Plant Molecular Biology 19: 401-404, 1992. © 1992 Kluwer Academic Pubfishers. Printed in Belgium. 401

Cloning and characterization of a gene involved in phytoene synthesis from tomato

John Ray 1, Philippe Moureau 1,4, Colin Bird 1, Alison Bird l, Don Grierson 2, Martin Maunders 2, 5, Mark Truesdale 3, Peter Bramley 3 and Wolfgang Schuch 1, I ICI Seeds, Plant Biotechnology Section, Jealott's Hill Research Station, Bracknell RG12 6EY, UK (* author for correspondence)," 2 AFR C Expression Unit, Department of Physiology and Environmental Science, University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough, LEI2 5RD, UK; 3Department of Biochemistry, Royal Holloway and Bedford New Colleges, Egham, TW20 OEX, UK; present addresses." 4 Medgenix Group, R & D Department, 6220 Fleurus, Belgium," S A TC, Cambridge Science Park, Cambridge, CB4 4WA, UK

Received 16 September 1991; accepted in revised form 31 January 1992

Key words." lycopene, prephytoene synthase, tomato, gene isolation

Tomato ripening is associated with a number of physiological and biochemical changes including degradation of chlorophyll in the plastids of the ripening tomato fruit and synthesis of lycopene, which is responsible for the red colour of ripe fruit. We have cloned the mRNAs for at least 19 proteins whose expression is increased during ripening [6, 8], and have investigated their func- tion using antisense RNA [1, 4, 9]. This has led to the identification ofpTOM5, a ripening-specific cDNA clone [6], as encoding an enzyme critical in carotenoid biosynthesis [ 1 ]. Further biochem- ical characterisation of plants expressing an- tisense RNA to pTOM5 has identified a block in the conversion between geranylgeranyl pyrophos- phate to prephytoene pyrophosphate indicating that the pTOM5 gene product is involved in phy- toene synthesis (P. Bramley et al., submitted for publication). This biosynthetic conversion repre- sents the first dedicated step of the carotenoid biosynthetic pathway. Using RFLP mapping, it has been demonstrated that two loci are present

in the tomato genome with sequences homolo- gous to pTOM5 [5]. Here we report the cloning and characterisation of genomic sequences with homology to pTOM5.

Genomic Southern hybridisations showed that pTOM5 hybridises to two Eco RI fragments of 6.5 kb and 8.5 kb (data not shown). In order to isolate these fragments, genomic libraries [2] were screened using pTOM5 cDNA insert. Five clones were isolated which contained approximately 25 kb of tomato DNA in overlapping clones (data not shown). The sequence of one of these clones, clone F, was determined in the region showing hybridisation to pTOM5. Comparison of the DNA sequence of this clone with pTOM5 re- vealed only limited homology. This ranged from 65~o in exon 1 to 89~o in exon 7 (see Fig. 1).

Despite extensive screening of genomic librar- ies (five independent experiments) we were un- able to isolate the gene coding for the pTOM5 mRNA using pTOM5 cDNA as a probe. We therefore screened the library using an oligonu-

The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Da- tabases under the accession numbers X60440 (clone F) and X60441 (GTOM5).

402

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Fig. 1. Structure of Clone F and GTOM5. The sequence of clone F was determined using Hind III and Hind III-Eco RI fragments cloned into M13. The sequence of GTOM5 was obtained from subclone GTOM5.7. The 3.3 kb Eco RI-Sal I fragment was cloned into M13. Sequencing was carried out using internal primers. The 3' region defined by the arrow was PCR amplified from Ailsa Craig genomic DNA. Barn HI sites were introduced into PCR primers to facilitate cloning of PCR products. The sequences of oligonucleotides were: TTTCTTTGAGGATCCGAACTTTGAAACATGATATCAGC covering the 3' end of intron4, and ATATTATATGGATCCACGGAAAGTTCTCTCAAAGGAGTTGAG from the extreme 3' end of the cDNA. Following PCR, the product was cut with Bam HI and the two resulting fragments were cloned into M13 and sequenced as above. The structure of the genomic clones is shown in relation to the structure of the pTOM5 cDNA [6].

cleotide CL100 (CATCTGTTCCGATGT- CATCGTCCG) which is specific to pTOM5. One phage was isolated which hybridised to CL100, the pTOM5 cDNA insert, but not CL99 (TTTTTTTTCTGATGACACAGCCAT), an oligonucleotide specific to clone F. Restriction mapping of DNA of this clone, GTOM5, identi- fied a 3.3 kb Eco RI-Sal I fragment which hybri- dised to pTOM5 cDNA. The DNA sequence of this gene was determined and found to be iden- tical in the exon sequences to the 5' portion of the pTOM5 cDNA. The 3' end of this gene, how- ever, was missing. Therefore, the 3' end of the gene was amplified by PCR using a 5' primer from exon 4 which is specific for GTOM5 and a 3' primer from the 3' end of the cDNA. A 1.4 kb fragment was isolated, sequenced and shown to encode the missing 3' end of the GTOM5 gene. The complete maps of these genes are shown in Fig. 1. The sequence of GTOM5 is compared to the sequence of clone F in Fig. 2.

The sequence of GTOM5 corresponding to pTOM5 is encoded by 6 exons varying in length from 51 to 612 bases. In contrast, sequences re- lated to pTOM5 found in clone F are encoded by 8 exons with the region having homology to the first exon of GTOM5 split into 3 exons. The po- sitions of the other introns in GTOM5 are con- served in clone F. However the size of introns found at identical positions varies between the genes. In addition, exon 1 of clone F contains one extra amino acid at position 14, and the homol- ogy at the C terminus breaks down 5 codons from the C terminus at a position with no homology to a consensus intron-exon boundary. This exon has 2 extra amino acids beyond the stop codon found in pTOM5. The regions corresponding to the 5' and 3' untranslated regions of pTOM5 do not show any homology to each other. Furthermore, the putative promoter sequences of both genes do not have any obvious homology.

In order to analyse the expression of the two

403

I0 20 30 40 SO 60 70 80 90 I00 110 120 130 140 150 I I l I l l I I l I I I l l I

TTTGcCTGTCTGTGGTCTTTT~TMTCTTTTTCTACA~GAGj~AGTGGGTMTTTTGTTT~GAGT~G~TTCTCTAGTGGGMTC~TAG~GTMTT~TTTTC~TAAAcTMGT~GTTTGG~GGTGAc~AA~A~ 150

NSE Y G L D G L E M S V A L L W V V S P C D V S N G T S F M E S V R E G N R F F D S

A~CAAAAATCTTGG~TTGTTT~CMCCM~GTTTTCT~CTcAG~TGTCTGTTGCCTTGT~TGGG~GTTTCT~CTTGTGAcGTCTC~TGGGAcMGTTTCATGGMTCAGTCcGGGAGGG~CcGTTTTTTT~CAT 300

* S H W K G G F K K R Q G W * S Q S L V S R H A V S K K E S R H R N L V S N E R I N R g G G K Q T H N G R K F S V R S A I L A T P S G E R T M T S E Q M V Y D C~GGCATAG~MTTTGGTGTCCMTGAGAGMTcM~GA~GTGGTGG~GC~CTMT~TGGACGG~TTTTCTG~cGGTcTGC~TTTTGGcTACTccATcTGGAGMCGGAcGA~GACATCG~cAGATGGTcTATGATG 450

K H I V D I Q V L F P V V L R Q A A L V K R Q L R S T N E L E V K P D I P I P G N L G L L S E A Y D R C G E V C A E Y A K TGGTTTTGAGGCAGGCAGcCTTGGTGM~GGCMCT~GATCTACcMTGAGT~GMGTG~CcGGA~CC~TTcCGGGG~T~TGGGCTTGTTGAGTG~GCATATGATA~GTGTGGT~TGTG~GAG~TGC~'aA~ 600

Y T F N L G T M L CGTTT~cT~G~T~GCTTCTTCMTCTATTCATTCGTTTACC~TTATTTGGT~GC~TMTTATG~TA~TGTTCATGT~TTGATG~c#'~aATGTTT~TCGGTGA~TTTGACTTTGAT~GGMC~TGC~ 750

D M T P E R R R A I W A I Y

ATGACTCCCGAGA~GMGGGC~TCTGGGCM~TGGT~GGTTCCGCCAGTTT~T~AGTTACGCGCAC~cACA~TGATT~TCGGGGGACGAGAAAAATAG~TGAGcTTT~GTTTTGAGGGGTCA~TGT~TAGG 900

T~TCC~GCTT~C~GcTTGAGATGTTTATTGTCA~TcATGcTC~CT~c~AAAACAcCTGA~a~AG~TTGAT~CTATTTACA~cT~TTATTTTcAGTTCTTTG~TGTTCA~TTTTTACcTATGG~CTGGTTTTCG 1050

CGA~GT~CTNA~TTC~TGTT~TA~AA~TCATTCcTCCCTTTTTCTCCACTTC~GCTTT~CTG~GTGTTG~AGGGG~CTCc~TTT~TGATTGCA~T~CG~TCTTGAGGTT~GTTTCTCAT~T 1200

H Q A I A W C R R T D E L V D G P N A S Y I T P A A L D R W E N R L E D V F N G R P F D M L D G A

~TCTGTTTAAACAGT~TG~TGCAG~G~CA~ATG~CTTGTTG~GGCCC~cGCATcATA~T~CCCCGG~GC~T~GATAGGTGGGAA~ATAGGC~TGTTTTC~TGGGCGGCCATTT~CATGcTC~TGGTGCT 1350

R L S D T V S N F P V D I Q

TTGTcC~TAcAGTTTCT~cTTTCC~TT~TTCAGG~Tc~CC~TTC~TGGTCT~TTGTTC~TTTcGGTTTG~GTcACTTTGCTG~GCTT~TM~GCT~CTT~GCC~GcGGA/V~TG~TG~GTTGMT 1500

CTC~GTTCT~TcTCC~TCTGTTTCTCTCGTCc~TACTACACATACTTCA~TCTG~TT~CATTT~T~TCTTTTGGTGTTG~TTGTATGTG~A~TG~cA~TCAT~T~G~CACAT~TT~ATT 1650

V W P F R D M I E G M R M D L R K S R Y K N F D E L Y L Y C Y Y V A G T

TGCTTTCTN~GCGT~TTGTCT~cCTTcC~TATATGTT~CAGCCATT~G~A~T~TTG~GG~TGCG~TGGAC~GA~Tc~GATAcAAA~ACTTC~C~C~CC~TTG~ATTATGTTGCTGG~CG 1800

V G L M S V P I M G I A P E S K A T T E S V Y N A A L A L G I A N Q L T N I L R D V G E D GTTGGGTT~TGA~TGTTCC~TTATGGGTATCGCCCcTG~TC~AAGGC~C~CAGAG~CG~T~TGCTGCTTTGGCTCTGGGGATCGcAAATC~TT~CT~CA~CTCAGAGATGTTG~G~GAGT~G~C~GCT~T 1950

G~TTTACGcAcAT~TTTTTTTTGcT~TATT~cA~TC~AAATA~GGAAAATGAGcTCTTCGGTTATCcGGTT~TATTTTTTTTATGTC~CAT~T~TMATG~T~GTATGAGTCGTTCTGGG~T~TTGCAG~CT 2100

c~T~AGCCGGTGTTGTG~TCcTGcTGTTTTGAGAGCTT~b~AGCTCATTAGT~GTCGT~GAGACG~G~TTcTTCATTGTGGc~TCTTTATTcCAcCTT~GTTGTGA~TTTTCATTATTGGTACATTT~GC~aACACCT 2250

~C~TTTATG~GGTGCCTTTTG~AA~TCAC~TACCTGTC~GTCGGCGTTT~TCACATTTCTTTGACA~TTG~TTTG~CATGA~TCAGCTC~GACA~TGACGAGCCATGATC~TTTCTTTCCTTTATTCTTTCTTT 2400

K A R R G R V Y L P Q D E L A Q A G L S D E D I F A G R V T D K W

G~GGTGCC~G~TT~AGGCTTCCGTTGTT~T~TA~TTGCTTTCcCTGCAGTGCCAG~G~G~G~TC~CTTGCCTC~GAT~TTAGCACAGGCAGGTCTAT~C~T~GA~TATTTGCTGG~GGGTGACC~T~TG 2550

Q WQ R I F M K K Q I H R A R K F F D,E A E K G V T E L S S A S R F P

GAG~TCTT~T~G~CA~A~CA~GGGC~G~GTTCTTTGATG~G~GAG~AGGCGT~CAG~TTG~CTCAGC~G~GATT~CCTGT~G~ATTCGT/~AACTCTT~TTT~TGA~u~T~TTCTTTTTTcGCGT~T 2700

NGATG~TATGGTTGcCTGTGTTGATG~TTTC~GGTCGATG~GTTGAGAC~GGGTTTTT~GTTTT~CG~TTTTAcGGGGTGCCATGTTATCTGC~C~T~TCTTAGGTAGTTG~cGG~GGG~G~TT~CTCA 2850

TGTTCAC~C~cC~Cc~GAAATG~CCTCGcA~GCTCG~T~N~TATTTGCT~GGCATGAcATTGNCG~NAT~A~'ATGTCT~GAT~TGGAAAAATC~TC~TT~ATC~M~GA~cATN~ATcT~G~ 3000

GCAC~cCGTGTTGTAAATGAGAAATTC~G~TcA~TCTT~GTTTTCTCTG~C~CC~C~CC~GG~cCT~TTGA~CTTGTCGTTCTCAG~TTTGCAcT~CATT~GTCGTGAGG~CCTGAAATGGCTTGG~ 3150

TG~TTCTGGA~T~CAAAAC~AT~T~G~GA~TC~T~C~CATTT~ACCC~C~TACCCTGG~TTG~TAc~C~c~TC~TG~CTT~T~TTCTCT~AcCTTTGTcTAC~TT~T 3300

L L R H E A S D V V W A S L V L Y R K I L D E I E A N D Y N N F T K R A Y V S K S K Q V D C I

GG~AAAAT~CCTCACTCGT~CTCGGTGTTTCCAGG~TGGGCATC~TGGTCTTG~CCGCAAAA~C~TGAG~TG~GCC~TGACT~C~CTTCAC~G~CA~TGT~GC~AGC~GTTGATTGCATT 3450

A H L S P K S S C P L A K T T Y C I C K I S C A S Y K T A S L Q R

ACC~TTGcATATGCAAAAT~TCTTGTGCCTcCNCAAAACTGCCTCTCTTCAAAGAT~GcATGAAAT~GA~TA~TA~GC~TG~cAT~GAAAA/~G~G~GAAATGTTGTTGTATTGANT~ 3600

TG~CAT~T~GGTTG~GT~TTc~TAT~TCTCTTG~GTTGTT~T~CTTCACTT~TCTcM~TCcT~GA~G~cTTTcCGT

Fig. 2. DNA sequence of Clone F and GTOM5. The sequence of GTOM5 and clone F corresponding to that of the cDNA clone pTOM5 is shown. The first and last 10 bases of each intron are overlined. Positions of amino acid changes in clone F are shown in bold face above the GTOM5-derived coding region. Positions of extra introns in clone F are marked with asterisks. Sequences corresponding to PCR primers are underlined.

404

the pTOM5 antisense fruit [1] encodes the same gene isolated here. Alterations in the structure or expression of the GTOM5 gene leading to re- duced expression would then account for the yel- low fruit phenotype. Further work is in progress to characterise the GTOM5 gene and its expres- sion in the r mutant.

Acknowledgements

Part of this work was supported by a SERC Col- laborative award to Professor D. Grierson. P.M. was a recipient of a training grant in Biomolecular Engineering from the EEC.

Fig. 3. Expression of GTOM5 and clone F in various tomato tissues and during normal and ethylene (C2Ho)-induced rip- ening. Autoradiograph of RNA dot blots of 1, 0.5 and 0.1 #g samples of poly(A) + RNA hybridised with CL99 (clone F- specific) and CL100 (GTOM5-specific). DNA controls (bot- tom panel) represent 0.25, 0.125 and 0.025 #g ofpTOM5 and clone F DNA.

genes oligonucleotides specific for GTOM5 (CLIO0) and clone F (CL99) were used in RNA dot blot hybridisations. RNA was isolated from a series of ripening tomatoes and dot hybridisa- tions were carried out in duplicate to the two oligonucleotides (Fig. 3). Expression of the gene represented by clone F could not be detected dur- ing normal ripening or ethylene promoted ripen- ing; nor was expression observed in root, leaf, stem or wounded leaf RNA. pTOM5, on the other hand, was expressed during ripening as shown previously [6]. This together with the structural analysis leads us to conclude that the gene rep- resented by clone F is not expressed and may represent a pseudogene.

These experiments have confirmed the pres- ence of two loci homologous to pTOM5 predicted from RFLP analysis [5]. The cloned fragments account for both Eco RI fragments seen in the Southern blots (6.5 kb: clone F; 8.5 kb GTOM5). It is interesting to speculate that the yellow flesh locus (r) on chromosome 3 which gives rise to yellow tomato fruit [3] similar in appearance to

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