The Tyrosine Aminomutase TAM1 Is Required for b-TyrosineBiosynthesis in Rice

Jian Yan,a,1 Takako Aboshi,b,2 Masayoshi Teraishi,b Susan R. Strickler,a Jennifer E. Spindel,c Chih-Wei Tung,c,3

Ryo Takata,b Fuka Matsumoto,b Yoshihiro Maesaka,b Susan R. McCouch,c Yutaka Okumoto,b Naoki Mori,b

and Georg Jandera,4

a Boyce Thompson Institute for Plant Research, Ithaca, New York 14853bGraduate School of Agriculture, Kyoto University, Kyoto 808-8502, Japanc Section of Plant Breeding and Genetics, School of Integrated Plant Sciences, Cornell University, Ithaca, New York 14853

ORCID IDs: 0000-0001-8597-9841 (J.Y.); 0000-0002-0121-0048 (S.R.S.); 0000-0002-9675-934X (G.J.)

Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plantspecies. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-b-tyrosine, anisomer of the common amino acid (S)-a-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar.Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with b-tyrosine being most prevalent intemperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed toencode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A pointmutation in TAM1 eliminated b-tyrosine production in Nipponbare. Rice cultivars that do not produce b-tyrosine havea chromosome 12 deletion that encompasses TAM1. Although b-tyrosine accumulation was induced by the plant defensesignaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects atphysiologically relevant b-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicotplants was inhibited by concentrations as low as 1 mM. As b-tyrosine is exuded into hydroponic medium at higher concentrations,it may contribute to the allelopathic potential of rice.

INTRODUCTION

Plants use a large variety of physical and chemical defenses toprotect themselves against pests, pathogens, and competitorsin the environment. Many species produce non-protein aminoacids, a diverse class of metabolites that can have toxic anddeterrent properties (Fowden, 1981; Bell, 2003). Although somenon-protein amino acids, e.g., homoserine and ornithine, areessential components of primary metabolism, most of the hun-dreds of known non-protein amino acids are secondary metab-olites with sporadic distribution in the plant kingdom and likelydefensive functions. Many non-protein amino acids are toxicanalogs of the standard 20-protein amino acids. Incorporation ofnon-protein amino acids can result in enzyme inhibition or mis-folding of proteins. In other instances, non-protein amino acidsserve as substrates for the biosynthesis of larger defensivemetabolites.

b-Amino acids, which have the amino group shifted from thea-carbon to the adjacent b-carbon, are relatively rare in bi-ological systems. However, they have been found as compo-nents of nonribosomal peptides, bacterial antibiotics, anticancerdrugs, and other natural products (Kudo et al., 2014). In partic-ular, the b-tyrosine and b-phenylalanine have been investigatedas building blocks for the biosynthesis of several medically rel-evant metabolites. b-Phenylalanine is a component of andrimid,an antibiotic from the bacterium Pantoea agglomerans(Ratnayake et al., 2011), as well as the anticancer drug taxolfrom yew trees (Taxus sp; Walker et al., 2004). b-Tyrosine isa pathway intermediate in the biosynthesis of several bacterialantibiotics: C-1027 from Streptomyces globisporus (Liu et al.,2002), myxovalargin from Myxococcus fulvus (Krug and Müller,2009), chondramide C from Chondromyces crocatus (Rachidet al., 2007), kedarcidin from Streptoalloteichus sp (Van Lanenet al., 2005), and maduropeptin from Actinomadura madurae(Van Lanen et al., 2007).b-Amino acids in microorganisms and plants can be formed

from a-amino acids by the action of aminomutases, whichcatalyze the reversible exchange of an amine group and a hy-drogen on adjacent carbons (Lohman and Shen, 2012). Oneclass of aminomutases has structural similarity to phenylalanineaminolyase (PAL) and tyrosine aminolyase (TAL), which catalyzethe deamination of L-phenylalanine and L-tyrosine, respectively,to form (E)-cinnamic acid and (E)-4-hydroxycinnamic acid. Forinstance, a PAL-like phenylalanine aminomutase (PAM) in Tsugacanadensis isomerizes (S)-a-phenylalanine to the (R)-b-isomer(Feng et al., 2011), and a TAL-like tyrosine aminomutase (TAM)from C. crocatus isomerizes (S)-a-tyrosine to (R)-b-tyrosine

1Current address: Key Laboratory of Plant Resources Conservation andSustainable Utilization, South China Botanical Garden, Chinese Acad-emy of Sciences, Guangzhou 510650, China.2 Current address: Faculty of Agriculture, Yamagata University, Tsuruoka,Yamagata 997-8555, Japan.3 Current address: Department of Agronomy, National Taiwan University,Taipei 10617, Taiwan.4 Address correspondence to gj32@cornell.edu.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Susan R.McCouch (srm4@cornell.edu), Yutaka Okumoto (okumoto.yutaka.4w@kyoto-u.ac.jp), and Georg Jander (gj32@cornell.edu).www.plantcell.org/cgi/doi/10.1105/tpc.15.00058

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(Wanninayake and Walker, 2013). The aminolyases and theaminomutases of this enzyme family both use a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) cofactor in the active siteto catalyze the reaction, either removing an amino group orshifting it to an adjacent carbon atom.

Like other plants that have been investigated, rice (Oryzasativa) produces a large variety of defense-related secondarymetabolites (Chen et al., 2014; Matsuda et al., 2015). Some ofthese metabolites have known allelopathic effects, i.e., they in-hibit the growth of neighboring plants after being exuded intothe soil or paddy water (Olofsdotter, 2001b). There is consider-able natural variation in this phenotype, with ;4% of tested ricecultivars being classified as strongly allelopathic (Belz, 2007). Insome cases, specific allelopathic compounds, including the di-terpenes momilactone A and B (Kato-Noguchi and Ino, 2003;Kato-Noguchi et al., 2010), phenolic acids (Chung et al., 2001;Rimando et al., 2001; Chung et al., 2002), and flavones (Konget al., 2004), have been identified in rice exudates and/or ex-tracts. Transposon knockout mutations of rice diterpene syn-thases reduced momilactone accumulation and compromisedthe allelopathic effects of rice root exudates on root growth oflettuce (Lactuca sativa) and barnyard grass (Echinochloa crus-galli ) (Xu et al., 2012a). Similarly, reducing rice PAL expressionusing an RNA interference approach attenuated the allelopathic

inhibition of barnyard grass root growth, suggesting the in-volvement of phenolic compounds (Fang et al., 2013). Given themultiple growth-inhibiting compounds that have been identifiedin rice exudates, it is likely that more than one biosyntheticpathway is required to generate strong allelopathic properties.Nevertheless, natural variation in the production of allelopathiccompounds has the potential to be incorporated into breedingprograms for production of rice cultivars that are naturally weed-suppressive (Olofsdotter, 2001a; Kong et al., 2011). Suchbreeding efforts will be facilitated by the identification of spe-cific rice genes that catalyze the formation of allelopathic com-pounds.Rice genome sequencing projects (International Rice Genome

Sequencing Project, 2005; Xu et al., 2012b), metabolic diversityamong cultivars, and new genetic mapping approaches facilitatethe identification of genes involved in the biosynthesis of sec-ondary metabolites. Genome-wide association studies demon-strate that, for many secondary metabolites, natural variationamong rice cultivars is associated with a small number ofquantitative trait loci (QTL) that have large effects on metaboliteabundance (Chen et al., 2014; Matsuda et al., 2015). Such large-effect QTL, in combination with information about the types ofenzymes that would be involved in the biosynthesis of particularmetabolites, make it possible to progress rapidly from the

Figure 1. Detection of b-Tyrosine in Rice.

(A) HPLC fluorescence detection chromatogram of derivatized rice foliar amino acids. Red, Nipponbare; black, Kasalath; green, amino acid standards(20 protein amino acids, meta-tyrosine, and norleucine). The arrow indicates the induced peak of interest.(B) Structures of (S)-a-tyrosine and (R)-b-tyrosine.(C) Relative abundance of (R)-b-tyrosine in dry rice seeds and rice seedlings, with and without jasmonic acid treatment. Mean 6 SE of n = 3. *P < 0.05,t test comparing control and jasmonic acid-treated samples.(D) Cultivars that contain b-tyrosine are relatively more abundant in a collection of 50 Japanese rice cultivars (JRC) than in a worldwide collection of69 rice cultivars (world rice collection [WRC]) from the NIAS Genebank (http://www.gene.affrc.go.jp). *P < 0.05, x2 test. b-Tyrosine data for each of the119 tested rice cultivars are in Supplemental Figure 4.

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identification of previously unknown rice metabolites to thecharacterization of genes and enzymes that are involved in theirbiosynthesis.

Here, we describe the identification of b-tyrosine, which ismore commonly associated with the biosynthesis of medicallyrelevant antibiotics in bacteria, in the vegetative tissue andseeds of rice, one of the world’s most important food crops. Onthe basis of natural variation among rice cultivars, we discov-ered a rice tyrosine aminomutase that converts the commonprotein amino acid tyrosine (a-tyrosine) into b-tyrosine. Consis-tent with a possible defensive function, we show that Pseudo-monas syringae growth is inhibited by b-tyrosine, and seedlings ofdicot species are much more sensitive to exogenous b-tyrosinethan rice and other tested monocots.

RESULTS

In a targeted effort to find previously unknown rice defensivemetabolites, we conducted a screen for non-protein amino acidsthat are induced by jasmonic acid, a plant defense signalingmolecule that is commonly associated with enhanced insectresistance. Liquid chromatography (LC) assays of rice cultivarNipponbare seedlings identified a peak in the LC chromatogramthat was distinct from the twenty protein amino acids (Figure1A). The area of this unknown peak increased in seedlings thathad been treated with jasmonic acid. Another tested rice

cultivar, Kasalath, did not appear to contain the compoundof interest (Figure 1A).Gas chromatography-mass spectrometry (GC-MS) and liquid

chromatography-mass spectrometry assays of Nipponbare andKasalath samples, as well as comparisons to authentic stand-ards (Supplemental Figures 1 and 2) showed that the unknownpeak in the chromatogram is b-tyrosine, an isomer of thecommon protein amino acid tyrosine (a-tyrosine) (Figure 1B).Liquid chromatography-tandem mass spectrometry comparisonof the purified Nipponbare metabolite samples to authentic (R)-b-tyrosine and (S)-b-tyrosine showed that the endogenous ricecompound is (R)-b-tyrosine (Supplemental Figure 3).Although b-tyrosine was originally detected in Nipponbare

leaves, subsequent assays showed that it is present in all partsof the rice seedlings, with the highest concentration in the leaves(Figure 1C). Jasmonic acid treatment of the seedlings increasedb-tyrosine content in the leaves, roots, and imbibed seed ma-terial. Dry rice seeds from plants that had not been elicited withjasmonic acid also contained significant amounts of b-tyrosine.When rice seedlings were grown hydroponically, b-tyrosine wassecreted into the medium.Unlike Nipponbare, the Kasalath rice cultivar does not con-

tain b-tyrosine, with or without jasmonic acid treatment. To in-vestigate the prevalence of b-tyrosine in rice more broadly, wescreened cultivars from a Japanese rice collection and a worldrice collection that were available from the National Institute of

Figure 2. Incorporation of [13C915N]Tyrosine into b-Tyrosine after Uptake via Leaf Petioles.

(A) Unlabeled and 13C915N-labeled tyrosine content in rice leaves that had taken up a 1 mM [U-13C9,

15N]tyrosine solution via the petioles. Mean 6 SE ofn = 3.(B) GC-MS chromatograms of unlabeled (m/z 218 and 280) and 13C9

15N-labeled (m/z 221and 289) tyrosine fragments, derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA).(C) Unlabeled and 13C9,

15N-labeled b-tyrosine content in rice leaves that had taken up a 1 mM [13C915N]tyrosine solution via the petioles. Mean 6 SE of

n = 3. ND, not detected.(D) GC-MS chromatograms of unlabeled (m/z 266 and 324) and 13C9,

15N-labeled (m/z 274 and 334) b-tyrosine fragments, derivatized with MSTFA.

b-Tyrosine in Rice 3 of 14

Agrobiological Sciences (NIAS) Genebank (https://www.gene.affrc.go.jp/databases-core_collections_en.php). Among 50 testedJapanese rice cultivars, 32 contained b-tyrosine (SupplementalFigure 4A). Including Nipponbare, eight out of 69 tested cultivarsin the world rice collection contained b-tyrosine (SupplementalFigure 4B), significantly fewer than in the Japanese rice collection(Figure 1D). Based on their phylogenetic relationship, rice cultivarscan be broadly classified into the indica, aus, aromatic, temperatejaponica, and tropical japonica populations (Garris et al., 2005).b-Tyrosine production was significantly overrepresented in thejaponica cultivars, with 36 of the 40 b-tyrosine-containing culti-vars coming from the two japonica groups. The presence ofb-tyrosine in the Korean cultivar Milyang 23 (Supplemental Figure4B) may be explained by its mixed indica and japonica ancestry.Nipponbare, a temperate japonica cultivar, was somewhat un-usual in having significantly increased b-tyrosine production inresponse to jasmonic acid treatment. Among the other 39b-tyrosine-producing cultivars, only three exhibit a significantb-tyrosine increase in response to jasmonic acid (SupplementalFigure 4).

As b-tyrosine can be formed through isomerization of a-tyrosine by PAL-like enzymes in bacteria (Christenson et al.,2003; Krug and Müller, 2009; Wanninayake and Walker, 2013),we conducted stable isotope labeling experiments to determinewhether this is also the case in rice. Exogenously added [13C915N]tyrosine was taken up efficiently via the petioles of detachedleaves of both Nipponbare, which produces b-tyrosine, andIR64, which does not (Figures 2A and 2B). Nipponbare, but notIR64, accumulated [13C9

15N]b-tyrosine in response to the up-take of [13C9

15N]tyrosine from the medium (Figures 2C and 2D).All isotope-labeled carbon atoms and the nitrogen were

conserved in the conversion of tyrosine to b-tyrosine by the riceseedlings, indicating TAM activity. More details of the chro-matographic analysis that confirmed the synthesis of [13C915N]b-tyrosine are provided in Supplemental Figures 5 and 6.Together, these results show that Nipponbare, but not IR64,contains TAM enzymatic activity that converts tyrosine tob-tyrosine.Recombinant inbred lines (RILs) derived from Nipponbare and

IR64 were used to map b-tyrosine accumulation. Among 147RILs derived from the Nipponbare x IR64 cross, 55 accumulatedb-tyrosine, and among 147 RILs derived from the IR64 x Nip-ponbare cross, 47 accumulated b-tyrosine (Supplemental Figure7). Genetic mapping using this population and scoring b-tyrosineas a binary trait (presence or absence) identified a highly signifi-cant QTL on chromosome 12 and a marginally significant QTL onchromosome 8 (Figure 3A). Crosses between indica and japonicarice almost always result in segregation distortion because of thenumerous sterility genes that segregate when these two varietalgroups are crossed (Spindel et al., 2013). Higher prevalence of theIR64 allele on chromosome 12 in the current mapping population(Supplemental Figure 8) can account for the fact that only aboutone-third of the RILs, rather than the expected 50%, containb-tyrosine. Mapping b-tyrosine as a quantitative trait, using onlythe 102 RILs that contain b-tyrosine, did not identify any addi-tional QTL (Figure 3A).The chromosome 12 QTL localized the presumed b-tyrosine

biosynthetic gene to an interval containing 25 genes annotated bythe Michigan State University Rice Genome Annotation Project(release 7; http://rice.plantbiology.msu.edu/; Kawahara et al., 2013;Supplemental Table 1). Among these genes, LOC_Os12g33610 (In-ternational Rice Genome Sequencing Project gene Os12g0520200),

Figure 3. Genetic Mapping of Rice b-Tyrosine QTL.

(A) b-Tyrosine QTL mapping with IR64 x Nipponbare recombinant inbred lines. Blue, binary model; red, quantitative model; black, combined.(B) Detection of LOC_Os12g33610 by PCR of genomic DNA from Nipponbare and IR64.(C) Three chromosome segment substitution lines have the Kasalath genotype at markers C1060 and C449 on the chromosome inserted in theNipponbare genetic background. These three lines do not produce b-tyrosine.

4 of 14 The Plant Cell

which was annotated as a PAL-like gene, stood out as a likelyTAM candidate because of the well-established sequence sim-ilarity between MIO cofactor-containing aminomutases andaminolyases (Lohman and Shen, 2012). PCR amplification withprimers designed to amplify LOC_Os12g33610 produced a prod-uct from Nipponbare genomic DNA, but not IR64 genomic DNA(Figure 3B).

Chromosome segment substation lines (CSSLs) that containsegments of Kasalath, which does not produce b-tyrosine (Fig-ure 1A), integrated into the Nipponbare genome (SupplementalFigure 9; Sasaki et al., 2013) were used in a second geneticmapping approach. Among 53 tested CSSLs, 50 had a pheno-type similar to that of Nipponbare, accumulating b-tyrosine andgenerally exhibiting higher accumulation in the leaves after jas-monic acid treatment (Supplemental Figure 9). Three CSSLs didnot have any detectable foliar b-tyrosine (Figure 3C). Consistentwith a role for LOC_Os12g33610 in b-tyrosine biosynthesis, allthree of these CSSLs had an introgression of the Kasalath ge-nome that covered the LOC_Os12g33610 region of chromo-some 12.

To confirm that LOC_Os12g33610 encodes TAM activity, thegene was transiently expressed in Nicotiana benthamiana, whichnormally does not contain b-tyrosine (Supplemental Figure 10).Transient expression over 6 d in N. benthamiana progressivelyincreased the amount of b-tyrosine, whereas plants transformedwith a control construct accumulated no detectable b-tyrosine

in the same time period (Figure 4A). For in vitro enzyme assays,the LOC_Os12g33610 protein was purified from N. benthamianaand was shown to catalyze the conversion of tyrosine to b-tyrosine(Figure 4B). The amount of b-tyrosine that accumulated in-creased with the reaction time (Figure 4C), the enzyme con-centration (Figure 4D), and the substrate concentration (Figure4E). The temperature optimum of the in vitro reaction was 29°C(Figure 4F) and the pH optimum was 10.5 (Figure 4G). The Km for

Table 1. Point Mutations in Rice TAM1 Eliminate b-TyrosineProduction in Vitro

Base Pair Change Amino Acid Change b-Tyrosine

Wild type Wild type YesG(371)A-A(400) deletion NoC-1246-T Q-560-M NoG(371)A-A(400); C-1246-T deletion; Q-560-M NoA-794-G Glu-265-Gly NoT-1409-C Val-470-Ala NoT-932-C Met-311-Thr NoA-464-G Asn-155-Ser NoT-249-C, T-353-C Ser-83-Ser, Leu-118-Pro NoG-640-A, C-724-T Ala-214-Thr, Pro-242-Ser NoC-2040-T Ser-680-Ser YesA-2068-G, T-2069-C Asn-690-Ser Yes

Figure 4. The Rice Gene LOC_Os12g33610 Encodes a Functional Tyrosine Aminomutase That Produces b-Tyrosine.

(A) Accumulation of b-tyrosine in N. benthamiana with transient expression of LOC_Os12g33610. Control, empty vector; ND, not detected. Mean 6 SE

of n = 3.(B) HPLC chromatogram from in vitro tyrosine aminomutase activity assays. Black, b-tyrosine standard; red, Tris buffer + tyrosine + enzyme; blue, Trisbuffer + tyrosine; green, Tris buffer+ enzyme. Inset picture shows an immunoblot. L, ladder; CP, crude protein; PP, His-tag purified protein.(C) to (E) Accumulation b-tyrosine in vitro increases with the time of the reaction (C), the enzyme concentration (D), and the substrate concentration (E).(F) and (G) The temperature optimum for the reaction is 29°C (F) and the pH optimum is 10.5 (G). Mean 6 SE of n = 3.

b-Tyrosine in Rice 5 of 14

tyrosine was calculated to be 0.58 mM and the Vmax was 5.1 31026 mM/s (Supplemental Figure 11). Several small deletionsand point mutations that were found during the cloning andsequence confirmation of LOC_Os12g33610 eliminated TAMenzymatic activity in N. benthamiana (Table 1). Many of thesepoint mutations are in regions of the protein that are highlyconserved in other PAL-like enzymes (Lohman and Shen, 2012).No cinnamic acid or b-phenylalanine was detected using phe-nylalanine as the substrate, indicating that LOC_Os12g33610does not encode a PAL or PAM enzyme (Supplemental Figure12). Similarly, the lack of 4-hydroxycinnamic acid productionfrom tyrosine showed that the enzyme is not a TAL. Therefore,we propose TAM1 as the name for the protein product ofLOC_Os12g33610.

To further confirm the in vivo function of TAM1, a TILLING(targeted induced localized lesions in genomes) approach(McCallum et al., 2000) was used to identify point mutations inthe endogenous Nipponbare gene. TAM1 DNA segments from1920 N-methyl-N-nitrosourea (MNU)-mutagenized plants wereamplified by PCR and cleaved with CelI nuclease to identifymismatches. Three sites with the GC-to-AT transition that istypical of MNU mutagenesis were identified, causing the aminoacid changes Thr-112-Ile, Gln-416-Stop, and Thr-560-Met(Supplemental Figure 13). Of the three point mutations, onlyGln-416-Stop eliminated b-tyrosine accumulation in Nippon-bare. Nevertheless, this targeted mutagenesis approach con-firms that TAM1 is necessary for the production of b-tyrosine inrice.The Nipponbare rice genome contains nine genes that are

annotated as PALs or PAL-like genes (http://rice.plantbiology.msu.edu/; Supplemental Data Set 1; Figure 5). Protein se-quence comparisons showed that LOC_Os11g48110 has 95%amino acid sequence identity to TAM1. However, in vitro as-says with the LOC_Os11g48110 protein product did not showany TAM activity. Similarly, protein products of three otherrice PAL genes (LOC_Os02g41680, LOC_Os04g43800, andLOC_Os02g41650) were tested in vitro and did not exhibit TAMactivity.Alignment of available Kasalath genomic sequence data

(Sakai et al., 2014) against the Nipponbare genome sequence(International Rice Genome Sequencing Project, 2005) showedthat Kasalath has a 30-kb deletion that includes the TAM1 re-gion on chromosome 12. For several of the tested cultivars fromthe Japanese and world rice cultivar collections, significantamounts of genomic sequence data are available in the NCBIshort read archive (http://www.ncbi.nlm.nih.gov/sra; accession

Figure 5. Cladogram of Nipponbare Proteins That Are Annotated asPhenylalanine Ammonia Lyases.

Gene names are from the Michigan State University rice annotationproject (http://rice.plantbiology.msu.edu/). The phylogenetic tree wascreated using MEGA6.06. Numbers at branches indicate bootstrap val-ues. For the amino acid sequence alignments, see Supplemental DataSet 1.

Table 2. TAM1 Gene Distribution and b-Tyrosine Production in Rice Cultivars

Cultivar Variety Group Origin b-Tyrosine Present TAM1 PresentTAM1 Coverage/GenomeCoverage NCBI/DDBJ Number

IR64 indica Philippines No No 0.000 SRR1328248Mansaku japonica Japan Yes Yes 1.056 SRR063630Kameji japonica Japan Yes Yes 1.011 DRR003658Omachi japonica Japan Yes Yes 1.013 DRR000720Nipponbare japonica Japan Yes Yes 1.000 SRR1043564Kasalath indica India No No 0.134 DRR008446Jena 035 indica Nepal Yes Yes 1.117 SRR1015932Keiboba indica China No No 0.000 SRR1015906Shoni indica Bangladesh No No 0.131 SRR1015933Tupa 121-3 indica Bangladesh No No 0.037 SRR1015927Surjamukhi indica India No No 0.066 SRR1015924Ratul indica India No No 0.131 SRR1015920Badari Dhan indica Nepal No No 0.035 SRR1015922Kluheenati indica Sri Lanka No No 0.138 SRR1015926Jaguary japonica Brazil Yes Yes 1.045 SRR1015930Rexmont japonica USA No No 0.876 SRR1015928Urasan 1 japonica Japan No No 0.094 SRR1015931Tupa 729 japonica Bangladesh No No 0.064 SRR1016472Milyang 23 indica/japonica Rep. Korea Yes Yes 1.081 SRR1239611Deejiaohualuo indica China No No 0.000 SRR1015904Hong Cheuh Zai indica China No No 0.000 SRR1015923

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number numbers in Table 2). We analyzed these short-readsequencing data to determine the relative coverage of the TAM1gene and the rest of the rice genome. Whereas rice cultivars thatcontain b-tyrosine have sequence coverage of TAM1 that issimilar to the rest of the genome, those that do not containb-tyrosine had few or no DNA sequencing reads that align withTAM1 (Table 2). As rice has nine PAL-like genes with similarDNA sequences (Figure 5), there is occasional spurious short-read alignment to TAM1, even in cultivars that do not have thisgene. Similar to Kasalath, other rice cultivars that do not pro-duce b-tyrosine have a deletion of the TAM1 gene. A similaranalysis of 50 resequenced rice genomes (Xu et al., 2012b),which included all major groupings of cultivated rice and the wildancestors Oryza rufipogon and Oryza nivara, showed that atleast 11 are likely to contain a TAM1 gene (Supplemental Table2). Seven of the cultivars with a predicted TAM1 are in thetemperate japonica phylogenetic group. The others are two auslandraces, one tropical japonica landrace, and one isolate ofO. rufipogon.

The induction of b-tyrosine by jasmonic acid in Nipponbare,as well as the sporadic distribution among the tested rice cul-tivars, suggested that this non-protein amino acid might havea defensive function. One hemipteran and two lepidopteranspecies were used to determine whether b-tyrosine has signif-icant negative effects on insects. b-Tyrosine was taken up intotobacco (Nicotiana tabacum) leaves via the petioles, and aphidreproduction was measured on leaves containing varyingamounts of b-tyrosine. Adult Myzus persicae (green peachaphid), a broad host range aphid species that feeds on manydicots and some monocots, were placed onto the tobaccoleaves that had taken up b-tyrosine via the petioles. Aphid re-production was inhibited, but only at 1 and 4 mM b-tyrosineconcentrations (Figure 6A), which are significantly higher thanthat which was observed in rice foliar tissue (Figure 1C). Whenlarvae of black cutworms (Agrotis ipsilon) and sugarcane borers(Diatraea saccharalis), two insects that are known to feed fromrice, were placed on artificial diet containing b-tyrosine, nosignificant growth inhibition was observed (Figures 6B and 6C),even at concentrations that were much higher than those foundin rice leaves. Thus, it is unlikely that b-tyrosine has an insect-deterrent function in rice.Pseudomonas syringae, a common pathogen of multiple plant

species, was used as a representative bacterial species to studythe effects of b-tyrosine. Whereas P. syringae was able to growefficiently on minimal medium with tyrosine or glutamate as theonly nitrogen source, the growth rate was significantly reducedwhen b-tyrosine was the only nitrogen source (Figure 7A). Whenb-tyrosine was added to minimal medium containing glutamateas the nitrogen source, P. syringae growth was significantly re-duced at a concentration of 10 mM b-tyrosine (Figure 7B). Asthis concentration is lower than that of b-tyrosine in Nipponbareleaves (Figure 1C), it is possible that the in vivo rice b-tyrosinecontent would be inhibitory to bacterial growth. A similar con-centration of tyrosine added to glutamate medium did not re-duce P. syringae growth (Figure 7C).As allelopathic effects have been observed in rice, we con-

ducted experiments to determine whether b-tyrosine couldcontribute to the growth inhibition of other plants. Arabidopsisthaliana root growth was significantly inhibited (Figure 8A), withan IC50 (50% inhibition of root growth) concentration of 4.4 mMb-tyrosine. Arabidopsis root growth inhibition by 5 mM b-tyrosinecould be partially rescued by exogenous addition of 40 mM ty-rosine (Figure 8B). b-Tyrosine root growth inhibition assays wereconducted with several other plant species and the IC20 (20%inhibition of root growth) was calculated for each species by linearregression across a range of b-tyrosine concentrations (Table 3;Supplemental Figure 14). Among the tested species, dicots ex-hibited growth inhibition at an ;100-fold lower b-tyrosine con-centration than monocots. Rice itself, both Nipponbare andKasalath, exhibited the highest b-tyrosine resistance, with nosignificant growth inhibition at any of the tested b-tyrosineconcentrations. The b-tyrosine inhibitory concentrations forArabidopsis and other dicots were similar to those that are foundin the hydroponic medium surrounding Nipponbare roots. Thissuggests that the b-tyrosine that is found in Nipponbare andother rice cultivars could contribute to the allelopathic effectsthat have been observed in cultivated rice.

Figure 6. Effects of b-Tyrosine on Insect Growth.

(A) M. persicae reproduction on tobacco leaves taking up solutionscontaining 0, 20, 100, 500, 1000, or 4000 mM b-tyrosine or 4000 mMtyrosine (control) via the petioles. Aphid reproduction and actual con-centration of b-tyrosine in the leaves on which aphids were feedingwere measured. Mean 6 SE of n = 5, *P < 0.01, t test relative to 0 mMb-tyrosine.(B) Black cutworm larval weight after 4 and 8 d on artificial diet con-taining 0, 100, 1000, or 10,000 mM b-tyrosine. Mean 6 SE of n = 6.(C) Sugarcane borer larval weight after 6 and 12 d on artificial dietcontaining 0, 100, 1000, or 10,000 mM b-tyrosine. Mean 6 SE of n = 6,*P < 0.01, t test relative to 0 mM control.

b-Tyrosine in Rice 7 of 14

DISCUSSION

Our results show that the non-protein amino acid b-tyrosine isan abundant secondary metabolite in some rice cultivars. Thegenome of Nipponbare, the first sequenced rice cultivar, containsthe TAM1 gene, which is required for b-tyrosine production.Presence of the TAM1 gene and production of b-tyrosine occurtogether, but are sporadically distributed among rice cultivars(Supplemental Figure 4 and Supplemental Table 2). A recent re-sequencing of 50 rice genomes included five isolates each of thepresumed wild ancestors of cultivated rice, O. rufipogon andO. nivara (Xu et al., 2012b). Whereas O. nivara clusters among theindica rice cultivars in a phylogeny of these sequenced genomes,O. rufipogon is more closely related to the japonica cultivars.Consistent with the observation that TAM1 and b-tyrosine aremost prevalent in japonica cultivars, we found the TAM1 gene inone out of five sequenced O. rufipogon genomes, but not in anyof the five sequenced O. nivara genomes (Supplemental Figure 2).Although the sample size is quite small and there are certainlyother possible explanations, TAM1 may represent a presence/absence genomic polymorphism that predates indica and ja-ponica rice domestication.

Rice TAM1 is a member of an MIO-containing class of ami-nolyases and aminomutases that has been investigated in Taxusand several bacterial species. By catalyzing the conversion of(S)-a-tyrosine to (R)-b-tyrosine, rice TAM1 stereospecificity issimilar to the Taxus and C. crocatus aminomutases, whichproduce (R)-b-phenylalanine and (R)-b-tyrosine, respectively(Walker et al., 2004; Feng et al., 2011), and distinct from thoseof P. agglomerans and S. globisporus, which produce (S)-b-phenylalanine and (S)-b-tyrosine (Christenson et al., 2003;Ratnayake et al., 2011). MIO-containing aminomutase enzymesare of interest for synthetic biology studies because b-phenylalanineand b-tyrosine are required for the biosynthesis of several mi-crobial antibiotics and anticancer drugs. Thus, our discovery ofthe rice TAM1 gene provides a new tool for the assembly ofthese biosynthetic pathways for the production of these medi-cally relevant natural products.

Another rice gene, LOC_Os11g48110, encodes a predictedprotein that is ;95% identical to TAM1 at the amino acid se-quence level (Figure 5). However, we were not able to detect anytyrosine aminomutase activity from the protein product, nor didany b-tyrosine QTL localize to LOC_Os11g48110 in the courseof our genetic mapping. Given the sequence similarity of the twogenes, it is possible that LOC_Os11g48110 also encodes anaminomutase, but perhaps with some other substrate. As MIOdomain-containing aminomutases that produce b-phenylalaninehave been identified in Taxus and several bacterial species(Feng et al., 2011), phenylalanine also is a possible substrate forthis as yet uncharacterized rice gene. Future research will de-termine whether cultivated rice contains not only TAM but alsoPAM enzymatic activity.In many plant species, the signaling molecule jasmonic acid

serves as an inducer for the production of metabolites thatprovide protection against insect herbivores (Howe and Jander,

Figure 7. Effect of b-Tyrosine on P. syringae Strain DC3000 Growth.

(A) Growth of P. syringae in mannitol medium with 0.2 g L21 of tyrosine, glutamate, or b-tyrosine as the only nitrogen source. Mean 6 SE of n = 3,*P < 0.001, t test relative glutamate at same day.(B) Growth of P. syringae in mannitol medium with varying concentrations of b-tyrosine and 0.2 g L21 glutamate as the nitrogen source. Mean 6 SE ofn = 3, *P < 0.001, t test relative to 0 mm b-tyrosine at the same time point.(C) Growth of P. syringae in mannitol medium with varying concentration of tyrosine and 0.2 g L21 glutamate as the nitrogen source. Mean6 SE of n = 3,*P < 0.001, t test relative to 0 mm tyrosine at the same time point.

Figure 8. Inhibition of Arabidopsis Root Growth by b-Tyrosine.

(A) Root elongation is inhibited by increasing b-tyrosine concentration.(B) Root growth inhibition by 5 mM b-tyrosine is partially rescued byexogenous addition of tyrosine. Mean 6 SE of n = 11 to 15; differentletters indicate significant differences, P < 0.05, Tukey’s HSD test.

8 of 14 The Plant Cell

2008). Although we initially identified b-tyrosine as a jasmonate-induced amino acid in the Nipponbare cultivar, this induciblephenotype appears to be an exception. In almost all other testedcultivars, there was no significant increase in b-tyrosine abun-dance after jasmonic acid treatment (Supplemental Figure 4).Growth of three insect species, M. persicae, A. ipsilon, andD. saccharalis, was not inhibited by b-tyrosine at concentrationssimilar to those found in Nipponbare (Figure 6). Thus, b-tyrosineis not likely to have a significant protective effect against insectherbivory in rice.

P. syringae, a generalist bacterial pathogen of plants, was notable to use b-tyrosine as a nitrogen source (Figure 7A). Mostlikely, these bacteria do not contain the necessary enzymes tocatabolize b-amino acids or perhaps amino acids with the (R)rather than the (S) configuration. Significant P. syringae growthreduction by 10 mM b-tyrosine in vitro (Figure 7B) suggestsinterference with endogenous bacterial metabolism. As theb-tyrosine concentration in rice leaves (Figure 1C) is consider-ably higher than the inhibitory concentration for P. syringae,there may be some protective effect for the rice plants. Patho-gens such as Xanthomonas oryzae, which are more specializedfor growing on rice, might be expected to exhibit a higher level ofresistance to b-tyrosine.

Root exudates of some rice cultivars show significant alle-lopathic effects (Belz, 2007). Consistent with this observation,several classes of growth-inhibiting metabolites have beenidentified in rice exudates (Chung et al., 2001, 2002; Rimandoet al., 2001; Kato-Noguchi and Ino, 2003; Kong et al., 2004;Kato-Noguchi et al., 2010), and it is likely that the effects ofseveral of these compounds are combined in the most allelo-pathic cultivars. Based on the observation that b-tyrosine isfound in Nipponbare hydroponic growth medium (Figure 1C)and can inhibit root growth of other plants, in particular dicots,at low concentrations (Table 3), there is some possibility thatb-tyrosine contributes to the allelopathic potential of rice.Further research, for instance, field experiments with the TAM1TILLING knockout mutant (Supplemental Figure 13), will beneeded to determine whether b-tyrosine has an allelopathicfunction.

In a recent study, RNA interference targeting PAL(LOC_Os02g41630) transcription was shown to attenuate in-hibition of barnyard grass root growth by a highly allelopathicrice cultivar (Fang et al., 2013). This effect was attributed toa general reduction in the abundance of rice phenolic compoundsin root exudates. However, given the DNA sequence similarities ofrice PAL and TAL genes, this expression silencing experiment alsomay have contributed to a reduction in b-tyrosine biosynthesis.Further research is needed to determine how b-tyrosine in-

hibits plant growth. A different tyrosine isomer, meta-tyrosine,may provide allelopathic properties to Festuca rubra (Chewingsfescue) (Bertin et al., 2007). Selection for Arabidopsis mutantsthat are resistant tometa-tyrosine identified a feedback-insensitivearogenate dehydratase and greatly increased phenylalanineaccumulation (Huang et al., 2010), suggesting that meta-tyrosineinterferes with aromatic amino acid metabolism. Although excesstyrosine can partially rescue the negative effects of b-tyrosine onArabidopsis growth (Figure 8), it remains to be determinedwhether the improved growth is due to altered b-tyrosine uptakeor direct competition between tyrosine and b-tyrosine in endog-enous plant metabolism.To date, b-tyrosine has been investigated most extensively as

a component of bacterial polyketide antibiotics and antican-cer drugs that have potential applications in human medicine(Lohman and Shen, 2012). Although we cannot rule out in-corporation into other biologically active metabolites in rice,b-tyrosine itself certainly accumulates as a biosynthetic endproduct. With a concentration of ;20 mg kg21 (Figure 1B), theb-tyrosine abundance in rice seeds is comparable to that ofother free amino acids (Saikusa et al., 1994). Thus, future re-search on rice b-tyrosine production should consider not onlythe likely antimicrobial and allelopathic functions in living plants,but perhaps also the as yet unknown metabolic role of thismetabolite in human diets.

METHODS

Plants and Growth Conditions

Rice (Oryza sativa) seedlings were grown in Cornell RiceMix (0.16m3 peat,0.34 m3 medium to coarse vermiculite, 2.3 kg lime, and 540 g Peters’Unimix Plus III [Griffin Greenhouse Supply]). Seeds were soaked in 1.2%sodium hypochlorite solution with shaking for 15 to 20 min, rinsed withsterile deionized four times, and soaked in fresh water overnight. Seedswere planted;1 cm deep, and pots were placed in a greenhouse at 26°Cwith 60%humidity. For normal growth, plants were watered from below tomaintain soil moisture. Unless otherwise noted, all plants were used forexperiments when they were 2 weeks old, with three full leaves.

Nicotiana benthamiana and Nicotiana tabacum were grown in Cornellmix (by weight, 56%peat moss, 35% vermiculite, 4% lime, 4%Osmocoatslow-release fertilizer [Scotts], and 1%Unimix [Peters]) andwere placed inConviron growth chambers with a 16:8-h light:dark photoperiod, 180mmol PAR m22 s21 light intensity, 23°C temperature, and 60% humidity.

Seeds from a world rice collection (Kojima et al., 2005) and Japaneserice collection (Ebana et al., 2008) were obtained from the National In-stitute of Agrobiological Sciences (NIAS) Genebank (https://www.gene.affrc.go.jp/databases-core_collections_en.php). Nipponbare/KasalathCSSLs (http://www.rgrc.dna.affrc.go.jp/ineNKNCSSL48.html) were pro-vided by the Rice Genome Research Center of the Japanese Ministry ofAgriculture, Forestry, and Fisheries. Mutant seed stocks for TILLING were

Table 3. Root Growth Inhibition by b-Tyrosine

Species IC20 (mM)a

Dicot speciesArabidopsis 0.6N. tabacum 0.8Medicago truncatula 2.7Brassica oleracea 2.8N. benthamiana 3.6Solanum lycopersicum 4.8

Monocot speciesBrachypodium distachyon 105Hordeum vulgare 154Setaria italica 382O. sativa cv Nipponbare >400O. sativa cv Kasalath >400

aConcentration at which root growth is inhibited by 20%.

b-Tyrosine in Rice 9 of 14

provided by the National Bio-Resource of the Ministry of Education,Culture, Sports, Science, and Technology (MEXT), Japan (Suzuki et al.,2008).

Chemicals and Solvents

[13C915N]Tyrosine was purchased from Cambridge Isotope Laboratories.

Jasmonic acid, (R) and (S)-Boc-b-tyrosine, andO-phthaldehydialdehyde-N-acetyl-L-cysteine reagent were purchased from Cayman ChemicalCompany, Sigma-Aldrich, and Wako Pure Chemical Industries, respectively.Other chemicals and solvents were obtained from Sigma-Aldrich and WakoPure Chemical Industries.

b-Tyrosine Structure Determination

Amino acid derivatives were analyzed using an HPLC-MS system. HPLC-MS was performed using an LCMS-2020 equipped with a ProminenceHPLC system (Shimadzu) in electrospray ionization (ESI) positive-ionmode. A reversed-phase column (Mightysil RP-18 GP2.0 3 50 mm i.d.;Kanto Chemical Co.) was run at 0.2 mL/min with a gradient of acetonitrilecontaining 0.1% formic acid in water containing 0.1% formic acid: 1%(0 to 2 min), 1 to 15% (2 to 9 min), 15 to 30% (9 to 14 min), and 30 to 80%(14 to 20 min). The column temperature was maintained at 40°C. TheMS was operated with nebulizer gas flow of 1.5 L/min, drying gas flow of15 L/min, ESI voltage of 1.8 kV, and temperature of 250°C.

For GC-MS analysis, solutions were dried under a nitrogen stream anddissolved in 100 mL of water. Then, 80 mL of an ethanol and pyridinemixture (v/v, 4/1) was added. A 10-mL aliquot of ethyl chloroformate wasadded and the mixture was shaken for 30 s until the evolution of CO2 gaswas complete. The derivatives were extracted with 200 mL of dichloro-methane and subjected to GC-MS. GC-MSwas performed with an Agilent6890N GC linked to an Agilent 5975B (Agilent Technologies) operated at69.9 eV with an HP-5ms capillary column (0.25 mm3 30 m, 0.25-mm filmthickness) with helium carrier gas at 1.0 mL/min in the splitless mode. Theoven temperature was programmed to increase from 178°C (10 minholding time) to 210°C at 10°C/min, from 210°C to 260°C at 30°C/min (10-min holding time), and then from 260°C to 290°C (5-min holding time) at30°C/min. The injector temperature was maintained at 240°C, the ionsource temperature at 300°C, and the quadrupole temperature at 150°C.ChemStation software (Agilent Technologies) was used for data acqui-sition. The amino acids were identified by comparing the retention timesand fragmentation patterns with those of authentic samples.

The b-tyrosine stereoisomer structure was determined using O-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) reagent (Buck andKrummen, 1987). Thirty milligrams of OPA (Wako Pure Chemical In-dustries) was dissolved in 1 mL ethanol and diluted with 22 mL of sodiumborate buffer (pH 10.0). Thirtymilligrams of NACwas dissolved in this OPAsolution. A 50-mL aliquot of the sample solution was mixed with 200 mL ofOPA-NAC reagent for 10 min at room temperature and analyzed byLCMS-IT-TOF (Shimadzu) using ESI positive ion mode. The CDL tem-perature was 250°C, the block heater temperature was 200°C, voltagewas 1.8 kV, nebulizer gas flow was 1.5 L/min, probe voltage was 4.50 kV,and ion accumulation time was 30 ms. HPLC separation of the reactionmixture was performed on a Mightysil RP-18GP column (50 3 2.0-mminside diameter). The solvent system consisted of 20 mM ammoniumformate (pH 6) (mobile phase A) and a 50% (v/v) mixture of acetonitrile andsolution A (mobile phase B). The diastereomeric b-tyrosine derivativeswere separated with a gradient of 10 to 30% B solution over 10 min and99% B solution was maintained for 5 min. (R) and (S)-Boc-b-tyrosine wasdissolved in dichloromethane:methanol (9:1) and cooled to 0°C. Tri-fluoroacetic acid (an amount equal amount to the solvent) was added andthe solution was stirred at room temperature. After the starting materialwas consumed, the solution was concentrated in vacuo.

Stable Isotope Labeling and Detection

Rice plants were inserted into 15-mL tubes containing 1 mM [13C915N]

tyrosine in water. Control plants were in tubes containing water only. Theplants were allowed to stand for 2 h, after which they were sprayed with1 mM jasmonic acid four times over 2 d. The plants were placed underdome covers and allowed to stand for 2 d. All leaves on each plant werecombined as one replicate for derivatization andGC-MS analysis of aminoacids.

Amino acid analysis byGC-MSwas performed as described previously(Joshi and Jander, 2009), with minor modifications. Leaves were frozen inliquid nitrogen in 2-mL tubes and ground to fine powder with 3-mm steelballs using a Harbil model 5G-HD paint shaker. Ground tissue was ex-tracted with methanol (500 mL per 100 mg fresh leaf tissue). The extractswere centrifuged at 18,000g for 10 min at room temperature. Super-natants (300 mL for each replicate) were dried to completion undernitrogen flow at 70°C. The residue was taken up in 25 mL N-methyl-N-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. Thesamples were heated for a further 50 min at 95°C, and GC-MS analysiswas performed using a Varian 1200L GC-MS (Agilent Technologies) witha DB-17 capillary column. Spectra of known amino acids were assignedby reference to a spectral library of amino acid standards and the NIST(National Institute of Standards) mass library.

Amino Acid Assays of Plant Material

Rice seedlings were grown in growth chambers as described above. Forelicitation, the leaves were sprayed with 1 mM jasmonic acid four timesover 2 d. Control plants were treated with deionized water. About 100 mgof rice tissue was weighed, frozen in liquid nitrogen in 2-mL micro-centrifuge tubes, and ground to fine powder with 3-mm steel balls usinga Harbil model 5G-HD paint shaker (Fluid Management). Ground tissuewas extracted with methanol (5 mLmg21 of fresh tissue) containing 40 mMnorleucine as an internal standard, the extracts were centrifuged at18,000g for 10 min at 25°C, and the supernatants were saved for analysis.

For amino acid analysis, samples were derivatized using the AccQ-Fluor reagent kit (Waters) according to the manufacturer’s directions.Five-microliter plant extracts were mixed with 35 mL of borate buffer, andthe reaction was initiated by the addition of 10 mL 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate reagent (AQC), followed by immediatemixing and incubation for 10 min at 55°C. Twenty microliters of eachsample was injected into a Nova-Pak C18 column (4.63 150 mm; Waters)and separated using a Waters 2790 HPLC pump system. Solvent A(containing sodium acetate and triethanolamine at pH 5.05) was pur-chased from Waters; Solvent B was acetonitrile:water (60:40). The gra-dient used for rice leaf analysis was 0 to 4 min, 15% B; 4 to 14 min, lineargradient to 23.2% B; 14 to 15 min, linear gradient to 26% B; 15 to 22 min,linear gradient to 31.4% B; 22 to 22.5 min, linear gradient to 100%B; 22.5to 26min, 100% B; 26 to 27 min, linear gradient to 15% B; 27 to 30 min,15% B. The flow rate was 1.0 mLmin21, and the column temperature was37°C. Eluted amino acid derivatives were detected using a Waters model2475 fluorescence detector, with an excitation wavelength of 250 nm andan emission wavelength of 395 nm. Data were recorded and analyzedusing Waters Empower software.

For analysis of b-tyrosine in different plant parts (Figure 1C), seedlingswere grownhydroponically for 2weeks and transferred to tubesfilledwith 4mLof hydroponic medium. Induction was performed by spraying 1 mM jasmonicacid (Cayman Chemical) once every 12 h for 2 d. Before the amino acidextraction, seedlings were separated into leaves, seed, and roots and crushedwith three stainless steel beads (3min on a shaker, 3000 strokes/minute) in 300mL of methanol. After centrifugation for 5 min at 3000 rpm, 5 mL of the su-pernatant was used in AQC derivatization as described above. The mediumwere dried up using a rotary evaporator and dissolved in a mixture of 5 mL ofmethanol and 70 mL of borate buffer. Dry seeds were extracted and the

10 of 14 The Plant Cell

samples were derivatized in the samemanner. The b-tyrosine AccQ derivativewas analyzed with a Shimadzu 2020 HPLC-MS system, as described above.

Preparation of Genomic DNA, RNA, and cDNA

Rice leaf material was harvested from seedlings, flash-frozen in liquidnitrogen, and stored at 280°C until sample preparation. After grinding ofthe frozen leaf material to a fine powder in a mortar filled with liquidnitrogen, DNA and RNA were extracted using the TRIzol reagent (LifeTechnologies) and SV Total RNA Isolation System (Promega), re-spectively, according to the manufacturers’ instructions. Nucleic acidconcentration, purity, and quality were assessed using a spectropho-tometer (NanoDrop 2000c; Thermo Scientific). Single-stranded cDNAwasprepared using SuperScript III reverse transcriptase and oligo(dT20)primers (Life Technologies).

Cloning and Expression of TAM Genes

The complete open reading frames of TAM1 and other rice PAL-like geneswere amplified from cDNA with the primer pairs listed in SupplementalTable 3, with the exception of LOC_Os11g48110. The LOC_Os11g48110could not be amplified from cDNA and instead was amplified from ge-nomic DNA. Subsequently, the intron section was deleted using anoverlap PCR method. PCR products were purified by Wizard SV Gel andPCR Clean-Up System (Promega), cloned as blunt fragments into theentry vector pDonr207, and then transferred to the destination vectorpMDC32 using the Gateway recombination system (Life Technologies).Both strands were fully sequenced to confirm that there were no errorsintroduced during the PCR amplification. The constructs were introducedinto the Escherichia coli strain DH5a. To overexpress rice TAM1 and PALin N. benthamiana, the destination vectors with the transgenes weretransfer into Agrobacterium tumefaciens strain GV3101. Liquid cultures ofthe bacteria harboring the expression constructs and a silencing sup-pressor (P38 carrying T-DNA constructs expressing the turnip crinklevirus capsid protein to reduce expression silencing; Thomas et al., 2003)were grown at 29°C to an OD600 of 0.6 to 0.7 with the antibiotics kana-mycin (50 mg mL21) and rifampicin (25 mg mL21). Bacterial cultures werecentrifuged at 3200g for 10 min, washed one time with infiltration buffer(10 mM MgCl2, 10 mM MES, and 200 mM acetosyringone), and re-suspended in infiltration buffer to OD600 of 0.45 for expression constructsand OD600 of 0.9 for the silencing suppression constructs. Both cultureswere incubated individually for 2 h. The construct-containing and P38-containing bacterial strains were mixed at a 1:1 ratio, and N. benthamianaleaves were infiltrated with the bacterial solution using a 1-mL syringe.Excess bacterial solution was wiped off with paper towel. Five days afterinfiltration, 8-mm-diameter leaf plugs were collected to confirm expres-sion of measurement of amino acids by HPLC fluorescence detectionusing the same protocol as that used for rice tissue.

Protein Purification and in Vitro Assays

Three days after infiltration of destination vector pYL436 with thetransgene into N. benthamiana, as described above, leaves were col-lected and ground in liquid nitrogen. About 1 cc 1-mm zirconia beads wasadded to each 15-mL tube, the 5 to 6 mL of ground tissue were extractedwith 2mL extraction buffer (100mMTris-HCl, pH 7.5, 100mMNaCl, 2mMEDTA, 2 mM EGTA, 1% Triton-X, 0.1% BME, 10% glycerol, 1 mMphenylmethylsulfonyl fluoride, 1 mM DTT, and 0.1% protease inhibitorcocktail [P8849; Sigma-Aldrich]) and shaken on a paint shaker 43 1 min,with 1 min incubation on ice between shaking periods. The extractionbuffer was centrifuged at 21,000g, 1 mL of the supernatant was collected,and 40 mL IgG Sepharose 6 Fast Flow (GE Healthcare) were added.Samples were placed in 2-mL tubes on a roller drum for 2 h at 4°C. After 2

h, tubes were centrifuged at 400g for 2 min at 4°C, the supernatantwas removed, and the beads were washed three times with 0.5 mL washbuffer (100 mM Tris-HCl, pH 7.5, 100 mMNaCl, 2 mM EDTA, 2 mM EGTA,0.5% Triton-X, 0.1% 2-mercaptoethanol, 10% glycerol, 1 mM phenyl-methylsulfonyl fluoride, 1 mM DTT, and 0.1% protease inhibitor). Beadswere washed with 1 mL cleavage buffer (50 mM Tris-HCl, 150 mM NaCl,1 mM EDTA, and 1 mM DTT), and 70 mL of cleavage buffer containing1.4 mL Turbo3C cleavage protease was incubated for 10 h with rotation at4°C. The supernatant was mixed with 5 mL washed GST beads (Clontech)and rotated at 4°C for 1 h. The supernatant was collected, mixed with25% glycerol, and used immediate for enzyme activity assays or stored at280°C. The protein concentration was determined using a Bradfordprotein assay kit (Bio-Rad). Proteins were transferred to a polyvinylidenefluoridemembrane (Millipore) for protein gel blot analysis. Antibodies usedwere as follows: mouse anti-MYC and mouse anti-rat IgG peroxidase(Santa Cruz Biotechnology).

The in vitro enzymatic activity of rice TAM1 was measured in assayswith purified recombinant protein and tyrosine or phenylalanine as thesubstrate. To measure time dependence, assays were performed with0.1 mM Tris-HCl buffer, pH 8.8, 0.05 mM potassium chloride, 0.2 mMtyrosine, and 0.49 mg enzyme solution for 0.5, 1, 1.5, 2, 4, and 6 h. Tomeasure temperature dependence, assays were performed with 0.1 mMTris-HCl buffer, pH 8.8, 0.05 mM potassium chloride, 0.2 mM tyrosine,and 0.49mg enzyme solution at 4, 12, 18, 22, 29, 38, 45, and 65°C. The pHdependence assays were performed with 0.05 mM potassium chloride,0.2 mM tyrosine, and 0.49 mg enzyme solution at pH 5, 6, 7, 8, 8.8, 9.5,10.5, 12.5, and 13.5 in 0.1 mM Tris-HCl buffer. To vary substrate con-centration, 200, 500, 1500, 2000, 2500, and 3500mM tyrosine were addedto 0.1 mM Tris-HCl buffer, pH 8.8, 0.05 mM potassium chloride, and0.49 mg enzyme, and 2.6, 7.9, 18.3, 28.3, 39.3, and 49.8 mg/mL enzymewas added to 0.1 mM Tris-HCl buffer, pH 8.8, 0.05 mM potassiumchloride, and 0.2 mM tyrosine. All of the assays were performed in 0.2-mLtubes, with a 12.5-mL total reaction volume. All samples were incubatedfor 6 h at 29°C, with the exception of experiments in which the reactiontimes and temperatures were specifically varied. The reaction tubes werecentrifuged at 2000g, and the supernatant was collected for amino acidanalysis. Five microliters of each extract was derivatized for amino acidanalysis using the AccQ-Fluor reagent kit (Waters). Reaction productswere detected by LC using an approach similar to that used for measuringamino acids in rice tissue. The gradient conditions were 0 to 23 min, lineargradient to 14% B; 23.1 to 26 min, linear gradient to 25% B; 26.1 to32 min, 100% B; 32.1 to 36 min, 14% B. The flow rate was 1.0 mL min21,and the column temperature was 32°C.

Pseudomonas syringae Bioassays

For in vitro growth experiments, P. syringae strain DC3000 was cultured at30°C in modified mannitol-glutamate medium (Kean et al., 1970; per liter:10 g mannitol, 0.2 g glutamate, 0.5 g KH2PO4, 0.2 g NaCl, 0.2 g MgSO4-7H2O; pH adjusted to 7.0 with 1 N NaOH prior to autoclaving). To testgrowth with other amino acids as nitrogen sources, glutamate was re-placed successively with 0.2 g L21 b-tyrosine or tyrosine. Cultures wereinoculated by diluting a fresh overnight culture of P. syringae 1:100 (50 mLinto 5 mL). OD600 was measured at 0, 24, 48, and 72 h after treatment. Forgrowth inhibition experiments, cultures were grown in mannitol-glutamatemedium and 0, 1, 10, 100, 500, and 1000 mM b-tyrosine or tyrosine.

Insect Bioassays

Tobacco seedlings were used for Myzus persicae bioassays. The de-tached tobacco leaves were inserted into 2-mL microcentrifuge tubescontaining 20, 100, 500, 1000, or 4000mM b-tyrosine.Water and 4000mMtyrosine were used as controls. After 3 h, five M. persicae adult aphids

b-Tyrosine in Rice 11 of 14

were put on the tobacco leaves. After 6 d, nymphs were counted and thetobacco leaves were harvested to determine the b-tyrosine and tyrosinecontent in the leaves on which the aphids had been feeding.

For black cutworm (Agrotis ipsilon) and sugarcane borer (Diatraeasaccharalis) experiments, eggs were obtained from Benzon Research.Eggs were hatched at 29°C, and five neonate caterpillars were placed ineach Petri dish (353 10 mm) with 0, 100, 1,000, or 10,000 mM b-tyrosineadded to artificial diet (Multiple Species Diet; Southland Products). After4 d for black cutworms and 6 d for the sugarcane borer, caterpillars wereweighed andmoved to fresh diet with the same concentration of b-tyrosine.After an additional 4 or 6 d, respectively, the caterpillars were weighedagain.

Agar Plate Bioassays with Arabidopsis and Other Seeds

To assess effects of b-tyrosine on the root growth of other plants, seedswere sterilized with 50% ethanol for 30 s, followed by 50%bleach for 2 min,and four rinses with sterile distilled water. Agar medium was preparedby mixing 250 mL water, 1.09 g Murashige and Skoog salts (Murashigeand Skoog, 1962), and 2 g Phytagar, with pH adjusted to 5.7 with 1 MKOH. b-Tyrosine at concentrations ranging from 0 to 400 mM was addedafter autoclaving. Seeds were planted on the agar medium in square Petridishes (1003 1003 15 mm), were placed at 4°C for 4 d, and then placedvertically in a growth chamber with a 16:8 h light:dark photoperiod, 180mmol photons m22 s21 light intensity, 23°C temperature, and 60% hu-midity. Root lengths were measured after 5 d for Medicago truncatula,barley (Hordeum vulgare), and Brassica oleracea, 8 d for Arabidopsisthaliana, Brachypodium distachyon, Setaria italica, and Solanum lyco-persicum, 11 d for N. tabacum and N. benthamiana, and 6 d for rice. Inseparate experiments, rescue of 5 mM b-tyrosine toxicity for Arabidopsiswas assessed by adding 40 mM tyrosine or phenylalanine to the agar.

Quantitative Trait Mapping

A population of 322 F9 RILs was developed by single-seed descent fromreciprocal crosses between Nipponbare and IR64. Total genomic DNAwas isolated and purified using the 96-plex DNeasy kit (Qiagen). The RILsand the two parental lines were genotyped using 96-plex genotyping-by-sequencing (GBS) as described previously (Elshire et al., 2011; Spindelet al., 2013). The GBS data were analyzed using the TASSEL 3.0 GBSpipeline, and the results aligned to the MSU v.7.0 rice genome usingBowtie2, as described by Spindel et al. (2013). Data were imputed usingPlaid impute (-m 15, -n 60, -w 5). PLUMAGE python scripts were usedpostimputation to perform a sequence error correction and remove singe-nucleotide polymorphisms (SNPs) with call rates #0.75, for a total SNPdata set of 86,528 SNPs and a genetic map consisting of ;1417 cen-timorgans. See Spindel et al. (2013) for details on the analysis pipeline andhttp://ricediversity.org/data for the molecular marker data set. A graphicrepresentation of the genetic map, produced using r/qtl, confirmed thatthere were no distortions in the genetic map. b-Tyrosine content wasmeasured by HPLC, as described above, in 147 RILs derived fromNipponbare x IR64 and 147 derived from IR64 x Nipponbare.

QTL mapping was performed using the above genotype and pheno-type data sets in R version 3.0.1 using the r/qtl package (v. 1.27-10). ThreeQTLmodels were tested: a binary model, a parametric quantitative model,and a combined binary+quantitative model. For the binary model, phe-notypes were coded as either 1 or 0, where 1 indicated presence ofb-tyrosine and 0 indicated absence. The QTL mapping script publishedwith Spindel et al. (2013) was used, except all references to the “normal”model were switched to “binary.” For the quantitative model, only in-dividuals expressing b-tyrosine were included in the analysis, and thequantitative phenotypes were used. The same QTL mapping script wasused to run the analysis, only with the model set to “normal.” The

combined model (model = 2part) is recommended for use when there isa large spike at one phenotype, “0” in our case, and automates theprocess of testing both a quantitative and binary model (for details, seeBroman and Sen, 2009). The QTL mapping script was modified to run thecombined model in accordance with Broman and Sen (2009). Figure 3Ashows the output of the combined model (rqtl plot () function). LODsignificance thresholds were determined using 1000 permutations.

Chromosome segment substitution lines from an existing collection oflines with Kasalath genomic segments inserted in Nipponbare (Yano,2001; Sasaki et al., 2013) were used for genetic mapping. Foliar b-tyrosinecontent was measured from seedlings with and without jasmonatetreatment, as described above. Lines that did not produce b-tyrosine dueto a Kasalath genomic insertion were identified based on the chroma-tograms.

Mutant Identification by TILLING

To identify point mutations in the LOC_Os12g33610 gene, DNA samplesof 1920 M1 plants originating from the treatment of Nipponbare seedswith MNU was used for TILLING (Till et al., 2003). DNA pools and mutantseed stocks were provided by the National Bio-Resource of MEXT, Japan(Suzuki et al., 2008). PCR primer pairs that were used to amplify theLOC_Os12g33610 region are described in Supplemental Table 3. PCRwas conducted using LA taq (with GC buffer) (Takara-Bio). Ampliconswere digested with CelI nuclease and were electrophoresed to find singlenucleotide polymorphisms. M1 plants that exhibited and extra band in thegels were sequenced to identify point mutations in LOC_Os12g33610.

Sequence Analysis and Phylogenetic Tree Reconstruction

Protein sequences for predicted Nipponbare rice PAL proteins weredownloaded from http://rice.plantbiology.msu.edu/. The Tsuga cana-densis PAM gene was downloaded from GenBank (GI:634380). A proteinalignment was implemented using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Based on the ClustalW alignment, a phylogenetic treewas constructed with MEGA6 (http://www.megasoftware.net/) usingneighbor joining. All positions with <90% site coverage were eliminated.Ambiguous bases were allowed at any position. A bootstrap resamplinganalysis with 1000 replicates was performed to evaluate the tree topology.

Bioinformatic Analyses of Rice Genomic Sequences

Illumina sequence reads were mapped to the Nipponbare genome withBowtie 2 v 2.2.3 (Langmead and Salzberg, 2012). Coverage of the Nip-ponbare genome and the TAM gene were calculated using Bedtools(Quinlan and Hall, 2010).

Accession Numbers

DNA sequences used in this article can be found at http://rice.plantbiology.msu.edu/index.shtml and in the NCBI Short Read Archive(see Supplemental Table 2 for accession numbers).

Supplemental Data

Supplemental Figure 1. Detection of an unknown rice amino acid.

Supplemental Figure 2. Total ion chromatogram from GC-MSanalysis of ethyl chloroformate derivatives.

Supplemental Figure 3. Identification of the b-tyrosine stereoconfi-guration by LC-MS/MS.

Supplemental Figure 4. b-Tyrosine abundance in rice cultivars, withand without jasmonic acid treatment.

12 of 14 The Plant Cell

Supplemental Figure 5. GC-MS profile of tyrosine and b-tyrosinestandards, derivatized with MSTFA.

Supplemental Figure 6. GC-MS profile confirms that tyrosine isconverted to b-tyrosine in rice.

Supplemental Figure 7. Mapping b-tyrosine as a quantitative traitusing recombinant inbred lines.

Supplemental Figure 8. Frequency of Nipponbare and IR64 molecularmarkers in the RIL population.

Supplemental Figure 9. Mapping b-tyrosine as a quantitative traitusing chromosome segment substitution lines.

Supplemental Figure 10. b-Tyrosine production by transient expres-sion in N. benthamiana.

Supplemental Figure 11. Enzymatic properties of rice TAM1.

Supplemental Figure 12. Rice TAM1 functions as a tyrosine amino-mutase.

Supplemental Figure 13. Identification of TAM1 mutations from a riceTILLING population.

Supplemental Figure 14. b-Tyrosine inhibition of plant root growth.

Supplemental Table 1. Genes in the mapping region of the b-tyrosineQTL.

Supplemental Table 2. Presence of TAM1 in 50 resequenced ricegenomes.

Supplemental Table 3. Primers used in this study.

Supplemental Data Set 1. Alignment of protein sequences that wereused for the phylogenetic tree construction in Figure 5.

ACKNOWLEDGMENTS

We thank Svetlana Temnykh, Sandra Harrington, and Fumio Onishi forassistance in generating recombinant inbred lines from crosses betweenNipponbare and IR64. This research was funded by US National ScienceFoundation Awards IOS-1139329 to G.J. and PGRP-1026555 to S.R.M.,by a grant from the Japan Science and Technology Agency to Y.O. andN.M., and by a fellowship from Science and Technology Star of Zhujiang,Guangzhou City (2013J2200082) to J.Y., S.R.S. was funded by theBoyce Thompson Institute.

AUTHOR CONTRIBUTIONS

J.Y. contributed to Figures 1A, 1B, 2, 3A, 3B, and 4 to 8, Tables 1, and 3,Supplemental Figures 1, 5, 6, 7, 10 to 12, and 14, Supplemental Tables 1and 3, Supplemental Data Set 1, and article writing. T.A. contributed toFigures 1C, 1D, and 3C, Tables 2 and 3, and Supplemental Figures 2 to 4and 9. M.T. contributed to Figure 3B and Supplemental Figures 9 and 13.S.R.S. contributed to Table 2 and Supplemental Table 2. J.E.S. and C.W.T.contributed to Figure 3A and Supplemental Figure 8. R.T. contributed toFigure 3B and Supplemental Figures 9 and 13. F.M. contributed to Figure1C and Table 3. Y.M. contributed to Figure 1C and Supplemental Figure3. S.R.M., Y.O., and N.M. contributed to experimental design and dataanalysis. G.J. contributed to experimental design, data analysis, andarticle writing.

Received January 20, 2015; revised March 19, 2015; accepted April 3,2015; published April 21, 2015.

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DOI 10.1105/tpc.15.00058; originally published online April 21, 2015;Plant Cell

and Georg JanderRyo Takata, Fuka Matsumoto, Yoshihiro Maesaka, Susan R. McCouch, Yutaka Okumoto, Naoki Mori Jian Yan, Takako Aboshi, Masayoshi Teraishi, Susan R. Strickler, Jennifer E. Spindel, Chih-Wei Tung,

-Tyrosine Biosynthesis in RiceβThe Tyrosine Aminomutase TAM1 Is Required for

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