Volume 14 Number 13 1986 Nucleic Acids Research Floral tissue of Petunia hybrida ( V 30 ) expresses only one member of the chakone synthase multigene family

Twenty Independent, petal-specific ctialcone synthase (CHS) cDNA clones have been Isolated from Petunia hybrida variety Violet 30 (V30). Sequence analysis shows that the largest of these clones contains the entire coding sequence. Using this clone In Southern blot analysis reveals the presence of multiple CHS gene copies In the genome of Petunia hybrida V30. Hybridization and sequence analysis of the CHS cONA clones shows that they are all copied from a single mRNA species. This indicates the presence of only one transcriptk>nal1y active CHS gene in petals. Finally we report the identification, cloning and partial characterization of this gene. INTRODUCTION Flavonolds represent a class of plant secondary metabolites, which play a central role In flower pigmentation and are thought to be involved in defence against environmental stress. The key-enzyme of flavonold biosynthesis is chalcone-synthase (CHS). This enzyme condenses 3 molecules of malonylCoA and 1 molecule of 4 coumaroyl-Co A to yield naringenln chalcone (1). Isomerizatlon and further substitution of this central intermediate leads to the synthesis of flavonols, flavonones, isoflavonoids and arrthocyanlns. The enzymes of the fiavonoid gtycoside pathway share a number of interesting features: (I) their synthesis is coordlnatety regulated (2,3), (II) In most plant species the synthesis is tissue-specific (4), (III) environmental stress (UV light and phytopathogens) leads to a general Induction of the pathway In otherwise non-expressing tissues (5-7). The characterization of the regulation phenomena mentioned above at the molecular level has just begun. cDNA and chromosomal CHS-clones have been Isolated from parsley (8) and P. hybrida (Roter traum) (9). Both systems, however, are genetically Ill-defined, complicating analysis of the genomic constitution. We therefore chose to clone CHS genes from a genetically well-defined variety of P.hybrida In this paper we describe the isolation and sequence analysis of a full size CHS cDNA clone from Petunia hybrida V30. Using this clone we have established that CHS genes comprise a muttigene family of which only one member is actively transcribed In floral tissue. MATERIA1 S AND METHODS Material Plant variety, plasmkjs, phages and bacterial strains are Bsted In Table I. C IRLPreu Limited, Oxford, England. 5229 at Prceton U niersity on July 3, 2015 http://narrdjournals.org/ D ow nladed from Nucleic Acids Research In vtlro translation of mRNA In vitro translation of poly A + mRNA from Petunia hybrida in rabbit reticulocyte lysates was carried out as described previously (17). Analysis of CHS-proteln bv Westem-blottinQ. Protein extracts from various tissues were prepared as described (17). Protein concentrations were determined using the Biorad Protein assay with bovine serum albumin as a standard. Analysis of CHSproteln by Westemblottlng was carried out as described previously (17), using an antiserum raised against parsley CHS and 1 25 l-proteln-A. Construction of a petal-soecific cDNA-librarv from P.hybrida (V30). PolyA +RNA isolated from petals of P. hybrida (V30) (17) was converted to ds-cDNA (18). After homopotymeric tailing with dC residues, the cDNA was annealed to Pst I cut, dG-talled pUC 9 DNA (PLBlochemlcals) and transformed to E. coli RR1 (19). Transformants were plated and screened by hybridization as described (20). Isolation of DNA Nuclear DNA from P. hybrida (V30) was Isolated from the top four leafs of 3 months old plants or from 3 weeks old seedlings by a published procedure (21). Large and small scale DNA isolations of X-phage were conducted as described (19). Plasmld DNA was isolated by the alkaline lysis method (19) Southern-blots and preparation ol probes Ten micrograms of P. hybrida DNA was digested to completion with EcoRI and electrophoresed on 0.8% agarose gels. The DNA was transferred to Gene-screen plus membranes and hybridized as recommended by the supplier (New England Nuclear). Copy numbers were estimated by running samples of cloned DNA in parallel and comparing autoradlographlc intensities of hybridized DNA. Copynumber calculations were based on a haploid genome size of 1.5 pg for P. hybrida. Southemblotting and hybridization analysis of cloned DNA fragments was carried out using Hybond-N membranes as recommended by the supplier (Amersham). Washing conditions of the blots are specified in the figure legends. P-labelled hybridization probes were prepared by nick-translation of restrlctionfragments purified from (sub)clones In M13 mp vectors. We preferred subclonlng in M13 because of the absence of cross-hybridization of this vector to P. hybrida DNA (unpublished data). DNA-seouencB analysis For a complete sequence analysis of cDNA clones, series of overlapping subclones in M13mp phage were constructed using nuclease Bal 31 (22). These were sequenced using the dldeoxychairrtermlnatlon method (23) with ^S-ATP. For sequence analysis of 3' and 5' ends, cDNA inserts were recloned In both orientations in M13mp10 and mp11. The synthetic oligo-nucleotide 5' dTTGAGACTGTTGTCCTCCA 3* was a gift of Dr. J.H. van Boom, State University of Leiden. Isolation and analysis of genomte CHS-clones P. hybrida (V30) nuclear DNA was digested to completion with EcoRI and ligated into XgtWES arms. Ugated DNA was packaged tn vitro using the Hohn and Murray procedure (19). A second library of P. hybrida (V30) DNA, partially digested with Sau3AI, In the phage vector EMBL3 was generated according to Dean el. al. (24). Both libraries, containing 5x10 (XgtWES) and 2x10 (XEMBL3) plaques 5230 at Prceton U niersity on July 3, 2015 http://narrdjournals.org/ D ow nladed from Nucleic Acids Research Figure 1: Analysis of CHS protein (A) and CHS translatable mRNA (B) levels in various tissues of P. hybiida (V30). A: Samples of 50 ug of protein, isolated from different tissues were analyzed for CHS content by SDS-gel electrophoresis followed by protein blotting as described (17). B: polyA+mRNA was translated In a rabbit reticulocyte lysate and labelled CHS immunoprecipatated with a parsley antiCHS serum. The product was electrophoresed on an SDS-acrylamlde gel and visualized by autoradiography as described previously (17). respectively, were screened by hybridization as described (19). Restrictlon-endonuclease maps of XgtWES inserts were generated by reckoning of the inserts In pUC18 and subsequent analysis of single and multiple restriction-enzyme digests. Restriction endonuclease maps of Inserts in XEMBL3 were generated by analysis of partial restriction-enzyme digests specifically labelled at the cosR site (25) RESULTS CHS expression: cholcs of tissue Previous studies from other laboratories have indicated that the chalcone synthase gene (CHS) occurs In at least two copies In the genome of several higher plants (9,26). The plant systems used In these studies are however genetically Hi-defined and heterozygosity of the CHS locus cannot be completely excluded. To circumvent these problems we chose to study an Inbred variety of Petunia hybrkia called Vlolet-30 (V30) which Is homozygous for all 91 markers tested Including 28 flavonoid-specific loci. Southern Wots of V30 nuclear DNA restricted with EcoRI and probed with an incomplete CHScDNA from the Petunia variety Roter Traum (9) reveals at least 8 hybridizing fragments ranging In size from 1.5 to 25 kb. (cf. Fig. 4). This suggests that CHS genes occur in multiple copies in the genome of Petunia hybrkia and raises the question which of these genes are actively transcribed. 5231 at Prceton U niersity on July 3, 2015 http://narrdjournals.org/ D ow nladed from Nucleic Acids Research