Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol151.Issue1.2000:

Plant Science 151 2000 1 – 8 A comparison of stilbene and chalcone synthases including a new stilbene synthase gene from Vitis riparia cv. Gloire de Montpellier P.H. Goodwin a, , T. Hsiang a , L. Erickson b a Department of En6ironmental Biology, Uni6ersity of Guelph, Guelph, Ont., Canada N 1 G 2 W 1 b Department of Plant Agriculture, Uni6ersity of Guelph, Guelph, Ont., Canada N 1 G 2 W 1 Received 13 May 1999; received in revised form 30 August 1999; accepted 30 August 1999 Abstract A stilbene synthase gene was cloned from Vitis riparia cv. Gloire de Montpellier after PCR amplification with primers designed to include the start and stop codons of stilbene synthase genes of V. 6inifera. The exon was very similar to that of other stilbene synthases, particularly those from V. 6inifera 99 nucleotide identity. An intron was found which interrupted the predicted codon for cysteine in the same location as in other stilbene and chalcone synthase genes. The intron showed high nucleotide identity 86 with an intron from a stilbene synthase gene of V. 6inifera. The V. riparia sequence was used in an evaluation of the relatedness of stilbene and chalcone synthases of plants. Five procedures involving distance, parsimony and maximum likelihood methods were used for constructing phylogenetic trees, and they yielded slightly to considerably different results. However, none of the trees were consistent with a previous hypothesis that stilbene and chalcone synthases cluster solely based on the genetic relatedness of the species, implying that stilbene synthase genes arose independently in plant families. In our analyses, stilbene and chalcone synthases of Vitis always clustered separately. The relatedness and origin of stilbene and chalcone synthases appears to be more complex than originally believed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Stilbene synthase; Chalcone synthase; Vitis riparia www.elsevier.comlocateplantsci

1. Introduction

Stilbenes are phenylpropanoid derivatives pro- duced by plants. The key enzyme for their synthe- sis is stilbene synthase, which catalyzes the addition of malonyl-CoA to 4-coumaroyl-CoA or cinnamoyl-CoA [1]. Stilbenes can be produced constitutively in wood of trees but can also be induced by plant stresses, such as wounding, ozone damage or disease [1]. Stilbenes are toxic to fungi and may contribute to disease resistance as phytoalexins. One reason for an increased interest in stilbene synthases in recent years is that the substrates for stilbene synthase are found widely in plants, and transfer of these genes to plants lack- ing stilbene synthase can increase their disease resistance. Expression of stilbene synthases has been reported in transgenic barley, oilseed rape, potatoes, rice, tobacco, tomatoes and wheat, and it has increased significantly disease resistance to a number of phytopathogenic fungi [2 – 6]. The cloning and sequencing of stilbene synthase genes revealed that they have many similarities to chalcone synthases, which are involved in flavonoid synthesis [7]. This appears to be related to the fact that both enzymes use the same sub- strates and share the same basic mechanism of catabolism [1]. However, each enzyme yields dif- ferent products, either stilbenes or naringenin chalcone, which differ in their ring structures. A comparison of stilbene and chalcone synthases showed 70 – 75 amino acid homology, an intron in a conserved location, and a conserved cysteine which is essential for the activity of both enzymes [1,8]. This has raised questions as to the evolution- Corresponding author. Tel.: + 1-519-824-4120, ext. 2754; fax: + 1-519-8370442. E-mail address : pgoodwinuoguelph.ca P.H. Goodwin 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 7 5 - 2 ary origins and relatedness of chalcone and stil- bene synthases. Studies of two different stilbene synthases of Pinus syl6estris showed that they had higher homology to chalcone synthase of P. syl6estris, than they did to the stilbene synthases of other plants [9,10]. Also, a consensus phylogenetic tree of chalcone and stilbene synthase proteins showed that stilbene synthases did not form a separate cluster but rather grouped with the chal- cone synthases of related families [11]. Since that report, there have been additional reported se- quences, including chalcone synthase sequences from Vitis 6inifera and a new stilbene synthase from V. riparia reported here, that were used in our analyses. Thus far, stilbene synthase genes have been cloned from a very limited number of plants, including two varieties of cultivated grapes V. 6 inifera, peanut Arachis hypogaea and two spe- cies of pines P. strobus and P. syl6estris [7,9,10,12 – 15]. The goal of this research was to re-examine the relatedness of stilbene synthases and chalcone synthases and to include a newly cloned stilbene synthase gene obtained from an- other species of Vitis, V. riparia. In our analyses, several different tree construction procedures in- volving distance, parsimony and maximum likeli- hood methods were used, and the results were compared.

2. Materials and methods