Plant Molecular Biology 22: 893-897, 1993. © 1993 Kluwer Academic Pubfishers. Printed in Belgium.

Update section

Short communication

893

The petunia homologue of the Antirrhinum majus candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene-forming enzyme

David Weiss 1, Arnold H. van der Luit, Johan T.M. Kroon, Joseph N.M. Mol and Jan M. Kooter* Department of Genetics, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, Netherlands (*author for correspondence)," 1present address." Department of Horticulture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

Received 29 January 1993; accepted in revised form 7 April 1993

Key words." Petunia hybrida, anthocyanin, flower pigmentation, flavonoid pathway, A2, candi

Abstract

The synthesis of anthocyanins in higher plants involves many enzymatic steps. Here we describe the isolation and characterization of a cDNA, ant17, which encodes a protein that has 73 ~o amino acid sequence identity with the candi gene product of Antirrhinum majus and 48 ~o with that of the maize a2 gene. This protein may therefore be involved in the synthesis of anthocyanins in the steps after the ac- tion of dihydroflavonol 4-reductase. This is consistent with the absence of ant17 expression in the reg- ulatory anthocyanin mutants of petunia an1, an2 and an11. Furthermore, ant17 is predominantly ex- pressed in corollas and anthers and is induced by gibberellic acid.

The most abundant pigments in flowers are an- thocyanins which are synthesized in a branch of the general phenylpropanoid pathway, the fla- vonoid pathway [9, 10]. Several enzymes of this pathway have been identified, including chalcone synthase (CHS), chalcone flavanone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihy- droflavonol 4-reductase (DFR) and UDP-glu- cose flavonoid 3-oxy-glucosyl transferase (UF3GT) for which the genes have been isolated from various plant species (reviewed in [6]).

The expression of the flavonoid genes in flow- ers is coordinately activated very early during co- rolla growth [2, 4, 12, 14]. However, the expres- sion of the genes that belong to the first and second half of the flavonoid pathway is controlled by different transcriptional regulators [ 1, 4, 8, 11, 12]. It is not well understood how all flavonoid genes are switched on simultaneously but it seems likely that the different regulators are produced upon a common signal. In petunia, evidence has been obtained that gibberellic acid (GA) is in-

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X70786.

894

volved [16]. Exogenous GA 3 transcriptionally activates the expression offlavonoid genes of both the first and second half of the pathway [ 17, 18] (D. Weiss et al., unpublished). GA may therefore act very early in the signal transduction pathway. In an attempt to obtain cDNAs of early GA- induced proteins, we differentially hybridized a V26 petunia corolla cDNA library with [32p]_ labelled first-strand cDNA probes. For the minus probe, poly(A) + RNA was used from young co- rollas that were grown for 24 h in sucrose. For the plus probe, RNA was used from corollas that were grown in sucrose medium for 24 h followed by a 4 h incubation in the presence of GA 3 [ 17]. After several rounds of purification and differen- tial hybridization one differential clone was ob- tained, which we called ant17. Using this clone as a probe on a corolla cDNA library we obtained longer ant17 cDNAs. Here we describe their characterization.

An antl 7 probe did not hybridize to any of the known flavonoid genes of petunia including phenylalanine ammonia-lyase (pal), chs, chi, dfr, f3h, and a rhamnosylation gene (rt), at high strin- gency conditions (data not shown). Anti7 could therefore be a novel cloned gene. The nucleotide sequence of the longest cDNA we had isolated from a corolla cDNA library was therefore de- termined. Translation of this sequence revealed several stop codons in all three reading frames. However, a shorter cDNA of 1533 bp did not contain the region with the stop codons and we could obtain an open reading frame (ORF) of 1290 nt corresponding to a polypeptide of 430 amino acids (Fig. 1). It turned out that the long- est antl7 cDNA contained a DNA segment downstream of nucleotide position 554 with the characteristics of an intron. Namely, the ORF of this cDNA could be restored by deleting the extra DNA segment using the consensus sequences of

CGTTGCTGTCGAGA~GC~GAG~GGAAATATTCATACAGAGATGGTG~T~AGTAGTTAC~CTCCTTC~GAGTTGA~GCTTGGCTAbJ~AGT M V N A V V T T P S R V E S L A K S

GG~TC~GGCCATCCCT~GGAGTATGTGAG~CAC~G~GAGTTG~TGG~TCGGAAACATCTTCGAGG~GAG~GAAAGATG~GGGCCTC~ 198 G I Q A I P K E Y V R P Q E E L N G I G N I F E E E K K D E G P Q

GTACC~C~TTGATTTGAAAGAAATTGACTCCGAGGAC~AGATTCGCGAGAAATGCCACCAGTTG~GAAA~AGCCATGG~TGGGGTGTCATG 297 V P T I D L K E I D S E D K E I R E K C H Q L K K A A M E W G V M

CACCTTGTG~TCAT~CATATCCGATGA~T~TC~TCGTGTC~GGTTGCTGGAGAGACCTTCTTTGATC~CCTGTTG~GAAAAGGAG~GTAT 3~ R L V N H G I S D E L I N R V K V A G E T F F D Q P V E E K E K Y

GCT~TGACC~GCC~TGGC~TGTCC~GGCTACGGCAGC~GCTAGCAAATAGTGCTTGTGGTCAGCTTGAGTGGGAGGATTATTTCTTCCATTGT 495 A N D Q A N G N V Q G Y G S K L A N S A C G Q L E W E D Y F F H C

• A AT A CCCTA GCTTTCCCTGAAGAC~GCGCGACTTGTCCATCT~CTAAAAATCCTACTGACTACACTCCAGC~C~GTG~TATGCC~GC G C GGG 594 A F P E D K R D L S I W P K N P T D Y T P A T S E Y A K Q I R A L

GC~CAAAGATTTTGACAGT~TTT~TATTGGGCTGGGGCTGG~G~GG~GACTAGAG~GG~GTTGGAGGCATGGAGGATCTGCTGCTTCAAATG 693 A T K I L T V L S I G L G L E E G R L E K E V G G M E D L L L Q M

~GATT~CTACTATCCC~GTGCCCCC~CCAG~CTAGCACTTGGCGTCG~GCTCATACAGATGTCAGCGCACTGACTTTCATCCTCCAC~TATG 792 K I N Y Y P K C P Q P E L A L G V E A H T D V S A L T F I L R N M

GTGCCCGGCTT~CTCTTCTATG~CAGT~T~CTGCT~GTGTGTGCCT~TTCTATCACATTGCA~TAGGGGACACCATTGAAATCCTA 891 V P G L Q L F Y E G Q W V T A K C V P N S I I M H I G D T I E I L

AGC~TGGT~GTAC~GAGCATCCTTCATAGAGG~TTGTG~TAAAGAGAAAGT~TTCTCATGGGCCATTTTCTGCGAGCCACCT~GGAG~G 9~ S N G K Y K S I L H R G V V N K E K V R F S W A I F C E P P K E K

ATCATCCTT~GCCCCTACCTGAGACTGTCACTGAGGCTGAGCCACCTCGATTCCCACCTCGCACCTTTGCACAGCATATGGCACAC~GCTCTTCAGG I089 I I L K P L P E T V T E A E P P R F P P R T F A Q H M A H K L F R

~GGATGAC~GGATG~CGCTGTTG~CACAAAGTCTT~TGAGGATG~TGGATACTGCTGCTG~CAT~GGTCCTC~G~GGAT~TCAGGAT 1188 K D D K D A A V E H K V F N E D E L D T A A E H K V L K K D N Q D

GCTGTTGCTGAG~TAAAGACATC~GGAGGATG~CAGTGTGGCCCTGCTGAGCAC~GATATC~GGAGGAT~ACAGGGTGCCGCTGCTGAG~C 1287 A V A E N K D I K E D E Q C G P A E H K D I K E D G Q G A A A E N

AAAGTCTTC~GGAG~T~TCAGGATGTTGCTGCTG~G~TCTAAATAGTTTCTCTTGCTGTCATAGTTTGATGGCTT~TATGAGACGTTTTTCAT 1386 K V F K E N N Q D V A A E E S K *

GTTTTTGATGATGTGTTGG~TCACGAGTGATCCCCG~TCTGTGTACTCTGC~TGTTTTCATTTTAGTAAAGCATACCCATTAAATTTC~GAAATA 1485

TTATCCTGCC (A) n 15~

Fig. I. Nucleotide sequence ~ d deduced ~ i n o acid sequence of antl 7 cDNA. The m o w head indicates the position of a pu- tative intron ~und in the longest cDNA that was an~ysed.

splice donor and acceptor sites (Fig. 1). Appar- ently the ant17 gene contains at least one intron.

The deduced amino acid sequence of ant17 re- vealed a 48~o identity with the A2 sequence of maize [13] and 73~o with the CANDI sequence of Antirrhinum majus (C. Martin, unpublished). An alignment of these three protein sequences is shown in Fig. 2A. ANT17 is 57 amino acids longer than CANDI and 34 amino acids longer than A2. Because of the significant similarities

895

between these three proteins it is likely that they have the same enzymatic activity. A2 and CANDI are considered to be involved in the conversion of leucoanthocyanidin, the product of the D F R re- action, to anthocyanidin, the first coloured com- pound of the pathway [6, 10, 12, 13]. These en- zymatic steps are not well characterized biochemically but involve a dehydration and an oxidation step [ 10, 13]. A2, CANDI, and ANT17 may encode the hydroxylase [ 12, 13]. This would

A

A2 MESSPLLQLPAARvEALSLSGLSAIPPEYvP`PADERAGLGDAFDLARTHANDHTAPRIFVVDISPFLD•S•QQQQRDECVEAVRA-AAADWGvMHIA•HG 99

i I III I 11 Ill IIIII l I I I i i I 1 1 I II IIILL II Anti7 M-VNAVVTTPS-RVESLAKSGIQAIPKEYVRPQEELNGIGNIFE .... EEKKDEGPQVFTIDLKEI--DSEDKEIREKC-HQLKK-AAMEWGVMHLVNHG 90

I II III I I]III IIIII II III II I I~I III I III I LI[ I I II I I IFI ~IIIII III Cand/ M--AI:)AIAPPS-RVELLSKSGIQSIPKEYIRPEEELKSIGDV~FA .... EEKNNDGPQLFVIDLAAI--NSEDEEVP.KKC-HDELKK/L~d~U3WGVIMHLINHG 90

A2 IPAELMDRLRAAGTAFFALP~QDKEAYANDPAAGRLQGYGSRLATNTCGQREWEDYLFHLVHPDGLADHALWPAYPPDYIAATRDFGRRTRDLA~TLLAI 199

I II I II IN II II fill I I l~IFi II Ill ~IIII II ] I II I II II I IN I Anti7 ISDELINRVKVAGETFFDQPvEEKEKYANDQANGNVQGYGSKLANSACGQLEWEDYFFHCAFPEDKRDLSIWPKNPTDYTPAT•EYAKQIRALATKILTV 190

Ill lllIlll III ll~llll [IIII IIL]III~IIII I IIIIIIIIIIII III III lllIl I II IIIIIIIII I I IIII I Caad/ VPDELTNRv~CJAGQEFFDLPVEEKEKH/LNDQASGN-VQGYGSKLANNASGQLEWEDYFFHCVFPEEKP`DMSIWPKDPADYIPATSEYAKQLRSLTTKILSV 190

A2 LSMGLLGTDRGDALE~LLTTTTTRT~L~DDDLLLQLKINYYPRCPQFELAVGVEAHTDV$A~$FILHNGVPGLQVLHGARWVIAR/~EPGTIIVHVGDALEI 299

II III I LII IIIII llllll lllIIIl IIIII~IIIII IIIII IIIII II I I II I II II A ~ I 7 LSIGL-GLEEGR-LEKEV ....... GGMEDLLLQMKINYYPKCPQPELALGvEA}~TDV~2LLT~ILHNMVPGLQLFYEGQWVTA/<CVPN$IItv/-~IGDTIEI 281

II II I I I I I I I I I II III I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I J l l ~ l l l I I I l I F I I I I l l l l l I l l l l l Cand/ LSLGL-GLEPER-LEKEV ....... GGIEDLILQb~<INYYPKCPQpDLALGVEAHTDVSALTFILHNMVPGLQLFYKGKWVTAKCVPNSIIMHVGDTIEI 281

A2 LSNGRYISVLHRGLVNREAVP`ISWVVFCEPPPDS~LLHPLPELVTEGHPARFIPRTFKQHLDRKLFKKKQQHKA---KAEEEDGGNGDHHRHEPPPQTN~ 396

IIII ~ I IIII II I II II IIIII I llll III I II II]I II Ill I F l II I I Anti7 LSNGKYKSILHRGVVNKEKVI~FSWAIFCEPPKEKIILKPLPETVTEAEPPRFPPRTFAQHMAEKLFRKDDKDAAVEHKvFNEDELDTAAE-HKVLKKDNQ 380

IIIIIIIIIIIII IIIIIII ill I II I I IIFIIII I IIII I llIll I 1111 I 1 II Cartdi LSNGKYKSILHRGL~EI~VP`ISWA~q~VE~HKDKVILKPL~EVV$E2~EPAMFTPRTFAEHLRSKLFREGEGE--VDKDG-GG~v~zIDRKQD-EKVVE 373

Ant17 DAVAENKDIKEDEQCGPAEHKDIKEDGQGAAAENKVFKENNQDVAAEESK 430

8

F3H M ..... TT ................................ F ............... AKE ........................................ 7

I II I II Anti7 MVNAVvTTPSRVESLAKSGIQAIPKEYVRPQEELNGIGNIFEEEKKDEGPQVPTIDLKEIDSEDKEIREKCHQLKKAAMEWGVMHLVNHGISDELINRVK l(]0

I I II I I I I il IIIIII 1 I EFE M ................... ENFP---IISLDKVNGV ...... ER ...... AATMEM ......... IKDACE ....... NWGFFELVNHGIPREVMDTVE 50

F3H ..... FFALPPEEKLRF--DMSGGKKGGF ...... IVSSHL ....... QGEVVQDWILEIVTYFSYPTRAILDYSRWPDKPEGWIAVTQKYSEKLMELACKL 87

li I III i r I I I I I II I I I II [ Anti7 VAGETFFDQPVEEKEKYANDQANGNVQGYGSKLANSACGQLEWEDYFFHCAFPEDKRDL ............. SIWPKNPTDYTPATSEYAKQIRALATKI 187

I I I II II I k ~ I II II EFE KMTKGHYKKCME--QILFKELVASK/LLEGVQILEVT ..... DMDWESTFFLKHLP--ISNI ............. SEVPDLDEEYP, EVMP, DFAKRLEKLAI~EL 128

F3H LDVL $ E ~MGLEKEALTKACVDMDQ - - - KVg-VNF yp KCP Ep D LTLGLKRHTDPG T I T LLLQD Q -VGGLQATKDNGKTW I TVQPVEGAFVVNLG DHGHFL SN 183

I III III b I I I II111 1 i II III I I l ILP I I i 11 Ill Antl7 LTVLSIGLGLEEGRLEKEVGGMEDLLLQMKINYYPKCPQPELALGVEAHTDvSALTFILHNM-VPGLQLFYE--GQWVTAKCVPNSIIMHIGDTIEILSN 284

E I I111 1 1 q 1 I II I I 1111 I I]I~ Ill I~ IE I I EFE LD LLCENLGLEKGY LKNAFYGSKGPNFGTKVSN Lp pCp Kp DL I KGLRAHTDAGG I I LLF QD'v-KVS GLQLLKD - - GQWI DVI~P MP, H S I VVNLGD Q LEVI TN 226

F3H GP, FKN/LDHQAVVqqSN S SRLS IATFQNPAP EAIVY - P LK I REGEK S I MD - EP I T FAE ..... MY BRKMS KD LE LAIRLKKQAKEQQLQA ............ E 264

I L I 111 U I l I I I F il i I III I PI I I I Antl7 GKYKSILHP`GVVNKEKVRFSWA~FC~PPKEKIILKPLPETVTEAEPPR~PPRTFAQHMAHKLFRKD-DKDAAVEH--KVFNEDELDTAAEHKVLKKDNQD 381

IIIII II I I I 1 1 I I I I I 1 I I II I I I~ EFE GKYKSVMHRVIAQKD~S LASFYNP GSDAV I Y - PAPALVEKEEAEK ...... NKQVYPK-F ............... VF-DDYMKLYAG- - - LKFQAKE 299

F3H VAAEKAKL ......... ESKPIEEILA

tl 1 1 i I Anti7 AVAENKD I KEDEQC GPAEHKD I KEDGQGAAAENKVFKENNQDVA2LEE SK

I I I I i I I EFE PRFE--AMK ....... AMETDVKMD ................ PIATV

282

430

320

Fig. 2. Alignment of the ANT17 amino acid sequence with that of CANDI from A. majus and A2 from Z. mays [13], and with flavanone 3-hydroxylase (F3H) [3] and ethylene-forming enzyme (EFE) [ 15] from petunia (B).

896

be consistent with the homology between ANT 17 and petunia flavanone 3-hydroxylase [3] and ethylene-forming enzyme [15] of 34~o and 31~o amino acid sequence identity, respectively (Fig. 2B).

To obtain additional evidence that ant17 is a structural flavonoid gene we examined the ex- pression pattern. The GA inducibility of the ant17 gene was first determined. Detached young co- rollas were first incubated in sucrose medium for 24 h to down-regulate flavonoid gene expression and then in sucrose medium containing 10/~M GA 3 for different time periods [ 17]. RNA was extracted and analysed on RNA blots. Hybrid- ization with an ant17 probe revealed a transcript of about 1.6 kb that was already present in stage 3 flower buds (To in Fig. 3A). Three to four hours after the transfer to GA-containing medium the transcript began to accumulate. The kinetics of ant17 induction by GA was very similar to that of chs (Fig. 3A) and of other flavonoid genes (not shown). This result was surprising since ant17 was initially identified as a rapidly GA-inducible gene in the differential cDNA screening. We do not have a plausible explanation for this finding but it was noticed that in the in vitro induction experiments, ant17 was sometimes more rapidly induced than other flavonoid genes for unclear reasons (data not shown).

Fig. 3B shows that ant17 is highly expressed in corollas (C) and, at a lower level, in anthers (A). Expression in sepals (SP), stem (ST), and leaves (L) was not detectable. In corollas, the expression is developmentally controlled similar to that of chs (Fig. 3C). Around stage 3 of corolla matura- tion, the transcripts rapidly accumulate. To ob- tain evidence that the ANT17 acts in the second half of the pathway, as suggested by the homol- ogy to A2 and CANDI, ant17 expression was analysed in the petunia mutants an1, an2 and an11. These regulatory mutants fail to express dfr and other late flavonoid genes [5, 7, 8] (Quat- trocchio et al., unpublished). Fig. 3D shows in- deed that also the ant17 gene is not expressed in these mutants. Taken together, the results suggest that the petunia ANT17 is the homologue of the Zea mays A2 and the A. majus CANDI, acting in

Fig. 3. Expression characteristics ofantl7. A. Gibberellic acid (GA3) inducibility. Detached corolla limbs were grown in vitro for 24 h in sucrose medium and then transferred to sucrose medium containing 10 #M GA 3. After different time periods (0, 3, 5 or 7 h) RNA was extracted and 10 pg total RNA was analyzed on northern blots [ 17 ]. The ant17 transcript is 1.6 kb in size, the chs transcript 1.4 kb. B. Expression in different tissues; C, corollas; A, anthers; SP, sepals; ST, stems; L, leaves. C. Expression during corolla development, stages 1-7. Expression in the corollas of the regulatory anthocyanin mu- tants of petunia, an1, an2 and anl 1. RNA from the wild-type petunia cultivar VR (WT) served as a positive control. All lanes contained 10 #g of total RNA.

the second half of the anthocyanin pathway after DFR. Additional evidence will be obtained by the inhibition of ant17 expression with antisense gene constructs.

Acknowledgements

We thank Dr Cathie Martin for providing the sequence of the candi gene and for helpful dis- cussions and Dr Ronald Koes for valuable com- ments. This work was supported by the Univer- sitair Stimulerings Fonds of the Vrije Universiteit, Amsterdam. J.M.K. is supported by a research fellowship from the Royal Netherlands Academy of Sciences.

References

1. Almeida J, Carpenter R, Robbins TP, Martin C, Coen ES: Genetic interactions underlying flower patterns in Antirrhinum majus. Genes Devel 3:1758-1767 (1989).

2. Beld M, Martin C, Huits H, Stuitje AR, Gerats A: Fla- vonoid synthesis in Petunia hybrida: Partial characteriza- tion of dihydroflavonol 4-reductase genes. Plant Mol Biol 13:491-502 (1989).

3. Britsch L, Ruhnau-Brich B, Forkmann G: Molecular cloning, sequence analysis and in vitro expression of fla- vanone 3fl-hydroxylase from Petunia hybrida. J Biol Chem 267:5380-5387 (1991).

4. Coen ES, Carpenter R, Martin C: Transposable elements generate novel spatial patterns of gene expression in An- tirrhinum rnajus. Cell 47:285-296 (1986).

5. Doodeman M, Gerats AGM, Schram AW, De Vlaming P, Bianchi F: Genetic analysis of instability in Petunia hybrida. 2. Unstable mutations at different loci as the result of transpositions of the genetic element inserted at the Anl locus. Theor Appl Genet 67:357-366 (1984).

6. Dooner HK, Robbins TP, Jorgensen RA: Genetic and developmental control of anthocyanin biosynthesis. Annu Rev Genet 25:173-199 (1991).

7. Gerats AGM, De VlamingP, Doodeman M, A1 B, Schram AW: Genetic control of the conversion of dihy- droflavonols into flavonols and anthocyanins in flowers of Petunia hybrida. Planta 155:364-368 (1982).

8. Gerats AGM, Farcy E, Wallroth SPC, Groot SPC, Schram A: Control of anthocyanin biosynthesis in Petu- nia hybrida by multiple allelic series of the genes An1 and An2. Genetics 106:501-508 (1986).

9. Harborne JB: Function offlavonoids in plants. In: Good- win TW (ed) Chemistry and Biochemistry of Plant Pig- ments, pp. 736-778. Academic Press, London (1976).

897

10. Heller W, Forkmann G: Biosynthesis. In: Harborn JB (ed) The Flavonoids, pp. 399-425. Chapman and Hall, London (1988).

11. Martin C, Carpenter R, Coen ES, and Gerats AGM: The control of floral pigmentation in Antirrhinum rnajus. In: Thomas H, Grierson D (eds) Developmental Mutants in Higher Plants, pp. 19-52. Cambridge University Press, Cambridge (1987).

12. Martin C, Prescott A, Mackay S, Bartlett J, Vrijlandt E: Control of anthocyanin biosynthesis in flowers of Anti- rrhinum majus. Plant J 1:37-49 (1991).

13. Menssen A, Hohmann S, Martin W, Schnable PS, Peter- son PA, Saedler H, Gierl A: The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a Dspm element in the A2 gene. EMBO J 9:3051-3057 (1990).

14. van Tunen AJ, Koes RE, Spelt CE, van der Krol R, Stuitje AR, Mol JNM: Cloning of the two chalcone fla- vanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. EMBO J 7:1257-1263 (1988).

15. Wang H, Woodson WR: Nucleotide sequence ofa cDNA encoding the ethylene-forming enzyme from petunia co- rollas. Plant Physiol 100:535-536 (1992).

16. Weiss D, Halevy AH: Stamens and gibberellin in the reg- ulation of corolla pigmentation and growth in Petunia hybrida. Planta 179:89-96 (1989).

17. Weiss D, van Blockland R, Kooter JM, Mol JNM, van Tunen AJ: Gibberellic acid regulates chalcone synthase gene transcription in the corolla of Petunia hybrida. Plant Physiol 98:191-197 (1992).

18. Weiss D, van Tunen AJ, Halevy AH, Mol JNM, Gerats AGM: Stamens and gibberellic acid in the regulation of flavonoid gene expression in the corolla of Petunia hybr- ida. Plant Physiol 94:511-515 (1990).