Cyclotides Associate with Leaf Vasculature and Are theProducts of a Novel Precursor in Petunia (Solanaceae)*□S
Received for publication, April 12, 2012, and in revised form, May 16, 2012 Published, JBC Papers in Press, June 14, 2012, DOI 10.1074/jbc.M112.370841
Aaron G. Poth‡§, Joshua S. Mylne‡1, Julia Grassl¶, Russell E. Lyons§, A. Harvey Millar¶2, Michelle L. Colgrave§,and David J. Craik‡3
From the ‡Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia, §CSIRO Division ofLivestock Industries, St. Lucia, Queensland 4067, Australia, and the ¶Australian Research Council Centre of Excellence in PlantEnergy Biology and Centre for Comparative Analysis of Biomolecular Networks, University of Western Australia, Crawley,Western Australia 6009, Australia
Background: Cyclotides are defense-related cyclic plant peptides.Results: Petunia cyclotides are encoded by novel cyclotide genes and occur in a discrete pattern in leaf architecture.Conclusion: Novel cyclotides exist in the Solanaceae and are abundant in vascular tissues.Significance:Cyclotide localization is consistent with an anti-herbivory role. Novel Solanaceae genes provide opportunities forexpressing designer cyclic peptides in major crop species.
Cyclotides are a large family of plant peptides that are struc-turally defined by their cyclic backbone and a trifecta of disul-fide bonds, collectively known as the cyclic cystine knot (CCK)motif. Structurally similar cyclotides have been isolated fromplants within the Rubiaceae, Violaceae, and Fabaceae familiesand share theCCKmotifwith trypsin-inhibitory knottins fromaplant in the Cucurbitaceae family. Cyclotides have previouslybeen reported to be encoded by dedicated genes or as a domainwithin a knottin-encoding PA1-albumin-like gene. Here wereport the discovery of cyclotides and related non-cyclic pep-tides we called “acyclotides” from petunia of the agronomicallyimportant Solanaceae plant family. Transcripts for petunia cyc-lotides andacyclotides encode the shortest knowncyclotidepre-cursors. Despite having a different precursor structure, theirsequences suggest that petunia cyclotides mature via the samebiosynthetic route as other cyclotides. We assessed the spatialdistribution of cyclotides within a petunia leaf section byMALDI imaging and observed that the major cyclotide compo-nent PhybAwas non-uniformly distributed. Dissected leafmid-vein extracts contained significantly higher concentrations ofthis cyclotide compared with the lamina and outer margins ofleaves. This is the third distinct type of cyclotide precursor, andSolanaceae is the fourth phylogenetically disparate plant family
to produce these structurally conserved cyclopeptides, suggest-ing either convergent evolution upon the CCK structure ormovement of cyclotide-encoding sequences within the plantkingdom.
Cyclotides are a family of backbone-cyclized plant peptidesfirst discovered inOldenlandia affinis from theRubiaceae plantfamily but since found in a growing number of plants from theViolaceae, Cucurbitaceae, and Fabaceae families (1). Cyclotidesare presumed to have a role in plant defense, given reports thatascribe insecticidal (2), molluscicidal (3), or anthelmintic (4)activities to isolated peptides. Since their initial discovery asthe active constituents of a uterotonic traditional medicine (5),a host of other bioactivities have been attributed to cyclotides,including anti-HIV (6), cytotoxic (7), and neurotensin inhibi-tory activity (8).The definitive structural feature common to cyclotides is the
cyclic cystine knot (CCK)4motif in which three disulfide bondsare entwined in a knotted conformation such that one disulfidebond is threaded through an opening bounded by two sectionsof the peptide backbone and the two disulfide bonds constrain-ing them (9). The cystine knot has been demonstrated to be thefeature that confersmost of their stability at high temperatures,in extremes of pH, and against proteolytic enzymes (10, 11).The CCK motif is very tolerant to sequence variation of thenon-Cys residues, as exemplified by the observation that itoccurs in two cyclic trypsin inhibitors, MCoTI-I andMCoTI-II(12), from a Cucurbitaceae plant that differ substantially insequence from other cyclotides and are closely related to someacyclic trypsin inhibitors from squash plants that are part of theknottin family. The stability and tolerance to sequence substi-tution has led to consideration of the CCK framework as a nat-
* This work was supported by an Australian Research Council (ARC) grant forcyclotide analysis (Grant DP0984390), an ARC Grant for development ofMALDI-MSI in plants (Grant DP0985873), and equipment purchased underARC Linkage Grant LE0775603 housed in the ARC Centre of Excellence inPlant Energy Biology at the University of Western Australia.
□S This article contains supplemental Table S1 and Figs. S1–S4.The nucleotide sequence(s) reported in this paper has been submitted to the Gen-
BankTM/EBI Data Bank with accession number(s) JQ886398, JQ886399, andJQ886400.
The protein sequence data reported in this paper will appear in the UniProtKnowledgebase under the accession numbers B3EWH5, B3EWH6, B3EWH7,and B3EWI4.
1 An ARC Queen Elizabeth II Fellow (Grant DP0879133) and the John S. MattickFellow.
2 An ARC Australian Professorial Fellow (Grant DP0771156).3 A National Health and Medical Research Council Professorial Fellow. To
whom correspondence should be addressed. Tel.: 61-7-3346-2019; E-mail:[email protected].
4 The abbreviations used are: CCK, cyclic cystine knot; MSI, mass spectromet-ric imaging; EST, expressed sequence tag; ACN, acetonitrile; RACE, rapidamplification of cDNA ends; AEP, asparaginyl endopeptidase; SFTI, sun-flower trypsin inhibitor.
ural combinatorial template (13) with applications in drugdesign (14).Several recent studies have demonstrated the suitability of
the CCK framework as a stable drug design scaffold, exempli-fied by the synthesis of modified cyclotides to incorporate bio-active peptide epitopes that would otherwise have short in vivohalf-lives. Examples include cyclotide-based vascular endothe-lial growth factor-A (VEGF) agonists (15) or antagonists (16)and inhibitors of tryptase � from human mast cells (17). Thesestudies highlight the potential value cyclotides have as peptidetherapeutics and provide an impetus for investigating their bio-synthesis in plants, potentially opening new opportunities forthe expression of “designer” cyclotides with pharmaceuticaltraits in plants.In Rubiaceae andViolaceae plants, cyclotides are products of
dedicated genes that comprise an endoplasmic reticulum signalsequence and a pro-region, followed by up to three cyclotidedomains, each flanked by an N-terminal pro-domain and aC-terminal tail (18, 19). Recently, we reported the occurrence ofcyclotides in the Fabaceae plantClitoria ternatea (20), and sub-sequently it was demonstrated that the Fabaceae cyclotides areencoded within a PA1b-like albumin where the cyclotide has“replaced” the first of its usual two domains (21, 22). TypicalFabaceae albumin-1 genes encode a PA1 pro-protein that ispost-translationally cleaved to liberate PA1b (a member of theknottin family) and PA1a albumins (23), whereas in the C. ter-natea albumin-1 gene, a cyclotide domain has replaced thePA1b knottin domain. Despite being encoded within itsunusual gene architecture, Cter M, the best characterized cy-clotide from C. ternatea, shares the key structural feature of aCCK motif with cyclotides derived from dedicated cyclotidegenes. Interestingly, another of the isolated peptides fromC. ternatea is identical in primary sequence to a previouslyreported cyclotide, Psyle F from Psychotria leptothyrsa fromRubiaceae (24).Although their gene expression does not appear to be
dynamically regulated (25), cyclotides are known to be differ-entially expressed within a plant. In Viola hederacea, cyclotidevhr1 is specific for only root tissue (26), whereas in O. affinis,Oak4 expression and its encoded peptide kalata B2 were absentfrom root tissue (25). Recent work has demonstrated that GFP-tagged cyclotide precursors accumulate in plant cell vacuoles(27). Several studies have reported insecticidal activity in cy-clotides (2, 21, 28) and provided the basis for further structure-activity studies (29), but little is known about the distribution ofcyclotides within individual plant tissues.Matrix-assisted laser desorption/ionization-mass spectro-
metric imaging (MALDI-MSI) is an analytical technique inwhich mass spectra are collected in a raster pattern across atissue section to generate an average mass spectrum, which,when overlaid upon an image of the sample, can reveal thespatial distribution and relative abundances of analytes (30).MALDI-MSI (31) has been applied in the study of animal andhuman tissues as a research tool as well as in amedical diagnos-tic capacity in the study of disease pathology (32–34) and tomonitor drug pharmacokinetics (35, 36). Recent examples ofplant MALDI-MSI providing insights through spatial informa-tion include the peptide analytes of developing soybean cotyle-
dons (37), secreted peptide hormones involved in plant devel-opment in Arabidopsis roots (38), and small-moleculeglucosinolate derivatives involved in plant defense from Arabi-dopsis leaves (39).Our discovery of cyclotides in Petunia arose from interrogat-
ing expressed sequence tag (EST) databases by tBLASTN withthe Cter A peptide sequence, which yielded many matches topotential cyclotide-encoding transcripts. Here we describe thecharacterization of cyclotides in Petunia x hybrida followingtheir isolation and tandem MS sequencing and report a novelarchitecture of their genes based upon cloning of three full-length cDNA clones and a wealth of other EST-derivedsequences.The discovery of cyclotides in the Solanaceae is significant
and exciting because this plant family includes many crop spe-cies, including potato and tomato, two of the largest food cropsby global yield with a combined world annual production ofmore than 450 million tons. Given the demonstrated potentinsecticidal activity of isolated cyclotides (2, 21, 28), knowledgeof the Solanaceae cyclotide gene architecture might enabletheir expression in important food crops to potentially providecrops protection from predation by herbivores. The combina-tion ofMALDI-MSI on cyclotide expression and localization inpetunia and MS analysis of leaf region extracts provided evi-dence of non-uniform distribution of the major cyclotide massconsistent with location in the vasculature of the leaf. A vascu-lar location is common for small molecule (39), physical (40),and peptidic (41–43) herbivory defense systems and wouldallow an additive role for cyclotides in reducing predation byherbivores. Exploitation of Solanaceae cyclotide genes mightthus allow production of novel, ultrastable therapeutics, lead tothe enhancement of the staple crops as “functional foods” (44–46), and/or reduce crop losses to insect attack.Previous studies investigating the expression of the cyc-
lotide-encoding gene Oak1 from Rubiaceae in the model plantNicotiana benthamiana reported the production of mainlymisprocessed peptides (27, 47). The discovery of a cyclotide-encoding gene from the Solanaceae has great potential toimprove the value ofN. benthamiana as a research tool to studycyclotide processing and also to study the effects of cyclotides inplant defense.
EXPERIMENTAL PROCEDURES
Materials—P. x hybrida seedlings were sourced from Pohl-mans Nursery (Gatton, Queensland, Australia). Solid phaseextraction cartridges and reverse phase HPLC columns werefrom Grace Vydac. All solvents and enzymes were supplied asdescribed previously (20).Extraction—P. x hybrida plants were rinsed extensively with
distilled water to remove soil prior to separation of the variousplant tissues. Fresh leaf (8.0 g) and root (5.3 g) samples werelyophilized prior to ball-milling using a RetschMM300 homog-enizer in three 30-s bursts at 25 Hz. Powdered plant sampleswere subsequently extracted in 60% acetonitrile (ACN), 1% for-mic acid with vortexing and probe sonication. Crude extractswere then centrifuged in a benchtop centrifuge at 4000� g, andthe supernatants were collected and diluted with 1% formicacid to give final solvent extract concentrations of 10% ACN,
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1% formic acid, and 100 g/liter or 80 g/liter wet plant weight forleaf and root materials, respectively.Separation—Crude extracts were separated on Grace C18-
Max solid phase extraction cartridges. Briefly, cartridges wereequilibrated following themanufacturer’s instructions using sixbed volumes of methanol followed by six bed volumes of 10%ACN, 1% formic acid. Crude extracts were applied to the car-tridges and washed with six bed volumes of 10% ACN, 1% for-mic acid. Bound peptide components were eluted from the car-tridges in a stepwise fashion, using increasing concentrations ofACN in 1% formic acid. Alternatively, crude extracts were sub-jected to preparative HPLC using a Grace Vydac C18 reversephaseHPLCcolumn (250� 20mm, 300Å, 15-�mparticle size)with a linear 1%/min ACN gradient as supplied by a ShimadzuLC-2010 HPLC system. Eluent was monitored at 214 nm, andfractions were collected manually.EnzymeDigestion—Enzymatic digestion of reduced and alky-
lated cyclotides was carried out prior to tandemMS analyses asdescribed previously (20). Briefly, cyclotides were cleaved toproduce linearized fragments following reduction and alkyla-tion to prevent reoxidation. Lyophilized crude leaf extract (1mg) was reconstituted in 150 �l of 100 mM ammonium bicar-bonate (pH 8.0) and reduced by the addition of 15 �l of 100mM
dithiothreitol and incubated at 60 °C for 30min under nitrogengas. To alkylate the sample, 15�l of 250mM iodoacetamide wasadded, and the mixture was incubated for 60 min at room tem-perature. The alkylated sample was digested by the addition of20 �l of 400 ng/�l endoproteinase Glu-C (P2922, Sigma) andincubated at 37 °C for 18 h. Each sample was quenched with 20�l of 5% formic acid and stored at 4 °C until further analysis.MALDI-MSI—Leaf and stem cryosections were applied
directly to indium tin oxide-coated glass slides (Bruker) anddried in a vacuum desiccator before undergoing further wash-ing and dehydration through submersion in cold 70% (v/v) iso-propyl alcohol for 30 s, and cold 96% (v/v) isopropyl alcohol for15 s before being returned to vacuum to dry for 20min.Washedslides were observed under an Olympus SZX7 stereomicro-scope. �-Cyano-4-hydroxycinnamic acid matrix was preparedat a concentration of 7mg/ml in 50%ACN, 0.2% trifluoroaceticacid and misted onto the surface of the dried sample slidesusing a Bruker Daltonics ImagePrep matrix sprayer. Sampleslideswere clamped into aBrukerDaltonicsMTPSlideAdapterII MALDI plate and analyzed using a Bruker Daltonics Ultra-Flex III MALDI-TOF instrument running flexImaging version2.1 software. Spectra were collected in linear positive ion modeusing a 100-�m raster across the leaf section over a mass rangeof 2500–5000 Da with signals of �1800 Da suppressed toremove matrix and polymer peaks. Following data analysis inflexImaging, positions on the leaf section corresponding topeaks of interestwere respottedmanuallywith two applicationsof the matrix solution prior to manual collection of MALDI-TOF spectra in reflectron positive ion mode using flexControlsoftware. Localization of specific m/z values was determinedover a window of �5 Da centered on the peak maxima.MALDI-TOF MS—MALDI-TOF analyses were conducted
using an Applied Biosystems 4700 TOF-TOF Proteomics Ana-lyzer. Samples were spotted 1:1 with matrix consisting of 5mg/ml cyano-4-hydroxycinnamic acid in 50% (v/v) ACN, 1%
(v/v) formic acid directly onto a stainless steel MALDI target.MALDI-TOF spectra were acquired in reflector positive oper-atingmode with the following parameters: source voltage set at20 kV, Grid1 voltage at 12 kV, mass range 800–5000 Da, focusmass 3000 Da, collecting 2000 shots using a random laser pat-tern and with a laser intensity of 5000. Spectra were externallycalibrated as described previously (48) by spotting cyano-4-hy-droxycinnamic acid matrix 1:1 with the ProteoMass MSCAL1peptide and protein MALDI-MS calibration kit calibrationmixture (Sigma) diluted 1:400.Static Nanospray—Reduced and endoproteinase Glu-C-di-
gested samples were subject to a cleanup step using C18 Zip-Tips (Millipore) to remove salts and elicit a solvent exchangefrom aqueous solution to 80%ACN, 1% formic acid. Samples (3�l) were transferred to nanospray tips (Proxeon, ES380), andnano-electrospray ionization was induced with a voltage differ-ential of 900 V applied to the tip on a QSTAR Pulsar i QqTOFmass spectrometer (Applied Biosystems). TOF spectra werecollected over the range m/z 400–2000. Product ion spectrawere collected (m/z 100–2000) using collision energy voltagesranging from 10 to 60 V. Both TOF and product ion data wereacquired using Analyst QS 1.5 software, and tandemMS spec-tra were manually assigned.Nano-LC-MS/MS—Reduced, carbamidomethylated, and
endoproteinase Glu-C-digested (linearized) cyclotide-contain-ing crude extracts were analyzed via LC-MS/MS with condi-tions as described previously (21).Cryosectioning—Petunia leaf tissue was prepared forMALDI
imaging with minor changes to a method described previously(37). Samples were cryosectioned using a Leica CM3050 cryo-tome with chamber temperature set at �19 °C and object tem-perature set at �17 °C. Frozen leaf tissue was floated on thesurface of optimal cutting temperature (OCT)medium appliedto the cryotome chuck and paradermal (adaxial longitudinal)cryosections sampled at 15-�m thickness.LC/MS Analysis—The relative quantitation of Phyb A (m/z
3069) among leaf parts was performed using a methoddescribed previously (39)with somemodifications. Leaveswereremoved fromP. x hybridaplants, flash-frozen in liquidN2, andlyophilized. These leaves were subsequently dissected to yieldmidvein, lamina, and peripheral leaf tissue samples, whichwerethen weighed in separate tubes, and 500 �l of water was addedper mg of dried plant tissue. Sealed sample tubes were placedinto a heater block set at 95 °C for 75 min, cooled to roomtemperature, and centrifuged at 4000 � g for 10 min. Samplesupernatants were introduced to a QSTAR Pulsar i QqTOFmass spectrometer (Applied Biosystems) equipped with a Tur-bospray ionization source, using an Agilent 1100 binary HPLCsystem (Agilent). Reversed phase separation of peptide analyteswas achieved using a linear gradient comprising solventA (0.1%formic acid) and solvent B (90% ACN, 0.1% formic acid (aque-ous)) at a flow rate of 200�l/min applied to a Jupiter C18 300-Åcolumn (Phenomenex) of dimensions 150mm� 2.0mmwith aparticle size of 5�m.TOF spectra were collected over the rangem/z 400–2000 and analyzed using Analyst QS 2.0 software.Cloning of PETUNITIDE Genes—We used tBLASTN to
search theNCBI dbEST database with the amino acid sequenceof Fabaceae cyclotide Cter A (GVIPCGESCVFIPCISTVIGC-
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SCKNKVCYRN). Several Petunia ESTs matched with Expect(E) values of�1e7, including FN039431, FN022914, FN016979,FN020412, FN022913, FN039432, FN017679, and FN017678.Further analysis of these ESTs indicated that they were likely toencode cyclotides. These sequences were used to retrieve sim-ilar Petunia EST sequences. An alignment of the following 32EST sequences was made: JI334153, JI334152, JI361658,JI335590, JI335591, DC242826, FN035504, FN039432,FN022913, FN017679, JI335742, FN006075, FN000250,FN001371, JI333116, FN002680, FN020915, FN001731,FN004316, FN003549, FN001318, FN005840, FN004550,FN005530, FN035505, FN039431, FN022914, FN016979,FN020412, FN017678, FN020916, and FN021459. Based onthis alignment, there appeared to be several different cy-clotide-encoding genes in Petunia. Against this alignment,primers that would amplify complete cyclotide domainswere designed for 5�- and 3�-RACE: JM532 (5�-CAT ATATAT GCC CCT CTC CT-3�) for 5�-RACE bound down-stream from the stop codon in 19 of 32 sequences aligned;JM533 (5�-GACGCACGCGTAATGGAT-3�) for 3�-RACEbound 30 bp upstream of the region encoding the cyclotidedomain in 23 of 32 sequences aligned; JM534 (5�-TCA CGTGTG TTT CTG CCA CT-3�) for 3�-RACE bound 4 bpupstream of the region encoding the cyclotide domain in 15of 32 sequences aligned; and JM535 (5�-TGT CAC GTGTGT TTC TGC AA-3�) for 3�-RACE bound in a similar loca-tion to JM534 that is 6 bp upstream of the cyclotide domainbut was specific for a different group of 6 among the 32sequences aligned.RNA was extracted from the leaves, flowers, and roots of
P. x hybrida using phenol/chloroform extraction, and selectiveprecipitation of RNA was performed using lithium chloride asdescribed previously (49). Between 500 ng and 1 �g of totalRNA was used to create 5�- and 3�-RACE libraries using theSMARTer RACE cDNAamplification kit (634923, Clontech) asper themanufacturer’s instructions. The three 5�-RACE librar-ies were PCR-amplified using JM532, whereas the three3�-RACE libraries were PCR-amplified using JM533, JM534,and JM535. The 5�- and 3�-RACE products were cloned intopGEM-T (Promega), sequenced, and aligned. These partialsequences suggested that up to five different transcripts had
been amplified. Using the transcript sequences tentativelynamed PETUNITIDE1 to -5, we designed the following primersin the 5�- and 3�-UTRs that would be specific and amplify thefull ORF: JM549 (5�-CAT ACT CAG TGA TTT CCC ACCA-3�) bound to the 3�-UTR of the transcript tentatively namedPETUNITIDE1; JM552 (5�-CAT ACT CAG CTA CAC ATAGTGC-3�) bound to the 3�-UTR of PETUNITIDE3; JM550 (5�-CAT ACC CAG TGA TTT TCC ACC A-3�) bound to the3�-UTR of PETUNITIDE4; JM551 (5�-CAT ACC CAG TAATTT CCT ACC A-3�) bound to the 3�-UTR of PETUNITIDE5;JM554 (5�-GCCAGCTACACATAGTGCT-3�) bound to the3�-UTR of most PETUNITIDE transcripts and is upstreamfrom the binding sites of JM550-JM552; and JM553 (5�-TGGCAA AGA TAA TAC TTT CA-3�) bound to the 5�-UTR ofboth PETUNITIDE2 and PETUNITIDE4.PCR amplification of the aforementioned 5�- and 3�-RACE
libraries with these primers yielded products of the expectedsizes that were subsequently cloned into pGEM-T andsequenced. This sequencing revealed three different PETU-NITIDE transcript products each encoding a full ORF. For eachPETUNITIDE transcript, at least three independent cloneswere obtained.
RESULTS
Searching for Cyclotide Sequences in Petunia TranscriptDatabases—Structurally homologous cyclotides have previ-ously been characterized from plants of the Rubiaceae,Violaceae, and Fabaceae families, with the investigated speciestypically having been selected on the basis of an identified bio-activity. To search for potential cyclotide-encoding geneswithin publicly available bioinformatic data, Fabaceae cyclotideCter A was used as a tBLASTN search string to interrogate theEST database at NCBI. Numerous putative cyclotide-encodingESTs from the genus Petunia matched the submitted proteinsequence (summarized in Fig. 1, with their accession numbersunder “Experimental Procedures”). We named these putativepetunia cyclotides Phyb A through Phyb L in accordance with apreviously established convention (50). PETUNITIDE1 to -3and the related ESTs appeared to encode precursor proteinspossessing an endoplasmic reticulum target signal and, towardthe end, a cyclotide domain containing six cysteines of typical
FIGURE 1. BOXSHADE alignment of cyclotide precursors in genus Petunia. A, PETUNITIDE sequences. B, consensus sequences derived from matched ESTs.Asterisks at the C termini of sequences indicate stop codons. Red bar denotes predicted signal motifs, and green bars denote cyclotide or acyclotide domains.Triangles denote prototerminal amino acids of encoded cyclotides. Disulfide connectivity is based upon previously characterized cyclotides. Sequencestranslated from EST data only are listed in supplemental Table S1 (note paired clones (F � R)).
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spacing, the highly conservedGlu in loop 1, a proto-N-terminalGly, and the usual proto-C-terminal Asx (i.e.Asn or Asp). Thisarrangement differs from cyclotide precursors from theViolaceae and Rubiaceae, which have longer regions betweenthe signal peptide region and the mature cyclotide domain(s),making the PETUNITIDE proteins essentially the same size asvery recently described precursors from the Rubiaceae plantChassalia chartacea, which are much shorter than previouslydescribed cyclotide precursors (supplemental Fig. S1).Commonly trailing the cyclotide domains’ proto-C-terminal
Asx is an Ile or Leu located two residues downstream (at P2�).This residue is consistently observed among the correspondingregions of Violaceae, Rubiaceae, and Fabaceae cyclotide genesand appears to be an important residue for processing (51). Inpreviously reported cyclotide genes, the amino acid at P1� istypically a Gly, but the Solanaceous precursors exhibit either aGly or Glu. We also observed that some ESTs encode PETU-NITIDE proteins that are punctuated with stop codons imme-diately following their cyclotide domains (Fig. 1; encoding puta-tive Phyb J, PhybK, and Phyb L).What effect this has on peptidematuration remained to be determined by analyzing the pep-tide profile of petunia. A list of ESTs that would encode theputative mature cyclotides and acyclotides is given in supple-mental Table S1.
Detection and Sequencing of Cyclotides and Acyclotides fromP. x hybrida—To confirm the synthesis and accumulation ofthe predicted cyclic and acyclic peptides, we prepared extractsof various P. x hybrida tissues and analyzed these directly usingMALDI-TOF MS. As shown in Fig. 2, A and D, both leaf androot extracts exhibited signals within the mass range m/z2800–3700 typical of cyclotides. A dominant signal observed atm/z 3069 (peak A) appeared in both leaf and root extracts andwas consistent with themass of the predicted cyclotide domainof PETUNITIDE1. After reduction and alkylation, a mass 348Da larger than peak A was observed atm/z 3417 (Fig. 2B), con-sistent with the alkylation of six cysteine residues. Followingdigestion with endoproteinase Glu-C, an additional increase inmass of 18Dawas observed. This signal corresponded to peakA� 366 Da and appeared atm/z 3435 (Fig. 2C), consistent with asingle peptide backbone cleavage, as would be expected for acyclotide. TandemMS characterization confirmed the identifi-cation of the peak atm/z 3435 as the bracelet cyclotide Phyb Awith sequence SCVWIPCVSAAIGCSCSNKICYRNGIGCGE,in agreement with the sequence of PETUNITIDE1 (Fig. 3A).The other dominant peak observed inMALDI-TOF analyses
of root extracts appeared at m/z 3388 (peak B), but no corre-sponding peak at �366 Da was observed in the endoproteinaseGlu-C-digested sample. Apart from the peak observed at m/z
FIGURE 2. MALDI-TOF MS of petunia leaf and root extracts reveals putative cyclotide masses. Following reduction of disulfide bonds, free thiols werealkylated, and peptide backbones were enzymatically digested with endoproteinase Glu-C. A, native leaf extract. B, reduced and carbamidomethylated leafextract. C, reduced, carbamidomethylated, and enzymatically digested leaf extract. D, native root extract. E, reduced and carbamidomethylated root extract.F, reduced, carbamidomethylated, and enzymatically digested root extract. G, the structures of native, alkylated, and digested Phyb A (peak a) and Phyb M(peak b) are schematically represented beside their corresponding spectra.
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3435 (PhybA), the dominant signal in the spectrum from endo-proteinase Glu-C-digested root extract was a peak atm/z 2978(Fig. 2F). Examination of the TOF-MS spectrum of the reducedand alkylated extract revealed a dominant peak at m/z 3736(Fig. 2E) corresponding to the addition of 348 Da (alkylation ofthe six Cys residues) to the peak atm/z 3388 in the native rootextract. Following endoproteinase Glu-C digestion, the peak atm/z 3736was noticeably absent, suggesting that proteolysis hadoccurred. A peak at m/z 3754 would be expected followingcleavage of a cyclic peptide backbone (at Glu in loop 1). How-ever, no such peak was observed, raising two possibilities: 1)that the peptide contained more than the single Glu in loop 1and/or 2) that the peptide was linear and was therefore cleavedinto more than one fragment. UnderMS/MS conditions, cyclicpeptides typically do not fragment as readily as linear peptides.Extensive fragmentation of the precursor atm/z 3736 (Fig. 3B)in tandemMS indicated its peptide backbone to be non-cyclic.The assigned peptide sequence was homologous to thecyclotide-like gene product predicted by EST FN005530,with sequence pQSISCAESCVWIPCATSLIGCSCVNSRCI-YSK, which we named PhybM, and was found to incorporate apyroglutamyl modification at its N terminus. The dominantpeak at m/z 2978 observed after endoproteinase Glu-C diges-
tion was subjected to tandemMS analysis, revealing its identityas the C-terminal portion of Phyb M.During LC-MS/MS analysis of reduced, alkylated, and endo-
proteinase Glu-C-digested root extracts, another acyclic pep-tidewas observed (Fig. 3C) with a parent ionmass and fragmen-tation pattern matching the cyclotide sequence encoded byEST FN001318 (STDCGEPCVYIPCTITALLGCSCLNKVC-VRP) and which we named Phyb K. The masses of the charac-terized aswell as putative cyclotides are reported inTable 1. Fig.4 illustrates an alignment of Phyb A with the cyclotide sharingthe highest sequence homology, cycloviolacin O17 from Violaodorata, alongwith the petunia acyclotides PhybKandPhybM,demonstrating the conserved cysteine spacing.Judging from LC-MS analyses, the abundance of petunia cy-
clotides in source plant material was within the range previ-ously reported for cyclotides in V. odorata and O. affinis (52),with Cter A in wet leaf material estimated to be 30.0 �g/g,whereas Cter K andCterMwere present in wet rootmaterial at2.3 and 7.6 �g/g, respectively.Determination of Cyclotide Distribution in Petunia Leaf
Tissue—To examine the spatial distribution of petunia cyclo-tides within plant tissue, we used MALDI-MSI to analyze aparadermal leaf section, generating an ion intensity map. As
FIGURE 3. MALDI-TOF/TOF analyses of cyclotide and acyclotide species isolated from petunia root. A, tandem MS analysis of the m/z 3435.47 precursor(Phyb A). B, tandem MS analysis of the m/z 3736.52 precursor (Phyb M). C, tandem MS analysis of the m/z 3600.67 precursor (Phyb K). D, tandem MS analysis ofthe m/z 2978.28 precursor (Phyb M fragment).
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shown in the average mass spectrum in Fig. 5A, numerouspeaks were detected in the range m/z 3000–3600. Analysis ofleaf extracts showed a single dominant peak atm/z 3069, whichwas sequenced and named Phyb A (Fig. 2A), and this peak wasobserved in the MALDI-MSI experiment along with peaks atm/z 3110, 3426, and 3463. Apart fromm/z 3069, none of theseother masses corresponded to isolated or predicted cyclotides(Figs. 1 and 2). LC-MS/MS analysis of leaf extracts was under-taken to determine if the MALDI-MSI peaks observed at m/z3110, 3426, and 3463were cyclotides; however, only a precursorat m/z 3424 (corresponding to m/z 3426 average mass inMALDI-MSI) was observed. Limited fragmentation of this pre-cursor was observed, but following a reduction step, a massincrease of 2 Da was observed (supplemental Fig. S2, A and B),indicating the presence of a single intramolecular disulfidebond, and leading to extensive fragmentation in subsequenttandem MS (supplemental Fig. S3). The sequence resultingfrom manual de novo mass spectral interpretation (DEEP-KRGTPEAKKKYSSVCVTNPTARICRY) was used in a BLASTsearch and found to be consistent with a translated EST(FN008610) from petunia encoding a sequence homologous tonuclear Photosystem II 5-kDa protein (PSII-T) described inother plant species. This identification was further bolsteredthrough the observation of a 210-Da increase inmass followingacetylation with acetic anhydride, consistent withmodificationof the four Lys side chains as well as the N-terminal primaryamines in the native peptide (supplemental Fig. S2C).Fig. 5B highlights multiple vascular features observed in a
dark field microscopy image of the leaf section analyzed in thisexperiment. In Fig. 5, C–F, the relative signal intensities ofselected peakmaxima (� 5Da) ranging from0% (black) to 100%(white) are superimposed upon the dark field leaf image (Fig. 5,C–F). Areas of increased signal intensity form/z 3069 and 3110peaks appeared to overlay with the vascular features (Fig. 5, CandD), whereas the spatial distributions and relative intensities
ofm/z 3426 and 3463 signals were not (Fig. 5, E and F). Signalsfor m/z maxima observed in the average spectrum, includingthe examples in Fig. 5, appeared to be differentially distributedacross the sample section and localized to distinct regions, withno evidence of “hot spots” or smearing. In Fig. 5G, the relativeintensities of signals for m/z 3426 and m/z 3069 are indicatedover a range from transparent (0%) to bright green or red(100%), respectively, and co-localization is indicated by yellowcoloration. The display of distinct green and red areas indicatedheterogeneous expression patterns, with the strongest signalsfor m/z 3426 appearing to present within areas upon the leafsection with the least vasculature, whereas for m/z 3069, thereverse is true. To confirm the localization pattern observed form/z 3069 in the MALDI-MSI experiment, the relative quanti-tation of Phyb A was determined via LC/MS in extracts of dis-sected petunia leaves. Approximately 2-fold higher concentra-tions of m/z 3069 (Phyb A) were detected in the midvein,compared with both the lamina and periphery of petunia leaves(Fig. 6B). Sixteen control signals were selected from the LC/MSdata, includingm/z 3424 (PSII-T), and their relative concentra-tions were similarly compared across leaf regions. In each case,there was either no statistically significant difference in theirconcentrations across the leaf or increased concentrations inthe lamina or periphery (or both) compared with the midveinextracts (supplemental Fig. S4).
DISCUSSION
Here we report the discovery and characterization of cy-clotides from P. x hybrida of the agronomically importantSolanaceae plant family. These peptides arise from the shortestknown cyclotide precursors and are distinct from previouslyknown precursors. This is the fourth architecturally distinctprecursor fromwhich cyclotides emerge, provoking interestingquestions about the evolutionary origin of their structurallyidentical CCK framework peptides. The new precursors pres-ent opportunities for designing synthetic peptides capable ofbeing cyclized efficiently in planta for a range of agricultural orpharmaceutical applications. Furthermore, we have confirmedenrichment of a cyclotide in the vasculature of leaves, a findingthat is consistent with a proposed general role of cyclotides inherbivory defense.Existence of Cyclotides in the Solanaceae—The discovery of
cyclotides within the Solanaceae plant family is an exciting andimportant development, given the significance of this plantfamily to human nutrition, and follows the recent landmarkdiscovery of cyclotide genes in amember of the Fabaceae family(21, 22). The Solanaceae is host to more than 3000 species,
TABLE 1Sequence alignment of cyclotides observed in P. x hybrida extracts
Da Da ppm DaPhyb A -GIGCGESCVWIPCVS-AAIGCSCSNKIC-YRN 3434.47 3434.47 3434.55 �23.29 3068.29Phyb K -STDCGEPCVYIPCTITALLGCSCLNKVC-VRP 1200.89 3599.67 3599.71 �11.11 3251.46Phyb M pQSISCAESCVWIPCAT-SLIGCSCVNSRCIYSK 1246.17 3735.51 3735.73 �58.89 3387.47
a Ile and Leu in Phyb M were assigned on the basis of homology to Phyb L.b Phyb K and Phyb M are acyclic.c Expt., experimental; Theor., theoretical; linearized peptide masses after reduction and carbamidomethylation of cysteines.d Theor., theoretical; native peptide masses.
FIGURE 4. Alignment of cylcoviolacin O17 with cyclotide Phyb A (86%homology) and acyclotides Phyb K and Phyb M from P. x hybrida. Disul-fide connectivity based upon previously characterized cyclotides is denotedby solid lines between boxed cysteines, and intercysteine loops are indicated.Loop 6 is not present in Phyb K or Phyb M, which are both acyclic.
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including staple crops, such as Solanum tuberosum (potato)and Solanum lycopersicum (tomato), which constitute two ofthe most important vegetable crops cultivated, with com-bined worldwide annual production exceeding 450 milliontons.Plant species previously investigated in the search for cy-
clotides have typically been selected on the basis of an identifiedbioactivity in their extracts, such as uterotonic activity inO. affinis (53) and C. ternatea (20), anti-HIV activity in Pali-courea condensata (54), hemolytic activity in Viola extracts,and trypsin inhibitory activity in Momordica cochinchinensis(12). In the current study, we examined P. x hybrida followingthe identification of ESTs from the genusPetunia via a databasesearch. Further experiments confirmed petunia cyclotides to bethe products of dedicated genes with a novel precursorstructure.
Structural and Evolutionary Implications of the Novel Pre-cursors fromGenus Petunia—The sequences of three cyclotide-encoding genes, named PETUNITIDE1 to -3, are shown in Fig.1 alongside the translated amino acid sequences deriving fromthe BLAST-matched ESTs, where they encode precursor pro-teins of 79 residues comprising an endoplasmic reticulum sig-nal sequence, a pro-region of 15 residues, a single cyclotide-encoding domain, and a six-residueC-terminal tail sequence. Adistinguishing feature of Solanaceae cyclotide precursors istheir relatively short (15 residue) N-terminal pro-regions com-pared with those from Rubiaceae (22–69 residues) andViolaceae (28–45 residues) cyclotide genes. In combinationwith their short C-terminal tails, the Solanaceae cyclotides areencoded by relatively short cyclotide-encoding precursors,similar in size to recently reported atypical cyclotide precursorsfrom Rubiaceae (55).
FIGURE 5. MALDI-MSI of a petunia leaf. The localizations of four distinct m/z signals are indicated with intensity scales relative to average spectrum maximuminset. A, average mass spectrum for the data acquired from the leaf section. B, dark field microscope image of a paradermal (adaxial longitudinal) cryosectionof P. x hybrida leaf. C, localization of m/z 3069. D, localization of m/z 3110. E, localization of m/z 3426. F, localization of m/z 3463. G, overlay of m/z 3426 (green)and m/z 3069 (red) signals.
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Some of the BLAST-matched Petunia ESTs appeared toterminate with stop codons directly C-terminal to the cyclo-tide-encoding domains, indicating that acyclic cyclotides(“acyclotides”) might be produced in planta. Accordingly, wecharacterized a peptidematching one of these predicted acyclicESTs.The first acyclotide characterized was violacin A from the
Violaceae plant Viola odorata, which we referred to at the timeas a “linear cyclotide” (56). Later, in O. affinis, the transcriptOak9 was found to encode kalata B20-lin, an acyclotide seem-ingly arising from a single nucleotide change that introduces astop codon (25). In two recent studies of Rubiaceae plantsHedyotis biflora and Chassalia chartacea, panels of novel“linear cyclotides” were characterized and referred to as “uncy-clotides” (55, 57). We prefer the term “acyclotide” for the fol-lowing two reasons. 1) This is in keeping with established prac-tices in nomenclature of organic compounds as either cyclic oracyclic (58). 2) Selectional restrictions on English language pre-fixes mean that the “un-“ prefix can be taken to confer twomeanings (cf. “unlockable”), and when added to the word“cyclic,” the resultant “uncyclic” can be construed to conveythat the item being described is “not cyclic” or alternatively thatit is “no longer cyclic.” Thus, the “a-“ prefix is unambiguous andconveys only one meaning to “acyclic”: that the item is “not
cyclic.” Interestingly, in some cases, the acyclotides have bio-logical activity comparable with that of their cyclic counter-parts (53), but in most cases, the linear homologues are devoidof the activity of the cyclic forms (59, 60).Fig. 7 illustrates a comparison of the gene structures of rep-
resentative cyclotide and related knottin- or acyclotide-encod-ing sequences, inwhichPETUNITIDE genes, in terms of overallstructure and size, can be seen to bear the most similarity torecently characterized CHASSATIDE genes identified withinRubiaceae plant Chassalia chartacea (55). Despite the lack ofpeptide evidence for cyclotide-like sequences in Poaceae, theyare likely to be produced as acyclic peptides due to their trun-cation by a predicted stop codon, as illustrated for “ZeamaysB,”and in this way bear similarity to genes encoding putative petu-nia acyclotides, including Phyb J, K, andL. Peptide evidencewasfound for hedyotide B2, an acyclotide found in Rubiaceae plantH. biflora, and the gene encoding it was found to have beentruncated at the C terminus of the cyclotide domain by a stopcodon (57), with the remainder of the gene exhibiting homol-ogy to other Rubiaceae cyclotide genes as indicated in Fig. 7, Aand B. Other acyclotides have also been characterized fromRubiaceae plants, including kalata B20-lin from O. affinis (25)and Psyle C from P. leptothyrsa (24).A single example of a linear cyclotide, violacin A, has been
described in the Violaceae plant V. odorata (56). However, thegene encoding violacin A is unique compared with other acy-clotide-encoding genes in that the premature stop codon doesnot appear after the entire peptide domain but rather appears totruncate an otherwise complete cyclotide gene. In this case, thenucleotide sequence immediately following the stop codon isreplete with sequence that would encode typical proto-C-ter-minal and CTR amino acids, suggesting that violacin A mightbe the result of a single nucleotide polymorphism.Solanaceae cyclotides are encoded by PETUNITIDE genes
that incorporate sequence motifs considered integral for cy-clotide biosynthesis and backbone cyclization in previouslydescribed cyclotide genes (51), including a proto-C-terminalAsx followed by a hydrophobic amino acid two residues C-ter-minal (e.g. –Asx-Xaa-Leu/Ile/Val–). It has been posited that anasparaginyl endopeptidase (AEP) would be the logical candi-date enzyme driving cyclotide biosynthesis (47, 51), due to thedemonstrated in vitro cleavage and transpeptidation (ligation)activity of jackbean AEP to produce mature concanavalin A(61) and its activity at a wide range of Asx-Xaa bonds (62). Alarge body of work has demonstrated that AEP canmature seedstorage globulins and albumins (63–68). In sunflowers (Aster-aceae), a gene encoding a napin-like preproalbumin storagePawS1 albumin gives rise to mature seed storage albumin aswell as small backbone-cyclized trypsin inhibitor embeddedupstream of the albumin (69). Following transformation ofPawS1 into an Arabidopsis aep null mutant, it was determinedthat AEP was required for cleavage reactions at the proto-Nterminus of SFTI, the proto-C terminus of SFTI, and theproto-N terminus of the PawS1 small albumin subunit (69), andbased on this, AEP was proposed as a good candidate enzymeformediating ligation of N and C termini of SFTI-1. This mightoccur through attack of the thioester acyl intermediate of AEP
FIGURE 6. Relative quantitation of Phyb A across petunia leaf regions.A, representative petunia leaf highlighting the regions sampled for LC/MSanalysis. Green highlight, midvein; red highlight, lamina; blue highlight, leafperiphery. B, relative extracted ion chromatogram peak intensities for signalat 1535.02� in LC/MS analyses (corresponds to m/z 3069 in MALDI experi-ments) of boiled plant extracts. Error bars, S.E. (n � 10). Asterisks denote sig-nificant differences at 0.001 � p � 0.01 (**) or 0.01 � p � 0.05 (*) followingone-way analysis of variance using Bonferroni’s multiple comparison test.Scale bar, 1 cm.
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by the freed glycine of SFTI-1, held close to the thioester by thedisulfide bond (61).Recently, it was discovered that the butterfly pea (C. ter-
natea) contains pea albumin-1-like genes in which a cyclotidedomain has “replaced” the first of the PA1 domains, and thiscyclotide domain is trailed by residues that would enable bio-processing via the same AEP-mediated mechanism (21, 22).One of the peptides characterized in the current study, Phyb
M, with the sequence pQSISCAESCVWIPCATSLIGCSCVN-SRCIYSK is supported by EST FN005530 and incorporates apost-translational N-terminal pyro-Glu modification. The firstpyroglutamyl modification of a linear cyclotide was reportedrecently in hedyotide B4 fromH. biflora, which was reported asa degradation product of a longer linear cyclotide, hedyotide B2(57). We did not detect peptide masses corresponding to non-pyro-Glu Phyb M, which suggests that it is not a degradationartifact from a longer mature peptide. N-terminal pyro-Glupeptides are known to be more resistant to degradation than
their corresponding N-terminal Gln homologs (70), so theincorporation of a modified N-terminal residue in lieu of back-bone cyclization may be an alternative strategy to provideenhanced stability toward exopeptidase activity. Thus, PhybMbridges an evolutionary gap between PhybK, an acyclotidewitha free N terminus, and Phyb A with its “complete” CCK motif.The discovery of all three peptide forms from P. x hybridamayrepresent “evolution in progress.”Despite the isolation of PETUNITIDE2 and PETUNITIDE3
transcripts, we found no mass spectrometric evidence for pep-tide masses for the cyclotides they would encode (Phyb B andPhyb C; Fig. 1). The sequences of these peptides as well as thoseencoded within the identified ESTs and the rest of the peptidescharacterized in this study are mostly homologous to manypreviously described cyclotides and incorporate permutationsof previously observed amino acids within loop regions. Anexception to this is the translated sequence of ESTs FN020915and FN020916 (GIPCGGSCVWIPCISGVQGCSCSNKIC-
FIGURE 7. Comparison of prototypic cyclotide and acyclotide-encoding gene structures in angiosperms. A, variation in cyclotide and related knottin genearchitectures among angiosperms. B, acyclotide and knottin domains. C, cyclotide domains. Signal sequence is shown in white boxes. Knottin and acyclotidedomains are shown in light blue boxes. Cyclotide domains are shown in orange boxes. N-terminal pro-domains are shown in green boxes. C-terminal repeats areshown in mauve boxes. V, Violaceae; R, Rubiaceae; P, Poaceae; F, Fabaceae; C, Cucurbitaceae; S, Solanaceae.
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YRN), in which the absolutely conserved Glu in loop 1 (Fig. 4),present in all previously characterized cyclotides, is replacedwith a Gly residue.The potentially wider prevalence of cyclotides among
Solanaceae plants remains to be elucidated; however, BLASTsearches using full-length PETUNITIDE sequences to query allGenBankTM nucleotide sequences, including EST databases,revealed only matches to the Petunia ESTs reported in thisstudy. This search confirms the uniqueness of the precursorsequence, especially considering the depth of EST coverageamong members of the Solanaceae (e.g. 334384 in tobacco,297142 in tomato, 249761 in potato, and 118054 in capsicum),and suggests that cyclotides evolved independently within theSolanaceae.Vascular Localization and Functional Significance—Trabi et
al. (26) investigated the tissue-specific distribution of a panel ofcyclotides from Viola hederacea by comparing LC/MS profilesof separate tissue extracts and demonstrated that cyclotides aredifferentially expressed among plant tissues. This phenomenonwas observed in a subsequent study of cyclotide localization inO. affinis plant tissues, which, in addition to examination ofextracted peptides via LC/MS, observed no cDNA encodingkalata B2 in root tissue (25). Complementary to these studies,recent work examined the subcellular location of cyclotidesduring their biosynthesis, the results of which indicate that theyare processed and accumulate within plant cell vacuoles (27).However, despite these advances, details on the intratissue dis-tribution of cyclotides are lacking.To examine the localization of cyclotides within petunia
leaves, we analyzed a tissue section using MALDI-MSI andobserved a number of peptide masses appearing in the massrange diagnostic of cyclotides (Fig. 5A). One of the signalsobserved was consistent with Phyb A and appeared to correlatewith the vascular structures of the prepared leaf section (Fig.5C). Through LC/MS analysis of dissected leaf extracts, relativequantitation of Phyb A was assessed in midvein, laminar, andperipheral leaf tissues. Phyb A was found to exist at �2-foldhigher concentrations within midvein tissue versus laminar orperipheral leaf tissue extracts (Fig. 6B). This distribution wasunique compared with the trends observed for 16 control m/zsignals, which were present either in equivalent abundanceacross the three leaf tissue areas or in higher abundance withinlaminar and/or peripheral leaf tissue extracts compared withmidvein extracts (supplemental Fig. S4). The size and directionof the -fold change in Phyb A abundancemight be of functionalsignificance in the context of plant defense, given a previousstudy of Arabidopsis thaliana in which it was demonstratedthat non-peptidic plant defensive glucosinolates were enrichedat the midvein and the outer lamina of leaves (39). In the Ara-bidopsis study, it was further observed thatHelicoverpa feedingpreference could be influenced by as little as 1.3-fold relativechanges in the concentration of indol-3-ylmethylglucosinolate,themajor glucosinolate present. The observed localization pat-tern for Phyb A (m/z 3069) primarily in the vasculature of theleaf section mirrors the glucosinolate study and places Solana-ceous cyclotides in the right location in leaves to be potentialmodulators of insect herbivory.
One of the limitations of MALDI-MSI is its inherent limiteddynamic range, which is instrument-, matrix-, and analyte-de-pendent. Few studies have quantified the dynamic range of thistechnique, but a recent investigation demonstrated linearity ofsignal intensity increasing with analyte concentration from thelimit of quantitation (femtomolar) over less than 2 orders ofmagnitude (71). Given the relative abundance of Cter A com-pared with other putative cyclotide signals in leaf extract (Fig.2A), it is therefore unsurprising that low abundance putativecyclotide signals in the extract were not detected duringMALDI-MSI, where the sample had not been deconvolutedthrough extraction, and the analyte was rather presented to theinstrument in a complete, complex sample matrix.Additional signals were observed during MALDI-MSI anal-
ysis of the leaf section that did not correspond to any of thecalculated peptide masses from PETUNITIDE genes or trans-lated EST sequences. Signals atm/z 3426 and 3463 appeared tobe abundant in areas of the leaf section distinct fromm/z 3069or 3110. Tandem MS of the major peak observed at m/z 3426(average) in the MALDI-MSI experiment (m/z 3424 monoiso-topic in ESI) following reduction of a single disulfide bond per-mitted de novo sequencing and its further identification asnuclear PSII-T 5-kDa protein. Although a fragment of a homol-ogous PSII-T protein has been sequenced from spinach (72),our work demonstrates the first mass spectral evidence of anynuclear PSII-T protein and describes a previously unreporteddisulfide bond. Given the conserved nature of the cysteines inhomologous nuclear PSII-T proteins (not shown) a disulfidebond could be expected in all such proteins. The even distribu-tion of PSII-T among all leaf areas, as shown in supplementalFig. S4O, is consistent with the ubiquitous nature of photosyn-thetic proteins in leaf tissue.Fig. 5G illustrates a differencemapof signal intensities for the
m/z 3426 (PSII-T) and 3069 (Phyb A) peptides and reflects thedifferential spatial expression of the two masses. The differen-tial localization of the various m/z signals from the MALDI-MSI experiment indicated that the intensity of any particularsignal was not significantly influenced by cell size or density andthat signals for each peptide were heterogeneous across thetissue sample. This validates the sample preparation method-ology and the suitability of the technique as a whole and dem-onstrates its ability to provide information on the spatial rela-tive abundance of peptide analytes.
CONCLUSIONS
Here we have demonstrated the first evidence that cyclotidesand acyclotides exist within the Solanaceae plant family as theproducts of a novel precursor structure. This work comple-ments previous characterizations of cyclotide-encoding genesfrom Violaceae, Rubiaceae, and Fabaceae plant families (73).Analysis of the Solanaceae cyclotide (PETUNITIDE) genesimplicates AEP in their proto-C-terminal processing, consis-tent with purported biosynthetic pathways of cyclotides in theliterature (47) and consistent with the demonstrated require-ment for AEP in processing the cyclic sunflower trypsin inhib-itor SFTI-1 (69). Petunia cyclotides and their encoding geneshave residues trailing the proto-C terminus consistent withthose shown previously to be important for their correct bio-
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synthesis. Similar to CHASSATIDE genes from Rubiaceae,PETUNITIDE genes are more compact than previously knowncyclotide precursors. Subtle differences between the sequencemotifs flanking the mature cyclotide sequences in Solanaceaeand phylogenetically distinct Rubiaceae or Violaceae precur-sors might explain the low yields of cyclic products followingexpression of both natural and designed cyclotides inSolanaceae plants (47, 51). Thus, the discovery of novel cy-clotide-encoding genes within the Solanaceae family mightenable their application as an alternative option for circularpeptide production compared with known cyclotide genes.PETUNITIDE genes might also be employed to enhance cropprotection within Solanaceae species important to humannutrition, such as potato, capsicum, and tomato, throughgenetic incorporation of custom cyclotide and/or acyclotide-encoding domains.Our data demonstrate that cyclotides associate with the vas-
cular features of petunia leaf tissues, which aligns with previ-ously characterized small molecule and peptidic mediators ofplant defense. Examples include glucosinolate (39) precursorsof toxic cyanocompounds in Arabidopsis, terpenes involved insquirt-gun defenses in Bursera sp. (40), pumpkin fruit trypsininhibitor (41), cysteine proteinase inhibitors in maize (42), anddefensins in capsicum (43). Hence, the localization of increasedconcentrations of cyclotides in these areas couldmodulate her-bivore feeding behavior and contribute to plant defense. Thiswork adds to the known pool of cyclotide-producing plant fam-ilies and provides an impetus for the further exploration ofSolanaceae species for cyclotides. Judging from the variation incyclotide gene structures now described, it seems likely thatfurther significant variations will be discovered in yet to bedescribed cyclotide-containing plant families. This combinedknowledge will be crucial to understanding their evolutionaryorigins as either the products of convergent evolution or poten-tially the action of transposable elements.
Acknowledgments—We thankAlun Jones and theMolecular andCel-lular Proteomics Facility (University of Queensland, Institute forMolecular Bioscience) for access to mass spectrometry instrumenta-tion and Dr. Anne Rae (CSIRO Plant Industries) for helpfuldiscussions.
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