Fruit Development, Ripening and Quality Related Genesin the Papaya Genome

Robert E. Paull & Beth Irikura & Pingfang Wu &

Helen Turano & Nancy Jung Chen & Andrea Blas &

John K. Fellman & Andrea R. Gschwend &

Ching Man Wai & Qingyi Yu & Gernot Presting &

Maqsudul Alam & Ray Ming

Received: 5 November 2008 /Accepted: 12 November 2008 /Published online: 18 December 2008# Springer Science + Business Media, LLC 2008

Abstract Papaya (Carica papaya L.) is the first fleshy fruitwith a climacteric ripening pattern to be sequenced. As amember of the Rosids superorder in the order Brassicales,papaya apparently lacks the genome duplication thatoccurred twice in Arabidopsis. The predicted papaya genes

that are homologous to those potentially involved in fruitgrowth, development, and ripening were investigated.Genes homologous to those involved in tomato fruit sizeand shape were found. Fewer predicted papaya expansingenes were found and no Expansin Like-B genes werepredicted. Compared to Arabidopsis and tomato, fewergenes that may impact sugar accumulation in papaya,ethylene synthesis and response, respiration, chlorophylldegradation and carotenoid synthesis were predicted. Similaror fewer genes were found in papaya for the enzymes leadingto volatile production than so far determined for tomato. Thepresence of fewer papaya genes in most fruit developmentand ripening categories suggests less subfunctionalization ofgene action. The lack of whole genome duplication andreductions in most gene families and biosynthetic pathwaysmake papaya a valuable and unique tool to study theevolution of fruit ripening and the complex regulatorynetworks active in fruit ripening.

Keywords Fruit growth . Expansins . Ethylene .

Respiration . Carotenoids . Chlorophyll . Volatiles .

Sugar accumulation

Introduction

The cultivated papaya (Carica papaya L.) is the mostimportant economic species in Caricaceae and is the onlymember of the genus Carica [19, 20]. Papaya andArabidopsis have both been placed in the Brassicales [8,9]. This classification is supported by morphological andmolecular characteristics [160, 161, 165, 166]. Whereas thecore Brassicales (including Arabidopsis and Brassica) andMoringaceae (Moringa oleifera) have dry siliques orcapsules, papaya has a fleshy berry fruit. Papaya is

Tropical Plant Biol. (2008) 1:246–277DOI 10.1007/s12042-008-9021-2

Communicated by Dr. Paul Moore

Electronic supplementary material The online version of this article(doi:10.1007/s12042-008-9021-2) contains supplementary material,which is available to authorized users.

R. E. Paull (*) : B. Irikura : P. Wu :H. Turano :N. J. Chen :C. M. WaiDepartment of Tropical Plant and Soil Sciences,University of Hawaii at Manoa,Honolulu, HI 96822, USAe-mail: paull@hawaii.edu

A. Blas :G. PrestingDepartment of Molecular Biosciences and Bioengineering,University of Hawaii at Manoa,Honolulu, HI 96822, USA

J. K. FellmanDepartment of Horticulture and Landscape Architecture,Washington State University,Pullman, WA 99164, USA

A. R. Gschwend : R. MingDepartment of Plant Biology,University of Illinois at Urbana-Champaign,Urbana, IL 61801, USA

Q. YuHawaii Agriculture Research Center,Aiea, HI 96701, USA

M. AlamAdvanced Studies in Genomics, Proteomics and Bioinformatics,University of Hawaii,Honolulu, HI 96822, USA

polygamous and is a herbaceous, single-stemmed and erectplant. Other than Carica papaya L., the other edible speciesin the family are Vasconcella candamarcensis Hook. f., V.monoica Desf., V. erythrocarpa Heilborn, V. goudotianaSolms-Laubach and V. quercifolia Benth. and Hook [187].These fruit are normally dry and lacking the juicy flesh ofC. papaya are mostly eaten cooked. Another edible species,V. pentagona, is called ‘babaco’. Isozyme and AFLPanalysis indicates that C. papaya is only distantly relatedto the Vasconcella species. The greatest diversity in C.papaya exists in the Yucatan-San Ignacio-Peter-Rio Mota-gua area of Central America. The wild population in thisarea has greater diversity than domesticated populations[134, 205].

The papaya fruit is a fleshy berry and fruit growthfollows a single sigmoid growth curve [224]. Duringdevelopment, all tissues in the gynoecium less than 1 mmare meristematic [170]. Later, the outer layer of theepidermis increases in size while the subepidermal layercontinues to divide both anticlinally and periclinally. Thecentral parenchyma of the pericarp increases in size anddivides with the placenta forming opposite the marginalvascular bundles. This meristematic activity lasts 28–42 days and determines final fruit size. Fruit growth showstwo major phases. The first lasts about 80 days post-anthesis, with a large increase in dry weight occurring justbefore fruit maturity. Fruit development takes 150–164 daysthat is extended by another 14–21 days in Hawaii in thecolder months [145, 155]. Mesocarp growth parallels seedand total fruit growth.

Papaya fruit shape is a sex-linked character and rangesfrom spherical to ovoid in female flowers to long,cylindrical or pyriform (pear-shaped) in hermaphroditeflowers. The fruit is normally composed of five carpelsunited to form a central ovarian cavity that is lined with theplacenta carrying numerous black seeds. Placentation isparietal with the seeds attached by 0.5–1 mm stalks. Theovarian cavity is larger in female fruit than hermaphrodite.The shape of the cavity at the transverse cut ranges fromstar-shape with five to seven furrows to smooth and circular[45].

Papaya ripening is climacteric with the rise in ethyleneproduction occurring at the same time as the respiratory rise[146]. Respiration is a critical factor in fruit growth anddevelopment, especially as it relates to fruit ripening. Fruitsare divided into two broad groups based on the role ofethylene in the ripening process and its relationship to therespiratory pattern. Climacteric fruits (e.g., banana, papaya,peach, tomato) demonstrate a peak in respiration andethylene production during ripening, including autostimu-latory (so-called System 2) ethylene production [27]. Thetiming of the autostimulatory ethylene peak [23, 108] andof other ripening events (skin color changes, carotenoid

synthesis, flavor development, softening) varies widelybetween species and cultivars [27, 35, 38, 86]. Non-climacteric fruit a gradual decline in the respiration rate,no marked peak in ethylene production and gradualchanges in other ripening parameters.

The papaya genome has been recently sequenced [132].The sequences represented 75% (277.4 Mb) of the(372 Mb) genome and 90% of the euchromatic regions.The assembled EST unigene set contained 16,362 unigeneswith 92.1% matching of the assembled whole shotgunsequences (WGS). The transcribed sequences matched only3.6% (13.4 Mb) of the whole genome and 48% of the genicregion in WGS [132]. This resource allows us to determinethe number of genes that are potentially involved in fruitgrowth and development, and in fruit ripening. Anunderstanding of the predicted gene number in each familyis the first step in determining which members of eachfamily are expressed and at what level during fruitdevelopment. In this review, we provide an overview ofthe number of predicted genes potentially involved in thedifferent phases of fruit growth, development and ripening.

Results and Discussion

Fruit Development

The papaya plant is a herb that can grow up to 9–10 m inheight and generally has only a single stem. Lateralbranching does occur if the apex is damaged or when theplant is tall. The lack of lateral shoot growth in plants hasbeen associated with auxin inhibition, abscisic acid (ABA)and strigolactone [82, 115, 221]. The plant growth regulatorABA plays a crucial role in the plant’s adaptation to stressand in seed maturation and dormancy, and fruit ripeningand senescence [221]. ABA and strigolactone are productsfrom the cleavage of carotenoids at one of its double bonds.Carotenoid cleavage is carried out by two groups of relatedenzymes. One group uses multiple carotenoid substratesand is referred to as carotenoid cleavage dioxygenase(CCD). The other group has C40-9-cis-epoxycarotenoid asthe preferred substrate and referred to as 9-cis-epoxycar-otenoid dioxygenase (NCED). The NCEDs are thought tobe involved in ABA synthesis while the role of CCDs isless clear, with some evidence to suggest a role in lateralshoot growth [14, 15], possibly by cleavage of carotenoidsto strigolactone or a related compound [82].

Two Arabidopsis genes CCD7/MAX3 and CCD8/MAX4control lateral shoot growth [14, 31, 82]. The citrushomolog gene (CsNCED1), likely plays a role in ABAsynthesis in leaves and fruits [163]. ABA has also beengiven a role in the regulation of citrus fruit skin coloration[162]. Citrus is a non-climacteric fruit and normally

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responds to ethylene by induced fruit coloration [3]. Papayais climacteric fruit though fruit mesocarp carotenoiddevelopment and fruit degreening are ethylene independent[121]. ABA via CCD and/or NCED may therefore play arole in fruit mesocarp carotenoid development and skindegreening. One CCD (CpCCD1) and two NCEDs(CpNCED1, CpNCED2) are predicted in papaya (Table 1).A partial open reading frame (ORF) was found for anotherpossible CpCCD (ABIM01019146.1) that had 67% identity(E-value 5e-37) to Arabidopsis CCD1 (NP_191911.1). Apapaya EST was not found for CpCCD1 or CpNCED1 anda chloroplast transit peptide was not found for CpNCED1.The Arabidopsis carotenoid cleavage family has fiveputative NCEDs and four putative CCDs [15] and tomatohas two (LeCCD1A and LeCCD1B) [180]. CpCCD1 had69% identity (E-value 0.0) with Arabidopsis AtNCED4,CpNCED1 has 73% identity (E-value 0.0) with ArabidopsisAtNCED5. CpNCED2 had 77% identity (E-value 0.0) withAtNECED3. The CpCCD and the CpNCEDs were allpredicted to have a carotenoid oxygenase motif.

Fruit Growth

The papaya fruit is a fleshy berry and fruit growth follows asingle sigmoid growth curve [224] and can weigh from 0.2 kgto 10 kg [136]. This plasticity in size provides a uniqueopportunity to study fruit development control. Tomato fruitcan similarly vary from small berries (~2 g) to large fruit(1,000 g) [113] with a single gene ORFX (QTL loci fw 2.2)being responsible for 30% of the difference [24, 52, 75].ORFX appears to act at or near the plasma membrane βsubunit of CKII kinase [52] and exerts its control throughearly fruit cell division [113]. One homolog to ORFX waspredicted in papaya (CpORFX; ABIM01013288.1) with 63%identity (E-value 2e-49) with ORFX from Solanum pennellii(AF261775.1) and 62% (E-value 1e-49) with Solanumlycopersicum (AAF74286.1). CpORFX shows homology tothe cysteine-rich PLAC8 domain of unknown function that isfound in animals and plants [123]. No papaya ESTs were

detected for CpORFX. The function of CpORFX in papaya isunknown though the wide range of papaya fruit size presentthe possibility it may have a similar role to that in tomato.

Fruit Shape

The shape of papaya fruit varies with fruit from femaleflowers being spherical to ovoid, while fruit from hermaph-rodite flowers being cylindrical or pear shaped [136]. Amutation in the quantitative trait locus (QTL), OVATE,changes the shape of tomato from round to pear-shaped[114]. This regulatory gene apparently has its impact earlyin flower development [104, 105]. A similar QTL ineggplant has a similar effect on shape [104]. OVATE is anegative regulatory hydrophilic protein with a putativebipartite nuclear localization signal [114]. Three OVATEproteins were predicted in the papaya genome with ho-mology to OVATE sequences in Arabidopsis (AAY78676.1)and Solanum lycopersicum (AAN17752.1). Papaya ESTswere only found for one of the gene (CpOVATE1,ABIM01006651.1) that had about 38% identity (E-value4e-06) to Solanum lycopersicum OVATE (AAN17752.1)and 39% identity (E-value 4e-35) with Arabidopsis(NP_178114.1). All three predicted papaya genes had the~70 aa plant specific motif (DUF623) of unknown func-tion, also reported for the tomato OVATE gene [114].CpOVATE 2 (ABIM01006789.1) has 31% identity (E-value2e-25) with Arabidopsis Ovate family (AtOFP8,NP_197466.1) and 58% identity (E-value 5e-21) with tomato(AAN17752.1). CpOVATE3 (ABIM01013535.1) which wason the same linkage group as the papaya sex related gene has67% identity (E-value 6e-22) with tomato OVATE protein(AAN17752.1) and an OVATE-like protein from tobacco(ABW05088) with 65% identity (E-value 2e-21).

Another major gene controlling the elongated fruit shapeof tomato is SUN that acts in a dosage dependent manner[214]. The gene belongs to the IQD family with AtIQD1being the only member with a known function. A homologto SUN was found in papaya (ABIM01022664.1). This

Table 1 Carotenoid cleavage dioxygenases predicted in the papayagenome, the whole genome shotgun sequence accession number inNCBI (WGS Accession), coding sequence for amino acids (CDS),amino acids (aa), whether a chloroplast transit peptide was predicted,presence of discrete portion of protein possessing its own function and

the superfamily (domain), similarity in peptide sequence with anotherspecies (homology), extent the sequences were invariant (identity), E-value was the expectation value for homology, and the number ofpapaya expressed sequence tags (EST) detected with at least 500 basesand 99% identity

WGS Accession cds aa Transit Peptide Domains Homology Identity, E-value ESTs

CpCCD1 ABIM01016290.1 1275 592 Yes Carotenoid cleavagedioxygenase 4a,

Citrus clementinaABC26011.1

72, 0.0 0

CpNCED1 ABIM01023760.1 1524 507 None 9-cis-epoxycartenoiddioxygenase 5,

Citrus clementinaABC26010.1

79, 0.0 0

CpNCED2 ABIM01015050.1 1857 618 Yes 9-cis-epoxycartenoiddioxygenase 3,

Citrus clementinaABC26013.1

77, 0.0 1

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papaya homolog had four introns, the same as SUN andwas 446 aa long versus 405 aa for SUN [214]. Thehomology of the predicted papaya gene was moderate(37%, E-value 4e-34) for SUN and 50% (E-value 5e-103)for Arabidopsis IQD11 (NP_196850.1) and 35% (E-value3e-32) for IQD12 (NP_196016.1). Another predictedpapaya homolog (ABIM01018776.1) was two-thirdsthe length and had only two introns possibly due to agap between the two sequenced contigs. This secondpredicted gene was 45% (E-value 4e-27) homologousto SUN and 53% (E-value 2e-29) to Arabidopsis IQD12(NP_196016.1). The variation in papaya shape from roundto elongated presents a unique model to ascertain the roleof SUN homologs in determining fruit shape.

Expansins

The cell wall proteins termed expansin are involved in cellwall relaxation and growth [129]. A number of expansingenes are recognized [56]. The expansins have been dividedinto two classes based on sequence homology: expansin A(EXPA), expansin B (EXPB) with additional groups that areEXPA-Like and EXPB-Like proteins (EXLA, EXLB). EXPAand EXPB normally have a secretory sequence of 20–30 aa,a EG-45 domain (~120 aa) with distant homology toglycoside hydrolases (GH45) though having no enzymaticactivity, and a potential carbohydrate binding domain (CBD)of 98 aa [49, 173].

Expansins have been shown to be expressed duringtomato fruit growth [168] and different isoforms areexpressed during fruit ripening [34, 167]. Papaya fruit havealso been shown to express an expansin (CpEXPA1). Fourhave been reported in banana fruit [12]. Immunoblots havealso detected expansins in ripening pear, persimmon, kiwifruit, strawberry and pineapple but not detected in pepper[168]. The papaya genome contains at least 15 CpEXPA,three CpEXPB and one CpEXPLA (Supplementary Table 1).Secretory sequences were not predicted on four of theCpEXPAs. All CpEXPs had similar intron positions andlengths to those described for Arabidopsis [49].

The papaya expansins were part of the large superfamilyof at least three subfamilies (Fig. 1) but did not appear tohave members in three of the clades (VI, VII, X) [173] andno EXLB were found. Sampedro et al., [173, 175] proposedthat the number in the last common ancestor for expansinEXLB was four, though none were predicted for papaya(Table 2). The monocot/dicot ancestor had 15 to 17expansin genes [173]. Papaya is classified as a basal cladein the Order Brassicales [165] with at least 19 expansingenes in close to the number predicted for the monocot/dicot ancestor (15) [173].

The expansins are believed to have arisen and diversi-fied early if not before colonization on land [49, 110].

Expansins are found in monocots, pines, ferns and mosses.The closely related expansin-like sequences are similarlywidely found and the absence of any EXLB predicted inpapaya was unexpected. One EXLB is found in Arabidopsisand rice, and four in poplar [173] and one predicted in pine[174] suggesting that EXLBs were present before theangiosperms/gymnosperms separation. The Arabidopsis(At4g17030) and Pinus EXLB (TC89171) are distantlyrelated [174] but no match was found to any predictedpapaya gene sequences or in the papaya EST database.When predicted papaya protein models were queried usingthe Arabidopsis expansin like-B sequence, homology wasfound to EXPLA and EXPB sequences predicted in papaya.A number of other papaya sequences were predicted with aEG45 motif though these peptides had less than 100 aminoacids and had no CBD motif.

Skin Color Changes

The pathway of chlorophyll breakdown during fruitripening is expected to be similar to that occurring duringsenescence and involves the removal of the phytol residueand the central Mg by chlorophyllase and a dechelatase.The product of these two activities is pheophorbide-a whichis the substrate for pheophorbide-a oxygenase (PAO), andred chlorophyll catabolite reductase (RCCR) [92]. Thebreakdown product from PAO and RCCR is then exportedfrom the degenerating chloroplast for further breakdown inthe cytoplasm. The predicted Mg dechelatase gene has notbeen identified [106].

One chlorophyllase (CpCLH) gene (ABIM01011000.1)was predicted in the papaya genome versus two genes inArabidopsis thaliana and three in Brassica oleracea [92].The papaya CpCLH protein had a predicted chloroplasttransit peptide of 21 aa and had 60% homology (E-value9e-18) to B. oleraceae BoCLH3 protein (Fig. 2) and 61%(E-value 4e-17) to AtCLH2. The CpCLH has a 2Fe-2Sferrodoxin, iron sulfur binding site and was expressed inpapaya based on ESTs. A magnesium cheltase subunitgene with three ESTs was predicted in papaya(ABIM01016494.1+ABIM01016495.1). This gene had91% identity (E-value 0.0) with soybean (CAA04526.1).

PAO genes have been identified by functional genomics.A single PAO gene is predicted for papaya (CpPAO,ABIM01011326.1). The predicted gene is expressed basedupon EST data and has a 72-aa chloroplast transit peptide.CpPAO was similar (E-value 2e-131) to the ArabidopsisAtPAO gene (At3g44880), first described as AcceleratedCell Death (ACD1) in Arabidopsis. ACD1 has homology totomato lethal leaf spot 1-like protein (AAL32300) and aputative cell death suppressor protein in Oryza sativa(AAK98735). All three species’s PAO genes contain apredicted iron-sulfur binding domain.

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Fig. 1 Phylogenetic tree ofexpansin super family proteinsequences from Arabidopsis,Popular and papaya aligned withClustalW and neighbor-joiningvalues. The tree was was con-structed with MEGA 4.0. Theexpansin and popular sequenceswere downloaded from http://www.bio.psu.edu/expansins/index.htm. Accessed 2008, Sept04. CpEXPA1 had been previ-ously described (ABD65309)and was used to number thepredicted CpEXPA. EXPAorthologous gene clades are in-dicated by bars and Romannumerals from [174]

250 Tropical Plant Biol. (2008) 1:246–277

As with a single gene in Arabidopsis (At4g37000),described as ACD2 [117, 213], a single RCCR gene waspredicted in papaya (CpRCCR, ABIM01012476.1). Thepapaya CpRCCR protein has a predicted chloroplasttransit peptide (61 aa) and papaya ESTs. RCCR hasslightly greater identity (74%, E-value 4e-92) to thetomato RCCR [154] than to the Arabidopsis ACD2(71%, E-value 1e-80).

Carotenoid Biosynthesis

Carotenoids serve several functions in plants [58, 76].During fruit ripening as chlorophyll is degraded, theunderlying carotenoids are unmasked and de novo biosyn-thesis occurs in the papaya mesocarp chromoplasts.Carotenoids are derived from the 5-carbon compoundisopentenyl pyrophosphate (IPP). The C40 backbone of allcarotenoids is assembled from two C20 geranylgeranylpyrophosphate (GGPP) molecules which in turn are eachderived from four C5 IPP molecules (see volatile productionbelow).

Synthesis of phytoene from two GGPP molecules is thefirst step in the carotenoid-specific biosynthesis pathwayand is catalyzed by phytoene synthase (PSY). PSY is a keyregulator in carotenoid biosynthesis and has been found tobe the rate-limiting enzyme in ripening tomato fruits,canola seeds and marigold flowers [10, 33, 73, 74, 91].PSY is expected to have close membrane-association asphytoene is lipid-soluble and is localized, along with itssubsequent end-products, inside the chloroplasts andchromoplasts [58]. Additionally, two forms of PSY, achromoplast- and chloroplast-specific form, have beenfound in tomato: PSY-1 and PSY-2, respectively [73].Desaturation of phytoene into z-carotene and lycopene ismediated by phytoene desaturase (PDS) and ζ-carotenedesaturase (ZDS), respectively. This desaturation convertsthe colorless phytoene into the pink-hued lycopene [58].Like PSY, the desaturases PDS and ZDS are closelyassociated with the plastid membrane but are not integralmembrane proteins [177]. Plastid transit peptides weredetected for PSY, PDS, ZDS and LCY-b for the papayagenome predicted genes. The published sequences for bothPDS and ZDS appear to be lacking part of the leadersequences, and do not start with a methionine as required bythe transit peptide prediction software. When the predictedpeptide sequences for PSY and ZDS from the papayagenome were used, both of which start with methionine,transit peptides were predicted.

Cyclization of lycopene via lycopene e-cyclase (LCY-e)or lycopene b-cyclase (LCY-b) results in a a- or b-carotene,respectively. Yamamoto [218] showed that 59.3% of thetotal carotenoids in yellow-fleshed papaya are comprised ofb-carotene or its derivatives, i.e., cryptoxanthin, indicatingthe b-ring cyclization pathway for synthesis of the yellow-pigmented carotenoids. The family of lycopene cyclases inplants shows evidence of multiple gene duplication eventsand divergence of catalytic function. Plant lycopene cyclasesshare a common phylogenetic origin with bacterial crtY andcrtL lycopene cyclases. Plant and bacterial lycopene cyclasesare polypeptides of ~400 aa, however, the plant lycopenecyclases have an additional 100 aa N-terminal transitsequence. Five regions of conserved amino acid sequencehave been identified: one putative dinucleotide-bindingregion and four motifs of unknown function [10, 58].

Several papaya genes that encode enzymes in thecarotenoid biosynthesis pathway have been previouslyidentified: phytoene synthase (PSY), phytoene desaturase(PDS), ζ-carotene desaturase (ZDS) and a chloroplast-specific lycopene β-carotene (LCY-b) (Table 3). BLASTnsearch of an EST database generated from a multiple-tissuetype cDNA library supports the activity of these singlegenes in papaya. Putative papaya homologs were identifiedfor lycopene ε-cyclase (LCY-e), β-ring carotene hydroxy-lase (CRTR) and zeaxanthin epoxidase (ZE) based on amino

CpCLH1

BoCLH3

AtCLH2

BoCLH2

SoyCLH1

SoyCLH3

CitrusCLH

SoyCLH2

AtCLH1

BoCLH1

0.000.050.100.150.200.250.300.35

Fig. 2 Phylogenetic relationship between predicted papaya chloro-phyllase and published sequences from NCBI aligned with ClustalWand neighbor-joining values. Tree was constructed with MEGA 4.0

Table 2 Expansin families in different plants, gene numbers and thelast common ancestor from Sampedro and Cosgrove [171]. Theestimates for papaya do not include incomplete sequences

Species EXPA EXPB EXLA EXLB

Last Common Ancestor 12 2 1 2Papaya 15 3 1 0Arabidopsis 26 6 3 1Popular 27 2 2 4Rice 34 19 4 1

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acid sequences obtained from GenBank (Table 3). Expres-sion of these putative papaya carotenoid biosynthesis geneswas supported by EST data in NCBI that include fruit EST[60].

Sugar Accumulation

Photosynthates are transported into fruits mainly as sucrosewith some fruits accumulating starch that is broken down tosugars during ripening while other fruit such as papayadepends upon a continued supply of sucrose for sweetnesswhen ripe. During early fruit growth the sucrose is thoughtto be unloaded through the symplast pathway involvingSUS (sucrose synthase) and SPS (sucrose phosphatesynthase) activities and metabolized in respiration and usedfor growth [144, 189, 223]. The activities of SUS and SPS,and their mRNA levels parallel the increase in growth.After fruit growth stops, sucrose accumulation is thought toinvolve a cell wall invertase to cleave the sucrose arrivingvia the phloem to hexoses, thus maintaining the sucrosesource to sink gradient. The hexoses are then taken up intothe fruit cells and vacuoles via hexose transporters [42, 144,212, 222, 223].

Sugars, mainly as sucrose, begin to accumulate inpapaya fruit about 110 days after anthesis during the last28–42 days of fruit development [43, 224]. Flesh total soluble

solids can be as low as 5% and up to 19%. The fact thatinvertase gene expression [225] and sugar (hexose) trans-porters [175] are upregulated just before and during sugaraccumulation suggests the participation of both invertasesand hexose transporter activities in fruit sugar accumulation.

Invertase (INV) is responsible for hydrolyzing sucroseinto fructose and glucose, and can be found in the cytoplasm,vacuole, and apoplast [188]. The family is classified intoacid, alkaline and cell wall invertase [29, 96, 203].Arabidopsis has one cell wall invertase and one acidinvertase [203]. In tomato, 16 cell wall invertase sequenceshave been found [77] while only two cell wall invertaseswere found in the papaya genome; CpCWINV1 andCpCWINV2 (Table 4). These two papaya cell wall invertaseshave the Glycohydrolase Family 32 (GH32) domain and thesecretory peptide occupied the first 26 aa. CpCWINV1 has100% homology in sequence with the published papayainvertase [225]. The second cell wall invertase (CpCWIN2)had high homology to the Arabidopsis cell wall INV1(NP_566464.1; At3g13790). The papaya acid invertaseCpAINV1 has the required GH32 domain but no signalpeptide and no papaya EST. This acid invertase predictionshowed moderate homology with the coffee invertase andArabidopsis cell wall invertase. Another predicted acidinvertase had less than 20% peptide sequence homologywith Japan pear and Arabidopsis acid invertases.

Table 3 Known and predicted papaya carotenoid biosynthesis genes,the whole genome shotgun sequence accession number in NCBI(WGS Accession), coding sequence for amino acids (CDS), aminoacids (aa), whether a chloroplast transit peptide was predicted,presence of discrete portion of protein possessing its own function

and the superfamily (domain), similarity in peptide sequence withanother species (homology), extent the sequences were invariant(identity), E-value was the expectation value for homology, and thenumber of papaya expressed sequence tags (EST) detected with atleast 500 bases and 99% identity

WGS Accession CDS aa ChloroplastTransitPeptide

Domains Homology Identity,E-value

EST

Previously ReportedPSY ABIM01042881.1 1744 438 Yes Isoprenoid_Biosynth_C1

superfamilyCarica papayaDQ666828.1

99, 0.0 3

PDS ABIM01021153.1 1251 417 Yes Pyridine nucleotide-sulfideoxidoreductase superfamily,PRK07233 Amino oxidase,pfam01593.

Carica papayaDQ779922.1

100, 8e-97 0

ZDS ABIM01018586.1 1680 470 Yes Pyr_redox superfamily; PRK07233,COG3349

Carica papayaDQ666829.1

100, 2e-154 0

LCY-b ABIM01002225.1 1512 503 Yes pfam05834, lycopene cyclasefamily

Carica papayaDQ415894.1

99, 0.0 2

PredictedCpLCY-e ABIM01012473.1 129 430 None Lycopene cyclase, FAD/NAD(P)

binding, pfam05834Arabidopsis thalianaNP_200513.1

71, 0.0 1

CpCRTR-b ABIM01017764.1 1041 346 None Carotene beta-ring hydroxylase,pfam04116

Citrus sinensisABB49053.1

62, 1e-82 4

CpZE ABIM01007866.1 1818 605 Yes Monooxygenase, zeaxanthinepoxidase, PRK07236

Nicotiana plumbaginifoliaQ40412.1

66, 0.0 1

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Sucrose synthase (SUS) catalyzes the reversible conver-sion of sucrose and UDP to UDP-glucose and fructose, andis only found in the cytoplasm [25, 124]. In Arabidopsis,five SUS genes are known and another predicted [25] withAtSUS1 having 67–72% homology to SUS genes fromother species. Citrus is reported to have three SUS genes[102] and papaya had four predicted SUS genes (CpSUS1to CpSUS4) (Table 4). All the predicted papaya SUSs hadsucrose synthase and glycosyl transferase (GT4) domains.The CpSUS1 has 99% (E-value 2e-142) homology with thereported partial sequence of SUS in papaya (AF420224)and 90% homology (E-value 0.0) with that of citrus(BAA89049), and, 85% (E-value 0.0) and 87% (E-value0.0) to Arabidopsis AtSUS1 and AtSUS3, respectively(Table 4). CpSUS2 showed similarity with citrus SUS2(BAA88904.1) and 85% homology (E-value 0.0) toAtSUS3 (NP_192137.1) while CpSUS3 had high homologywith onion (ABV90637.1; E-value 0.0) and tobacco(ABA64521.1; E-value 0.0). The fourth papaya sucrosegene (CpSUS4) had high homology with AtSUS5(NP_138534.2). Another SUS gene was predicted in papaya

with a GT4 domain and a sucrose synthase domains but thesequence was not complete and only about half theexpected peptide length.

Three sucrose phosphate synthase (SPS) genes werepredicted in papaya (CpSPS1, CpSPS2, CpSPS3). All hadGT4 and SPS domain (Table 4). The CpSPS1 and CpSPS2showed homology with the sucrose synthase in citrus andArabidopsis. In higher plant, three families of SPS aredescribed and the functional differences between the familiesare not obvious [107]. Four putative SPS genes occur inArabidopsis, two genes belong to Family A and one geneeach belongs to Family B and Family C. The CpSPS1 had87% homology (E-value 0.0) to citrus SPS and 72% toArabidopsis’s AtSPS1F (NP_ 196672.3). The second papayaSPS, CpSPS2 was similar to Arabidopsis AtSPS2F(NP_797528.1) with a homology of 77% (E-value 0.0) and80% (E-value 0.0) to the citrus SPS (BAA23213.1). TheAtSPS4F gene in Arabidopsis had a homology of 73% (E-value 0.0) with the CpSPS3 in papaya.

Papaya contains at least four hexose transporters genes(CpHT1 to CpHT4). In contrast to papaya, tomato contains

Table 4 Predicted cell wall (CW) and acid invertases (INV), sucrosephosphate synthase (SPS) and sucrose synthase (SUS) found in thepredicted in the papaya genome, the whole genome shotgun sequenceaccession number in NCBI (WGS Accession), coding sequence foramino acids (CDS), the number of introns in the nucleotide sequence(introns), amino acids (aa), presence of discrete portion of protein

possessing its own function and the superfamily (domain), similarityin peptide sequence with another species (homology), extent thesequences were invariant (identity), E-value was the expectation valuefor homology, and the number of papaya expressed sequence tags(EST) detected with at least 500 bases and 99% identity

WGS Accession cds Introns aa Domain Homology Identity,E-value

EST

CpCWINV1 ABIM01017629.1 4061 5 579 GH32 papaya, AAL16015.1 99, 0.0 4CpCWINV2 ABIM01007212.1 2851 2 329 GH32 Arabidopsis, NP_566464.1,

At3g13790 AtCWINVI69, 9e-84 1

CpAINV1 ABIM01023808.1 2134 3 455 GH32 Arabidopsis NP_566464.1,At3g13790 AtCWINVI

55, 2e-140 0

CpAINV2 ABIM01008005.1 1769 2 122 GH32 Japanese pear BAF35859.1 81, 8e-24 0CpSPS1 ABIM01011233.1 to

ABIM01011236.17336 19 1049 Glycos-Transf,

S6pp CBSArabidopsis NP_797528.1,AtSPS2F/ AtSPS1

72, 0.0 6

CpSPS2 ABIM01023167.1 +ABIM01023166.1

6486 11 908 Sucrose synthase,Glyco_transferase

Arabidopsis NP_196672.3,At5g20280.1 AtSPS1F

77, 0.0 0

CpSPS3 ABIM01020295.1 7499 13 1021 GT1, S6PP Arabidopsis NP_ 001031609.1,At4g10120.1 AtSPS4F

73. 0.0 0

CpSUS1 ABIM01015572.1 3548 12 805 Sucrose SynthaseGlyco_transferase

papaya AF420224 99, 2e-142 7

Arabidopsis, At3g43190 AtSUS4 87, 0.0CpSUS2 ABIM01011863.1 6486 14 788 Sucrose synthase, GT1 Arabidopsis NP_192137.1,

At4g02280.1 AtSUS378, 0.0 0

CpSUS3 ABIM01015610.1 5375 12 859 GT1, S6PP Arabidopsis NP171984.2,At1g04920 AtSPS3F

77, 0.0 1

CpSUS4 ABIM01022037.1 4399 13 862 SucroseSynthase,Glyco_transferase

Arabidopsis NP138534.21,At5g37180.1 AtSUS5

74, 0.0 1

CpSUS5 ABIM01020824.1 toABIM01020827.1

7657 8 392 Glycos_Transf_GTB,sucrose synthase

Arabidopsis NP_192137.1,At4g02280 AtSUS3

80, 8e-87 2

Tropical Plant Biol. (2008) 1:246–277 253253

three named loci (HT1, HT2, and HT3) and seven putativeloci [61, 79], Arabidopsis contains four named loci (SGB1,TMT1, TMT2, TMT3) and five putative loci [39]. A fifthpapaya hexose transporter was indicated, but the genomicsequence of the ORF was incomplete. Papaya ESTs werefound for two of the predicted hexose transporters CpHT2and CpHT3 (Table 5). The number of introns varied widely,CpHT1, CpHT2, CpHT3, and CpHT4 had two, 12, 11, andone intron, respectively. The predicted peptide for CpHT3had a 60% chance of being localizing to the plastid. Theremaining papaya hexose transporters did not show specificlocalization or export. Papaya contains at least threeincomplete hexose transporter genes or pseudogenes thathad only seven to 11 of the 12 expected transmembranedomains while having domains for MFS and sugartransporter. One of the pseudogenes (ABIM01025390.1)had four ESTs identified. Two of the three pseudogenes hadsecretory sequences for the endoplasmic reticulum.

Respiration

Plant respiration differs from most animal respiration inpossessing a number of additional components including i)the presence of an alternative oxidase to Cytochrome Coxidase that is cyanide insensitive, ii) an internal rotenone-insensitive NAD(P)H non-proton pumping dehydrogenase,and iii) external NAD(P)H non-proton pumping dehydro-genases [64, 118, 206]. Alternate oxidase (AOX) is aterminal quinol oxidase that is non-proton pumping, andtransfers an electron to oxygen while dissipating the energyas heat [128]. AOX is resistant to cyanide [90] and sensitiveto salicylhydroxamic acid (SHAM) [178]. AOX activity istaxonomically widespread and found in all kingdoms [128].In dicots, two types of AOX, AOX1 and AOX2 are found,while monocots seem to posses only AOX1 [54]. AOXs

have been associated with a number of physiologicalfunctions such as thermogenesis in Sacred Lotus duringflowering [210]. Other roles include balancing carbonmetabolism and electron transport, as may happen duringclimacteric fruit ripening [53, 195], control of reactiveoxygen species generation, O2 scavenging and resistance totoxins and pathogenicity [128, 133]. Though AOX1 andAOX2 are present in multigene families in most plants [128]only two genes for alternative oxidase AOX1 was predictedfor papaya, CpAOX1 (ABIM01002926.1), and CpAOX2(ABIM010 10551.1 + ABIM01010553.1). The predictedCpAOX1 protein had a predicted signal peptide of 22 aminoacids, two transmembrane regions, and was supported byfinding papaya ESTs. The two characteristic AOX conservedcysteine residues occurred at Cys 105 and Cys 155. Twointrons similar to Oryza sativa AOX (AB004813) werepresent in the gene. The predicted CpAOX1 gene has 79%(E-value 1e-138) and 71% (E-value 1e-139) identity to thetwo Arabidopsis genes Q9ZRT8 and Q39219, respectively.CpAOX2 similarly has two transmembrane regions. Nosecretory sequence was predicted though EST data suggestedit is expressed. CpAOX2 has 88% identity (E-value 1e-109)to Vigna unguiculata AOX (Q93X12) and 82% (E-value 1e-102) with Arabidopsis (O22049).

Plant mitochondria can oxidize NADH and NADPHwithout proton pumping [80, 130]. These dehydrogenasesare referred to as Type II NAD(P)H [130, 131]. Type IIdehydrogenases operate in parallel to Type I proton-pumping multi-subunit complex I. Both types are found inthe electron transfer chains of several bacteria, and infungal and plant mitochondria. Type II dehydrogenases arefound on the external (NDB) and internal (NDA) faces ofthe inner mitochondrial membrane and transfer electronsfrom NAD(P)H to quinone. It has been proposed that thereare four distinct types of NAD(P)H dehydrogenases: two on

Table 5 Predicted hexose transporters in the papaya genome, thewhole genome shotgun sequence accession number in NCBI (WGSAccession), coding sequence for amino acids (CDS), the number ofintrons in the nucleotide sequence (introns), amino acids (aa), whethera secretory peptide was predicted (secretory) and predicted location(ER endoplasmic retriculum), presence of discrete portion of protein

possessing its own function and the superfamily (domain), similarityin peptide sequence with another species (homology), extent thesequences were invariant (identity), E-value was the expectation valuefor homology, and the number of papaya expressed sequence tags(EST) detected with at least 500 bases and 99% identity

WGS Accession cds Introns aa Secretory Domains Homology E value EST

CpHT1 ABIM01004384.1 2700 2 503 ER Sugar transporter conserved site_2,Sugar transporter conserved site_1,12TMHMM

Q07423, Ricinuscommunis

0 2

CpHT2 ABIM01021724.1,ABIM01021723.1

6879 12 543 ER Sugar transporter conserved site_2,12TMHMM

AAF74569,Arabidopsis thaliana,

0 3

CpHT3 ABIM01019606.1 10663 11 537 Plastid Sugar transporter conserved site_2,MFS, 12TMHMM

AAF74567, Solanumtuberosum

1e-114 1

CpHT4 ABIM01025307.1 1569 1 522 ER Sugar:hydrogen ion symporter,Sugar transporter, 12TMHMM

NP_172592, Arabidopsisthaliana,

0 0

254 Tropical Plant Biol. (2008) 1:246–277

either side of the inner membrane. One to oxidize NADHand the other for NADPH [130, 159]. However, Rasmussonet al., [158] reported two Type II dehydrogenases for potatolocated on the inner and outer surfaces of the innermitochondrial membrane. Only three ORF with similarityto Type II dehydrogenase genes were found in the papayagenome. The ORF for an internal NAD(P)H dehydroge-nase, CpNDA1 (ABIM01007569.1) had 67% identity (E-value 0.0) to Arabidopsis NDA2 (At2g29990, NP_180560.1). A papaya EST was found for CpNDA1. Onegene was also predicted, CpNDB1 (ABIM0101 0022.1 +ABIM01010023.1) that had 79% identity (E-value 0.0) toArabidopsis NDB4 (At2g20800, NP_179673.1). A mito-chondrial transit peptide was predicted for the CpNDB1protein but not for CpNDA1. All had predicted ORFs motifsfor the pyridine-redox superfamily.

Ethylene Synthesis

Arabidopsis and papaya show evolutionary similarity in thenumber of genes associated with ethylene synthesis, receptorsand response pathways. Papaya had four S-adenosyl-L-

methionine synthases (SAMS), while twelve are reported forArabidopsis [217] and eight in tomato [137]. The eightTomato 1-Amino- Cyclopropane-1- Carboxylate Synthase(ACS) (LeACS) gene so far characterized differ in tissue-specific expression, developmental control, and ethyleneinduction [2, 137]. Two of the papaya sequences fromdifferent regions of the papaya genome had the closestBLASTp homology to Catharanthus roseus SAM2 (Table 6).Unlike other SAMs which do not have introns, one of thesesequences appeared to have three. This may be due to aproblem with the protein prediction, since correspondinggaps also appeared in the amino acid alignment with C.roseus SAM2. A tBLASTn search using the assemblednucleotide sequence showed no match between amino acids97 and 102, plus one large region of low homology betweenamino acids 254 to 328; otherwise, there was very goodhomology with many SAMs genes.

The papaya genome was predicted to contain seven ACSgenes, each with aminotransferase I and II domains whichwere predicted to be an ACS domain (Table 6). Two othersequences translated into the aminotransferases I and IIdomains was found. However, the closest BLASTp

Table 6 Predicted ethylene synthesis related genes in papaya, thewhole genome shotgun sequence accession number in NCBI (WGSAccession), coding sequence for amino acids (CDS), the number ofintrons in the nucleotide sequence (introns), amino acids (aa),presence of discrete portion of protein possessing its own function

and the superfamily (domain), similarity in peptide sequence withanother species (homology), extent the sequences were invariant(identity), E-value was the expectation value for homology, and thenumber of papaya expressed sequence tags (EST) detected with atleast 500 bases and 99% identity

WGS Accession Length Introns a.a. Domains Homology Identity EST

CpSAM1 ABIM01013924.1 20359 0 396 S-AdoMet_synt_N,M,C Carica papaya,AAN07179.1

99 21

CpSAM2 ABIM01011585.1 49518 3 328 S-AdoMet_synt_N,M,C Catharanthus roseus,CAA95857.1

68 0

CpSAM3 ABIM01017452.1 11573 0 390 S-AdoMet_synt_N,M,C Nicotiana tabacum,AAR15895.1

91 0

CpSAM4 ABIM01020562.1 70657 0 393 S-AdoMet_synt_N,M,C Catharanthus roseus,CAA95857.1

95 10

CpACS1 ABIM01025946.1 7684 3 484 aminotransferase 1 & 2 Carica papaya,AAC98809.1, ACS1

98 3

CoASC2 ABIM01028192.1 10151 3 488 aminotransferase 1 & 2 Carica papaya,CAJ34918.1, ASC2

98 2

CpACC3 ABIM01026980.1 20136 3 442 aminotransferase 1 & 2 Carica papaya, CAB86187.1 99 0CpASC4 ABIM01001561.1 9407 3 586 aminotransferase 1 & 2 A. thaliana,

ACS12/NP_199982.263 3

CpACS5 ABIM01007946.1 71660 3 552 aminotransferase 1 & 2 Rosa sinensis, AAQ88100.1 77 1CpACS6 ABIM01019463.1 38240 3 466 aminotransferase 1 & 2 Prunus salicina,

ABW03081.180 0

CpACS7 ABIM01028551.1 10531 3 472 aminotransferase 1 & 2 S. lycopersicum,AF179247.1

70 0

CpACO1 ABIM01020907.1 3567 2 235 2OG-FeII_Oxy Carica papaya,AAC98808, ACO1

100 90

CpACO2 ABIM01019610.1 60279 3 310 2OG-FeII_Oxy Carica papaya,AAL78058.1, ACO2

100 101

CpACO3 ABIM01013582.1 33423 2 310 2OG-FeII_Oxy P tremula x P tremuloides,AAN87846.1

79 0

Tropical Plant Biol. (2008) 1:246–277 255255

matches to these two sequences were not annotated as ACS,and their homology to non-ACS aminotransferases washigh and were not included in the papaya ACS list. Threesequences matched papaya sequences already deposited inGenbank, including CpACS1 and CpACS2 (98% and 98%identity, respectively). All seven putative ACS sequencescontained the conserved dodeca-peptide binding site [219],although, one had an Asn substitution for the key Lys thatbound either PLP or the 2-aminobutyrate of adenosylme-thionine [219]. The possible existence of nonfunctionalsequences with ACS homology in the papaya genomewould not be novel; Arabidopsis has at least one nonfunc-tional gene (AtACS1) shown to encode a protein with noenzyme activity [202] that is nevertheless included in thegene family. In addition, AtACS3 is not even transcribed,and AtACS10 and AtACS12 encode aminotransferasedomains but have no ACS activity; none of these areincluded in the ACS family [217].

A minimum of three ACO genes were predicted inpapaya (Table 6), with six additional sequences that showedpartial homology to ACO-like genes or genes-encoding the2-oxoglutarate FeII oxygenase domain. This domainencompasses more than ACO’s, but it is the motif thatdescribes ACO’s. As with the ACS, papaya has two well-studied CpACO genes, with multiple Genbank representa-tives. The CpACO genes identified (Table 6) reflectBLASTp matches to CpACO1 and CpACO2 from C.papaya, plus a translated supercontig with 79% identity toa poplar protein shown to have ACO activity [7]. The

sequence that matched CpACO1, lacks the first 83 aa fromACO1 (AAC98808), due to sequencing ambiguities, butotherwise matched the remaining 235 aa. All threesequences retained the three conserved subdomains ofACOs and 2-oxoglutarate FeII oxygenases [201], as wellas the HxD site for enzymatic activity. CpACOs share themotif KxxR within the conserved C-terminal site that wasidentified as essential for Petunia hybrida ACO1 enzymeactivity [220]. The protein encoded on (ABIM01013582.1)CpACO3 differs at this site by three amino acids, which ismore than any of the ACOs from 24 different plants,including papaya (LPKEPRFR vs. the consensusQAKEPRFE). Five other predicted models had between42 and 71% homology to Arabidopsis proteins with 2-oxoglutarate FeII oxygenase domains, but these Arabidop-sis proteins are not clearly involved in ACO activity.

Ethylene Response

Papaya ripening has both ethylene insensitive and ethylenesensitive components [47, 122] as has been found fortomato [150]. At least three ethylene receptor genes(CpETR1 to CpETR3) were predicted in the papayagenome. All three encode a protein with the expectedprotein domains, though CpETR1 had no receiver domain(Table 7). CpETR1 was homologous (100%, E-value 0.0)to the papaya receptor cDNA sequence (AF311942.1)deposited by the late Dr. Hamid Lazan and his group(2000) from Malaysia. This sequence has 78% homology

Table 7 Predicted papaya ethylene receptors, structure and phylogenetic relationship with known Arabidopsis ethylene receptors. The whole genomeshotgun sequence accession number in NCBI (WGS Accession), coding sequence for amino acids (CDS), the number of introns in the nucleotidesequence (introns), amino acids (aa), presence of discrete portion of protein possessing its own function and the superfamily (domain), similarity inpeptide sequence with another species (homology), extent the sequences were invariant (identity), E-value was the expectation value for homology,and the number of papaya expressed sequence tags (EST) detected with at least 500 bases and 99% identity

WGSAccession

cds Intron aa Domains Homology Identity,E-value

ESTs

CpETR1 ABIM01020836 25248 4 629 GAF domain, His Kinase A (phosphoacceptor) domain, 3 transmembranedomains

Carica papaya AAG41977 100%, 0.0 9

CpETR2 ABIM01015727 56390 5 738 GAF Domain Clp amino terminal domain, His Kinase A (phosphoacceptor)domain, Response regulator receiver domain, 3 transmembrane domains

Prunus persica ethylenereceptor Q9M7M1 ETR1

88%, 0.0 4

CpETR3 ABIM01030637 5560 9 767 GAF domain, Response regulatory domain, His-kinase domain, 3transmembrane domains

Lycopersicon esculentumethylene receptor homologAF118844_1

67%, 0.0 8

AtERS1

CpETR1

CpETR2

AtETR1

CpETR3

AtEIN4

AtETR2

0.05

Table 7 Predicted papaya ethylene receptors, structure and phyloge-netic relationship with known Arabidopsis ethylene receptors. Thewhole genome shotgun sequence accession number in NCBI (WGSAccession), coding sequence for amino acids (CDS), the number ofintrons in the nucleotide sequence (introns), amino acids (aa), presenceof discrete portion of protein possessing its own function and the

superfamily (domain), similarity in peptide sequence with anotherspecies (homology), extent the sequences were invariant (identity), E-value was the expectation value for homology, and the number ofpapaya expressed sequence tags (EST) detected with at least 500 basesand 99% identity

256 Tropical Plant Biol. (2008) 1:246–277

(E-value 0.0) with the Arabidopsis ERS1 (At2g40940,NP_181626.1) (Table 7). Arabidopsis ETR1 (At1g66340,NP_176808.3) has the greatest homology with CpETR2(86%, E-value 0.0). Based upon gene and protein structure,both CpETR1 and CpETR2 fall into the ethylene receptorsubfamily 1 [94] with CpETR3 being in subfamily 2.Subfamily 1 receptors maybe required for most ethyleneresponses [208]. Originally, we reported four two-compo-nent ethylene receptors [132], however, a more thoroughanalysis indicated that one of the previously reportedCpETRs lacked the expected GAF domain and a fullhistidine domain [88, 208]. This partial sequences(ABIM01010980) was 139 aa long peptide with 75%homology (E-value 3e-75) to apple ethylene receptor. Afifth potential gene (ABIM01025304.1) was short (224 aa).It only had the DUF623 domain of unknown function andlow homology to the nearest ethylene receptor(AAQ56281) of Litchi chinensis. Papaya’s three predictedethylene receptors were fewer than the five ETRs found inArabidopsis [176] and the six in tomato [100]. Sinceethylene receptors seem to be a negative regulator of action[93], degradation plays a significant role in the control ofripening [99]. The relatively small complement of papayaethylene receptors means that fewer interactions may occurbetween receptors and that their role in ripening should bemore easily discerned.

Ethylene receptors are disulfide linked dimers and ethylenebinding involves a copper cofactor [164]. The gene RAN(Response to Antagonist) plays a role in making the ethylenereceptor apoprotein functional by transporting a copper ion tothe ethylene receptor site in the membrane spanning regions.As with Arabidopsis, only one RAN homolog was found inpapaya (Table 8), CpRAN had 76% homology (E-value 0.0)to Arabidopsis RAN1 (NP_ 200892.1).

The ethylene response pathway following ethylenebinding involves a RAF-related kinase CTR1 that has arole in the negative response by forming a complex with thereceptor [93]. Ethylene binding inhibits CTR1 kinaseactivity [141] and thus relieves the repression of theethylene response pathway [5, 6]. A signal is thentransmitted from the positive regulator EIN2 to EIN3/EILsand induces transcription of ethylene response factors(ERF). AtCTR1 is one of six Arabidopsis MAPK kinasesand three LeCTR1-like genes in tomato [1, 78] with noevidence that more than one (AtCTR1, LeCTR1) beinginvolved in the ethylene signal transduction pathway. Atleast one CpCTR1 gene was found in the papaya genome(ABIM01019395 + ABIM01040871) that had 56% homol-ogy with LeCTR1 (E-value 0.0). A second, but incompleteCTR sequence, was also found (ABIM01001640) (Table 8).One possible CpEIN2 gene was found, although thenucleotide and amino acid sequence were short (Table 8),

Table 8 Ethylene response genes predicted in the papaya genomeThe whole genome shotgun sequence accession number in NCBI(WGS Accession), coding sequence for amino acids (CDS), thenumber of introns in the nucleotide sequence (introns), amino acids(aa), presence of discrete portion of protein possessing its own

function and the superfamily (domain), similarity in peptide sequencewith another species (homology), extent the sequences were invariant(identity), E-value was the expectation value for homology, and thenumber of papaya expressed sequence tags (EST) detected with atleast 500 bases and 99% identity

WGS Accession cds Introns aa Domains Homology Identity,E-value

EST

CpRAN1 ABIM01008271 9019 8 ATPase, coupled totransmembrane movementof ions, phosphorylativemechanism Heavy-metal-associateddomain

NP_199292.1 RAN1Arabidopsis thaliana

76%, 0.0 4

CpCTR1 ABIM01019395 17660 13 793 CTR1-like protein tyrosine kinase AAR89821 Lycopersiconesculentum

56%, 0.0 4

ABIM01040871 1813CpCTR2 ABIM01001640 44125 7 1022 Protein tyrosine kinase ABE80154 Medicago

truncatula88%, 2e-114 0

CpEIN1 ABIM01024839 14603 7 1294 EIN2 AAR08678Petunia x hybrida

86%, 1e-22 2

CpEIN2 ABIM01006170 34454 1 601 EIL2 Ethylene insensitive 3 ABK35086 Prunus persica 61%, 1e-87 0CpEIN2 ABIM01011208 9335 1 665 EIN3-binding F-box protein 2 ABC24972 Lycopersicon

esculentum57%, 0.0 7

CpEIL1 ABIM01017539 9817 0 603 EIL4, Ethylene insensitive 3 AAP04000Nicotiana tabacum

70%, 0.0 10

CpEIL2 ABIM01012325 17077 1 601 EIL2, Ethylene insensitive 3 ABK35086 Prunus persica 70%, 0.0 2

Tropical Plant Biol. (2008) 1:246–277 257257

the alignments with CTR1-like kinase and the proteinkinase family had fewer gaps and about one hundred aminoacid matches. Four possible EIN3/EIL1 genes were alsofound, with two (CpEIL1, CpEIN3) having high EIN2/EILhomologies to published sequences and these wereexpressed as papaya ESTs. Arabidopsis has nine EIN3and EIN3-like genes [28] and tomato has at least five [186].

Eight ERF genes were found with high homlogy andanother twelve predicted ERF-like sequences that had lowhomology matches or had no significant protein domains(Supplementary Table 2). One hundred and twenty-twoethylene responsive binding factors in the AP2/ERFsuperfamily have been found in Arabidopsis [135].CpERF1 falls into group IX that has 17 members inArabidopsis with eight in group VIII. Using the conserved60 amino acid sequences for AP2/ERF [135], an additional92 potential genes with this sequence were found in papayathat suggested more ERF maybe present in the papayagenome (data not shown).

Isothiocyanates

The glucosinolates are found almost exclusively in theorder Brassicales [161] and have a sulfonated oxime with aβ-thioglucose residue. Papaya synthesizes high levels ofbenzylglucosinolates (BG) and benzylisothiocyanates(BITC) with BG being the only glucosinolate found inpapaya [26]. BITC is produced by the removal of theglucose moiety by the action of a β-thioglucosidase(myrosinase). Myrosinase is separated from the isothiocya-nate, in specialized myrosin cells where myrosinase isbrought into contact with its substrate BG followingphysical damage. Myrosinase expression and activityincreases are fourfold at harvest, then decline slightlyduring ripening [169]. Both BG and BITC levels arehighest in the seeds, followed by the peel and pulp, anddecrease during fruit development [119, 169, 193].

Genes were predicted for all steps except for the firsthydroxylase in the biosynthesis of BG. In Arabidopsis, theP450 monooxygenase CYP79A2 is involved in the conver-sion of phenylalanine to the aldoximine [83, 101, 211]. Nopapaya gene with homology to CYP79A2 was found. WhenArabidopsis CYP79A2 was used to search the papayadatabase, homology was only found to two closely relatedP450s, CYP79B2 and CYP79B3 (Table 9). CYP79B2 andCYP79B3 both act on tryptophan and may be involved inauxin synthesis [101]. It is possible that the papayaCYP79B2 and CYP79B3 homologs have broader substratespecificity as found for some other P450 monooxygenases[83], and that can convert in papaya phenylalanine to thealdoximine. A single P450 gene (CpCYP83B1) waspredicted in papaya for the reduction and cysteine additionstep with 70% homology (E-value 0.0) to Arabidopsis

CYP83B1 gene. After these two reduction steps, twopredicted aminotransferases C-S lyases (CpCSL1, CpCSL2)were found with high homology to the Arabidopsis homologand Brassica napus SUR1. A single glucosyl transferasegene was found in papaya with high homology (55%, E-value 2e-138) to Brassica napus UGT74B1 [84]. The nextstep, which involves sulfate transfer, had two predictedgenes (CpPAPS1, CpPAPS2), though CpPAPS2 had only apartial sulfotransferase domain characteristic of this genefamily [152].

Two myrosinase genes have been reported in Arabidop-sis [157, 215] though only one (CpMYR) was predicted forpapaya, which is in agreement with a recent report [169].The predicted CpMYR had 98% homology (E-value 3e-146) to the published papaya myrosinase sequence with aglucosyl hydrolase family 1 (GH1) domain. Only onemyrosinase binding protein was found with low homology(37%, E-value 4e-20) to the Arabidopsis MBP2. A singlemyrosinase-associated protein was also predicted in papaya,as for Brassica napus [157].

Laticifers and Protease

The papaya fruit, as do the other aerial parts of this plant, has adense network of laticifers [170]. The articulated laticifersbegin as a column of cells that form vessel-like structuresthat retain the usual organelles. The milky latex is about 85%water with the 15% being composed of 25% insoluble matterof unknown composition. The soluble fraction containscarbohydrates (~10%), salts (~10%), lipids (~5%), andbiomolecules mainly proteins (~40%) [65]. The biomole-cules include cysteine proteinases (papain, chymopapain),cystatin, β-1,3-glucanase, chitinase, lipases and other pro-teins. The latex is thought to function as an induced defensemechanism [172, 182]. The latex is harvested from largefruit green varieties by scratching the skin, allowing theexuded latex to coagulate and dry, then the dried latex iscollected by scrapping. The latex is purified and itsproteolytic activity is used for meat tenderization and chill-proofing of beer [65]. During fruit ripening proteinaseactivity declines [147] and some of that laticifers break andrelease latex under the cuticle disrupting this barrier andincreasing fruit water loss rate [148].

Four cysteine proteinases account for 80% of theenzyme fraction [65]. The proteinases are papain, chymo-papain, caricain (proteinase omega) and glycyl endopepti-dase (proteinase IV). Papaya proteinases are synthesized asproezymes with a signal sequence. The prosequence iscleaved and proteinase activated. All four proteinases aremembers of the peptidase C1A subfamily of cysteineproteinases. Papain has been most intensively studied [65]though a minor component (5–8%) of the endopeptidases inpapaya latex [18, 21]. One gene for a propapain (CpPAPA)

258 Tropical Plant Biol. (2008) 1:246–277

Tab

le9

Predicted

papaya

glycosinalates

synthesisgenesandamyrosinasegene.N

oho

molog

toCYP79

A2,

thefirststepin

biosyn

thesis,w

asfoun

din

papaya.T

hree

closelyrelatedCYP79

genes

werepredicted.

The

who

legeno

meshotgu

nsequ

ence

accessionnu

mberin

NCBI(W

GSAccession

),coding

sequ

ence

foram

inoacids(CDS),thenu

mberof

intron

sin

thenu

cleotid

esequ

ence

(introns),am

inoacids(aa),p

resenceof

discretepo

rtionof

proteinpo

ssessing

itsow

nfunctio

nandthesuperfam

ily(dom

ain),sim

ilarity

inpeptidesequ

ence

with

anotherspecies(hom

olog

y),extent

thesequ

enceswereinvariant(identity

),E-value

was

theexpectationvalueforho

molog

y,andthenu

mberof

papaya

expressedsequ

ence

tags

(EST)detected

with

atleast50

0basesand99

%identity

WGSAccession

cds

Intron

saa

Dom

ains

Hom

olog

yIdentity,

E-value

EST

CYP79

ABIM

0102

3062

.117

811

558

cytochromeP45

0,family

79,subfam

ilyB,

polypeptide2;

oxyg

enbind

ing

Arabido

psisthaliana

CYP79

B2,

NP_1

9570

5.1

60,0.0

0

ABIM

0101

9933

.120

501

534

cytochromeP45

0,family

79,subfam

ilyB,

polypeptide3;

oxyg

enbind

ing

Arabido

psisthaliana

CYP79

B3,

NP_1

7982

0.2

58,.0.0

0

ABIM

0102

3066

.117

721

559

cytochromeP45

0,family

79,subfam

ilyB,

polypeptide3,

oxyg

enbind

ing

Arabido

psisthaliana

CYP79

B3,

NP_1

7982

0.2

0

CpC

YP83

B1

ABIM

0100

7450

.116

452

477

P45

0,83

B1

Arabido

psisthaliana

CYP83

B1,

NP_1

9487

8.1

70,0.0

3

C-S

Lyase

ABIM

0101

9595

.142

166

421

Aspartate/ty

rosine/aromatic

aminotransferase

Arabido

psisthaliana

,BAB10

727.1

80,0.0

2CpC

SL1

ABIM

0101

9594

.1CpC

SL2

ABIM

0101

5665

.118

866

420

Aspartate/ty

rosine/aromatic

aminotransferase

Brassicarapa

subsp.

pekinensis

SUR1,

ACH41

754.1

61,4e-144

2

Thioh

ydroximate

S-glycosyltransferaseUGT74

B1

ABIM

0100

2346

.114

751

453

thiohy

drox

imateS-glucosyltransferase

Glycosyltransferasefamily

28N-terminal

domain.

Brassicana

pus,AAL09

350.1

55,2e-138

0

CpPAPS1

ABIM

0102

3999

.110

761

344

sulfotransferase_1

superfam

ilyArabido

psisthaliana

ST2a,

NP_5

6817

7.1

60,6e-126

1

CpPAPS2

ABIM

0100

1554

.120

004

126

Partialsulfotransferase_1

superfam

ily.

Brassicarapa

ST5a,ACH41

751.1

781e-39

0MyrosinaseCpM

YR

ABIM

0100

5474

.136

6313

492

Glucohy

drolaseGH1

Caricapa

paya

thioglucoside

glucoh

ydrolase,ACC95

418.1

98,3e-146

1

MYRBinding

Protein

ABIM

0101

9550

.110

861

191

Jacalin

-likelectin

domain.

Proteins

containing

thisdo

mainarelectins.

Arabido

psisthaliana

MBP2

(Myrosinasebind

ingprotein2),

NP_1

7561

5.1

37,4e-20

MYRAssociatedProtein

ABIM

0100

7310

.113

884

343

SGNH_p

lant_lipase_lik

e,,aplantspecific

subfam

ilyof

theSGNH-fam

ilyof

hydrolases

Arabido

psisthaliana

epith

iospecifier

mod

ifier,ABB90

255.1

52,3e-100

12

Tropical Plant Biol. (2008) 1:246–277 259259

precursor was predicted (ABIM02027591.1). The encodedprotein has a 26 aa ER secretory sequence and 95% identity(E-Value 2e-162) to the published papaya cDNA sequence(P00784) (Table 10). A papaya ESTwas found for CpPAPA.

A single papaya chymopapain gene (CpCHYP) waspredicted that had 99% identity (E-value 0.0) to chymopa-pain isoforms I, III, and V (X97789, AJ131996, andAJ131998 respectively), and 100% to chymopapain iso-form II and IV (AJ0131995 and AJ1319970) (Table 10).Chymopapain is distinguished from papain by the proteo-lytic activity remaining after papain is removed [95]. Fiveprochymopapain isoforms (I to V) were identified fromsequenced leaf cDNAs [194], all the isoforms have a freecysteine at position 251. The translated isoform cDNAsdiffer in one or two amino acid substitutions. At position222, a cysteine is replaced by a tyrosine in isoforms III andV, while at position 266, a valine is replaced by aphenylalanine in isoforms II, III, and V. These single aminoacid substitutions require only a single base change. Thepredicted CpCHYP protein had the nine amino acidsreported for isoforms III, IV and V. It is possible that thechymopapain isoforms differ because of errors in PCR,sequencing or translation. The CpCHY protein, possiblyisoform III, had 66 ESTs. A chymopapain isoform III ESTwas also reported by Devitt et al. [60].

A number of other proteinases have been reported inpapaya latex [16, 65, 182]. Two caricain genes werepredicted (proteinase omega) and one glycyl endopeptidasegene (Table 10). The predicted gene numbers are in agree-ment with those reported [65], though one of the caricaingene cDNA has not been deposited in Genbank. TheCpCARP1 was 100% identical (E-value 2e-69) to the de-scribed sequence, although it is only 130 aa long due toincomplete sequencing of the WGS. A glutamine cyclo-transferase (glutamine cyclase) gene was predicted in papaya(CpGTR) as previously reported from papaya latex [65, 140].The predicted papaya gene (Table 10) was longer than thepredicted 288 aa peptide from the cDNA (AF061240) inGenBank though analysis suggested intron boundaries mayhave not been accurately predicted.

One cystatin (CpCYST) and two kunitz-type trypsininhibitors (CpTINH1, CpTINH2) were predicted in thepapaya genome in agreement with the published data [17].The cystatin encoded the 125A domain and was classifiedas a member of the CY superfamily. Papaya ESTs werefound for both the cystatin and trypsin inhibitors (Table 10).

At least 27 β-1,3-glucanase proteins (GH17) werepredicted in papaya and activity has been detected inpapaya latex [65]. The papaya carbohydrate active enzymeswill be the focus of a paper in preparation. In addition to β-1,3-glucanase, chitinase II (GH19) has been reported forpapaya latex. A gene with 61% identity to the partial cDNA(P81241) was found in the papaya genome (Table 10).

Other than the genes encoding proteinases and thoselaticifer proteins described above, the genes encoding otherproteins potentially found in papaya latex have not beenwell described. A search of the papaya genome for latexassociated protein genes found six genes including one forcaspase reported as a rubber latex abundant protein andlysophospholipase (Supplementary Table 3). In addition, anumber of potential latex genes were found whosepredicted protein had allergen domains.

Lignin Synthesis

Lignins are amorphous heteropolymers resulting from theoxidative coupling of p-hydroxycinnamyl alcohols, p-coumaryl, coniferyl, and sinapyl alcohols by both laccasesand plant peroxidases in the cell wall. This lignificationprocess is crucial to xylem formation as it confers resistanceagainst the tensile forces in the xylem, imparts waterimpermeability, and protection against chemical and bio-logical degradation [51, 63, 126, 143].

The papaya genome had three predicted phenylalanineammonia lyase (PAL) lignin biosynthesis genes, comparedto the four Arabidopsis genes and two in Populus [89, 156].CpPAL1 and CpPAL2 genomic sequences had two predictedexons, whereas CpPAL3 had only one exon. The PAL genesin Arabidopsis have from one to three exons. Papaya PALgene amino acid sequences were ~85% homologous to theArabidopsis sequences and about 88% homologous to thePopulus PAL sequences (Supplementary Table 4). CpPAL1and CpPAL2 both contained the PAL and PAL-HALconserved domains found in Arabidopsis and PopulusPAL genes, but CpPAL3 had only a partial sequence ofthe conserved domain. It was possible that the second exonof CpPAL3, actually has the remainder of the conserveddomain, but it was located on a separate scaffold.

Two candidate trans-cinnamate 4-hydroxylase (C4H)genes were identified in papaya (Supplementary Table 4)while two genes are identified in Populus and one inArabidopsis [89, 156]. As in the Arabidopsis C4H gene,CpC4H1 consists of three predicted exons, while CpC4H2had two. Papaya’s class I C4H gene, CpC4H1, was verysimilar to Arabidopsis and Populus class I C4H genes, withan amino acid sequence homology of 86% and 93%,respectively. CpC4H2, a class II C4H gene, shared signifi-cant homology (83%) to the Populus class II C4H gene.Both papaya C4H genes contained the cytochrome p450conserved domain.

Similar to Arabidopsis, papaya had four candidate 4-coumarate: CoA ligase (4CL) genes, whereas Populus hasone [63, 89, 156]. Cp4CL1, Cp4CL2, and Cp4CL3, allbelong to the class I 4CL genes. The class I papaya 4CLgenes had five predicted exons, similar to the four and fiveexons in the class I 4CL genes in Arabidopsis. The class II

260 Tropical Plant Biol. (2008) 1:246–277

Tab

le10

Papain,

chym

opapainandotherproteinasespredictedgenesoccurringin

papaya

latex,

thewho

legeno

meshotgu

nsequ

ence

accessionnu

mberin

NCBI(W

GSAccession

),coding

sequ

ence

foram

inoacids(CDS),am

inoacids(aa),whether

asecretorysequ

ence

was

predicted(secretory)its

locatio

n(ERendo

plasmic

retriculum

)andaa

leng

th,presence

ofdiscrete

portionof

proteinpo

ssessing

itsow

nfunctio

nandthesuperfam

ily(dom

ain),similarity

inpeptidesequ

ence

with

anotherspecies(hom

olog

y),extent

thesequ

enceswereinvariant(identity

),E-value

was

the

expectationvalueforho

molog

y,andthenu

mberof

papaya

expressedsequ

ence

tags

(EST)detected

with

atleast50

0basesand99

%identity

Nam

eWGSAccession

cds

aaSecretory

Dom

ains

Hom

olog

yIdentity,

E-value

EST

Papain

CpPAPA

ABIM

0102

7591

.117

1431

8ER26

Peptid

aseC1A

+I29

Papainprecursor

PAPA

1_CARPA

P00

784

95,2e-162

1

Chy

mop

apain

CpC

HYP

ABIM

0102

4208

.114

6936

1ER26

Peptid

aseC1A

+I29

Chy

mop

apainIsoform

III

AJ131

996.1

99,0.0

66

Caricain

CpC

ARP1

ABIM

0102

7561

.180

0213

0no

neI29superfam

ilyProteinaseom

ega,

Papaya

AAU07

805.1

100,

2e-69

0

CpC

ARP2

ABIM

0102

5565

.115

4033

6ER26

Peptid

aseC1A

+I29

PAPA

3_CARPA

Caricain

(proteinaseom

ega)

69,2e-119

1

Glycylendo

peptidase

CpG

LP1

ABIM

0102

1038

.114

8334

8no

nePeptid

aseC1A

+I29

ProteinaseIV,

PAPA

4_CARPA

,P05

994

99,0.0

Glutaminecyclotransferase

CpG

TR1

ABIM

0101

7768

.148

0032

2no

neGlu_cyclase_2

superfam

ilyGlutaminecyclotransferase

precursorpapaya

AAC27

745.1

59,3e-92

1

Cystatin

CpC

YST

1ABIM

0101

9193

.115

9899

none

CY

superfam

ily,I25A

Cysteineproteinase

inhibitor,

papaya

cystatin

CAA50

437.1

98,2e-157

9

Trypsin

inhibitor

CpT

INH1

ABIM

0100

1601

.159

719

8ER26

InhibitorI3,Kun

itztype

LSPI_CARPA

,latexserine

proteinase

inhibitorP80

691

39,3e-24

1

CpT

INH2

ABIM

0100

1397

.159

121

7ER25

STI-Soy

bean

tryp

sininhibitor

Latex

proteinase

inhibitor,

LSPI_CARPA

55,5e-50

1

β-1,3-G

lucanase

Atleast27

genespredicted

Chitin

aseII

ABIM

0101

2647

.196

9ER29

Glycoside

hydrolaseGH19

,lysozymelik

eChitin

aseII,Arachishypo

gaea

CAA57

773.1

75,4e-121

2

Chitin

aseIIpapaya

GH

193C

QL

61,4e-85

Tropical Plant Biol. (2008) 1:246–277 261261

papaya 4CL gene had six predicted exons, while theArabidopsis class II 4CL gene have seven exons. The fourpredicted papaya Cp4CL genes encoded proteins with 65–75% amino acid homology to the class I 4CL genes inArabidopsis and 68–78% in Populus. Cp4CL4 belongs tothe class II 4CL genes although it shared less than 50%homology with the class II genes found in Arabidopsis. Allpapaya 4CL genes contained the acetyl-CoA synthaseconserved domain as found in the Arabidopsis and Populus4CL genes. Though Cp4CL4 had low homology withArabidopsis and Populus 4CL genes, it still contained theexpected conserved domain. Further studies are needed toascertain the role of this gene.

Three hydroxycinnamoyl-CoA shikimate/quinatehydroxycinnamoyltransferase (HCT) candidate genes werefound in papaya (Supplementary Table 4), whereas onlyone HCT gene is present in Arabidopsis and Populus [89,156]. CpHCT1 contained two exons, as does the Arabi-dopsis HCT gene, but CpHCT2 has ten predicted exons andCpHCT3 had five. The CpHCT1 predicted protein had 83%homology to both Arabidopsis and Populus HCT aminoacid sequences. CpHCT2 and CpHCT3 had considerablyless homology, below 50%. CpHCT1 and CpHCT2contained a complete transferase conserved domain, butCpHCT3 only has a partial conserved domain. The lowsequence similarity and broken conserved domain sug-gested CpHCT2 and CpHCT3 may have functions otherthan those of bonafide HCT genes.

Expression data showed only one P-coumaroyl shiki-mate 3′-hydroxylase/coumaroyl 3-hydroxylase (C3H) genepresent in papaya, which is fewer than the two C3H genesreported by Ming et al. [132]. Arabidopsis and Populus alsoonly have one C3H gene [63, 89, 156]. CpC3H1 encoded aprotein with 83% homology to the amino acid sequence ofthe Arabidopsis C3H gene and an 89% homology to thePopulus C3H gene. CpC3H1, as with the Arabidopsis C3Hgene, had three predicted exons and also contained thecomplete cytochrome p450 conserved domain.

The papaya genome was predicted to have two trans-caffeoyl-CoA 3-O-methyltransferase (CCOMT) genes, asdoes Populus, but Arabidopsis only contains one [63, 89,132, 156]. CpCCOMT1 was predicted to have five exonsand CpCCOMT2 had one; Arabidopsis CCOMT genecontains four exons. The CpCCOMT1 predicted amino acidsequence only matched about 60% to the amino acid se-quences of CCOMT genes in Arabidopsis and Populus.CpCCOMT2 amino acid sequence had a much higher ho-mology, greater than 90% to both Arabidopsis and Populusamino acid sequences. Both papaya CCOMT genes had theS-adenosylmethionine-dependent-methyltransferase con-served domain, though CpCCOMT2 was a partial domain.

Whereas Arabidopsis has two cinnamoyl-CoA reductase(CCR) genes, only one CCR gene was present in papaya

and Populus [63, 89, 132, 156]. Three exons were predictedfor CpCCR1, but the two Arabidopsis CCR genes have fourand five exons. The CpCCR1 predicted protein had about a75% to 80% homology to the Arabidopsis CCR amino acidsequences and 87% homology to the Populus CCR aminoacid sequence. CpCCR1 contained a partial AdoHcyase, S-adenosyl-L-homocysteine hydrolase conserved domain asalso found in the Arabidopsis and Populus CCR genes.

Four candidate coniferylaldehyde 5-hydroxylase(CA5H), also called ferulate 5-hydroxylase (F5H), geneswere identified in papaya [132]. Populus has two CA5Hgenes and Arabidopsis has one [63, 89, 156]. CpCA5H1and CpCA5H2 both had two exons, CpCA5H3 had three,and CpCA5H4 had seven predicted exons. CpCA5H1 andCpCA5H2 had the closest homology to Arabidopsis andPopulus CA5H amino acid sequences, with 79% and 78%homology, respectively. CpCA5H3 and CpCA5H4 pre-dicted protein amino acid sequences were not as similar,with about 70% similarity to Arabidopsis and PopulusCA5H amino acid sequences. All of the papaya CA5Hgenes contained a complete cytochrome p450 conserveddomain (Supplementary Table 4).

Papaya and Arabidopsis genomes both contained onecaffeic acid 3-O-methyltransferase (COMT) gene while thePopulus genome contains two [89, 132, 156]. CpCOMT1contained five exons, compared to four exons in theArabidopsis COMT gene. The CpCOMT1 predicted aminoacid sequence was about 76% homologous to that of bothArabidopsis and Populus. The BLASTp results showed agap of 16 amino acids in the papaya sequence after aminoacid 264, compared to both the Arabidopsis and Populussequences. CpCOMT1 contained a complete S-adenosyl-methionine-dependent methyltransferase conserved do-main, as well as a dimerisation domain, as do theArabidopsis and Populus COMT genes.

Two candidate cinnamyl alcohol dehydrogenase (CAD)genes were identified in papaya, the same number as foundin Arabidopsis, with Populus having one CAD [63, 89,156]. CpCAD1 had six and CpCAD2 had seven predictedexons. CpCAD1 predicted protein has the highest aminoacid sequence homology to Arabidopsis and Populus CADproteins, with 78% and 81% similarity, respectively.CpCAD2 homology to Arabidopsis and Populus CADamino acid sequences was lower. The Arabidopsis CADgenes have five exons. Both CpCAD1 and CpCAD2 containthe alcohol dehydrogenase GroES-like conserved domainand the zinc-binding dehydrogenase conserved domain, asseen in Arabidopsis and Populus CAD genes.

Polyphenoloxidases and Peroxidases

Flavonoid oxidation occurs widely in plants and linked toprotection against UV damage, a deterrent for herbivores,

262 Tropical Plant Biol. (2008) 1:246–277

pathogenicity, scavengers of free radicals, and lignindegradation [51, 55, 109, 125, 126, 153, 196, 197]. Theoxidation, especially of o-diphenols in the presence ofmolecular oxygen, leads to the highly reactive semiqui-nones and quinones that interact with phenols, amino acidand proteins yielding brown products. The brown productsoccur in seed coats, and in other tissues followingenvironmental stress and physical injury, and are a majorproblem during food processing. Polyphenol oxidases(PPO) and peroxidases (POD) are the two major groupsof enzymes involved in oxidation. PPOs are divided intotyrosinase and laccase, with the tyrosinases includingmonophenolase and cathechol oxidase that oxidizes o-diphenols. Laccase oxidizes o-diphenol and p-diphenols.Cathechol oxidase is localized in the chloroplast whilelaccases and peroxidases are secreted [51, 126, 153].

In papaya, browning reactions occur during seed coatmaturation, in the skin following chilling injury andfreezing, and occasionally on the cut mesocarp surfacefollowing minimal processing. Phenolic compounds inpapaya skin decline during ripening to one quarter of thatat the mature green stage [192]. Chilling injury of colorbreak fruit is associated with the development of a darkolive skin scald while in riper fruit a lighter browncondition occurs [46] possibly due to lower amount ofphenol substrate in riper fruit. During papaya processinginto juice [44] and with frozen chunks [40, 41], colorchanges have been reported with increases in PPO andPOD activities.

Nine laccase genes were predicted in the papaya genome(Table 11). The papaya laccases (CpLAC1 to CpLAC9) allhave the three laccase Cu-oxidase domains and werepredicted to be secreted. All but four of the predictedlaccase genes had papaya ESTs and greater than 66% to79% identity with known laccase sequences. CpLAC2 andCpLAC3 were found on the same WGS sequence and were91% identical, and may be either a recent duplication or asequencing error. CpLAC9 was predicted to encode aprotein of 431 aa versus 545–593 aa for CpLAC1 toCpLAC8. CpLAC9 maybe only a partial sequence, since ithad only two and not three Cu-oxidase domains normallyfound in laccase genes [51, 126, 127].

The brown reactions reported during chilling injury andprocessing is probably due to PPO activity [41] and at leastfive PPO genes (CpPPO1 to CpPPO5) were predicted(Table 11). The activity of PPO declines slightly duringripening [41] and six isoforms were reported for hermaph-rodite fruit with only four isoforms found for female papayafruit. The difference between the number of isoforms [40]and the predicted gene number may reflect alternativesplicing, post-translation processing or one of the partialPPO-like sequences may be a real gene (data notpresented). All CpPPOs predicted proteins had predictedor potential chloroplast transit peptides of 43–46 aa. Intomato, PPOs are encoded by a seven-member PPOmultigene family on chromosome 8 [196]. The seventomato PPOs show differential expression during develop-ment and following stress [197]. Papaya PPOs were also

Table 11 Polyphenol oxidase (tyrosinase, dicopper containing) and laccase (multi copper oxidase) genes predicted in the papaya genome, thewhole genome shotgun sequence accession number in NCBI (WGS Accession), coding sequence for amino acids (CDS), the number of introns inthe nucleotide sequence (introns), amino acids (aa), whether a secretory sequence was predicted (secretory) its location (ER endoplasmicretriculum) and aa length, similarity in peptide sequence with another species (homology), extent the sequences were invariant (identity), E-valuewas the expectation value for homology, and the number of papaya expressed sequence tags (EST) detected of at least 500 bases and 99% identity

WGS Accession CDS aa Secretory Homology Identity,E-value EST

Laccase Cu-oxidase SuperfamilyCpLAC1 ABIM01016852.1 2124 545 None LAC6 Arabidopsis NP182180.1 70, 0.0 0CpLAC2 ABIM01015850.1 2224 547 ER, 23 Diphenol oxidase Tobacco AAC49536.1 77, 0.0 2CpLAC3 ABIM010158201 2473 558 ER, 23 Diphenol oxidase Tobacco AAC49536.1 78, 0.0 1CpLAC4 ABIM01015697.1 2741 557 ER, 22 Diphenol oxidase Tobacco AAC49536.1 78, 0.0 0CpLAC5 ABIM01007467.1 2162 562 ER LAC11 Arabidopsis NP_195946.2 78, 0.0 1CpLAC6 ABIM01005823.1 2483 579 Poss ER, 31 LAC5 Arabidopsis NP_181568.1 79, 0.0 1CpLAC7 ABIM01012580.1 2829 566 ER, 23 Tomato laccase ACB10229.1 69, 0.0 0CpLAC8 ABIM01015449.1 2376 593 ER, 22 Liriodendron tulipifera laccase AAB17194.1 66, 0.0 0CpLAC9 ABIM01001394.1 2361 431 ER, 32 LAC13 Arabidopsis NP_196330.3, Only 2 Cu oxidase motifs 75, 2e-150 1Tyrosinase SuperfamilyCpPPO1 ABIM01007553.1 1809 602 chloro, 43 Apple polyphenol oxidase BAA21676.1 57, 2e-172 0CpPPO2 ABIM01002511.1 1848 615 chloro, 46 Poplar polyphenol oxidase AAU12257.1 61, 0.0 0CpPPO3 ABIM01025488.1 1794 597 chloro, 43 Poplar polyphenol oxidase AAK53414.1 56, 0.0 1CpPPO4 ABIM01013357.1 1459 365 Poss Plastid Cherimoya polyphenol oxidase ABJ96144.1 56, 7e-74 1CpPPO5 ABIM01013359.1 1305 365 Poss Plastid Cherimoya polyphenol oxidase ABJ96144.1 40, 5e-101 1

Table 11 Polyphenol oxidase (tyrosinase, dicopper containing) andlaccase (multi copper oxidase) genes predicted in the papaya genome,the whole genome shotgun sequence accession number in NCBI(WGS Accession), coding sequence for amino acids (CDS), thenumber of introns in the nucleotide sequence (introns), amino acids(aa), whether a secretory sequence was predicted (secretory) its

location (ER endoplasmic retriculum) and aa length, similarity inpeptide sequence with another species (homology), extent thesequences were invariant (identity), E-value was the expectation valuefor homology, and the number of papaya expressed sequence tags(EST) detected with at least 500 bases and 99% identity

Tropical Plant Biol. (2008) 1:246–277 263263

clustered with two on each supercontigs 27 and 62.Supercontig 27 and 62 are not on the same linkage groupsuggesting that papaya PPO’s may not be located in a smalllocus on the same chromosome. Papaya PPOs did showsignificant homology with published PPO sequences and fitreadily within the superfamily (Fig. 3).

Peroxidases (POD) are secreted glycoproteins that areinvolved in cell elongation, cell wall modification andpathogen defenses [143]. The Arabidopsis genome has 73genes for POD [204] and rice has 138 [143] while only 33were predicted for papaya (Supplementary Table 5). Allpapaya PODs, CpPOD1 to CpPOD33 had a 22 to 26 aminoacid secretory sequences and molecular weights from 279to 437 amino acids with 47 to 83% identity to knownPODs. The presence of the secretory sequence to theapoplast suggested they would be classified as Class IIIPODs. Most of the predicted papaya PODs fell into therange 320–360 aa, similar to that reported for other plantPODs. No papaya ESTs were found for 18 of the predictedpapaya genes. The absence of ESTs did not mean that thegenes were not expressed, many Arabidopsis PODs areonly expressed at low levels and often in all organs [204].RT-PCR does detect the Arabidopsis PODs that have noESTs reported in the databases. Five papaya POD isoformshave been reported to increase during ripening [181] with aMW range similar to that of the predicted genes.

Volatile Production

All plant parts emit volatiles [57] with multiple functions.Of the many roles in plants, ecological interactions such asdefense against herbivores, pathogens, and as attractants foranimals to disperse pollen and seeds are presumed to beprincipal functions. [148, 149, 199]. Additionally, volatilesas components of flavor and aroma perception substantially

contribute to the utility of plant parts as human foodstuffs.Odorants are volatile and interact with human olfactoryreception [36] but they must be somewhat lipohilic as wellas water soluble, possesses adequate vapor pressure, andoccur in sufficient concentration for olfactory receptorinteraction. At least one hundred and sixty-six compoundshave been identified in papaya fruit volatiles [70, 71, 119,151, 179]. The most commonly identified papaya volatileshave been methyl butanoate, ethyl butanoate, 3-methyl-1-butanol and 1-butanol. The esters of lower fatty acids areconsidered to contribute much to the typical papaya flavor[151]. The amounts and relative content of volatiles hasbeen shown to vary with the stage of ripeness [71, 98], forexample, linalool production increases nearly 400 fold withonly a sevenfold increase in benzyl isothiocyanate duringripening [71]. When the fruit has reached the edible ripestage butanol, 3-methylbutanol, benzyl alcohol and α-terpineol are at their maximum concentrations [4]. One ofthe two volatiles that most closely resembled those ofpapayas is linalool, a product of the plastid synthesispathway. The other papaya volatile is methyl benzoate,described as having papaya qualities on odor assessment[119]. The sweaty odor quality of some papaya cultivarsis due probably to the production of methyl butanoate[119]. The Hawaii variety had very little methyl butanoate(0.06%) [70] while the Sri Lankan variety had 48.3%[119]. In addition, benzyl isothiocyanate contributes apungent off-odor. The amount of each volatile componentvaries both with cultivar and locality [72, 119].

Most volatile esters are described as fruity [37] and themost likely precursors are amino acids and lipids. Theprecursors of volatile esters, with branched alkyl chain, arevaline, isoleucine and other amino acids from threonine[138, 216]. Aliphatic esters and alcohols are producedfrom free fatty acids such as linoleic and linolenic [22].

Fig. 3 Polyphenoloxidase rela-tionship with other describedpolyphenol oxidases (PPO) fromNCBI, aligned with ClustalWand neighbor-joining values.The tree was constructed withMEGA 4.0

264 Tropical Plant Biol. (2008) 1:246–277

The pathways come together in the formation of aldehydesthat are reduced to alcohols by alcohol dehydrogenase[185] and ester formation by alcohol acyltransferase [59,69].

Single genes were found in the papaya genome for allthe enzymes in the biosynthesis from threonine to 3-keto-3-methylvalerate (Table 12). At least three branched chainamino transferase genes were found that could convert 3-keto-3-methylvalerate via isoleucine to 3-oxo-methylpenta-noic acid. The next step to 2-methyl butanal involvespyruvate decarboxylase and three genes encoding thisenzyme were predicted from the genome data; all werepredicted to have the characteristic domains. Four branchedchain alpha keto acid dehydrogenases (alcohol dehydro-genases) were predicted with three that had papaya EST.The interconversion between 3-methyl butanol and 2-methylbutyl acetate possibly involved a single predictedalcohol acyl transferase gene and four predicted carbox-ylesterases genes (Table 12).

Straight chain ester biosynthesis commonly occurs fromfatty acid with linoleic acid being a common precursor. Thefirst enzyme is lipoxygenase of which nine genes werepredicted in papaya with another possible partial sequencefound (Table 13). Except for two of the predicted lip-oxygenase, all had papaya ESTs. The predicted gene for thepreviously described papaya lipoxygenase (CpLOX1) hadeight ESTs and a chloroplast transit peptide. The otherCpLOXs did not have predicted chloroplast transit peptides,suggesting they may be located in the cytoplasm (Table 13).

One papaya hydroperoxide lyase gene (P450 superfamilymember) was found and no isomerasewas predicted (Table 13).Hex-3-enal to hex-2-enal isomerase has been reported to beunstable and no sequences are in the gene databases. Theisomerase possibly occurs in the cytoplasm [140, 226]. Thedehydrogenation of hex-3-enal to hex-3-enoic acid by alde-hyde dehydrogenase could involve upwards of five genes. Twoof the aldehyde dehydrogenase had predicted mitochondrialtransit peptides and may be involved in volatile production. Anumber of alcohol dehydrogenase genes were predicted thatcould convert hex-3-enal to hex-3-enol (Table 13).

Two volatiles with fruity/floral properties are 2-phenyl-acetaldehyde and 2-phenylethanol derived from phenylala-nine. The first enzyme in the pathway is a bifunctionalphenylacetaldehyde synthase possibly combining decarbox-ylation-amine oxidation leading to phenylacetaldehyde[97]. The last step is the reduction of two phenylacetalde-hyde to 2-phenylethanol [199]. The reduction involvesNADPH and 2-phenylacetaldehyde reductase. Genes werepredicted in papaya for both steps in biosynthesis(Table 14). Two predicted phenylacetaldehyde synthase(CpPALDS1, CpPALDS2) genes were found having highhomology to the deposited gene sequences for Rose andPetunia (Table 14). A single gene with moderate homology

(48%, E-value 3e-68) to the tomato gene was predicted forthe reduction to 2-phenylethanol [199].

Terpenoids, widely distributed in lichens, algae, andhigher plants [13, 30, 62, 178, 190, 99], represent the mostdiverse family of natural products, as there are over 40,000different structures identified thus far. Many terpenoids arenon-volatile and constitute important elements of plantfunctional processes, i.e. plant growth regulation, redoxreactions, membrane structure, and photosynthesis [56].Volatile and flavor compounds from essential oils ofvarious terpene classes are produced in leaves and fruitwhile carotenoids give the fruit skin and flesh its uniquecolor. Biosynthesis occurs through two different pathways:the acetate-mevalonate pathway in the cytoplasm [30] andthe non-mevalonate pathway in the plastids [111, 112]. Themevalonate pathway leads to sesquiterpenes and triterpenes(sterols) while monoterpenes, diterpenes, tetraterpenes(carotenoids) and polyterpenes are formed in the plastids.

Genes were predicted for most of the steps in terpenebiosynthesis occurring both in the plastids (Table 15) and inthe cytoplasm (Table 16). Two DXP synthases werepredicted with the shorter predicted peptide lacking achloroplast transit peptide (Table 15). Another partial DXPsynthase, one-third the length of the expected peptide wasfound with 78% homology (E-value 3e-77) to the rubberTPP enzyme domain (BAF98289.1). A full length linaloolsynthase gene (ABIM01025787.1) was predicted with twopartial sequences (ABIM0102980.1, ABIM01019472.1). Nogeranyl-geranyl diphosphate (GGPP) synthase was foundthat had a chloroplast transit peptide. A short sequence(ABIM01003036.1) had low homology (61%, E-value 1e-26, 105 aa) to AAQ84170.1 GGPP synthase that catalyzesthe farnesyl diphosphate to GGPP conversion was found.

Cytoplasmic terpene synthesis from mevalonate todimethylally diphosphate has a single gene for each step(Table 16). Two geranyl diphosphate genes were predictedwith significant homology to published sequences. Two fulllength squalene synthases were predicted along with twopartial sequences (ABIM01018750.1, 191 aa; ABIM010207788.1, 261 aa) that had high homology to a tomatohomologue (ABU40771.1) and Panax quinquefolius se-quence (CAJ58418.1).

The cleavage of carotenoids derived from the terpenesynthesis pathway can lead to the production of volatiles.Carotenoid cleavage can occur at any conjugated doublebonds by various enzymes such as CCD and NCEDs(discussed above in the Fruit Growth section) to form analdehyde or ketone in each product. The products includeabscisic acid, strigolactone and various aroma compounds[207]. While Arabidopsis contains nine CCD, five of whichare directly involved in abscisic acid synthesis, the otherCCDs lead to volatiles and non-volatile apocarotenoids [183]with one CCD able to cleave multiple substrates at different

Tropical Plant Biol. (2008) 1:246–277 265265

Tab

le12

Predicted

papaya

genesforthebranched

chainestersbiosynthesispathway.T

thewholegenomeshotgunsequence

accessionnumberin

NCBI(W

GSAccession),coding

sequence

foram

ino

acids(CDS),am

inoacids(aa),presenceof

discreteportionof

proteinpossessing

itsow

nfunctio

nandthesuperfam

ily(dom

ain),sim

ilarityinpeptidesequence

with

anotherspecies

(hom

ology),extentthe

sequenceswereinvariant(identity),E

-value

was

theexpectationvalueforhomology,andthenumberof

papaya

expressedsequence

tags

(EST

)detected

with

atleast5

00basesand99%

identity

Sub

strate

Enzym

eWGSAccession

cds

aaDom

ain

Hom

olog

yIdentity,

E-value

EST

Threonine

Threon

inedeaminase

ABIM

0100

2164

.147

3959

8PA

LP,

PRK09

224,

Arabido

psisthaliana

NP_1

8761

6.1

76,0.0

12-ketobu

tyrate Acetolactatesynthase

ABIM

0123

825.1

6524

257

ACT,

iLvH

,Arabido

psisthaliana

NP_8

5017

2.1

smallsubu

nit

82,8e-124

0

2-acetoh

ydroxy

butyrate

Acetohyd

roxy

acid

reductoisomerase

ABIM

0102

2972

.131

3845

9Ado

Hcyase,

IlvC

Sup

erfamily,

Arabido

psisthaliana

NP_1

9142

0.1

85,0.0

0

2,3-dihy

drox

y-3-methy

lvalerate

ABIM

0101

2960

.189

5261

7PRK00

911,

IlvD

-EDD

Arabido

psisthaliana

NP_1

8903

6.1

83,0.0

02-keto-3-m

ethy

lvalerate

Aminotransferase

isoleucine

Aminotransferase

ABIM

0101

9595

.119

5133

7BranchedchainAA

transferase,,

PLPDE-IV

Sup

erfamily

RiceNP_0

0104

2537

.165

,1e-126

7

ABIM

010115

18.1

1472

315

PLPDE-IV,Aminotransferase,

Arabido

psisthaliana,NP_2

0059

3.2

69,8e-126

1ABIM

0101

9463

.159

3041

7BCAT_b

eta_family,PLPDE_IV

Branched

Chain

Aminotransferase

Arabido

psisthaliana

NP_5

6692

3.1

76,0.0

1

2-ox

o-3-methy

lpentano

icacid

Pyruva

tedecarbox

ylase

ABIM

0101

8822

.124

9164

0COG39

61,TPP_enzym

e_N

Sup

erfamily,

TPP_P

DC_IPDC,.1

Arabido

psisthaliana

NP_1

9503

366

,0.0

2

ABIM

0100

2684

.133

2658

9TPP_enzym

e_N

Sup

erfamily,

TPP_P

DC_IPDC,

Citrus

sinensisAAZ05

069.1

87,0.0

1

ABIM

0101

8795

.124

9160

5TPP_enzym

e_N,TPP_P

DC_IPDC,

Arabido

psisthaliana

NP_2

0030

7.1

85,0.0

72-methy

lbutanal

Alcoh

oldehyd

rogenase

ABIM

0102

3823

.123

9935

4Alkenal

redu

ctase,

Arabido

psisthaliana

NP_1

9719

9.1

73,4e-152

1branched

chainalpha

ketoacid

dehyd

rogenase

ABIM

0100

2736

.128

5146

5TPP_enzym

esSup

erfamily,

TPP_E

1_PDC_A

DC_B

CADC

RiceNP_0

0106

6324

.172

,5e-180

1

ABIM

0101

0666

.141

3347

7AcoA,TPP_enzym

esSup

erfamily,

TPP_E

1_PDC_A

DC_B

CADC,

RiceABA95

968.2-alph

asubu

nit

76,0.0

0

ABIM

010118

66.1

+ABIM

010118

65.1

5264

501

2-ox

oacid_

dhsuperfam

ily,PRK1185

6Arabido

psisthaliana

NP_8

5052

7.1

62,7e-171

3

3-methy

lbutanol

⇓Alcoh

olacyl

tran

sferase

ABIM

0102

1279

.119

7635

5BCAT_b

eta_family,PLPDE_IV

Sup

erfamily.

Arabido

psisthaliana

NP_1

7247

8.1

6.75

0e-139

1

ABIM

0100

7428

.113

7445

7Transferase

Sup

erfamily,AtAcetyl

transferaselik

eArabido

psisthaliana

NP_1

8923

3.1

36,1e-79

0

⇑Carbox

ylesterase

ABIM

0102

0297

.110

0233

3Esterase_lip

aseSup

erfamily,CXE

Actinidia

deliciosa

ABB89

020.1

57,4e-110

0ABIM

0102

2048

.110

4134

6Esterase_lip

aseSup

erfamily,CXE

Actinidia

deliciosa

ABB89

015.1

62,3e-117

3ABIM

0102

3942

.194

231

3Esterase_lip

aseSup

erfamily,CXE

Malus

pumila

ABB89

002.1

67,4e-125

1ABIM

0101

3294

.197

532

4Esterase_lip

aseSup

erfamily,CXE

Malus

pumila

,ABB89

010.1

58,2e-101

32-methy

lbutylacetate

266 Tropical Plant Biol. (2008) 1:246–277

positions [207]. Only one CpCCD was predicted for papayaand another partial CCD ORF was found. Tomato containstwo closely related CCDs, LeCCD1A and LeCCD1B thatgenerate the aldehydes and ketones including geranylacetone, pseudoionone and β-ionone [183]. CpCCD mayserve a similar role in papaya ripening and generate ketonevolatiles such as 6-methyl-5-hepten-2-one in tomatoes [207]and as detected in papaya fruit volatiles [71].

Conclusions

The genome of papaya (372 Mbp) is three times larger thanthat of Arabidopsis (125 Mbp) and two-fifths the size of thetomato genome [11]. Papaya is predict to code for 24,746genes, which is 10% to 20% fewer than in Arabidopsis(31,114) and 35% fewer than the estimated 38,000 non-transposon genes in tomato. Unlike the tomato genome, the

Table 14 Predicted papaya genes involved in volatile produced fromphenylalanine. The whole genome shotgun sequence accessionnumber in NCBI (WGS Accession), coding sequence for amino acids(CDS), the number of introns in the nucleotide sequence (introns),amino acids (aa), presence of discrete portion of protein possessing its

own function and the superfamily (domain), similarity in peptidesequence with another species (homology), extent the sequences wereinvariant (identity), E-value was the expectation value for homology,and the number of papaya expressed sequence tags (EST) detectedwith at least 500 bases and 99% identity

Accession cds Introns aa Domains Homology Identity,E-value

EST

Phenylacetaldehyde SynthaseCpPALDS1 ABIM01011918.1 1485 0 494 Aminotrans_1_2_superfamily

GadB carboxylase, Group IIpyridoxal 5 P decarboxylase

Rose hybrid cultivar,ABB04522.1

60, 1e-178 0

CpPALDS2 ABIM01015898.1 8856 11 480 Aminotransf_1_2 SF, GadB, AromaticL-amino acid decarboxylase

Petunia x hybridABB72475.1

81, 3e-171 1

Phenylacetaldehyde ReductaseCpPALDR1 ABIM01000864.1 1665 5 281 AdoHcyase Super-family NAD dependent

epimerase/dehydratase familyABR15768.1 Solanumlycopersicum

48, 3e-68 3

Table 13 Predicted papaya genes potentially involved in straightchain esters biosynthesis pathway. The whole genome shotgunsequence accession number in NCBI (WGS Accession), codingsequence for amino acids (CDS), the number of introns in thenucleotide sequence (introns), amino acids (aa), whether a secretorysequence was predicted (secretory) its location (ER endoplasmic

retriculum) and aa length, presence of discrete portion of proteinpossessing its own function and the superfamily (domain), similarityin peptide sequence with another species (homology), extent thesequences were invariant (identity), E-value was the expectation valuefor homology, and the number of papaya expressed sequence tags(EST) detected with at least 500 bases and 99% identity

Substrate Enzyme CDS aa Secretory Domain Homologs Identity, E-value EST

Linoleic acid LipoxygenaseCpLOX1 ABIM01007255.1 4847 925 chloro PLAT_LH2, Lipoxyg Papaya AAR84664.1 97, 0.0 8CpLOX2 ABIM01005441.1 4664 816 none PLAT_LH2, Lipoxyg Prunus dulcis CAD 10779.2 67, 0.0 2CpLOX3 ABIM01008628.1 3856 854 none PLAT_LH2, Lipoxyg Corylus avellana CAD 10740.1 71, 0.0 3CpLOX4 ABIM01008664.1 3664 855 none PLAT_LH2, Lipoxyg Corylus avellana CAD 10740.1 71, 0.0 2CpLOX5 ABIM01010727.1 3480 992 none PLAT Lipoxyg, Zantedeschia aethiopica AAG18376.1 71, 0.0 4CpLOX6 ABIM01025187.1 3710 788 none PLAT, Lipoxyg Populus deltoides LOX1, AA257445.1 61, 0.0 1CpLOX7 ABIM01011535.1 4692 867 none PLAT Lipoxyg Corylus avellana CAD 10740.1 70, 0.0 2CpLOX8 ABIM01012839.1 3786 849 none PLAT Lipoxyg Strawberry, CAE17327.1 71, 0.0 2CpLOX9 ABIM01012840.1 3957 849 none PLAT Lipoxyg Arabidopsis CAC19365.1 73, 0.0 0CpLOX10 ABIM01002936.1 3548 692 none PLAT Lipoxyg, partial Cotton AAK50778.1 54, 4e-75 0

ABIM01002935.1 2x Lipoxyg motifs13-hydroperoxide linoleic acidHydroperoxidase lyase

ABIM01029563.1 1952 495 chloro P450 Superfam Guava AAK15070.1 66, 0.0 2Hex-3-enal Aldehyde dehydrogenaseCpALDH1 ABIM01002498.1 3158 480 none LuxC, AldeDH Arabidopsis NP_195348.2 67, 0.0 0CpALDH2 ABIM01013559.1 7286 487 none LuxC Arabidopsis ALDH3H NP_17508.1 73, 0.0 0CpALDH3 ABIM01015730.1 2244 491 none LuxC AldedH Maize AAR21278.1 57, 1e-165 0CpALDH4 ABIM01014117.1 4011 533 mito LuxC AldedH Tobacco CAA71003.1 82, 0.0 1CpALDH5 ABIM01019111.1 3694 536 mito LuxC, Aldedh Lotus corniculatus AAO72532.1 85, 0.0 2Hex-3-enoic acid

Tropical Plant Biol. (2008) 1:246–277 267267

Tab

le15

Predicted

papaya

genesfortheterpeneplastid

biosyn

thesispathway.The

who

legeno

meshotgu

nsequ

ence

accessionnu

mberin

NCBI(W

GSAccession

),coding

sequ

ence

foram

ino

acids(CDS),am

inoacids(aa),whether

atransitsequ

ence

was

predicted(Transit)

itslocatio

n,presence

ofdiscrete

portionof

proteinpo

ssessing

itsow

nfunctio

nandthesuperfam

ily(dom

ain),

similarity

inpeptidesequ

ence

with

anotherspecies(hom

olog

y),extent

thesequ

enceswereinvariant(identity

),E-value

was

theexpectationvalueforho

molog

y,andthenu

mberof

papaya

expressedsequ

ence

tags

(EST)detected

with

atleast50

0basesand99

%identity

Sub

strate

Enzym

eWGSAccession

cds

aaTransit

Dom

ain

Hom

olog

yIdentity,

E-value

EST

Pyruv

ate+Glyceraldehyd

e-3-ph

osph

ate

↓DXPsynthase

ABIM

0100

5151

.11161

718

chloro

TPP-D

XS

Tom

atoAAD38

941.1

85,0.0

0ABIM

0101

6399

.159

8854

9no

neTPP-D

XS.TPP_enzym

eAtDXPS3

68,9e-163

10ABIM

0101

6400

.1NP_0

0107

8570

.11-deox

y-D-xylulose-5-ph

osph

ate

↓DXPreductoisomerase

ABIM

0101

4958

.141

3138

4no

neDXP_reductoisom

_sup

erfamily,

PRK05

447

Rub

berBAF98

290.1

92,0.0

0

2-C-m

ethy

l-d-erythrito

l-4-ph

osph

ate

↓2C

-methylerythritol,

2,4-cyclod

iphosphatesnthase

ABIM

0102

5949

.174

7523

6chloro

MeC

DP_syn

thase,

Rub

berBAF98

295.1

74,1e-95

9

ABIM

0102

5947

.1ABIM

0102

5948

.1Isop

entyldipho

sphate

(IPP),dimethy

lally

diph

osph

ate

↓Isom

erase

Geranyl

diph

osph

ate

↓Linaloo

lsynthase

ABIM

0102

5787

.139

3085

2Isop

reno

id_B

iosyn_

C1

Arabido

psisthaliana

NP56

4772

.146

,0.0

0Linaloo

lGeranyl

diph

osph

ate

↓farnesyl

diphosphatesynthase

ABIM

0100

7640

.138

0934

2Trans_IPPS_H

T,Isop

reno

id_b

iosyn_

C1

Rub

berAAB07

264.1

85,4e-178

0Farnesyldiph

osph

ate

↓GGPPsynthase

Geranylgerany

ldiph

osph

ate

↓Terpenesynthase

ABIM

0100

7557

.140

5chloro

Isop

reno

id_b

iosyn_

C1

Arabido

psisthaliana

NP_5

6477

2.1

51,3e-81

1Diterpenes

-Phy

toene-Chlorop

hyll&

Carotenoids

268 Tropical Plant Biol. (2008) 1:246–277

Tab

le16

Predicted

papaya

genesforthecytoplasmic

terpenes

biosyn

thesispathway.The

who

legeno

meshotgu

nsequ

ence

accessionnu

mberin

NCBI(W

GSAccession),coding

sequ

ence

for

aminoacids(CDS),am

inoacids(aa),presence

ofdiscrete

portionof

proteinpo

ssessing

itsow

nfunctio

nandthesuperfam

ily(dom

ain),similarity

inpeptidesequ

ence

with

anotherspecies

(hom

olog

y),extent

thesequ

enceswereinvarant

(identity

),E-value

was

theexpectationvalueforho

molog

y,andthenu

mberof

papaya

expressedsequ

ence

tags

(EST)detected

with

atleast50

0basesand99

%identity

Sub

strate

Enzym

eWGSAccession

cds

aaDom

ain

Hom

olog

yIdentity,

E-value

EST

Mevalon

ate↓Mevalon

atekinase

ABIM

010115

27.1

4225

387

ERG12

Rub

berAAL18

925.1

79,7e-171

25-ph

osph

omevalon

ate

↓phosphom

evalon

atekinase

ABIM

0100

5009

.123

911

507

ERG8

Rub

berAAL18

926.1

76,0.0

0ABIM

0100

5006

.15-diph

osph

atem

evalon

ate

↓DiPM

Decarbox

ylase

ABIM

0102

2218

.153

9641

9MVD1

Rub

berBAF98

285.1

84,0.0

0Isop

entyldipho

sphate

(IPP)

↓polyisoprenesynthase

ABIM

0100

3036

.140

776

Isop

reno

id_b

iosyn_

C1

Pop

ulus

alba

BAD98

243.1

50,3e-10

0Dim

ethy

lally

diph

osph

ate

↓IpdiP

Isom

erase

ABIM

0100

5339

.175

5437

0Trans_IPPS_H

T,Quercus

robu

rCAC20

852.1

78,3e-167

1Geran

ylDiP

synthase

ABIM

0100

5340

.1Isop

reno

id_b

iosyn_

C1

ABIM

0100

5341

.1ABIM

0100

4175

.188

229

3Isop

reno

id_b

iosyn_

C1

Rub

berBAF98

300.1

51,1e-76

1Geranyl

diph

osph

ate

↓farnesyl

diphosphatesynthase

ABIM

0100

7640

.138

0934

2Trans_IPPS_H

T,Isop

reno

id_b

iosyn_

C1

Rub

berAAM98

379.1

85,4e-178

0Farnesyldiph

osph

ate

↓Squalenesynthase

ABIM

0102

0788

.155

2941

5Trans_IPPS_H

T,Isop

reno

id_b

iosyn_

C1

Pan

axqu

inqu

efoliusCAJ584

18.1

65,5e-93

0ABIM

0102

0788

.155

9041

9Trans_IPPS_H

T,Isop

reno

id_b

iosyn_

C1

Glycine

max

BAA22

559.1

87,2e-144

0Squ

alene–>triterpenes

↓Squalenemon

oxyg

enase

ABIM

0101

9466

.139

9461

2PRK05

192,

SE,

RiceNP_0

0104

9463

.184

,0.0

10Sterols

ABIM

0102

1285

.140

6648

2PRK05

192,

SE

Medicag

otrun

catula

CAD23

249.1

80,0.0

1

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papaya genome is largely euchromatic [132, 209]. Thelower gene number in papaya may reflect that, unlikeArabidopsis [32], the papaya genome has not undergone arecent whole-genome duplication and has had feweropportunities for fractionation of linkage arrangements bypost-duplication gene loss [132, 142]. This suggests thatgene structure, function, and arrangement in papaya mayresemble the ancestral angiosperm more closely than dopreviously sequenced angiosperms [116]. Comparison ofthe five sequenced clade members suggests a minimalangiosperm gene set of 13,311. Fewer introns have beenfound in papaya genes than in their orthologs in Arabi-dopsis (8,319 versus 11,718) and rice (6,874 versus 7,011).Approximately 2,000 transcription factors in over 60families have been identified in Arabidopsis [85], withpapaya having fewer members in the majority of families,but a significantly higher number of predicted MADS-boxproteins (171 versus 141). The comparison of geneexpression and activity during fruit ripening suggests thatthe same regulatory pathways and similar controls occur[81]. The lack of whole genome duplication and reductionsin most papaya gene families and biosynthetic pathwaysmakes papaya a valuable tool for the study of the complexregulatory networks active in fruit ripening. The example,fewer gene numbers for ethylene synthesis, receptors and inthe ethylene response pathways may provide insights as tohow ripening is controlled.

Materials and Methods

Gene Prediction

Papaya unigenes from complementary DNA were alignedand used in gene prediction software [133]. Spliced align-ments of proteins from the plant division of GenBank, andtranscripts from related angiosperms, were generated. Genepredictions were combined with spliced alignments ofproteins and transcripts to produce a reference gene set.Gene prediction software Augustus, GlimmerHMM andSNAP were used. Ab initio gene prediction softwareFgenesh, Genscan and TWINSCAN were trained onArabidopsis. Predicted proteins were aligned to the plantdivision of GenBank and transcripts from related angio-sperms (Arabidopsis thaliana, Glycine max, Gossypiumhirsutum, Medicago truncatula, Nicotiana tabacum, Oryzasativa, Zea mays). Gene predictions were combined withspliced alignments of proteins and transcripts to produce areference gene set using the evidence-based combinerEVidenceModeler (EVM). Where predicted genes werenot indicated and to confirm a prediction, known cDNAand protein sequences were downloaded for Arabidopsis,poplar, tomato and other species as required. Local searches

were made using the papaya WGS, EST and predicted genedatabases using the BLAST search application in BioEdit[87]. The potential papaya candidate genes indicated in theBioEdit search were then cross-checked against NCBIBLAST and homology confirmed and conserved domainsdetermined.

Similarity Searches and Alignment

Similarity searches were carried out by online BLASTsearch [120]. Best ORF predicted were BLASTp againstnon-redundant database to determine similarity with otherproteins. Proteins with low E-score of ≥50 were acceptedfor comparison, with emphasis on proteins with E-scores≤100 favored. Similar proteins that had published annota-tion that was not predicted were identified.

Sequences were aligned using the multiple sequencealignment program, CLUSTAL W version 1.6 [200] usingdefault parameters (gap penalty of 15, gap length penalty of6.66). Gaps and positions with ambiguities were excludedfrom the phylogenetic analysis. Phylogenetic analysis wasperformed using MEGA 4.0 [191] using neighbor-joiningmethods [171]. The corresponding whole genome shotgunreads (WGS) sequence was identified by using the BLASTroutine on the genomic sequence identifying the ORF withBLASTn.

Predicted Peptide Domains

Protein domains were predicted using InterProScan againstprotein databases (PRINTS, Pfam, ProDom, PROSITE,SMART).

Papaya ESTs

TheGenBank accession numbers of the papaya ESTs from thegenome project are EX227656 to EX303501 [132]. TheNCBI GenBank contains 77,128 papaya EST entries (2008September 08). The genes reported herein, were doublechecked against this database. EST sequences were identifiedby taking the genomic nucleotide sequence of the regionidentified for the ORF and using BLASTn against the ESTdatabase for Caricia papaya. Matches were screened byaligning the EST sequence with the genomic nucleotidesequence using the ALIGN program. Matches of greater than99% for more than 500 sequential bases were generally used.EST matches were further screened by Prosite and Inter-proscan to insure that they encoded the expected gene.

Targeting Sequences

Predotar was used as the primary screen for N-terminalputative targeting sequences [184] for mitochondrial,

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plastid and ER targeting. MitoProt [50], ChloroP [66, 67]and TargetP [67] were also used to verify predictions withall programs having a low false positive rate. The absenceof a predicted transit or secretion sequence did not meanthat it was not present, as the programs only predicts about85% of the sequences [68].

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