Condensed tannin biosynthesis genes are regulated separately from other flavonoid biosynthesis genes in apple fruit skin Adam Matthew Takos, Benjamin Ewa Ubi 1 , Simon Piers Robinson, Amanda Ruth Walker * Commonwealth Scientific and Industrial Research Organisation, Division of Plant Industry, Adelaide Laboratory, PO Box 350, Glen Osmond, South Australia 5064, Australia Received 16 June 2005; accepted 29 September 2005 Available online 25 October 2005 Abstract Although the molecular basis of anthocyanin biosynthesis has been studied in several types of fruit, little is known about how structural flavonoid genes are regulated to balance the requirement for condensed tannin (CT) and flavonol synthesis. We cloned three genes encoding enzymes involved in CT synthesis from cDNA of skin of the red apple variety Malus  domestica Borkh. cv. ‘Cripps Red’; two leucoanthocyanidin reductases (MdLARs) and an anthocyanidin reductase (MdANR). We measured steady-state transcript levels of the CT genes and the other structural genes of the flavonoid pathway by Real Time PCR and correlated this with flavonoid accumulation in skin throughout early fruit growth and during ripening. Transcripts of the MdANR gene were abundant in both green and red skin sectors early in development which correlated with accumulation of CTs in these tissues. The MdLAR1 gene transcripts were detected in green skin early in development but transcripts of MdLAR2 were present only in juvenile fruit. All the CT transcripts were much less abundant during fruit ripening when the majority of anthocyanin accumulated and transcripts of the other pathway genes were at a high level. The results indicated that transcription of the genes for synthesis of CTs in apple fruit skin was differentially regulated from the other flavonoid pathway genes which were regulated in a coordinate fashion to control synthesis of anthocyanins. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Anthocyanin; Tannin; Flavonoid; Regulation; Apple; Proanthocyanidin 1. Introduction Anthocyanins, condensed tannins (CTs) and flavonols are secondary metabolites called flavonoids that are found in most higher plants and are synthesized from common precursor compounds. Anthocyanins are responsible for most of the red and purple colours in flowers and fruits and also occur in leaves. During fruit development anthocyanin acts as a signal of ripening to birds and animals and therefore assists in seed dispersal [1,2]. CTs, also referred to as proanthocyanidins, are polymers of flavonoid molecules which, when oxidised, cause brown pigmentation of plant seeds. CTs may act as feeding deterrents in reproductive tissues and developing fruit [3,4] and also impart astringency to fresh fruit, fruit juices and wine. Flavonols, although colourless, can behave as co-pigments to anthocyanins in vivo [5], are required for pollen tube formation in some plants [6] and may also function as UV-B-protectants [7]. Anthocyanins, CTs and flavonols are all antioxidants and may have beneficial effects for human health when incorpo- rated in the diet [8]. Biochemical and genetic analysis in a range of plants has enabled the isolation of the structural genes of the flavonoid pathway (shown in Fig. 1) and ordering of the enzymes that they encode [9]. The pathway can be described as consisting of ‘common flavonoid’ genes required for synthesis of precursors of the different classes of flavonoids and ‘branch-specific flavonoid’ genes for each flavonoid class. It has been demonstrated recently that there are two possible branch pathways to formation of CTs in plant cells: (i) leucoantho- cyanidin reductase (LAR) synthesizing the 2,3-trans-flavan-3- ol [10] and (ii) a reaction catalysed by the product of the Arabidopsis BANYULS gene [11] which has been demonstrated to encode anthocyanidin reductase (ANR) to synthesize the 2,3- cis-flavan-3-ol [12]. In apple fruit (Malus  domestica Borkh.) www.elsevier.com/locate/plantsci Plant Science 170 (2006) 487–499 * Corresponding author. E-mail address: mandy.walker@csiro.au (A.R. Walker). 1 Current address: Department of Genetics and Biotechnology, Faculty of Science, University of Calabar, P.M.B. 1115-Calabar, Nigeria. 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.10.001