(Fig. 7A). The levels of two sesquiterpenes, d-elemeneand b-caryophyllene, were also reduced in af leaves.Analyses of extracts from isolated type VI trichomeglands, which are the main source of tomato leaf

Figure 4. Field-grown af plants aresusceptible to natural populations ofinsect herbivores. A, Number of fleabeetles (mean 6 SE) on wild-type (WT)and af plants. The inset shows a fleabeetle feeding on a leaf of the af mu-tant. B, Number of flea beetle feedingsites (mean 6 SE; see inset in A) oneach host genotype. Data in A and Bwere determined for 30 replicateplants per genotype 9 d after trans-plantation of seedlings to the field plot.C, Number of Colorado potato beetles(CPB; mean 6 SE) on each host geno-type. Beetles were counted on 18replicate wild-type and af plants 40 dafter plants were transplanted to thefield. The inset shows a Colorado po-tato beetle feeding on the af mutant.D, Number of blister beetles (mean 6 SE)on each host genotype. Beetles werecounted on 11 replicate wild-type andaf plants 30 d after plants were trans-planted to the field. The inset shows ablister beetle feeding on the af mutant.Asterisks represent significant differ-ences between wild-type and af plants(unpaired Student’s t test: **P , 0.01,***P , 0.001). [See online article forcolor version of this figure.]





terpenoids (Kang et al., 2010a, 2010b), yielded similarresults (Fig. 7B).

To investigate the genetic basis of the terpenoiddeficiency in af trichomes, we measured the level ofb-phellandrene (the major monoterpene) in progenyfrom a cross between af and its parental line (cv RedCherry). Analysis of the anthocyanin phenotype inboth F1 and F2 (260 F2 plants) confirmed that af seg-regates as a monogenic recessive mutation (197 AC+

plants and 63 AC2 plants; x2 = 0.04, P = 0.8415) and

further showed that the AC+ and AC2 phenotypeswithin this population are unambiguous (Fig. 2). Theb-phellandrene content in type VI glands of F1 plantswas approximately one-third of that in glands from thewild-type parent but significantly greater than that ofaf plants (Supplemental Fig. S3A), suggesting that afis semidominant with respect to the terpenoid phe-notype. Analysis of b-phellandrene levels in leaf-dipextracts from 56 randomly selected F2 individuals (34AC+ plants and 22 AC2 plants) showed that the

Figure 5. The Af gene encodes CHI. A, Fine genetic mapping of Af delimited the target gene to an interval between markersSGN-U571424 and CT93 on chromosome 5. Numbers in parentheses indicate the number of recombination events identifiedbetween markers and the target gene. B, Physical map of the region defined by markers SGN-U571424 and C2_At3g55120.Black boxes indicate exons within three predicted genes (arrows). The asterisk denotes the location of the mutation identified inthe last exon of CHI1. C, Wild-type (WT) and af-derived nucleotide sequence (top) encoding the C terminus of CHI1 and thecorresponding deduced amino acid sequence (bottom). Stop codons are indicated by boldface letters and depicted in thepredicted amino acid sequence by asterisks. D, Enzymatic activity of CHI1WT and CHI1af. Purified recombinant CHI1WT andCHI1af proteins (0.125 mg) were mixed with substrate (naringenin chalcone), and the amount of the substrate was measuredspectrophotometrically (A340) at various times thereafter. Control reactions in which enzyme was omitted were performed inparallel and used to correct for nonenzymatic turnover of the substrate. E, Western-blot analysis of CHI protein levels in wild-type and af leaves. Crude leaf extract was separated into supernatant (sup.) and pellet fractions by centrifuging at 21,000g for25 min at 4˚C. The resulting supernatant (10 mg of protein) and a proportional amount of detergent-solubilized protein in thepellet fraction were blotted and probed with a polyclonal antibody against tomato CHI1 (top). The polyvinylidene difluoridemembrane was stained with Coomassie blue after western-blot analysis (bottom). Arrows denote CHI1 and the large subunitof Rubisco (RbcL).


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amount of b-phellandrene in AC+ plants was consistentlygreater than that in AC2 individuals (SupplementalFig. S3B). The mean level of b-phellandrene in AC2

(af/af) plants was 30.5% of that in AC+ (Af/2) siblings.This finding establishes a genetic linkage between theanthocyanin and terpenoid phenotypes.

The terpenoid deficiency in af trichomes could resultfrom the loss of CHI1 function or, alternatively, a sec-ondary mutation closely linked to the Af locus. To dis-tinguish between these possibilities, we measured theterpenoid content in leaves of theCHI1::CHI1 transgeniclines described above. The level of the major monoter-pene (b-phellandrene) in type VI glands isolated fromfour independent complemented (AC+) lines rangedbetween 39% and 97% of that in wild-type trichomes

Figure 6. Chemical and genetic complementation of the af mutant.A, Three-week-old af seedlings were supplied through the cut stemwith 0.1 mM naringenin (+ Nar) or a mock control (2 Mock). Antho-cyanin accumulation in leaf veins was visualized 3 d after treatment.Photographs in the bottom row (bar = 1 mm) show magnified views ofthe images in the top row (bar = 2 mm). B, Anthocyanin accumu-lation in CHI1::CHI1-complemented transgenic lines. Anthocyaninswere extracted from leaf tissue of the wild type (WT), af, four com-plemented transgenic lines (CHI1-2, CHI1-3, CHI1-4, and CHI1-9),and one noncomplemented transgenic line (CHI1-20). A photographof the resulting extract is shown above the spectrophotometricquantification of each genotype. FW, Fresh weight. C, Western-blotanalysis of CHI protein levels in CHI1::CHI1-complemented trans-genic lines. Soluble protein (20 mg) from leaves of the indicatedgenotype was blotted and probed with a polyclonal antibody againsttomato CHI1. The large subunit of Rubisco (RbcL) was used as aloading control as described in the legend to Figure 5E.

Figure 7. af leaves are deficient in trichome-borne terpenoid pro-duction. Levels of the indicated compound were measured in leaf-dipextracts (A) and isolated type VI glands (B). Each data point representsthe mean 6 SE of four biological replicates from wild-type (WT) and afplants. Asterisks represent significant differences between wild-typeand af plants (unpaired Student’s t test: *P , 0.05, **P , 0.01,***P , 0.001). FW, Fresh weight.







(Fig. 8A). Similarly, the level ofb-caryophyllene in theselines ranged between 93% and 110% of wild-type levels(Fig. 8B). In all four complemented lines, terpenoidlevelswere significantly greater than those in the congenicaf parent. The terpenoid content of glands isolated fromthe noncomplemented (AC2) control line (CHI1::CHI1-20)was comparable to that in the af mutant. Analysis of ter-penoid levels in leaf-dip extracts obtained from the samegenotypes gave similar results (Supplemental Fig. S4A).These findings indicate that the terpenoid deficiency of afleaves is caused by the defect in CHI1.

DISCUSSION

Af Encodes a CHI

Mutations affecting anthocyanin pigmentation havebeen instrumental not only as markers in classicalplant genetics but also as tools to identify flavonoid

biosynthetic genes and associated regulatory loci inmany plant species (Grotewold, 2006). Several muta-tions in tomato cause complete loss of anthocyaninpigments, which, depending on environmental condi-tions, typically accumulate in hypocotyls, stems, abaxialleaf surfaces, and cortical cells at the base of trichomes(von Wettstein-Knowles, 1967). Here, we provide sev-eral independent lines of evidence to demonstrate thatanthocyanin deficiency in the afmutant results from lossof function of an isoform (CHI1) of CHI. The small de-letion identified in the CHI1 gene from af plants abol-ishes CHI enzymatic activity and also prevents theaccumulation of CHI1 protein. Transgenic comple-mentation experiments showing that the anthocyanindeficiency is restored by expression of the wild-typeCHI1 gene from its native promoter showed that the afmutation is responsible for the defect in flavonoid pro-duction. Consistent with a biochemical role for SlCHI1in the cyclization of naringenin chalcone to the corre-sponding flavanone (naringenin), exogenous naringeninrestored anthocyanin accumulation in vascular tis-sues of the af leaves. A search of the tomato genome(http://solgenomics.net) identified seven genes anno-tated as CHI or CHI-like sequences. Phylogenetic analy-ses showed that only two of these, CHI1 and CHI2, areclosely related to canonical CHIs that catalyze thecyclization of chalcones to tricyclic (S) flavanones(Supplemental Fig. S5). The five remaining CHI-likegenes in tomato are predicted to encode polypeptidesmost closely related to noncatalytic CHI-fold familyproteins, which bind fatty acid substrates (Ngaki et al.,2012). The apparent lack of CHI2 expression, togetherwith the strong flavonoid deficiency of the af mutant,indicates that CHI1 is the major isoform for flavonoidproduction in tomato leaves. As expected, we foundthat af plants are deficient in the production of othernaringenin-derived metabolites, including rutin andseveral flavonol glycosides. The af mutant should pro-vide a useful genetic tool to assess the physiologicalroles and nutritional importance of flavonoid produc-tion in tomato. Because low levels of some flavonoids(e.g. rutin) were detected in af leaves, we cannot ex-clude the possibility that another CHI isoform plays aminor role in flavonoid biosynthesis or that naringeninchalcone is converted nonenzymatically to naringeninat very low levels (Bednar and Hadcock, 1988).

Mutations affecting the phenylpropanoid and fla-vonoid pathways can have pleiotropic effects on plantgrowth, development, and fertility (Taylor andGrotewold, 2005; Peer and Murphy, 2007; Schilmilleret al., 2009a). Although af abolishes the activity ofCHI1 and eliminates anthocyanin production, obviousdefects in the growth and development of the mutantwere not apparent. This finding is consistent with theview that flavonoids are not essential for the normalgrowth and development of tomato under optimizedgrowth conditions.We did notice, however, that the fruitsize of af plants was smaller than that of cv Red Cherry,which is the parental line employed for the x-ray muta-genesis used to generate themutant (Burdick, 1958). This

Figure 8. Terpenoid accumulation in type VI glands from CHI1::CHI1transgenic lines. Levels of b-phellandrene (A) and b-caryophyllene (B)were measured in type VI glands isolated from leaves of the indicatedgenotypes. Transgenic lines (T2 generation) were homozygous for thetransgene and correspond to the same lines described in the legend toFigure 6. Each data point represents the mean 6 SE of three indepen-dent plants per line. Asterisks represent significant differences betweenaf and other genotype plants (unpaired Student’s t test: **P , 0.01,***P , 0.001). WT, Wild type.


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observation raises thepossibility that the “wild-type” line(LA0337; cv Red Cherry) that we used for our studies isnot isogenic with the af accession LA1049. These consid-erations, togetherwith the fact that the x-raymutagenesisis likely to have introduced othermutations, highlight thenecessity of using genetically complemented lines forcomparative analyses of af-related phenotypes.The crystal structures of the functional monomers of

CHI from alfalfa (Medicago sativa) and Arabidopsis(Arabidopsis thaliana) have provided detailed informa-tion about the active-site topology of the enzyme,whichappears to be highly conserved in a wide range of plantspecies (Jez et al., 2000; Ngaki et al., 2012). As expected,residues involved in enzyme catalysis and the formationof the active site are well conserved in tomato CHI1(Supplemental Fig. S6). Although theC-terminala-helix(a7) that is truncated by the af mutation does not par-ticipate directly in substrate binding or catalysis, it doesform an a-helical segment (helix-turn-helix a6-a7) thatprovides structural support to the enzyme’s active site(Jez et al., 2000; Ngaki et al., 2012). Our results show thatthis mutation not only abolishes the in vitro activity ofthe enzyme but also prevents the accumulation of theprotein in af tissues. Accordingly, af is a true null mutantwith respect toCHI1 function.Our ability to detectCHI1transcripts but not the corresponding CHI1 protein in afleaves suggests that truncation of a7 destabilizes theprotein in planta.

Modulation of Terpenoid Production by CHI1

Previous studies showed that af plants, unlike severalother anthocyanin-deficient mutants tested, are highlysusceptible to attack by native populations of insectherbivores (Rick et al., 1976). Of particular interest wasthe finding that increased susceptibility of af plants toinsect attack was associated with a “lack of foliagearoma” and a striking reduction in the density of typeVItrichomes. Based on the results of genetic cosegregationtests, Rick et al. (1976) concluded that the anthocyanindefect and altered reactivity to herbivore attack aremostlikely conditioned by a single gene (af). Our resultsconfirm and extend thesefindings in severalways. First,we demonstrate that glandular trichomes from af leavesare deficient in monoterpenes and sesquiterpenes thatare derived from the MEP and mevalonic acid path-ways, respectively (Schilmiller et al., 2009a, 2010). Thispleiotropic metabolic defect explains the lack of leafvolatiles and presumably accounts for the aroma-related phenotype of af leaves. We also found that thesize and density of type VI glands on af leaves are re-duced in comparison with wild-type plants. Under ourstandard growth conditions, the reduction in trichomedensity was not as severe as that reported by Rick et al.(1976). These differences may reflect plant growth con-ditions (e.g. light intensity) or plant developmentalstage, which can have strong quantitative effects on tri-chome characteristics. Finally, ourfield studies performedin East Lansing, Michigan, like those conducted in Davis,

California (Rick et al., 1976), showed that the afmutant ishighly susceptible to attack by native populations of fleabeetles (Epitrix spp).Wealsoobserved that other species ofherbivorous beetles were more prevalent on af comparedwith wild-type plants grown in the same test plot. In-creased susceptibility to insect herbivores has been dem-onstrated for other trichomemutants of tomato, includinghairless and odorless2, which are also deficient in the pro-duction of trichome-borne terpenoids (Kang et al., 2010a,2010b). These collective observations are consistent withthe view that tomato leaf volatiles act as repellents ofherbivorous beetles (Rick et al., 1976) and,more generally,highlight the importance of glandular trichomes in medi-ating tomato-herbivore interactions (Kennedy, 2003; Liet al., 2004; Howe and Jander, 2008; Peiffer et al., 2009;Bleeker et al., 2012).

Given the well-established role of CHI in flavonoidbiosynthesis and the distinct nature of flavonoid andterpenoid metabolic pathways, the ability of the wild-type CHI1 gene to complement the terpenoid-deficientphenotype of af was unexpected. Indeed, the pleiotro-pic phenotypes of af led De Jong et al. (2004) to proposethat af likely defines a regulatory gene such as TRANS-PARENT TESTA GLABRA1, which controls bothanthocyanin production and trichome developmentin Arabidopsis (Walker et al., 1999). There are severalhypotheses to explain why the loss of CHI1 results indecreased production of terpenoids in type VI glands.For example, a flavonoid pathway intermediate or endproduct whose synthesis depends on CHI1 may pro-mote, either directly or indirectly, terpenoid production.This idea is consistent with studies showing that fla-vonoids can modulate gene expression and varioushormone responses (Peer and Murphy, 2007; Ringliet al., 2008; Pourcel et al., 2013).

An alternative hypothesis is that the accumulation ofnaringenin chalcone (the substrate of CHI1) or anotherupstream pathway intermediate inhibits terpenoidproduction in gland cells. This potential mechanism ofmetabolic cross regulation is analogous to the recentlydiscovered role of a MEP pathway intermediate(methylerythritol cyclodiphosphate) in controlling theexpression of stress-responsive nuclear genes (Xiaoet al., 2012). Naringenin chalcone, like other open-chainflavonoids within the chalcone family, possesses as anelectrophilic a,b-unsaturated carbonyl group that con-tributes to the broad biological activities of chalcones,including the inhibition of various biosynthetic andregulatory proteins (Sahu et al., 2012).We speculate thatreduced CHI activity in glandular trichomes of the afmutant may lead to increased levels of naringeninchalcone, which could potentially react with glutathi-one to alter cellular redox status ormodifyCysgroups inproteins. In this context, it is noteworthy that naringeninchalcone accumulates in tomato fruit peel, indicatingthat CHI is a rate-limiting step in flavonol biosynthesisin this tissue (Muir et al., 2001). Similarly, regulation ofCHI1 expression in type VI glands by developmental orenvironmental cues could provide a mechanism tocontrol metabolic flux through both the flavonoid and






terpenoid pathways. Measurement of naringenin chal-cone levels in typeVI glands fromwild-type andmutantlines will provide a critical test of this hypothesis. It isalso possible that the regulatory effect of CHI1 on ter-penoid production is not dependent on isomerase ac-tivity per se but rather on the ability of CHI1 to bind arelated small molecule. Interestingly, one of the mosthighly expressed genes in hop (Humulus lupulus) glan-dular trichomes encodes a CHI-like protein that is in-active as a CHI (Wang et al., 2008).

Our genetic analyses suggested that although af isfully recessive with respect to the anthocyanin pheno-type, the mutation appears to be semidominant withrespect to terpenoid production in type VI trichomes.Given our uncertainty about the genetic relationshipbetween afandcvRedCherry (see above), this conclusionawaits confirmation through detailed characterizationof genetically complemented af lines. Nevertheless, a semi-dominant effect of af on terpenoid production wouldsuggest that a single functional allele of CHI1 is insuffi-cient to meet the metabolic demand for terpenoid pro-duction in cells of the trichomegland.Haploinsufficiencyin plants often involves multiprotein complexes associ-ated with metabolic processes (Meinke, 2013). Indeed,there is evidence that CHI and other flavonoid biosyn-thetic enzymes assemble into multiprotein complexes(Burbulis andWinkel-Shirley, 1999). The dual cytosolic/nuclear localization of CHI has also led to the suggestionthat CHI-derived compounds may regulate gene ex-pression in Arabidopsis (Saslowsky et al., 2005). Thesecollective studies raise the possibility that CHI1 maymodulate terpenoidproduction through interactionwithproteins involved in terpenoidproductionorwithproteinsrequired for the expression of genes encoding terpenoidbiosynthetic enzymes. Given the prevalence of transcrip-tion factor binding to exons (Stergachis et al., 2013), wecannot exclude the possibility that the af deletionmutationindirectly affects the activity of regulatory proteins thatcontrol the expression of terpenoid pathway genes.

Flavonoid and terpenoid biosynthesis dominate themetabolic activity of glandular trichomes inmany plantspecies (Lange et al., 2000; Aziz et al., 2005; Nagelet al., 2008; Wang et al., 2008, 2009; Sallaud et al., 2009;Chatzopoulou et al., 2010; Dai et al., 2010; Schilmilleret al., 2010; McDowell et al., 2011; Bleeker et al., 2012;Tissier, 2012). Although genes encoding many of therespective biosynthetic enzymes have been identifiedand characterized in detail, it remains to be determinedhow metabolic flux through these two apparently dis-parate pathways is coordinated. That such coordinationexists is supported by studies showing that the emissionof phenylpropanoid/flavonoid- and terpenoid-derivedcomponents of scent in snapdragon (Antirrhinummajus)flowers is controlled by similar developmental andenvironmental (e.g. diurnal) cues (Dudareva et al.,2000, 2003).More intriguingly, recent transgenic studiesprovide evidence for the existence of transcription factorsthat promote metabolic flux through both pathways(Bedon et al., 2010; Ben Zvi et al., 2012). Our characteri-zation of pleiotropic metabolic defects in the tomato af

mutant is consistent with the emerging role of secondarymetabolites as lineage-specific signals that coordinateresponses to changing environments (Clay et al., 2009;Jander and Clay, 2011; Xiao et al., 2013). Currently, wehave no evidence that the afmutation impairs terpenoidproduction in cell types other than type VI glands. It ispossible, therefore, that the modulation of terpenoidmetabolism by CHI1 reflects a mechanism to coordinatethe development and biosynthetic capacity of this spe-cialized defensive structure (Kliebenstein, 2013). Theenhanced susceptibility of af plants to attack by coleop-teranherbivores further suggests thatflavonoid-terpenoidcross-regulation impacts species interactions in an eco-logically meaningful way (Stam et al., 2013). Futurestudies are needed to address themechanisms bywhichsecondary metabolites, or the enzymes that producethem, exert specific effects on seemingly unrelatedmetabolic pathways.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Tomato (Solanum lycopersicum) ‘Red Cherry’ (LA0337) was used as the wildtype for all experiments. Seeds for the wild type, af (LA1049), and the IL5-2introgression line (LA4055) were obtained from the C.M. Rick TomatoGenetics Resource Center (University of California, Davis). Seedlings weregrown in Jiffy peat pots (Hummert International) and maintained in a growthchamber as described previously (Chen et al., 2006; Kang et al., 2010b). Three-to 4-week-old plants were used for metabolite profiling and analysis of tri-chome morphology and density.

Metabolite Analysis

For quantification of anthocyanins, leaves from 4-week-old plants wereplaced in a 2-mL microcentrifuge tube (Dot Scientific) with 1 mL of methanolcontaining 1% (v/v) hydrochloric acid and incubated overnight at 4°C. An-thocyanin pigments in the resulting extract were measured spectrophoto-metrically at 530 and 657 nm. Anthocyanin levels were calculated as previouslydescribed (Rabino and Mancinelli, 1986).

Leaves from 4-week-old plants were used to prepare leaf-dip or type VItrichome extracts as described previously (Kang et al., 2010b). For the analysisof flavonoids and other nonvolatile compounds, single leaflets were placedin 1 mL of methanol:acetic acid:water (9:1:10) containing 10 mM propyl-4-hydroxybenzoate as an internal standard and incubated overnight in thedark with gentle shaking. Alternatively, a defined number of type VI glands,collected with a Pasteur pipette, were dissolved in 100 mL of the same buffer.For the analysis of terpenoid compounds, single leaflets were incubated for5 min (with gentle shaking) in 1 mL of methyl tertiary-butyl ether containingtetradecane (10 ng mL21) as an internal standard. Alternatively, type VI glandscollected with a Pasteur pipette were dissolved in 100 mL of methyl tertiary-butyl ether containing the internal standard. Quantification of metabolites wasperformed as described previously using HPLC/time-of-flight MS for non-volatile metabolites and GC-MS for volatile terpenes (Kang et al., 2010b).

Trichome Density and Morphology

A dissecting microscope (Leica MZ16) equipped with KL 2500 LCD lightsources and a Leica DFC 290 camera was used to document trichome mor-phology, size, and density. Scanning electron microscopy was performed asdescribed previously (Kang et al., 2010b). All measurements were performedon wild-type and af plants grown together in the same growth chamber.

Analysis of Insect Herbivory in Field Plots

Field experiments were performed in the summer of 2008 and 2009. Four-week-old plants grown in the greenhouse were transferred to a test plot at the


Kang et al.



Department of Plant Pathology Research Farm, Michigan State University, inEast Lansing. Wild-type and af plants were grown in alternating rows, with100-cm spacing between plants within and between rows. Plants werewatered manually every other day for 1 week, after which they were allowedto grow under natural conditions. Plants were monitored twice per week forthe presence of naturally occurring insect herbivores as well as for feedingdamage by herbivores. Leaf damage inflicted by flea beetle attack wasassessed by counting the number of feeding sites (holes) on all leaves of theplant.

Map-Based Cloning of Af

Fine-mapping of Af was performed with an F2 population derived from across between af (LA1049) and IL5-2 (LA4055) and was facilitated by the to-mato genome sequence (Tomato Genome Consortium, 2012). A population of1,255 F2 plants was scored for the anthocyanin phenotype (AC+ or AC2) andsubsequently genotyped with PCR-based conserved ortholog set markers lo-cated within the introgressed region of chromosome 5 (Tomato-EXPEN 2000map; http://solgenomics.net). Fifty-six plants showing recombination be-tween markers C2_At3g25690 and C2_At1g69210 were identified. These re-combinants were further genotyped with marker C2_At3g55120 as well asthree new markers developed in this region of chromosome 5. Specifically,S. pennellii EST sequences generated in the Solanum Trichome Project(http://www.trichome.msu.edu) were used to convert the RFLP markersT1790 and CT93, and the tomato unigene SGN-U564842, to PCR-based mark-ers. Primer sequences used for mapping are listed in Supplemental Table S1.The SGN-U571424 marker is located in the last exon of Solyc05g010300. TheC2_At3g55120 marker that cosegregated with the target locus (CHI1) islocated in the third intron of CHI1. Genomic DNA extraction and PCR con-ditions were as described previously (Kang et al., 2010a).

PCR Analysis

RNA extracted from leaves (Plant RNeasy Kit; Qiagen) was used for cDNAsynthesis (Thermoscript RT-PCR system; Invitrogen) according to the manu-facturer’s protocol. Full-length cDNAs corresponding to SlCHI1 and SlCHI2were amplified by PCR (DNA Engine Dyad Thermal Cycler; Bio-Rad) usingthe primer set (CHI1 and CHI2) listed in Supplemental Table S2. A cDNAencoding elongation factor4A was PCR amplified using the elF4A primer(Supplemental Table S2) and used as a loading control. RT-PCR (25 mL)contained 2 mL of cDNA, 1 mL of a 10 mM solution of each primer, 0.75 mL of10 mM deoxyribonucleotide triphosphate mix, 5 mL of 53 KAPA buffer,and 0.5 mL of KAPA DNA polymerase (KAPAHiFi HotStart; Kapa Bio-systems). The amplification protocol included an initial 4-min denaturationstep at 95°C, followed by 25 cycles in which the template was denatured for20 s at 98°C, annealed for 15 s at 52°C, and extended for 40 s at 72°C, followedby a final incubation for 2 min at 72°C. Amplified products were separatedon a 1% agarose gel. Full-length genomic DNA corresponding to SlCHI1 andSlCHI2 from wild-type and af plants was PCR amplified using the primer setslisted in Supplemental Table S2. The PCR-amplified fragments were cloned intothe pGEM-T-Easy plasmid vector (Promega). Automated nucleotide sequencingwas performed at the Michigan State University Genomics Technology SupportFacility (http://rtsf.msu.edu/).

Characterization of Recombinant CHI1

Plasmid pGEM-T containing the SlCHI1 cDNA isolated from wild-type oraf plants was PCR amplified using the CHI1-NS primer set (SupplementalTable S2). The resulting 0.7-kb DNA fragment was digested with NdeI andSalI, gel purified, and ligated into vector pET28b (Novagen) to generate ex-pression constructs pET-CHI1WT and pET-CHI1af, which encode the normaland af mutant forms of CHI1, respectively. These constructs were transformedinto Escherichia coli strain BL21. A single Escherichia coli colony was inoculatedinto 3 mL of Luria-Bertani medium containing 30 mg mL21 kanamycin, andthe culture was grown overnight at 37°C. A 100-mL aliquot of this culturewas used to inoculate 50 mL of Luria-Bertani medium containing 30 mg mL21

kanamycin. The culture was grown to log phase (A600 = 0.4–0.8) at 37°C, atwhich point isopropyl-thio-b-D-galactopyranoside (Sigma-Aldrich) was addedto a final concentration of 0.5 mM. The culture was grown for an additional 7 hat 20°C. Cells were harvested by centrifugation at 7,000g and resuspended in1 mL of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM

imidazole, 10 mM b-mercaptoethanol, 10% (v/v) glycerol, and 1% (v/v)Tween 20. After sonication and centrifugation, the cleared lysate was ap-plied to a nickel-affinity column (nickel-nitrilotriacetic acid agarose resin). Thecolumn was washed with 3 column volumes of lysis buffer followed by a finalwash with lysis buffer containing 35 mM imidazole. CHI1-His protein waseluted from the column with lysis buffer containing 250 mM imidazole butlacking Tween 20. Fractions containing CHI1 were pooled and buffer ex-changed to 5 mM HEPES (pH 5.5), 25 mM NaCl, and 1 mM D/L-dithiothreitolwith an Amicon Ultra-15 10-kD filter (Millipore). Purified CHI1 proteinswere quantified with the Pierce BCA Protein Assay Kit (Thermo Scientific)according to the manufacturer’s instructions. Purified recombinant CHI1enzymes were determined to be more than 95% pure as determined bySDS-PAGE.

The enzymatic activity of recombinant CHI1 was measured spectropho-tometrically as described previously (Jez and Noel, 2002). Briefly, 1-mL reac-tions containing 100 mM naringenin chalcone, 50 mM HEPES (pH 5.5), 5% (v/v)ethanol, and a known amount of purified CHI1 were incubated at 25°C. ThepH was optimized to minimize spontaneous conversion of naringenin chal-cone to naringenin. Naringenin chalcone was purchased from ChromaDex.The time-dependent decrease in A360 of reactions containing or not containingCHI1 was monitored with a DU 800 spectrophotometer (Beckman Coulter).Absorbance values recorded in the absence of enzyme were subtracted fromvalues recorded in the presence of CHI1.

Antibody Production and Western-Blot Analysis

Total leaf protein was extracted with buffer containing 100 mM Tris-HCl(pH 6.8), 20 mM EDTA, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride, and2% (v/v) 2-mercaptoethanol as described (Chen et al., 2007; Gonzales-Vigilet al., 2011). Rabbit polyclonal antibodies raised against SlCHI1 were obtainedfrom a commercial vendor (Cocalico Biologicals). Purified recombinant SlCHI1(0.5 mg of protein) was used as the antigen according to the vendor’s standardprotocol. Western-blot analysis was performed as described previously(Schilmiller et al., 2007). Anti-CHI1 antibodies were diluted 1:5,000 in TTBSbuffer (50 mM Tris [pH 7.4], 500 mM NaCl, and 0.05% Tween 20) containing1% nonfat milk. Blots were washed three times with TTBS buffer and thenincubated with a peroxidase-conjugated donkey anti-rabbit secondary anti-body (1:15,000 dilution; Thermo Scientific). CHI1 protein-antibody complexeswere visualized with the SuperSignal West Pico chemiluminescent substrate(Thermo Scientific) according to the manufacturer’s instructions.

Naringenin Treatment

Naringenin (Sigma) was supplied to af seedlings through their cut stem asdescribed previously (Howe et al., 1996). Briefly, 3-week-old seedlings grownunder standard conditions were excised at the base of the stem with a razor.The cut end of the stem was placed immediately into a 50-mL conical tubecontaining a solution of 0.1 mM naringenin (dissolved in 0.6% [v/v] ethanol).Control af seedlings were placed into the same solution lacking naringenin.Seedlings were incubated in this condition for 3 d prior to photographingleaves.

Transgenic Plants

Plasmid pGEM-T containing an SlCHI1 genomic clone was reamplifiedwith the CHI1-XS primer set (Supplemental Table S2). The resulting fragmentwas digested with XbaI and SacI to release a 2.4-kb fragment that was clonedinto the XbaI and SacI sites of the pBI-TS binary vector, which contains thecauliflower mosaic virus 35S promoter and the nopaline synthase terminator(Koo et al., 2006; Schilmiller et al., 2007), to generate pBI-35S::CHI1. The CHI1promoter region (approximately 1.5 kb) was amplified from a tomato bacterialartificial chromosome clone (LE-HBa0147F10) by PCR (KAPAHiFi HotStart;Kapa Biosystems) using the CHI1Pro-SX primer set (Supplemental Table S2).The resulting fragment was digested with SbfI and XbaI and cloned into theSbfI and XbaI sites of pBI-35S::CHI1 to replace 35S promoter. The resultingconstruct (pBI-CHI1::CHI1) was introduced into Agrobacterium tumefaciensstrain AGLO and used to transform af cotyledon explants as described pre-viously (Li et al., 2003, 2004). The presence of the transfer DNA insert in in-dependent primary (T0) transformants was confirmed by PCR using theM13R-CHI1Pro primer set (Supplemental Table S2) to amplify a fragmentspanning the pBI-TS vector sequence and the CHI1 promoter region.




http://solgenomics.net

http://www.trichome.msu.edu





http://rtsf.msu.edu/







Regenerated T0 transgenic plants containing the CHI1::CHI1 transgene werepotted in soil and transferred to a growth chamber for preliminary bio-chemical analyses. These plants were subsequently transferred to a green-house for the collection of seeds (T1 generation) from individual lines. T1seedlings exhibiting an AC+ phenotype were selected from lines in which theratio of AC+ to AC2 was 3:1. These AC+ plants were grown in a greenhousefor the collection of T2 seeds. Lines in which all T2 progeny exhibited the AC+

phenotype were selected as being homozygous for the transgene.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. RT-PCR for CHI1 and CHI2 from wild-type andaf leaves.

Supplemental Figure S2. Affinity purification of CHI1WT and CHI1af

expressed in Escherichia. coli.

Supplemental Figure S3. b-Phellandrene levels in F1 and F2 plants de-rived from a cross between the afmutant (LA1049) and its reported wild-type parent (cv Red Cherry; LA0337).

Supplemental Figure S4. Terpenoid accumulation on the leaf surface ofCHI1::CHI1 transgenic lines.

Supplemental Figure S5. Phylogenetic tree of CHI and CHI-fold proteinsfrom cultivated tomato.

Supplemental Figure S6. Amino acid sequence alignment of CHIs fromseveral plant species.

Supplemental Table S1. Description of PCR-based markers for geneticmapping.

Supplemental Table S2. Description of oligonucleotide primers used forPCR.

ACKNOWLEDGMENTS

We thank Eric Czuprenski, Razi Shaha, and Eric Kastory for assistancewith plant growth and genetic mapping experiments,Dr. Cornelius Barry forhelpful advice with genetic mapping, Ray Hammerschmidt and Gary Zehr forproviding field space, the Michigan State University Center for AdvancedMicroscopy for assistance with scanning electron microscopy, the MichiganState University Mass Spectrometry and Metabolomics Core for GC-MS re-sources, the SOL Genomics Network for bacterial artificial chromosome clonesand bioinformatics resources, and the C.M. Rick Tomato Genetics ResourceCenter (University of California [Davis]) for kindly providing tomato seedstocks.

Received December 2, 2013; accepted January 13, 2014; published January 14,2014.

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the flavonoid biosynthetic enzyme chalcone … flavonoid biosynthetic enzyme chalcone isomerase...

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