Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)

Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)
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  Plant Molecular Biology 24: 743-755, 1994. © 1994 Kluwer Academic Publishers Printed in Belgium 743 Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape Vitis vinifera L.) Francesca Sparvoli 1 Cathie Martin 3, Attilio Scienza 2, Giuseppe Gavazzi 1 and Chiara Tonelli 1,, 1Dipartimento di Genetica e di Biologia dei Microorganismi, Universit~ degli Studi di Milano, Via Celoria 26, 20133 Milano, Italia (*author for correspondence), 2 Istituto di Coltivazioni A rboree, Universith degli Studi di Milano, Via Celoria 2, 20133 Milano, Italia, 3Department of Genetics, John Innes Institute, Colney Lane, Norwich NR4 7HU, UK Received 22 September 1993; accepted in revised form 11 January 1994 Key words: anthocyanins, cDNA cloning, flavonoids, gene expression, genomic organization, stilbenes, Vitis vinifera L. Abstract Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydoxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:ftavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Anti- rrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65~o (UFGT) to 90~o (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome, The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed. Introduction Flavonoids are secondary metabolites wide- spread among plants and involved in many plant functions such as UV protection, defence against pathogen attack, legume nodulation and pollen viability [14, 27, 35, 40]. Anthocyanins, a fla- vonoid subclass, are the main pigments in flow- ers and fruits, where they serve as insect and animal attractants respectively, thus playing an The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X75969 CHS), X75963 CHI), X75964 DFR), X75965 F3H), X75966 LDOX), X75967 PAL), StSy X76892 StSy) mad X75968 UFTG).  744 important role in the ecology of pollination and seed dispersal. Flavonoid biosynthesis has been extensively studied in many plants like maize (Zea mays), petunia (Petunia hybrida) and snapdragon (Anti- rrhinurn majus). Mutants with a block in antho- cyanin pigmentation are viable and readily de- tectable. Their study has disclosed the existence of two classes of genes affecting anthocyanin bio- synthesis. One class includes the structural genes of the pathway (Fig. 1), common to different spe- cies [32, 43, 47, 59, 64, 66, 67] and a second class that includes genes that regulate the activity of the biosynthetic genes, conditioning the spatial and temporal accumulation of pigments. This last class includes the C1 and the R gene families in maize encoding products related to the Myb and Myc families of transcription factors, respectively [5, 6, 11, 41, 50, 63] and in snapdragon, the Delila gene the protein product of which shows exten- sive homology to the products of the R gene fam- ily [25]. Flavonoid biosynthesis that appears PhenylNanine citosol vacuol Aurones -.4--.--- Chalcones Flavones -.~----- Flavanones I F3H Flavonols ~ Dihydroflavonols Leucoanthocyanidins +u ox ~ Dehydratase Anthocyanidins UFGT Anthocyanin 3-o-glucosides RT Anthocy~min 3-o-natinosides PAL C4H 4CL Cinamic acid ~ 4-Coumaric acid --.....Ib-4-Coumaroyl-CoA CHS ins / .... ~.-...,u,~ ,NN - Coumarin • &, tilbenes Viniferins Fig. i. Schematic representation of the general phenylpropanoid metabolism and of the branching pathways for flavonoid and stilbene production. The enzymic activities for each step are indicated by capital letters. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; StSy, stilbene synthase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydoxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; UFGT, UDP glucose:flavonoid 3-O-glucosyl transferase; RT, rhamnosyl transferase.  regulated both developmentally as well as in a tissue-specific way, is induced by a variety of en- viromental stimuli including light, fungal elicitors, UV radiation, interactions with microorganisms and wounding [9, 29, 34, 37, 48, 63]. When pars- ley cell cultures are treated with fungal elicitors or UV light, the enzymes of the general phenylpro- panoid metabolism are rapidly induced [16, 39]. Similar results are observed in many legumes after elicitor treatment [8, 54]. In other systems, such as peanut (Arachis hypogaea L.), pine (Pinus sylvestris) and grape (Vitis vinifera L.), StSy in addition to flavonoid enzymes is also induced by UV light and elicitors since stilbenes are phytoal- exins in these species [19, 22, 23, 38]. StSy is active only in some plant species. It is closely related to CHS, the first enzyme committed to flavonoid biosynthesis as both enzymes utilize 4-coumaroyl-CoA and malonyl-CoA as substrate, but StSy determines a different type of ring clo- sure leading to a C14 stilbene compound and the release of 4 molecules of CO2 while CHS releases only 3 CO2 molecules. The presence of anthocyanins in grape berries is important both for the taste of wine and for fermentative processes involved in wine produc- tion. However, knowledge of the genetics of fla- vonoid, particularly of anthocyanin biosynthesis, is still scanty. Most of the studies have been con- ducted on the quantity and type of these com- pounds present in the berries, since the variabil- ity in the class and amount of anthocyanins represents a good chemotaxonomic approach to the identification and classification of different grape varieties and species [44]. More is known about stilbene biosynthesis. In grape, stilbenes (resveratrol and its oligomeric derivatives vin- iferins) have antimicrobial activity against Botry, tis cinerea and Plasmopara viticola, the main pathogens of this species [ 12]. Liswidowati et al. [38] demonstrated that StSy is rapidly and tran- siently synthesized in grape cell cultures after elicitation with B. cinerea. A clone for grape StSy has been isolated [45] and it has recently been shown that StSy, when expressed in transgenic tobacco plants, confers an increased disease re- sistance against B. cinerea [28]. 745 This report deals with the characterization of the structural genes required for anthocyanin and stilbene biosynthesis in grape, cDNA clones cod- ing for PAL, CHS, StSy, CHI, F3H, DFR, LDOX and UFGT were isolated by means of heterologous probes. These clones were then used as probes to describe the genomic organization of each gene, to determine whether the correspond- ing enzyme activity is coded by a single copy gene or by a multigene family. We also report on how these genes are regulated in young seedling in response to light treatment and show that the expression of genes for ftavonoid synthesis, but not PAL and StSy, is coordinately enhanced by light. Materials and methods Plant material and growth conditions Material was derived from manual self-pollination of individual plants of the following V. vinifera L. varieties: Lambrusco a Foglia Frastagliata (Lam- brusco f.f.), Marzemino, Teroldego, Kolor (Pinot Nero x Tenturier) and Gamay Tenturier. All genotypes were kindly provided by Dr Luigi De Micheli of the Istituto Agrario of San Michele all'Adige, Trento, Italy. Seeds were stored at 4 °C on stratification. Germination was achieved by washing the seeds from sand, followed by sterilization with a solu- tion of sodium hypochloride (770) for 20 min, sclerification with a diluted solution of nitric acid (1.25 7o) and incubation overnight in water with gentle stirring, at room temperature. Seeds were then washed, plated in a plastic box on 3 mm Whatman paper and left at 25 °C in the dark. After 12-14 days, well developed seedlings (0.5-2 cm) are obtained. Light treatment consisted of exposure to continuous white light from fluores- cent lamps F36T12/CW/HO (fluence rate 66 W/m2). Anthocyanin determination Anthocyanins were extracted using 1 ~o HC1 in ethanol; the extracts were centrifuged and the  746 supernatants used to determine the absorbance at 530 nm. Anthocyanin concentration was ex- pressed as absorbance value at 530 nm per gram of fresh weight. Mean values represent 4-6 inde- pendent replicates. Construction of cDNA library Total RNA was extracted from 14 day old seed- lings (Lambrusco f.f.) grown in continous light for 48 h. Poly(A)+ RNA was prepared using the 'poly(A) + quick mRNA purification kit' (Strat- agene), cDNA was prepared using 5 #g of poly(A + ) RNA according to the instructions of the Amersham cDNA synthesis and cloning kits. The first strand was synthesized using the AMV reverse transcriptase, while for the second-strand RNAse H and the Klenow fragment of DNA polymerase were used. The cDNA was ligated to Eco RI adaptors, then it was cloned in the Eco RI site of 2gtl0, packaged and plated on selective NM514 cells. The resultant library contained ca. 6 x 10 6 p.f.u. Screening of the cDNA library In all cases, duplicate filters were hybridized in 3 x SSC (0.3 M NaC1, 0.03 M sodium citrate), 0.5 To sodium dodecyl sulphate (SDS), 0.02~o Fi- coll, 0.02~o polyvinyl-pyrrolidone (PVP), 50 mg/ml salmon sperm DNA, 1 #g/ml poly(A) at 52 °C, and then washed in 3 x SSC, 0.5~o SDS, at 52 °C. PAL A PAL cDNA clone (pJAM824) from A majus [43], was used to screen about 100 000 recombinant clones in order to identify the ho- mologous grape clone. Washing of filters allowed the isolation of several strongly hybridizing clones. None of them represented a full-length cDNA. The longest clone was subcloned into the Eco RI site of pBSKS and named pBS204. CHS A CHS clone (pcLC46) from maize [66] was used to isolate the homologous grape clone. Approximately 100 000 p.f.u, were analysed and two types of full-length cDNA clone were isolated and subcloned into the Eco RI site of pBSKS. One of the two clones, named pBS305, was used for this work. CHI About 100 000 recombinant clones were probed with a CHI clone (pJAM823) from A majus [43]. A strongly hybridizing clone of the expected size (1 kb) was isolated, subcloned into the Eco RI site of pBSKS and named pBS407. F3H About 100 000 p.f.u, were screened with the clone pJAM239 from A majus coding for F3H [43]. Several clones were isolated. The long- est one was a full-length clone, that was sub- cloned into the Eco RI site of pB SKS and named pBS710. DFR A DFR clone (pJAM212) ofA. majus [43] was used to probe 100 000 recombinant phages. One strongly hybridizing full-length clone was isolated, subcloned into the Eco RI site ofpB SKS and named pBS510. LDOX The LDOX cDNA was isolated by prob- ing the library (100 000 p.f.u.) with the A majus clone pJAM322 coding for the function corre- sponding to the Candica locus [43 ]. Several clones were isolated. Amongst them one of the expected size (1.4 kb) was subcloned into the Eco RI site of pBSKS and named pBS603. UFGT About 100 000 recombinant clones were screened with the A majus clone pJAM338 cod- ing for the UFGT [43] and a strongly hybridiz- ing partial length clone (0.5 kb) was isolated. The insert was subcloned into the Eco RI site of pBSKS and named pBS801. StSy About 100 000 p.f.u, were screened using a synthetic oligonucleotide (G474: 5'ATGGCT- TCAGTTGA3') corresponing to the 5' region of a grape cDNA clone [46]. Several different clones were selected and one of them, named pBSK111, was subcloned into the Eco RI site of pBSKS.  All clones were sequenced following the pro- cedure described previously [56] using specific oligonucleotides as primers. 747 according to the manufacturer's instructions. Hy- bridization and filter washing were the same as for Southern blots. DNA extraction and Southern blots DNA was extracted from dark grown seedlings (Lambrusco f.f.) as previously described [62] with the addition of 1 ~o PVP to the extraction buffer. About 5 #g of genomic DNA, digested with a 6-fold excess of restriction enzyme, was sepa- rated by electrophoresis on an agarose gel and then transferred to a Biodyne B nylon membrane (Pall) according to the manufacturer's instruc- tions. DNA probes and filter hybridization were performed as previously described [63]; filters were washed in 0.1 x SSC, 0.2~o SDS at 65 °C. RNA extraction and northern blots Total RNA was extracted from seedlings (Lam- brusco f.f.) grown in darkness or exposed to light for a different number of hours. The method used the same extraction buffer as for DNA extraction and RNA was selectively separated from DNA by an overnight precipitation in a solution con- taining 2 M LiC1 and 0.8 M Urea. Approximately 10 #g of total RNA were separated on a forma- ldehyde gel [55] and after electrophoresis trans- ferred to a Biodyne B nylon membrane (Pall) Results Accumulation of anthocyanins in grape seedlings In general, synthesis of anthocyanins in response to light, as well as tissue-specificity of their accu- mulation, are genotype-dependent processes [7, 15, 42, 43, 63]. Here we report the analysis of different varieties of grape for their ability to ac- cumulate anthocyanins in darkness and for their tissue-specific production of anthocyanins. When exposed to continuous light, all varieties accumu- lated pigments in their hypocotyl while only three of the five varieties tested (Marzemino, Kolor, Gamay Tenturier) did it also in darkness (Table 1). Kolor and Gamay Tenturier also pro- duced anthocyanins in roots, the latter also in darkness. In order to investigate the role of light in promoting anthocyanin synthesis, the kinetics of pigment accumulation under white light were determined in Lambrusco f.f. In seedlings antho- cyanin production starts after 12 h of irradiation (0.48 OD530 per g fresh weight (f.w.) and increases up to 96 h of irradiation (12.98 OD530 per g f.w.). Different light qualities have also been employed in order to ascertain how grape seedlings respond in terms of anthocyanin production to different Table 1. Analysis ofanthocyanin tissue-specific distribution in seedlings (2 weeks old) of different grape varieties grownin darkness or irradiated with white light for 72 h. Values are referred to the percentage of plantlets containing anthocyanins. Variety Light Dark Number of seedlings analysed hypocotyl root hypocotyl root Lambrusco f.f. 100 Teroldego 100 Marzemino 100 Kolor 100 (Pinot Nero x Tenturier) Gamay Tenturier 100 0 0 0 167 0 0 0 172 0 5.5 0 135 68 37.7 0 117 68.6 53.2* 53.2* 140 * Pigmentation was present both in the hypocotyl and roots.
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